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FOOD AND BEVERAGE CONSUMPTION AND HEALTH SERIES
GARLIC CONSUMPTION AND HEALTH
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FOOD AND BEVERAGE CONSUMPTION AND HEALTH SERIES
GARLIC CONSUMPTION AND HEALTH
MIHAIL PĂCURAR AND GAVRIL KREJCI

EDITORS
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CONTENTS
Preface vii
Chapter 1 A Comprehensive Survey of Garlic Functionality 1
Alejandra Cardelle-Cobas, Ana Cristina Soria,
Marta Corzo-Martínez and Mar Villamiel
Chapter 2 Medicinal Properties of Garlic:
Importance of its Antioxidant Activity 61
Perla D. Maldonado, Daniel Limón, Sonia Galván-Arzate,
Abel Santamaría and José Pedraza-Chaverrí
Chapter 3 Cancer Chemoprevention by Garlic Constituents:
Potential Cellular and Molecular Targets 119
Molay K. Roy, Lekh R. Juneja and Tojiro Tsushida
Chapter 4 Trace Elements in Garlic 155
A. Gonzálvez, S. Armenta and M. de la Guardia
Chapter 5 Characterization and Discrimination of
Mediterranean Bulb-Producing Garlic 181
Giovanni Figliuolo and Stefania Mang
Chapter 6 Inhibitory Effects of Diallyl Sulfides from Garlic
(Allium Sativum L.) on Family X DNA Polymerase
Activity, and Human Cancer Cell Growth 199
Yoshiyuki Mizushina, Masayuki Nishida,
Yuko Kumamoto-Yonezawa, Isoko Kuriyama
and Hiromi Yoshida
Chapter 7 Garlic: A Promising Antidote to Heavy Metal Toxicity 215
Rajdeep Chowdhury and Keya Chaudhuri
Chapter 8 Phase Properties and Lipid Composition of Microsomal
Membranes from Storage Leaf of Garlic: Modifications
Induced by Sprouting Radioinhibition 231
Mónica B. Pérez and Clara A. Croci
vi Contents
Chapter 9 Health Effects of Garlic and Prostate Cancer 245

J. Arunakaran
Chapter 10 The Influence of Garlic (Allium sativum L., Alliaceae)
Extracts on the Pharmacodynamic Effects of Drugs 255
Biljana Bozin, Neda Mimica-Dukic and Isidora Samojlik
Index 267
PREFACE
Garlic (Allium sativum L.) is the edible bulb from a plant of the Allium genus,
commonly used for flavoring in cooking and for its beneficial effects for human health.
Despite the numerous therapeutic effects attributed to garlic, the chemistry behind its health-
promoting effects is still poorly understood. This book attempts to address this problem, as
well as the effects of garlic on cardiovascular diseases and the side effects that may develop
from the consumption of garlic. This book also briefly overviews the occurrence and
chemistry of garlic constituents, and reviews the latest evidence in accordance with anticancer
properties of garlic and/or garlic constituents, with special emphasize on the mechanism of
action behind their chemoprevention effects. Furthermore, the research advances on the
chemistry and pharmacology of garlic and the potential and molecular mechanism of garlic
mediated attenuation of heavy metal toxicity are discussed. Other chapters in this book
explore the relationship between garlic consumption and prostate cancer, among other
diseases and the effects of gamma rays on storage leaf of garlic bulbs in terms of phase
properties of microsomal membranes and their lipid and fatty acid composition.
Chapter 1 - Garlic (Allium sativum L.) is the edible bulb from a plant of the Allium genus,
commonly used for flavouring in cooking and for its beneficial effects for human health. Although
garlic cloves are usually eaten raw or cooked, different garlic dietary supplements including dried
or powdered formulations, oils and liquid extracts have being recently incorporated into the
market to satisfy the demand of consumer for garlic bio-active compounds.
Despite the numerous therapeutic effects attributed to garlic, the chemistry behind its
health-promoting effects is still poorly understood. Garlic is a major source of sulfur-
containing compounds, particularly S-alk-(en)yl-L-cysteine sulphoxides (Cs), being alliin the
major one. Volatiles such as allicin, and lipid-soluble sulphur compounds such as diallyl
sulphide, diallyl disulphide, diallyl trisulphide, dithiins, ajoene and others, are originated from
ACSOs by different metabolic pathways after tissue damage of garlic by cutting, crushing or
bitting. These compounds provide to garlic its characteristic odour and flavour, as well as most of
its biological properties. The effect of garlic on cardiovascular diseases, including
hypocholesterolemic, anti-hypertensive, antithrombotic, and anti-hyperglycaemic activities, is one
of its most extensively studied benefits. Garlic intake has also been described to reduce the risk for
developing several types of cancer, especially those of the gastrointestinal tract (colon and
stomach). Other bioactivities previously described in garlic include antimicrobial, antioxidant,
antiasthmatic, immunomodulatory and prebiotic effects.
viii Mihail Păcurar and Gavril Krejci
Recently, it has been demonstrated that additional garlic constituents such as organo-
selenium compounds, steroid saponins and sapogenins (e.g. β-chlorogenin), vitamins B6 and
B12, flavonoids (e.g. allixin), lectins and N-fructosyl-aminoacids, may contribute, along with
organo-sulphur compounds, to the above mentioned biological effects of this vegetable.
Despite garlic can cause side effects, including gastrointestinal distress, allergic and
asthmatic reactions, and interfere with a few medications, its use as therapeutic agent seems
to be safe, since these adverse effects appear with an excessive and prolonged consumption.
Thus, the efforts of research should be directed to determine the effective intake to note the
beneficial properties as well as the most suitable preparation to avoid undesirable effects.
Chapter 2 - Garlic (Allium sativum) is among the oldest of all cultivated plants. It has
been used as a medicinal agent for thousands years. This remarkable plant has multiple
beneficial effects such as antimicrobial, antithrombotic, hypolipidemic, antiarthritic,
hypoglycemic, antitumor and antioxidant activities. A large number of studies have
demonstrated the antioxidant activity of garlic by using different preparations, including fresh
garlic extract, aged garlic extract, garlic oil, and a number of organosulfur compounds
including alliin, allicin, and S-allylcysteine. These studies have been carried out both under in
vivo - in diverse experimental animal models associated to oxidative stress - and in vitro
conditions - using several methods to evaluate capacity of extracts or compounds to scavenge
reactive oxygen species or to induce oxidative damage -. Derived from these experiments, the
protective effects of garlic have been associated with a prevention or amelioration of
oxidative stress. In addition, it has been shown that several garlic preparations and/or
organosulfur compounds derived from garlic are able to directly scavenge reactive oxygen
species, including hydrogen peroxide, superoxide anion, hydroxyl radical, and peroxynitrite.
Moreover, it has been shown that garlic is able to inhibit inducible nitric oxide synthase,
which in turn may contribute to decrease nitrosative damage. In addition, as supporting
evidence of the protective properties of garlic compounds, in this review we are including
some original data of the ameliorative action of S-allylcysteine on the aberrant circling
behavior exerted by 6-hydroxydopamine in rats. In summary, the antioxidant activity of garlic
has been clearly characterized in in vivo and in vitro studies, thus emphasizing its potential
use as a therapeutic agent against different disorders.
Chapter 3 - Plant products or their metabolites have been utilized for both nutritional and
medicinal purposes throughout the history of human civilization. In particular, Allium
vegetables such as onions and garlic are among the oldest agricultural products in use as
spices for food preparation as well as a remedy for several ailments. In general, garlic, a rich
source of some phytochemicals, has been known for its medicinal uses as an antibiotic,
antithrombotic and antineoplastic agent [1]. Accumulated evidence suggests that garlic and
some of its constituents are effective in suppressing the incidence of several diseases
including cardiovascular diseases, diabetes, obesity, gastrointestinal disorders and cancer.
Among these diseases is cancer in advanced metastasized stage, which is considered
incurable. Therefore, prevention rather than treatment has become a strategy to minimize the
occurrence and development of this deadly health disorder.
Recent knowledge in cell technology and a growing understanding of the cellular and
molecular etiology of cancer have made it easier than ever to assess the efficacy of a
prospective molecule against the proliferation of cancer. In numerous studies, garlic extract as
well as some of its active constituents, particularly organosulfur compounds, have been
investigated for their value in inhibiting, retarding or reversing the dysfunctional cell-
Preface ix
signaling pathway in cancer cells. It appears that garlic and/or its organosulfur constituents
exert anticancer effects through multiple mechanisms that include modulation of carcinogen
metabolism, inhibition of DNA adduct formation, up regulation of detoxifying enzymes [2]
and DNA repair systems, and regulation of cell proliferation, apoptosis and immune
responses [3]. There has been convincing evidence showing that garlic constituents such as S-
allylmercaptocysteine, diallyl disulfide, and S-trityl-L-cysteine modulate NF-kappaB [4], AP-
1 [5], Akt [6], and MAPK [7] signaling pathways by regulating a number of downstream
signaling molecules in the anticancer mechanisms.
Based on the available evidence, this chapter briefly provides an overview of the
occurrence and chemistry of garlic constituents. This chapter also lists the latest evidence in
accordance with the anticancer properties of garlic and/or garlic constituents with special
emphasis on the mechanism of action behind their chemoprevention effects. This
contribution, furthermore, provides a realistic broad view of areas of future research for
promoting garlic as a functional food ingredient.
Chapter 4 - The State-of-arte of the scientific literature concerning the presence of trace
elements in garlic samples has been evaluated trough the discussion of methods proposed for
both, sample preparation and measurement, of all studied elements. Special attention has been
paid to the method of choice and the most appropriated and safe preparation strategy. Data
concerning 41 elements in different origin garlic samples are also presented.
Chapter 5 - Allium ampeloprasum is bulb-producing garlic less common than A. sativum
and poorly investigated for agricultural and pharmaceutical use. This species is cultivated as
landrace mixed with Common garlic (CG) in rural farmer fields of the Mediterranean
Countries. In USA, because of its spectacular size, this domesticated garlic is named as
“Elephant garlic” or “Great headed garlic” (GHG). The Mediterranean region is the
geographic area of evolution and differentiation of the primary gene-pool of A.
ampeloprasum (wild Leek) represented by an array of different citotypes. Following the
domestication, in the above mentioned region, have been isolated and selected garlic types
with a ploidy level greater than 4X yielding big bulbs. Few of these clones, along with
humans, migrated to the New World since the 20th century. Overall, the wild forms of A.
ampeloprasum, GHG, cultivated Leek and Kurrat, upon a taxonomic point of view, given the
variable plant morphology and ploidy, are considered members of the same species-complex.
Great headed garlic compared with A. sativum (soft-neck form) shows differences at
morphologic, cytogenetic and molecular level. Great headed garlic bulb yield is higher than
A. sativum. Differences in the heritability of yield related traits between CG and GHG reflect
both different ploidy levels and genetic backgrounds. From a breeding perspective differences
in heritability are relevant to select the best clones. The heritable traits more correlated with
yield are plant height within A. sativum and neck diameter within A. ampeloprasum.
Moreover, karyotype analysis and DNA markers are perfect tools to determine genetic
relationships among A. ampeloprasum and its relatives. The genetic profile at diagnostic
sequences such as the M13 DNA repeated sequence, nucleotide variation at Internal
Transcribed Spacer (ITS) of the ribosomal gene together with morphological traits indicate
that different citotypes from various origins are probable members of the same species-
complex (A. ampeloprasum). The M13 polymorphism can be relevant to distinguish with a
single analysis A. ampeloprasum plant materials from A. sativum.
Chapter 6 - Diallyl sulfides are characteristic flavor components obtained from garlic.
These compounds are thought to be responsible for their epidemiologically proven anti-
x Mihail Păcurar and Gavril Krejci
cancer effect on garlic eaters. Diallyl sulfides are selectively inhibitors of mammalian family
X DNA polymerases (pols) such as pol β, pol λ and terminal deoxynucleotidyl transferase
(TdT), in vitro, and the order of their effect is as follows: Sample-A (the purified fraction
from garlic consisted of diallyl trisulfide, diallyl tetrasulfide and diallyl pentasulfide
[molecular ratio: 5.3 : 3 : 1]) > diallyl trisulfide > diallyl disulfide > diallyl monosulfide,
suggesting that the number of sulfur atoms in the compounds may play an important
structural role in enzyme inhibition. The inhibitory effect of Sample-A on rat pol β, human
pol λ and calf TdT is dose-dependent, and 50 % inhibition was observed at a concentration of
9.7, 34.5 and 37.1 μM, respectively. The compound influences neither the activities of other
eukaryotic pols such as α, γ, δ, ε, η, ι and κ, prokaryotic pols, nor the other DNA metabolic
enzymes tested. The inhibitory effect of N-terminal truncated pol λ lacking a BRCA1 C-
terminus (BRCT) domain and a proline-rich region (residues 245–575, Del-2 pol λ) is
stronger than that of intact pol λ (i.e., residues 1–575, Full-length pol λ) and truncated pol λ
lacking a BRCT domain (residues 133–575, Del-1 pol λ). Sample-A-induced inhibition of
pol X activities is competitive with respect to both the DNA template-primer and the dNTP
substrate. The suppression of human cancer cell (promyelocytic leukemia cell line, HL-60)
growth has the same tendency as the inhibition of pol X family among compounds. Allicin
and alliin, which are organosulfur compounds containing garlic, hardly affect their activities.
In conclusion, diallyl sulfides are suggested to bind to the pol β-like region of family X pols,
and have anti-cancer activity.
Chapter 7 - Garlic for centuries has been well known for its medicinal attributes in
addition to its other virtues. Garlic in different forms has antioxidant properties. These
properties are shown to be due to the existence of compounds such as water soluble
organosulfur compounds, S-allylcysteine and lipid soluble compounds like diallyl sulfide. It
shows phenomenal ameliorating properties against heavy metal poisoning due to its
possession of chemicals containing organo-sulfur groups, volatile oils, enzymes,
carbohydrates and amino acids. With the threat of heavy metal poisoning increasing every
day and lead, mercury, cadmium, arsenic, and copper poisoning gradually attaining alarming
proportions, garlic was extensively exploited to treat the metal-induced toxicities. Recent
supportive evidences indicate that garlic contain compounds capable of detoxifying lead,
cadmium, methlymercury, phenylmercury and arsenic. The restorative property of garlic was
attributed to its antioxidant activity and/or chelating efficacy. The clastogenic effects of the
heavy metals were also pronouncedly reduced by dietary administration of garlic. Fatal
effects with respect to body metal burden, oxidative stress and mitochondrial injury were
effectively reduced by garlic. The curative effect of garlic was superior to those of 2,3-
dimercapto-1-propanol (BAL) and D-penicillamine(PEN), 2,3-dimercaptosuccinic acid
(DMSA) and N-acetyl-DL-penicillamine (APEN), and the current remedies. In this
commentary, the research advances on the chemistry and pharmacology of garlic and the
potential and molecular mechanism of garlic mediated attenuation of heavy metal toxicity are
discussed.
Chapter 8 - The aim of the present work was to evaluate the effect of gamma rays on the
storage leaf of garlic bulbs in terms of phase properties of microsomal membranes and their
lipid and fatty acid composition in order to correlate these features with sprouting inhibition
induced by gamma irradiation.
Preface xi
Garlic bulbs were irradiated 30 days after harvest with an average dose of 60 Gy of Co-
60 gamma rays. Rough and smooth microsomal membranes were isolated by
ultracentrifugation from tissues of irradiated and non-irradiated storage leaves. The integrity
of the microsomes was corroborated by transmission electron microscopy. Wide-angle X-ray
diffractograms of both fractions were recorded along 240 days of storage using PW 1700
diffractometer. Lipids were separated by thin layer chromatography. The fatty acid
composition of major lipid fractions was studied by gas-liquid chromatography.
The diffractograms featured peaks at Bragg spacing of 4.15 Å and 3.75 Å, revealing the
presence of a gel (crystalline) phase, while the characteristic peak of the liquid-crystalline
phase (4.6 Å) was not observed in both sorts of membranes. Irradiation was found to bring
about modifications in the intensity of 4.15 Å and 3.75 Å peaks from smooth microsomal
membranes, but not in the behavior along the period studied. Data from the rough microsomal
fraction were erratic and unreliable. Parallel to these changes, radiation induced significant
modifications in the level of smooth microsomal membrane triacyglycerols in relation to
phospholipids and their fatty acids.
These findings indicate that the storage leaf tissues of garlic are radiosensitive in terms of
physical and chemical properties of their smooth microsomal membranes. The significance of
the results in relation to proving the application of the radioinhibition process in garlic bulbs
is presented.
Chapter 9 - Garlic (Allium sativum) has been used for thousands of years for medicinal
purposes. Garlic ranks highly among foods that help to prevent disease, largely due to its high
content of organosulfur compounds and antioxidant activity. Aged garlic extract (AGE) has
been found to prevent atherosclerosis and protect against cardiovascular disease, increase
circulation and immunity. AGE has been shown to prevent various kinds of cancer,
neurodegenerative disease and has antiaging effects in improving memory, endurance and
learning [1].
Garlic traditionally has been used as a natural antibiotic thought to protect against
infection, to lower blood pressure and to treat atherosclerosis, asthma, arthritis, cancer and
circulatory problems.
It grows in the form of bulb. Sanskrit records show its medicinal use about 5000 year
ago. It has been used for at least 3000 years in Chinese medicine. The Egyptians,
Babylonians, Greeks and Romans used garlic for healing purposes. In 1859, Pasteur noted
garlic’s antibacterial activity and it was used as an antiseptic to prevent gangrene during
World War I and World War II.
Historically garlic has been used around the World to treat hypertension, infections and
snake bites etc. Currently, garlic is used for reducing cholesterol levels and cardiovascular
risk as well as for its antineoplastic and antimicrobial properties.
Garlic has a high concentration of sulfur containing compounds. The thiosulfinates,
including allicin, appear to be the active substances in garlic. Allicin is formed when alliin a
sulphur containing amino acid comes into contact with the enzyme alliinase when raw garlic
is chopped, crushed or chewed. Dried garlic preparations containing alliin and alliinase must
be enteric coated to be effective because stomach acid inhibits alliinase. Because alliinase
also is deactivated by heat, cooked garlic is less powerful medicinally. The antimicrobial,
hypolipidemic, antioxidant and anti-thrombotic effects that have been attributed to garlic are
thought to be related to allicin and other breakdown products. The antineoplastic effects may
be related to the sulfur compounds or to other unknown components [1].
xii Mihail Păcurar and Gavril Krejci
Allicin, S- Allyl cysteine (SAC) and Ajoene are the three active compounds found in
varying amounts in flesh garlic. Allicin a chemical formed when garlic is crushed appears to
have antibacterial properties. SAC has been shown to be effective against the initiation of
tumors in animals. Ajoene appears to be an anti-blood clotting agent.
Garlic supplementation contains allicin, an odorless precursor of the garlic smell in the
active compounds of allicin and ajoene. The enzyme alliinase is needed to convert alliin to
allicin and ajoene. Fresh garlic in large quantities can lower cholesterol levels. Garlic thins
the blood; it may lower blood pressure [2].
Garlic has been used throughout the world to treat coughs, tooth ache, earache, dandruff,
hypertension, hysteria, diarrhoea, dysentery, diphtheria, vaginitis and many other conditions.
Garlic contains a complex mixture of oil and water soluble organosulfur compounds. Oil
soluble compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl
trisulfide (DATS) are effective as antiproliferative against various tumors. The water soluble
compounds S-allyl cysteine, S-ethyl cysteine and S-propyl cysteine have little effect.
Chapter 10 - Garlic (Allium sativum L., Alliaceae) has played one of the most important
dietary and medicinal roles in many cultures for centuries. It has been used as a spice and
condiment and, due to its potential benefits, in preventive and curative treatments.
Epidemiological, clinical and preclinical studies have shown the close relationship between
dietary habits, including garlic intake, and the occurrence of diseases. A wide array of
therapeutic effects such as hypolipidaemic, antiatherosclerotic, hypoglycaemic, anticoagulant,
antihypertensive, antimicrobial and hepatoprotective action have been reported. However, the
most common complication of garlic use is prolonged bleeding and side effects including
halitosis, nausea, hypotension, headache and bloating. Drug-herb interactions with
hypoglycaemics, cardiovascular medications and monoamine oxidize inhibitors also have
been reported. With respect to this, the aim of the study was to examine the influence of polar
extracts derived from garlic on pentobarbitone- and thiopentone-induced sleeping time,
midazolam-induced impairment in motor coordination and analgesic effect of codeine in
mice. The examined extracts were obtained from the immature garlic plant, ground and air-
dried garlic bulbs (prepared as an aged garlic extract) and fresh garlic bulbs, with determined
content of total phenolics, flavonoid glycosides and aglycones. The animals were divided into
four groups (three extracts’ groups and a control) according to peroral pretreatment regime
with a particular extract during five consecutive days. Tested drugs were administered 2 h
after the application of particular extract on the fifth day of experimental procedure, and the
measurements were done according to survey protocol. Pretreatment with all extracts
produced changes in both pentobarbitone- and thiopentone-induced sleeping time. Also,
examined extracts exhibited notable changes in induction time after the application of both
hypnotic drugs. All three extracts reduced motor impairment caused by midazolam and
decreased the analgesic effect of codeine. Because garlic alone does not induce sleep, motor
discoordination or analgesic effect, and because its use produces changes in tested drug
effects, the interaction between drugs and phytopreparations containing garlic should be
additionally examined/confirmed in humans.
In: Garlic Consumption and Health ISBN: 978-1-60741-642-5
Editors: M. Pacurar, G. Krejci, pp. 1-60 © 2010 Nova Science Publishers, Inc.
Chapter 1
A COMPREHENSIVE SURVEY OF GARLIC

FUNCTIONALITY
Alejandra Cardelle-Cobas, Ana Cristina Soria,

Marta Corzo-Martínez and Mar Villamiel*
Instituto de Fermentaciones Industriales (CSIC),
c/Juan de la Cierva, 3 28006 Madrid Spain
ABSTRACT
Garlic (Allium sativum L.) is the edible bulb from a plant of the Allium genus, commonly
used for flavouring in cooking and for its beneficial effects for human health. Although garlic
cloves are usually eaten raw or cooked, different garlic dietary supplements including dried or
powdered formulations, oils and liquid extracts have being recently incorporated into the
market to satisfy the demand of consumer for garlic bio-active compounds.
Despite the numerous therapeutic effects attributed to garlic, the chemistry behind its
health-promoting effects is still poorly understood. Garlic is a major source of sulfur-
containing compounds, particularly S-alk-(en)yl-L-cysteine sulphoxides (Cs), being alliin
the major one. Volatiles such as allicin, and lipid-soluble sulphur compounds such as
diallyl sulphide, diallyl disulphide, diallyl trisulphide, dithiins, ajoene and others, are
originated from ACSOs by different metabolic pathways after tissue damage of garlic by
cutting, crushing or bitting. These compounds provide to garlic its characteristic odour and
flavour, as well as most of its biological properties. The effect of garlic on cardiovascular
diseases, including hypocholesterolemic, anti-hypertensive, antithrombotic, and anti-
hyperglycaemic activities, is one of its most extensively studied benefits. Garlic intake has
also been described to reduce the risk for developing several types of cancer, especially those
of the gastrointestinal tract (colon and stomach). Other bioactivities previously described in
garlic include antimicrobial, antioxidant, antiasthmatic, immunomodulatory and prebiotic
effects.
Recently, it has been demonstrated that additional garlic constituents such as
organo-selenium compounds, steroid saponins and sapogenins (e.g. β-chlorogenin),
*
Corresponding author: e-mail: mvillamiel@ifi.csic.es , Tel: +34 915622900, Fax: +34 915644853
2 Alejandra Cardelle-Cobas, Ana Cristina Soria, Marta Corzo-Martínez et al.
vitamins B6 and B12, flavonoids (e.g. allixin), lectins and N-fructosyl-aminoacids, may
contribute, along with organo-sulphur compounds, to the above mentioned biological
effects of this vegetable.
Despite garlic can cause side effects, including gastrointestinal distress, allergic and
asthmatic reactions, and interfere with a few medications, its use as therapeutic agent
seems to be safe, since these adverse effects appear with an excessive and prolonged
consumption. Thus, the efforts of research should be directed to determine the effective
intake to note the beneficial properties as well as the most suitable preparation to avoid
undesirable effects.
INTRODUCTION
Genus Allium is formally classified in the family Liliaceae, represented by 280 separate
genera and 4000 species. However, recent taxonomic revisions have seen members of this
genus placed in the family Alliaceae. Of the approximately 700 species Allium, the edible
members, including onion (A. cepa L.), garlic (A. sativum L.), chives (A. schoenoprasum L.),
leek (A. porrum L.) and Welsh onion (A. fistulosum L.) are highly prized (Fenwick & Hanley,
1985). Among them, garlic is one of the oldest cultivate plants. Its possible ancestor appears
to be A. longicuspis, a native in the mountainous regions of central Asia, which later spread to
China, the Near East, and the Mediterranean regions before moving west to Central and
Southern Europe, Northern Africa (Egypt) and Mexico (Lutomski, 1987). Today, garlic
cultivation is distributed throughout most regions of the temperate world.
Garlic has been used as spice and food ingredient in cooking all over the world because
of it combines well with an enormous range of foods, adding its own aroma and flavour as
well as enhancing the flavours of the foods with which it is mixed (Woodward, 1996).
Besides to be used like food, garlic has long been used in folk medicine with protective and
curative purposes.
The earliest indication of the use of garlic is in clay models in Egyptian cemeteries, dated
to as early as 3,750 B.C. (Woodward, 1996). It was part of the staple diet of the Egyptian
pyramid builders and several cloves of garlic were also found in the tomb of Tutankamen.
The pharaohs believed that by taking garlic to the afterlife, the food there would always be
well seasoned. The Codex Ebers, an Egyptian medical papyrus dated to about 1550 B.C. and
translated in 1937, contains over 800 therapeutic formulas of which 22 mention garlic as an
effective remedy for a variety of ailments including heart problems, headache, bites, worms
and tumors (Block, 1985). Garlic is also mentioned in the literature of Ancient Israel (The
Talmud) and in the Bible during the time of the exodus. The Romans also extolled the virtues
of garlic. Pliny the Elder, a Roman naturalist, described in his Historia Naturalis how garlic
could be used for gastrointestinal disorders, dog and snake bites, scorpion stings, asthma,
madness, convulsions, tumors and constipation. Dioscorides, a chief physician to the Roman
army in the first century A.D., prescribed garlic as a vermifuge or expeller of intestinal
worms. Likewise, in Babylonian and Greek civilizations, use of garlic has been recorded by
Hippocrates, “the Father of Medicine”, as an effective laxative and diuretic, by Aristophanes
and Galen as excellent for the treatment of uterine tumors, and by Aristotle as a cure for
rabies. During the first Olympic Games in Greece in 776 B.C., athletes ingested garlic as
stimulant (Fenwick & Hanley, 1985; Block, 1985). In China, garlic tea has long been
recommended for fever, headache, cholera, dysentery and prolonging longevity (Srivastava et
A Comprehensive Survey of Garlic Functionality 3
al., 1995) and in India, garlic has been used for centuries for the treatment of hemorrhoids,
rheumatism, dermatitis, abdominal pain, cough and as an antiseptic lotion for washing
wounds and ulcers, due to its antibacterial properties. Indeed, the realisation in 1858 by the
French Louis Pasteur that garlic had potent antibacterial properties later led to its use in the
First and Second World Wars, when penicillin and sulfa drugs were scarce, as an antiseptic to
disinfect open wounds and prevent gangrene.
Nowadays, garlic is being still employed in folk medicine for over the world for the
treatment of various ailments such cardiovascular diseases, cancer and microbial infections
(Ali et al., 2000).
THE CHEMISTRY OF GARLIC

Some of the nutritional and chemicaI properties of garlic bulbs are given in Table 1.
Garlic has been analysed for moisture, carbohydrates, protein, fat, minerals, vitamins, energy,
ash, pH, acidity and essential oil contents (Haciseferogullari et al., 2005). Protein content was
found to be considerably higher than that in other vegetables such as bean and pea
(Cemeroglu & Acar, 1986), but crude oil content was considerable lower. Garlic moisture
was also low as compared to caper bud and caperberries fruits (Ozcan & Akgül, 1998; Ozcan,
1999) and other vegetables (Cemeroglu & Acar, 1986). Among minerals, garlic is known to
contain high levels of potassium (21 g/kg), phosphorous (6 g/kg) followed by magnesium (1
g/kg), sodium (532.78 mg/Kg), calcium (363.61 mg/Kg) and iron (52.91 mg/Kg). In addition,
garlic also contains the minerals selenium and germanium. The amount of these minerals in
the bulb depends on the content of the respective minerals in the soil where the bulb is grown.
Vitamins like riboflavin, thiamine, nicotinic acid, vitamin C and vitamin E are other
important chemical constituents.
The biological effects of some of these constituents in intact garlic, such as lectins (the
most abundant proteins in garlic), prostaglandins, fructan, pectin, adenosine, vitamins B1, B2,
B6, C and E, biotin, nicotinic acid, fatty acids, glycolipids, phospholipids and essential amino
acids, have been studied for over several decades (Fenwick & Hanley, 1985). Recently,
special attention has been given to certain steroid saponins and sapogenins such as β-
chlorogenin. Several studies have demonstrated the importance of their biological and
pharmacological activities such as antifungal, antibacterial, antitumor, anti-inflammatory,
antithrombotic and hypocholesterolemic properties (Matsuura, 2001; Lanzotti, 2006). Since
β-chlorogenin is bioavailable in vivo and detected in blood, this indicates that β-chlorogenin
may be a bioactive compound in garlic. Other characteristic chemical constituents of garlic
include allixin and organo-selenium compounds. These chemical compounds are reported to
exhibit several biological effects, including cholesterol reduction, cancer prevention and
others (Amagase, 2006).
However, despite the fact that the above mentioned compounds contribute in part to
garlic bioactivity, evidence from several investigations suggests that the biological and
medical functions of garlic are mainly due to their high content in organo-sulphur compounds
(Augusti & Mathew, 1974; Wargovich et al., 1988), which likely work synergistically with
other compounds such as organo-selenium compounds.
Table 1. Nutritional value and properties of garlic. Values expressed per 100 g of raw garlic.
Properties Values Minerals Values Vitamins Values
Energy 119 kcal Potassium 446 mg Thiamin (Vit. B1) 0.16 mg
Moisture 70 % Phosphorus 134 mg Riboflavin (Vit. B2) 0.02 mg
Protein 4.3 g Magnesium 24.1 mg Niacin (Vit. B3) 1.02 mg
Carbohydrate 24.3 g Sodium 19 mg Piiridoxin (Vit. B6) 0.32 mg
Fiber 1.2 g Calcium 17.8 mg Folic acid 4.8 μg

Fat 0.23 g Iron 1.2 mg Ascorbic acid (Vit. C) 14 mg
Alcohol 0g Zinc 1.1 mg Carotenoids (β-carotenes)) 5 μg
Ash 2.3 %
Iodine 4.7 μg Vitamin A Traces
pH 6.05
Selenium 2 μg Vitamin E (Tocopherols) ) 0.011 μg
Acidity 0.172 %
Intact garlic cloves contain only a few medicinally active compounds (Block, 1992;
Lawson, 1993). The primary sulphur-containing constituents in whole garlic are the S-
alk(en)yl-L-cysteine sulfoxides (CSs, 1.8%) and γ-glutamyl-S-alk(en)yl-L-cysteine
peptides (0.9%), both non-volatile and, therefore, odour-free sulphur compounds (Figure
1). It has been stimated that S-allyl-L-cysteine sulphoxide (alliin [1]) and S-methyl-L-
cysteine sulphoxide (methiin), the major CSs in garlic, together with S-(2-
carboxypropyl)glutathione, γ-glutamyl-S-allyl-L-cysteine, γ-glutamyl-S-(trans-1-
propenyl)-L-cysteine and γ-glutamyl-S-allyl-mercapto-L-cysteine, make up more than
82% of the total sulphur content of whole garlic (Sugii et al., 1964; Fenwick & Hanley,
1985; Sendl, 1995). The γ-glutamylcysteine peptides are biosynthetic intermediates for
corresponding CSs (Lancaster & Shaw, 1989). On prolonged storage or during
germination, the enzyme γ-glutamyl transpeptidase acts on γ-glutamylcysteine peptides to
form thiosulfinates (Sendl, 1995) such as S-allyl-cysteine (SAC [2]), which is also
present in intact garlic and contributes heavily to the health benefits of some garlic
preparations (Amagase et al., 2001). The thiosulfinates other than SAC (e.g. allicin [3])
as well as other oil-soluble components such as ajoenes [4] (e.g. E-ajoene and Z-ajoene),
vinyldithiins [5] (e.g. 2-vinyl-(4H)-1,3-dithiin and 3-vinyl-(4H)-1,2-dithiin), and sulfides
(e.g. diallyl sulphide, DAS [6], diallyl disulphide, DADS [7], and diallyl trisulphide,
DATS [8]), provide to garlic its characteristic odour and flavour as well as most of their
biological properties (Lanzotti, 2006), but they are not naturally occurring compounds in
intact garlic. When garlic is cut, crushed, chewed, dehydrated or otherwise processed, the
vacuolar enzyme, alliinase, is released and rapidly lyses the cytosolic CSs (mainly alliin),
which are converted into hundreds of organo-sulphur compounds in a short period of
time. First, it is formed the reactive intermediate allylsulfenic acid (R-SOH), which
immediately condenses to form the odoriferous alkyl alkane- thiosulfinates, among
which, allicin represents 70-80% of total. Then, allicin (allyl 2-propene thiosulfinate) and
other thiosulfinates such as allyl methane thiosulfinate, which are very unstable products,
instantly undergo a number of transformations, giving rise to other sulphur-compounds
derivatives (e.g. products [4-10]), depending on environmental and processing conditions
(as temperature, pH and solvent polarity) (Block, 1985; Reuter & Sendl, 1995; Amagase,
2001) (Figure 1). Sulphur-containig compounds in commercial garlic preparations vary,
depending on their manufacturing processes. Likewise, the variety of garlic determines
the composition and quantity of each CS identified in garlic, which, in turn, determine the
odour, flavour variation and biological activities observed for garlic.
In addition to odoriferous oil-soluble compounds, less odorous water-soluble
organosulphur compounds such as SAC and S-allylmercaptocysteine (SAMC) have
shown to be biologically active in several areas. The non-volatile sulphur-containing
compounds SAC and SAMC are present in several garlic preparations, although the
content varies considerably (Lawson, 1993; Imai et al., 1994).
Given such chemical diversity, garlic has received considerable attention from both
chemist and biologist alike as new source of bioactive compounds.
8
4
5
Figure 1. Formation of organo-sulphur compounds during metabolic pathways in processed garlic.
Reprinted from Trends in Food Science & Technology, 18, 609-625. Biological properties of onions
and garlic by Corzo-Martínez et al. (2007); with permission from Elsevier.
GARLIC CONSUMPTION AND GARLIC SUPPLEMENTS

The worldwide trade of garlic has increased in the last years due to changes in consumer
habits. The global production displayed an increase of 35% over the period 1998-2003 (from
9.1 to 12.1 million tons), which resulted in an increase of 13% in the yield and of 18% in the
cropped area (from 0.95 to 1.125 million hectares). According to FAO 2005, global
production of garlic is close to 15 million tons and it is estimated that the cropped area has not
undergone great changes in recent years.
Several products of garlic are available in the international market and their popularity has
increased in the last decade. The strong odour of fresh garlic has influenced to the consumers
towards these commercial products as an optimal choice for increasing daily garlic intake.
The variety and manufacturing process of garlic are important considerations when
choosing a garlic supplement, since, as indicated previously, they can markedly influence the
composition of the garlic product and, therefore, its biological effects and toxicity (Fenwick
& Hanley, 1985; Kritchevsky, 1991; Banerjee et al., 2003). Garlic products that contain the
most safe, effective, stable, and odourless components are the most valuable as dietary
supplements. Documentation of the safety and effectiveness is crucial in the evaluation of all
garlic products that are proposed for use health promotion (Amagase, 2001).
Garlic supplements can be classified into four groups: garlic essential oil, garlic powder,
garlic oil macerate and garlic extract (Table 2). Garlic essential oil is obtained by steam distillation
of garlic and consists of a variety of sulfides such as DAS, DADS and DATS (Block, 1985; Yan
et al., 1992). Commercially available garlic oil capsules generally contain vegetable oil and a
small amount of garlic essential oil because of pungent odors. Garlic powder is mass-produced as
a flavouring agent for condiments and processed foods. Garlic cloves are sliced or crushed, dried
and pulverized into powder. Garlic powder is thought to retain the same ingredients as (crushed)
raw garlic, mainly alliin; however, amounts may vary significantly (Amagase, 2001). Oil
macerates were originally developed for use as condiments. There are two types of oil macerate
products on the market and both are packaged in soft gel capsules. One is made by simply mixing
a garlic flavoring powder with vegetable oil. Its constituents are almost the same as the capsule
and tablet forms of garlic powder. Another one is made by grounding raw garlic into vegetable oil.
This type of product contains leftover alliin and allicin-decomposed compounds such as dithiins,
ajoene and sulfides and, therefore, it has a strong garlic odor. For garlic extract, whole or sliced
garlic cloves are soaked in an extracting solution (e.g. purified water and diluted alcohol) for
varying amounts of time. After separation of the solution, the extract is generally concentrated and
used. Powdered forms of the extract are also available. These aqueous or alcoholic extracts contain
primarily water-soluble sulphur-compounds. In particular, KYOLIC aged garlic extract (AGE) is
one of the most popular brands on the market. AGE is obtained by storage at room temperature of
sliced and soaked in a water/ethanol mixture raw garlic for longer than 20 months (Amagase,
2006). It contains mainly the water-soluble sulphur-compounds SAC and SAMC, as well as small
amounts of oil-soluble sulphur compounds.
One of the most important considerations in the above mentioned products is their
standardization, which is the key to delivering consistent quality and efficacy of garlic
products to consumers. It was initially thought that allicin was the main active substance in
vitro of garlic; however, its effects in vivo are questionable. Several studies have revealed that
the bioavailability of allicin is poor due to its great instability, not being detected in the blood
or urine after the oral ingestion of raw garlic (Lawson et al., 1992). Currently, it is well
known that allicin is simply a transiet compound that is rapidly descomposed to other
compounds. These findings clearly indicate that allicin does not contribute to the in vivo
effects of garlic. Though no garlic supplement on the market can contain allicin due to its
instability and high reactivity, some garlic powder products contain alliin and the enzyme,
alliinase, and, therefore, could generate a certain amount of allicin (the so-called "allicin
potential"). However, only a very small amount of allicin (< 5%) has been produced in
simulated gastric fluid compared with water (Freeman & Kodera, 1995), demonstrating that is
not generated in appreciable amounts. Therefore, allicin cannot be an appropriate marker
compound to the standardization of garlic supplements. SAC is a stable water-soluble
organosulphur compound and, unlike allicin, can be detected in the plasma, liver and kidney
after oral intake (Nagae et al., 1994). SAC is the only reliable human compliance marker used
for studies involving garlic consumption because it is detectable and increases quantitatively
in the blood after oral intake of garlic products (Steiner & Li, 2001). Because it is found in
many preparations, it might be used for standardization of garlic preparations and/or to
compare various sources. AGE is the only product standardized for SAC.
Table 2. Garlic supplements on the market.
Garlic supplement Main compounds and characteristics

Aged garlic extract Commercially available as concentrated extract and in
powdered form.
Mainly water-soluble compounds (e.g. SAC, SAMC or
saponins) and small amount of oil-soluble sulphur compounds.
Well standardized with SAC.
Well-established safety.
Garlic powder Commercially available in capsule and tablet forms.

Alliin and a small amount of oil-soluble sulphur compounds.
Standardized with allicin. Not well standardized.
Garlic essential oil Commercially available as oil capsules.

Only 1% oil-soluble sulphur compounds (e.g. DAS, DADS or
DATS) in 99% vegetable oil.
Garlic oil macerate Commercially available as soft gel capsules.

Oil-soluble sulphur compounds (dithiins, ajoene and sulfides)
and alliin.
EFFECTS RELATED TO CARDIOVASCULAR DISEASE

Cardiovascular disease is a complex disfunction characterized by multiple factors.
Nowadays it is the most important cause of death in the developed countries and consequently,
most research efforts were conducted to prevent it, thus, most breakthrough discoveries from
natural products have been in the cardiovascular area (Gilani et al., 1997). There are many
factors associated with cardiovascular diseases, among which can be included: elevated blood
cholesterol and triglycerides levels; increased platelet activity, which can give rise to
arteriosclerotic plaques formation; elevated blood homocysteine; alteration on glucose
metabolism; hypertension; and obesity. These cardiovascular disease risk factors are mainly
determined by uncontrollable causes (heredity, gender and age) and lifestyle-related causes
(smoking, inactivity, stress and diet), which are possible to be modified. For this reason, a
potential approach to the prevention and treatment of cardiovascular disease could be based on
the diet. Epidemiologic studies indicate that diets rich in fruits, vegetables, and spices provide
phytochemicals associated with lower risk of all-cause cancer and, primarily, with
cardiovascular-disease death. One source of such phytochemicals is garlic, which in the
prevention and treatment of cardiovascular diseases is well-known through the world.
Preparations of garlic and its chemical constituents have been investigated for possible effects
on the cardiovascular diseases mentioned above. In 2000, in the third National Health and
Nutrition Examination Survey garlic was listed more frequently than other dietary supplements
(Radimer et al., 2000). These supplements include garlic powder tablets, oil of steam-distilled
garlic, oil of macerated garlic, ether-extracted oil of garlic and aged garlic extract (AGE). Some
studies suggest that even the uncontrollable factors which cause the cardiovascular disease can
actually be controlled or modified (Gómez del Arco et al., 1997; Waleh et al., 1998). For
instance, S-allylcysteine (SAC) (one of the garlic active compounds, the major sulphur
compound in AGE), for example, has been shown to regulate transcriptional factors that are
required for gene expression (Geng et al., 1997). Thus, Chuah et al. (2007) found that SAC is
protective in myocardial infarction because it regulates the expression of a protein which is
responsable for the H2S production in the heart. Hence, dietary modification may help keep
undesirable genes suppressed and desirable genes activated.
The role of garlic and its chemical constituents in preventing cardiovascular disease has
been extensively acclaimed by several authors.
Effects on Levels of Serum Lipids (Cholesterol and Triglycerides)
Cholesterol is an extremely important biological molecule that has roles in membrane

structure as well as being a precursor for the synthesis of the steroid hormones and bile acids.
Both, dietary cholesterol and that synthesized de novo are transported through the circulation
in lipoprotein particles, being stored as cholesteryl esters in cells.
The synthesis and utilization of cholesterol must be tightly regulated in order to prevent
over-accumulation and abnormal deposition within the body. Slightly less than half of the
cholesterol in the body derives from biosynthesis de novo. Biosynthesis in the liver accounts
for approximately 10%, and in the intestines approximately 15%, of the amount produced
each day. Cholesterol synthesis occurs in the cytoplasm and microsomes from the two-carbon
acetate group of acetyl-CoA (King & Marchesini, 2007) as shown in Figure 2.
Of particular clinical importance is the abnormal deposition of cholesterol and

cholesterol-rich lipoproteins in the coronary arteries. Such deposition, eventually leading to
atherosclerosis, is the complex interaction of serum cholesterol with the cellular components
of the arterial wall. Cholesterol is the pathogenic substrate of many cardiovascular diseases
and it continues to be the leader cause of death in developed countries (Fabris et al., 1994).
Diseases related to atherosclerosis, such as ischemic heart disease (IHD) and stroke, are
mainly, associated with elevated serum lipids (Medical Research Council Working Party,
1988) but also with male gender, age, hypertension, cigarette smoking, diabetes, etc.
Thus, total serum cholesterol is an important factor in the development of these diseases.
Cholesterol present in the β-lipoprotein (LDL, Low Density Lipoprotein) and pre-β-
lipoprotein (VLDL, Very Low Density Lipoprotein) fractions finds its way into the arterial
wall, whereas α-lipoprotein (HDL, High Density Lipoprotein or commonly known as “good
cholesterol”) helps to reduce the serum cholesterol (Vinay et al., 2008).
Several in vitro studies have indicated that garlic and its constituents inhibit certain
enzymes involved in the cholesterol and fatty acids biosynthesis in cultured rat hepatocytes
and human hepatyc cells (Gebhardt, 1993; Liu & Yeh, 2001; Yeh & Liu, 2001). It has also
been shown that more water soluble compounds like SAC present in AGE are less cytotoxic
and more efficient in inhibiting cholesterol biosynthesis than the lipid-soluble sulphur
compounds such as DAS (Yeh & Liu, 2001).
The antihyperlipidemic effect of garlic has been extensively studied and different trials
carried out in animals, mainly rats and rabbits, have demonstrated that different commercially
available garlic preparations, such as garlic essential oil and raw garlic, decrease significantly
the content of total serum cholesterol (Chang & Johnson, 1980), LDL and VLDL and also
significantly increases the level of HDL. In a study with cholesterol-fed rabbits, it was shown
that AGE reduces vessel wall cholesterol accumulation and arteriosclerotic plaques
development in arterial wall (Effendy et al., 1997; Campbell et al., 2001). Also, in a more
recent study, Ashraf et al. (2005) demonstrated that a dietary supplementation of garlic and
turmeric reduced the atherogenic properties of cholesterol and maintained the NO-mediated
endothelial function in rats.
An increase in HDL/LDL ratio is a preventive effect of the development of IHD. However,
garlic’s antiatherosclerotic activity is probably due to its direct effect on the processes occurring in
the vascular wall as it does not depend on blood cholesterol lowering. Some studies as those
carried out by Lau et al. (1987) and Campbell et al. (2001) verified this theory. Cholesterol
reduction (as well as the other risk factors) can be considered as an indirect approach to the
treatment of atherosclerosis, but the effects observed at the arterial wall level provide a promising
basis for the development of direct antiatherosclerotic therapy (Alexander et al., 1997).
In studies carried out in humans, raw garlic, its powder extracts or its oil extracts have
shown their capacity to reduce the cholesterol and triglycerides blood levels due to the intake
of high fat meals (Bordia et al., 1974; Basksh et al., 1984).
Figure 2. Cholesterol biosynthesis pathway.

Thus, in volunteers with normal blood levels of lipids, Bhushan et al. (1979) reported that
eating 10 g of fresh garlic per day for two months significantly decreases (15%) serum
cholesterol levels. Augusti (1977) found a diminution of 29% cholesterol levels among
hypercholesterolemic patients. In another studies carried out with patients with coronary
artery disease, medication with garlic essential oil during five months produced a 10% of
diminution on serum cholesterol and a 21% on triglicerydes (Damnau, 1941).
In a broad metha-analysis, Silagy and Neil (1994) concluded that garlic decrease
cholesterol levels about 12% (triglycerides too) after 4 weeks of treatment, remained then
unchanged for the rest of the experiment. Moreover, these authors found a maximal reduction
of cholesterol with raw garlic (3 garlic cloves daily) or with garlic oil (8 mg daily).
Although the most of the studies carried out in this area have revealed the cholesterol-
lowering effects of raw garlic and garlic supplements, such as garlic essential oil and AGE
(Lau et al., 1987; Warshafsky et al., 1993; Neil et al., 1996), more recent publications have
shown different results. Thus, Mulrow et al. (2000) reported that garlic powder is ineffective
in lowering blood-cholesterol levels probably due to varied levels of allicin potential in the
garlic-powder supplements used in the clinical studies (Lawson & Wang, 2001). As above
indicated, the amount of allicin is not a constant during the elaboration of the different garlic
supplements (Amagase et al., 2001)
Active Compounds and Anti-Cholesterolemic Pathway by Garlic Derivatives
Organo-sulphur compounds are the main active substances responsible for the
hypolipidemic and hypocholesterolemic effects of garlic, as much in humans as in
experimentation animals (Yeh et al., 1997; Liu & Yeh, 2002). Several decades ago, Gebhardt
(1993) reported the multiple inhibitory effects of garlic extracts in several different steps in
cholesterol biosynthesis pathway in human hepatic cells. According to him, defined
compounds (allicin) present in water soluble extracts of garlic inhibit the biosynthesis of
cholesterol in hepatocytes, thus contributing to the reduction of serum cholesterol. Thus, it
was demonstrated that allicin extracted from garlic decreases total serum lipids, cholesterol
and phospholipids contents in rats fed allicin as compared to control animals (Augusti &
Mathew, 1974). Some allicin-derived compounds in garlic that have demonstrated to possess
a beneficial effect on cardiovascular variables are ajoene, methyl ajoene, DAS, DATS, 2-
vinyl-4H-1,3-dithiin and SAC. Methiin and flavonoid quercetin (Glasser et al., 2002) have
also shown to have the ability to reduce serum cholesterol levels and arteriosclerosis
severity. Moreover, other no sulphur components of garlic, such as steroid saponins, have
also demonstrated to be able to reduce serum cholesterol concentrations (Koch, 1993).
All these compounds may exert their hypocholesterolemic effect by three different
mechanisms; by inhibiting hepatic cholesterol biosynthesis (Gebhardt et al., 1994; Gupta & Porter,
2001; Singh & Porter, 2006), by enhancing cholesterol turnover to bile acids and its excretion
through gastrointestinal tract (Srinivasan & Sambaiah, 1991), or, in the case of plant saponins, by
inhibiting cholesterol absorption from intestinal lumen without changing HDL cholesterol levels
in hypercholesterolemic animal models (Matsuura, 2001; Slowing et al., 2001).
Conversely to the above mentioned studies, Lawson et al. (1998) found negative results
possibly due to the preparations with reduced bioavailability of allicin. Recently, Gardner et
al. (2007) have reported that neither raw garlic nor powdered garlic and AGE supplements, in
reasonable doses, have statistically significant effects on LDL cholesterol or other plasma
lipid concentrations in adults with moderate hypercholesterolemia. Therefore, although garlic
appears to hold promise in reducing parameters associated with cardiovascular disease, more
in-depth investigations are required (Rahman & Gordon, 2006).
Anti-Hypertensive Effect
Hypertension (systolic blood pressure (SBP) ≥ 140 mm Hg; diastolic blood pressure
(DBP) ≥ 90 mm Hg), a typical lifestyle-related disease, has been considered the most
important risk factor for chronic circulatory disease (Japanese Ministry of Health and
Welfare, 2005) and is one of the major risk factors of atherosclerosis (Srivastava et al., 1995),
affecting an estimated 1 billion individuals worldwide (Chobanian et al., 2003). Primary
management should include relevant lifestyle modifications such as increased exercise,
weight loss and dietary changes which could incorporate dietary supplementation. Garlic has
played an important dietary as well as medicinal role in human history (Lawson, 1998). Blood
pressure reducing properties of garlic have been linked to its hydrogen sulphide production
(Benavides et al., 2007) and allicin content (Banerjee et al., 2003; Higdon & Lawson, 2005)
which has angiotensin II inhibiting and vasodilating effects, as shown in animal and human
cell studies (Kaye et al., 2000; Al-Qattan et al., 2003; Mohamadi et al., 2000; Sharifi et al.,
2003; Al-Qattan et al., 2006; Benavides et al., 2007). Preliminary studies in humans and
reviews on garlic preparations and blood pressure have been inconclusive. Das et al. (1995)
founded some evidences that suggested garlic reduces blood pressure by inhibiting platelet
nitric oxide synthase. Nitric oxide (NO) is an important local vasodilatador which controls
several physiological functions of the cardiovascular system. Three kinds of NO synthases
(NOSs): neuronal constitutive NOS (ncNOS), inducible NOS (iNOS) and endothelial
constitutive NOS (ecNOS), are responsible for NO biosynthesis. A meta-analysis published in
1994 reported promising results in subjects with mild hypertension but found insufficient
evidence to recommend garlic for clinical therapy (Silagy & Neil, 1994). Later, anti-
hypertensive effect of garlic was determined in multiple studies with hypertensive rats using
AGE, aqueous garlic extracts and garlic powder (Fallon et al., 1998; Al-Qattan et al., 1999;
Harauma & Moriguchi, 2006). In contrast, other investigations carried out with ethanolic
extracts of garlic in hypertensive rats reported that oral administration of extracts during a
normal salt diet or during a high salt diet do not influence blood pressure (Kivirantava et al.,
1989).
Currently, many medical supplies and health foods have been researched and developed
to prevent or improve hypertension (Harauma & Moriguchi, 2006). The increasing use of
these alternative and complementary therapies for hypertension (Ernst, 2005; Yeh et al.,
2006) make it timely to provide an updated systematic review and meta-analysis of trials
investigating the effect of garlic preparations on blood pressure (Ried et al., 2008). Inclusion
of additional data from studies published since 1994 has enabled subgroup meta-analyses of
hypertensive and normotensive subjects. This systematic review and meta-analysis suggests
that garlic preparations are superior to placebo in reducing blood pressure in individuals with
hypertension. Many clinical trials find no significant antihypertensive effect despite form,
dose or duration of treatment (Valli & Giardina, 2002). Future large scale long-term trials are
needed to investigate whether standardised garlic preparations could provide a safe alternative
or complementary treatment option for hypertension in clinical practice.
Active Compounds and Anti-Hypertensive Pathway by Garlic Derivatives
Several investigations have allowed the determination of the mechanism by which garlic
exerts its anti-hypertensive action. Some studies of garlic effect on muscular contraction in
vitro have concluded that its hypotensive action may be, at least partly, due to a direct
relaxant effect on smooth muscles (Aqel et al., 1991). On the other hand, other studies have
suggested that garlic may also exert an indirect vasodilator effect, inducing the NO and
hydrogen sulphide synthesis, both potent vasodilators. The latter is synthesized from
sulfhydryl-containing amino acids present in large amounts in garlic extracts, such as cysteine
(that it is the most abundant) and the S-alk(en)yl derivatives as SAC, SEC (S-ethylcysteine)
and SPC (S-propylcysteine) (Liu & Yeh, 2002). Likewise, a recent study with several rat
models of hypertension has indicated that quercetin and its methylated metabolite
isorhamnetin can reduce blood pressure and prevent angiotensin II-induced endothelial
dysfunction by inhibiting the overexpression of p47 (phox), a regulatory subunit of the
membrane NADPH oxidase, and the subsequent increased superoxide production, resulting in
a highest NO bioavailability (Sanchez et al., 2007).
A novel drug assayed in hypertensive rats has been recently synthesised through the
reaction of the pharmaceutical drug Captopril with allicin (Figure 3). The reaction product,
called allylmercaptocaptopril (CPSSA), provides better protection against hypertension, since
it has the Captopril ability to inhibit the angiotensin-converting enzyme (ACE) and the allicin
ability to reduce serum cholesterol and triglycerides levels (Miron et al., 2004).
Figure 3. A novel drug (allylmercaptocaptopril) recently synthesised through the reaction of the
pharmaceutical drug Captopril with allicin. Reprinted by permission from Macmillan Publishers Ltd:
American Journal of Hypertension, 17, copyright (2004).
Anti-Hyperglycaemic or Anti-Diabetic Potential
Diabetes Mellitus, often referred to simplify, as diabetes, is a disease in which the body
does not produce or properly use insulin. Insulin is a hormone that is needed to convert sugar,
starches and other food into energy needed for daily life. Thus, diabetes resulting in
abnormally high blood sugar levels (hyperglycemia). Its cause continues to be a mystery,
although both genetics and environmental factors such as obesity and lack of exercise appear
to play roles.
The relationship between diabetes Mellitus and atherosclerosis is likely based on the
interactions between arterial cells and atherogenic glycosylated LDL lipoproteins originated
during diabetes development, that play a key role in the initiation of an atherosclerotic lesion,
inducing cholesterol accumulation in arterial cells (Ide & Benjamin, 2001) and other more
severe atherosclerotic manifestations at cellular level that lipoproteins from no diabetic
subjects (Winocour, 1994; Sobenin et al.,1994).
The garlic effectiveness as hypoglycaemic agents has been scarcely investigated and the
existing data are controversial, having not found evidence of its effectiveness in all cases
(Sheela & Augusti, 1992; Mansell et al., 1995).
The hypoglicemic effects of garlic and its individual components have been demonstrated
in animal models (Jain et al., 1973; Zacharias et al., 1980; Sheela & Augusti, 1992) whereas
other researchers found no significant alteration of hyperglycaemia in animals (Swanston et
al., 1990). Recently, it has been reported that long-term absorption of natural flavonoids as
quercetin could be useful to prevent advanced glycation of collagens, which contributes to
development of cardiovascular complications in diabetic patients (Urios et al., 2007). Type II
diabetes Mellitus is characterized by premature accelerated atherosclerosis development
leading to early invalidization and high mortality in this category of patients (Krolewski et al.,
1991; Burchfiel et al., 1993). In a study on the use of natural remedies for type II diabetes
Mellitus treatment in a diabetic women group from United States, garlic appeared among the
most used vegetables (Johnson et al., 2006) and in a recent double-blinded placebo controlled
study with a new garlic-based formulation (namely, time-released garlic powder tablets
Allicor), Sobenin et al. (2008), established that this product is recommended for the treatment
of type II diabetes Mellitus along with dietary treatment and/or sulfonylurea derivatives to
achieve better metabolic control. In addition garlic supplement may improve the other risk
factors (reduction of serum triglycerides, inhibition of cholesterol synthesis, etc). Thus, the
use of this vegetable is suggested in conjunction with anti-diabetic drugs to increase their
therapeutic potential and to minimize their oral dosage.
Active Compounds and Anti- Hyperglycaemic Pathway by Garlic Derivatives
The bioactive constituents from garlic, such as methiin and S-allyl cysteine sulphoxide
(SACS) (Sheela & Augusti, 1992), exert their anti-diabetic action by 3 different ways: (i)
stimulating the insulin production and secretion by pancreas, (ii) interfering with dietary
glucose absorption, and (iii) favouring the insulin saving (Srinivasan, 2004a, 2004b).
Anti-Platelet or Anti-Thrombotic Effect
As it is known, platelets (or thrombocytes), are the cells circulating in the blood that are
responsible for mantain the haemostatic integrity of blood vessels and the stop of bleeding
after injury (Ali et al., 2000) through vasoconstriction, clot formation and blood coagulation.
High levels of platelets may increase the risk of thrombosis: the formation of a clot or
thrombus into a blood vessel obstructing the flow of blood through the circulatory system
(see Figure 4). Therefore, it is evident that platelet circulation is much related to certain
cardiovascular diseases (Becker, 1999).
Garlic and its components are known to possess antiplatelet activity which has been
demonstrated mostly in vitro (Lawson et al., 1998) and several platelet inhibitors have been
isolated and characterized from this vegetable. The inhibitory effects of garlic extracts as well as
allicin, ajoene and other individual garlic compounds on thrombus formation and platelet
aggregation has been also investigated (Srivastava, 1986; Mayeux et al., 1988; Apiz-Castro et
al., 1992). Cavagnavaro et al. (2007) studied the effect of cooking on garlic antiplatelet activity
and its content in thiosulfinates. Their results suggested that allicin and thiosulphinates are
responsible for the in vitro antiaggregatory activity and that crushing garlic before moderate
cooking can reduce the loss of activity. This partial loss of antithrombotic effect in crushed-
cooked garlic may be compensated by increasing the amount consumed.
Figure 4. Formation of a clot or thrombus into a blood vessel obstructing the flow of blood through the
circulatory system. Reprinted by permission from Macmillan Publishers Ltd: Nature, 451, 914-918,
copyright (2008).
The study carried out by Chang et al. (2004) showed that the alkenyl thiosulfate sodium
2-propenyl thiosulfate (2PTS) obtained from boiled garlic has the potential to prevent
cardiovascular disease by inhibiting platelet aggregation in dogs and humans in vitro. As
these compounds are not volatile, these compounds are considered heat-stable platelet-
inhibitory factors.
Aqueous and organic garlic extracts are also able to inhibit platelet aggregation induced
by a number of physiologically important aggregating agents, as collagen and adrenaline, and
the thromboxanes synthesis in vivo (Mohammad & Woodward, 1986) by several
mechanisms, such as inhibition of several steps of the arachidonic acid pathway in platelets
(Ali et al., 2000), which is the thromboxanes precursor. Due to the variations in methods of
preparation, the different garlic products commercially available may show different
inhibitory effect on platelet aggregation (Lawson et al., 1992).
It was found that garlic oil administration to healthy subjects and patients with coronary
artery disease (CAD) inhibited platelet aggregation ex vivo. Though garlic components leave
the body quickly, a slow building up of the active ingredients may take place. This was
evident from the observation that though a 2-3 fold higher dose was not effective in inhibiting
platelet aggregation when administred once, whereas lower dose became effective in long-
term administration (Bordia et al., 1996).
Two clinical studies reported reductions in platelet aggregation of 16.4% and 58%
respectively with garlic oil obtained from 9-10 g fresh garlic cloves (Boullin, 1981; Barrie et
al., 1987). In a randomized double-blind study of normal healthy subjets, the effect of three
different doses of AGE compared with placebo on platelet aggregation and adhesion were
measured after 6 weeks of supplementation. AGE supplementation reduced platelet function,
and this inhibitory effect was selective, affecting collagen and epinephrine but not ADP-
induced aggregation. Not all studies show a favourable effect of garlic on platelet function. A
placebo-controlled, double-blind, randomized study on healthy men showed no effect of
garlic extract on platelet aggregation, serum tromboxane and platelet activating factor (Morris
et al., 1998).
Active Compounds and Anti- Platelet Pathway by Garlic Derivatives
Antiplatelet activity is substantially affected by genotype, environment and storage

duration of vegetable. It has been reported by several epidemiologic studies that, in garlic, the
antiplatelet activity is determined, in part, by the native concentration of organo-sulphur
compounds and genotypically determined sulphur content of the bulb (Goldman et al., 1996).
These compounds have structural similarity to ajoene, considered the major antiplatelet
compound in garlic extracts. In addition, other no sulphur compounds, such as β-chlorogenin
and quercetin, have been also shown to inhibit platelet aggregation (Rahman et al., 2006).
The mechanism of platelet aggregation inhibition is associated at least with reduction of
tromboxane formation from exogenous arachidonate (Srivastava, 1986) and perturbation of
the physicochemical properties of platelet plasma membrane (Apiz-Castro et al., 1983).
Gillian et al. (2006), in a preliminary study, reported the mechanisms that may be involved in
the inhibition of platelet aggregation by AGE when platelets are stimulated with adenosine
diphosphate (ADP). These authors founded that the mechanism involved appear to be
multiple in nature, involving membrane fluidity changes, inhibition of phospholipase C,
inhibition of calcium mobilization, increase in NO and cAMP (cyclic adenosine
monophosphate) production, and inhibition of TXA2 (tromboxane A2), all of which can lead
to an inhibition of platelet aggregation.
The different results obtained are probably due to the use of different garlic preparations
and variable amounts of the active constituents in garlic in these studies (Rajaram, 2003).
Effect on Hyperhomocysteinemia
Homocysteine (Hcy) is a sulphur-containing amino acid formed during metabolism of

methionine, an essential amino acid derived from the diet. The determination of total plasma Hcy has
become a very useful tool because moderately elevated values of circulating Hcy constitute an
important risk factor for the development and progress of occlusive vascular affections as it is shown
in Figure 5 (Fischer et al., 2000). In addition, hyperhomocysteine is a risk factor for ischaemic heart
disease (IHD) in diabetic patients (Okada et al., 1997).
Figure 5. Homocysteine: a risk factor for cardiovascular disease.
Hcy exists in normal human plasma in several different forms. Approximately 70% is
bound to plasma proteins, mainly albumin, through disulphide bounds. The remaining
homocysteine circulates as a free thiol compound, reduced or combined by oxidation with
other thiols, as cysteine, resulting in mixed disulphide, or another molecule of Hcy, to form
the dimmer homocystine (Mansoor et al., 1992b). Hence measurement of total plasma
homocysteine as a cardiovascular risk factor involves assay of bound, free, reduced and
oxidized forms. The concentration of total Hcy is regulated by disulphide-disulphide
exchange and thiol-disulphide exchange reactions. Cysteine plays an essential role in
modulating thiol-disulphide exchange (Ozkan et al., 2002), whereas protein-bound cysteine
and cysteinylglycine participate in disulphide-disulphide exchanges (Mansoor et al., 1992a).
There are several factors that cause increase of Hcy. Hyperhomocysteinemia can be
congenital, due to hereditary metabolic affections (Mudd & Levy, 1983), or acquired and to have a
multifactor origin. The commonest cause of acquired hyperhomocysteinemia is the folate, vitamin
B6 and/or B12 deficiency (Durand et al., 1996; Jacobsen, 1996; Ubbink et al., 1996; Sumner et al.,
1996) and the drugs consumption that interfere with these vitamins metabolism.
Because garlic contains vitamins B6 and B12 and a large amount of aminothiol
compounds, such as SAMC, DAS, diethyl disulphide (DEDS) and dipropyl disulphide
(DPDS) (Liu & Yeh, 2000), it was thought that garlic intake may be an effective way to
reduce plasma homocysteine levels.
Hyperhomocysteinemia has been reported in several individuals with genetic defects in
enzymes such as cysthatione β-synthase (Clarke et al., 1991; Aguilar et al., 2004) and N5,
N10-methylenetetrahydrofolate reductase (Aguilar et al., 2004; Takenata, 1993). Conversely,
folic acid supplementation is effective in reversing elevated homocysteine level (Doshi et al.,
2002; Boers, 2000; Moat et al., 2004).
Garlic contains a variety of aminothiol compounds that may interact with free and
protein-bound homocysteine. Yeh et al. (2005) indicated that a reduction in plasma level of
Hcy could not be attributed to disulfide-disulfide exchange and thiol-disulphide exchange
among aminothiol compounds and Hcy. Several recent studies (Yeh et al., 2005; Yeh & Yeh,
2006; Weiss et al., 2006; Ide et al., 2006) have demonstrated the effectiveness of AGE to
reduce the plasma concentration of Hcy in rats with hyperhomocysteinemia induced by severe
folic acid deficiency, but the action mechanism is not yet known with absolute certainty. Yeh
and Yeh (2006) established that the reduction in total Hcy of several folate-deficient rats was
accompanied by a proportional decrease in protein-bound and free Hcy, resulting in an
unchanged protein-bound: free homocysteine ratio. AGE added to the diet does not alter
plasma concentrations of other aminotiol compounds: cysteine gluthatione and
cysteinylglycine. These data, together with the increase of S-adenosylmethionine and the
decrease of S-adenosylhomocysteine concentrations in the liver, suggest that the
hypohomocysteinemic effect of AGE most likely steams from impaired remethylation of
homocysteine to methionine and enhanced transsulfuration of homocysteine to cystathione.
Smoking, alterations in serum lipid profiles, hypertension and diabetes are the risk factors
that are conventionally associated to the early appearance of cardiovascular disease.
However, many patients with clinical manifestations of premature arteriosclerosis do not
show any of these risk factors. In the last ten years, new risk factors for arteriosclerotic
vascular disease such as hyperhomocysteinemia have been described, which have allowed to
develop new measures of prevention. Cardiovascular risk is further increase by a combination
of hyperhomocysteinemia, hypertension and smoking (Boers, 2000). It has been documented
that plasma total-homocysteine levels in patients with cardiovascular disease are significantly
higher than those of normal subjects (Ueland et al., 1992). Similarly, patients with myocardial
infarction had increased levels of homocysteine as compared to other free of infarction
(Stampfer et al., 1992) The risk for cardiovascular diseases caused by hypercholesterolemia is
associated with atherosclerosis. However, the mechanism underlying homocysteine-induced
cardiovascullar diseases is still controversial (Yeh & Yeh, 2006). It has been suggested that
homocysteine may impair production of endothelium-derived relaxing factor, stimulate
proliferation of smooth cells, retard endothelial NO activity, and induce cardiovascular
fibrosis (Massy et al., 1994; Tsai et al., 1994; Das, 2003; Tyagi, 1999).
Endothelial dysfunction (ED) due to decreased bioavailable NO by increased vascular
oxidant stress plays a critical role in the vascular pathobiology of hyperhomocysteinemia.
AGE can minimize intracellular oxidant stress and stimulates NO generation in endothelial
cells. Weiss et al. (2006) carried out a placebo-controlled, blinded, cross over study to
examine whether AGE prevents macro- and micro ED during acute hiperhomocysteinemia
induced by an oral methionine challenge in healthy subjets and the results allowed to
conclude that AGE may at least partly prevent a decrease in bioavailable NO during acute
hiperhomocysteinemia.
In addition, Nagatoshi et al. (2006) demonstrated the effectiveness of AGE in the
homocysteine inhibition and, hence, in modulation of formation of early atherosclerotic
lesions in a study carried out with human cells.
Evidences, here showed, from different clinical trials point toward garlic having, mostly,
a role to play in either preventing or delaying cardiovascular disease. However, more research
is still required to convince health workers, consumers, and regulatory bodies.
EFFECTS ON CANCER AND MUTAGENESIS

Numerous scientific reports imply that vegetable intake may affect cancer incidence. In
reviews of epidemiologic studies there is convincing evidence that high consumption of
certain vegetables reduces the risk of colorectal, stomach, lung and esophageal cancers; in
addition, there is evidence for cancers of the breast and bladder (World Cancer Research
Fund, American Institute for Cancer Research, 1997). Garlic is one of the most ancient spice
plants reputed to have an effect on cancer. As recorded around 1550 B.C. in the Ebers
Papyrus, garlic was applied externally for the treatment of tumours by ancient Egyptians and
internally by Hippocrates and Indian physicians (Hartwell, 1967, 1968; Block, 1985).
However, the modern era of the use of garlic as anticancer agent begins in the 1950s when
Weisberger and Pensky (1958) demonstrated in vitro and in vivo that thiosulfinate extracts
from garlic inhibited the tumour cells growth. Since these investigations, many
epidemiological and laboratory studies have been developed to evidence the chemopreventive
or anticarcinogen effects of garlic and related Allium species. Interestingly, China provides an
ideal “Field Laboratory” for epidemiological studies of cancer incidence. Stomach cancer was
found to rank higher for males and females in cancer mortality (Wang et al., 1985; Lau et al.,
1990) than other cancer incidence in China (Mei et al., 1982). They suggested that garlic
consumption may inhibit nitrate reduction by bacteria. Subsequently, the lower gastric nitrite
(a nitrosamine precursor) concentration may reduce the risk of developing stomach cancer.
Likewise, You et al. (1989) identified that smoking, salty foods and moldy grains are
associated with increased risk of stomach cancer (You et al., 1989). A significant reduction of
stomach cancer risk was found to be associated with increasing consumption of garlic,
scallicens and Chinese chives (You et al., 1988). In addition, it has been also shown an
inverse relationship between garlic consumption and the incidence of sarcoma (Lau et al.,
1990) and carcinoma in colon (Lau et al., 1990; Steinmetz et al., 1994), oesophagus (Lau et
al., 1990; You et al., 1998), prostate (Hsing et al., 2002), bladder, liver (Lau et al., 1990;
Lamm & Rings, 2001), lungs (Le Marchand et al., 2000), mammas (Lau et al., 1990; Challier
et al., 1988), and skin (Lau et al., 1990).
Several investigations have shown that both water- and lipid-soluble sulphur compounds
from garlic provide anticarcinogen benefits, however, generally, the lipid-soluble sulphur
compounds such as DAS and its metabolites, diallyl sulphoxide (DASO), diallyl sulfone
(DASO2), DADS and DATS are the most effective antitumorogenic agents. Although the
question of how these compounds result in chemoprevention has not yet been fully answered,
several mechanisms of action have been proposed (Knowles & Milner, 2001; Griffiths et al.,
2002; Thomson & Ali, 2003) (Figure 6).
Oxidative
activation
Procarcinogen
Excretion
+ Garlic increases the
Carcinogen detoxifying enzymatic
systems activity
Initiation Garlic affects carcinogen
DNA binding -
Garlic has an effect
DNA Binding on DNA repair
Garlic prevents chromosomal mechanisms
aberrations and protects DNA - DNA repair
from activated mutagens +
Mutation
+ Apoptosis
Garlic induces
apoptosis
Launched cell
Garlic inhibits
Promotion cell division -
Cell proliferation
Progression
Neoplastic cell
Figure 6. Modes of action by which garlic and its derivatives exert their anticarcinogenic activity.
Garlic compounds can alter the carcinogen metabolism either increasing the detoxifying
enzymatic systems activity that increase the carcinogen polarity, facilitating its excretion from
the body (Guyonnet et al., 1999), or inhibiting the procarcinogens activation by cytochrome
P450 (Dion & Milner, 1997; Khanum et al., 2004). Glutathione-S-transferase (GST) is a well-
known detoxifying enzyme in Phase II metabolism of drugs that removes harmful
electrophiles by conjugating them with glutathione. Therefore, GST can play a detoxifying
role in metabolism of carcinogens that may be electrophilic in nature. Sparnins and coworkers
(1986, 1988) studied the effect of oral administration of allyl methyl trisulfide (AMTS) on
GST, a detoxifying enzyme, in the liver, forestomach, small intestine and lung of mice. They
observed that 96 h after oral administration of AMTS, GST activity was increased in all
tissues and, in addition, benzo[a]pyrene induction of forestomach tumors was suppressed.
Similarly, three other garlic-derived compounds (allyl methyl disulfide, DATS and DADS)
stimulated GST activity in these organs. In contrast, saturated (propyl) derivatives did not
affect GST activity in these organs of mice. These results suggest that allyl groups are
important for the stimulation of GST. Such anticarcinogenic activity of DADS against
benzo(a)pyrene in mice has been also reported by Srivastava et al. (1997). Similarly,
Sumiyoshi and Wargovich (1989) reported that the oral administration of DAS (400 mg/Kg)
stimulated mouse hepatic GST activity. They also reported elevated colonic GST activity. In
both the liver and colon, the increased GST activity was DAS dose-dependent. In an earlier
study, Wargovich and Goldberg (1985) also found that DAS affects aflatoxin B1 metabolism
and DNA binding and prevents nuclear damage to colon epithelial cells in vivo induced by
chemical carcinogens such as DMH (1,2-dimethylhydrazine) and NMBA (N-nitroso
methylbenzylamine), by inhibiting the conversion of procarcinogens to ultimate carcinogens

in the liver.
Manson et al. (1997) also studied the effect of oral administration of garlic oil to rats on a
number of drug metabolizing enzymes in liver tissues. They reported that garlic oil induced
phase II enzymes such as GST and the conjugating enzyme, gamma-glutamultranspeptidase.
In other study, Singh et al. (1998) observed that treatment of mice with DADS and
DATS, which are potent inhibitors of benzo(a)pyrene-induced forestomach and pulmonary
tumorogenesis, resulted in a statistically significant increase in forestomach and lung
NAD(P)H: quinone oxidoreductase (NQO) activity, an enzyme implicated in the
detoxification of actived quinone metabolites of benzo(a)pyrene. In addition, DADS and
DATS were much more potent inducers of forestomach NQO activity than DAS, which is a
weaker inhibitor of benzo(a)pyrene-induced tumorogenesis than the former compounds.
Ajoene has been also shown to be able to inhibit aflatoxin B1-, benzo(a)pyrene- and 4-nitro-
1,2-phenylenediamine-induced mutagenesis in vitro models as well as prevent in vivo skin
tumor of mouse by 12-Ο-tetradecanoylphorbol-13-acetate (Tadi et al., 1991; Ishikawa et al.,
1996; Nishikawa et al., 2002).
Anticarcinogen compounds from garlic have also an anticlastogenic effect, preventing the
chromosomal damage (Lau et al., 1990; Khanum et al., 2004). Several authors have studied
the anticlastogenic effects of garlic. In several studies with mice, Choudhary et al. (1997)
have observed that aqueous garlic extract administered orally either alone or in combination
with mustard oil significantly reduced the frequency of chromosomal aberrations resulting
from intravenous injection of sodium arsenate, a strong clastogen. It has been suggested that
trivalent arsenate induces toxicity by binding to thiol ions which ultimately leads to inhibition
of certain enzymatic reactions. Therefore, the sulphur-containing compounds in crushed
garlic may be the principal factors responsible for the significant reduction of the clastogenic
effects of sodium arsenate by crude garlic extract (Sharma & Talukder, 1987; Choudhary et
al., 1997a, 1997b). Chowdhury et al. (2008) found several evidences, including reduction of
intracellular ROS level in human tumor cells, inhibition of tissue lipid peroxide generation,
and increase of total tissue sulfhydryl groups, glutathione and antioxidant enzymes level,
which indicated that AGE can be a potential protective regimen for arsenic mediated toxicity.
Garlic compounds can also inhibit the tumor growth, by inhibition of cell division and
induction of apoptosis (Perchellet et al., 1990; Izzo et al., 2004). Apoptosis, also known as
programmed cell death, is a means by which living organisms control abnormalities in cells.
It is of interest that in numerous human pathological conditions including cancers, apoptotic
signalling cascades are often impaired (Rose et al., 2005). Both garlic extracts and their
phytochemical constituents can induce apoptosis in several in vitro cell culture models. From
the available data, activation of the proteolytic enzymes, changes in intracellular redox
homeostasis, generation of reactive oxygen species (ROS) and the activation of stress
signaling cascades are all implicated in the apoptotic response of cancer cells to garlic sulphur
compounds. Li et al. (1995) investigated the effect of AGE and two of its components, SAC
and SAMC, on human breast cancer cells. They observed an anti-proliferative response of
these compounds and an alteration in glutathione level without significant concurrent changes
in the glutathione metabolizing enzymes (Li et al., 1995). In a more recent study, Katsuki et
al. (2006) reported that AGE has chemopreventive effects on DMH-induced colon
carcinogenesis through modulation of cell proliferation. Likewise, studies have shown that
SAMC can inhibit cell proliferation in human erythroleukaemia cell lines as well as in human
colon cancer cells (Sigounas et al., 1997; Shirin et al., 2001). Xiao et al. (2003) later found
that SAMC exerts anti-proliferative effects by arresting cells in mitosis and triggering
apoptosis. Similarly, garlic-derived sulfides (DAS, DADS and DATS) have also been shown
to be potent inducers of apoptosis in cancer cells. Many reports have shown that DAS has
antitumor efficacy in cultured carcinoma cell lines, such as lung cancer cells and mouse skin
tumors (Wargovich et al., 1992; Hong et al., 2000; Arora & Shukla, 2003). Likewise, Xiao et
al. (2006) have observed that DATS induces apoptosis in human prostate cancer by activation
of pro-apoptotic proteins. Both, DADS and DATS, induce apoptosis in cultured human
neoplastic and non-neoplastic lung cancer cells (Sakamoto et al., 1997; Hong et al., 2000) and
human leukaemia HL-60 cells exposed to DADS undergo apoptotic cell death (Kwon et al.,
2002). At micromolar concentrations, DADS also inhibits cell proliferation and induces
apoptosis in vitro in estrogen receptor positive and negative breast cell lines, as well as in
human gastric cell lines (Li et al., 1998; Nakagawa et al., 2001). Moreover, DADS has been
shown to inhibit cell proliferation in human colorectal cells by inducing the pro-apoptotic
gene NAG-1 (Bottone et al., 2002) and it has been reported to be as effective as the colon
anticancer compound 5-fluorouracil in nude mice at equivalent doses (Sundaram & Milner,
1996; Singh et al., 1996). Ajoene has been also shown to exhibit antitumor activities either in
vitro on breast cancer, hepatocellular, gastric and colon carcinoma, or in vivo on
hepatocarcinoma and sarcoma, through both cell cycle blockage and apoptosis of tumor cells
(Li et al., 2002). Another interesting property of ajoene is its selective cytotoxic action on
neoplastic (vs. normal) cells (Li et al., 2002; Dirsch et al., 1998). Indeed, ajoene induces
apoptosis in human leukemic HL60 cells but not in peripheral mononuclear cells of healthy
donors (Dirsch et al., 1998). Recently, Terrasson et al. (2006) have demonstrated a cytotoxic
effect of Z-ajoene against a large spectrum of cell lines (astrocytoma, lymphoma,
neuroblastoma, etc.) by inducing apoptosis. This effect was mediated by accumulation of pro-
apoptotic proteins in Z-ajoene-treated cells which was likely due to both increase in gene
transcription and in inhibition of their proteolysis by proteasome enzymes. These authors also
investigated a new activity of Z-ajoene against human cytomegalovirus (HCMV), a DNA
virus of the herpesvirus family that has been associated with several tumor cells including
those from glioblastoma and colorectal cancers. Data demonstrated a potent anti-HCMV
activity of Z-ajoene in vitro that was mediated by an increase of apoptotic cells after
infection. Regarding to allicin, it has been determined that this lipid-soluble volatile organo-
sulphur compound, but not its precursor alliin, inhibits proliferation of human mammary,
endometrial, and colon cancer cells through induction of apoptosis, cell cycle blockage and
transient drop in the intracellular glutathione level (Hirsch et al., 2000; Oommen et al., 2004).
Recently, a number of researchers have focussed on garlic antimutagenic activity,
observing that certain sulphur compounds such as DAS have an effect on DNA repair
mechanisms, protecting the DNA from activated mutagens and preventing, thus, the initiation
of carcinogenesis (Wargovich et al., 1988; Hong et al., 1991; Khanum et al., 2004).
Another mechanism of action is the effective stimulation of the immune response. To
date, this latter action mechanism is thought to be the most important direct anticarcinogen
action of garlic (Lamm & Riggs, 2001), which has been documented in cultures of different
cancerous tissues, including colon, prostate, bladder and stomach (Pan et al., 1985; Knowles
& Milner, 1997). Given the importance of this mode of action, it will be treated more in depth
later.
Moreover, it is accepted that phytochemicals of garlic (and other foods) with antioxidant
properties minimize DNA damage by reacting with free radicals and in this way they could
prevent cancer (Perchellet et al., 1990). However, in some studies antioxidants increase
incident of cancers instead of lowering it. It is therefore likely that antioxidants are acting in
different way than expected. One of the possibilities is that they are disrupting specific
pathways or inhibit enzymes that are important in carcinogenesis (Jankun et al., 2003). In
particular, the pro-inflammatory enzyme lipoxygenase, is a regulator of human cancer
development and it is overexpressed in a variety of tumors including breast, colorectal and
prostate cancer, and cancer cell lines (Pidgeon et al., 2002) and that its inhibition trigger
tumor cell apoptosis, reduce tumor cell motility and invasiveness, or decrease tumor
angiogenesis and growth (Nie et al., 2001). Belman et al. (1989) investigated the inhibition of
soybean lipoxygenase (LOX) by onion and garlic components. They found that the di- (1-
propenyl) sulphide was the only irreversible inhibitor. DATS, allyl methyl trisulfide and
DADS were competitive inhibitors, while 1-propenylpropyl sulphide and ajoene were mixed
inhibitors. Sendl et al. (1992) also studied LOX inhibitory activity of garlic. They used
extracts of wild garlic (Allium ursinum) and garlic (Allium sativum) with defined chemical
compositions to assess their inhibitory potential on LOX. The inhibition rates as IC50 values
of these extracts showed a good correlation with the %-content of the major sulphur-
containing compounds (thiosulfinates and ajoene).
In addition to organo-sulphur compounds, eruboside-B, a steroid saponin isolated from
garlic bulb, and allixin (phytoalexin), are largely responsible for the anticarcinogenic activity
of garlic (Yamasaki et al., 1991; Matsuura, 1997). Allixin, being a phenolic compound, is an
effective inhibitor of phospholipid metabolism stimulated in vitro by the tumor promoter
(Kodera et al., 1989). Garlic is also rich in flavonols, particularly kaempferol, which have
antineoplastic effects by helping in the detoxification of carcinogenic compounds, by
inducing apoptosis (Brisdelli et al., 2007), by inhibiting bioactivating enzymes (Lautraite et
al., 2002; Muto et al., 2001) and due to its antioxidant and anti-inflammatory activities
(Mutoh et al., 2000; Raso et al., 2001). Moreover, garlic is one of the best natural sources of
germanium. It is of interest to note that this trace metal has also been reported to prevent and
cure cancer. Garlic is also an excellent source of selenium (Se), which has potential
therapeutic value in cancer treatment (Bolton et al., 1982; Lawson, 1993; El-Bayoumy et al.,
2006). Epidemiological studies have indicated a relationship between Se intake and the
incidence of certain cancers. Se-enriched garlic has higher anticarcinogenic activity than the
common plant (Ip et al., 1992). This increased effect of cancer prevention is achieved at least
partly by S substitution with Se. The pure Se-compounds have proved to be superior
anticancer agent than their corresponding S-analogues. For example, diallyl selenide is at
least 300 times more active than DAS in the reduction of tumours of mammal cancer (El-
Bayoumi et al., 1996). Se-methyl selenocysteine is the major organo-Se-compound in garlic
bulb and, along with γ-glutamyl-Se-methyl selenocysteine, the major Se-compound
possessing anti-cancer activity (Block et al., 2001). In mammary tumor model, Se-methyl
selenocysteine was shown to be the most effective Se-compound so far in reduction of tumors
(Whanger, 2004). Identification and quantification of Se-compounds in Se-enriched Allium
are particularly important in order to study the anti-cancer mechanisms in detail. For this
reason, new analytical techniques are necessary to gain more insight in the identification of
Se-compounds (Arnault & Auger, 2006).
Large-scale gene expression analysis in combination with functional assays yields a

considerable amount of information on anticarcinogenic and antimutagenic potential of garlic
active components. Thus, for example, data from cDNA array studies reveal that the
antiproliferative effects of DADS may be related to changes in gene expression of aggrecan 1,
tenascin R, vitronectin and cadherin 5 (Knowles & Milner, 2003). Likewise, it has been
recently reported that the response to garlic and its components depends on the consumer’s
genetic backgrounds (nutrigenetic effects), DNA methylation and histone regulation
(nutritional epigenomic effects), ability to induce or repress gene expression patterns
(nutritional transcriptomics effects), occurrence and activity of specific proteins
(nutriproteomic effects), and/or dose and temporal changes in cellular small-molecular-weight
compounds (metabolomics effects). Knowledge about each of these variables and the
identification of biomarkers that can be used to predict who will and will not respond to garlic
or other Allium foods will be essential for the development of tailored strategies for reducing
cancer burden and for effective intervention to occur (Milner, 2006).
ANTIOXIDANT PROPERTIES
Research studies evidence that plant-based diets, in particular those rich in vegetable and
fruits, provide a great amount of antioxidant phytochemicals, such as vitamins C and E,
phenolic compounds (flavonoids), vegetable pigments (antocianins and carotenoids), as well
as thiols (as sulphur compounds) (Yang et al., 2004; Sharma et al., 2005; Dimitrios, 2006).
As antioxidants, all of these are compounds able to slow down, stop or reverse oxidation of
nucleic acids (DNA), proteins and lipids by scavenging oxidizing agents such as reactive
oxygen species (ROS) (Wilson & Demming-Adams, 2007). These oxidation processes play
an important role in aging and in a wide range of common diseases, including cancer and
cardiovascular, inflammatory and neurodegenerative diseases, such as Alzheimer’s disease
and other age-related degenerative conditions (Borek, 1997; Gutteridge, 1993; Richardson,
1993). It has been demonstrated that endogenous levels of ROS increase during chronic
infection and inflammation, strenous physical exercise, hypermetabolic states seen in stress,
trauma and sepsis, and during exposure to exogenous sources of ROS such as tobacco smoke,
UV light or polluted air (Borek, 2001).
Among garlic-derived products, AGE is the preparation with the highest antioxidant
activity, even more than fresh garlic and other commercial garlic supplements (Imai et al.,
1994). This is due to its own extraction procedure, since the long-term extraction of garlic
ages the extract, modifying unstable molecules with oxidant activity such as allicin (Freeman
& Kodera, 1995) and increasing stable and highly bioavailable water-soluble organo-sulphur
compounds content such as SAC and S-allylmercaptocysteine (SAMC), which have potent
antioxidant activity (Imai et al., 1994). SAC and SAMC are the major organo-sulphur
compounds found in AGE, nevertheless, this garlic preparation has other compounds with
antioxidant effect, such as stable lipid-soluble allyl sulphides derived from allicin (e.g. DAS,
DATS, DADS and diallyl polisulphides) (Awazu & Horie, 1997; Amagase et al., 2001);
tetrahydro-β-carboline derivatives, which are formed during the natural aging process
(Ichikawa et al., 2006); flavonoids (as allixin); saponins; and essential micronutrients
(selenium, Se) and macronutrients, as lectins, whose antiperoxide effect has been
demonstrated in the liver, kidney and heart of rats (Rajasree et al., 1999; Amagase et al.,
2001; Borek, 2001). Another recently identified antioxidant compounds of AGE are N-
fructosyl glutamate, N-fructosyl arginine (Ryu et al., 2001) (whose antioxidant activity is
comparable to that of ascorbic acid) and N-fructosyl lysine (Moreno et al., 2006), Amadori
rearrangement products, originated during the first steps of the Maillard reaction as a result of
processing and storage, mainly to high temperatures.
Phytochemicals in AGE may act in synergistic or additive way. In addition to scavenging
ROS (Awazu & Horie, 1997; Borek, 2001), they exert their antioxidant action by enhancing
the activities of the cellular antioxidant enzymes superoxide dismutase (SOD), catalase and
glutathione peroxidase (Awazu & Horie, 1997), and increasing glutathione in the cells (Liu et
al., 1992). This is an important defence mechanism in living cells, since, in addition to
protecting against oxidative stress and being a cofactor for the antioxidant enzyme glutathione
peroxidase, is one of the detoxification systems of the body and induces the detoxifying
enzyme glutathione-S-transferase (GST). Thus, they provide additional protection to own
antioxidant defences of organism against oxidant damage, decreasing the risk of injury to
vital molecules and helping to prevent, thus, the onset and progression of diseases
(Gutteridge, 1993; Borek, 1997). According to this, a study carried out by Kempaiah and
Srinivasan (2004) showed that the sulphur compounds in garlic are effectively able to protect
the endogenous thiol pool. In this study, rats were given a high-fat diet with or without garlic,
and blood levels of triglycerides and thiols such as glutathione were assessed. Food intake per
se was not affected by garlic. The high-fat diet increased the levels of blood triglycerides,
decreased the levels of thiols such as glutathione and increased lipid oxidation. Authors found
that all of these adverse effects of the high-fat diet were effectively reduced by regular
addition of garlic to the diet. When garlic was added to the high-fat diet, total endogenous
thiols increased by 16 per cent, glutathione increased by 28 per cent and the level of catalase,
which is depleted under oxidative stress, also increased.
Particularly, due to its antioxidant action, AGE decreases the risk of cardiovascular and
cerebrovascular disease inhibiting the lipid peroxidation and oxidation of LDL, which play an
important role in the initiation and progression of arteriosclerosis (Steinberg, 1997; Amagase
et al., 2001; Lau, 2006). Oxidation of lipids can also cause direct effects such as
destabilization of lipid membranes, e.g. of red blood cells (Yang et al., 2004; Kempaiah &
Srinivasan, 2004). Thus, AGE protects the erythrocytes membrane against oxidative stress
inhibiting the formation of abnormally dense erythrocytes, which are believed to play an
important role in the clinical manifestations (painful crisis and anaemia) of sickle cell
anaemia patients (Ballas & Smith, 1992). It also inhibits free radical and mutations-mediated
DNA damage, decreasing, therefore, the onset and development of tumors (Borek, 1997).
Moreover, AGE has radioprotective effects (Lau, 1989), protecting against ionising radiation
and UV light-induced damage. Likewise, AGE limits the biosynthesis of pro-inflammatory
enzymes such as inducible nitric oxide synthetase (NOS), cyclooxygenase (COX) and
lipoxygenase (LOX) (Janssen-Heininger et al., 2000). Chronic over-production of either COX
or LOX causes excess inflammation and increased endogenous levels of ROS, contributing to
chronic pro-inflammatory diseases such as cardiovascular disease, diabetes, arthritis
rheumatoid and others (Goodsell, 2005). COX and LOX also play physiological roles in
processes such as growth, development, wound healing and senescence. The messengers
produced by LOX can either stimulate or prevent the programmed cell death or apoptosis. For
this, an over- production of this enzyme could give rise to an insufficient cell death, which
could lead to development of cancer (Hannun, 1997), or to an excessive cell death, which is
involved in neurodegenerative diseases, such as Alzheimer’s disease or dementia. The
synthesis of these pro-inflammatory enzymes (COX and LOX) is regulated by gene
regulatory factors (transcription factors), whose expression is, in turn, controlled by reduction
(via antioxidants) and oxidation (via ROS). One of these transcription factors is nuclear factor
kappa B (NF-κB), a master control gene of the immune/inflammatory response (Janssen-
Heininger et al., 2000). Under normal conditions, NF-κB remains inactivated by another
factor, its I-κB inhibitor. However, when NF-κB is stimulated, this is under insufficient levels
of antioxidants, particularly sulphur-containing ones (such as those present in garlic)
(Janssen-Heininger et al., 2000), or an excess of ROS, more COX/LOX is synthesized and
inflammation is triggered. Lang et al. (2004) found that allicin can inhibit the production of
pro-inflammatory cytokine messengers in a study of inflammatory bowel disease, apparently
by inactivating the pro-inflammatory factor NF-kB via its I-κB inhibitor. By virtue of
sulphur-based antioxidants found in garlic, NF-kB was maintained in its inactive state, thus
preventing the synthesis of excess COX/LOX. The role of garlic in preventing age-related
diseases has been also investigated extensively over the last 10-15 years. It is now accepted
that aging and age-related diseases are, at least in part, caused by free radical reactions. Thus,
because of its strong antioxidant properties, AGE has been suggested that it can prevent age-
related chronic diseases of the cardiovascular, immune and brain systems, which can cause
loss of autonomy, dependence and high social costs for individuals and society. In fact, it can
inhibit the thrombus and cataract formation, improve blood circulation and energy levels,
rejuvenate skin, and prevent arthritis and cancer. Moreover, other studies have demonstrated
that it promotes neuronal cells survival by inhibition of the pro-inflammatory enzyme LOX
and protection against oxidative damage, increasing cognitive functions, memory and
longevity and slowing down age-related impairment of learning behaviour and memory
(Moriguchi et al., 1997). However, more experimental evidence is required to confirm this
last hypothesis (Sumi et al., 2001). Due to this neurotrophic activity attributed to AGE, the
garlic potential as natural alternative for the treatment of neurodegenerative diseases, such as
Alzheimer’s disease or dementia, has been recently studied (Chauhan, 2005, 2006).
IMMUNOMODULATORY ACTIVITY
Garlic has been suggested as a promising candidate for maintaining the homeostasis of
immunomodulatory activity (Burger et al., 1993; Kyo et al., 2001; Lamm & Riggs, 2001).
Since the immune dysfunction plays an important role in the development and progress of
several diseases, modification of immune functions by garlic can contribute to their treatment
and prevention.
Several studies have been carried out on animal models to examine the effect of different
garlic components and formulations on immunomodulatory activity. AGE has shown to exert
an anti-allergic effect (Kyo et al., 1997, 2001), as it may directly and/or indirectly modify the
functions of mast cells, basophiles and lymphocytes, which play a leading role in the allergic
cascade reactions, including inflammation.
Patya et al. (2004) found that multiple intraperitoneal administration of synthetic allicin
elicited a marked antitumor effect in mice inoculated with B-16 melanoma and MCA-105
fibrosarcoma. They postulated that such immune-stimulatory effect of allicin was mediated
by its activation of the proto-oncogene p21ras, which has been identified as a key molecular
switch involved in regulating lymphocyte activation.
The pharmacologic effect of AGE to inhibit the tumor cell growth through immune
stimulation has also been described (Lamm & Riggs, 2001; Kyo et al., 2001). The recognized
toxicity of effective therapies against cancer and the absence of toxicological effects observed
for garlic treatment have made garlic a valuable alternative therapy for cancer (Lamm &
Riggs, 2001).
Garlic appears to be effective for restoration of the immune suppression by different
agents such as chemotherapy, UV irradiation and physical and psychological stress (Reeve et
al., 1997; Ushijima et al., 1997; Kyo et al., 1999; Kyo et al., 2001; Dwight et al., 2006). Age-
related deterioration of learning behaviour (Zhang et al., 1997), and abnormal impairment of
immune response, as occurs with acquired immunodeficiency syndrome (AIDS) (Lamm &
Riggs, 2001), have been reported to be improved by the immunomodulatory effect of this
vegetable.
The component in garlic that is responsible for the effective immune stimulation is not
known conclusively, and it is likely that multiple ingredients are immunologically active.
Nakata & Fujiwara (1975) identified a carbohydrate in the garlic extract that appeared to be
responsible for the antitumor immunity. In a later study, Hirao et al. (1987) isolated a protein
fraction from garlic with a clear immune-stimulating effect in vitro. However, these
compounds are not the only active ingredients, since results of other studies suggest that
several low-molecular-weight sulfur compounds from garlic such as DAS, SAC, etc have also
immune-stimulating properties (Sundaram & Milner, 1996; Geng et al., 1997).
EFFECTS ON MICROORGANISMS
Effects of garlic on different categories of microbes are discussed in the following. In
folk medicine, garlic has long been associated with the treatment of viral, bacterial, fungal,
and parasitic infections. Nowadays, the antimicrobial properties of garlic have been the focus
of several recent studies. It is apparent from recent chemical characterisation of their sulphur
compounds that the therapeutic effects, particularly with regards to the antimicrobial
properties, are due to the allicin-derived compounds (Rose et al., 2005). However, some
proteins, saponins and phenolic compounds can also contribute to this activity (Griffiths et
al., 2002). Due to the great antimicrobial activity that garlic possesses, this vegetable could be
used like natural preservatives, to control the microbial growth (Pszczola, 2002).
Antiviral Activity
Garlic has long been stated to possess antiviral properties; however, hardly any work has
been done to investigate these properties. Nagai (1973) reported in vivo antiviral effect of
garlic in mice against intranasally-inoculated influenza virus. Garlic extract also enhaced the
production of neutralizing antibodies when it was inoculated with the influenza vaccine.
Weber et al. (1992) reported the effectiveness in vitro of allicin and its various transformation
products against Herpes Simplex Virus 1 and 2, Vesicular Stomatitis Virus, Vaccinia Virus
and Parainfluenza Virus type 3. Garlic extract was effective against each virus tested, and, at
the highest concentration tested (1 g/mL), the infectivity of each virus was substantially
reduced (Weber et al., 1992). Moreover, garlic extract also shows in vitro activity against
human cytomegalovirus, human rhinovirus type 2, Human Immunoeficiency Virus (HIV),
viral pneumonia and rotavirus (Tsai et al., 1985; Meng et al., 1993). Allicin, ajoene, DATS,
allyl methyl thiosulfinate and methyl allyl thiosulfinate have been reported to possess
antiviral activity, being ajoene the most effective of them all (Hughes et al., 1989; Weber et
al., 1992). In the case of HIV, it is thought that ajoene acts by inhibiting the integrin-
dependent processes (Tatarintsev et al., 1992) and DADS has also proven effective against
HIV-infected cells (Shoji et al., 1993). The antiviral activities of various commercial garlic
products against herpes simplex virus type 1 and parainfluenza virus type 3, including garlic
powder tablets and capsules, oil-macerated garlic, steam-distilled garlic oils, garlic aged in
aqueous alcohol and fermented garlic oil, have been also studied. Antiviral activities of these
commercial products seem to be dependent upon their preparation process and those products
with the highest levels of allicin and other thiosulfinates have the best antiviral activities
(Weber et al., 1992).
Antibacterial Activity
Garlic has been used for centuries in various societies to combat infectious diseases.
Louis Pasteur (1858) and Lehmann (1930) provided the first modern scientific evidences on
medicinal an antibacterial use of garlic extract. More recently, a number of studies have
proven the garlic effectiveness to inhibit the growth of gram-positive, gram-negative and
acid-fast bacteria, as well as toxin production.
The antibacterial activity of garlic is widely attributed to allicin. This is supported by the
observation that if garlic extract is stored at room temperature its antibacterial effectiveness is
greatly reduced. This reduction occurs to a much lesser extent if the extract is stored at 0-4ºC,
suggesting thermal instability of the active components (Harris et al., 2001). Because of its
relative instability and high reactivity, allicin may not have antibacterial activity in vivo. The
allicin-derived organo-sulphur compounds such as DAS, DADS and ajoene (Naganawa et al.,
1996), as well as other thiosulfinates isolated from oil-macerated garlic, as 2-propene-1-
sulfinothioic acid S-(Z,E)-1-propenyl ester [AIIS(O)SPn-(Z,E)], 2-propenesulfinothioic acid
S-methyl ester [AIIS(O)SMe] and metanesulfinothioic acid S-(Z,E)-1-propenyl ester
[MeS(O)SPn-(Z,E)] (Yoshida et al., 1999), are also largely responsible for the antibacterial
activity of garlic.
The antibacterial effect of garlic apparently results from thiol-disulphide exchange
reactions between these sulphur compounds and free thiol groups of bacterial enzymes such
as alcohol deshydrogenase, thioredoxin reductase, trypsin, other proteases and RNA and
DNA polymerases (needed for the replication of the bacterial chromosomes). This disruption
can affect to cell essential metabolism and, therefore, to bacterial virulence and growth
(Jonkers et al., 1999; Bakri & Douglas, 2005).
The bacterial strain Staphylococcus aureus causes pus-producing infections, such as

boils, as well as pneumonia and urinary tract infections (Todar, 2005). Cultures of this strain,
as well as Streptococcus (including S. viridans and S. haematyticus), Vibrio cholerae,

Pseudomonas, Proteus vulgaris, Klebsiella pneumoniae, Salmonella enteriditis (the
bacterium responsible for salmonella food poisoning), Mycobacterium, Clostridium and
Micrococcus, are effectively inhibited by fresh garlic, vacuum dried powdered garlic
preparations and garlic oil. Garlic has been also shown to inhibit the bacterial growth of
Bacillus (including B. typhosus, B. dysenteriae, B. enteriditis, B. subtilis, B. megaterium, B.
pumitus, B. mycoides, and B. thurigiensis), Sarcina lutea, Serratia marcescens amd
Escherichia coli (a common toxin-producing) (Cavallito & Bailey, 1944; Johnson & Vaughn,
1969; Delaha & Garagusi, 1985; Tsao et al., 2003; Zhou, 2003; Benkeblia, 2004).
Chowdhury et al. (1991) also investigated the ability of garlic to inhibit antibiotic-resistant
strains of bacteria. They showed that garlic extract was effective in vitro against Shigella
dysenteriae, S. flexneri, S. sonnei and E. coli, being the minimum inhibitory concentration of
extract 5 μL/mL. Promising in vivo activity was also shown against drug-resistant S. flexneri.
Moreover, several authors have used multiple resistant strains of bacteria to investigate
antibiotic potential of garlic. They found that garlic was more effective than any of the test
antibiotics (penicillin, ampicillin, doxycycline, streptomycin and cephalexin) against clinical
strains of Staphylococcus, Escherichia, Proteus, Pseudomonas and Klebsiella bacteria (Bakri
& Douglas, 2005; Lai & Roy, 2004). Moreover, DAS and DADS have been shown to be
potent therapeutic agents for the treatment of infections originated by S. aureus resistant to
methicilin (Tsao & Yin, 2001; Tsao et al., 2003) and allicin has demonstrated to exert
bacteriostatic effects on some vancomycin-resistant enterococci. An inhibitory synergism was
observed when used in combination with vancomycin (Jonkers et al., 1999). It is thought that
allicin modifies the sulfydryl groups on the enzymes of the TN1546 transposon, which
encodes vancomycin resistance, enhancing susceptibility to vancomycin.
Recently, it has been reported that garlic extracts inhibits the growth of oral pathogens,
concretely Streptococcus mutans and S. sobrinus and Porphyromonas gingivalis and
Prevotella intermedia (gram-positive bacteria), considered as the main bacteria responsible
for dental caries and adult periodontitis, respectively (Kim, 1997; Bakri & Douglas, 2005;
Groppo et al., 2007).
The use of garlic extracts as effective agents for inhibition of the growth of Helicobacter
pylori, which is responsible for serious gastric diseases as ulcers and even stomach cancer
development, has been also proposed. Cellini et al. (1996) demonstrated that aqueous garlic
extract effectively inhibited sixteen clinical isolates and three reference strains of
Helicobacter pylori. The concentration of garlic extract required for 90% inhibition of the
microbes was 5 mg/mL. More recently, several studies have shown that H. pylori could be
efficiently controlled, even better than the commercial antibiotics for H. pylori, when ethanol
and acetone were used for extraction instead of water (O’Gara et al., 2000; Sivam, 2001;
Canizares et al., 2002, 2004). Epidemiological studies have demonstrated that allicin, allyl-
methyl and methyl-allyl thiosulfinate, isolated from acetonic garlic extracts, as well as DAS
and DADS can reduce the risk of gastric neoplasia induced by H. pylori, and inhibit the
gastritis due to this bacterium (You et al., 1998). Likewise, a number of studies have reported
that garlic exerts a differential inhibition between beneficial intestinal microflora and
potentially harmful enterobacteria (Rees et al., 1993). Inhibition observed in E. coli was more
than 10 times greater than that seen in Lactobacillus casei for the same garlic extract dose
(Skyrme, 1997). This behaviour is not clear, but may be due to a greater sensitivity of
enterobacteria to allicin possibly because of the different composition and the increased
permeability to allicin of their cell membrane (Miron et al., 2000).
Antifungal Activity
Several in vitro and in vivo studies have shown the great effectiveness of garlic against a
broad spectrum of yeasts (Davis & Perrie, 2003) and fungi, including Epidermophyton and
Trichophyton, two of the three filamentous fungal genera classified as dermatophytes
(Schmidt & Marquardt, 1936), Candida, Torulopsis, Cryptococcus, Rhodotorula and
Trichosporon (Tansey & Appleton, 1975). Likewise, Adetumbi and Lau (1986) reported that
aqueous extract of dehydrated garlic preparation inhibits the growth of the dimorphic fungus
Coccidioides immitis and in vitro fungal spore germination.
Aqueous extract of garlic has been successfully used in treating cryptococcal meningitis,
which is caused by the fungus Cryptococcus neoformans (Singh & Singh, 1997). Davis et al.
(1990) reported a significant in vivo response to intravenous injection of garlic extract in two
patients with C. neoformans and three patients with other types of meningitis. In these cases,
plasma titres of anti-C. neoformans activity rose two-fold over pre-administration titres. In a
later report, Davis et al. (1994) investigated the use of a concentrated garlic extract that
contained 34% allicin, 44% total thiosulfinates and 20% vinyldithiins. This extract displayed
significant in vitro fungicidal and fungistatic activity against 3 different isolates of C.
neoformans, as well as an in vitro synergism with amphotericin B. This in vitro synergistic
activity of garlic with amphotericin B, one of the main antifungal drugs, was also reported by
Shen et al. (1996) in a later study. Likewise, garlic has proven to be more effective than
nystatin in retarding growth of the fungi, including Aspergillus and Penicillium (Srivastava,
1984). Moreover, aqueous extract of garlic has been also demonstrated to inhibit the growth
of other zoopathogenic fungi such as Histoplasma capsulatum, a fungus that produces a
disease similar to tuberculosis, dermatophytes that cause athletics’ foot and ringworm and
Candida albicans, commonly involved in vaginitis (Srivastava et al., 1995). Venugopal and
Venugopal (1995) also studied the ability of garlic to treat ringworm. They concluded that
garlic could be used as an effective antidermatophytic agent, and suggested that advance
extraction and purification steps could prove garlic to be as effective as standard antifungal
drugs.
Such antifungal activity of garlic extracts depends on their concentration in allicin and its
breakdown sulphur products such as DAS, DADS, DATS, and ajoene. Tansey and Appleton
(1975) determined the activities of DATS, DATeS, DADS and DAS against three species of
Candida and three of Aspergillus, which were ordered as follows: DATeS> DATS> DADS>
DAS. Ajoene also possesses antifungal activity against Aspergillus. Reimers et al. (1993),
studying the antifungal activity of ajoene, observed that the addition of ajoene to some fungal
growth mixtures, including Aspergillus niger, Candida albicans and paracoccidioides,
resulted in inhibition at concentrations lower than that experienced with allicin, suggesting
that ajoene has stronger activity than allicin. Such findings are in agreement with those
obtained in an earlier study by Yoshida et al. (1987). Likewise, in a recent study, allicin has
demonstrated to synergize the fungicidal activity of Cu2+ ions against various strains of
fungus, by inducing Cu2+ complexation with a plasma membrane protein (Ogita et al., 2006).
Tadi et al. (1990), studying the antifungal activity of AGE and its major constituents, SAC
and SAMC, found no in vitro activity. However, when AGE was administrated to infected
mice, the number of organisms was reduced up to 80%.
Adetumbi et al. (1986) and Lemar et al. (2002) reported that reduction of Candida
albicans growth by garlic extracts is due to the inhibition of lipids, proteins and nucleic acid
synthesis. Active compounds of garlic have also shown to destroy fungal cells by inhibiting
of succinate dehydrogenase and decreasing, thus, the oxygen uptake (Szymona, 1952),
reducing the organism growth, changing the lipid profile of the cell membrane (Ghannoum,
1988) and inhibiting the synthesis of the fungal cell wall by the alilamines. These compounds
inhibit the squalene monoxygenase, an enzyme involved in the formation of fungal cell wall,
besides being essential for the cholesterol synthesis (Gupta & Porter, 2001).
In addition to sulphur compounds, a great variety of antifungal proteins and peptides
have been isolated from several Allium species, such as the peptide Ace-AMP1 from onion
seeds (Phillippe et al., 1995), the protein allivin from bulbs of the round-cloved garlic (Wang
& Ng, 2001), and chitinases from garlic, leek (Allium porrum) and chive (Allium tuberosum)
(Van Damme et al., 1993; Lam et al., 2000). Likewise, it is necessary to consider certain
steroid saponins, such as eruboside-Β, isolated from the garlic bulb that also exhibit
antifungal activity for Candida albicans (Matsuura et al., 1988).
Therefore, garlic and its derivatives appear to meet all criteria for being considered
antifungal agents, since, in addition to their effectiveness against a broad spectrum of fungi
and yeast, they are cheap and safe.
Antiparasitic Activity
Literature on the antiparasitic capacity of garlic focuses mainly on protozoan parasites.

African tripanosomiasis, amoebiasis and giardiasis are all serius threats to humans and
livestock in vast regions of Africa, South America and Asia. Due to the occurrence of
unpleasant side effects and increasing resistance to the synthetic pharmaceuticals
recommended for the treatment of these diseases, garlic has been investigated as a potential
alternative. Results of a clinical study (Lun et al., 1994) carried out on patients with
tripanosomiasis, amoebiasis and giardiasis demonstrated that DATS, an allicin breakdown
product, is effective against Tripanosoma brucei (ssp. brucei, ssp. rhodesiense, ssp.
gambiense, ssp. evansi, ssp. congolense and ssp. equiperdum), Entamoeba histolytica,
Giardia lamblia and Giardia intestinalis.
Moreover, several studies have demonstrated that garlic extracts are also effective against
Opalina ranarum, O. dimidicita, Balantidium entozoon, Leishmania, Leptomonas and
Crithidia (Reuter et al., 1996).
In China, DATS, easily synthesised and more stable than the extremely volatile allicin, is
commercially available as a preparation, called Dasuansu, prescribed for the treatment of
giardiasis (Lun et al., 1994) and infections by Entamoeba histolytica and Trichomonas
vaginalis (Lang & Zhang, 1981). In addition, ajoene and other organo-sulphur compounds
from garlic are also effective antiprotozoals.
OTHER BENEFITS
The prebiotic effect of garlic and other plant sources has recently received considerable
attention (Sharma et al., 2006; Mussatto & Mancilha, 2007). Fructans are non-reducing
water-soluble saccharides which are naturally present in garlic and are used by garlic as a
carbohydrate reserve for osmoregulation, adaptation to low temperature photosynthesis, and
protection from freezing stress (Darbyshire & Henry, 1981; Chow, 2002; Fujishima et al.,
2005). Concentrations ranging from 125 to 235 mg/g on a wet weight basis have been
reported for garlic fructans, which make up 96% of total non-structural garlic carbohydrates
(Losso & Nakai, 1997).
Fructo-oligosaccharides (FOS) are fructans consisting of β (2→1) linked fructosyl units
with a terminal sucrosyl moiety, which are obtained either by hydrolysis from inulin or from
sucrose by transfructosylation. FOS have been described to selectively stimulate the growth
and/or activity of beneficial bacteria (bifidobacteria and lactobacilli) in the colon, and thus
improve host health (Ernst & Feldheim, 2000). In a study by Cardelle-Cobas et al. (2008),
FOS with degree of polymerisation (DP) from 3 to 7 including 1-kestose, neokestose,
nystose, etc were determined in commercial dehydrated garlic. The presence of FOS in garlic
with a DP higher than 7 is well known (Darbyshire & Henry, 1981). Although no
identification has been done, polyfructosaccharides with a DP as high as 38 or even 50 have
also been described (Darbyshire & Henry, 1981; Losso & Nakai, 1997). However, highly
polymerized fructans are not efficiently utilized by bifidobacteria (Losso & Nakai, 1997).
In addition to their prebiotic character, garlic FOS present other important beneficial
properties to the health of consumers. They have been associated with a lower risk of
infections and diarrhea, with an improvement of the immune system response (Mussatto &
Mancilha, 2007) and with a non-cariogenic effect (Yun, 1996). FOS have also been described
to increase ferrum, calcium and magnesium absorption (Hidaka et al., 1991) and to decrease
the levels of cholesterol, phospholipids and tryglicerides in serum (Yun, 1996). As many
oligosaccharides are not digested by humans because the human body lacks the enzymes
required to hydrolyze the β-links formed among the units of some monosaccharides, these
garlic components are suitable for use in sweet, low caloric diet foods, and for consumption
by individuals with diabetes (Mussatto & Mancilha, 2007).
SAFETY
Adverse Effects
Despite the extensive research supporting the numerous beneficial biological properties
of garlic and garlic supplements, several papers dealing with their adverse effects and toxicity
and interactions with different drugs and chemicals have also been published (Tattelman,
2005).
Garlic pungent smell, reflected in both breath and body odors, is the most common
adverse effect associated with the intake of small amounts of garlic. Long-term
supplementation of garlic and/or consumption of excessive amounts of this vegetable may
cause other less frequent undesirable effects such as gastrointestinal upsets (indigestion,
diarrhea, etc), flatulence and changes in the intestinal flora (Ackermann et al., 2001). The use
of certain garlic preparations such as enteric-coated garlic supplements, designed to deliver
allicin (1-5 mg depending on the product label claim) directly into the intestinal tract, has also
been reported to be hazardous for stomach mucosa (Hoshino et al., 2001; Amagase et al.,
2001). The effect of several of these garlic preparations (raw garlic powder, boiled garlic
powder and pulverized enteric-coated garlic product) directly delivered into the stomach, as
described by Hoshino et al. (2001), is shown in Figure 7.
Allergic reactions to garlic are rare but might cause contact dermatitis,
rhinoconjunctivitis, asthma, urticaria, etc in susceptible individuals (Lybarger et al., 1982;
Añibarro et al., 1997; Asero et al., 1998; Kao et al., 2004). Burns and contact dermatitis are
the most noted adverse effects after topical application of raw or crushed garlic (Parish et al.,
1987; Canduela et al., 1995; Davis, 2005; Friedman et al., 2006). Most of allergic symptoms
are hypothesized to occur due to garlic’s primary allergens: allicin, diallyl disulfide, and
allylpropyl disulfide (Farrell & Staughton, 1996), being diallyl sulphide the most allergenic
compound when it is topically applied.
A study on a group of workers exposed to garlic and clinically diagnosed with asthma
and rhinitis, revealed IgE-mediated allergy as the cause of their occupational allergy
(Añibarro et al., 1997). Although very few papers try to identify allergenic proteins in garlic,
a combination of proteomics and immunologic methods has been used to identify alliin lyase
(a glycoprotein) as a major allergen of garlic (Kao et al., 2004).
a c
b d
Figure 7. (a) Stomach mucosa immediately after the administration of sample. Three white spots were
observed on the stomach mucosa, indicating that the sample was administered to three sites. Stomach
mucosa 24 h after direct administration of (b) raw garlic powder, (c) boiled garlic powder and (d)
pulverized enteric-coated garlic product. Whereas redness of mucosa was noted in (c) and (d), ulcer-
like erosion of mucosa was observed in (b). Reprinted with permission of The Journal of Nutrition,
2001, 131, 1109S-1113S.
Drug and Chemical Interactions
Several studies have shown contradictory results related to garlic’s interaction with drugs
(Piscitelli et al., 2002; Gallicano et al., 2003). Due to its antithrombotic properties, it has been
suggested that patients taking anti-clotting drugs such as Warfarin use caution when taking
raw garlic or certain garlic supplements, since their anticoagulant activity may be enhanced
and originate prolonged bleeding (Ackermann et al., 2001). High doses of garlic should
therefore be avoided prior to surgery (Burnham, 1995). However, recent clinical trials have
reported the safety of aged garlic extract as a complementary therapy for several drugs,
including Warfarin, Aspirin, statins (cholesterol-lowering drugs), etc (Macan et al., 2006;
Budoff et al., 2004).
It has been reported that the intake of a garlic powder supplement reduced the blood
concentrations of Saquinavir and Ritonavir, protease inhibitors used as antiviral HIV drugs,
due to the stimulation of P450 isozymes (Piscitelli et al., 2002; Gallicano et al., 2003). Unlike
garlic-powder products that contain oil-soluble sulfur compounds derived from allicin (DAS,
DADS, etc), the water-soluble AGE active components neither cause P450-induced
contraindications nor produce severe gastrointestinal toxicity (Amagase, 2006).
Horie et al. (2001) reported that AGE may protect the small intestine against the side
effects (nauseas, vomits, diarrhea, stomatitis and gastrointestinal ulceration) induced by
antitumor drugs. AGE and diallyl disulfide in steam-distilled garlic oil have been shown to
protect against the cardiotoxic effects and oxidative injuries caused by doxorubicin, an
antineoplastic agent widely used in cancer therapy (Kojima et al., 1994; Awazu & Horie,
1997; Dwivedi et al., 1998). The utility of AGE against liver damage caused by different
environmental chemicals and medicinal substances, all of them producing free radicals, has
also been proved (Nakagawa et al., 1988; Wang et al., 1998).
It has been recently reported that cooking garlic with meat seems to reduce the
production of carcinogenic chemicals that may occur in meat as a result of cooking methods,
such as grilling, that expose meat to high temperatures (Wilson et al., 2005). Diallyl sulfide,
the garlic phytonutrient responsible for garlic’s pungency, may help prevent cancer by
inhibiting the effects of one such carcinogen: 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine (PhIP). The production of the liver enzymes that transform PhIP into activated
DNA-damaging compounds is decreased by DAS. In addition, DAS signals the genes
responsible for producing two protective antioxidant enzymes (glutathione-S-transferase and
superoxide dismutase), which help to protect the body against harmful compounds such as
those produced from PhIP.
Dosage, Administration Route and Formulation Type
Conditions of extraction have shown to greatly affect the chemical composition of garlic
preparations (Khanum et al., 2004) (Figure 8). A desirable extraction process should
eliminate the toxic compounds while retaining the most active components. However, to
further establish a garlic formulation as a safe and effective treatment, dosage and
administration route should be taken into account.
It has been taken for granted that garlic is safe in a wide range of doses. However, several
studies have reported that the use of high concentrations and/or prolonged administration of
garlic may cause undesirable effects. In a study by Agusti (1996), prolonged feeding of high
levels of raw garlic to rats resulted in anaemia, weight loss and failure to grow due to lysis of
red blood cells. A significant loss of the normal cellular architecture of the heart, liver and
kidneys after 30 days feeding of garlic homogenate at a dose of 1 g/kg/day was also reported
by Banerjee et al. (2001, 2002). Chronic administration of garlic powder (50 mg/day) also
resulted in inhibition of spermatogenesis in rats, reflecting the antiandrogenic nature of garlic
(Dixit & Joshi, 1982).
Figure 8. Major organosulfur compounds present in different garlic preparations based on the extraction
method. Reprinted from Trends in Food Science & Technology, 18, 609-625. Biological properties of
onions and garlic by Corzo-Martínez et al. (2007); with permission from Elsevier.
However, the toxic effects of garlic may be appreciably reduced at lower concentrations.
Oral dosages recommended in the literature to promote health in adults are 4 g (1-2 cloves) of
raw garlic per day, one 300-mg dried garlic tablet (standardized to 1.3% alliin or 0.6% allicin)
2-3 times per day, or 7.2 g of aged garlic extract per day (Tattelman, 2005).
Although a number of researchers have shown the inhibitory effect of AGE on tumour
growth in a dose related manner (Belman, 1983; Lamm & Riggs, 2001), repeated injections
have been described to become toxic (Lamm & Riggs, 2001). Different outcomes depending
on the administration route have also been reported by Lau et al. (1986), with intratumoral
injections of garlic being more effective than intraperitoneal admissions for the treatment of
mouse bladder tumours. Recently, a reversal of antioxidant effect has also been described
with an increase in the dose of raw garlic homogenate (Banerjee et al., 2002).
The above mentioned dosage-dependent toxicity can not be explained fully, but it could
be related with the ability of some allicin-derived sulfur compounds present in garlic to cross
the cell membranes and spontaneously combine with the SH-groups of amino acids and
proteins, thus interfering with the cell metabolism. In moderate amounts, human cells are not
poisoned by these garlic compounds as they contain glutathione, a sulphur-containing amino-
acid that combines with the allicin derivatives, preventing cell damage. However, at higher
doses, interaction between garlic compounds and enzymes in the body could inhibit their
activity, explaining garlic toxicity (Banerjee et al., 2003; Stephen, 2005).
The study of the bioavailability and metabolic fate of the active ingredients (or their
metabolites) in garlic preparations is essential, since their concentration and their effects in
vitro may not determine their effectiveness in vivo. Dried garlic preparations are required to
be enteric coated to be effective because allicin formation is inhibited by a low
gastrointestinal pH (Tattelman, 2005; Li et al., 2007). However, microencapsulation can give
rise to a significant loss in bioactivity and, as previously mentioned, can cause gastrointestinal
upsets (Hoshino et al., 2001).
Similarly, oil-based preparations are presumably less efficacious because of the
instability of sulfur compounds in this media (Freeman & Kodera, 1995). Compounds such as
allicin, sulfides, ajoene, vinyldithiins, etc have not been found in blood or urine, even after
consumption of a large amount of garlic and, therefore, are likely not to be the active
compounds per se. The instability and/or metabolism of these compounds could contribute to
the inconsistent results found in several clinical studies on hypocholesterolemic effect of
garlic oil and garlic powder products (Breithaupt-Grögler et al., 1997; Berthold et al., 1998).
SAC, the water-soluble organosulfur compound used to standardize AGE can be detected
in plasma, liver and kidney after oral intake; its bioavailability being higher than 87% for the
different animals tested (Nagae et al., 1994). N-acetyl-SAC, a metabolite of this compound
due to the action of N-acetyltransferase, was also identified in urine. The usefulness of these
compounds as adequate markers for clinical studies involving garlic is therefore proved
(Steiner & Li, 2001).
With regards to processing conditions, the deactivation by heat of alliinase has
questioned the therapeutic efficacy of cooked garlic. In a study with rats, Prasad et al. (1996)
demonstrated that garlic subjected to a cooking temperature of 100 ºC for 20 min preserves its
bioactive compounds (sulfur compounds, dietary fibre and essential trace elements such as
selenium and copper), antioxidant potential and protein profile. The decrease in the total
content of antioxidants is, however, significant after heating at 100 ºC for more than 40 min.
Several studies on the effect of controlled storage of commercial dehydrated garlic
samples on Maillard reaction evolution have been carried out (Cardelle-Cobas et al., 2005;
Moreno et al. 2006). In general, dehydrated garlic exhibited a very slow progress of the
reaction which did not lead to any noticeable change in its antioxidant activity upon storage.
Therefore, processing and storage conditions should be taken into account to determine the
quality and effectiveness of the different garlic products marketed.
CONCLUSION
Although used primarily today as a food flavouring agent in cooking, there is good
evidence that garlic may be beneficial for a wide variety of conditions and diseases.
Nowadays, the trend towards the use of natural remedies with fewer side effects has also
promoted garlic consumption as an alternative therapy for certain diseases. However, before
garlic can be considered as a safe and effective therapy, further research into several
questions is required.
Despite garlic cloves are usually eaten raw or cooked, different garlic dietary
supplements including dried or powdered formulations, oils and liquid extracts have been
recently incorporated into the market to satisfy the demand of consumer for garlic bioactive
compounds. However, it is worth noting that these components are highly dependent on the
garlic preparation and, therefore, no single garlic dietary supplement may cover the wide
range of biological activities here reported (Figure 9). Furthermore, several aspects such as
garlic variety, growing location, manufacturing processing and storage conditions, etc may
also affect the content of garlic active components, their stability and health benefits.
Anticarcinogenic and
antimutagenic activities
Antimicrobial activity
Antioxidant (antiprotozoal, antifungal,
activity antibacterial, antiviral)
Immunomodulatory
Other beneficial effects activity
(prebiotic, relief of side effects
Garlic (Allium sativum)
of drugs and chemicals)
Effects related to
cardiovascular disease
(hypolipidemic, hypocholesterolemic,
anti-hypertensive, anti-diabetic, anti-
thrombotic, anti-hyperhomocysteinemia)
Figure 9. Summary of the multiple health-promoting effects of garlic.
Future research should also be done to standardize the content of active compounds in
garlic supplements. This would help to establish the effective dosage and type of garlic
(dehydrated, aged, etc) most appropriate for the health-promoting effect wanted.
The search for active preparations with undesirable pungent odour and taste kept at a
minimum would allow the use of this vegetable and its derivatives as functional ingredients
with therapeutic function in many processed foods. For instance, they could be employed in
the manufacturing of highly consumed products (e.g. fast foods or ready-to-eat foods) with
the aim of providing them with antioxidants, prebiotics, mineral nutrients, etc of usefulness in
the prevention of nutritional deficiencies.
Garlic has been extensively studied in vitro and in vivo using animal models. However,
human clinical trials are scarce and they are often of short duration and including a small
number of patients (Fleischauer & Arab, 2001). Therefore, there is a need to gain reliable
scientific credibility based on well designed trials of the actual and potential health benefits
ascribed to standardized preparations of garlic with known active components (Tattelman,
2005).
Finally, garlic products are marketed both as foodstuffs and as herbal medicinal products.
Whereas garlic consumption is generally accepted as safe, the lack of toxicity of garlic
supplements is now to be guaranteed prior to their use as bioactive products. At the current
time, a standardized regulation of nutrition and health claims on foods is being introduced
with the purpose of defining a set of generally applicable criteria for the scientific
substantiation of these claims (Asp & Bryngelsson, 2008). This would assure that the
consumer benefits without risk from all the nutritional and health-promoting effects of an old
natural remedy: garlic.
ACKNOWLEDGMENTS
This work has been funded by Ministry of Education and Science of Spain (project
AGL2007-63462, and by Consolider CSD2007-00063 INGENIO 2010). A.C.Soria thanks
CSIC and the EU for a postdoctoral I3P contract. A. Cardelle-Cobas thanks MEC for an FPU
grant and M. Corzo-Martínez thanks CSIC for a predoctoral I3P grant.
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Chapter 2
MEDICINAL PROPERTIES OF GARLIC: IMPORTANCE

OF ITS ANTIOXIDANT ACTIVITY
Perla D. Maldonado1, Daniel Limón2, Sonia Galván-Arzate3, Abel

Santamaría4 and José Pedraza-Chaverrí5*
1
Laboratorio de Patología Vascular Cerebral, Instituto Nacional de Neurología y
Neurocirugía Manuel Velasco Suárez, México D.F. 14269, México.
2
Laboratorio de Neurofarmacología, Departamento de Farmacia, Facultad de Ciencias
Químicas, Benemérita Universidad Autónoma de Puebla, Puebla 72570, México.
3
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía
Manuel Velasco Suárez, México D.F. 14269, México.
4
Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y
Neurocirugía Manuel Velasco Suárez, México D.F. 14269, México.
5
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de
México, México D.F. 04510, México.
ABSTRACT
Garlic (Allium sativum) is among the oldest of all cultivated plants. It has been used
as a medicinal agent for thousands years. This remarkable plant has multiple beneficial
effects such as antimicrobial, antithrombotic, hypolipidemic, antiarthritic, hypoglycemic,
antitumor and antioxidant activities. A large number of studies have demonstrated the
antioxidant activity of garlic by using different preparations, including fresh garlic
extract, aged garlic extract, garlic oil, and a number of organosulfur compounds including
alliin, allicin, and S-allylcysteine. These studies have been carried out both under in vivo
- in diverse experimental animal models associated to oxidative stress - and in vitro
conditions - using several methods to evaluate capacity of extracts or compounds to
scavenge reactive oxygen species or to induce oxidative damage -. Derived from these
experiments, the protective effects of garlic have been associated with a prevention or
*
Corresponding author: Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de
México, México D.F. 04510, México. Tel.: (+5255) 5622-3878; E-mail address: pedraza@unam.mx
62 Perla D. Maldonado, Daniel Limón, Sonia Galván-Arzate et al.
amelioration of oxidative stress. In addition, it has been shown that several garlic
preparations and/or organosulfur compounds derived from garlic are able to directly
scavenge reactive oxygen species, including hydrogen peroxide, superoxide anion,
hydroxyl radical, and peroxynitrite. Moreover, it has been shown that garlic is able to
inhibit inducible nitric oxide synthase, which in turn may contribute to decrease
nitrosative damage. In addition, as supporting evidence of the protective properties of
garlic compounds, in this review we are including some original data of the ameliorative
action of S-allylcysteine on the aberrant circling behavior exerted by 6-hydroxydopamine
in rats. In summary, the antioxidant activity of garlic has been clearly characterized in in
vivo and in vitro studies, thus emphasizing its potential use as a therapeutic agent against
different disorders.
INTRODUCTION
Garlic (Allium sativum) is a perennial bulbous plant belonging to the family
Amaryllidaceae, and is closely related to the common onion. It has been cultivated since
ancient times and used for culinary and medicinal purposes by many cultures during centuries
[Hahn, 1996; Block, 1985]. Garlic is a source particularly rich in organosulfur compounds
which are responsible for its flavor and aroma, as well as its potential health benefits [Reuter
et al., 1996].
The principal garlic organosulfur compounds in several garlic presentations are shown in
Table 1. In raw garlic cloves there are two main compounds: 1) γ-glutamyl-S-alkyl-L-
cysteines and 2) S-alkyl-L-cysteine sulfoxides [Lawson, 1996]. The most abundant
organosulfur compound in raw garlic cloves is alliin (S-allylcysteine sulfoxide), which is
present at 10 mg/g fresh garlic or 30 mg/g dry weight [Lawson, 1998]. When garlic cloves are
sliced, or when the powder of dried cloves becomes wet in a non-acid solution, the L-cysteine
sulfoxides, which are odorless, are very rapidly converted into a new class of compounds, the
thiosulfinates, which are responsible for the odor of freshly chopped garlic. This is because
cysteine sulfoxides, which are located only in the clove mesophyll storage cells, come in
contact with the enzyme allinase or alliin lyase (E.C. 4.4.1.4), which is located only in the
vascular bundle sheath cells. Allinase is active at pH 4–5.8, but is immediately inhibited at
acidic pH values below 3.5 or by cooking. Furthermore, microwave heating destroys allinase
activity in 1 min [Song and Milner, 1999]. Due to the abundance of alliin, the main
thiosulfinate formed upon crushing garlic is allicin (Figure 1) [Lawson, 1996]. Crushing
garlic does not change its γ-glutamyl-S-alkyl-L-cysteines content. Allicin breaks down to
form a variety of fat-soluble organosulfur compounds (Figure 2), which depends on garlic
treatment; e.g. diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS)
are obtained by steam-distillation whereas ajoene and vinyl dithiins are obtained by
maceration with vegetal oil (Figure 2) [Block, 1985]. In addition, under in vivo conditions, S-
allyl mercaptocysteine (SAMC) is formed when ajoene, DADS or DATS react with cysteine
(Figure 2) [Lawson and Wang, 2005].
Medicinal Properties of Garlic: Importance of its Antioxidant Activity 63
Table 1. Main organosulfur compounds found in garlic presentations
Garlic Presentation Organosulfur compound

Raw garlic cloves S-alkyl-L-cysteine sulfoxides:
S-allylcysteine sulfoxide (allin)
S-methylcysteine sulfoxide (methiin)
S-trans-1-propenylcysteine sulfoxide (isoalliin)
Cycloalliin
γ-glutamyl-S-alkyl-L-cysteines:
γ-glutamyl-S-trans-1-propenylcysteine
γ-glutamyl-S-cis-1-propenylcysteine
γ-glutamyl-S-allylcysteine
γ-glutamyl-S-methylcysteine
Others: γ-glutamyl-methionine
γ-glutamyl-S-allylmercaptocysteine
Garlic powder Alliin
γ-glutamyl-L-cysteines
Chopped garlic S-alkyl-L-cysteine sulfoxides: cycloalliin
γ-glutamyl-S-trans-1-propenylcysteine
γ-glutamyl-S-cis-1-propenylcysteine
γ-glutamyl-S-methylcysteine
Thiosulfinates:
Allyl 2-propenethiosulfinate (alliicin)
Allyl methyl thiosulfinates
Allyl trans-1-propenyl thiosulfinate
Methyl trans-1-propenyl thiosulfinate
Methyl methanethiosulfinate
Others: γ-glutamyl-methionine
γ-glutamyl-S-allylmercaptocysteine
S-allylmercaptocysteine
Aged garlic extract S-alkyl-L-cysteine sulfoxides: alliin
γ-glutamyl-S-1-propenylcysteine
Others:
S-allylcysteine S-methylcysteine
S-1-propenylcysteine γ-glutamylcysteine
S-allylmercaptocysteine
Steam-distilled garlic Diallyl disulfide Allyl methyl tetrasulfide
oil Diallyl trisulfide Dimethyl trisulfide
Allyl methyl trisulfide Monosulfides
Allyl methyl disulfide Pentasulfides
Diallyl tetrasulfide Hexasulfides
Oil macerated 2-vinyl-(4H)-1,3-dithiin Allyl methyl trisulfide
Garlic 3-vinyl-(4H)-1,2-dithiin E-ajoene
Diallyl disulfide Z-ajoene
Diallyl trisulfide
O
S spontaneus S
2 OH S
- H2O
O NH2 2-propenesulfenic acid Allicin
Allinase
S +
COOH (crushing)
CHCNH2COOH 2 H2O
Alliin CH3COCOOH + 2 NH3
Aminoacrilic acid
Piruvic acid
Figure 1. The formation of allyl 2-propenethiosulfinate (allicin) takes places when S(+)-allyl-L-
cysteine sulfoxide (alliin), which is located only in the clove mesophyll storage cells, come in contact
with the enzyme alliinase (alliin lyase, EC 4.4.1.4), which is located only in the vascular bundle sheath
cells.
S S
S S S CH3
S
O allyl methyl trisulfide
steam distillation diallyl trisulfide
S +
S +
Allicin
S S
S
diallyl disulfide diallyl sulfide
vegetal oil
cysteine
O
cysteine S COOH
S S S
S
Ajoene NH2
S-allyl mercaptocysteine
+
S S
S
S
2-vinyl-4H-1,2-dithiin 3-vinyl-4H-1,2-dithiin
Figure 2. Structures of the main organosulfu compound in garlic oils. Garlic oils are the result of
converting the water-soluble 2-propenethiosulfinate (allicin) of crushed cloves to oil-soluble sulfides by
the use of steam (steam-distilled oil) or by incubation in a common plant oil (oil-macerate).
Many garlic preparations and organosulfur garlic compounds are currently under
investigation for their potential to prevent and treat chronic diseases [Lawson and Gardner,
2005]. One of the better known garlic preparations is aged garlic extract (AGE), which is
formed during garlic aging (up to 20 months). During this time, unstable and highly odorous
compounds in fresh garlic are converted into more stable and less odorous compounds
[Amagase et al., 2001]. Interestingly, AGE chemical composition is different from that of
garlic powder [Lawson, 1998]. For instance, the most abundant compounds in AGE are:
alliin, γ-glutamyl-L-cysteines, γ-glutamyl-S-allylcysteines, S-allylcysteine (SAC), and
SAMC, whereas in garlic powder the most abundant compounds are: alliin and γ-glutamyl-L-
cysteines [Lawson, 1998] (Table 1). Moreover, it has been shown that AGE exhibits
antioxidant properties in vitro [Borek, 2001; Ide and Lau, 1999a; Kim et al., 2001; Yamasaki
and Lau, 1997] and in vivo [Borek, 2001; Dillon et al., 2002]. In addition, AGE is a
commercially available garlic preparation that has been widely studied for its high antioxidant
content and its health-protective potential [Borek, 2001; Ide and Lau, 1999a; 2001; Kim et al.,
2001; Morihara et al., 2002; Yamasaki and Lau, 1997].
Another well known garlic preparation is garlic oil, this formulation is a mixture from
oil-macerated and steam-distilled chopped garlic. It contains oil-soluble allyl sulfides derived
from allicin, such as DADS, DATS, allyl methyl trisulfide, allyl methyl disulfide, diallyl
tetrasulfide, allyl methyl tetrasulfide, dimethyl trisulfide, monosulfides, pentasulfides and
hexasulfides (Table 1) [Pentz and Siegers, 1996].
Different types of garlic supplements are available commercially, and each type provides
a different profile of organosulfur compounds depending on how it was processed [Lawson
and Gardner, 2005]. A considerable number of in vivo and in vitro studies have been
performed in order to test the antioxidant properties of garlic preparations. In this kind of
studies, the effect of garlic preparations on oxidative stress markers was evaluated, including
lipid peroxidation (Lpx) and protein carbonylation. The effect of garlic on changes in the
antioxidant system has been also determined. In addition, the specific scavenging capacity of
different garlic preparations or organosulfur compounds on different reactive oxygen (ROS)
and nitrogen (RNS) species has been characterized. Also, the effect of garlic on cell cultures
has been examined. In this regard, several reviews related with the antioxidant properties of
garlic have been written in recent years and the reader is referred to these sources for
additional information and for in-depth reviews of specific topics [Lawson, 1998; Amagase et
al., 2001; Borek, 2006; Ahmad and Ahmed, 2006; Rahman, 2003; Rahman and Lowe, 2006;
Atmaca, 2004; Banerjee et al., 2003a; Banerjee and Maulik, 2002].
1.1. Garlic Powder, Garlic Extracts, Garlic Homogenates, and Allicin
The following is a compilation of studies evidencing the therapeutic potential of garlic

powder, extracts and compounds in several experimental models accompanied by oxidative stress.
1.1.1. In vitro studies
It has been shown that garlic powder is able to inhibit Cu2+-induced low density lipoprotein
(LDL) oxidation [Lewin and Popov, 1994; Pedraza-Chaverri et al., 2004b] and to scavenge hydroxyl
radical (OH•) [Lewin and Popov, 1994; Pedraza-Chaverri et al., 2006; Torok et al., 1994],
superoxide anion (O2•⎯) [Pedraza-Chaverri et al., 2006], hydrogen peroxide (H2O2) [Pedraza-
Chaverri et al., 2006], and peroxynitrite (ONOO⎯) [Pedraza-Chaverri et al., 2007b]. In addition, the
commercial garlic powder known as Kwai® inhibits lipid hydroperoxide formation [Yang et al.,
1993] and Siegers et al. [1999] showed that garlic powder inhibited the O2•⎯ production in human
granulocytes activated with phorbol myristyl acetate. Pedraza-Chaverri et al. [2006; 2007b] also
described that the ability of heated extracts of garlic powder to inhibit Cu2+-induced-LDL oxidation
in human serum, and to scavenge O2•⎯, OH•, and ONOO⎯, was unaffected, while its ability to
scavenge H2O2 was decreased [Pedraza-Chaverri et al., 2006; 2007b].
Garlic aqueous extract reduces OH• production [Yang et al., 1993; Prasad et al., 1996;
Kim et al., 2001], inhibits Lpx [Yin and Cheng, 1998; Prasad et al., 1996] and lipid
hydroperoxide formation [Yang et al., 1993; Prasad et al., 1996], and scavenges ONOO⎯
[Pedraza-Chaverri et al., 2007b], hypochlorous acid [Pedraza-Chaverri et al., 2007a] and O2•⎯
[Kim et al., 2001]. Also, it prevents Cu2+-induced LDL oxidation [Pedraza-Chaverri et al.,
2004b]. Moreover, Horie et al. [1989] found that the ethanol-soluble fraction of garlic extract
protects against Lpx in isolated microsomes. Furthermore, Zasukhina et al. [2003] found that
garlic extract (170 μg/ml medium) added 24 h before mutagen treatment (irradiation, CdCl2
or 4-nitroquinolone-1-oxide) inhibited O2•⎯ and OH• formation, and protected of DNA
damage in CdCl2-treated human RD cells and leukocytes. In addition, it has been shown that
when aqueous extract of raw garlic is heated, a reduced ability to decrease OH• production
[Prasad et al., 1996] and to inhibit Lpx [Yin and Cheng, 1998] can be obtained.
Recently, Gorinstein et al. [2005] studied the effects of extracts of liophylized garlic and
found that the antioxidant potential (evaluated with β-carotene, 1,1'-diphenyl-2-
picrylhydrazyl -DPPH-, nitric oxide, and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate -
ABTS-) of Polish, Ukrainian, and Israeli garlic samples subjected to 100oC for 20 min is
preserved. The antioxidant ability decreases significantly after 20 min of cooking at 100oC. In
this regard, it has been shown that heating decreases raw garlic ability to inhibit Lpx [Yin and
Cheng, 1998] and the OH• scavenging ability [Prasad et al., 1996]. However, Pedraza-
Chaverri et al. [2006] did not find any change in OH• scavenging ability. The H2O2 and
ONOO⎯ scavenging ability of heated raw garlic or boiled garlic cloves were unaffected
[Pedraza-Chaverri et al., 2006; 2007b]. In contrast, extracts of pickled garlic or microwave-
treated garlic are less effective to scavenge ONOO⎯ [Pedraza-Chaverri et al., 2007b]. In
addition, the ability to scavenge hypochlorous acid was eliminated in extracts of microwave-
treated garlic cloves but not in heated extracts of raw garlic or extracts of boiled garlic cloves
[Pedraza-Chaverri et al., 2007a].
Allicin has been shown to possess antioxidant properties [Okada et al., 2006]. It
scavenges OH• [Prasad et al., 1995], O2•⎯ [Chung, 2006] and peroxyl radical [Okada et al.,
2005], prevents Lpx [Prasad et al., 1995], inhibits methyl linoleate oxidation and shows
reactivity with DPPH [Okada et al., 2005]. Also, allicin inhibits both native (isolated from
allicin-treated groups) and oxidized LDL degradation by isolated mouse macrophages [Gonen
et al., 2005]. The in vitro data described in this section are summarized in Table 2.
1.1.2. In vivo studies
Heart
Rietz et al. [1993] studied the cardiac ischemia/reperfusion in Langendorff heart

preparations of rats. The authors observed that the size of the ischemic zone, as well as
ischemia/reperfusion-induced arrhythmias, were decreased by feeding 2% garlic in the diet by
8 weeks. Consistently, Banerjee et al. [2002a] showed that garlic homogenates (0.125, 0.25
and 0.5 g/kg/day/30 days) prevented oxidative stress and heart injury induced by
ischemia/reperfusion. This treatment ameliorated the increase in thiobarbituric acid-reactive
Table 2. In vitro antioxidant activity of garlic extracts and garlic-derived compounds
Garlic preparation References
Aqueous extract of garlic powder Lewin & Popov, 1994

Pedraza-Chaverri et al., 2004b; 2006; 2007
Torok et al., 1994
Siegers et al., 1999
Heated extracts of garlic powder Pedraza-Chaverri et al., 2006; 2007b
Aqueous extract of garlic Pedraza-Chaverri et al., 2004b; 2006; 2007a; b
Yang et al., 1993
Prasad et al., 1996
Yin and Cheng, 1998
Zasukhina et al., 2003
Okada et al., 2005
Kim et al., 2001
Heated aqueous extract of raw Pedraza-Chaverri et al., 2006; 2007a; b
garlic Prasad et al., 1996
Yin and Cheng, 1998
Extracts of lyophilized garlic Gorinstein et al., 2005
Allicin Prasad et al., 1995

Okada et al., 2005; 2006
Gonen et al., 2005
Chung et al., 2006.
Ethanolic extract of garlic Horie et al., 1989
substances (TBARS) and the decrease in glutathione (GSH) levels, as well as the antioxidant
enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx).
In addition, Banerjee et al. [2003b] found that the oral administration of raw garlic
homogenate in three different doses (125, 250, 500 mg/kg/day) for 30 days prevented
isoproterenol-induced myocardial necrosis in rats. This protective effect was associated with
the preservation of heart SOD activity and reduction in plasma TBARS. Moreover, chronic
administration of raw garlic homogenates (250 and 500 mg/kg daily, orally, for 30 days)
protected the heart against the increase in myocardial TBARS and the decrease in endogenous
antioxidants (SOD, GPx and CAT) induced by adriamycin [Mukherjee et al., 2003]. The
adriamycin-induced damage in mice was also studied by Thabrew et al. [2000], who found
that a diet supplemented with 20 or 100 mg/kg of garlic prevented the increase in
malondialdehyde (MDA) and the decrease in GPx in erythrocytes.
Of further consideration, Zalejska-Fiolka et al. [2007] recently studied the effect of garlic
supplementation on antioxidants parameters in erithrocytes, as well as Lpx and atherosclerotic
processes in oxidized oil-fed rabbits; they found that the addition of garlic in such diets
inhibited atherosclerotic changes in the aorta wall, which was related with the homeostatic
activity of antioxidant enzymes (GPx and total SOD) and Lpx (low concentration of MDA in
erythrocytes).
Liver
Sener et al. [2005a] studied the effect of aqueous garlic extract (500 mg/kg) on the
hepatic ischemia/reperfusion injury in rats. They found a protective effect which was
associated with an increase in hepatic GSH levels and a decrease in tissue MDA levels and
myeloperoxidase (MPO) activity. In the same year it was reported that aqueous garlic extract
(250 mg/kg for 28 days) ameliorated liver fibrosis and oxidative damage (preventing the
increase in ROS, MDA and MPO activity and increasing hepatic GSH levels) induced by
biliary obstruction in rats [Gedik et al., 2005].
Another recent study showed that aqueous garlic extract (300 mg/kg for 14 days)
attenuated hepatitis and oxidative stress induced by galactosamine/lipopolysaccharide
(DGaIN/LPS) (300 mg/kg-30μg/kg) in rats. The hepatic damage was evidenced by a
significant increase in the activities of marker enzymes such as alanine transaminase,
aspartate transaminase, and lactate dehydrogenase, increase of bilirubin, lipid peroxides, and
MPO activity levels in serum. The antioxidant enzyme activities (SOD, CAT and GPx) and
GSH in liver homogenate were significantly decreased in the DGalN/LPS, whereas the
pretreatment of rats with garlic extract reversed these altered parameters near to normal
control values [El-Beshbishy, 2008].
In a previous study, Pal et al. [2006] found that fresh garlic homogenates (0.25
g/kg/day/28 days oral) protected against isoniazid- and rifampicin-induced hepatic damage,
decrease in GSH and increase in Lpx in rats. Furthermore, Wang et al. [1996] previously
showed that fresh garlic homogenates (5 g/kg, given 2 h before acetaminophen injection)
prevented acetaminophen-induced hepatotoxicity, which was associated with amelioration in
liver GSH depletion. In the same study, these authors found that the following organosulfur
compounds derived from garlic had hepatoprotective effects when given 1 h before the
hepatotoxic compound: DAS (0.2 mmol/kg), allyl mercaptan (0.2 mmol/kg), methyl allyl
sulfide (2 mmol/kg), diallyl sulfoxide (0.2 mmol/kg), diallyl sulfone (0.2 mmol/kg), N-
acetylcysteine (0.2 mmol/kg), cysteine (2 mmol/kg), SAC (2 mmol/kg), S-methylcysteine (2
mmol/kg), and S-ethylcysteine (2 mmol/kg). Interestingly, Bruck et al. [2005] found that both
hepatic injury and inducible nitric oxide synthase (iNOS) liver expression induced by
concanavalin A were attenuated by allicin pretreatment (21 mg/kg/day orally for 7 days, the
last allicin dose was administered 1 h prior concanavalin A injection) in Balb/c mice.
On the other hand, Vimal and Devaki [2004] demonstrated that oral pretreatment of
allicin (0.3 mg/kg body weight, in 1% gum acacia solution) for 15 days prevented the D-
galactosamine-induced hepatitis, the decrease in the activities and levels of the antioxidant
enzymes SOD, CAT, GPx and glutathione-S-transferase (GST), the reduction in GSH levels,
and the increase in Lpx in liver.
Brain
Saleem et al. [2006] studied the protective effect of a pretreatment with aqueous garlic
extract (500 mg/mL/kg of body weight, i.p.) 30 minutes before the induction of middle
cerebral artery occlusion in rats, which was associated with the prevention in GSH depletion,
decrease in glutathione reductase (GR), GPx, GST, SOD, and CAT activities, as well as the
increase in MDA levels.
Lung
Ameen et al. [2003] found that garlic extract (1% body weight/6 days/week i.g. for up to
180 days) attenuated chrysotile-mediated pulmonary toxicity and Lpx in rats. Garlic also
modulated GSH levels and GST activity. Furthermore, allicin (0.1 mg at the beginning of
reperfusion) improved pulmonary blood flow and decreased pulmonary vascular resistance
after lung ischemia/reperfusion injury in rats [Batirel et al., 2002].
Kidney
Kabasakal et al. [2005] showed that the administration of aqueous garlic extract (500
mg/kg administered 15 minutes prior to renal ischemia and immediately before the
reperfusion period) ameliorated renal ischemia/reperfusion injury in rats. This protective
effect was associated with restoration of GSH levels and prevention of ROS and MDA
formation, as well as MPO activity and collagen content. In addition, Durak et al. [2007]
found that the supplementation with 5% aqueous garlic extract (20 ml/kg/day) in drinking
water was able to ameliorate cyclosporin A-induced renal damage and increase in MDA in rat
kidney. A diet supplemented with 2% of garlic by 2 weeks in rats also ameliorated
gentamicin-induced acute renal failure by preventing the decrease in renal Mn-SOD and GPx
activities, and the increase in Lpx in kidney [Pedraza-Chaverrí et al., 2000].
Kalayarasan et al. [2008b] have recently demonstrated the promising role of the Nrf2-
mediated antioxidant driven defense force of aqueous garlic extract (200 mg/kg) and S-
allylcysteine (100 mg/kg) on potassium dichromate-induced apoptosis and oxidative stress in
hepatocytes of Wistar rats, characterized by restored activities of liver maker enzymes to
normal status and also by the activities of enzymatic antioxidants, non enzymatic antioxidants
and GSH levels.
Another recent study demonstrated and supported the use of garlic powder as a
renoprotective agent by ameliorating K2Cr2O7-induced renal injury, and this effect was
associated with its antioxidant properties as it prevented by 44-71% the alterations in markers
of renal injury (blood urea/nitrogen, serum creatinine, proteinuria, urinary excretion of N-
acetyl-beta-d-glucosaminidase), by 55% the histological damage, and by 47-100% the
increase in markers of oxidative and nitrosative stress (GST, MDA, and protein carbonyl
content [Pedraza-Chaverri et al., 2008].
A diet for 4 weeks supplemented with 2% garlic powder given to female Wistar rats
prevent by 40-59% the alterations in different markers of renal injury, by 33% the histological
damage, and by 38-75% the increase in markers of oxidative and nitrosative stress in a model
of cisplatin-induced nephrotoxicity (7.5 mg/kg). These data supported the capacity of garlic
powder to ameliorate cisplatin-induced renal injury associated with its antioxidants properties
and its use as a renoprotective agent [Razo-Rodríguez et al., 2008]. Finally, another group has
recently found that aqueous garlic extract is able to ameliorate cadmiun-induced
nephrotoxicity [Suru, 2008].
Bladder
Zeybek et al. [2006] studied the effect of aqueous garlic extract (250 mg/kg, i.p. for 3 days)
on protamine sulfate-induced bladder injury in rats. The protective effect of garlic was associated
with amelioration of Lpx and the increase in GSH levels. In addition, Saglam et al. [2006] found
that aqueous garlic extract (500 mg/kg) ameliorated water avoidance stress-induced degeneration
of the urinary bladder and prevented the increase in bladder MDA levels and the decrease in GSH.
Testis
Unsal et al. [2006] demonstrated that garlic aqueous extract (5 ml/kg) is able to prevent
the increase in MDA levels and xanthine oxidase activity, as well as histologic damage
induced by testicular torsion/detorsion.
Multiorgan dysfunction
Sener’s group [2003] found a protective effect of aqueous garlic extract (500 mg/kg, i.p.
immediately after burn injury) in a model of thermal injury in rats, which was associated with a
prevention in the following alterations: decrease in GSH levels, increase in MDA and protein
oxidation levels, and MPO activity in liver, intestine and lung at 2 and 24 h post-burn injury.
Shortly thereafter, Sener et al. [2005b] showed that aqueous garlic extract (125 mg/kg; i.p.,
injected along with nicotine hydrogen bitartrate for 21 days) exerted a protective effect on chronic
nicotine toxicity in rats. This protective action was evaluated in aorta, heart, kidney and urinary
bladder, and was associated with restoration of GSH levels and decrease in MDA levels, as well as
with MPO activity. By the same time, Omurtag et al. [2005] studied the effect of aqueous garlic
extract (125 mg/kg, i.p. for 30 days) on naphthalene-induced oxidative stress in liver, kidney, lung
and brain from Balb/c mice. They found that all the oxidative markers evaluated (GSH and MDA
levels, MPO activity and collagen content in tissues) were reversed by garlic treatment. More
recently, sodium arsenite-induced oxidative stress has been shown to be ameliorated by aqueous
garlic extract [Chowdhury et al., 2008], whereas garlic methanolic extract was able to ameliorate
streptozotocin-induced oxidative stress in liver and intestine [Rajani-Kanth et al., 2008].
Genotoxic- and carcinogen-induced oxidative damage
Kumaraguruparan et al. [2005] studied the N-methyl-N'-nitro-N-nitrosoguanidine-induced

genotoxicity and oxidative stress in mice. They found that aqueous garlic extract (125 mg/kg i.g.
pretreatment for 5 days followed by the genotoxic 1.5 h after the final feeding) exerted a protective
effect on genotoxicity in a mechanisms associated with modulation of Lpx, GSH, and the GSH-
dependent enzymes (Gpx and GST) in stomach and liver. In addition, Bhuvaneswari’s group
[2004] described the effect of aqueous garlic extract administration (125 mg/kg body weight given
for 5 days by gavage and followed by 7,12-dimethylbenz[a]anthracene 1.5 h after the final
feeding) and tomato extract on 7,12-dimethylbenz[a]anthracene-induced genotoxicity and
oxidative stress in albino mice. Garlic administration reduced genotoxicity, and only in
combination with tomato, significantly reduced Lpx, enhanced GSH levels, and Gpx and GST
activity in liver. Also, Chandra-Mohan et al. [2004] found that aqueous garlic extract (250
mg/kg/day for 5 days) ameliorated the frequencies of 7,12-dimethylbenz[a]anthracene-induced

bone marrow micronuclei and Lpx in mice. These changes were associated with antioxidant-
enhancing effects: GSH, GPx, and GST in liver and erythrocytes. Furthermore, Arivazhagan’s
group [2004] demonstrated that aqueous garlic extract (250 mg/kg, i.g. 3 times/week starting 1
week before the carcinogen exposure and continuing for 2 weeks after the final exposure of the
carcinogen) was able to ameliorate gastric carcinogenesis induced by N-methyl-N'-nitro-N-
nitrosoguanidine in rats. This protective effect was associated with the prevention of TBARS
increase and GST decrease in liver, GSH decrease in plasma, erythrocytes and liver, GPx decrease
in erythrocytes and liver, and vitamin C decrease in plasma. Finally, Khanum et al. [1998] proved
that feeding rats with fresh garlic (4% by 23 weeks) decreased azoxymethane-induced damage,
which was associated with and increase in hepatic GPx activity and a tendency to decrease Lpx.
Lipid-induced oxidative damage
In 2005, Gonen et al. demonstrated that a diet supplemented with allicin (9 mg/kg/day)
reduced the atherosclerotic plaque and the LDL susceptibility to Cu2+-induced oxidation in
apolipoprotein E-deficient and LDL receptor knockout mice. Also, Heinle and Betz [1994]
found that a 5% garlic diet ameliorated the increase in plasmatic levels of cholesterol and the
decrease in GPx, GR, GST, and glucose 6-phosphate dehydrogenase activities in liver, heart
and aorta induced by a 2% cholesterol diet for 4 weeks in rats. Furthermore, Kempaiah and
Srinivasan [2004a] also studied the effect of garlic feeding (2% garlic powder diet for 8
weeks), and they found that this diet ameliorated the GSH depletion, the decrease in GR
activity in erythrocytes, and the decrease in antioxidant enzymes (GR, GST, CAT, and SOD)
activities in liver induced by hypercholesterolemia. The same garlic diet prevented the
following changes induced by a fat diet: decrease in total thiols and GSH, increase in Lpx in
erythrocytes and plasma, and GSH decrease in liver [Kempaiah and Srinivasan, 2004b].
Gorinstein et al. [2006] studied the effect of a garlic-based diet (25 mg/kg body weight)
obtained from raw or boiled garlic and their aqueous extracts over a period of 30 days in rats
with a high cholesterol diet. They found that the decrease in blood lipids was associated with
an increase in plasma antioxidant activity. Garlic boiled for 20 min had the same bioactivity
as raw garlic in its antioxidant spectra.
Finally, Durak et al. [2002a; 2002b] found that garlic extract feeding (1.5 ml/kg/day) was
able to reduce the plaque surface area in the aortas, the MDA levels, and the susceptibility to
oxidation and to increase the blood antioxidant potential and antioxidant status in cholesterol
fed-rabbits, thus providing a promising background to garlic as a protective agent. All the in
vivo studies described in this section are summarized in Tables 3, 4 and 5.
In general terms, the sequential description of this evidence is clearly pointing out to
alleviative actions of garlic in all its presentations. In particular, garlic powder and aqueous
garlic extract have both been extensively studied in different experimental paradigms, and the
findings of these investigations have served to claim that these preparations exerted
antioxidant effects mainly through their properties to reduce peroxidative damage, as well as
to increase the activity of endogenous antioxidant systems.
1.2. Aged Garlic Extract (AGE) and Hydrophilic Organosulfur Compounds
Table 3. Garlic powder ameliorates oxidative damage in animal

experimental models in vivo.
Experimental model Garlic powder dosage Reference
Cardiac ischemia/reperfusion Diet with 2% wild garlic Rietz et al., 1993

powder
Adriamycin-induced toxicity Diet with garlic (20, 100 Mukherjee et al., 2003
mg/kg)
Gentamicin-induced Diet with 2% raw garlic Pedraza-Chaverri et al., 2000
nephrotoxicity powder.
Potassium dichromate- Diet with 2% raw garlic Pedraza-Chaverri et al., 2008
induced nephrotoxicity powder
Cisplatin-induced Diet with 2% raw garlic Razo-Rodríguez et al., 2008
nephrotoxicity powder
Rats fed 2% cholesterol diet Diet supplemented with Heinle and Betz, 1994
for 4 weeks 5% garlic powder
Rats with Diet with 2.0% garlic Kempaiah and Srinivasan, 2004a
hypercholesterolemic diet powder
Rats with high-fat diet Diet with 2% garlic Kempaiah and Srinivasan, 2004b
powder
Rats-fed cholesterol diet Diet with lyophilized Gorinstein et al., 2006
raw or boiled garlic and
their aqueous extracts
(25 mg/kg)
Cholesterol-fed rabbits Diet plus garlic extract Durak et al., 2002a;b
AGE is known to scavenge H2O2 [Ide et al., 1996; Ide and Lau, 1999] and O2•⎯ [Dillon et al.,
2003; Maldonado et al., 2003a]. Consistently, Wei and Lau [1998] observed a protective effect of
AGE (1-8 mg/ml) in endothelial cells challenged by H2O2 or the O2•⎯ generator system
xantine/xanthine oxidase. The following AGE compounds present these properties: a) SAMC,
scavenges OH• and 1O2 [Pedraza-Chaverri et al., 2004]; b) alliin scavenges OH• [Kourounakis and
Rekka, 1991], O2•⎯ [Chung, 2006] and H2O2 [Ide et al., 1996] and to inhibits Lpx [Ide et al., 1996];
and c) Nα-(1-deoxy-D-fructos-1-yl)-L-arginine scavenges H2O2 [Ryu et al., 2001; Ide et al.,
1999]. The later also inhibits the Cu2+-induced LDL oxidation and the dose-dependently peroxides
release during the co-incubation of macrophages with oxidized LDL [Ide et al., 1999].
Recently, new compounds derived from AGE known as tetrahydro-beta-carbolines (1-
methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid and 1-methyl-1,2,3,4-tetrahydro-
beta-carboline-1,3-dicarboxylic acid) have been identified. These compounds show strong
H2O2 scavenging activities, while inhibit 2,2’-azobis(2-amidinopropane)hydrochloride-
induced Lpx and nitrite production from murine macrophages induced by lipopolysaccharide
[Ichikawa et al., 2002; 2006], although they are not present in raw garlic. Furthermore, the
following compounds with strong antioxidant activity have been isolated from 80% ethanol
extract of garlic skin: trans-coumaric acid, N-trans-coumaroyloctopamine, and N-trans-

feruloyloctopamine [Ichikawa et al., 2003].
Table 4. Aqueous garlic extracts ameliorates oxidative

damage in experimental models in vivo.
Experimental model Aqueous garlic extract dosage References

Hepatic 500 mg/kg Sener et al., 2005a
ischemia/reperfusion injury
Biliary obstruction-induced 250 mg/kg Gedik et al., 2005
liver fibrosis
Galactosamine/lipopolysacc 300 mg/kg for 14 days El-Beshbishy, 2008
haride (DGaIN/LPS)-
induced hepatitis
Protamine sulfate-induced 250 mg/kg Zeybek et al., 2006
bladder injury
Ischemia/reperfusion injury 5 ml/kg (Kastamonu garlic) Unsal et al., 2006
after testicular torsion and
detorsion
Nicotine-induced toxicity 125 mg/kg Sener et al., 2005b
Thermal injury-induced 500 mg/kg Sener et al., 2003
oxidative damage
Naphthalene-induced 125 mg/kg Omurtag et al., 2005
toxicity
N-methyl-N'-nitro-N- 125 mg/kg Kumaraguruparan et al.,
nitrosoguanidine-induced 2005
genotoxicity
Potassium dichromate- 200 mg/kg and SAC 100 mg/kg Kalayarasan et al., 2008b
induced hepatotoxicity
Focal cerebral ischemia 500 mg/kg Saleem et al., 2006
Renal ischemia/reperfusion 500 mg/kg Kabasakal et al., 2005
injury
Cadmiun-induced 1 ml/100 g Suru, 2008
nephrotoxicity
N-methyl-N'-nitro-N- 250 mg/kg Arivazhagan et al., 2004
nitrosoguanidine-induced
gastric carcinogenesis
7,12- 125 and 250 mg/kg Bhuvaneswari et al., 2004
dimethylbenz[a]anthracene- Chandra-Mohan et al., 2004
induced genotoxicity
Sodium arsenite-induced 20 mg/kg Chowdhury et al., 2008
oxidative stress
Cyclosporin A-induced 1 g /kg Durak et al., 2007
nephrotoxicity
Water avoidance stress- 500 mg/kg/day Saglam et al., 2006
induced degeneration of the
urinary bladder
Table 5. Raw garlic homogenates, fresh garlic homogenates and allicin ameliorate
oxidative damage in experimental models in vivo.
Experimental model Garlic presentation References

Isoproterenol-induced Raw garlic homogenate (125, Banerjee et al., 2003b
myocardial necrosis 250 and 500 mg/kg)
Adriamycin-induced Raw garlic homogenate (250 Thabrew et al., 2000
cardiotoxicity in rats and 500 mg/kg
Ischemia/reperfusion- Raw garlic homogenate (125, Banerjee et al., 2002a
induced cardiac injury 250 and 500 mg/kg)
Acetaminophen-induced Fresh garlic homogenate (5 Wang et al., 1996
hepatotoxicity g/kg)
Azoxymethane-induced Diet with fresh garlic (4%) Khanum et al., 1998
damage
Isoniazid and rifampicin- Fresh garlic homogenate Pal et al., 2006
induced liver injury (250 mg/kg)
Streptozotocin-induced Methanolic extract of garlic Rajani Kanth et al., 2008
oxidative damage in liver (500 mg/kg)
and intestine
D-galactosamine-induced Allicin Vimal and Devaki, 2004
hepatitis (0.3 mg/kg)
CCl4-induced acute liver S-allyl cysteine (SAC) 50-200 Kodai et al., 2007
injury mg/kg
Chrysotile-mediated Garlic extract Ameen et al., 2003
pulmonary toxicity 1% body weight
Lung ischemia/reperfusion Allicin Batirel et al., 2002
injury (0.1 mg)
Experimental model of Diet with allicin Gonen et al., 2005
atherogenesis (9 g/kg/day)
AGE (1, 2.5 and 5 mg/ml/24 h) also prevents Lpx [Ide and Lau, 1997; 1999a; Wei and Lau,
1994; Yamasaki and Lau, 1997; Wang et al., 1998; Ide et al., 1996; Imai et al., 1994; Yang et al.,
1993], GSH depletion [Ide and Lau, 1999a] and enhances the GSH content [Geng and Lau, 1997;
Wang et al., 1999] in bovine pulmonary artery endothelial cells treated with oxidized LDL. This
protective effect was associated with an increase in GPx, SOD and CAT activities and a decrease
in H2O2 and O2•⎯ generation. Also, Ide and Lau [1999a] showed that AGE suppressed H2O2
production from J774 cells-induced by oxidized LDL, interferon-γ and lipopolisacharide. Wang et
al. [1998] found that the incubation of rat liver slices with AGE (1-5% 30 min before the
hepatotoxin) protected them against the toxicity and Lpx induced by bromobenzene. Dillon et al.
[2003] observed that AGE diethyl ether extract (10% v/v) reduced Cu2+- and 15-lipoxygenase-
induced LDL oxidation. AGE (1-4 mg/ml) is also able to ameliorate the H2O2-induced toxicity
and oxidative damage (increase in TBARS) in pulmonary aorta endothelial cells [Yamasaki and
Lau, 1997]. Furthermore, AGE (0.1%) reduced the methotrexate-induced damage, and this
protective effect was associated with the decrease in the cromatin condensation, DNA
fragmentation, caspase-3 activation, and cytochrome c release; as well as with the preservation of
intracellular GSH levels induced by methotrexate in IEC-6 cells [Li et al., 2008].
It has been shown that other hydrophilic organosulfur compounds have also antioxidant
properties; for instance, N-acetylcysteine (NAC) protects cerebellar granule cells against 4-
hydroxy-2-nonenal-induced cell death [Arakawa et al., 2006]; NAC and S-ethylcysteine
protect human LDL against Cu2+- and amphotericin B-induced LDL oxidation and glycation,
scavenge O2•⎯ and show a marked Cu2+-chelating capability [Ou et al., 2003]; S-ethylcysteine,
S-methylcysteine and S-propylcysteine are able to protect partially oxidized LDL and plasma
samples (obtained from diabetic patients) from further glucose-induced oxidation, while
decreasing the loss of α-tocopherol levels in oxidized LDL and plasma and increasing CAT
and GPx activities in plasma [Huang et al., 2004]. Nishimura et al. [2006] also showed that S-
methylcysteine and S-propylcysteine are able to ameliorate Cu2+-induced LDL oxidation.
Pinto et al. [1997] determined the effect of SAMC and SAC exposures on early phases of
GSH metabolism. They demonstrated that prostate carcinoma cells presented an increase in
GSH level within 30 min of exposure to SAMC or SAC. Since the changes in GSH
concentration are associated with detoxification of xenobiotic substances, as well as cell
protection from damage due to oxidation of sulfhydryl groups in the presence of garlic
derivatives, the significance of elevated levels of GSH has been interpreted in terms of activities
of the enzymes governing its synthesis and utilization. On the other hand, polyamines -
particularly spermine - are likely to play an essential role in cell division and differentiation. A
depletion of GSH by sulfhydryl blocking agents induces oxidative stress in tissues and produces
a marked induction of ornithine decarboxylase, the regulatory enzyme for polyamine
biosynthesis (conversion of ornithine to putrescine). In this regard, transiently depleted
concentrations of putrescine and spermine after treatment with SAMC have been found in some
studies. In summary, the reduced growth of LNCaP cells and reduced spermine concentrations
due to SAMC have been suggested to involve the inhibition of ornithine decarboxylase activity,
either by elevating intracellular GSH (which in turn, inhibits the induction of ornithine
decarboxylase), or by direct reaction with ornithine decarboxylase, or both.
Colon cancer is known to be one of the leading causes of cancer morbidity and mortality
worldwide. The use of garlic and its constituents for the chemoprevention or treatment of
gastric or colon cancer has received increasing attention in recent years. There is evidence
indicating that SAMC also inhibits growth, arrests cells in G2-M, and induces apoptosis in
both SW-480 and HT-29 human colon cancer cell lines, an effect likely associated with an
increase in intracellular levels of GSH and activation of JNK1 [Xiao et al., 2003]. In contrast,
treatment of these cells with equimolar concentrations of SAC had no effect on the inhibition
of growth or cell cycle progression, nor in inducing apoptosis. The inhibitory effects on cell
proliferation obtained with SAMC were consistent with findings obtained with allyldisulfide
derivatives. Moreover, previous reports showed that DADS, ajoene ([E,Z]-4,5,9-
trithiadodeca-1,6,11-triene-9-oxide) and SAMC produced significant decrease in the number
of cells in the G1 phase and a corresponding accumulation of cells in G2-M phase in
erythroleukemia (HEL), promyelocytic leukemia (HL60), and colon carcinoma (HCT15, SW-
480, and HT-29) cultured cells. In contrast, human umbilical vascular endothelial cells and
smooth-muscle cells were found arrested in G1 when treated with these compounds. The
inhibitory effects of SAMC on HT-29 cells were less pronounced than those on SW-480
[Shirin et al., 2001]. A question remains on whether the mechanistic effect of SAMC is
exerted by a direct action on microtubules or on other protein controlling the M phase.
Finally, it has also been suggested that the anticarcinogenic effect of allylsulfide
derivatives is mainly mediated by the induction of GSH since this endogenous tripeptide is
able to detoxify several carcinogens, exerts intracellular antioxidant effects, and regulates
DNA and protein synthesis. In this regard, treatment of SW-480 or HT-29 cells with SAMC
has been shown to be accompanied by enhanced cellular levels of GSH [Shirin et al., 2001].
However, it is unlikely that the cell cycle arrest and apoptotic effects of SAMC can be merely
explained by an increase in GSH. Simultaneously, SAMC, SAC, and other garlic compounds
may, by enhancing cellular levels of GSH, exert protective actions in the early phases of
carcinogenesis by scavenging free radicals. Thus, specific allylsulfide compounds inhibit the
initiation of the carcinogenic process whereas others, like SAMC, might, through their
antiproliferative effects, inhibit tumor promotion and/or progression. Altogether, the evidence
derived from these cell culture studies suggest that SAMC may be useful in chemoprevention
of colon cancer when used alone or in combination with other compounds, e.g., sulindac or its
derivatives. Nonetheless, further studies are required to evaluate the efficacy of SAMC in
experimental animal models [Shirin et al., 2001]. The above data are summarized in Table 6.
Liver
Wang et al. [1999] proved that the pretreatment of rats with AGE (2 or 10 ml/kg) was
able to prevent bromobenzene-induced damage in liver slices which was associated with the
increase of hepatic GSH levels. Nakagawa et al. [1988] found that AGE (100 mg/kg/day, by
gavage 2 h before toxic), and SAMC (200 mg/kg orally 0.5 h after toxic) protect liver against
carbon tetrachloride (CCl4)- or acetaminophen-induced damage in mice. This protective
effect was associated with the suppression in the reduction in GSH levels, as well as the
increase in LPx. Consistently, Sumioka et al. [1998] found that SAMC (100 mg/kg, i.g. given
2 and 24 h before toxic) administration ameliorates liver injury induced by acetaminophen in
mice. The pretreatment with SAMC suppressed the increase in hepatic Lpx and the decrease
in hepatic reduced coenzyme Q9. In addition, S-propylcysteine (1 g/L for 4 weeks in the
drinking water) ameliorated acetaminophen-induced hepatotoxicity in mice [Hsu et al., 2006]
through a mechanism associated with the amelioration of oxidative stress and the increase in
GPx activity in liver.
Heart
Sangeetha and Quine [2006] found that alliin (40 and 80 mg/kg/day/35 days) was able to
ameliorate the isoproterenol-induced (a) cardiac damage, (b) Lpx and (c) the decrease in
GSH, vitamins C and E levels, as well as GR and GST activities. Moreover, Kojima et al.
[1994] found that AGE administration (0.05 ml, i.p. six times/week/40 days) was able to
ameliorate the doxorubicin-induced cardiotoxicity in mice evaluated by the structural,
functional and oxidative (Lpx) damage of heart.
Table 6. In vitro antioxidant effect of aged garlic extract

(AGE) and hydrophilic organosulfur compounds.
AGE Dillon et al., 2003

Maldonado et al., 2003a
Ide and Lau, 1999a,b; 1997
Wang et al., 1998
Wei and Lau, 1998
Geng and Lau, 1997
Ide et al., 1996
Imai et al., 1994
Diethyl ether extract of AGE Dillon et al., 2003
Alliin Ide et al., 1996
Kourounakis and Rekka, 1991
S-allylcysteine Medina-Campos et al., 2007
Chung, 2006
Kim et al., 2006b
Nishimura et al., 2006
Perez-De La Cruz et al., 2006
Huang et al., 2004
Maldonado et al., 2003b
Ho et al., 2001
Kim et al., 2001
Numagami and Ohnishi, 2001
Ide and Lau, 1999a,b;1997
Yamasaki & Lau, 1997
Ide et al., 1996
Imai et al., 1994
S-allylmercaptocysteine Pedraza-Chaverri et al., 2004a
Ide et al., 1996
Imai et al., 1994
Shirin et al., 2001
Xiao et al., 2003
N-acetylcysteine Arakawa et al., 2006
Ou et al., 2003
S-ethylcysteine, Nishimura et al., 2006
S-methylcysteine, and Huang et al., 2004
S-propylcysteine
THbetaCs derivatives from AGE Ichikawa et al., 2002; 2006
N-alpha-(1-deoxy-D-fructos-1-yl)-L-arginine Ryu et al., 2001

from AGE. Ide et al., 1999
Compounds isolated from 80% ethanol extract Ichikawa et al., 2003
of garlic skin
Kidney
SAMC has been demonstrated to attenuate gentamicin-induced oxidative and nitrosative

stress and renal damage in rats [Pedraza-Chaverri et al., 2004]. AGE (1.2 ml/Kg/12 h/ 6 days)
prevented the renal alterations induced by gentamicin. The protective effect of AGE was
associated with the decrease in the oxidative stress and the preservation of Mn-SOD, GPx,
and GR activities [Maldonado el at., 2003a].
Brain
Numagami et al. [1996] showed that AGE (0.08–0.5 ml/kg, i.p. 30 min before ischemia)
was able to ameliorate ischemia/reperfusión brain damage in rats.
Augusti and Sheela [1996] have shown that alliin ameliorates (200 mg/kg/day, i.p.) the
diabetic condition of alloxan-treated rats, an effect that was associated with an increase in GSH
levels and decrease in Lpx in heart, kidney and liver, as well as with an increase of SOD and CAT
activities in liver. Helen et al. [2003] found that alliin (100 mg/kg/day/21 days) ameliorated Lpx
and prevents the decrease in CAT and SOD activities and in vitamins A, C and E levels induced
by nicotine in heart, liver and lung of rats. The above data are summarized in Table 7.
Table 7. Aged garlic extract (AGE) and garlic hydrophilic components ameliorate
oxidative damage in experimental models in vivo.
Experimental model Garlic presentation References

Doxorubicin-induced AGE Kojima et al., 2006
cardiotoxicity (0.05 ml/mouse)
Isoproterenol-induced Alliin Sangeetha and Quine, 2006
myocardial infarction (40 and 80 mg/kg)
Bromobenzene-induced AGE Wang et al., 1999
liver injury (2 or 10 ml/kg)
CCl4-and acetaminophen- AGE (100 mg/kg) Nakagawa et al., 1988
induced liver damage SAC (100 mg/kg)
SAMC (200 mg/kg)
Acetaminophen-induced SAMC (200 mg/kg) Sumioka et al., 1998
liver injury
Ischemic brain damage AGE (0.5 ml/kg) Numagami et al., 1996
SAC (300 mg/kg),
Gentamicin-induced AGE (1.2 ml/kg) Pedraza-Chaverri et al., 2004
nephrotoxicity SAC (250 mg/kg) Maldonado et al., 2003a,b
SAMC (100 mg/kg)
Alloxan-induced diabetes Alliin Augusti and Sheela, 1996
(200 mg/kg)
Nicotine-induced Alliin Helen et al., 2003
oxidative stress (100 mg/kg)
SAC: S-allylcysteine, SAMC: S-allylmercaptocysteine.
1.2.3. S-allylcysteine (SAC)
The two major hydrosulfur compounds obtained from AGE are SAC and SAMC, which
in comparison with other metabolites, are more stable once synthesized (up to two years).
This is probably why, besides its widely proved antioxidant properties, these two compounds
have been widely employed in toxic models involving oxidative stress.
In vivo and in vitro studies have served to claim that these compounds are able to suppress
the skin, esophageal, stomach, colon, liver, lung and breast cancer growth. In addition, they
have shown to inhibit the proliferative effects of several cancer cell lines derived from colon,
lung, leukemia, skin, breast and androgen-independent prostate cancer (PCas). However, in
regard to their structure, the chemical reactivities of SAMC and SAC differ in that the first
contains a disulfide bond capable of cross-reacting with free sulfhydryl groups, whereas the
later is a thioether unable to undergo the same reaction [Pinto et al., 1997]. This fact might serve
to explain the differences that have been reported in their effects this far.
SAC: an antioxidant compound abundant in AGE
SAC is synthesized from γ-glutamyl-S-allylcysteine by the action of γ-glutamyl-S-

transferase (Figure 3). This compound is stable [Lawson, 1998] and easily absorbed by
gastrointestinal tract after oral administration [Kodera et al., 2002]. One of its advantages in
regard to other garlic compounds, such as allicin and DAS, is its limited toxicity established
by its higher lethal oral dose [Amagase et al., 2001].
Pharmacokinetic studies demonstrate fast absorption and distribution phases followed by
a slow elimination phase for oral administration, as well as fast distribution and slow
elimination phases for i.v. administration [Nagae et al., 1994; Yan and Zeng, 2005].
Pharmacokinetics of SAC in humans by oral garlic administration revealed a half-life of 10 h
and clearance time of 30 h [Kodera et al., 2002], suggesting a high bioavailability. After its
oral administration, SAC is absorbed by gastrointestinal tract, and its higher concentrations
are detected in plasma and kidney up to 8 h post-intake [Nagae et al., 1994; Yan and Zeng,
2005]. More detailed information on pharmacokinetics of SAC can be obtained from a recent
compilation of our group [Herrera-Mundo et al., 2009].
Figure 3. γ-Glutamyl-S-allylcysteine is converted to S-allylcysteine (SAC) by a γ-glutamyltransferase

(Taken from Herrera-Mundo et al., 2009).
SAC as an antioxidant
SAC has been reported to effectively scavenge O2•⎯ [Kim et al., 2001; Maldonado et al.,
2003b; Medina-Campos et al., 2007], H2O2 [Ide and Lau, 1999; Maldonado et al., 2003b;
Medina-Campos et al., 2007; Ho et al., 2001], OH• [Kim et al., 2001; Chung, 2006;
Numagami and Ohnishi, 2001; Medina-Campos et al., 2007], and ONOO⎯ [Medina-Campos
et al., 2007; Kim et al., 2006b]. Recently, Medina-Campos et al. [2007] showed that SAC
also scavenges hypochlorous acid (HOCl) and singlet oxygen (1O2). This group revealed that
SAC scavenges HOCl in a similar manner than lipoic acid, but it resulted more efficient to
scavenge 1O2 than lipoic acid and GSH, suggesting the potential of SAC as a protective agent
in events associated with excessive formation of 1O2 and HOCl. In addition, SAC prevents
Lpx [Imai et al., 1994; Ide and Lau, 1997; Chung, 2006], protein oxidation [Maldonado et al.,
2003b] and nitration [Kim et al., 2006b].
Antioxidant and Protective Effects of SAC in In Vitro and In Vivo Models
In consideration to the many antioxidant properties characterized in SAC, this compound

has been tested in regard to its pharmacological effects in a considerable number of toxic
models. The following is a compilation of studies evidencing the therapeutic potential of SAC
in experimental paradigms coursing with oxidative stress.
In vitro studies
The in vitro effects of SAC have been collected from various models. For instance,
SAC has been shown to ameliorate the ONOO⎯ induced hemolysis [Morihara et al., 2005],
Lpx in H2O2-induced endothelial cell injury [Ide and Lau, 2001], the oxygen and glucose
deprivation-induced damage in cerebrocortical cultures [Kim et al., 2006b], and the
oxidation of LDL [Ide et al., 1997; Ide and Lau, 2001; Higuchi et al., 2003; Ho et al., 2001;
Huang et al., 2004]. SAC also protected oxidized LDL and plasma samples (obtained from
diabetic patients) from glucose-induced oxidation in a mechanism related with a prevention
α-tocopherol depletion and an enhancement in CAT and GPx activities in plasma [Huang et
al., 2004].
Of note, SAC has been tested as a neuroprotective agent against the neuronal damage
induced by tunicamycin and β-amyloid peptide in cerebellar granule and hippocampal cell
cultures of rats. Neuronal survival seen in these models was associated with the reduction
of intracellular ROS formation [Kosuge et al., 2003]. Protective effects of SAC were also
reported in β-amyloid peptide-induced apoptosis, which were related with a reduction in
O2•⎯ and H2O2 generation [Peng et al., 2002]. In further support of this evidence, Ito et al.
[2003a] reported protective effects of SAC on amyloid β-peptide induced toxicity in
hippocampal cultures and in PC12 cells. These actions seem to be selective as SAC
produced no effects on tunicamycin- and β-amyloid-induced toxicity in granule cerebellar
culture, nor against 4-hydroxy-2-nonenal-induced toxicity in both cultures [Ito et al.,
2003b]. As an experimental approach to characterize the type of cell death produced by
amyloid beta peptide, the increase in cleaved caspase-12 by the peptide in cultured
organotypic hippocampal slices has been recently described to be reversed by SAC [Ishige
et al., 2007], thus suggesting that this antioxidant may be acting on caspase-dependent
signaling pathways to prevent cell death.
SAC may also block the H2O2-induced nuclear factor kappa B (NFkB) activation in
human T cells [Geng et al., 1997] and endothelial cells [Ho et al., 2001], thus accounting
for protective signaling. By the same years, Ide and Lau [1999b] found that SAC decreased
toxicity and prevented GSH depletion induced by oxidized LDL in endothelial cells.
Shortly thereafter, Kim et al. [2001] reported that SAC also inhibits NO• production and
iNOS expression, while reduces NFkB activation in macrophages.
Inhibition of glucose formation and methylglyoxal derived advanced glycation
endproducts are attributable to SAC, which also exhibited a potent Amadori activity when
compared to pyridoxamine - an inhibitor of advanced glycation end products -. SAC limited
the formation of carboxymethyllysine - a non-crosslinked advanced glycation endproduct
derived of oxidative processes -. Nevertheless, further studies are needed to assess whether
SAC may protect against glycation and free radicals in diabetes and ageing processes
[Ahmad et al., 2007].
Recently, Chu’s group [2006] described the ability of SAC to suppress androgen-
independent prostate cancer cell proliferation and invasive events. The inhibitory effect of
this molecule was clearly associated with induction of mesenchymal to epithelial transition
and restoration of E-cadherin expression at transcription and protein levels, as well as the
reduction of E-cadherin repressor expression, thus demonstrating a novel anticancer effect.
Table 6 summarizes the in vitro antioxidant properties of SAC.
In vivo studies
Heart
Isoproterenol-induced myocardial necrosis has served as an approach to evaluate

cardioprotective drugs and myocardial alterations in ischemic disorders. It has been
demonstrated that oral pretreatment with SAC (150 mg/kg) produced a cardioprotective effect
on isoproterenol-induced myocardial infarction in rats. The authors [Padmanabhan and
Prince, 2006] suggested that this protective effect is linked to the antioxidant actions of SAC
(decreased Lpx levels in plasma and heart). SAC also improved the antioxidant status by
preventing the depleted activity of the antioxidant enzymes SOD, CAT, GPx, GR and GST,
and protected nonenzymatic antioxidants such as GSH and ascorbic acid. These promising
effects of SAC have been recently corroborated in the isoproterenol-induced cardiac damage
since oral pretreatment with SAC (100 and 150 mg/kg, for 45 days) was able to enhance the
activity of mitochondrial (isocitrate dehydrogenase, succinate dehydrogenase, malate
dehydrogenase and α-ketoglutarate dehydrogenase) and respiratory chain (NADH
dehydrogenase and cytochrome c oxidase) enzymes. Moreover, SAC decreased the lysosomal
enzymes activity (β-glucuronidase, β-N-acetyl glucosaminidase, β-galactosidase, cathepsin-D
and acid phosphatase) induced by isoproterenol [Padmanabhan and Mainzen-Prince, 2007].
Cardioprotective effects of SAC have also been found in an acute myocardial infarction
model. SAC (50 mg/kg), given as a pretreatment, decreased mortality and infarct size, while
increased left ventricular cystathionine-gamma-lyase expression (an enzyme responsible for

hydrogen sulfide production in the heart), thus suggesting that SAC protects from myocardial
infarction by means of a hydrogen sulfide production-related pathway [Chuah et al., 2007].
In addition, doxorubicin, a potent anticancer drug, has been shown to be effective against
a broad range of human neoplasms. Its clinical use, however, is limited by its many
cardiotoxic effects, which are likely to be the result of free radical generation and Lpx. SAC
administration (30 mg/kg, i.p. for 5 days, starting 2 days prior to the doxorubicin
administration) reduced the doxorubicin-induced toxicity in the heart and liver of mice.
Doxorubicin induced a mortality rate of 58 %, while SAC treatment reduced this parameter
down to 30%, suggesting that SAC may ameliorate the adverse effects of doxorubicin in
cancer chemotherapy [Mostafa et al., 2000]. Altogether, these findings suggest that SAC is a
promising tool to explore therapeutic alternatives in cardiovascular diseases.
Liver
Liver has been reported to be another important target for the protective actions of SAC.
Oral SAC treatment (100 mg/kg/day, administered 2 h before CCl4) was found to protect liver
from CCl4- and acetaminophen-induced damage in mice [Nakagawa et al., 1988]. The authors
suggested that amelioration of hepatic injury by SAC was associated with the prevention of
both GSH depletion and increase in Lpx. The effect of SAC in acetaminophen-induced
hepatotoxicity in Balb/cA mice has been also characterized [Hsu et al., 2006]. In this study,
SAC (1 g/L into drinking water for 4 weeks and previous to acetaminophen treatment) was
suggested to exert protective effects though the amelioration of oxidative stress as follows:
increase of GPx activity, as well as prevention of MDA formation and GSH depletion. These
data demonstrated that SAC is a potential protective agent against acetaminophen-induced
hepatotoxicity.
On the other hand, CCl4 has been shown to induce hepatic injury (in liver microsomal P-
450 system) by means of its reactive metabolite trichloromethyl radical (zCCl3). SAC (200
mg/kg/i.g., 30 min before the CCl4-injection) was shown to be more effective than other
sulfide compounds (N-acetylcysteine and L-cysteine) for attenuating CCl4-induced liver
damage [Kodai et al., 2007]. Derived from these findings, three possible mechanisms to
explain this protective effect were suggested: 1) SAC might be detoxifying CCl4-generated
radicals and/or modifying the metabolic activation of CCl4 2) SAC might scavenge ROS
produced by secondary infiltrating inflammatory cells; or 3) SAC might act as a mimetic drug
of GSH. These mechanisms were proposed in light that SAC attenuated both the histological
damage and the Lpx in liver. Therefore, SAC has been suggested to be a more promising
therapeutic tool than other sulfide compounds to ameliorate chronic inflammatory diseases,
including liver fibrosis.
Lung
The lung is another organ demonstrated to be sensitive to the attack of ROS. Although
CCl4 is mostly metabolized by hepatic cytochrome P450, it can induce systemic inflammation
and fibrosis in some other organs. Experiments employing SAC (50, 100, or 200 mg/kg i.g.
everyday for 8 weeks) revealed that this agent is more effective than other sulfide compounds
(N-acetylcysteine and L-cysteine) to reduce CCl4-induced interstitial pulmonary fibrosis in
rats [Mizuguchi et al., 2006], in a mechanism likely associated with the reduction of Lpx,
leukocytes infiltration, iNOS expression and ROS formation in the lung, thus suggesting that
SAC might have a potential use in the prevention of interstitial pulmonary fibrosis.
Kidney
Nephrotoxic models have been also employed to characterize the protective properties of
SAC. In 2003, Maldonado’s group reported that SAC (250 mg/kg i.p. 24 h before the first
gentamicin dose and 125 mg/kg/12h i.p. for 4 days along with gentamicin treatment) is able
to ameliorate gentamicin-induced acute renal failure in rats. These protective actions were
associated with the prevention of oxidative damage (protein carbonyl content) and the
preservation of Mn-SOD, GPx, and GR activities.
The induction of renal injury by ischemia/reperfusion is largely associated with oxidative
damage. In a recent study of our group [Segoviano-Murillo et al., 2008], SAC was tested in
animals subjected to right nephrectomy in a model involving 40 min of ischemia and 6 h of
reperfusion. SAC (100 mg/kg/i.p., 30 min before nephrectomy, 15 min before ischemia,
immediately before reperfusion, and 2 h after reperfusion) ameliorated ischemia/reperfusion-
induced histological damage, as well as the expression of renal injury markers, including
blood urea nitrogen, and serum creatinine. These effects were explained through a reduction
of Lpx (assessed by positive immunostaining for 4-hydroxy-2-nonenal). Moreover, our
results were consistent with the effect of other antioxidants in similar models and suggested
that the antioxidant properties of SAC are responsible for renal protection.
On the other hand, chronic renal failure has been associated with oxidative and
nitrosative stress - specifically with an unbalance in O2•⎯ and NO• metabolism -. In particular,
rats with 5/6 nephrectomy constitute a widely employed model for studying chronic renal
failure since these rats present progressive renal damage, proteinuria, oxidative and
nitrosative stress, and hypertension. In this model SAC (200 mg/kg/i.p., everyday for 30
days) was reported to reduce hypertension and renal damage [Cruz et al., 2007]. The
renoprotective effect of SAC was again associated with its antioxidant properties, since SAC
prevented the increase in 3-nitrotyrosine and poly(ADP-ribose) levels, while recovered the
SOD activity. In contrast, the antihypertensive effect of SAC was more related with a
reduction in the renal expression of gp91phox and p22phox, two subunits of NADPH
oxidase, as well as with the ability of SAC to modulate NO• production. Derived from these
findings, we concluded that SAC may be useful to ameliorate hypertension and to delay the
progression of renal damage, although further studies should be performed to clarify the
precise mechanisms by which SAC decrease the iNOS expression, gp91phox and p22phox
levels, and the increase in SOD activity.
Carcinogens-induced oxidative damage
Carcinogens have been shown to act through mechanisms associated with ROS formation.
Therefore, the use of antioxidant molecules, such as SAC, to explore these mechanisms,
represents an important tool for basic and clinical research. The protective effect of SAC (100
mg/kg, three times per week starting on the day following the first exposure to N-methyl-N'-
nitro-N-nitrosoguanidine, during 3 weeks) in gastric carcinogenesis induced by N-methyl-N'-
nitro-N-nitrosoguanidine and saturated NaCl in rats was explained by an increase in Lpx in

stomach and a decrease of this process in liver, plasma and erythrocytes [Velmurugan and
Nagini, 2005]. Moreover, a protective effect of SAC (200 mg/kg/i.g., every other day, for 6
weeks) on hepatic carcinogenesis induced by N-nitrosodiethylamine in rats has been associated
with the following changes: decreased Lpx and increased content of vitamins C, E, and A in
plasma, as well as increased GSH levels and enhanced activities of GPx, SOD, and CAT in
erythrocytes [Sundaresan and Subramanian, 2003].
In regard to metastatic cancer, this represents one of the main causes of cancer-related death
as it rarely responds to available treatments. Chu and et al. [2007] recently characterized the
effect of SAC on CWR22R, a human androgen-independent prostate cancer xenograft, in nude
mice. SAC was able to inhibit the growth of CWR22R, with no detectable toxic effect on nude
mice, and this effect was assumed to be associated with a reduction in serum prostate-specific
antigen level and a proliferation rate of xenografts, accompanied by an inhibition of invasion
through the restoration of E-cadherin and γ-catenin expression. Furthermore, the apoptotic rate
of SAC-treated tumours was increased, coursing with a decrease in anti-apoptotic protein Bcl-2
and an increase in cleaved caspase-3. Results of this study suggested that SAC might be
considered of potential therapeutic value for suppressing androgen-independent prostate cancer.
In further support to the interesting evidence described above, Sundaram and Milner [1996]
compared the effects of DADS – another garlic-derived organosulfur compound – and SAC to
inhibit the in vitro growth of human tumor cell lines. Results of this study in three tumor lines
(HCT-15 from colon, A549 from lung and SK MEL from skin) demonstrated that garlic
compounds, including SAC, may exhibit cytostatic properties.
The administration of SAC in drinking water (1 g/L for 4 weeks) has been shown to be
able to prevent the streptozotocin-induced decrease in GSH levels and in CAT and GPx
activities, and the increase in MDA in liver and kidney of rats [Hsu et al., 2004b]. Consistent
with these findings, SAC also ameliorated the alterations in hepatic GPx induced by a lipid-
enriched diet in mice [Lin et al., 2004].
Central nervous system
SAC is known to contain a thioallyl group that has partially served to explain its
antioxidant capacity. The molecular structure of this agent is likely to exert beneficial effects
as this is a nucleophilic group that can easily donate a proton to an electrophilic species, thus
neutralizing it. Consequently, SAC has been shown to be able to decrease the reactivity of
various ROS and RNS [Medina-Campos et al., 2007]. Numagami et al. [1996] demonstrated
that AGE compounds presenting a thioallyl group (particularly SAC) exhibited a strong
antioxidant capacity in a model of cerebral ischemia in rats. Indeed, SAC reduced the infarct
volume and brain edema, while prevented ONOO⎯ formation and Lpx. More recently, SAC
(300 mg/kg, i.p.) produced a protective effect on cerebral ischemic injury in rats by means of
the inhibition of extracellular signal-regulated kinase activity [Kim et al., 2006b]. The fact
that SAC can cross the blood brain barrier turned it soon of potential interest to be tested in
neurotoxic models. In fact, the prophylactic impact and rescue properties of AGE compounds
(mainly SAC) in ischemia/reperfusion injury are being recently discussed and reinforced
[Sener et al., 2007].
Previously, Moriguchi’s group [1997] reported protective actions of AGE compounds
when tested in cerebral atrophy models. In this study, SAC prevented the cerebral atrophy
caused by neuronal cell loss in senescence-accelerated mice and increased survival and
axonal branching from rat hippocampal neurons. Based on these findings, the authors
suggested that thioallyl group is a key factor accounting for neurotrophic activity. Moreover,
SAC accelerated the axonal branching, thereby suggesting that this molecule is not only
acting as an antioxidant agent, but also as a neurotrophic agent. Remarkably, in contrast to
SAC, other garlic compounds having a thioallyl group (DAS and DADS), were tested in the
same model, resulting in no protection. These results suggested that only SAC exerted
neurotrophic activity. The explanation for the preferential efficacy of SAC, in contrast with
other agents, could be related with the fact that SAC is a hydrosoluble compound, whereas
DAS and DADS are liposoluble. The relevance of this fact is significant since this property
might be accounting for the SAC-induced protective effects in reducing edema formation,
infarct area, Lpx, motor dysfunction and loss of memory skills after middle cerebral artery
occlusion assayed to produce global ischemic damage [Numagami and Ohnishi, 2001]. Thus,
the thioallyl group in SAC and other AGE molecules is also likely to be responsible of
antioxidant and neuroprotective effects, as suggested by further evidence demonstrating that
these agents contributed to reverse the learning and memory deficits seen in two strains of
senescent-accelerated mice [Nishiyama et al., 2001]. Despite the fact that anti-aging effects
are more often seen in animal models when employing AGE instead of SAC alone
[Moriguchi et al., 1994; 1996], the several positive properties of SAC should not be ignored
at all. In this regard, when SAC is administered as a dietary supplement to senescence-
accelerated mice, it has been shown to reverse the learning deficit, as well as the cognitive
disorders associated to this model [Nishiyama et al., 2001].
Another potential factor accounting for protection is dosage. When increasing acute doses
of SAC (100, 300 and 600 mg/kg, i.p.) were tested against cerebral ischemia in rats
[Numagami and Ohnishi, 2001], only the higher doses (300 and 600 mg/kg) exerted
protective effects (prevention of increased water content in the brain). These findings have
served as a basis for other groups to consider dosage to characterize the acute effects of SAC
in other toxic models. For instance, Pérez-Severiano et al. [2004a] tested different doses of
SAC (150, 300 and 450 mg/kg i.p.) in an in vivo neurotoxic model evoked by quinolinic acid,
a well-known excitotoxic and pro-oxidant molecule currently tested in rodents and primates.
In this study, SAC protected against the striatal oxidative damage induced by quinolinate by
preventing ROS formation and Lpx at a dose of 300 mg/kg; however, a higher dose (450
mg/kg) produced oxidative toxicity itself. Authors explanained that 300 mg/kg SAC is a dose
probably producing an optimum level of thioallyl groups, thereby reducing the oxidative
toxicity; however, when the balance of thiols is disrupted by an excess of these groups in the
system, then the result is a non-specific oxidative damage. This consideration is supported by
proposals from other groups that an excess of thiol availability might be responsible for
further reactions with NO• through nitrosylation events, ultimately leading to the formation of
highly toxic nitrosothiols and the consequent oxidative damage [Jara-Prado et al., 2003].
Furthermore, the protective effects of SAC on the quinolinate-induced toxicity model
have been recently confirmed under in vitro conditions by Dairam et al. [2008]. These authors
compared the effects of curcumin and SAC on quinolinate-induced oxidative stress in rat
brain homogenates, showing that SAC possesses antioxidant and metal-binding (Fe2+ and
Fe3+) activities that might be accounting for its neuroprotective features.
Our group has also tested the effects of SAC on the oxidative and mitochondrial function
alterations produced in vivo by 3-nitropropionic acid in striatal tissue of rats [Herrera-Mundo
et al., 2006]. The pretreatment with SAC in this model resulted in protection of striatal tissue
by reduction in Lpx accompanied by a recovery of the mitochondrial reductant capacity.
These findings supported previous results of Pérez-De La Cruz et al. [2006] demonstrating
similar protective effects of SAC on the oxidative toxicity produced by 3-nitropropionic acid
in vitro in isolated brain synaptosomes. Since 3-nitropropionic acid is a well-known
mitochondrial toxin capable of irreversibly blocking succinate dehydrogenase (Complex II)
activity and depleting the energy metabolism responsible for neuronal functions [Herrera-
Mundo et al., 2006], the implications of these findings emphasize the neuroprotective
potential of SAC, pointing out to the design of therapeutic strategies for treatment of
neurodegenerative disorders based on the properties of this agent.
In regard to the effects of SAC on behavioral markers of neurotoxicity, this antioxidant
has been demonstrated to be able to reduce, or even block, the rotation behavior produced by
an intrastriatal infusion of quinolinic acid [Pérez-Severiano et al., 2004a], the learning deficit
produced by β-amyloid peptide [Pérez-Severiano et al., 2004b], and the early hyperkinetic
pattern of motor activity evoked by the systemic adminsitration of 3-nitropropionic to rats
[Herrera-Mundo et al., 2006]. The relevance of this evidence is enhanced when considering
that the assessed behavioral tasks are known to represent major integrative functions of the
Central Nervous System, therefore implying that SAC exerts a broad spectrum of
neuroprotective actions, a question that has been recently investigated in other neurotoxic
models. Indeed, our group has recently tested the antioxidant properties of SAC in three in
vitro neurotoxic models of calcium-induced oxidative damage [Pérez-De La Cruz et al.,
2008]. These models (combining excitotoxicity, oxidative damage and energy depletion) were
assessed in brain synaptosomal fractions, and the role of intracellular calcium as the main
inducer of Lpx was tested in the presence of different potentially protective agents.
Altogether, our results demonstrated that SAC was one of the most consistent antioxidant
agents tested, even against that oxidative activity generated in the cytoplasmic domain.
In further support of the last observations, an interesting and quite recently explored
effect of SAC has been investigated, opening a new potential research line that might offer
support to the use of this agent in neurodegenerative disorders. García et al. [2008] have
recently described evidence demonstrating antioxidant and neuropreventive effects of SAC in
two experimental models of Parkinson’s disease. Remarkably, in these experiments, SAC was
able to prevent not only Lpx and mitochondrial dysfunction from synaptosomal fractions of
1-methyl-4-phenyl-1,2,3,6-tetrahydropiridinium (MPTP)-treated mice and 6-
hydroxydopamine (6-OHDA)-treated rats, but also partially prevented the dopamine depletion
in the striatum of both models, which in turn accounted for total prevention of aberrant kinetic
patterns in MPTP-treated mice, as well as asymmetric behavior in 6-OHDA-treated rats. The
later markers (neurochemical and behavioral data) were obtained at 7 and 15 days after the
last administration of the respective toxins, and revealed properties of SAC beyond its
antioxidant capacity, since also exhibited features as a neuroprotective agent in compromised
dopaminergic functions. For these experiments, the schemes of SAC administration were 120
mg/Kg i.p. for 5 days in MPTP model, and 300 mg/kg i.p. for 3 days in 6-OHDA model.
Table 8 summarized the evidences of the in vivo antioxidant capacity of SAC.
In consideration to the promising results of this first approach using SAC in Parkinson’s
disease models, more recently our group has characterized, in a more detailed manner, the
dose-dependent course of the protective actions of SAC as a confirmatory action of this agent
on the asymmetric behavior of 6-OHDA-treated rats. Doses of SAC tested this time were 75,
300 and 600 mg/kg i.p., and aberrant movements (360° turns) were assessed 15 days post-
lesion (intranigral unilateral injection of 6 μg/μl 6-OHDA to rats). Original results of this
complementary series of experiments are shown in Table 9.
Table 8. S-allylcysteine (SAC) ameliorates oxidative

damage in experimental models in vivo.
Experimental model SAC dosage References

Isoproterenol-induced cardiac damage 100 and 150 Padmanabhan and Prince, 2006
mg/kg Padmanabhan and Mainzen-Prince, 2007
Acute myocardial infarction model 50 mg/kg Chuah et al., 2007
Doxorubicin-induced heart and liver 30 mg/kg Mostafa et al., 2000
toxicity
CCl4- and acetaminophen-induced 100 mg/kg Nakagawa et al., 1988
damage
Acetaminophen-induced liver damage 1 g/L Hsu et al., 2006
CCl4-induced damage 200 mg/kg Kodai et al., 2007
CCl4-induced lung fibrosis 50, 100, 200 Mizuguchi et al., 2006
mg/kg
GM-induced nephrotoxicity 125 mg/kg Maldonado et al., 2003
Ischemia/reperfusion-induced renal 100 mg/kg Segoviano-Murillo et al., 2008
damage
Chronic renal failure (5/6 nephrectomy 200 mg/kg Cruz et al., 2007
model)
Nitrosoguanidine and saturated sodium 100 mg/kg Velmurugan and Nagini, 2005
chloride induced gastric cancer
N-nitrosodiethylamine-induced hepatic 200 mg/kg Sundaresan and Subramanian, 2003
carcinogenesis
Streptozotocin-induced diabetes 1 g/L Hsu et al., 2004b
Lipid-rich diet induced damage 1 g/L Lin et al., 2004
Ischemic brain damage 300 mg/kg Numagami and Ohnishi, 2001
Numagami et al., 1996
Focal cerebral ischemic injury 300 mg/kg Kim et al., 2006b
Amyloid-beta peptide-induced damage 300 mg/kg Perez-Severiano et al., 2004b
Senescence-accelerated mice 40 mg/kg Nishiyama et al., 2001
Quinolinic acid-induced neurotoxicity 300 mg/kg Pérez-Severiano et al., 2004a
Dairam et al., 2008
3-nitropropionic-induced neurotoxicity 300 mg/kg Herrera-Mundo et al., 2006
Pérez-De La Cruz et al., 2006
Calcium-induced oxidative damage 10-1000 μM Pérez-De La Cruz et al., 2008
Parkinson disease models (MPTP and 120 and 300 Garcia et al., 2008
6-hydroxydopamine) mg/kg
Table 9. The effect of different doses of S-allylcysteine (SAC) on the asymmetric

(rotation) behavior induced by an unilateral intranigral injection of 6-OHDA (6 μg/μl)
to rats at day 15 post-lesion.
6-OHDA SAC dose Total number of turns Percent of reduction vs. 6-OHDA
- - 0±0 -
- 300 0±0 -
+ - 54 ± 20 -
+ 75 26 ± 12 52
+ 300 3±1 95
+ 600 20 ± 6 63
Circling behavior was challenged in animals by amphetamine (5 mg/kg, s.c.) 15 days after the
intranigral lesion was practiced to animals, and recorded for 80 min. Mean values ± SEM, and
percent values vs. 6-Hydroxydopamine (6-OHDA)-treatment are presented.
As expected, these findings confirmed our previous observations that 300 mg/kg of SAC
are sufficient to completely prevent asymmetric behavior in 6-OHDA-lesioned rats. The
lower dose of SAC, represented by 75 mg/kg, was able to reduce circling behavior by a half,
thus evidencing the dose-dependency of the protective event. However, the higher dose (600
mg/kg) surprisingly increased the 6-OHDA-induced rotation behavior with respect to the 300
mg/kg dose group. Of course, the later effect has been assumed to be unspecific, and can be
explained by a possible autoxidation process of thiol containing-compounds, such as SAC,
thereby enhancing oxidative activity [Jara-Prado et al., 2003]. Nevertheless, it is clear enough
that, at certain concentrations, the promising antioxidant and neuroprotective properties of
SAC in this and other models deserve more detailed mechanistic characterization, such as that
mentioned by Kim et al. [2006b] in regard to the ability of this agent to prevent brain
ischemic injury by its proved capacity to scavenge ONOO⎯, while inhibiting the activity of
extracellular signal-regulated kinase.
Final considerations for SAC properties
SAC exerts protective effects in different experimental models through its action as a
ROS and RNS scavenger, since it decreases oxidative stress and preserves the antioxidant
enzymes activity, as well as nonenzymatic compound levels. In further consideration to all its
properties, SAC is suggested to show prophylactic capabilities at a clinical level. Therefore,
the evidence collected in this section supports the positive effects attributed to SAC, as it is
able to reduce cardiovascular disease, stroke and neurodegenerative damage. Further research
is needed to clarify the protective mechanisms of this antioxidant in several experimental
models, as well as to support its potential use for clinical purposes.
1.3. Garlic oil and lipophylic organosulfur compounds
The following represents a compilation of evidences dealing with the antioxidant and
protective properties of garlic oil and lipophylic compounds at an experimental level.
Several years ago, it was demonstrated that garlic oil prevents Lpx [Hikino et al., 1986].
Consistently, Chen et al. [2003] found that garlic oil suppressed the increase of Lpx in rat
liver homogenates induced by FeSO4. Lohani et al. [2003] observed a significant reduction in
asbestos fibers-induced damage after treatment of human mesothelial cells with 5 µM but not
with 10 µM of DAS. In addition, DADS inhibits Lpx [Dwivedi et al., 1998; Fanelli et al.,
1998] and lipid hydroperoxide formation [Yang et al., 1993]. Also, diallyl polysulfides (tri,
tetra, penta, hexa, hepta sulfide) inhibited Lpx induced by ascorbic acid and iron in rat liver
microsomes [Horie et al., 1992]. Diallyl pentasulfide also inhibits the Lpx induced by
doxorubicin in beef heart [Awazu and Horie, 1997]. Thereafter, Ou et al. [2003] found that
DAS and DADS protect human LDL against Cu2+- and amphotericin B-induced LDL
oxidation and glycation. These compounds were also able to scavenge O2•⎯ and showed
marked Cu2+-chelating capability. Huang et al. [2004] showed that DAS and DADS delayed
further glucose-induced oxidation in already partially oxidized and glycated samples (LDL
and plasma) isolated from non-insulin-dependent diabetes patients. In addition, these garlic
compounds were able to prevent glucose-induced decrease in (a) plasma antioxidant enzymes
CAT and GPx activities and (b) α-tocopherol content in LDL. Altogether, these results
suggest that DAS and DADS could protect partially oxidized and glycated LDL or plasma
against further oxidative and glycative deterioration, which might benefit patients with
diabetic-related vascular diseases.
Recently, Pari et al. [2007] reported that DATS (5-50 µg/ml) was able to reduce Cd-
induced cell death, Lpx and ROS increase in Vero cells. Moreover, it has been found that
DATS, 2-vinyl-4H-1,3-dithiin, and 3,4-dihydro-3-vinyl-1,2-dithiin are able to ameliorate Cu2-
induced human LDL oxidation [Nishimura et al., 2006].
Following this line of findings, Liu et al. [2006] found that NO• production and iNOS
expression-induced by lipopolisacharide in macrophages was suppressed by DADS and
DATS but not by DAS. DADS and DATS were also able to suppress H2O2 production and
NFkB activation, and the inhibitory effect of DATS on LPS-induced iNOS expression is
likely attributed to its antioxidant potential to inhibit nuclear factor-kappa B (NF-κB)
activation. Consistently, Chang and Chen [2005] demonstrated that DAS, DADS and allyl
methyl sulfide (AMS) suppressed the NO• and prostaglandin E2 production in
lipopolysaccharide-activated macrophages, AMS exhibiting the least inhibition. Furthermore,
DAS and DADS, but not AMS, inhibited iNOS expression. These authors also reported that
DAS inhibited the production of cytokines in stimulated macrophages, and this inhibition was
closely associated with the suppression of NO• and prostaglandin E(2) production. DADS
also repressed the production of stimulated tumour necrosis factor-α (TNF-α) and IL-10 and
increases the production of activated IL-1beta and, to a lesser extent, IL-6. Only the decrease
in IL-10 production was associated with the DADS-induced NO• inhibition. Yet, the DAS-
and DADS-suppressed NO• production was independent of TNF-α. By contrast, AMS,
slightly suppresses the stimulated TNF-α but enhances IL-10 production, and such
modulation was closely associated with the decrease in NO• production. Moreover, Prasad et
al. [2004] examined on human polymorphonuclear leukocytes whether 5-lipoxygenase - the
key enzyme involved in biosynthesis of leukotrienes - is a possible target of DAS and other
compounds. They showed that DAS (non-phenolic compound) inhibited 5-lipoxygenase,
although the high inhibitory effect was observed with quercetin, eugenol and curcumin
(compounds with one or more phenolic ring and methoxy groups in their structure).
In 2004, Gong et al. observed in HepG2 cells, that the inhibition of both ERK and p38
MAPK pathways almost completely blocked the DAS-induced Nrf2 nuclear translocation and
heme oxygenase (HO-1) mRNA expression. This is relevant since Nrf2 has been recognized
as an important antioxidant-driven force. Moreover, the addition of NAC, a ROS scavenger,
blocked the increase of ROS production induced by DAS, as well as DAS-induced ERK
activation, Nrf2 expression and nuclear translocation, and ho-1 expression. Due to these
results, authors suggested that DAS addition to HepG2 cells stimulates a transient increase of
ROS, a signal that induces ERK activation and its interaction with p38 MAPK pathways;
which subsequently stimulates Nrf2 protein synthesis and its nuclear translocation to finally
enhance ho-1 gene transcription and protein synthesis.
Recently, DATS > DADS > DAS were reported to suppress ox-LDL-induced E-selectin
and vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells,
suggesting that the effects of DATS and DADS are likely to be dependent on the
phosphoinositide 3-kinase (PI3K)/dephosphorylated protein kinase B (PKB), or the protein
kinase A (PKA)/cAMP responsive element binding protein (CREB)-signaling pathway in an
adhesion molecule-specific manner [Lei et al., 2008].
DAS also inhibits alcohol-induced cytochrome P450 and decreases ROS production in
thymocytes [Huentelman et al., 1999]. Shimada et al. [2006] found that DADS treatment
reduced the ethanol-induced CYP2E1 enzyme activity and protein expression in isolated
human hepatocytes. Also DADS prevented the increase in MDA formation and caspase-3
activity, and completely prevented GSH depletion induced by ethanol. These data clearly
show that DADS reduces ethanol-induced toxicity in human hepatocytes by reducing
CYP2E1 activity and/or stabilizing the cellular GSH content, supporting a key role of DADS
in preventing hepatotoxicity via CYP2E1 activation. The in vitro data described in this
section are summarized in Table 10.
Heart
Saravanan and Prakash described in 2004 that garlic oil (75 mg/kg per day, orally for a
period of 60 days previous toxic) prevented isoproterenol-induced myocardial infarction, the
increase in Lpx and the decrease in GSH levels and in SOD, CAT, GPx, GST and GR
activities.
Liver
Augusti et al. [2005] found that garlic oil (100 mg/kg) treatment prevented CCl4-induced
liver damage in rats. A bit earlier, Fukao et al. [2004] reported that DADS and DATS (10
μmol/kg for 14 consecutive days) pretreatment decreased the CCl4-induced acute liver injury,
an effect that was associated with an increase in GST and quinine reductase activities. Of
note, the major protective effect was observed with DATS, and thus, it was proposed that
DATS could be one of the key factors in garlic oil that protects the body from the injury
caused by free radicals encountered in daily life.
In 1996, Hu et al. found that DAS (50-200 mg/kg, 1-3 h after acetaminophen injection)
protected against acetaminophen hepatotoxicity in rats. In support, Lin et al. [1996] showed
that diallyl sulfone (25 mg/kg 1 h before, immediately after or 20 min after) protected against
acetaminophen hepatotoxicity, and a dose of 5 mg/kg 1 h before prevented GSH depletion in
liver.
Table 10. In vitro antioxidant activity of garlic oil

and lipophylic organosulfur compounds.
Garlic oil Hikino et al., 1986

Chen et al., 2003
DAS, DADS Lei et al., 2008
Shimada et al., 2006
Chang and Chen, 2005
Gong et al., 2004
Huang et al., 2004
Prasad et al., 2004
Lohani et al., 2003
Ou et al., 2003
Huentelman et al., 1999
Dwivedi et al., 1998
Fanelli et al., 1998
Yang et al., 1993
DATS Lei et al., 2008
Pari et al., 2007
Liu et al., 2006
Nishimura et al., 2006
Awazu and Horie, 1997
Horie et al., 1992
2-vinyl-4H-1,3-dithiin Nishimura et al., 2006
3,4-dihydro-3-vinyl-1,2-dithiin
Diallyl tetrasulfide Awazu and Horie, 1997
Diallyl pentasulfide Horie et al., 1992
Diallyl hexasulfide
Diallyl heptasulfide
DAS: Dially sulfide, DADS: Diallyl disulfide, and DATS: Diallyl trisulfide.
More recently, it has been reported that DAS (250 mg/animal and 500 mg/animal, i.g.)
supplementation for 1 week is able to protect against 7,12-dimethyl benz(a)anthracene-
induced oxidative stress, characterized by restored antioxidant enzyme levels and Lpx in liver
from mice. Furthermore, DAS restored significantly the downregulation of antiapoptotic Bcl-
2 and upregulation of pro-apoptotic Bax proteins observed with 7,12-dimethyl
benz(a)anthracene [Prasad et al., 2008].
Chittezhath et al. [2006] reported that DAS and allyl methyl sulfide reduced the liver
content of glutamate pyruvate transaminase and alkaline phosphatase, which were elevated
after irradiation to animals. Also, there was a decrease in the levels of lipid peroxides in
serum and liver, and an enhancement of the GSH content in these animals.
Garlic oil also prevents acute ethanol-induced fatty liver, and dramatically prolongs the
drunken time while shortens the walking time. In addition, it suppresses the elevation of
MDA levels, restored the GSH levels and enhanced the SOD, GR and GST activities in liver.
Hence, these protective effects should be associated with the antioxidant activity of garlic oil
[Zeng et al., 2008].
Murugavel et al. [2007] observed that diallyl tetrasulfide (40 mg/kg body weight, i.g. for
3 weeks) protected against cadmium-induced oxidative damage in the liver. Diallyl
tetrasulfide indeed reduced the cadmium accumulation and different markers of oxidative
stress (Lpx and protein carbonyl levels). Further, it restored the levels of hepatic antioxidant
defense (prevented the decrease in SOD, CAT, GPx, GST, GR, and glucose-6-phosphate
dehydrogenase activities, as well as the levels of GSH, vitamin C and vitamin E).
DAS also attenuated warm hepatic ischemia/reperfusion-induced injury, and the
hepatoprotective effect of DAS was associated with the decrease in Lpx level and in situ O2•⎯
generation and the increase in GSH level. Additionally, DAS increased HO-1 protein
expression - an enzyme that confers cytoprotection against oxidative stress -, and decreased
the CYP2E1 protein levels and activity in the liver [Shaik et al., 2008].
Lung
In the lung, DAS (120 mg/kg/i.p.) has been shown to be able to prevent the decrease in
GSH levels, SOD, CAT, and GPx activities, and the increases in Lpx levels and MPO activity
induced by bleomycin, a cancer inductor. Moreover, DAS reduced the bleomycin-induced
activation of iNOS and NF-κB, while decreased the augmented levels of the early
inflammatory cytokines TNF-α and IL-1β in lung tissue. Derived from these findings it was
postulated that DAS might be a promising agent for the treatment of idiopathic pulmonary
fibrosis, or at least for preventing or delaying the development of fibrosis during
antineoplastic therapy [Kalayarasan et al., 2008a].
Brain
Gupta et al. [2003] described a neuroprotective effect of garlic oil (23 and 46 mg/kg, i.g.,
90 min before subjecting mice to global cerebral ischemia) on ischemia/reperfusion-induced
cerebral injury. These authors found that garlic oil decreases mitochondrial Lpx, cerebral
infarct size, and the impairment of short-term memory and motor coordination in mice.
Also in mice, garlic oil decreased ROS formation, MDA content and DNA damage
induced by Tributyltin (a toxin that can be consumed by contaminated seafood). Garlic oil
was responsible also of preventing the depletion of cortical thymocytes and damage to
nucleoli and mitochondria. Of additional consideration, in FL human amniotic cells, garlic oil
prevented the tributyltin-induced cytotoxic effects and the increase in intracellular ROS
generation. The authors concluded that garlic oil could be an effective agent as food
supplement to reduce the toxicity of tributyltin [Liu and Xu, 2007].
Yamada et al. [2006] found that ajoene (0.5 mg/day) and oil-macerated garlic extract
(containing 0.5 mg ajoene/day) treatments reduced the mortality, cerebral injury and the
increase in blood serum TBARS levels from stroke-prone spontaneously hypertensive rats.
Interestingly, Kim et al. [2006a] also showed that dietary SAC reduces mortality with
decreased incidence of stroke in the same experimental model of hypertensive rats.
Returning to DAS, this agent has been shown to inhibit cytochrome P450, while
potentiates the selective dopaminergic neurone degeneration in C57/bl mice [Vaglini et al.,
2004]. Furthermore, DAS prevented EtOH-induced decrease in long-chain unsaturated
membrane fatty acids. However, fertile chicken eggs expossed to DAS promoted abnormally
low brain mass [Miller et al., 2000].
Kidney
In kidney, DAS administration (50 mg/kg/day/4 days, i.g) was able to ameliorate
gentamicin-induced nephrotoxicity, oxidative stress (protein carbonyl content) and nitrosative
stress (3-nitrotyrosine immunostaining) in rats [Pedraza-Chaverri et al., 2003b]. Consistently,
in a previous report DADS (50 mg/kg/day/4 days, i.g.) was able to ameliorate gentamicin-
induced nephrotoxicity, oxidative stress, and the decrease in the activity of the antioxidant
enzymes GPx, GR, and Mn-SOD in rats [Pedraza-Chaverri et al., 2003a].
On the other hand, garlic oil administration (50 or 100 mg/kg by 1 week) was able to
ameliorate the nephrotoxicity induced by iron-nitrilotriacetate in rats [Iqbal and Athar, 1998].
Pari et al. [2007] found that DATS (40 mg/kg/day) was able to reduce Cd-induced renal
damage and oxidative stress (Lpx and protein carbonyl content), and to prevent the decrease
in the nonenzymatic (GSH, ascorbic acid, vitamin E) and enzymatic (SOD, CAT, GPx, GST
GR, and glucose-6-phosphate dehydrogenase) antioxidants.
Al-Ghamdi et al. [2004] reported that DAS reduced the H2O2-mediated LLC-PKI cell
death and cytochrome P4502E1 activation. It was therefore proposed that cytochrome
P4502E1 activation occurs possibly due to OH• and contributes to H2O2-mediated LLC-PK1
cell necrosis by acting as a source of iron, while perpetuating the generation of OH• via the
Fenton reaction, thus suggesting that cytochrome P4502E1 inhibition may be considered a
novel approach for the prevention of tubular injury caused by oxidative damage.
Effects on detoxifying enzymes
Previous studies have shown the ability of DAS, DADS, and DATS to alter levels of
hepatic drug-metabolizing enzymes, including cytochromes P450, epoxide hydrolase, GST,
and NAD(P)H quinone oxidoreductase 1 both under in vitro and in vivo conditions
[Cherrington et al., 2003; Chen et al., 2004; Zhang et al., 2006].
In this regard, Fukao et al. [2004] found that allyl sulfides of garlic oil altered both phase
I and phase II enzymes. DAS (100 μmol/kg for 14 consecutive days) increased the phase I
enzyme cytochrome P-450 (CYP) 2E1 activity; whereas DATS (10 μmol/kg for 14
consecutive days) and DADS (100 μmol/kg for 14 consecutive days) did the same with GST,
quinone reductase, and GPx. DAS also induced Phase II enzymes [Brady et al., 1988;
Wargovich et al., 1988]. A greater induction of CYP2B1/2 mRNA levels was observed in
male than in female rats when animals were treated with DAS [Cherrington et al., 2003].
Moreover, DAS induced CYP2B10 in mice [Cheng et al., 2005] and specifically activated
CYP2B1/2 gene in liver through a nuclear accumulation of a DNA-protein complex binding
NR1 [Zhang et al., 2006].
Recently, Fisher et al. [2007] found that in Wistar-Kyoto rats (expressing little
constitutive androstane receptor) garlic oil, DAS, and DADS induced higher CYP2B1/2
mRNA levels in males than in females. Conversely, DAS induced NAD(P)H quinone
oxidoreductase 1 levels equally in Wistar-Kyoto males and females, indicating constitutive
androstane receptor-independent induction in rats. DAS also induced NAD(P)H quinone
oxidoreductase 1 in WT mice, and this induction was completely absent in Nrf2-/- mice.
Interestingly, DAS-mediated induction of CYP2B10 mRNA was independent of the Nrf2
presence or absence. These studies clearly indicate that garlic oil and DAS activate
constitutive androstane receptor and Nrf2 to induce drug-metabolizing enzymes (CYP2B and
NAD(P)H quinone oxidoreductase 1).
Of further consideration, Tsai et al. [2005] reported that DADS and DATS increased
GST mRNA, protein expression and activity in hepatocytes, and that GPE I (enhancer
element) is responsible for this upregulation. Consistently, this group found that DADS and
DATS are both able to induce GST protein and mRNA expression in Clone 9 cells through
the increase of the activator protein-1 (AP-1)-DNA binding activity. Later on, the
phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated
kinase (ERK), but not of p38, was stimulated in the presence of DADS and DATS. These
results indicate that the effectiveness of DADS and DATS on GST expression is likely to be
related with the JNK-AP-1 and ERK-AP-1 signaling pathways, and thus, that DADS and
DATS enhance the binding of AP-1 to GPE I [Tsai et al., 2007].
DAS (0.5 or 2 mmol/kg) has been also shown to increase the 7-pentoxyresorufin O-
dealkylase activity, whereas DADS and DATS (0.5 mmol/kg) increase the placental form of
GST activity in liver, lung and jejunum tissues. DAS, DADS, and DATS enhanced all the
cytochrome P450 2B1 and the placental form of GST proteins in liver, lung, and jejunum.
However, only DADS and DATS increased the placental form of GST mRNA levels in liver
and lung, whereas DAS increased cytochrome P450 2B1 mRNA levels in the liver. These
findings suggest that allyl sulfides of garlic oil differentially induce cytochrome P450 2B1
and the placental form of GST, and the up-regulation of these two biotransformation enzymes
is tissue-specific [Lii et al., 2006].
Carcinogens-induced oxidative damage
Garlic oil-derived organosulfur compounds such as DAS, DADS, and DATS, provide
significant protection against carcinogenesis, and this protection is likely to be related with
their antioxidant properties and the induction of phase II detoxification enzymes.
Abdel-Wahhab and Aly, in 2003, found that garlic oil (5 mg/kg body weight i.g. for 15
days), prevented the oxidative changes in liver and kidney induced by an aflatoxin-
contaminated diet in rats. Garlic oil increased GSH levels and SOD activity in liver and
decreased MDA levels in liver and kidney. Gued et al. [2003] also found that DAS (50 or 320
mg/kg i.p. for 4 days) ameliorated the increase in lipid hydroperoxides in breast and liver
tissues in a model of cancer induced by diethylstilbestrol in rats. Recently, it has been shown
that garlic oil pretreatment (50–100 mg/kg for a week) significantly and dose-dependently
prevented ferric nitrilotriacetate-induced hepatic damage, as well as tumor promotion. Garlic
oil attenuated the increase in hepatic Lpx and H2O2 production, while preserved GSH levels
and antioxidant enzymes activity. In addition, it also preserved ornithine decarboxylase
activity and DNA synthesis, suggesting that garlic oil has the potential to be used as a
chemopreventive agent [Agarwal et al., 2007].
Khanum et al. [1998] proved that feeding rats with fresh garlic (4%) or diet supplemented
with garlic oil (one garlic oil capsule per individual diet cup by 23 weeks) decreased
azoxymethane-induced damage, which was associated with an increase in hepatic GPx
activity and a tendency to decrease Lpx. Consistently, Sengupta et al. [2006] found that DAS
protects against azoxymethane-induced colon carcinogenesis in rats, which was associated
with cyclooxygenase-2 and iNOS inhibition.
Green et al. [2005] reported that the administration of diethylstilbestrol in female rats,
produced DNA adducts in microsomes, mitochondria, and nuclei in the breast, whereas DAS
treatment (200 mg/kg/i.p.) to these rats inhibited by 86%, 93% and 100 % the DNA adducts
in microsomes, nuclei, and mitochondria, respectively. This study provided evidence that
DAS alters diethylstilbestrol-induced breast cancer, possibly by means of a direct inhibition
of the cytochrome P450 enzymes activity. In addition, Wilson et al. [2007] found that DAS
attenuated the 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine-induced formation of lipid
peroxides and DNA strand breaks in normal breast epithelial cells, thereby suggesting that the
organosulfur compound may be used in the chemoprevention of this heterocyclic amine that
causes mammary carcinomas in female rats and mice. Moreover, Yu et al. [2003] observed
that DADS and DATS inhibited the epoxide-forming oxidant of dimethyldioxirane and were
able to directly inhibit the RNA polymerase enzyme in vitro. The authors proposed that the
protective effects of DADS and DATS on tumor induction may be mediated by a competitive
epoxidation/inhibition mechanism that prevents the formation of carcinogen epoxide, and
consequently, the binding of the carcinogen to DNA to initiate carcinogenesis.
DAS, DADS, and DATS were also shown to induce apoptosis in glioblastoma cells due
to a triggered production of ROS and increased intracellular Ca2+ that induced apoptosis
through a p38 MAPK phosphorylation and redox-sensitive JNK1 activation pathway. The
relevance of ROS production was evidenced by ascorbic acid pretreatment, since it attenuated
this effect, the p38 MAPK phosphorylation, and the JNK1 activation [Das et al., 2007]. In
addition, DAS and DADS decreased the viability and induced apoptosis in human malignant
neuroblastoma SH-SY5Y cells. In this case, apoptosis was associated with an increase in
intracellular Ca2+, an increase in Bax:Bcl-2 ratio, a mitochondrial release of cytochrome c, an
increase in cytosolic Smac/Diablo, and a down regulation of apoptosis inhibitor proteins and
NFk-B. Moreover, the caspase-9 and caspase-3 activations indicated the involvement of
intrinsic apoptotic pathways [Karmakar et al., 2006]. Shortly thereafter, Kim et al. [2007]
showed that DATS inhibited growth in vitro and in vivo by causing apoptosis in human
prostate cancer cells (LNCaP, LNCaP-C81, LNCaP-C4-2). DATS-induced apoptosis in
LNCaP cells correlated with the collapse of mitochondrial membrane potential, the modest
increase in protein levels of Bak, and a down-regulation of Bcl-2 and Bcl-xL protein levels.
DATS treatment also increased ROS formation in LNCaP cells, thereby suggesting that the
mitochondria-mediated cell death by DATS was associated with ROS generation and is
regulated by Bax/Bak, but independent of Bcl-2 or Bcl-xL [Kim et al., 2007]. In addition,
Xiao et al. [2006] reported that oral gavage of DATS significantly retarded growth of PC-3
xenografts in athymic mice without causing weight loss. Tumors from DATS-treated mice
exhibited a markedly higher count of apoptotic bodies. Consistent with the results in PC-3
cells, the DATS-mediated suppression of PC-3 xenograft growth correlated with the induction
of the proapoptotic proteins Bax and Bak.
Recently, it was reported that DAS increases ROS formation, induce apoptosis, and
promote cell cycle arrest mainly at G2/M phase in Colo 320 DM colon cancer cells. DAS also
decreased alkaline phosphatase and lactate dehydrogenase activities, exhibiting
antiproliferative and cytotoxic effects, respectively. Later on, DAS upregulated the NFk-B
expression, promoted the expression of caspase-3 and the suppression of ERK-2 activity in
Colo 320 DM cells. This study supported the consideration that DAS may be a drug with
potential therapeutic uses to treat cancers [Sriram et al., 2008].
Of recent description, Iciek et al. [2007] found that DADS increased hepatic sulfane
sulfur levels, and γ-cystathionase and mercaptopyruvate sulfotransferase activities, but do not
affect the hepatic GSH levels in healthy mice. Moreover, DADS corrected the concentrations
of GSH and sulfane sulfur, while ameliorated the γ-cystathionase activity that had been
lowered in the livers of Ehrlich ascites tumor-bearing mice. In contrast, DADS had not effect
on sulfane sulfur and GSH levels, nor on γ-cystathionase activity in Ehrlich ascites tumoral
cells. Altogether, these results suggest that DADS efficiently and selectively enriches
hepatocytes in sulfane sulfur with concomitant activation of enzymes implicated in its
formation and transfer, and so it can be helpful for chemotherapy.
Gastrointestinal damage
In 2004, Khosla et al. found that garlic oil (0.25 and 0.5 mg/kg, 30 min before ethanol
administration) ameliorated the ethanol-induced gastric ulcers and Lpx, and prevented the
decrease in antioxidant enzyme levels (GPx, CAT, and SOD). Chiang et al. [2006] also
showed that the activity of iNOS in the intestinal mucosa was significantly suppressed by a
treatment with garlic oil (50 or 200 mg/kg body weight for 2 weeks before endotoxin
administration) and DATS (0.5 mmol/kg body weight i.g. for 2 weeks before endotoxin
administration) in endotoxin-induced intestinal mucosal damage in rats. The high dose of
garlic oil tested also lowered the peripheral levels of nitrate/nitrite and iNOS activity in the
intestinal mucosa. Finally, both the higher garlic oil dose and DATS significantly raised the
content of non-protein-reduced thiols in the intestinal mucosa.
Prasad et al. [2006] described modulatory effects of DAS against testosterone-induced

oxidative stress in mice. Remarkably, DAS (250 and 500 mg/mouse) treatment reduced the
apoptotic cell population preceded by a decrease in ROS levels and a restoration of
mitochondrial transmembrane potential, followed by decreased DNA fragmentation. DAS
was also able to prevent testosterone-induced Lpx and the decrease in the antioxidant
enzymes CAT, SOD, GR, and GST in prostate and liver.
In addition, garlic oil (100 mg/kg/day for 21 days) resulted effective to prevent the
nicotine-induced oxidative damage (Lpx and formation of conjugated dienes and
hydroperoxides), as well as the decrease in antioxidant enzymes (CAT, GPx and SOD) in
liver, lungs, heart and kidney of rats [Helen et al., 1999].
Anwar and Meki [2003] studied the effect of garlic oil (10 mg/kg i.p. for 15 days) on
oxidative stress in a diabetes model induced by streptozotocin in rats. Garlic oil decreased
hepatic Lpx, increased GST activity in liver and erythrocytes, and increased SOD activity in
liver and kidney. In other study on diabetes induced by streptozotocin in rats, Liu et al. [2005]
found that garlic oil (100 mg/kg body weight) and DATS (40 mg/kg body weight) treatment
were both able to ameliorate the diabetic condition. The in vivo data described in this section
are summarized in Tables 11 and 12.
When considered together, this evidence clearly demonstrate that these compounds –
particularly DATS – exhibit consistent anti-cancer properties that, in turn, can be associated
with the modulation of signaling pathways further leading to tumoral processes. Of relevance,
these actions are likely to be, in general terms, dependent on the modulation of Phase I and II
detoxifying enzymes, which in turn are responsible for handling of carcinogens. In addition,
garlic oil and garlic oil-derived compound have been shown a protective effect in several
experimental models associated to oxidative stress.
Table 11. Antioxidant effect of garlic oil in in vivo models.
Experimental model Garlic oil dosage References

Isoproterenol-induced myocardial 75 mg/kg Saravanan and Prakash, 2004
infarction
CCl4-induced liver damage 100 mg/kg Augusti et al., 2005
Acute ethanol-induced oxidative 50, 100 and 200 mg/kg Zeng et al., 2008
estress
Ischemia/reperfusion-induced 23 and 46 mg/kg Gupta et al., 2003
cerebral injury
Iron nitrilotriacetate-induced 50 and 100 mg/kg Iqbal and Athar, 1998
nephrotoxicity
Aflatoxin-induced toxicity The oil soluble extract Abdel-Wahhab and Aly, 2003
of garlic (5 mg/kg)
Ferric nitrilotriacetate-induced 50–100 mg/kg Agarwal et al., 2007
hepatic damage
Nicotine-induced 100 mg/kg Helen et al., 1999
lipoperoxidation
Ethanol-induced gastric injury 0.25 and 0.5 mg/kg Khosla et al., 2004
Endotoxin-induced intestinal 50 or 200 mg/kg Chiang et al., 2006
mucosal damage DATS (0.5 mmol/kg)
Streptozotocin-induced diabetes 100 mg/kg Liu et al., 2005
DATS (40 mg/kg)
Streptozotocin-induced diabetes 10 mg/Kg Anwar and Meki, 2003
DATS: Diallyl trisulfide.
1.4. Effect of Garlic on the Enzymatic Antioxidant System
Several studies have described the effect of garlic and garlic compounds on the
antioxidant system. As it is described below, an effect has been observed in most of these
studies, depending on the garlic presentation or garlic compound used, as well as the dose
and time of treatment, and the species used.
Table 12. Antioxidant effect of garlic oil-derived compounds in in vivo models.
Experimental model Garlic compound References

CCl4-induced acute liver injury DADS and DATS Fukao et al., 2004
(10 mmol/kg)
Acetaminophen-induced DAS Hu et al., 1996
hepatotoxicity (50 mg/kg)
Acetaminophen-induced DASO2 Lin et al., 1996
hepatotoxicity (5 and 25 mg/kg)
7,12-dimethylbenz(a) anthracene- DAS (250 and 500 mg/animal) Prasad et al., 2008
induced oxidative stress
Irradiation-induced oxidative stress DAS (250 μg/animal) Chittezhath et al., 2006
Cadmium-induced oxidative damage Diallyl tetrasulfide Murugavel et al., 2007
(40 mg/kg )
Hepatic ischemia/reperfusion-induced DAS (1.75 mmol/kg) Shaik et al., 2008
injury
Bleomycin-induced toxicity DAS (120 mg/kg) Kalayarasan et al., 2008a
Stroke-prone spontaneously Ajoene (0.5 mg/day) and oil- Yamada et al., 2006
hypertensive rats macerated garlic extract (0.5 mg
ajoene/day)
Gentamicin-induced nephrotoxicity DAS and DADS Pedraza-Chaverri et al.,
(50 mg/kg) 2003a,b
Cadmium-induced nephrotoxicity DATS Pari et al., 2007
(40 mg/kg)
Diethylstilbestrol-induced breast DAS Gued et al., 2003
cancer (50 and 320 mg/kg)
Diethylstilbestrol-induced breast DAS Green et al., 2005
cancer (200 mg/kg)
2-amino-1-methyl-6- DAS Wilson et al., 2007
phenylimidazo[4,5-b] pyridine- (100 μM)
induced toxicity
Garlic compounds induce apoptosis in DAS Sriram et al., 2008
cancer cells DADS Das et al., 2007
DATS Kim et al., 2007
Karmakar et al., 2006
Xiao et al., 2006
Testosterone-induced oxidative stress DAS Prasad et al., 2006
(250 and 500 mg/mouse)
DAS: Dially sulfide, DADS: Diallyl disulfide, DATS: Diallyl trisulfide, and DASO2: Diallyl sulfone
A diet supplemented with 2% garlic by 2 weeks reduced CAT activity and

expression, while supressed H2O2 generation in liver and kidney, but had no effect on
SOD and GPx activities in both organs from rats. Of note, Lpx in liver, kidney and urine,
as well as total antioxidant status in plasma and circulating GPx and SOD activities were
unchanged in these animals [Pedraza-Chaverri et al., 2001]. By the same time, Banerjee
et al. [2001] studied the effect of chronic ingestion of garlic homogenates (250, 500, and
1000 mg/kg/day/30 days by gavage) on the antioxidant status in liver and kidney of
normal rats. They found a decrease in liver and kidney TBARS and GPx activity, and an
increase in SOD activity without changes in CAT activity at a dose of 250 mg/kg. CAT
and SOD activities decreased without changes in TBARS at a dose of 500 y 1000 mg/kg.
Also, they observed ultrastructural damage in both tissues at a dose of 1000 mg/kg.
Furthermore, Banerjee et al. [2002b] studied the effect of chronic ingestion of garlic
homogenates (125-2000 mg/kg/day/30 days by gavage) on the antioxidant status in heart
of normal rats. They described an increase in CAT and SOD, and a decrease in GPx
activities and TBARS at lower doses. In contrast, ultrastructural heart damage was found
at 1000 mg/kg, and a clear toxic effect at 2000 mg/kg.
In 2004, Hsu et al. administered NAC, SAC, SEC, SMC and SPC (1 g/L in the
drinking water for 4 weeks) to Balb/cA mice and found that all these compounds
enhanced CAT and GPx activities in kidney and liver, while reduced the Fe2+- and
glucose-induced Lpx in plasma, kidney and liver.
Previously, Chen et al. [2003] found that garlic oil (30, 80 and 200 mg/kg, i.g. thrice
weekly for 6 weeks) dose-dependently increased GST, GR, SOD and decreased GPx
activities and TBARS in liver. In addition, Wu et al. [2001] studied the effect of garlic oil
(200 mg/kg), DAS (20 and 80 mg/kg), DADS (80 mg/kg) and DATS (70 mg/kg)
administration thrice weekly for 6 weeks on the antioxidant system. Garlic oil, DADS,
and DATS enhanced GSH levels in erythrocytes. DADS and DATS enhanced liver GR
and GST activities and decreased GPx activity. Garlic oil also induced an increase in GR
activity and a decrease in GPx activity. However, garlic oil, DAS, DADS and DATS had
no effect on GST, GR and GST activities in erythrocytes, but enhanced GST expression
in liver. In previous studies, Chen et al. [1999] studied the effect of DAS (50 and 200
mg/kg/day/8 days, i.g.) and garlic homogenates (2 or 4 g/kg/day/7 days, i.g.) on
antioxidant enzymes in liver, lung, kidney, brain, and heart from rats. The activity and
content of CAT decreased in liver and heart, and remained unchanged in lung, kidney,
and brain; SOD and GPx activities remained unchanged after DAS administration in rats.
DAS administration (100 mg/kg/day/3 days, i.g.) to mice resulted in a similar decrease in
CAT activity, whereas garlic homogenates decreased hepatic CAT activity in rats and
mice (5 g/kg/day/3 days, i.g.). These authors also found that diallyl sufone, a DAS
metabolite, administered to mice (50 mg/kg, i.g. for 3 days) had no effect on antioxidant
enzymatic system [Chen et al., 1999].
Finally, it seems pertinent to claim that DADS plays an important role in the
modulation of the GSH related antioxidant system. The effects of garlic oil, DADS (200
mg/kg) and DAS (100 mg/kg) administration thrice weekly for 7 weeks on some
antioxidant enzymes in rat liver have been studied. Authors of these investigations found
that (a) DAS and DADS enhanced GST activity, (b) garlic oil and DADS decreased GPx
activity, (c) DAS and DADS increased GR activity, (d) garlic oil increased SOD activity,
and (e) these three organosulfur compounds enhanced GSH content in erythrocytes but no
in liver [Sheen et al., 1999; Wu et al., 2001].
CONCLUDING REMARKS
Altogether, the data presented in this review clearly shows that garlic, in several
presentations, as well as in different organosulfur compounds found, have all antioxidant
activity in a variety of experimental models under in vivo and in vitro conditions. Of course,
these data may be relevant for humans. Indeed, several researchers have found a clear
antioxidant effect in humans with different garlic presentations.
Dillon et al. [2002] showed that the administration of AGE (5 ml for 14 days) to humans
decreased the urinary excretion of 8-iso-prostaglandin F2a (biomarker of oxidative stress in
vivo). In addition, Durak et al. [2004] found that the administration of aqueous garlic extract
to volunteers (10 g garlic/day for 4 months) improved blood lipid profile, enhanced blood
antioxidant potential and decreased MDA levels in subjects with high blood cholesterol.
Furthermore, Dhawan and Jain [2004] administered garlic pearls containing 250 mg of garlic
oil twice a day for 2 months to patients with hypertension, and found a significant decline in
both systolic and diastolic blood pressures, a reduction in oxidized-LDL and urinary
concentrations of 8-iso-prostaglandin F2α levels, and a moderate increase in the total
antioxidant status. Finally, Dhawan and Jain [2005] demonstrated that the ingestion of garlic
pearls containing garlic oil (2.5%) for a period of 8 weeks decreased the DNA damage in
patients with essential hypertension, where DNA damage was measured by the decrease in
the urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). These studies clearly and
strongly suggest that the ingestion of garlic may help in the prevention or treatment of
diseases associated with oxidative stress in humans.
ACKNOWLEDGMENTS
This work was supported by CONACYT Grants 48812 (J.P.), 52222-M (P.D.M.) and
48370-Q (A.S.), and DGAPA IN 207007 (J.P.).
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Chapter 3
CANCER CHEMOPREVENTION BY GARLIC

CONSTITUENTS: POTENTIAL CELLULAR
AND MOLECULAR TARGETS
Molay K. Roy1*, Lekh R. Juneja1 and Tojiro Tsushida2

1
Taiyo Kagaku Co., Ltd, Takaramachi, Yokkaichi, Mie 510-0844,
2
National Food Research Institute, Kannondai, Tsukuba, Ibaraki, 305-0842
ABSTRACT
Plant products or their metabolites have been utilized for both nutritional and
medicinal purposes throughout the history of human civilization. In particular, Allium
vegetables such as onions and garlic are among the oldest agricultural products in use as
spices for food preparation as well as a remedy for several ailments. In general, garlic, a
rich source of some phytochemicals, has been known for its medicinal uses as an
antibiotic, antithrombotic and antineoplastic agent [1]. Accumulated evidence suggests
that garlic and some of its constituents are effective in suppressing the incidence of
several diseases including cardiovascular diseases, diabetes, obesity, gastrointestinal
disorders and cancer. Among these diseases is cancer in advanced metastasized stage,
which is considered incurable. Therefore, prevention rather than treatment has become a
strategy to minimize the occurrence and development of this deadly health disorder.
Recent knowledge in cell technology and a growing understanding of the cellular
and molecular etiology of cancer have made it easier than ever to assess the efficacy of a
prospective molecule against the proliferation of cancer. In numerous studies, garlic
extract as well as some of its active constituents, particularly organosulfur compounds,
have been investigated for their value in inhibiting, retarding or reversing the
dysfunctional cell-signaling pathway in cancer cells. It appears that garlic and/or its
organosulfur constituents exert anticancer effects through multiple mechanisms that
include modulation of carcinogen metabolism, inhibition of DNA adduct formation, up
regulation of detoxifying enzymes [2] and DNA repair systems, and regulation of cell
proliferation, apoptosis and immune responses [3]. There has been convincing evidence
*
Corresponding author: E-mail: mroy@taiyokagaku.co.jp
120 Molay K. Roy, Lekh R. Juneja and Tojiro Tsushida
showing that garlic constituents such as S-allylmercaptocysteine, diallyl disulfide, and S-

trityl-L-cysteine modulate NF-kappaB [4], AP-1 [5], Akt [6], and MAPK [7] signaling
pathways by regulating a number of downstream signaling molecules in the anticancer
mechanisms.
Based on the available evidence, this chapter briefly provides an overview of the
occurrence and chemistry of garlic constituents. This chapter also lists the latest evidence
in accordance with the anticancer properties of garlic and/or garlic constituents with
special emphasis on the mechanism of action behind their chemoprevention effects. This
contribution, furthermore, provides a realistic broad view of areas of future research for
promoting garlic as a functional food ingredient.
1. INTRODUCTION
The term chemoprevention is defined as the use of medicine or natural agents to inhibit
the development or progression of cancer cells by either blocking or repairing the damage
occurring at a molecular level or by arresting or reversing the pathways that result from such
damage. Previous clinical/experimental trials for chemoprevention in humans/lower animals
with premalignancy lesions or at risk for developing tumors have shown promising results.
However, the main obstacle of chemoprevention is its adverse side effects. It is proposed that
natural phytochemicals would generate less toxicity compared to the chemically-synthesized
chemopreventive molecules. Phytochemicals such as alkaloids, monoterpenes, flavanoids,
isoflavones, saponins, and organosulfur compounds, though non-nutritive in nature, are
considered protective against diseases. These chemicals are known to be produced by plants
for their own protection, but their pharmacological effects are of great interest and are being
explored extensively. Several thousand molecules of plant origin have been reported for their
activity in cancer chemoprevention. Among them, a dozen molecules are being tested
clinically as chemopreventive drugs for their major cancer targets. These compounds include
green tea catechins, the grape flavonol resveratrol, and garlic-originated organosulfur
compounds (OSCs).
Garlic has a long history of medicinal use, dating back to 1500 B.C., when garlic was
used as a remedy for heart disease, headaches, and tumors in most cultures of the world. In
ancient India, it was used to treat parasites and gastrointestinal disorders. In ancient China and
Egypt, garlic was employed to enhance respiration, digestion, and sexual performance. People
of ancient Rome used garlic to treat cardiovascular problems, gastrointestinal diseases, and
musculoskeletal disorders. In Greece, it was used to empower the performance of soldiers and
athletes. Use of garlic throughout the ancient history of human culture was similar to the use
of other medicinal plants, based on historical experience. With the advent of modern science,
active basic research is exploring garlic’s mechanism of action in disease preventive
performance seen throughout its history. Today, a simple search on SCOPUS using the key
word ‘garlic’ revealed 6576 hits, of which one-third was related to cancer.
2. CHEMISTRY OF GARLIC
Table 1 shows the nutrient composition, including minerals and vitamin C in fresh garlic.
Apart from moisture (63%), carbohydrates (23–28.6%), protein (4.4–6.4%), and oil (0.07%)
Cancer Chemoprevention by Garlic Constituents: Potential Cellular… 121
[8], garlic contains a high level of minerals such as phosphorous, calcium, potassium, sulfur,
zinc, iron, and selenium. Among the vitamins, garlic contains a substantial amount of
riboflabin, thiamine, vitamin C, and nicotinic acid. The oil part of garlic is a rich source
allicin and different types of thio-organic compounds. The amount of the minerals as well as
vitamins and other nutrients in garlic depends mostly on soil, climate, and farming conditions.
Besides these, different growth stages significantly influence the level of garlic constituents.
Table 1. Vitamin/mineral composition of garlic
Mineral/vitamin Total amount/100g wet weight

germanium 14 μg
calcium 50–90 μg
copper 0.02–0.03 μg
iron 2.8–3.9 μg
potassium 100–120 μg
magnesium (43–77 μg
chromium 0.3–0.5 mg
manganese 0.2–0.6 mg
boron 0.3–0.6 mg
barium 0.2–1 mg
aluminum 0.5–1 mg
sodium 10–22 mg
phosphorous 390–460 mg
zinc 1.8–3.1 mg
selenium 15–35 μg
thiamine 0.25 mg
riboflavin 0.08 mg
vitamin C 5 mg
nicotinic acid 0.5 mg
retinal 15 μg
selenium 15–35 μg
thiamine 0.25 mg
riboflavin 0.08 mg
vitamin C 5 mg
Source: [9]
Thiol compounds present in garlic mostly contribute to its health benefit effect in
humans. However, processing conditions and extraction methods significantly affect the level
of different thiol compounds. Crushing garlic releases an enzyme, allinase, which converts
allin to allicin, the source of the characteristic garlic odor. Allicin is considered the most
physiological functional component of crushed garlic. Processing garlic by steam distillation
yields numerous sulfide derivatives including diallyl disulfide, dimethyl-, methyl allyl
suffides; dimethyl-, dipropyl- allyl propyl- and methyl allyl disulfides; dimethyl-, diallyl- and
methylallyl trisulfides; methyl propyl trisulfide, diallyl thi-sulfinate, and sulfur dioxide. Ethyl
alcohol extraction at room temperature yields the oxide of diallyl disulfide, called allicin,
where at subzero temperatures yields alliin. Extraction with diethyl ether provides a good
yield of major volatiles, such as allyl methyl sulfide, diallyl sulfide, allyl methyl disulfide,
diallyl disulfide, and AMT, whereas extraction with n-hexane results in poor recoveries.
Thermal treatment at various pHs significantly affects the stability and generation of garlic
volatile compounds. The formation of 3-vinyl-4H-1,2 dithiin and 2-vinyl-4H, 1,3 dithiin,
which are decomposed from allicin, reach their highest levels around pH 5.5. The formation
of allyl methyl sulfide, allyl (Z)-propenyl disulfide, dimethyl disulfide, methyl(E) propenyl
disulfide, allylmethyl trisulfide, and diallyl trisulfide is favored in neutral or weakly acidic
conditions, whereas the formation of diallyl sulfide, allyl methyl disulfide, allyl propyl
disulfide, diallyl disulfide, and methyl propyl disulfide is favored around pH 9.0. Among the
aforementioned garlic constituents, some compounds (Figure 1) such as allin (responsible for
the typical garlic odor), alline (odorless compound), ajoene (naturally occurring disulfide),
diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), S-allylcysteine
(SAC), organosulfur compounds and allyl sulfur compounds are under extensive investigation
for their health benefits.
3. ANTICANCER PROPERTIES OF GARLIC

3.1. Case Control Study
Several case-control/epidemiological studies have provided evidence for an association

between increased consumption of garlic with decreased risk of cancer development in
humans. For instance, You et al. [10] investigated an association of garlic consumption and
decreased risk of gastric cancer in a population based case-control study involving 564
patients with stomach cancer and 1,131 controls. Subjects in the highest quartile of Alliume
intake experienced only 40% of the risk of those in the lowest quartile. Protective effects were
seen for garlic, onions, and other allium foods. A case-control study conducted in high-and
low-risk areas of Italy to evaluate reasons for the striking geographic variation in gastric
cancer mortality within the country, found that reduced GC risk was associated with
increasing intake of raw vegetables, fresh fruit and citrus fruits. The study also revealed
lowered risk of cancer was associated with the consumption of spices, olive oil and garlic
[11]. A population-based, case-control study of laryngeal cancer was conducted in Shanghai,
China, during 1988–1990, in which 201 incident cases (177 males, 24 females) and 414
controls (269 males, 145 females) were interviewed. Cigarette smoking was the major risk
factor while the intake of fruits, certain dark green/yellow vegetables, and garlic where found
to be protective against the development of cancer suggesting dietary factors play an
important etiologic role against the development of cancer [12]. A prospective cohort study
conducted on 41,837 women aged 55–69 years via the State Health Registry of Iowa revealed
an inverse association of garlic consumption in occurrence of colon cancer [13].
Accumulated evidence from the epidemiological data of the abovementioned case-control
study, through the period 1987–1994, in three geographical locations, East Asia, Europe and
North America shows an inverse relation between garlic consumption and occurrence of
cancer. Such studies are continued to examine the effect of garlic consumption on gastric
cancer risk in a large population. In a recent report, Setiawan et al. [14] have demonstrated a
negative dose-response association between intake of garlic stalks and risk of stomach cancer
in Qingdao (odd ratio=0.30; 95% confidence intervals: 0.12-0.77) confirming protective
effects of allium vegetables (especially garlic and onions) against stomach cancer. Garlic-
induced anticancer effect is mainly attributed to its rich organosulfur constituents. Thus, much
research interest has focused on the anticancer properties of its OSCs in various animal and
human models, and cell culture, through the last two decades.
3.2. Inhibition of Carcinogenesis in Animal Models
Model animals are used to investigate effect and mechanism of action(s) of numerous
drugs/phytochemicals against the development and progression of various diseases including
cardiovascular disease, cancer and diabetes. Until recently, numerous investigators have
examined the efficacy of garlic or its components against the progression of carcinogenesis in
various animal models. In most of the studies, chemical carcinogens were used to induce
cancer on skin, lung, esophagus, stomach, liver, duodenum and small intestine, pancreas,
colon, bladder, prostate and mammary gland. In the rat, tumors can occur within 15 weeks of
carcinogen administration. Searching on Pubmed online databases, we have identified over
100 studies wherein garlic extract or its components were used to investigate the progress of
carcinogenesis in either chemical carcinogen-induced tumorogenesis or artificially
transplanted tumors in a model animal. In most of the studies, a positive association between
garlic consumption and inhibition of carcinogenesis was observed. A few of them, however,
identified no association or positive association between garlic consumption and occurrence
of carcinogenesis.
Table 2. Organosulfur compounds of garlic
Organosulfur Compounds (OSCs) Total amount/ wet weight

Gamma-glutamyl-S-alk(en)yl-L-cysteines 5–16 mg/g
alliin 3–5 mg/g
Allicin 5 μg/g
S-allylcysteine (SAC) 91 μg/g
Ajoene 60–150 μg/g
Di allyl sulfide (DAS) 30–100 μg/g
Di allyl di sulfide (DADS) 530–610 μg/g
Di allyl trisulfide (DATS) 900–1100 μg/g
Allyl methyl sulfide (AMS) 3.8–4.6 μg/g
Allyl methyl disulfide (AMDS) 100 μg/g
Allyl methyl trisulfide (AMTS) 250–270 μg/g
Di methyl disulfide (DMDS) 2.4–2.5 μg/g
Di methyl trisulfide (DMTS) 15–19 μg/g
Propyl methyl disulfide (PMDS) 0.7–0.8 μg/g
Source: [9]
SH
S
O O
S S
OH
HO N 1,3-diallyltrisulf ane (DATS)
H
NH 2 O
gamma-glutamyl cysteine O
S S
O S
(E)-1-allyl-2-(3-(allylsulf inyl)prop-1-enyl)disulf ane

S OH (Ajoene)
O NH 2
(2S)-3-(allylsulf inyl)-2-aminopropanoic acid (Alliin) O
S OH
O
NH 2
S
S-Allyl cysteine (SAC)
S
S-allyl prop-2-ene-1-sulfinothioate (Allicin)

O
S
S S OH
S
NH 2
1,2-diallyldisulf ane (DADS)
s-allylmercapto cysteine (SAMC)
diallylsulf ane (DAS)
Figure 1. Chemical structure of commonly studied organo sulfur compounds
In 1983, Belman et al. reported that mice bearing skin cancer treated with garlic oil at a
dose of 10–10,000 μg/week exhibited reduced tumor yield and had a lower incidence of PMA
initiated epidermal tumors [15]. Similarly, mice when given an oral administration of allyl
methyl trisulfide (AMT), a constituent of garlic oil, 96 and 48 hr prior to benzo(a)pyrene (BP)
adminstration, AMT inhibits the occurrence of forestomach tumors in mice [16]. Lau et al.
[17] examined the efficacy of garlic extract (GE) in inhibiting tumor growth in mice. In the
experiment, C3H/He mice were transplanted subcutaneously in the hind limb with 5 x 104
tumor cells. GE given through IP route to transplanted mice exhibited significant therapeutic
effect in inhibiting tumor growth comparing to the mice received GE through IL route. Garlic
at a dose of 400 mg/kg body weight was found effective in reducing incidence of chemical
carcinogen induced carcinogenesis in the uterine cervix of virgin young adult Swiss albino
mice from 73% in control group to 23% in test group. The decline of incidence was
statistically significant [18].
DAS (diallyl sulfoxide), which constitutes about 30–100 μg/g of garlic has been shown to
inhibit carcinogenesis induced by several chemical carcinogens, including 1,2-
dimethylhydrazine, benzo(a)pyrene, /V-nitrosomethylbenzylamine. In the early 1990, Hong et
al. demonstrated that DAS inhibited metabolic activation of NDMA could be due to the
competitive inhibition and inactivation of cytochrome P450IIE1 in rat lung and nasal mucosa
microsomes [19]. Wargovich and associates reported that pretreatment with DAS completely
prevented the formation in rat oesophagus of both malignant and premalignant lesions by V-
nitrosomethylbenzylamine (NMBzA), an exceptionally potent and selective oesophageal
carcinogen in this species [20]. Ludeke et al. have reported that mice receiving DAS exhibited
dose-dependent inhibition of DNA methylation by NMBzA in various rat tissues. DAS at a
dose of 200 mg/kg b. w., decreases in the extent of DNA methylation ranged from 26% in
oesophagus to 78% in lung, in addition to the reduced activation of the carcinogen [21]. A 1%
solution of DAS given to hamster prior to, during and after 7,12-dimethylbenz[a]anthracene
(DMBA) treatment resulted in a significant reduction in the level of unscheduled DNA
synthesis (UDS), the frequency of y-glutamyl transpeptidase (rGT) histochemical lesions and
tumor frequency, served as indices of anticarcinogenesis [22]. DAS pretreatment through
either of oral or parenteral route significantly reduced the MNNG induction of biomarkers,
such as nuclear aberrations (NA) and ornithine decarboxylase (ODC) activity in the glandular
stomach mucosa of the Wistar rat suggesting DAS may potentially inhibit MNNG-induced
gastric cancer [23]. Baer et al. [24] showed that mice receiving DAS exhibited greater
resistance against radiation injury in the colon with reduction of nuclear damage and
suppressing the proliferative response induced by radiation exposure. The reduction in the
severity of tissue damage is considered to be due to the activation or amplification of a DNA
repair process prior to the onset of the injury. In a contrary observation, DAS was found
ineffective in the prevention of post-initiation phases of nitrosomethylbenzylamine-induced
esophageal carcinogenesis in the Sprague Dawley rat [25]. In the study, DAS, though
ineffective in the post-initiation phase in terms of chemoprevention, also did not promote
esophageal cancer in this study.
Carcinogens require metabolic activation for their carcinogenic effect. Several previous
studies have examined the effect of garlic components on the activation of some carcinogens.
In a study, DAS and its putative metabolites diallyl sulfoxide and diallyl sulfone (DASO2)
were used to examine the inhibition of P-450 2E1 mediated p-nitrophenol hydroxylase
activity in liver microsomes from acetone-pretreated male Sprague-Dawley rats.
Preincubation of the microsomes with DASO2 inactivated p-nitrophenol hydroxylase activity
in a process that was time- and NADPH-dependent and was accompanied by a loss of
microsomal P-450-CO binding spectrum [26]. Tadi et al. investigated effects of ajoene and
DAS on the metabolism and DNA binding of aflatoxin B1 (AFB1) using rat liver 9000xg
supernatant as the metabolic activation system. They have observed that ajoene and DAS at
100 mg/ml; inhibited [3H]AFB1 binding to calf thymus DNA and adduct formation. The
compound also decreased the formation of both organ-soluble and water-soluble metabolites
of [3H]AFB1 [27]. Allixin, a phytoalexin isolated from garlic, was also investigated for its
effects on the binding of [3H] AFB1 to calf thymus DNA and on the formation of metabolites
of [3H]AFB1. The data indicated that the effect of allixin on AFB1-induced mutagenesis and
binding of metabolites to DNA may be mediated through an inhibition of microsomal P-450
enzymes [28]. In a in vivo study, mice were given tobacco-specific 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone (NNK) to induce lung tumor, and the effects of DAS on the
tumorigenicity and the metabolism of NNK in A/J mouse lung were examined. Female A/J
mice at 7 weeks of age were pretreated with DAS (200 mg/kg body wt in corn oil, p.o) daily
for 3 days. The study found that DAS pretreatment inhibit the pulmonary metabolism of
NNK, and reduced the rates of formation of keto aldehyde, keto alcohol, NNAL-N-oxide, and
NNK-N-oxide by 70-90% [29].
Garlic extract or its active constituents are also tested against various model carcinogens.
In a study, selenium enriched garlic was tested for its efficacy against DMBA induced
carcinogenesis, wherein animals given the selenium-enriched garlic (final concentration 3
ppm Se in the diet) developed the fewest mammary tumors induced by DMBA. DADS was
found more active than DAS or allyl methyl sulfide in inhibiting DMBA induced
carcinogenesis in the experimental animals [30]. Consumption of garlic powder also
significantly depressed the in vivo binding of DMBA to mammary cell DNA. The activity of
glutathione S-transferase (GST) in mammary and liver tissue from rats fed 2% dietary garlic
powder was higher than observed in tissues from rats fed the basal diet [31]. Organosulfur
compounds such as Isothiocyanic acid isobutyl ester (IAIE), dipropyl trisulfide (DPT), allyl
mercapton (AM), dimethyl trisulfide, DAS, DATS, allyl methyl sulfide, AMT, and dipropyl
sulfide of allium vegetables like garlic and onion exerted enhancing effects on the
development of diethylnitrosamine (DEN)-induced neoplasia of the liver in male F344 rats. In
the study, promotion of rat hepatocarcinogenesis was characterized by increased cell
proliferation with increased poly-amine biosynthesis [32, 33]. These cancer-promoting effects
of garlic components may be due to the doses used in the study. Because, a low dose of
garlic, 20 mg/kg b. w./day significantly reduced DEN-induced hepatocarcinogenesis in
Wistar rats [34]. According to Brady et al., a DAS 200 mg/kg b. w. of rats, equivalent to 14 g
DAS, or 14,000 garlic cloves in 70 kg adult, altered the normal balance of cytochrome P450
activities in rats. Thus, a larger dose of garlic or its any components might be cytotoxic.
Garlic extract/OSCs are shown to prevent 1,2-dimethylhydrazine (DMH) induced
carcinogenesis in model animal. In 1987, Wargovich et al. have reported that DAS, a flavor
component of garlic given to C57BL/6J mice reduced incidence of DMH induced colon
carcinoma by 74% [35]. Braddy et al. showed that a higher dose of DAS (200 mg/Kg b.w.)
may suppress the elevation of P450IIE1, an isozyme of cytochrome P-450 activating
oxidative metabolism of DMH [36]. Cheng et al. investigated dose depended effects of garlic
to inhibit 1,2-dimethylhydrazine (DMH) colon carcinoma in rats, and found that 4.76 g/m2
body surface/day or oral intake of approximately 10 g of garlic/day by an 80 kg human might
enough for an equivalent effect in human [37]. In a recent study, diet containing 4% aged
garlic extract (AGE) reduced the number of colon tumors and aberrant crypt foci induced by
DMH in rats [38].
In vivo effect of garlic extracts or its components on carcinogenesis induced by other
carcinogens is also studied since long before. In 1991, Hong et al. observed that p.o.
administration of DAS in rats might inhibit carcinogenesis by suppressing rat nasal
microsomes induced metabolism of several carcinogens, such as N-nitrosodimethylamine
(NDMA), N-nitrosodiethylamine (NDEA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK) [19]. OSCs such as DAS, allyl mercaptan (AMC), and allyl methyl disulfide
(AMDS) exhibited marked inhibition of NDEA-induced neoplasia of the forestomach when
the compounds were administered p.o. 96 and 48 h prior to NDEA. A significant reduction in
the pulmonary adenoma formation also was observed in some additional experiments,
wherein test compounds were given p.o. either 15 min or 1 h prior to NDEA [39]. Kwak et al.
have shown that garlic oil might suppress the expression of hepatic P4502E1, a key enzyme
responsible for metabolic activation of carcinogens [40]. DAS suppressed vinyl carbamate
(VC)- and NDMA-induced mutagenesis/tumorigenesis through inhibition of the cytochrome
P-450 IIE1 isoform, which is responsible for activation of these carcinogens in female ICR
mice [41]. DAS given to rodents prevented chemopreventive effect induced by NDMA in
several organ sides, wherein DAS resulted in a significant decrease of cytochrome P450 2E1-
dependent p-nitrophenol hydroxylase and NDMA demethylase activities [42]. A diet
containing 5% allin-riched garlic powder, significantly reduced the carcinogeneic effects
including DNA damage, DNA alteration induced by alfatoxin B1, DMH or NDMA in rats
[43]. Feeding toads with AFB1 together with fresh garlic or garlic oil showed a significant
reduction in tumor incidence from 19% in control groups to 3% and 9% in animals given
AFB1 plus garlic and AFB1 plus garlic oil, respectively [44]. An aged garlic extract, reduced
doxorubicin (DOX) induced cardiotoxicity in rats by reducing thiobarbituric acid-reactive
substance (TBARS) level from 332.5 ± 67.0 nmol in DOX treated mice to 221.3 ± 31.6 nmol
in rats given garlic extract plus DOX [45]. Pretreatment with DAS or DAS-metabolites
(DAS-O2) significantly protected rats/mice from acetaminophen (APAP)-induced liver
toxicity in a time- and dose-dependent fashion by suppressing metabolic activation of APAP
[46, 47]. Pretreatment with ajoene (20-100 mg/kg b.w), exhibited a hepatoprotective effect
against acetaminophen-APAP induced liver injury in mice and suppressed the rise in serum
glutamic pyruvic transaminase activity, and the reduction in the hepatic reduced glutathione
level, the decrease in hepatic protein thiol content resulting from acetaminophen
administration [48]. When a fresh garlic homogenates (FGH, 5g/kg b.w) was administered to
Swiss-Webster mice 2 hr prior to, or immediately after, an acetaminophen (APAP) treatment
(0.2 g/kg), APAP-induced hepatotoxicity was essentially prevented as indicated by serum
levels of alanine aminotransferase and lactate dehydrogenase and by liver histopathology
[49]. In a recent study, a combination of various phytochemicals/nutraceuticals containing
garlic extract was given in vivo models for 28 days. An APAP exposure of 400mg/kg b.w. ip
injections of APAP for 24 h caused a massive liver injury in control animals. Exposure to
PNM for 28 days significantly reduced animal mortality and all the APAP-induced
biochemical events such as ALT leakage, MDA accumulation, DNA fragmentation and Bcl-
xL expression [50]. On the other hand, in a human study, garlic extracts administration prior
to APAP exposure found no discernible effect on oxidative metabolism but was associated
with a slight increase in sulfate conjugation of drug suggesting garlic extract has limited
potential as a chemopreventive agent [51].
3.3. Inhibition of Cancer Cell Proliferation in Vitro
Displaying uncontrolled cell growth is one of the hallmarks of cancer cell etiology. Thus,
the most fundamental and basic steps for evaluating anticancer efficacy of a prospective
compound include studying proliferation inhibition activity. Until recently, there have been
numerous studies showing garlic extract or its active constituents efficiently inhibit the
proliferation of various cancerous cells, in several in vivo or in vitro assay systems (Table 3).
In 1990, Scharfenberg et al., for example, evaluated the effects of ajoene (4,5,9-trithiadodeca-
1,6,11-triene-9-oxide), which arises from alliin, on the proliferation of tumorgenic lymphoid
cell line derived from a Burkitt lymphoma (BJA-B) by comparing the effect on human
primary fibroblasts (FS4), and a permanent, non-tumorgenic cell line derived from baby
hamster kidney cells (BHK21). The study revealed that ajoene, at a dose of 2-50 μM
significantly suppressed the proliferation of tumorgenic BJA-B cells. On the other hand, FS4
or BHK cells were found less sensitive to the compound [52]. Welch and colleagues
examined the effect of S-allyl cysteine (SAC), a derivative of aged garlic extract on the
proliferation and differentiation of LA-N-5 human neuroblastoma cells in vitro. Cells exposed
to 600 μg/ml of SAC inhibited human neuroblastoma cell growth by 50% at the end of 2 days
exposure [53].
Using canine mammary tumor cells CMT-13 cells, Sundaram et al. compared the
proliferation inhibitory activity of water soluble organosulfur compounds, S-allyl-cysteine
(SAC), S-ethyl-cysteine (SEC) and S-propyl-cysteine (SPC) with that of oil-soluble
organosulfur compounds (DAS, DADS, DATS). The study revealed that the activity of oil-
soluble compounds were much stronger than a water-soluble compound. A one micro M
DADS substantially reduced the growth of the cells comparing to the effect of a 1 mM SAC.
Exploring the mechanism(s) underlying the activity, the study demonstrated that addition of
glutathione prior to DADS exposure markedly decreased the severity of the growth inhibition.
Treatment with DL-buthionine-SR-sulfoxamine, a specific inhibitor of glutathione synthesis,
attenuated the growth inhibition caused by DADS. These results show that some organosulfur
compounds found in garlic are effective inhibitors of the growth of the neoplastic CMT-13
cell. The inhibitory effects of these compounds are modified by intracellular glutathione [54].
Owing to the fact that different cancer cells show differential sensitivity, Takeyama et al.
investigated the effect of SAC on the proliferation of nine human and one murine melanoma
cell lines. In the study, doses of 1.2–10 mM SAC significantly suppressed the proliferation as
assessed by a [3H]thymidine incorporation assay, where as three control human
lymphoblastoid cell lines were not inhibited by SAC concentrations <5 mM [55].
Sakamoto et al. compared the antiproliferative effects of DATS and DADS on cultured
human neoplastic (A549) and nonneoplastic (MRC-5) lung cells, wherein addition of 10 μM
DATS reduced A549 growth by 47%, whereas 10 μM DADS decreased growth by only 20%
[56]. Siegers et al. showed that neither of garlic powder nor the Garlic extract was able to
suppress the growth of human hepatoma HepG2 or human colorectal carcinoma Caco2 cells
at concentrations of up to 1000 μg/ml. However, a combination of both garlic powder
preparation and a garlic extract (reported as 8–10% L(+)-alliin enriched) was effective in
suppressing the proliferation of the cancer cells. Results suggest that antiproliferative effects
of garlic may be due to breakdown products of alliin, such as allicin or polysulfides, rather
than alliin itself, since the addition of an alliinase system (garlic powder) to an alliin enriched
preparation without alliinase (garlic extract) potentiated the effects observed with the two
preparations alone [57]. Matsuura et al. investigated the effect of aged garlic extract (AGE)
on the growth and angiogenesis of three different colorectal cancer cell lines—HT29, SW480,
and SW620—wherein the invasive activities of SW480 and SW620 cells were inhibited by
AGE [58]. In a very recent study, Zhang et al. have shown that a local application of allicin
via gastroscopy on progressive gastric carcinoma results in cell growth inhibition
characteristics of occurrence of apoptosis and arrest of cell cycle, suggesting allicin can be
locally applied to suppress the progression of gastric carcinoma [59].
3.4. Cell Cycle
Reproducing new cells is fundamental to all living organisms. Traditionally, cell cycle
defines the duplication of a cell comprising of highly regulated series of events that result into
reproduction of two cells. A cell population while in normal proliferation cycle contains cells
in three different phases- G0/G1, S, and G2/M. Fluorescence labeling of the nuclei define the
amount of DNA cells have while it complete a cycle, and such a analysis indicates cells in
G0/G1 phase has 1x DNA, while the cells in G2/M phase has 2x DNA. Since the cells in the
S-phase synthesize DNA, they possess DNA between 1x and 2x.
Table 3. Antiproliferative effect of garlic extract/OSCs on various cancer cells/local
Garlic extract/ Cell/local cancer Proliferation Major effects Reference

OSCs inhibition (IC50)
Garlic HepG2 330μg/mL [57]
powder/extract Caco-2 480 μg/mL [57]
(1:10)
Z-ajoene HL60 5.2–10 μmol/L G2/M arrest [60, 61]
KB 15.8 μmol/L [60]
Hela 17.9 μmol/L [60]
Bel-7402 18.4 μmol/L [60]
HCT 19.6 μmol/L [60]
BGC-823 24.7 μmol/L [60]
MCF-7 26.1 μmol/L [60]
B16F10 62 μmol/L Caspase-3 [62]
activation
SMAC SW480 150 μmol/L Apoptosis, G2/M [63]
arrest
DADS SW480 56 μmol/L Apoptosis, G2/M [63]
arrest
HL-60 >25 μmol/L Apoptosis
N18 27.6 μmol/L Apoptosis, ROS, [64]
Ca+2 release
DATS HCT-15 11.5 μmol/L Apoptosis, G2/M [65]
arrest
DLD-1 13.5 μmol/L Apoptosis, G2/M [65]
arrest
S-trityl-L-cysteine SW480 0.9 μmol/L Apoptosis, G2/M [63]
(trityl-cys) arrest
Table 4. OGCs that induce cell cycle arrest in various cancer cell lines
Garlic/components Doses used Cells Arrested Reference

phase
DADS 0, 25 or 50 μM HCT-15 S phase [68]
50 μM TM6 cell S phase [71]
50 μM HCT-15 G2/M [70], [72]
100 μM HCT-29 G2/M [72]

50 μM SH-SY5Y G2/M [73]
200 μM Caco-2 and HT-29 G2 [74]
100-200 μM A549 G2/M [75]
Table 4. (Continued)
Garlic/components Doses used Cells Arrested Reference

phase
HL-60 (transplanted) G1 [77]
25-40 μM PC-3 G2/M [76] 25-40 μM
S- 46 μM, 90 μM HEL, OCIM-1 G2/M phase [69]
allylmercaptocysteine
(SAMC)
Methylselenocysteine TM6 cell line S phase [71]
(MSC)
Aqueous extract HT-29 G2/M [78]
Allicin 16-40 μM MCF-7) G0/G1, G2/M [79]
S- 200-300 SW-480 and HT-29 G2/M [80]
allylmercaptocysteine μM
(SAMC)
Ajoene 10 μM HL-60 G2/M [81, 82]
1-50 μM SMC G1 [83]
20 μM HL-60, U937, G2/M [61]
DATS HGC BGC823 G1/S [84]
100 μM J5 G2M [85]
G2M [86, 87]
Allitridi 9 g/ml MGC803 and G2/M [88]
SGC7901
Water extract (GEL, 1% HepG2 G2/M [89]
GES)
Flow cytometry is used to monitor DNA content and trace the dynamic changes in DNA
cycle in proliferating cells and offer a hint for designing clinical treatment protocol, monitor
prognosis and elucidate the mechanisms of anti-tumor drugs. In 1992, Xie et al. reported the
effect of a garlic extract on the proliferation and cell cycle in tumor cells. In the study, they
have shown that garlic oil treatment to S180 tumor cells rapidly decreased the cells in S-phase
while the number is increased in G1 phase. This observation suggest that garlic oil may
blockade cells to progress from G1 phase to S phase and result in accumulation of cells in G1
phase and directly inhibit the synthesis of DNA and the cell cycle [66]. In 1996, Xu et al.
have observed that an exposure of water extract of selenium-enriched garlic (Se-garlic) to a
transformed mammary epithelial cell culture model resulted in growth inhibition, GI phase
cell cycle arrest and apoptotic DNA double strand breaks [67]. In 1997, Knowles et al.
observed that DADS treatment (0, 25 or 50 μM) with cultured human colon tumor cells
(HCT-15) results in significant decrease in the proportion of cells in the G1 phase while
increase the percentage in the S phase [68]. However, Sigounas et al. were the first who have
reported that a garlic component, such as S-allylmercaptocysteine (SAMC) inhibits the
growth of cancer cells line by arresting cell cycle progression at G2/M phase. In the study,
they have observed that SAMC inhibited 50% growth of erythroleukemia cell lines, HEL and
OCIM-1 at a dose of 0.046 mM for OCIM-1 cells and 0.093 mM for HEL [69]. Since then
several other investigators have reported a similar effect of a garlic extract/component on
different cancer cells. DADS at a concentration of 50 μM inhibited cyclin B1/p34cdc2 kinase
complex activity in human colon tumor cells (HCT-15) with the increase of cells in the G2/M
phase accompanied by an increase in cyclin B1 protein expression [70]. A 100 μM DADS or
an aqueous extract of garlic also arrested G2/M phase in HT-29 cells with an increased
expression of epidermal growth factor receptor and integrin-beta6, and the effect was
consistent with G2/M phase cell cycle arrest [72, 78]. Wu et al. have reported that DADS
induced cell cycle arrest at G2/M phase in A549 lung cancer cells in a time- and dose-
dependent manner accompanied with the increase of intracellular reactive oxygen species
(ROS) [75]. Druesne et al. showed that DADS at 200 μM could inhibit cell proliferation
through the inhibition of HDAC activity, histone hyperacetylation and increase in
p21waf1/cip1 expression and the effect was associated with an accumulation of cells in the
G2 phase of cell cycle [74]. DADS exposed to prostate cancer cells PC-3 is shown to arrest
cell cycle at G2/M phase [76]. Apart from these observations, DADS given to mice
transplanted with HL-60 cells, increased cell population at G1 phase, from 25.4% in control
mice to 63.4% in the treatment [77].
Shirin et al. reported that the garlic derivative S-allylmercaptocysteine (SAMC) inhibits
growth, arrests cells in G2-M, and induces apoptosis in human colon cancer cells [80]. S-
allylmercaptocysteine (SAMC) inhibited the growth of two human colon cancer cell lines,
SW-480 and HT-29 by cell cycle progression in G2/M phase [80]. Methylselenocysteine
(MSC), arrested cell cycle of mammary cell (TM6 cell line) at S-phase by decreasing the
activity of CDK2, and without affecting the level of cyclin E and cyclin A [71]. The pure
allicin at a concentration of 16-40 μM or water extract of garlic powder with equivalent
allicin concentrations inhibited 50% growth of MCF-7 cells. The growth inhibition was
accompanied by accumulation of cells in the G0/G1 and G2/M phases of the cell cycle (MCF-
7 cells) and not by a significant increase in cell death [79].
Ajoene at a concentration of 1–50 μM suppressed the proliferation of rat SMC cells by
arresting cell cycle at G1 phase [83]. A concentration of 20 μM ajoene treated with Leukemia
cell lines U937 and HL60 inhibited cell proliferation. The proliferation inhibition of U937
cells was associated with accumulation of cell at G2/M cell cycle checkpoint by 153%
comparing to the control cells [61]. HL-60 cells when treated by a 10 μM ajoene exhibited
significant accumulation of cells in G2/M phase [81, 82].
DATS, another critical organic allyl sulfur component of garlic, inhibited proliferation of
PC-3 and DU145 cells, but not a normal prostate epithelial cell line (PrEC) by enrichment of
the G2/M cell population [86]. The DATS-induced cell cycle arrest in PC-3 cells was found
to be associated with increased Tyr15 phosphorylation of cyclin-dependent kinase 1 (Cdk1)
and inhibition of Cdk1/cyclinB1 kinase activity. Wu et al. used DAS, DADS and DATS to
investigate their modulatory effects on cell viability and cell cycle in human liver tumor cells
(J5). They observed that DATS was more effective than DADS or DAS, and a 100 μM DATS
accumulated 78% cells in G2/M phase comparing to DADS induced accumulation (19.4%),
DAS accumulation (14.4%) and control cells (14.5%) [85]. On the other hand, Li et al. have
shown that DATS inhibited the growth of gastric cancer HGC cell line BGC823 cells by
inducing cell cycle arrest at G1/S phase [84].
Ha et al. examined the effect of allitridi, a garlic preparation on cell cycle of human
gastric cancer (HGC) cell lines MGC803 and SGC790. They observed that treatment with
allitridi at a concentration of 3, 6 or 9 μg/ml for 24 h, decreased the percentage of G0/G1
phase cells with a significant increase of G2/M phase cells compared with the cells in control
group [88]. Latina (GEL) and Sulmona (GES), the two different water extract garlic
preparation of Italy were examined to assess their effect on the proliferation and cell cycle of
HepG2 hepatoma cells. In the study, GEL and GES suppressed cell proliferation by arresting
cell cycle progression at G2/M phase dependent on p53/p21 expression [89].
3.5. Induction of Apoptosis
Apoptosis is a genetically controlled and inherit mode of cell death, critical to normal
embryonic development and maintenance of tissue homeostasis. Dysregulation of apoptosis
underlies numerous pathological conditions including cancer. Cancer cells oppose the normal
cell turnover to support malignant growth. Thus, designing therapeutic approach to trigger
apoptosis in cancer cells is critical for treating cancer. Primitive features characteristic to
apoptotic cell death includes membrane blebing, oligonucleosomal DNA degradation, and
DNA ladder formation. Defining cancer cells undergoing apoptosis has become an excellent
tool to explore anticancer properties of various phytochemicals including green tea catechins
[90], grape resveratrol [91], carbazole alkaloids [92], and organosulfur compounds.
Numerous publications indicate that garlic extract/constituents induced cell cycle arrest or
antiproliferative activity is closely related with occurrence of apoptotic cell death. Sundaram
et al. were the first who have shown that DADS induced proliferation inhibition of HCT-15
cells was due to the occurrence of apoptosis as determined by morphological changes and
DNA fragmentation assay [93]. Sakamoto and colleagues compared the antiproliferative
effects of DATS and DADS on cultured human neoplastic (A549) and nonneoplastic (MRC-
5) lung cells, and revealed that DATS was more effective comparing to DADS and a 1 μM
DATS significantly induced apoptosis the cells [56]. SAMC induced proliferation inhibition
and G2/M phase cell cycle arrest in erythroleukemia cell lines (HEL and OCIM-1) resulted in
fragmented DNA characteristic of apoptosis [69]. MSC induced S-phase cell cycle arrest in
TM6 cells follows an apoptotic cell death after 48 h of exposure to the cells [71]. Allicin
induced cell cycle arrest at S-G2/M phase was shown correlated with the occurrence of
apoptosis in promyelocytic leukemia HL-60 cells and erythroleukemia K562 cells [94].
Induction of apoptosis by other organosulfur compounds/extract such as ajoene [95, 96],
garlic oil [97], DAS, DADS or garlic extract [98] are reported. In several studies, several
studies garlic components are shown to induce apoptosis through mitochondrial dependent
pathway often termed as intrinsic pathway, wherein activation of mitochondria is
accompanied by the release of proapoptotic factors, such as procaspases, cytochrome c,
apoptotic protease-activating factor 1 (Apaf-1), endonuclease G and apoptosis-inducing
factor. The proapoptotic factors such as Apaf-1, cytochrome c, and procaspase-9 in
combination with ATP form a complex called apoptosome that activates caspase-9, which
cleave procaspase-3 and generate active caspase-3, the central executioner of apoptosis.
Mitochondria-dependent caspase cascade is governed by several other factors. Among them,
TNF-receptor proteins, bax, bid, Smac are shown as positive modulators, on the other hand
BCl-2, BCl-xL act as a negative modulators (Figure 2).
DADS, a major oil soluble garlic constituent is shown to induce apoptosis in a number of
cell lines via classical mitochondira dependent pathway. Nakagawa et al. have reported that
DADS induced growth inhibition and induction of apoptosis in MDA-MB-231 breast cancer
cells was accompanied by up-regulation of Bax protein (142%), down-regulation of Bcl-X(L)
protein (38%) and activation of caspase-3 (438%) compared with the values in control cells
[99]. DADS induced apoptosis in HL-60 cells and concurrent activation of caspase-3 and
cleavage of poly(ADP-ribose) polymerase (PARP), and molecular DNA fragmentation were
blocked by a caspase-3 inhibitor or antioxidant suggesting ROS may contribute to apoptosis
induction at least in part alongside with a mitochondrial dependent apoptosis in HL-60 cells
[100]. DADS treatment to human bladder cancer T24 cells resulted in increased caspase-3
activity, PARP cleavage, CAD induction, chromosomal DNA breaks, the feature
characteristics of mitochondrial dependent apoptosis. The numbers of apoptoic cells were
significantly reduced when the cells were exposed to catalase, an antioxidant enzyme,
suggesting ROS may contribute to the induction of apoptosis [101]. Lin et al. further explored
the DADS-induced apoptosis using mouse-rat hybrid retina ganglion cells (N18), wherein
DADS increased the levels of Ca2+ and decreased mitochondrial membrane potential with
the increase release of cytochrome c, cleavage of pro-caspase-3. A caspase-3 inhibitor or an
intracellular calcium chelator completely blocked the apoptosis suggesting Ca2+ modulates
the cell death induced by DADS [64]. Release of Smac is one of the hallmarks in
mitochondrial dependent apoptosis. Smac deactivates activity of apoptosis inhibitory proteins
(AIPs), which prevents caspase cascades initiated by cytochrome c. Recently, DADS is
shown to induce mitochondrial release of Smac into the cytosol accompanied with the release
of cytochrome c, and activation of calpain, caspase-9, and caspase-3 in human glioblastoma
T98G and U87MG cells. Other apoptotic events such as overexpression of Bax, down-
regulation of Bcl-2 and some B1RC proteins are also demonstrated. Pretreatment of cells with
ascorbic acid attenuated ROS production, p38 MAPK phosphorylation, and JNK1 activation
induced by DADS. Pretreatment with JNK1 inhibitor 1 also significantly reduced cell death
[102]. DAS and DADS mediated cytochrome c release and caspase-3 activation in human
malignant neuroblastoma SH-SY5Y cells was associated with an increase in [Ca2+]i, increase
in Bax:Bcl-2 ratio, mitochondrial release of increase in cytosolic Smac/Diablo, and down
regulation of inhibitor-of-apoptosis proteins (IAP). In the study, DAS and DADS mediated
increased of calpain and caspase-3 was shown to produce 145 kD spectrin break down
product (SBDP) and 120 kD SBDP. In addition, DADS mediated capase-3 activation cleaved
cytosolic ICAD to release and translocation of CAD to the nucleus for nuclear DNA
fragmentation [103].
Other garlic components, such as SMAC, DATS, and ajoene are also studied to
investigate their roles in induction of apoptotic as a possible connection of anticancer
mechanisms. Shirin et al. found that SAMC induced growth inhibition and induction of
apoptosis in SW-480 and HT-29 was associated with an increase in caspase3-like activity.
These affects of SAMC were accompanied by induction of jun kinase activity and a marked
increase in endogenous levels of reduced glutathione [80]. The anti-proliferative and
apoptotic effect of SAMC to gastric cancer cells was associated with the induction of Bax,
p53, and caspase-9. Mitochondrial cytochrome c activation and caspase-3 activation
demonstrated that the activation of caspases accompanies the apoptotic effect of SAMC,
which mediates cell death [104]. Dirsch et al. have reported that ajoene induced a dissipation
of the mitochondrial transmembrane potential, release of cytochrome c, activation of
caspase-3 with a reduced expression of Bcl-xL in leukemia indicating an activation of a
mitochondria-dependent caspase cascade [105].
DATS mediated apoptosis induction involves with the increase of caspase-3 activity [65]
in HCT-15 and DLD-1 colon cancer cells, increase of oxidative stress and transmembrane
mitochondrial potential in Caco-2 and HT-29 colon carcinoma cell lines [106], and induction
of Bax and Bac [107] proteins in PC-3 cells. DATS-mediated suppression of HUVEC
survival was associated with apoptosis induction characterized by accumulation of subdiploid
cells, cytoplasmic histone-associated DNA fragmentation, and cleavage of caspase-3 and
poly-(ADP-ribose)-polymerase [108], inactivation of AKT and mitochondrial translocation of
BAD in PC-3 and DU145 human prostate cancer cells [6]. Kim et al. have shown that DATS
induced apoptosis was associated with a collapse of mitochondrial membrane potential,
modest increase in protein level of Bak, and down-regulation of Bcl-2 and Bcl-xL protein
levels LNCaP cells. In the cells, DATS induced apoptosis was significantly attenuated by
knockdown of Bax and Bak proteins and by pretreatment with antioxidant N-acetylcysteine,
suggesting the mitochondria-mediated cell death by DATS is associated with ROS generation
and regulated by Bax/Bak proteins [109]. DATS induced apoptosis in glioblastoma cells was
due to production of ROS, increase in ER stress, decrease in mitochondrial membrane
potential, and activation of stress kinases and cysteine proteases [102]. Zhang et al.
investigated the inhibitory effects of hepatic targeted polybutyllcyanoacrylate nanoparticles of
DATS (DATS-PBCA-NP) on orthotopic transplanted HepG2 hepatocellular carcinoma in
nude mice, wherein growth of the transplanted carcinoma was reduced, with the down-
regulated expression of proliferation cell nuclear antigen (PCNA) and Bcl-2 proteins [110].
Mechanism underlying ajoene induced apoptosis induction also involves several feature
characteristics of intrinsic apoptotic pathway including mitochondrial membarane
permeabilization, cytochrome c release followed by caspase-3 activation. Ajoene-induced
DNA fragmentation in HL-60 promyelocytic leukemia cells, MGc-803 gastric mucoid
adenocarcinoma cells and Molt-4 T lymphocyte leukemia cells was associated with the
inhibition of proto-oncogene bcl-2 expression [96]. Ajoene induced apoptosis in HL-60 cells
is shown associated with the activation of a mitochondria-dependent caspase cascades
involving the activation of an initiator caspase, caspase-8. In addition, over expression of
BCl-xL clearly diminished the caspase activity and apoptosis [105]. Li et al have shown
ajoene induced apoptosis in HL-60 cells was accompanied with caspase-3 and BCl2
inactivation [111]. Moreover, ajoene is shown to enhance apoptosis inducing activity of some
chemotherapeutic drugs such as, cytarabine and fludarabine in human CD34-positive resistant
myeloid leukaemia cells by enhancing their BCl2 inhibitory and caspase-3 activation activity
[112, 113], suggesting ajoene may possess omnipresent activities against leukemia.
Growth inhibitory effects of allicin to SiHa cells (human cervical cancer cell line) was
demonstrated through the induction of apoptosis characterized by formation of apoptotic
bodies, nuclear condensation and a typical DNA ladder in cancer cells, activation of caspases-
3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase [114]. On the other hand, allicin
may induce apoptosis via a caspase-independent pathway. In an important observation, Park
et al. demonstrated that allicin treatment with gastric epithelial cells resulted in morphological
changes, DNA fragmentation, hypodiploid DNA contents mediated via translocation of Bax
to mitochondria and subsequent release of AIF and PKA appears to be involved in allicin-
induced apoptosis in the cells [115]. In addition, allicin induced growth inhibition and elicited
apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c
release into the cytosol, activation of caspase 9/3, and DNA fragmentation in HL60 cells.
These events were, however prevented in cells pretreated with cyclosporine A, an
mitochondrial permeability transition pore (mPTP) inhibitor widely used to investigate
mitochondira mediated apoptosis in a variety of cell line [116]. The activation of the
mitochondrial apoptotic pathway was further evidenced by GSH depletion and by changes in
the intracellular redox status. In addition, allicin induced apoptosis in LM-8 cell is related to
down-regulate Bcl-2 protein expression and up-regulate Bax protein expression [117]. The
accumulated evidence suggests that allicin may inhibit the growth and induce apoptosis in
different cells with different mechanisms showing its diverse effect in suppressing the growth
of diverse types of cancers.
Survival factors Death Stimuli

FasL
Fas/
C D95
FADD
BAK BCl-xL
tBID
BAD
RAS
Caspase-8/10 BCl-2
JNK
PI3K AKT
tBID BAD BAX

AIF
BAK Smac/
ROS
Diablo
Apap-1
Caspase-3 Caspase 9 Cyt c
P53
Apoptosis
DNA Damage/Stress
Apoptosis
Figure 2. Classical apoptotic pathway and cell survival factors in mammalian cells
An oil-soluble allyl sulfur compounds are more effective antiproliferative agents than
their water-soluble counterparts. The ability of these compounds to suppress proliferation is
associated with a depression in cell cycle progression and the induction of apoptosis. This
depression in cell division coincides with an increase in the percentage of cells blocked in the
G2/M phase of the cell cycle. A depression in p34cdc2 kinase may account for this blockage
in cell division [118]. In a recent report, Kim et al. [119] demonstrated that thiosulfinates
increased the activation of initiator caspase-8 and -9, and the effector caspase-3 in PC-3 cells.
Thiosulfinates stimulated Bid cleavage, decreased the expression of the anti-apoptotic protein
Bcl-2 and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also
increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in
PC-3 cells.
3.6. Inhibition of Tumor Angiogenesis
Angiogenesis is a normal physiological process involved in growth, development and

wound healing. However, the process is also an obligatory factor for the genesis and spread of
solid cancers. Tumor angiogenesis constitute a network of blood vessel to supply oxygen,
nutrient to cancer growths. In addition, the network sends signals to surrounding normal
tissues to enable the growth of new vessel so that tumor can spread in a new location.
Stimulation of angiogenesis is performed by several and proteins/growth factors. Among
them, FGF, EGF, GC-SF, IL-8, PDEGF, TGFa, TNF, VEGF, adenosine, and PGE are well
defined for their role in stimulating angiogenesis.
Garlic extract and its components are shown to target the angiogeniss pathway while they
suppress progression of cancer in cellular and animal models. A study by Sukula et al.
observed the effects of DAS on the life span of ehrlich ascites (EA) tumor bearing Swiss
albino mice, cytotoxicity and angiogenesis. Results revealed that DAS increased life span was
associated with the suppression of tumor growth and inhibition of angiogenesis in the EA
tumor bearing mice [120]. An herbal preparation containing garlic extract exerted significant
inhibitory effect on tumor angiogenic activity and on growth on L-1 sarcoma [121]. Mousa et
al. [122] demonstrated alliin induced inhibition of fibroblast growth factor-2 and vascular
endothelial growth factor (VEGF)-induced angiogenesis in human endothelial cells and
inhibition of ex vivo neovascularization in chick-chorioallantoic membrane model, wherein
the anti-angiogenic effects of alliin were mediated, at least in part, by increase in cellular
nitric oxide and p53 protein expression. Matsuura et al. described aged garlic extract (AGE)
as a good chemopreventive agent because of its antiproliferative action on colorectal
carcinoma cells and inhibitory activity on angiogenesis [58]. AGE suppressed proliferation of
colorectal cancer cells (HT29, SW480, and SW620), and reduced the invasive activities of
SW480 and SW620 cells as assessed by the Matrigel chemoinvasion assay. Additional tests
indicated that AGE increased the adhesion of the endothelial cells to collagen with the
reduction of capillary like tube formation in a three-dimensional collagen matrix assay. Xaio
et al. [108] observed that DATS treatment significantly disrupted the capillary-like tube
formation and migration by HUVEC that was accompanied by suppression of vascular
endothelial growth factor (VEGF) secretion, down regulation of VEGF-Receptor 2
expression, inactivation of Akt and activation of ERK1/2. Recently, Thejass et al. [123] have
shown garlic components (DADS and DAS) mediated inhibition of endothelial cell
proliferation and migration was associated with the reduction of matrix metallopro-teinases 2
and 9. In addition, an increase in the level levels of circulating anti-angiogenic factors, tissue
inhibitor of metalloproteinase and interleukin-2 levels was observed when DAS was
administered to C57BL/6 mice injected with B16F-10 melanoma cells [124]. Attenuation of
cell migration and the induction of cell death by AGE was also documented in rat sarcoma
cells [125]. In addition, SAMC administration (300 mg/kg) to CB-17 SCID/SCID mice
implanted with PC-3 cells reduced the number of lung metastasis per lung by 85.5% and
completely abolished adrenal gland metastasis [126]. Similarly, an ip injection of ajoene (5–
25 μg/g body weight) significantly inhibited pulmonary metastasis in C57BL/6 mice injected
with B16/BL6 melanoma cells Taylor et al. [127] . Based on the finding of the above studies
it is concluded that garlic extract and its components are able to affect tumor angiogenesis
and metastasis.
3.7. Modulation of Cell Signaling
Cell signaling is a coordinated communication system exists in every functional cell to

govern over cellular activities. Cell signaling enable cells to response the environmental
changes correctly and precisely for developing or repairing cells and tissue homeostasis.
Errors or mismatching in cellular information systems results in the occurrence of diseases
like cancer, diabetes, obesity, and immune disorders.
Through the last, several years there have been extensive studies exploring cell-signaling
network in the context of human diseases and coordination between diverse types of cells in
higher organisms. In each signaling network, cell receives information from the environment
through a class of protein called receptors. Receptor molecules while it binds to specific
ligands transmit signal through a diverse intermediate molecules to gene, which, in turn, code
for protein molecules required to stimulate a specific response they receive via ligand
molecules. In cancerous cells, several signaling pathways are shown to be deregulated. Thus,
there are studies showing how a chemopreventive molecule fixes the abnormal cell signaling
so that a tumor lost its control over its uncontrolled growth. Through last few years, garlic
constituents have shown to modulate several signaling pathways.
3.7.1. Nuclear factor kappa B
Nuclear factor-kappaB (NF-kappaB) is an oxidative stress–sensitive transcription factor

that plays a critical role in the regulation of a variety of genes encoding proteins in cell
growth, innate immunity, and cell death. In an inactive form, NF-kappaB exists in cytosol by
forming a complex with the inhibitory protein IkappaBalpha. An enzyme, IkappaBkinase
phosphorylates IkappaBalpha protein to dissociate the protein from NF-kappaB, which, in
turn, results in activated NF-kappaB. The activated NF-kappaB is then translocated into the
nucleus where it binds to specific sequences of DNA called response elements (RE). The
DNA/NF-kappaB complex then recruits other proteins, such as RNA polymerase to transcribe
downstream DNA into mRNA, which, in turn, translated into protein to modulate cell
function. Activation of NF-kappaB has been associated with several aspects of tumorigenesis,
including cancer cell proliferation, prevention of apoptosis, and increases of angiogenesis and
metastasis potential.
In a study, Drisch et al. [95] have shown that, ajoene, an important garlic component in
crashed garlic activates NF-kappaB-DNA binding to induce cell death in HL-60 cells, the
effect was however prevented by an antioxidant treatment. On the other hand, Karmakar et
al., in a recent report have shown that garlic components such as, (DAS) and diallyl disulfide
(DADS) induced cell death to human malignant neuroblastoma SH-SY5Y is associated with
the reduced NF-kappaB activation. Similarly, Ban et al. found that treatment with
thiacremonone resulted in inhibition of NF-kappaB activation in SW620 and HCT116 human
colon cancer cells [4].
These differential mechanisms of garlic component to prevent the growth of cancer cells
might depend on the type of cancer cells, and their oxidative stress status. It is considered that
substances causing oxidative stress may induce cell apoptosis via activation of NF-kappaB.
This notion of hypothesis is supported by a very recent study, wherein Sriram et al. [128]
have shown that garlic component induced apoptosis in Colo 320 DM human colon cancer
cells is associated with the induction of ROS, and increased activation of NF-kappaB.
3.7.2. Mitogen activated protein kinases
Mitogen activated protein kinases, also known as MAPKs, have received increasing
attention as a target for cancer prevention and therapy. There are three major types of MAPKs
in mammalian cells: the extracellular signal regulated protein kinases (ERK), the p38
MAPKs, and the c-Jun NH2-terminal kinases (JNK). Each of the MAPK pathway consists of
a cascade in which a MAP3K activates a MAP2K that activates a MAPK (ERK, JNK, and
p38), resulting in the activation of NF-kappaB, cell growth, and cell survival. ERKs cascades
play a critical role in transmitting signals initiated by growth factor induced tumor promoters
such as EGF, PDGF. On the other hand, P38/JNK kinase cascades are believed to be
modulated by stress related tumor promoters, such as UV.
Administration of DATS to Human hepatoma HepG2 cells resulted in activation of three
major mitogen-activated protein kinases (MAPKs)--extracellular signal-regulated protein
kinase, c-Jun N-terminal kinase, and p38 [2]. Wen et al. have shown that inhibitors specific to
MAPKs may suppress DATS induced MAPKs activation in HepG2 cells and enhance the cell
death [7]. Wu et al. proposed that modulation of MAPKs and production of reactive oxygen
species (ROS) may play pivotal roles in apoptosis induction by most GCs and ITCs [3]. Karl
et al. observed that DAS supplementation significantly reduced the DMBA induced protein
expressions of PI3K/Akt and p38MAPK without affecting the expression JNK1 and ERK1/2
[129] and suppress anti-carcinogenic effect induced by DMBA. Xaio et al. found that DATS
treatment caused an activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not
c-Jun NH2-terminal kinase (JNK) or p38 mitogen-activated protein kinase (p38 MAPK). In
addition, DATS-mediated apoptosis induction and inhibition of HUVEC tube formation was
partially but significantly attenuated by pharmacologic inhibition of ERK1/2 but not JNK or
p38 MAPK [108]. A transient increase of the phospho-p38 and phospho-p42/44
(phosphorylated p38 MAPK and phosphorylated p42/44 MAPK) was associated with
induction of apoptosis in human nasopharyngeal carcinoma cells [130].
DAS, DADS, and DATS treatment of glioblastoma cells triggered production of ROS
that induced apoptosis with the phosphorylation of p38 MAPK and activation of the redox-
sensitive JNK1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production,
p38 MAPK phosphorylation, and JNK1 activation. Pretreatment with JNK1 inhibitor 1 also
significantly reduced cell death suggesting major garlic components such as DAS, DADS,
DATS inhibit cancer cell proliferation via modulating ROS sensitive MAPKs pathyways
[102]. In a recent study, SB203580, an inhibitor of p38, inhibited DADS induced apoptosis
and p38 activation in HL-60 showing p38 signaling are likely involved in DADS induced
cancer preventing mechanisms [131].
3.7.3. Activator protein 1
Transcription factor activator protein-1 also known as AP-1 is a protein dimer composed
of members of the basic region leucine zipper protein superfamily, specifically, the Jun, Fos.
A high level of AP-1 activity has been shown to be associated with the tumor promotion and
progression of various types of cancers, such as lung, breast, and skin cancer. Son et al. have
shown that allicin pretreatment to human umbilical vein endothelial cells (HUVECs) resulted
in the decrease of AP-1 activation and phosphorylation of the c-Jun NH2-terminal kinase
(JNK) induced by gamma irradiation [5], which is often used to induce carcinogenesis in
model experiments. On the other hand, an increased expression of a phase II detoxicification

enzyme, glutathione S-transferase pi (GSTP) protein and mRNA have been shown to be
upregulated by DATS and DAS in clone-9 cells via activation of AP-1 and ERK 1/2 MAPKs.
These observations suggest that different garlic components have differential mechanisms to
provide their chemopreventive supports [132].
3.8. Miscellaneous
3.8.1. Phase II/anti-oxidative enzymes
Phase II enzymes play important roles in protecting cells against the stress of ROS or
toxic substances. In general, phase II enzymes are considered to detoxify the products of
Phase I, wherein carcinogens are catabolized to form highly reactive intermediary molecules.
Phase II enzymes include glutathione transferases (GST), quinone reductase (QR), gamma-
glutamyl-cysteine synthase, epoxide hydrolase (EH), UDP-glucuronosyl transferase (UGT)
and thioredoxin reductases (TR). Toxicity effect of a carcinogen largely depends on the
balance of Phase I and Phase II enzymes. Experimental evidence suggests that garlic or its
components enhance detoxification process by modulating the induction of several Phase II
enzymes. Prevention of BP-induced carcinogenesis in mice by some garlic components is
shown associated with elevation of hepatic and target organ total GST activity [133]. Hu et al.
have shown that DAS, DADS and DATS administration to A/J mice results in expression of
Alpha (mGSTA3-3, mGSTA1-2, mGSTA4-4), Mu (mGSTM1-1) and Pi class GST
(mGSTP1-1) in the liver, lung and forestomach [134-136]. On the other hand, DADS
remarkably increased hepatic GST, QR, and UGT activity comparing to DAS, DADS, DPS or
DPDS in male SPF Wistar rats [137]. Subsequently, garlic extract or its OSCs DAS and
DADS were found to be potent inducers of quinone reductase activity in the liver,
forestomach and/or lung of mice exposed to BP mutagen [138, 139]. Similarly, garlic oil
given to rat resulted in increased microsomal ER activity by 1.4–2 fold against 1-chloro-2,4-
dinitrobenzene exposure to the animals.
The mechanism by which garlic extract or its component induces antioxidant enzymes or
antioxidant effect against toxins is still being explored. It is widely considered that the ability
of OGCs to induce phase II enzymes is largely dependent on the activity of its sulfhydryl
groups [140]. On the other hand, transcription factor Nrf2 (nuclear factor-E2 related factor 2)
is shown to play an essential role in the antioxidant response element (ARE)-mediated
expression of phase II enzymes. Chen et al. showed a positive correlation between OSC-
mediated induction of Phase II enzymes, activation of anti-oxidant response element and
accumulation of transcription factor nuclear factor E2-related factor 2 in HepG2 hepatoma
cells [2]. Tsai et al. have documented an essential role for GSTP enhancer I element (GPE I),
but not GPE II, in DADS mediated and DATS-mediated induction of Pi class GST in Clone 9
liver cells [132, 141]. In a recent study, Kalayarasan et al. have investigated the role of garlic
extract or SAC in the prevention of geneotoxicity induced by Chromium in the hepatocytes of
Wistar rats. In the study, AGE and SAC induced restoration of superoxide dismutase,
catalase, glutathione peroxides activity was shown associated with increased expression of
Nrf2 [142].
Garlic extracts or its components have been shown to activation of non-enzymatic

system. Non-enzymatic antioxidant action includes reducing power (scavenging of free
radicals, inhibition of lipid peroxidation), metal ions chelating ability, and interacting with
biomembranes and/or with other antioxidants. Yin et al. have shown that nonenzymatic
antioxidant activities of DAS, DADS, SEC and NAC were mainly associated with their
reducing power and interactions with biomembranes [143]. Using a DCFH-DA, a ROS
sensitive cell permeable probe, Roy et al. further proven the ability of GE to suppress
oxidative stress in cellular model [144]. S-allylmercaptocysteine (SAMC) and S-allylcysteine
(SAC) induced growth inhibition to human prostate carcinoma (LNCaP) cells was associated
with an increase in reduced glutathione concentrations in LNCaP cell [145]. SAMC given to
mice before exposure to carcinogen (APAP) significantly suppressed the reduction of reduced
glutathione, in addition to the suppression in the increase in hepatic lipid peroxidation and the
decrease in hepatic reduced coenzyme Q9 (CoQ9H2) [146]. SAC administration to NDEA-
induced carcinogenesis resulted in the inhibition of tumor incidence, modulated the lipid
peroxidation, and increased the reduced glutathione, glutathione dependent enzymes,
superoxide dismutase, and catalase [147]. All of the above evidence suggests that garlic
extracts and its OSCs prevent cells from the loss of antioxidant capacity in chemical induced
carcinogenesis.
3.8.2. DNA polymerase activities
DNA polymerase (DNA pol) catalyses the addition of deoxyribonucleotides to the 3′-
hydroxyl terminus of primed double-stranded DNA (dsDNA) molecules. Over expression of
DNA pol and its increased synthesis has been a characteristic feature of cancer cell
proliferation. Thus, prevention of DNA pol in cancer cells has become a strategy for retarding
the growth of cancer cell. The human genome encodes 14 pols to conduct cellular DNA
synthesis. Depending on amino acid homology, DNA polymerases are classified into A, B, X
and Y families. The family X includes pol-beta, -delta, -I, -gamma and terminal
deoxynucleotidyl transferase (TdT). These pols possess nucleotidyl transferases activities and
catalyze DNA polymerization in a distributive manner.
In a recent report, Nishida and colleagues [148], for the first time have shown that garlic
constituents, particularly diallyl compounds inhibit the activities of family X pols such as –
beta, -delta, and TdT and suppress the DNA synthesis to inhibit the proliferation of cancer
cells. Investigating the mechanism behind the garlic constituents induced Pol X activities the
author proposed that DADS components specifically binds to Pol –beta like region of the X-
family, thereby inhibit the activities of the polymerases to inhibit DNA synthesis in cancer
cells.
3.8.3. Cyclooxygenase
Cyclooxygenase, a synonym of prostaglandin synthase/prostaglandin synthetase, is an

enzyme that plays important roles in the synthesis of prostanoids, including prostaglandins
and prostacyclin. Inhibition of cox enzyme may reduce the symptoms of inflammation and
pain. Currently, three cox isoenzymes—cox 1, cox 2 and cox 3—are identified in mammalian
cells. Among them, cox 2, which is regulated by mitogens, tumor promoters, cytokines, and
growth factors, is shown over-expressed in various pre-malignant and malignant lesions
including colon, liver, pancreas, breast, lung, bladder, skin, stomach, head and neck, and
esophagus. Thus, COX-2 inhibition has become an effective strategy for chemoprevention.
Through the last few years, chemopreventive activity of various dietary factors such as,
resveratrol, genistin, lutein, green tea catechins are shown to be associated with the inhibition
of COX 2 activity or reduced COX 2 expression. Garlic extracts and its OSCs also shown to
suppress the Cox 2 expression/activity in various cancer cells. In 1980, Vender et al. have
shown that garlic oils inhibit fatty acid oxygenase in sheep vesicular gland preparations with
the decreased formation of prostaglandin E2 (PGE2) and PGD2. [149]. Since then several
authors have reported garlic extract or OSCs suppressed the COX2 activity in in vivo/in vitro
models. Dirsch et al. have shown that ajoene dose-dependently inhibits the release of LPS -
induced prostaglandin E2 in RAW 264.7 macrophages accompanied with an inhibition of
COX-2 enzyme activity [150]. Other OSCs such as DAS, monosulfides (DAMS), disulfides
(DADS) and trisulfides (DATS) are shown to suppress COX-2 gene expression in HEK 293T
cells, wherein DATS found stronger than DAMS and DADS [151]. Thiacremonone another
member of garlic derived OSCs was shown to inhibit COX-2, iNOS gene expression in colon
cancer cells [4]. In a recent study, Lee et al have shown DAS prevents IL-1beta and MSU
crystal induced COX-2 upregulation in synovial cells and chondrocytes and ameliorates
crystal induced synovitis potentially through a mechanism involving NF-kappaB [152].
3.8.4. LOX/iNOS
Increased activity of lipoxygenase (LOX) or inducible nitrogen synthase (iNOS) is

associated with inflammatory and carcinogenic processes. Garlic-derived OSCs have been
shown to suppress the LOS/iNOS activity/expression in various experimental models. Since
the report published by Belman et al. reporting inhibition of soybean lipoxygenase by onion
and garlic oil constituents [153], numerous investigators have shown that OSC suppression of
LOX and iNOS activity is associated with the inhibition of cancer cell growth.
3.8.5. Modulation of P450 2B1/2E1
DAS, an important constituent of garlic, induces the activation of in cytochrome P450

2B1 while it inhibits the activity of P450 2E1. Such a selective effect of DAS on P450
enzymes is of considerable interest toward the understanding of dietary effects on xenobiotic
metabolism. In a study, Pan et al. [154] have investigated the mechanism of DAS induced
P450 2B1 induction. Immunoblot analysis revealed that DAS increased in the level of P450
2B1/2 protein. The level of P450 2B1/2 mRNA in rat liver also increased markedly, reaching
a maximum at 12 h after the DAS treatment. Hybridization with the isozyme-specific
oligonucleotide probes revealed that the mRNA levels of both P450s 2B1 and 2B2 were
induced. In contrast, the level of P450 2E1 mRNA in the liver of DAS-treated rats was not
changed. The transcription of P450 2B1/2 genes was blocked completely by alpha-amanitin,
an inhibitor of RNA polymerase II suggesting the induction of P450 2B1/2 in rat liver by
DAS is mainly due to transcriptional activation. In the DAS-treated rats, P450 2B1 induction
was found at 12 h after the treatment while the levels of P450 2B1/2 mRNA increased 66-fold
in the duodenum and 23-fold in the stomach. DAS treatment, however, did not change the
levels of P450 2B1/2 mRNA in the lung and nasal mucosa.
3.8.6. Inhibition of tumor cell adhesion and invasion
Metastasis is one of the major causes of cancer-related death since the condition rarely
responds to available treatments. It has long been proposed that garlic inhibits tumor growth
more effectively during the initiation rather than the promotion phase of breast carcinogenesis
and has minimal effect on late stage tumor growth. Through last few years, several reports
have shown garlic constituent influences tumor cell adhesion and invasion. Using colony-
forming, wound-closure as well as matrigel-invasion assays, Chu et al. [155] observed that S-
allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) suppressed invasive abilities of
PCa cell with a restoration of E-cadherin expression at transcription and protein levels.
Howard et al. [126] have shown that SAMC given to androgen-independent prostate
cancer mouse model reduces the number of lung and adrenal metastases by inhibiting the
growth of primary tumors and up regulation of E-cadherin. SAMC prevents dissemination by
decreasing tumor cell intravasation in mice is the first report showing in vivo antimetastatic
properties of garlic. Very recently, Gaptor et al. have shown that SAC significantly reduced
anchorage-dependent and -independent growth of MDA-MB-231 breast tumor cells in a dose-
and time-dependent fashion [156]. They have also shown that a sub-lethal dose of SAC alters
mammary tumor cell adhesion and invasion through components of the extracellular matrix.
SAC induced increased expression of E-cadherin and reduced MMP-2 expression were
considered, at least partially for inhibition of a mammary tumor cell invasion and cell motility
by SAC, since E-cadherin and MMP-2 plays vital role in cancer metastasis. Using a wound
closure assay, the authors have shown that SAC reduced the migration of MDAMB-231 cells
in a dose- and time-dependent manner.
4. CONCLUSION
In summary, there is convincing and substantial evidence derived from multiple levels of
studies involving case-control/epidemiological, animal, and cell culture demonstrating the
protective effects of garlic extracts and their active organosulfur constituents against cancer.
Research over the last two decades has also demonstrated that OSCs not only inhibit
chemically-induced cancer but also suppress cancer cell proliferation in vivo and in vitro via
modulating multiple pathways in cell cycle arrest, apoptosis induction, and inhibition of
angiogenesis governed by several cell-signaling molecules. Future research may focus on
clinical trails of these compounds to prevent and treat cancer in humans. For this purpose,
toxicity and pharmacokinetic issues must be addressed through proper investigation. From a
‘neutraceutical’ point of view, garlic extracts or some of their organosulfur compounds may
be promising in a wide range of health treatments, such as antihypertensive [157], antidiabetic
[158], hypolipidemic [159], hepatoprotective [160], and platelet antiaggregatory activity
[161], which have been substantiated by modern research. Garlic has a strong spicy flavor.
Delivering GE/OSCs with low pungency might be a challenge because most of the health
benefit is attributed to garlic’s OSC components. Taiyo Kagaku Co., Ltd. has a modern, state-
of-the-art micronutrient delivery system. Here, we take up the challenge of attaining
palatability and bioavailability by masking undesirable tastes and flavor for delivering this
gift of nature to mankind.
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Chapter 4
TRACE ELEMENTS IN GARLIC
A. Gonzálvez, S. Armenta and M. de la Guardia*

Department of Analytical Chemistry, Research Building, University of Valencia,
50th Dr. Moliner St., E-46100 Burjassot, Valencia, Spain
ABSTRACT
The State-of-arte of the scientific literature concerning the presence of trace elements
in garlic samples has been evaluated trough the discussion of methods proposed for both,
sample preparation and measurement, of all studied elements. Special attention has been
paid to the method of choice and the most appropriated and safe preparation strategy.
Data concerning 41 elements in different origin garlic samples are also presented.
Keywords: garlic, trace elements, review
1. INTRODUCTION
Garlic (Allium Sativum) is classified by the Codex Alimentarius as a bulb vegetable, a
pungent highly flavoured food derived from fleshy scale bulb, and it is one of the most
popular vegetables in the world with an annual production of about three million metric tons.
Garlic has been used since ancient times (1550 BC) as food, seasoning product and also in the
treatment of many diseases. In fact, nowadays, it is well known that Allium species are a rich
source of phytonutrients, useful for the treatment or prevention of a number of diseases,
including cancer, coronary heart disease, obesity, hypercholesterolemia, diabetes type 2 and
hypertension among others [Augusti, 1996].
The major growing areas for garlic are USA, China, Egypt, Korea, Russia and India
[FAO, 2004]. It is widely consumed by humans and there are about 300 varieties of garlic
cultivated worldwide.
*
Corresponding author: E-mail address: miguel.delaguardia@uv.es
156 A. Gonzálvez, S. Armenta and M. de la Guardia
Garlic contains more than 200 different compounds which provide different biological
activity. In this sense, scientific attention has been focused on polar organic compounds, such
as thiosulfinates, volatile sulfur compounds, sapogenins, saponins, flavonoids and
degradation products.
Moreover, the metallic profile of garlic is an important parameter not only because of the
metabolic role of some elements but also the quality of such samples is also related to the
concentration of trace metals, being also necessary to verify, that it is free from heavy metals
in toxic quantities for the health of the consumers.
There are a number of factors influencing the concentration of heavy metals in plants and
soils. These factors include climate, irrigation, atmospheric deposition, the nature of the soils
on which the plant is grown and the degree of maturity of the plant at the time of harvesting
[Voutsa et al., 1996]. Among of them, heavy metal contamination derived from
anthropogenic sources is one of the severest, this can strongly influence trace element
speciation and hence bioavailability.
So, the study of trace elements in garlic is important from two different points of view.
On one hand, it is necessary for healthy commissions and regulations which should control
that the concentration of heavy metals in garlic is lower than the maximum residue level
allowed. On the other hand, it is also important for producers due to the possibility to use
trace element for the classification and authentication of garlic denomination of origin [Smith
et al., 2005].
In short, this chapter summarizes the different analytical methods, including also a review
of sample treatments, proposed in the literature for the analysis of trace and ultra trace
elements in garlic.
2. EVOLUTION OF THE SCIENTIFIC LITERATURE ON TRACE

ELEMENTS DETERMINATION IN GARLIC
Figure 1 depicts the evolution of the published papers on trace elements determination in
garlic, indexed in the Analytical Abstract data base for the whole period. It evidences 3
different production rates; the first one corresponds to the period comprised from 1946 till the
mid eighties in which the productivity rate was 0.15 papers per year. The scientific
productivity increased till 2.5 papers per year from 1985 till 2004. This increase in the
number of published papers is probably related to the important improvements in the
available instrumentation for atomic and ionic spectrometry that took place at the end of the
eighties and the beginning of the nineties. Finally, from 2004 till nowadays the scientific
productivity has increased till 5.3 papers per year, which means that the trace element
determination in garlic is still an interesting research topic for the scientific community.
A deeper revision paying special attention on the reviews published on elemental
composition in garlic can be found in Table 1. Sakai et al. reviewed in 2001 the speciation
analysis of As, Se and Sb by means of liquid chromatography coupled to inductively coupled
plasma mass spectrometry (ICP-MS) in environmental samples. This review emphasized the
advantages of this hyphenated techniques in comparison with different analytical techniques,
such as, atomic absorption spectrometry (AAS) and inductively coupled plasma optical
emission spectrometry (ICP-OES) for the speciation analysis [Sakai et al., 2001].
Trace Elements in Garlic 157
90
Electrochemical
Molecular
80 Techniques
Cummulated number published
Techniques
7,69%
7,69%
Nuclear
70 Techniques
12,82%
60
Atomic
50 spectrometry
64,10%
Hyphenated
40 Techniques
7,69%
30
20
10
0
1946
1951
1956
1961
1966
1971
1976
1981
1986
1991
1996
2001
2006
Year
Figure 1. Evolution of the scientific literature about elemental composition of garlic.

Source: Analytica. Abstracts 1946-2008. Inset: Percentage distribution of the analytical tools employed
for the traces element determination in garlic.
Table 1. Review articles published on elemental composition in garlic.
Topic Elements considered References

Speciation analysis by LC-ICP-MS in As, Se, Sb Sakai et al., 2001
environmental and biological samples
Selenium hyphenated techniques (MS) Se Goenaga Infante et al, 2005
in dietary sources
Selenium speciation in garlic and onion Se Arnault et al, 2006
Selenium speciation from food source Se Dumont et al, 2006
Volatile compounds of garlic and onion Se Lanzotti et al, 2006
Analysis elemental composition of Li, B, Na, Mg, Al, P, Cl, Gonzálvez et al, 2008
seasoning products K, Ca, Sc, V, Cr, Mn, Fe,
Co, Ni, Cu, Zn, Ga, Ge,
As, Se, Br, Rb, Sr, Mo, Cd,
Ba, Pb, Bi, La, Ce, Sm, Eu,
Dy, Yb, Lu, Hg, Th
Special attention has been paid to the speciation analysis of seleno-compounds present in
garlic. In this sense, a review of the different approaches for the Se speciation based on MS
techniques (hyphenated) in food supplements was published in 2005 by Goenaga Infante et al.
[Goenaga Infante et al., 2005]. Arnault et al. in 2006 summarizes different analytical techniques
also for the speciation analysis of Se, based mainly in the combination of a chromatographic
technique with the use of ICP–OES or ICP–MS detectors [Arnault et al., 2006]. Dumont et al.
focuses on the Se speciation in garlic samples from the point of view of its role in health issues.
Mainly based on the Se content in different food sources worldwide and the extent to which their
consumption is reflected in the Se content of human tissues and body fluids [Dumont et al., 2006].
H He
Li Be B C N O F Ne
Na Mg Al Si P S Cl Ar
K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr
Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe
Cs Ba Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn
Fr Ra Rf Ha
La Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu
Ac Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr
Not determined
1-5
5-15
15-25
More 25
Figure 2. The most abundant element determined in garlic.
Another aspect reviewed is dealing with volatile sulphur compounds present in garlic and
onion [Lanzotti et al., 2006], which are responsible for the pungent of these vegetables.
Gonzálvez et al. in 2008 reviewed the different analytical procedures including the sample
pre-treatment normally used to determine the trace element profile in different spices and
vegetables; such as garlic and onions [Gonzálvez et al, 2008].
Another aspect that should be analyzed concerns the different elements that have focused the
interest of the scientific researchers in garlic analysis. In this sense, Figure 2 shows the periodic
table of the elements highlighting the most frequently analyzed elements. Cu, Zn, Mn and Fe have
been determined in garlic samples more than 25 times, followed by Na, Mg, Ca, Se, Cd and Pb
which have been studied between 15 and 25 times. Moreover, elements such as Al, P, Cl, K, Sc,
Ti, V, Co, Ni, Ge, As, Br, Rb, Sr, Mo, Sb and Hg have been studied between 5 and 15 times.
3. ANALYTICAL METHODS FOR TRACE ELEMENTS

DETERMINATION IN GARLIC
Nowadays, the trace element analysis in garlic samples encompasses a number of
analytical techniques based on inductively coupled plasma, absorption and fluorescence
atomic spectrometry, X-ray fluorescence spectrometry, hyphenated techniques, nuclear
techniques, electrochemical techniques and also, molecular spectroscopy based techniques.
Additionally the advances in available instrumentation allows the analysis of almost all the
element of the periodic table.
Table 2. Overview of the use of inductively coupled plasma based techniques for the determination of trace elements in garlic
Technique Sample pre-treatment Elements Analytical features References

ICP-OES Mo, Zn, Mn, Al, Cd, Fe, Mg, Ca, Pb, Cu, Ni, Ti, Co, K, RSD=4-20% Liu et al., 1989
Na, Li, Cr, B, Te, As, Ba, P, Bi, Tl, Rb, Sr
Carbonization 18 elements Wu et al., 1992
Wet digestion 30 elements Abe et al., 1993
Al, Ca, Cd, Ce, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Si, Zn RSD=<±1-10% Ward et al., 1994
Wet digestion Al, As, B, Cd, Cr, Co, Fe, Hg, Mn, Pb, Sn, Zn Bargagli et al., 1997
19 elements Zhang et al., 1998
Ag, Al, Au, Ba, Br, Be, Ca, Cd, Ce, Cl, Co, Cr, Cs, Cu, Awadallah et al., 1999
Eu, Fe, Ga, Hg, K, La, Li, Mg, Mn, Na, Ni, P, Pb, Rb,
Sb, Sc, Se, Sr, Th, Ti, V, Zn
Wet digestion Al, Cu, Fe, Mn, Sr, Zn Li et al., 2004
Microwave-assisted digestion Al, B, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, K, Li, Mg, Hacıseferoğulları et al,
HNO3 Mn, Na, Ni, P, Pb, Sr, Zn 2005.
Microwave-assisted digestion Al, As, B, Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Wang et al., 2006
P, Pb, S, Se, Sr, Zn
ICP-MS Al, Ca, Cd, Ce, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Si, Zn RSD=<±1-10% Ward et al., 1994
Microwave-assisted digestion As, Cu, Ni, Mg, Zn, Se Roychowdhury et al.,
HNO3-H2O2 2003
Microwave-assisted digestion Se LOD= 0.05 µg L-1 Wysocka 2003
RSD 3-7%
Pb, Hg, Cd, As, U, Cr, V, Cu, Zn, Mo, Pd, Sn, Sn, Ti, Raman et al., 2004
W
Microwave-assisted digestion Li, B, Na, Mg, P, S, Ca, Ti, Mn, Fe, Cu, Ni, Zn, Rb, Sr, Smith et al., 2005
Mo, Cd, Ba
Note: ICP-OES: Inductively Couples Plasma Optical Emission Spectrometry, ICP-MS: Inductively Coupled Plasma Mass Spectrometry.
In this sense, the inset in Figure 1 gives an overview of the most popular techniques
employed for the determination of trace element in garlic. The most common tools for trace
element determination and metal speciation are atomic spectrometric based techniques
including flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption
spectrometry (GFAAS), atomic fluorescence spectrometry (AFS), inductively coupled plasma
optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry
(ICP-MS). Other analytical techniques also used for trace element analysis are those based on
X-Ray, like X-Ray Fluorescence and Particle induced X-ray emission (PIXE).
3.1. Inductively Coupled Plasma Based Techniques
The major advantage of ICP-OES and ICP-MS in trace element analysis is their
simultaneous multielemental determination capability. ICP-OES is one of the most widely
used techniques for multi element analysis at ppm level and ICP-MS at ppb levels.
Table 2, summarizes the different methodologies based on ICP published in the scientific
literature regarding the determination of trace elements in garlic.
The first precedent of trace elemental analysis in garlic by plasma emission was made
using a direct-current plasma and it was developed by Liu et al. in 1989. Authors determined
Mo, Zn, Mn, Al, Cd, Fe, Mg, Ca, Pb, Cu, Ni, Ti, Co, K, Na, Li, Cr, B, Te, As, Ba, P, Bi, Tl,
Rb, Sr obtaining precision values ranging from 4 to 20% [Liu et al., 1989].
Later, multi-elemental (18 elements) analysis of garlic was undertaken by ICP-OES
providing accurate results [Wu et al., 1992]. The results were tested by analysis of a cabbage
standard reference material. Abe et al. analyzed the trace elemental composition (30
elements) of healthy food samples by means of ICP-OES after digestion with HNO3 in a
Teflon vessel digestion bomb [Abe et al., 1993]. Ward et al. demonstrated that the exposure
of crops to motor vehicle activities provided significant increases of heavy metal
concentrations of 40–80% which can be removed by washing plant surface [Ward et al.,
1994]. This analysis was done in different types of food samples by using ICP-OES and ICP-
MS with quality control assessment using 4 biological reference materials.
The concentrations of several trace elements (Al, As, B, Cd, Cr, Co, Fe, Hg, Mn, Pb, Sb,
Zn) were determined in different samples, like mosses, plants and organs of small
mammalians from a geothermal area in Tuscany [Bargagli et al., 1997]. It was demonstrated a
deposition of Hg, As, B and Sb in biological samples collected within a few hundred meters
of geothermal power plants. However, this contamination levels in vegetables grown in the
geothermal field were not a risk for the consumer’s health.
A methodology based on ICP-OES was successfully developed for the analysis of 19
elements in garlic and its products [Zhang et al., 1998], being determined until 36 elements in
Egyptian garlic samples [Awadallah et al., 1999] and Al, Cu, Fe, Mn, Sr, and Zn in Chinese
garlic’s [Li et al., 2004]. Additionally, Wang et al. determined the trace elemental
composition of different parts of the garlic plant by ICP-OES and alternatively by AAS and
AFS after microwave assisted sample pre-treatment. Different elements such as, Al, As, B,
Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, P, Pb, S, Se, Sr and Zn were determined and it
was found that tender leaves and bulbs of garlic cumulate Cu, Fe, Mn, S, Se and Zn [Wang et
al., 2006].
Table 3. Overview of the use of atomic spectroscopy techniques for the determination of trace elements in garlic

FAAS Zn, Cu, Mg, Mn, Ca Chun et al. 1984
Fe, Cu, Zn , Mn, Cr, Ni, Co Khan et al., 1985
Cd, Pb, Cu, Zn, Fe, Mn Sattar et al., 1989
Dry ashing Mg, Cu, Zn, Fe Falandysz et al., 1993
Wet digestion with HNO3-HClO4 Se RSD=1.5-1.8% Zhang et al., 1994
Cd, Pb, Cr, Zn, Mg, Cu, Ni, Fe Bulinski et al., 1995
Wet digestion with HNO3-HClO4 Fe, As, Pb, Hg, Cd, Cr, Ni Masud et al., 1998
Wet digestion Cu, Zn, Mn, Fe, Mg, Ca Lavilla et al., 1999
Ag, Al, Au, Ba, Br, Be, Ca, Cd, Ce, Cl, Co, Cr, Cs, Awadallah et al., 1999
Cu, Eu, Fe, Ga, Hg, K, La, Li, Mg, Mn, Na, Ni, P,
Pb, Rb, Sb, Sc, Se, Sr, Th, Ti, V, Zn
Dry ashing with HNO3-HClO4 Cu, Zn Onianwa et al., 2001
Wet digestion with HNO3-HClO4 Cu, Fe, Ca, Mg, Zn, Ni, Co, Cd, Mn, Pb Han et al., 2002
Wet digestion with HNO3/H2O2 Ca, Mg, Na, K, Fe, Zn, Co, Cu, Cr, Ni, Pd, Cd, Ba, Sahito et al., 2002
Al
Pre-concentration by Te LOD=0.03 µg L-1 Kaplan et al., 2005
coprecipitation RSD=3.5%
Microwave-assisted digestion Al, As, B, Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Wang et al., 2006
Ni, P, Pb, S, Se, Sr, Zn
Wet digestion with HNO3 - Cu, Zn Iyaka, 2007
H2SO4 -H2O2
Microwave-assisted digestion K, Na, Ca, Cd, Cu, Mn, Pb, Mg, Fe RSD=1.02-3.30% Zhao et al., 2007
Dry ashing with HCl-HNO3 Cr, Ni, Cu, Zn, Cd, Pb Yang et al., 2007
HG-AAS Hydride generation/ SPE Chitosan Se (IV), Se (VI) LOD= 20 ng L-1 Jiang et al., 1999
RSD= 2-5 %
Dry ashing HCl-HNO3 As Yang et al.,, 2007
HG-IAT- Microwave-assisted digestion with Te LOD=0.9ng L RSD= 7% Matusiewicz et al.,
FAAS HNO3-H2O2 2007
CV-AAS Dry ashing with HCl-HNO3 Hg Yang et al., 2007
ETAAS Wet digestion with Al LOD= 4 pg López et al., 2000
HNO3-V2O5
Wet digestion with Cr LOD= 1.0 pg García et al, 2000
HNO3-V2O5 RSD= 5-5.2%
Wet digestion with HNO3-HClO4, Na, Mg, P, Fe, Cu, Zn, Mn, Al, Co, I, Pb, As Alam et al., 2001
H2SO4-catalyst mixt, HNO3-HCl,
H2SO4-Cr2O3
Microwave-assisted digestion Se LOD= 0.6 µg L-1 Wysocka et al., 2003
RSD=3-7%
Wet digestion with HNO3-HS2O4 Ge LOD= 0.051 ng McMahon et al., 2005
Microwave-assisted digestion Se LOD=1.3 µg L-1, LOQ=3.3 ug Izgi et al., 2006
L-1
Microwave-assisted digestion Se (IV), Se (VI), total Se LOD=0.010 mg L-1, RSD= Saygi et al., 2007
SPE Diaion HP-2MG 2.8–8.3%
HG- Wet digestion with HNO3- HF Ge LOD= 0.004 µg L-1 Tao et al., 1993
ETAAS LOD= 0.03 µg L-1
AFS Microwave-assisted digestion Se, As, Hg Wang et al., 2006
HG-AFS pre-reduction with thiourea Se (VI) LOD= 10 pg mL-1 Qiu et al., 2006
Note: AAS: Atomic Absorption Spectrometry, FAAS: Flame Atomic Absorption Spectrometry, HG-AAS: Hydride Generation Atomic Absorption
Spectrometry, HG-IAT-FAAS: hydride generation, integrated atom trap flame atomic absorption spectrometry (FAAS), CV-AAS: Cold vapor Atomic
Absorption Spectrometry, ETAAS: Electrothermal Atomic Absorption Spectrometry, HG-ETAAS: Hydride Generation Atomic Absorption Spectrometry,
AFS: Atomic fluorescence spectrometry, OES: Optical emission spectrometry.
On the other hand, the tremendous advantages offered by ICP-MS have allowed a
considerable increase in the use of this methodology in the last decade. This methodology
presents excellent selectivity and it offers also a high sensitivity and the possibility of
performing isotopic determinations.
A methodology based on ICP-MS after microwave assisted digestion of samples was
developed for the analysis of As, Cu, Ni, Mn, Zn, Se in onion, garlic and cumin
[Roychowdhury et al., 2003]. ICP-MS and electrothermal atomic absorption spectrometry
(ETAAS) have been used for the determination of the total concentration of Se in garlic after
microwave digestion [Wysocka et al., 2003]. Studied garlic samples originated from two
different geographical regions differed in selenium content as a function of the soil
composition: from Eastern Poland, where the soil is poor in selenium, and from Irapuato in
Mexico, where the soil is rich in selenium.
Furthermore, Raman et al. provided an evaluation of metal composition in botanical
supplements by analysis of Pb, Hg, Cd, As, U, Cr, V, Cu, Zn, Mo, Pd, Sn, Sb, and Ta using
ICP-MS [Raman et al., 2004].
Moreover, the trace elemental profile of garlic samples, as it has been aforementioned in
the Introduction section, is also useful for the determination of the geographical origin of
garlic based on their differences in mineral composition. In this sense Smith et al. [Smith et
al., 2005] determined the geographical origin of different garlic samples using elements such
as Li, B, Na, Mg, P, S, Ca, Ti, Mn, Fe, Cu, Ni, Zn, Rb, Sr, Mo, Cd and Ba determined by
ICP-MS, employing discriminant analysis as multivariate statistical analysis.
3.2. Absorption and Fluorescence Atomic Spectrometry
AAS is a sensitive and highly selective spectrometric tool suitable for the determination
of selected elements at trace and ultra-trace levels. Those techniques can be subdivided in
flame absorption atomic spectrometry (FAAS), electrothermal atomic absorption
spectrometry, (ETAAS) and additionally cold vapour atomic absorption spectrometry
(CVAAS), used for Hg determination and hydride generation atomic absorption spectrometry
(HGAAS), suitable to do the determination of element forming covalent hydrides.
The main strategies developed for trace element determination in garlic samples are
summarized in Table 3.
As it has been aforementioned, FAAS has been commonly used in the determination of
trace elemental composition of several samples. In this sense, Se was the first element
determined by this technique in garlic after wet digestion, with a relative standard deviation
of 1.5-1.8% [Zhang et al., 1994].
Other elements, such as: K, Na, Al, As, Ba, Ca, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pd, Pb, Mg,
Fe, Zn, have been also determined by FAAS since 1998 till now using in all cases acid
digestion prior to sample analysis [Iyaka et al., 2007; Sahito et al., 2002; Han et al., 2002;
Lavillaet al., 1999; Masud et al.,1998].
Moreover, the use of microwave assisted digestion in the determination of trace elements
in garlic could be done rapidly and effectively, displaying a low background. The trace
elemental composition of different parts of the garlic plant was determined by ICP-OES and
alternatively by AAS and AFS after microwave assisted sample pre-treatment [Wang et al.,
2006]. Al, As, B, Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, P, Pb, S, Se, Sr and Zn were
determined in garlic samples and it was evidenced that tender leaves and bulbs cumulate
several elements. On the other hand, a procedure to determine K, Na, Ca, Cd, Cu, Mn, Pb,
Mg, Fe in garlic by FAAS after microwave assisted digestion was described by Zhao in 2007
with recoveries ranging from 96 % to 104 % and relative standard deviation values from 1.02
% to 3.30 % [Zhao et al., 2007].
Different techniques have been employed for the determination of Cr, Ni, Cu, Zn, Cd, Pb,
As and Hg in garlic. Except for As and Hg atomic absorption spectrometry was used after dry
ashing muffle digestion. In the case of As, hydride generation was coupled to atomic
absorption spectrometry for sample introduction and for Hg it was used cold vapour
generation [Yang et al., 2007].
Hydride generation with atom trap (HG-IAT) and flame atomic absorption spectrometry
(FAAS) has been employed to determine Te in garlic. The detection limit was 0.9 ng mL−1.
The sensitivity enhancement, compared with FAAS, was 222 times and the sensitivity can be
further improved by increasing the collection time. The precision for this procedure was 7.0%
(n=6) for Te [Matusiewicz et al., 2007].
The first precedent on the use of ETAAS for the trace elemental composition of garlic is
that published in 1993 by Tao et al. [Tao et al., 1993] in which Ge was analysed using (FI)
hydride generation after pre-concentration. This methodology provided limits of detection of
0.004 and 0.03 µg L-1.
The level of aluminium in garlic has been determined using ETAAS. The accuracy and
precision of the proposed method were verified against certified reference materials obtaining
a range of precision from 1.10 to 4.07% [López et al., 2000]. Chromium level was also
analysed in garlic by ETAAS [García et al., 2000]. The proposed method is suitable to
determine Cr in spices and is useful for routine control analyses of this kind of products
because of its sensitivity, versatility and speed. Detection limit (1 pg), analytical precision
(5.0–5.2%) and accuracy (97.95%) obtained by this method were acceptable for the
determination of Cr in foodstuff.
Bangladesh and Indian garlic samples were analysed by ETAAS, determining Na, Mg, P,
Fe, Cu, Zn, Mn, Al, Co, I, Pb and As after different wet digestion treatments. The analytical
methodology employed was verified using reference materials obtaining ± 2.0% of accuracy
[Alam et al., 2001].
ETAAS has been also used for the determination of the total concentration of Se in garlic
samples from different geographical regions [Wysocka et al., 2003]. The quantification was
accomplished by using the standard addition technique and the spectral interferences were
eliminated using Zeeman background correction. The analytical results revealed differences
in Se content among garlic samples of various origins.
Ge has been analyzed by McMahon et al, after dry ashing mineralization. It was found
experimentally that Ca, Co, Cu, Mg, Ni, Pb and Zn do not interfere Ge determination. The
method was optimized with detection limits from 3.3 to 125 µg L−1 [McMahon et al., 2005].
Another methodology for the determination of Se in garlic was developed by Izgi et al,
after pre-concentration with 3,3-diaminobenzidine (DAB) reagent on activated carbon.
Artificial solutions which represent garlic samples were used to understand the interference
effects in the selenium signal by ETAAS. Data obtained by this procedure were compared
with k0-INAA providing agree results for both methodologies [Izgi et al., 2006].
The determination of Se, has been also improved by using ETAAS after solid phase
extraction for speciation of Se (IV) and Se (VI). The method is based on the solid phase
extraction of selenium (IV) ammonium pyrrolidine dithiocarbamate (APDC) chelate on the

Diaxion HP-2MG. In this case, the absorption capacity of the sorbent was 5.20 mg g-1 Se (IV)
and the limit of detection of Se (IV) was 0.010 µg L-1 [Saygi et al., 2007].
Only two precedents [Wang et al., 2006; Qiu et al., 2006] were found about the use of
atomic fluorescence spectrometry (AFS) in the determination of Hg, As and Se in garlic in spite
of the high sensitivity and selectivity which can be found by AFS for elements such as Sb, Te or
Bi.
A novel methodology was developed for Se speciation analysis of cultured garlic samples
[Qiu et al., 2006]. In this study, thiourea was novelly developed as a reduction reagent for on-
line pre-reduction of Se (VI) before conventional hydride generation (HG) by KBH4/NaOH–
HCl. After thiourea on-line pre-reduction, the HG efficiency of Se (VI) was greatly improved.
The detection limit of Se (VI) was 10 pg mL−1 when using on-line TU pre-reduction followed
by HG-AFS.
Table 4. Overview of the use of hyphenated techniques for the determination of trace
element in garlic.
Technique Sample pre- Elements Analytical References

treatment features
HPLC-AFS water extraction- SeCys, Se (IV), LOD=2.1, 2.9, Liang et al.,
Electrochemical SeMet, Se (VI) 4.3, 3.5 ng mL-1 2005
vapor generation
water extraction- UV SeCys, Se (IV), LOD= 5.2, 6.7, Liang et al.,
TiO2 photocatalysis SeMet, Se (VI) 25.9, 10.3 ng 2005
reduction mL-1
pre-reduction with SeCys, Se (IV), LOD= 0.06, Qiu et al.,
thiourea SeMet, Se (VI) 0.08, 0.05, 0.04 2006
ng mL-1, RSD=
5%
HPLC-ICP- Ultrasound assisted Se Montes-
MS Microwave-assisted Bayon et al.,
digestion 2005
Organic extraction
HCl, ammoniun
acetate and protease
HPLC-FDP Organic extraction 2- LOD= 1.5, 2.25, Bernard et al.,
with methanol methyltiophene, 4.5 ng g-1 1994
CS2,
ethanesulfonic
sulfonate
Note HPLC: High-performance liquid chromatography, HPLC-AFS: HPLC-Atomic Fluorescence
Spectrometry, HPLC-ICP-MS: HPLC- Inductively Coupled Plasma Mass Spectrometry, HPLC-
FDP: HPLC-Flame Photometric Detector,
3.3. X-Ray Fluorescence Spectrometry
X-ray fluorescence (XRF) is the emission of secondary X-rays from a material that has
been excited with high-energy X-rays or gamma rays. The phenomenon is widely used for
elemental analysis and chemical analysis, particularly in the investigation of a wide range of
solid materials, such as metals, glass, ceramics and building materials, and for research in
geochemistry, forensic science and archaeology.
Trace element analysis via X-ray fluorescence spectrometry (XRF) and proton induced x-
ray emission (PIXE) techniques provides non-destructive, reproducible, efficient and high
sensitive alternatives for multi-element characterization of major and minor components.
Ni, V, Cr and Ba were determined in garlic cultivated in some areas of Kiev by means of
X-ray spectrography [Barannik et al., 1970]. Toxic metals; such as: Hg, Pb, and As can be
detected in food samples by using XRF and PIXE. In addition, other essential or neutral trace
elements Na, K, Mg, Ca, Cu, Cr, V, Zn, Mo, Fe, Mn, Ni, Se, Cl, S, and P were also detected
[Flocchini et al., 1997].
3.4. Hyphenated Techniques
The combination of chromatography, in its various modes, and sensitive spectroscopy

detection methods has coalesced into hyphenated techniques. Because advantages of the
hyphenated techniques mainly a high degree of automation, a good reproducibility and a short
analysis time, they have become the method of choice for speciation analysis.
Different hyphenated techniques have been described for the determination of trace
element in garlic as it can be seen in Table 4. The combination of chromatography with those
techniques before mentioned has been a method of choice for speciation of Se [Liang et al.,
2005; Qiu et al., 2006].
Selenium speciation in garlic samples has been made by HPLC coupled with atomic
fluorescence spectrometry (AFS), in a system in which online photolysis of organic selenium
species by UV-irradiation was followed by pre-reduction of selenium in high oxidation state
by UV/TiO2 PCRD (UV–UV/TiO2 PCRD), and electrochemical selenium generation and
fluorescence measurement [Liang et al., 2005].
Thiourea pre-reduction followed by HG has been used as an interface between ion-pair
high performance liquid chromatography and atomic fluorescence spectrometry,
selenocystine, selenomethionine, selenite and selenate can be measured simultaneously and
quantitatively. The limits of detection were 0.06, 0.08, 0.05 and 0.04 ng mL−1, respectively,
and the relative standard deviations for all the 4 considered species were lower than 5% [Qiu
et al., 2006].
Furthermore, several sample extraction techniques have been evaluated in order to obtain
the highest Se extraction efficiency in garlic [Montes-Bayon et al., 2006]. Se extraction yields
were calculated based on the ICP-MS determination of the total Se content in the
corresponding extracts and in the plant tissue after its microwave digestion. The action of
ultrasounds allowed the reduction on the extraction time while maintaining good Se
recoveries. The accuracy of total Se determination was controlled by analyzing a reference
material (aquatic plant, BCR-670). On the other hand, speciation studies of the extracts were
carried out by using ion-pairing reversed phase and size exclusion/ion exchange liquid
chromatography columns. The two separation mechanisms were suitable to isolate the main
extractable Se species which were identified as Se-methyl selenocysteine and Se-methionine
in both systems.
Furthermore, a HPLC- flame photometry method has been described for the analysis of
sulphur compounds like 2-methylthiophene, CS2, ethanesulfonic sulfonate in garlic samples
[Bernard et al., 1994].
3.5. Nuclear Techniques
Instrumental neutron activation analysis (INAA) allows the simultaneous determination

of trace elements giving sensitive and reliable results for seasoning product samples, in the
range of very low concentrations. It does not require any dissolution nor sample preparation
and is scarcely affected by interferences if samples are properly measured. Nevertheless, it
requires big facilities a long analysis time and laboratories with the adequate safety conditions
to use a nuclear reactor.
Table 5 resumes the published papers on nuclear techniques for trace elemental analysis
of garlic samples.
Table 5. Overview of the use of nuclear techniques for the determination of trace
elements in garlic.
Technique Sample Elements Analytical References

treatment features
INAA Ca, Co, Mo, Zn, Fe, Rb, Br, Baicheva et al.,
Se, Cr, Sc, Cs, Ce, Sb 1988
18 elements Zaidi et al., 1992
Na, K, Mg, Cl, Al, Ca, Sc, Cr, Al-Jobori et al.,
Mn, Fe, Co, Zn, Br, Rb, Sr, Sb 1992
Br, Cl, Co, Cr, Cu, Fe, Hg, K, Singh et al.,
Mn, Mo, Na, P, Rb, Sb, Sc, 1995
Se, Sr, Th, Zn
Ag, Al, Au, Ba, Br, Be, Ca, Awadallah et al.,
Cd, Ce, Cl, Co, Cr, Cs, Cu, Eu, 1999
Fe, Ga, Hg, K, La, Li, Mg, Mn,
Na, Ni, P, Pb, Rb, Sb, Sc, Se,
Sr, Th, Ti, V, Zn
Irradiation 2, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, Miyamoto et al.,
40 minutes Cl, Br, Mn, Fe, Co, Zn, Sb, Al, 2000
and 36 hours Sc, La, V, Ce, Sm, Eu, Dy, Yb,
Lu
2 and 4 days, Br, Cl, Co, Cr, Cs, Cu, Fe, Hg, RSD= Singh et al., 2006
2 K, Mn, Mo, Na, P, Rb, Sb, Sc, <±5-10%
and 4 weeks Se, Sr, Th, Zn accurate
up to 3 <±10%
months
measurements
Technique Sample Elements Analytical References

treatment features
IPAA Irradiation 2 Na, K, Rb, Cs, Mg, Ca, Sr, Ba, Miyamoto et al.,
hours Cl, Br, Mn, Fe, Co, Zn, Sb, Al, 2000
Sc, La, V, Ce, Sm, Eu, Dy, Yb,
Lu
PAA multi-channel Ca, Ti, V, Cr, Fe, Ni, Cu, Zn, Rong et al., 1992
analyser with Ge, Se, Sr, Cd, La, Pb
Ge(Li)
detector
dry acid
digestion
Note: INAA: Instrumental neutron activation analysis, IPAA: Instrumental photon activation analysis,
PAA: Proton activation analysis
Table 6. Overview of the use of electrochemical techniques for the determination of

trace elements in garlic.
Technique Sample pre- Elements Analytical References

treatment features
CSV Microwave-assisted Se RSD= 7-12.6% Inam et al., 1999
digestion HNO3-
HClO4
DPV Pre-concentration Te (IV) LOD= 5.6 nM, Khoo et al., 2000
with poly(3,3'- RSD=1.82-2.56%
diaminobenzidine)-
digestion HNO3-
HCl
AdSV Use of U, Sb, V, LOD-ng L-1 Sander et al., 1996
Chloroanilinic acid Mo
as complex reagent
CSA Wet digestion with Se LOD=0.40 ppb Zvonimir et al.,
pre-concentration 2005
step with prior
deaeration
Wet digestion with Se LOD= 1.15 ppb Zvonimir et al.,

pre-concentration 2005
step without
deaeration
dASCP Dissolved with HCl Mn LOD= 8 ng Kg-1 Dugo et al., 2005
Note: CSV: Cathodic stripping voltammetry; DPV: Differential pulse voltammetry; AdSV: Adsorptive
stripping voltammetry; dASCP derivative anodic stripping chronopotentiometry; CSA:
Chronopotentiometric stripping analysis
INAA methods were used to compare the concentration level of Ca, Co, Mo, Zn, Fe, Rb,
Br, Se, Cr, Sc, Cs, Ce and Sb of garlic invaded with D. dipscasi and uninvaded garlic
[Baicheva et al., 1988].
Additionally, the concentration of 18 trace elements (essential, toxic and non-essential) in
garlic and different spices consumed in Pakistan have been analysed by INAA. The
comparison of values obtained with Canadian and Egyptian data evidenced variations in trace
element composition [Zaidi et al., 1992]. Samples of 20 types of vegetable from various
locations in Iraq were analyzed by INAA for Na, K, Mg, Cl, Al, Ca, Sc, Cr, Mn, Fe, Co, Zn,
Br, Rb, Sr, and Sb [Al-Jobori et al., 1992] and nutritive trace elements were determined in a
typical vegetarian diet and its various components in India by INAA [Singh et al., 1995].
INAA, ICP-OES and AAS have been applied to the determination of Ag, Al, Au, Ba, Br, Be,
Ca, Cd, Ce, Cl, Co, Cr, Cs, Cu, Eu, Fe, Ga, Hg, K, La, Li, Mg, Mn, Na, Ni, P, Pb, Rb, Sb, Sc, Se,
Sr, Th, Ti, V and Zn in food samples obtaining consistent results [Awadallah et al., 1999].
INAA and instrumental photon activation analysis (IPAA) were used to determine 26
trace elements in garlic and other vegetables. High elemental correlations exist in some sets
of elements, reflecting characteristics of chemical nature of elements and of their
physiological functions [Miyamoto et al., 2000].
The nutritive potential of black pepper, garlic and onion, in terms of trace element
contents, has been evaluated using NAA. Trace elemental concentrations from different
irradiation periods or from different photo peaks of the same radionuclide were determined in
short as well as in long-term irradiations [Singh et al., 2006].
Table 7. Overview of the use of molecular techniques for the determination of trace
elements in garlic.

UV-Vis 2-(-Br-2-pyridylazo)-5- Cu, Zn Zhang et al.,
spectroscopy diethylaminophenol) 1992
with Triton X-100
Ge LOD= 12 ug L-1, Zou et al.,
RSD=8-4% 2000
Chromogenic reganet Ge Pan et al.,
trimethoxyphenylfluron 2001
e
reaction Se RSD=0.9-2.2 % Bao et al.,
thiocyanate/rhodamine 2001
B/Tween-20
Reduction of azure I by Se LOD= 0.015 mg L-1 Fan et al.,
NA2S and citric acid RSD= 3.1% 2005
Micellar solubilization Ge RSD= 4.3% Deng et al.,
high-pressure airtight 2005
digestion
Proton activation analysis can be used for the determination of Ca, Ti, V, Cr, Fe, Ni, Cu,
Zn, Ge, Se, Sr, Cd, La and Pb in garlic. Garlic ashes were wrapped in Al foil and bombarded
with 12.4 MeV protons for 1 hour and the activities were measured by using a multi channel
analyser with a Ge (Li) detector [Rong et al., 1992].
3.6. Electrochemical Techniques
One of the most important quantitative voltammetric techniques is stripping voltammetry,

which is composed of three related techniques: anodic, cathodic, and adsorptive stripping
voltammetry. Anodic stripping voltammetry is very useful for metals that form amalgams
with mercury; such as: Bi, Cd, Cu, Ga, In, Pb, Tl, Sb and Zn. In the case of cathodic stripping
voltammetry, several analytes have been analyzed as bromide, chloride, iodide, sulphide or
thiocyanate.
Table 5 highlights the methods available for electrochemical determination of Se, Te, U,
Sb, V, Mo in garlic.
Mercury drop electrode using cathodic stripping voltammetry (CSV) allows the
determination of Se in garlic samples without any separation procedure or pre-concentration
technique [Inam et al., 1999].
For the determination of Te concentration in garlic Khoo et al., developed a flow based
methodology employing a wall-jet cell at the poly(3,3-diaminobenzidine) (PDAB) film
modified gold electrode. The optimized method for the continuous flow mode provided a
detection limit of 5.6 × 10−9 mol L−1 for 10 min pre-concentration. Typical relative standard
deviations for six consecutive determinations were 1.82-2.56% for Te(IV) (10 min pre-
concentration) [Khoo et al., 2000].
Adsorptive stripping voltammetric (AdSV) determination of U, Sb, V, and Mo in water
and garlic has been described using chloranilinic acid as the complex forming reagent
obtaining detection limits in the ng L-1 range [Sander et al., 1996].
Chronopotentiometric stripping analysis (CSA) provides a better selectivity than
voltammetric stripping due to reduced influence of the capacity current. Accuracy of time
measurement is higher than current measurement. Nevertheless the technique has been only
applied once for Se determination in garlic [Zvonimi et al., 2005]. Mercury film electrode
comparing to commonly used mercury drop electrodes provides an enhanced better sensitivity
due to significant surface and volume ratio. Applying different procedures for the sample
pretreatment and hexavalent selenium electroinactivity, the content of Se6+, the sum of Se0,
Se2- and Se4+ , as well as total selenium were determined [Zvonimi et al., 2005].
Differential anodic stripping chronopotentiometry (dASCP) was used for the
determination of Mn in garlic. The accuracy of the optimised chronopotentiometric method
was tested by the analysis of certified reference materials and was also compared with
graphite furnace atomic absorption spectroscopy. The obtained results confirmed that dASCP
is a valid tool for rapid and reliable Mn determination in vegetables, particularly prone for
routine analysis [Dugo et al., 2005].
3.7. Molecular Techniques
The determination of trace element concentration based on its molecular absorption in the
UV-Vis range has been frequently used as a base of quantitative analytical methods due to the
possibility to form chemical complexes that have strong absorption bands in the UV/Vis
region of the electromagnetic spectrum. An additional advantage to UV/Vis absorption is that
in most cases it is relatively easy to adjust experimental and instrumental conditions so that
Beer’s law can be obeyed. Methods for the analysis of waters, wastewaters, clinical and food
samples relying on the absorption of UV/Vis radiation are among some of the most frequently
employed analytical procedures.
Table 7 describes methods found for the determination of trace elements in garlic
samples by UV-Vis.
Cu and Zn were simultaneously determined in garlic with 2-(5-bromo-2-pyridylazo)-5-
diethylaminophenol in the presence of Triton X-100. For Cu it was used the absorbance
difference at 509 and 569 nm, and for Zn the difference at 530 and 573 nm [Zhang et al.,
1992].
Molecular techniques have been applaied for the determination of Ge in garlic samples
through by the formation of a complex with 1mM salicylfluorone/ 10 mM
cetyltrimethylammonium bromide (2:1) measured at 506.2 nm [Zou et al, 2000] or by using
the mixture of 11.25M of phosphoric acid, 50 g L-1 Triton X-100 and 3mL of ethanolic 0.4 g
L-1 trimethoxyphenylfluorone making the absorbance measurements at 505 nm [Pan et al.,
2001]. Ge was also determined by a micellar spectromphotometry. Good recoveries from
90% to 105% were found and a good precision defined by a relative standard deviation less
than 4.3% [Deng et al., 2005].
Se has been determined based on the formation of an absorption complex with 1mM
rhodamine B in the presence of 1% Tween-20 which absorbs Se at 596 nm obtaining a
precision in terms of relative standard deviation of less than 2.2 % [Bao et al., 2001].
On the other hand, Se (IV) analysis based on its catalytic effect on the reduction of azure
I by Na2S at pH 7.0 citric acid sodium hydrogen phosphate buffer has been made by UV-Vis,
obtaining a limit of detection of 0.015 mg L-1 [Fan et al., 2005].
4. SAMPLE TREATMENT
There are various sample treatment procedures normally used for trace elemental analysis
of garlic, and they can be selected according to the analytical characteristic, required by of the
analyst: accuracy, sensitivity, detection limit, specificity, and interferences. Other factors to
take into account are the costs of the complete analysis, instrumental availability, and time of
analysis.
4.1. Digestion Methods
Digestion of garlic sample is usually a crucial step before the analysis of trace elements.
The destruction of the organic matter can be done by wet, dry ashing or microwave- assisted
treatment. The selected digestion procedure depends on what the residue solution will be
used for and on limitations based on cost, time, and number of samples to be analysed.
4.1.1. Dry ashing
Dry ashing methods present the disadvantage of possible losses of volatile elements,
analyte reactions with the crucible material or combustion residues and also risks of sample
contamination. However it is clear that after dry ashing of garlic sample it could be dissolved
the remaining sample ashes in a reduced volume of acid solutions. So, providing a sample
pre-concentration way.
Dry ashing technique has been for the determination of Mn by ETAAS through digestion
of garlic samples with concentrated nitric acid in a muffle furnace at 550 ºC for 2 h [Dugo et
al., 2005] or by using a temperature gradient up to 550ºC and leaving this temperature during
10 h for the determination of Cu and Zn by AAS [Onianwa et al., 2001]. A special dry ashing
digestion was developed for Hg and As determination using a sulfonitric (H2SO4–HNO3)
mixture containing V2O5 in the case of As analysis and a V2O5 –HNO3–H2SO4 mixture for
Hg at 450ºC for 12 h in a muffle furnace [Yang et al., 2007].
In fact, it must be emphasized that dry ashing assures a complete elimination of the
organic matter and provides high pre-concentration factors but digestion procedures are
tedious and prone to produce looses or contaminations.
4.1.2. Wet digestion
In wet ashing, organic matter is oxidized by the addition of acids and oxidants or their
combinations. In general, wet digestions in open vessels usually require constant operator
attention and are prone to systematic errors, such as contamination and loss of volatile
elements.
100000
10000
1000
100
10
0.1
0.01
1E-3
1E-4
1E-5
Li B NaMgAl P Cl K CaSc V CrMnFeCoNi CuZnGaGeAsSeBrRbSrMoCdSbTeBaPb Bi LaCeSmEuDyYb LuHgPb Bi ThSbTe
Figure 3. Concentration ranges of elements determined in garlic from the published studies. Data
reported correspond to different samples of different origin analysed by different techniques. So, for
details it must be read trough the text and there references (a total of 23 references).
Wet digestion can be used prior to the analysis by means of techniques such as atomic
absorption spectrometry (ETAAS, FAAS) or electrochemical techniques (CSA). The
combination of HNO3 and HF at high-temperature (180 ºC) for 2 hours has been used in trace
elemental analysis of garlic [Lavilla et al., 1999].
The use of a multiplace block digestion for closed sample mineralization has been
proposed for Cr determination in spices. In this case a portion of homogenized sample was
treated with HNO3 and V2O5 as a catalyst and heated at 60ºC for 30 min and at 120ºC for 60
min. The digestion procedure provides a rapid and complete sample mineralization, without
analyte losses and or contamination. Moreover, mineralization of a large number of samples
can be carried out at the same time with considerable reduction of reagents use and
preparation time [García et al., 2000].
The mixture of nitric acid and perchloric acid heated at 150 ºC during 20 min provides
clear and colourless solutions suitable for Se determination by CSA [Zvonimir et al., 2005].
4.1.3. Microwave-assisted treatment
Microwave-assisted digestion it nowadays the most popular treatment used for trace
element determination in atomic spectroscopy techniques. It has clear advantages over the
traditional acid digestion using convective heating systems in terms of recovery, precision,
time and looses of volatile elements. It reduces the possibility of cross contamination and the
consumption of reagents.
The combination of different acids, sulphuric, nitric and hydrochloric is a very frequent
method for sample digestion before their quantitative trace elemental analysis
[Roychowdhury et al., 2003]. The mixture of some acids such as HNO3 (10), HNO3 and HCl
(10/5), HNO3 and H2O2 (10/2), HNO3 and HClO4 (10/1) were investigated to select the
optimum conditions for the digestion of garlic sample. According to recovery results (garlic
44–48% and onion 71–78%) of digested sample with different acid combinations, HNO3 plus
H2O2 mixture was selected [Izgi et al., 2006].
4.2. Organic Extraction
Ionic species, including metal ions, are quite insoluble in organic solvents. If the charge
on the metal ion is neutralized or the ion is bounded to a large organic moiety, the metal
become soluble in an organic solvent and, consequently, can be extracted from the aqueous
phase. It can be achieved either by formation of metal chelates, metal–organic complexes, or
by ion pairing. The formation of metal chelates is the most common extraction technique used
for metal extraction.
Solvent extraction has been widely used in the determination of trace elements in garlic
by hyphenated techniques such as HPLC-ICPMS or HPLC-FDP and extraction with HCl,
ammonium acetate [Montes Bayon et al., 2005] or methanol [Bernard et al., 1994] were used.
4.3. SPE Extraction for Pre-Concentration
Trace elements can be concentrated from solution by solid-phase extraction (SPE). SPE
usually involves passing the solution through a column, cartridge, or disk containing a solid
material that more or less specifically binds the ions present in solution. The solid-phase
extractant may be an ion-exchange resin, a chelating resin, or other material designed to bind
all cations, or anions, or to bind specific groups of ions.
An adsorbed Diaion HP-2MG column was used for Se determination in garlic by ETAAS
[Saygi et al., 2007] and Jiang, employed Chitosan column for Se speciation by means of
HGAAS [Jiang et al., 1999].
5. TRACE ELEMENT CONTENT IN GARLIC

From the 81 papers published in the literature about trace elements in garlic the analytical
results of the 41 elements (Li, B, Na, Mg, Al, P, Cl, K, Ca, Sc,V, Cr, Mn, Fe, Co, Ni,Cu, Zn,
Ga, Ge, As, Se, Br, Rb, Sr, Mo, Cd, Sb, Te, Ba, Pb, Bi, La, Ce, Sm, Eu, Dy, Yb, Lu, Hg, Pb,
Bi, Th, Sb, Te) determined are summarised in Figure 3.
As it can be seen potassium, ranging from 0.3 to 2.1% (w/w) is the element present in
garlic sample with the highest concentration. Only P present concentration levels ranging
1000 ppm and 0.1%. Na, Mg, Al, Cl, Ca and Fe have been determined at concentration levels
ranging from 0.1 to 10000 ppm.
On the other hand Sm, Eu, Dy, Yb, Lu, Hg, Th are at ppt level For the main part of
elements determined in garlic there is a big fluctuation of their content which can vary from
few ppb to 100 ppm, as a function of the origin and production conditions of samples
considered.
6. CONCLUSIONS
As it can be evidenced trough the scientific literature available, trace elements in garlic is
a subject of interest in research groups based on the general use of garlic as food and
seasoning products. ICPMS and ICP-OES offer convenient tools in profiling of single,
multielement and speciation analysis of the different elements present in garlic and seasoning
products. Although atomic absorption spectrometry techniques, AAS, nuclear techniques and
electrochemical method were also used, ICP-OES and ICPMS were the unique techniques
selected for determination of a big number of heavy metals in a same sample. The particular
advantage of ICP-MS when compared to other techniques is its high sensitivity and the
possibility to do isotope determination.
On considering the sample preparation procedures, it is clear that total digestion of garlic
made trough microwave assisted treatment in closed reactor is the method of choice to avoid
accuracy errors in multielement trace determinations.
7. ACKNOWLEDGMENTS
A. Gonzálvez acknowledges the F.P.U (ref AP2007-04566) grant provided by the
Ministerio de Ciencia e Innovacion to carry out this study.
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Chapter 5
CHARACTERIZATION AND DISCRIMINATION OF

MEDITERRANEAN BULB-PRODUCING GARLIC
Giovanni Figliuolo and Stefania Mang

Dept. Biologia – Università degli Studi della Basilicata – V.le Ateneo Lucano, 11 85100
Potenza, Italy
ABSTRACT
Allium ampeloprasum is bulb-producing garlic less common than A. sativum and
poorly investigated for agricultural and pharmaceutical use. This species is cultivated as
landrace mixed with Common garlic (CG) in rural farmer fields of the Mediterranean
Countries. In USA, because of its spectacular size, this domesticated garlic is named as
“Elephant garlic” or “Great headed garlic” (GHG). The Mediterranean region is the
geographic area of evolution and differentiation of the primary gene-pool of A.
ampeloprasum (wild Leek) represented by an array of different citotypes. Following the
domestication, in the above mentioned region, have been isolated and selected garlic
types with a ploidy level greater than 4X yielding big bulbs. Few of these clones, along
with humans, migrated to the New World since the 20th century. Overall, the wild forms
of A. ampeloprasum, GHG, cultivated Leek and Kurrat, upon a taxonomic point of view,
given the variable plant morphology and ploidy, are considered members of the same
species-complex. Great headed garlic compared with A. sativum (soft-neck form) shows
differences at morphologic, cytogenetic and molecular level. Great headed garlic bulb
yield is higher than A. sativum. Differences in the heritability of yield related traits
between CG and GHG reflect both different ploidy levels and genetic backgrounds. From
a breeding perspective differences in heritability are relevant to select the best clones.
The heritable traits more correlated with yield are plant height within A. sativum and neck
diameter within A. ampeloprasum. Moreover, karyotype analysis and DNA markers are
perfect tools to determine genetic relationships among A. ampeloprasum and its relatives.
The genetic profile at diagnostic sequences such as the M13 DNA repeated sequence,
nucleotide variation at Internal Transcribed Spacer (ITS) of the ribosomal gene together
with morphological traits indicate that different citotypes from various origins are
probable members of the same species-complex (A. ampeloprasum). The M13
polymorphism can be relevant to distinguish with a single analysis A. ampeloprasum
plant materials from A. sativum.
182 Giovanni Figliuolo and Stefania Mang
INTRODUCTION
The genus Allium is a large group comprising about 750 species, widely distributed from
the dry subtropics to the boreal zone (Fritsch and Friesen, 2002). Among the most widespread
bulb producing garlic species, A. sativum (Common garlic) and A. ampeloprasum (Leek,
Kurrat, Great headed garlic, Pearl onion) have economic significance for direct use and
breeding. A. ampeloprasum and its allies share the Mediterranean region and the Southwest
Asia as a center of origin, while A. sativum evolved in Central Asia (McCollum, 1987).
Different taxa within the Allium genus can be efficiently classified by applying genome and
phylogenetic analyses especially to assess controversial classifications based on morphology,
ecology or cytology (Avise, 1994).
The purpose of this article was to synthesize the current knowledge on A. ampeloprasum
species-complex, investigate wild and cultivated garlic germplasm from the Mediterranean
region and also identify tools able to efficiently distinguish A. ampeloprasum complex from
A. sativum.
DISTRIBUTION AND CLASSIFICATION OF WILD A. AMPELOPRASUM

SPECIES-COMPLEX
A. ampeloprasum gene-pool is actually defined as an assemblage of different species,
subspecies and botanic varieties. Floristic classification of the wild gene-pool is mainly based
on ecology and morphology. A. ampeloprasum L. [syn.: A. porrum L. subsp. Eu-
ampeloprasum Breistr.; A. scorodoprasum L. subsp. Babingtonii (Borrer) Nyman] (broad leaf
wild leek) widely distributed in the Mediterranean Isles, continental Europe and regions on
the border of the Mediterranean Sea (from Turkey to Portugal and Tunisia) is growing on the
margins of farmer fields, in dry semi-natural grasslands as well as in abandoned farmer fields.
McCollum (1987) reports its centre of origin in the regions of Northern Africa and South-
West Asia.
Widely distributed only across the Mediterranean basin is A. commutatum Guss. [(syn.:
A. ampeloprasum L. subsp. Ampeloprasum var. commutatum (Guss.) Fiori; A. ampeloprasum
L. var. lussinense Haracic and A. bimetrale Gandoger)] (Island garlic) which is reported as
localized in the Mediterranean islands and coasts, where it grows on rocks and sea-cliffs
(Pignatti, 1982). However, the reported morphological differences between A. ampeloprasum
L. and A. commutatum Guss. are small and mainly related to the shape, dimension and
number of bulblets, outer flower’s tepal apex and stamen shape (Pignatti, 1982).
A. ampeloprasum L. subsp. Ampeloprasum var. bulbiferum Syme (syn.: A. ampeloprasum
L. subsp. Ampeloprasum var. bulbilliferum J. Lloyd) is distributed only in France (Brittany)
and South England (Cornwall) (Boscher and Auger, 1991).
A. ampeloprasum var. babingtonii (Borrer) Syme, covering a broad range of habitats,
including stream sides, sea cliffs, open woodland, peaty heath and roadsides (Treu et al.,
2001), is locally spread across the northern limit (S.W. England, Ireland and Channel Islands)
of the wild leek (A. ampeloprasum) distribution area; this type is apparently relic of former
cultivation, has numerous bulbils and few flowers (Walters et al., 1998).
Characterization and Discrimination of Mediterranean Bulb-Producing Garlic 183
Figure 1. Differentiation based on morphological traits between Great headed garlic clones and
Common garlic sampled from farmer fields in South Italy.
A. atroviolaceum Boiss. [(syn.: A. ampeloprasum var. atroviolaceum (Boiss.) Regel]

widely distributed in the Eastern Mediterranean with its Eastern limit in Apulia (Italy) -
growing in dry uncultivated fields and on road sides - is morphologically close to the wild
Leek. The pearl-onion group (A. ampeloprasum var. sectivum Lued.) is another group of the
complex distributed in the Atlantic and Temperate regions of the Europe (Fritsch and Friesen,
2002) and cultivated under small-scale agriculture (Hanelt, 2001).
Ploidy level among the wild specimens belonging to several different regions of the
Mediterranean basin shows a series of six ploids, from diploids (2X) to heptaploids (7X), and
karyotype analysis demonstrated that A. ampeloprasum from Croatia is similar to the Iberian as
well as to the Israeli A. ampeloprasum var. ampeloprasum and var. truncatum (Pejčinović, 1997).
The pairing pattern of meiotic chromosomes ranges from bivalent types to uni-, tri- (rarely) and
multivalents. This mixed pattern of chromosome pairing at meiosis – quite usual for autopolyploid
plants - implies the production of gametes with unbalanced chromosome number as demonstrated
by the variable degree of flower sterility. The vegetative reproduction ensures the transmission of
heterozygote genomes, while seeds from selfing can produce genotypes either with a reduced level
of heterozygosis or aneuploid (in Leek) and thus, less fit to the environment. Among the diploid
species closely related with A. ampeloprasum, A. sativum and A. longicuspis is A. macrochaetum
Boiss & Haussk subsp. tuncelianum (syn.: A. tuncelianum (Kollmann) Özhatay, Mathew &
Şiraneci) investigated as possible wild progenitor of CG endemic to Anatolia (Ipek et al., 2008)
and natural in the eastern regions (Syria and Israel).
Sardinia Malta Tremiti Basilicata
Figure 2. Inflorescence color variation of Allium ampeloprasum (wild Leek) from different
Mediterranean sites grown ex-situ at University of Basilicata (South Italy) since 2003.
Similar to A. ampeloprasum is Allium iranicum Wendelbo (syn.: A. ampeloprasum L.

subsp. iranicum Wendelbo). A. iranicum differs from A. ampeloprasum only by its long
stipitate bulblets born on slender stolons beneath the leaf sheaths, their acute and sub-acute
inner and outer perianth segments and glabrous filaments (Ghaffari, 2006). Chromosome
number of A. iranicum is tetraploid, probably allotetraploid, with four members of satellite
chromosomes (a pair of A and B type respectively) as demonstrated by the predominant
formation of bivalents in meiosis. The same author showed that A. ampeloprasum and A.
commutatum (which could be diploids) are not present in Iran and suggested further
investigations in order to identify the hybrid origin of A. iranicum.
The wild forms are greatly diverging from the domesticated varieties (Leek, Kurrat and
Great headed garlic) at morphologic level.
Taxonomic investigation of the Allium species is rather difficult due to the notable
polymorphism exhibited by the taxa. The taxonomic classification criterion is generally
dictated by the structure of the generative organs, but this is unsatisfactory for taxon
identitification given that the reproductive system of Allium is very labile. A core of
specimens was previously investigated by Figliuolo and Di Stefano (2007) to better
characterize morphology, growing habits and genetics of the wild Leek from different origins
of the Mediterranean region (Sardinia, Tremiti, Malta islands, and South Italy).
When the morphology of this core plant collection was analyzed in a common
environment the wild Leek specimens displayed dry leaves at the anthesis while the GHG
clones had a longer stay green. In opposite to the wild specimens the GHG had at anthesis
green leaves. The inflorescence and plant sizes were positively correlated with the size of the
bulblets. In particular, inflorescence size ranged from 7 x 6 – 6 x 6 cm to 4.5 x 3 cm revealing
a compact shape except for the Tremiti Island specimens having a more lax inflorescence
(Figure 2).
Flowers, composed of 6 tepals with tricuspidate stamens bearing an anther just outside
the rim of the corolla, in all specimens presented the tepal margins slightly denticulate. Tepal
average size was 2.5 x 5 mm with a filament ranging from 6 (Tremiti) to 7 mm (GHG).
Nevertheless, the accessions were variable for tepal shape and colour. Tepal shape was more
round in Tremiti specimens and elliptical shaped in all other specimens (GHG included).
Inflorescence color was ranging from lilac (Basilicata specimens, Tremiti, one clone of
GHG), light lilac (Malta, the second clone of GHG) to white with lilac stipe at the center of
the tepal (Stintino, Sardinia) (Figure 2). Umbels were composed of hundreds of protandrous
flowers.
Figure 3. Allium ampeloprasum (Great headed garlic) cloves compared with A. sativum (sigle bulb
type).
The underground bulb was composed of two cloves with a developed apex embedded by
a tunic ranging in color from brown (Stintino, Sardinia) to light cream (Tremiti and GHG)
(Figure 4). Bulblets (helmet shaped) growing together with the bulb and embedded within the
tunic from the middle part of the bulb to the bottom were variable in shape and number.
However, the helmet shape length can be more (Sardinian accession) or less developed
(Tremiti accession) (Figure 4). The most common ploidy level was tetraploid (4X)
(specimens from Tremiti and south Basilicata) while diploids were from Northern Sardinia. In
some umbels of tetraploid specimens seed production was observed. The plants produced a
single bulb as observed also for GHG and CG when the wild germinating cloves were not
exposed to the required growing degree days. The stalk length, depending on the variable
natural environments, ranged from 40 cm (on arid sea-cliffs) to 1.20 cm (in fertile soils). Plant
size was related with bulb size under green house conditions.
A. ampeloprasum as a Vegetable Crop
The domesticated gene-pool of A. ampeloprasum is mainly represented by three

cultivated forms: Great headed garlic (A. ampeloprasum L. subsp. ampeloprasum var.
holmense Asch. & Graebn.), Leek [A. ampeloprasum L. subsp. ampeloprasum var. porrum
(L.) J. Gay (syn.: A. porrum L.)] and Kurrat [(A. ampeloprasum Schweinf. Var. kurrat, (syn.:
A. kurrat Schweinf. ex Krause)].
Figure 4. Colour, bulblets shape, bulb and clove variation in wild Mediterranean Allium ampeloprasum
from different geographic origins.
Few minor crops belonging to ampeloprasum complex should be also mentioned: Pearl-
onion (A. ampeloprasum var. sectivum Lued.), Prei-anak - a tetraploid leek grown in
Indonesia but probably introduced from Europe (Kik et al., 1997) - and Mushuu-ninniku (a
Japanese tetraploid garlic-like) (Ariga et al., 2002). Also, the Persian Leek (A. ampeloprasum
var. persicum Mousavi and Kashi), has been reported as a cultigen native to the Middle East,
tetraploid (2n=4X=32), closely related to A. ampeloprasum and - based on morphological
traits - different from the sympatric wild form A. iranicum (Mousavi et al., 2006).
The high level of genetic polymorphism characterizing the wild gene-pool of the
ampeloprasum species-complex is correlated with the wide range of eco-geographic
adaptability. Domestication produced a great divergence of plant morphology, as
demonstrated by the big cloves of GHG or by the lack of bulbs in Leek and Kurrat. Cultural
practices also play a significant role in promoting phenotypic plasticity in different climatic
contexts (Astley et al., 1982).
Great headed garlic
Great headed garlic (syn.: Buffalo, Big Tex, Tahiti, Elephant or Giant garlic) is a leek-
like plant - with large cloves and a flavour milder than the CG - not yet appreciated by the
wide market. Its origin is very likely from rare cross events between wild leeks. It is
cultivated as a landrace often confused with CG (A. sativum) in Southern Italy, in the
Mediterranean Islands (Figliuolo et al., 2001) and probably in the Eastern Mediterranean
regions. The plant is large with a bulb exceeding 15 cm in diameter and weighing up to 450 g,
containing 4-6 cloves which can reach 10 cm tall by 3 cm across (Figure 3). The bulb
wrappers are extremely white and they cure out to be very thin and flaky being intact only on
freshly harvested bulbs. After few months wrappers disappear leaving bare the cloves without
affecting their storability (Figure 3). Bulbs of this vegetable have been introduced during the
20th century, for the first time, in Oregon after purchasing a stock from the local Eastern
European immigrant gardeners (Orin, 2002) and also in South America (Argentina)
(Lampasona et al., 2003; Hirschegger et al., 2006). The high ploidy level usually hexaploid
(6X) and rarely octoploid (8X) (Hirschegger et al., 2006) is associated with big plant and bulb
size (McCollum, 1987; Hanelt, 1990; Figliuolo et al., 2001; Fritsch and Friesen, 2002). Great
headed garlic stores longer than CG even when separated into individual cloves. It produces
bottom bulbils (helmet shaped corms) that have hard shells with sharp pointed tops that store
longer than bulbs like the wild A. ampeloprasum species-complex. The bulblets are attached
to the bottom of the bulb but grow up on its sides and are often incorporated into bulb
wrappers several layers deep. The crop is propagated by cloves although in few cases seed
production has been reported (Hirschegger et al., 2006). One commercial cultivar is available
in Northern America whereas in the Mediterranean Countries it is still cultivated for self-
consumption either as landrace or selected clone by few garlic-lovers. Within the
Mediterranean region is possible to find both GHG landraces and wild forms of
ampeloprasum species-complex (Figliuolo et al., 2001). The GHG is more responsive than
CG to environmental climatic factors and to fertility inputs. In South Italy GHG is planted in
late Autumn and harvested at the end of the Spring together with CG. However given its long
life cycle, GHG yield could be higher if harvested during the Summer.
The seed stalk production indicates that the plant has received a sufficient winter chill (<
10°C, > 0°C) for 6-8 weeks and consequently, will produce a segmented bulb. This type of
garlic under Dutch climatic conditions produces only small bulblets instead of big cloves
(Astley et al., 1982). Great headed garlic contains less allicin, therefore it is less bitter and
flavored than CG and appears to be more resistant to diseases. It is preferred on pizza and his
big cloves are usually cut into little slices. It can be also used in quick stir fry dishes, salad
dressing (raw) and roasted whole to produce a large amount of a smooth textured paste with
very little garlic “zing”. While little and bitter cloves of CG are preferred entirely or as little
debris for sauces, salads and some pasta recipes. The volatile oil of GHG was demonstrated to
have a beneficial effect on epidermal damage in mouse (Nguansangiam et al., 2003) and an
antimicrobial effect in humans (Rattanachaikunsopon and Phumkhachorn, 2008 and 2009)
The germplasm of GHG can improve the gene-pool of clove-producing garlic by
significantly increasing in Mediterranean environments the garlic yield for commercial
purposes. The number of accessions of cultivated hexaploid GHG is very small and more
collecting is needed to increase the germplasm available to carry out a better assessment of
the effective population size and genetic variation.
The evaluation of a living collection of GHG would be relevant for clonal selection as
well as to identify mitochondrial genome variation of breeding interest (Engelke et al., 2004).
In addition, this germplasm can be relevant to better explore the genetic basis of the hybrid
vigor within the genus Allium.
Leek
Leek is a herb, whose edible part is an etiolated pseudo-stem formed by leaf sheaths,
multiplied by seeds, brought from the Eastern Mediterranean into Europe in the Middle Age
(Astley et al., 1982). Leek worldwide distribution is mainly due to its tolerance to low
temperatures. Also its life-cycle does not require stringent photoperiodic conditions and is not
affected by a bulb resting stage. Two morphological types are known: with very thin pseudo-
stems (in Bulgaria and Turkey) and with thick pseudo-stems (in most European countries).
Seed sowing in Spring will produce marketable plants by Autumn and the cold hardiness
permits harvesting throughout the Winter, when few other vegetables are available (Astley et
al., 1982). In opposite to GHG, in Europe, commercial cultivars are available along with
breeding programs based on inter-specific crosses and gene transfer.
Leek is tetraploid (4X), allogamous with up to 20% self-fertilization (Ved Brat, 1965). Its
wild ancestor is supposed to be a tetraploid species, although allopolyploidy is not excluded
(Khazanehdari et al., 1995). Meiotic chromosome pairing irregularities give rise to

segregation errors correlated with aneuploid seedlings at a frequency between 4.3% - 8.4%
(Khazanehdari and Jones, 1997). Protandry offers a partial barrier to self-pollination because
pollen from flowers can fertilize receptive stigma of more advanced flowers in the same
umbel (Clement et al., 2007). Clement et al. (2007) also suggest to regenerate gene-bank
accessions of Leek by coupling suitable insect pollinator with caged plants. This system
would maintain the genetic integrity of each accession reducing the risk of contamination by
pollen from neighboring accessions, yet under conditions that encourage high rates of cross-
pollination. Inbreeding depression, correlated with selfing (De Clercq et al., 2003) can
severely reduce the vigor of important agricultural traits (seedling emergence, plant growth
and plant fresh yield) (Berninger and Buret, 1967; De Clercq et al., 2003). Cytoplasmic male
sterility (CMS), carried by gene loci in the mitochondrial genome, is proposed as the best
mechanism in hybrid seed production (Engeleke, 2004). As little variability exists in the
mitochondrial genome of Leek and Kurrat (Kik et al., 1997) it is necessary to search new
genetic resources for breeding programs.
Moreover, the whole range of the botanical entities above described, forming the
Mediterranean A. ampeloprasum species-complex could be used as genetic resource in
marker assisted breeding (Kik et al., 1997; Figliuolo et al., 2001; Fritsch and Friesen, 2002)
and for in-vitro technologies (protoplast fusion, embryo rescue and chromosome doubling
techniques). The improvement of a CMS system for Leek by using A. fistulosum as well as
Onion (A. cepa) and the ampeloprasum species-complex is in progress (Peterka et al., 2002;
Engeleke et al., 2004; Peterka et al., 2005).
Kurrat
Kurrat is a crop of the Near and Middle East, very popular and widely grown in Egypt.
Similar to Leek but without stem, Kurrat allows successive cuttings. It is a tetraploid (4X),
resistant to yellow stripe virus, completely interfertile with Leek and reproduced by seeds
(Kadry et al., 1955; Astley et al., 1982). Its cultivation can be traced back three or four thousand
years to the early civilization in the Near East (Hassan, 1989). It is used fresh, for seasoning and
is rising popularity in the USA. Breeding programs attempting to transfer the yellow stripe
resistance to Leek (van Dijk, 1994; Rabinowitch, 1997; Peterka et al., 2005) have been carried
out as well as in vitro shoot proliferation to promote clonal propagation of plants bearing
desired traits (Mohamed-Yasseen et al., 1994 and Mohamed-Yasseen et al., 1995). Very similar
to Kurrat is the Tarée group cultivated in northern Iran (van der Meer, 1997).
DISCRIMINATION OF CULTIVATED BULB-PRODUCING GARLICS

The Mediterranean region is also the secondary centre of diversity of CG soft-neck type (A.
sativum L.) (Vavilov, 1951), whose wild progenitor is A. longicuspis Regel (Vvdensky, 1946)
(generally diploid), a species endemic to central Asia (McCollum, 1987). Some studies report
variation for ploidy level within cultivated garlic populations and relationships between ploidy
level and type of reproduction system (Etoh, 1985). The presence of several ploidy levels,
within the Mediterranean garlic gene-pool, reflects not only macroscopic variation of morpho-
physiologic traits but also different degree of fertility and genetic divergence. For instance, one
accession of tetraploid and self-fertile garlic was collected into a field of Campania (Italy)
(Bozzini and De Luca, 1991). The hope is to promote garlic improvement for yield, quality and
adaptation by using, as parents, clones with fertile flowers (Yanagino et al., 2003).
Clones of cultivated garlic appear variable for morpho-physiologic traits making it
possible to distinguish plant materials based on their plant structure and yield-related traits
(Conti and Ferraresi, 1974; Pooler and Simon, 1993; Avato et al., 1998; Figliuolo et al, 2001;
Baghalian et al., 2006).
Landraces of CG and GHG are cultivated for clove production and sometime, in Southern
Italy, these two species are grown in the same field as a clone mixture under common cultural
practices. Heterogeneous environmental conditions and bulb exchange within and between
rural communities could have been enhanced the garlic population diversity. In addition,
GHG is managed as A. sativum and therefore is often confused with CG.
If GHG and CG are treated with the same agricultural technologies, at the end of the cultural
cycle GHG bulbs are not very big and its morphological traits tend to converge with those of CG.
Furthermore, it is known that polyploid types have a longer life cycle and, harvesting them
together with the diploid types, will not allow the fully expression of GHG yield potential. It must
be pointed out what already consolidated by the USA gardeners which found that GHG needs a
different agronomic treatment because of its better yield response to high water and nitrogen
inputs (Orin, 2002). However, depending upon the environmental conditions (e.g.: day length and
cold requirement) either species can yield a single bulbous head.
The correct classification of commercial bulbs from these two species is difficult and the
correct distinction can be achieved with:
a). discriminant multivariate analysis of morphological descriptors (Figliuolo et al.,

2001);
b). karyotype and flow cytometry analyses (Hirschegger et al., 2006);
c). molecular analysis using species-specific markers (Figliuolo and Di Stefano, 2007).
Figliuolo et al. (2001), in order to differentiate the two clove producing cultivated garlic
species and to explore the extent of quantitative trait genetic variation, carried out in Southern
Italy a field trial, adopting a spaced plant grown, with an environmental background specific for
CG, by analyzing sixteen morphological quantitative traits. Morphological traits distinguished
the GHG consistently with karyotype classification. Great headed clones were all hexaploids
while CG was confirmed as diploid. Although the best discriminant model was based on 15
traits, the 100% rate of correct classification was already maintained by four traits (leaf basal
width, total number of leaves, clove diameter and neck height) (Figliuolo et al., 2001).
The different genetic background between the two species is reflected both in differences
in their respective matrix correlation structures and in the heritability of quantitative traits.
The correspondence between the two correlation matrices for the two species was r = 0.88 (P
< 0.01) showing that, on average, for each species, traits are correlated with similar measures.
In both species the number of cloves per bulb is negatively correlated with the clove weight (r
= 0.58) (P < 0.01) and thus, plants with high number of cloves have small cloves. However,
the number of cloves per bulb is little (CG) or not (GHG) correlated with bulb weight. The
“plant height”, “leaf length” and “number of leaves” are more correlated with “neck
diameter” in GHG than in CG.
In short, for GHG the vegetative development (plants with big neck diameter and high
number of leaves) is strongly correlated with yield traits. The most heritable traits correlated
with yield are plant height (h2 = 0.62) within CG and neck diameter (h2 = 0.75) within GHG.
Additionally, the high value of the broad sense heritability for characters highly
correlated with yield such as plant height for A. sativum and neck diameter for A.
ampeloprasum respectively allows an easy field screening of garlic clones.
It appears that in A. sativum selection over time for two different types had been
achieved: the first with high number of small cloves per bulb and the second with a big bulb
with a relatively low number of big cloves. The selection of specimens having heavy bulbs
composed of a high number of big cloves might be possible given that the number of cloves
per bulb and bulb weight are little or uncorrelated.
It is worth to stress that different environmental conditions can generate different data,
especially for garlic which is widely distributed over a large range of latitudes at world level.
For instance, Baghalian et al. (2006), in Iran, did not found plant height significantly
correlated with yield, in opposite to what demonstrated in the Italian environment by Conti
and Ferraresi (1974) and Figliuolo et al. (2001). Pre-replication factors, due to different clove
size and quality, determined by the environmental effect on the clove source, may play a
significant effect on yield traits. Such pre-replication factors, for instance, determine both a
significant positive within clone correlation and a correlation between clove and bulb weight
(Conti and Ferraresi 1974). At inter-specific level an efficient distinction between A. stivum
gene-pool and A. ampeloprasum species-complex is achieved with PCR (Polymerase Chain
Reaction) of the M13 nuclear repeated DNA sequence.
DISCRIMINATION TROUGH DNA NATURALLY

OCCURRING VARIATION
Several tools such as karyotype analysis and molecular markers when used to asses the
phylogeny of A. ampeloprasum and its relatives allowed a steady phylogenetic classification
(Havey, 1991; Maaβ and Klaas, 1995; Lampasona et al., 2003; Ipek et al., 2008). Thus,
minisatellite, random amplified and single nucleotide polymorphisms are the most achievable
markers to assess controversial classifications based on morphology, ecology or cytology.
The genetic analysis of chloroplastic sequences of all cultivated forms (Havey, 1991) confirm
Vvedensky’s taxonomy (1946), and, in particular, the close genetic similarity between A.
ampeloprasum and A. sativum.
For phylogenetic analyses the well known diagnostic gene sequence data of ITS (Internal
Transcribed Sequence) are commonly used. Also in this study the above mentioned sequences
have been evaluated using the maximum likelihood method (Felsenstein, 2007).
Over 588 ITS nucleotide sites, 105 were polymorphic with a transition-transversion ratio
equal to 1.8. The highest number of polymorphic sites were found in the complex A.
ampeloprasum (101 variable sites) and the minimum was recorded in A. sativum (1
polymorphic nucleotide). Heterozygote sites were unevenly recorded among the gene-pools.
On average 0.5 heterozygous sites were observed within A. sativum, only 1 heterozygote site
in cultivated Leek, 0.8 heterozygous sites within the wild leek from islands (Sardinia, Tremiti
and Malta), 3.6 sites within the wild Leek specimens from south Italy, and the maximum
number of 10 heterozygote sites in GHG.
Upon a genetic point of view GHG was classified as a member of the wild Leek gene-
pool (Figure 5). The wild Leek from Sardinia (W_SR1 and W_SR2) was the most rich in
sequence variation and thus the most differentiated from the A. ampeloprasum species-
complex as well as from A. sativum (P < 0.01). Sardinian specimens are also diploids
(2n=2X=16) (Figliuolo and Di Stefano, 2007). The commercial Leek cultivar was related
with A. ampeloprasum species-complex but significantly differentiated (P < 0.01). Closely
related with the wild and cultivated Leeks was A. sativum (either Chinese or Mediterranean
types). Although it can be inferred a common ancestral sequence for both CG and Leek-like
garlic, phylogenetic analyses assessed statistically significant (P < 0.05) sequence difference
between the two gene-pools (Figure 5). The two GHG clones sampled from geographically
distant farms (about 50 Km) had identical ITS sequence.
The high number of heterozygote sites (Figure 6) is consistent with the plant hybrid vigor
and with the hypothesis that the nuclear genome of GHG could be the result of chromosome
number increase following rare events of cross pollination between unrelated specimens of
wild Leeks within the species-complex A. ampeloprasum. The most likely origin of the
hexaploid (2n=6X=48) GHG specimens used in this study may have been arose, for instance,
from the union of unbalanced gametes (n=3X) produced from genetically different tetraploid
ancestors (2n=4X). Less likely gamete combinations, involving less common citotypes of A.
ampeloprasum species-complex, could have been also possible. The M13 marker pattern
(Figure 7) allowed us to exclude that GHG is an inter-specific hybrid involving genomes of
A. sativum and A. ampeloprasum gene-pool given the absence of the 1000 bp fragment
(specific to A. sativum) into the genome of GHG specimens. The high number of
heterozygote sites over the ITS sequence excludes that GHG can have arisen from selfing,
from crosses between relatives or from somatic polyploidization.
Legend Origin
Wild leek
W_ML Malta
W_TR Tremiti
W_SR1 Sardinia
W_SR2 Sardinia
W_S_IT1 South IT
W_S_IT2 South IT
W_S_IT3 South IT
Cultivated leek
LEEK_Cv Holland
GHG1 South IT
GHG2 South IT
Common Garlic
SfN_IT1 Soft neck IT
SfN_IT2 Soft neck IT
StN_CH Hard neck CH
SnB_CH Single bulb CH
Figure 5. Maximum likelihood phylogeny (*=P<0.05 or ** = P<0.005, branch length significantly

positive) of Wild leek, Great headed garlic, Cultivated Leek (A. ampeloprasum) and Common garlic (A.
sativum) from different origins, based on nucleotide polymorphisms over the ITS DNA sequence
(NCBI Acc. No. FJ890323 to FJ890395).
Figure 6. Alignement of ITS sequences including wild Leeks from isles (Malta and Sardinia) and
cultivated Great headed garlic (South Italy) covering 40 nucleotides showing the presence of
heterozygous sites (stars) only in Great headed garlic.
In autopolyploid species plant vigor is related with the relative degree of heterozygosis
which is maintained in natural populations by cross pollination. Great headed garlic, from a
breeding point of view, can be considered the first generation (F1) bearing the maximum of
heterozygosity. This hybrid, if sexually fertile, will generate second and third generations
with a reduced level of heterozygosis (Bingham, 1979). However, as observed in a wide array
of plant families, polyploidy and self-compatibility are not strongly associated, and there is no
evidence that plant species and families having a functional self-incompatibility system also
have fewer polyploid taxa (Mable, 2004). It is more likely that polyploids carrying multiple
alleles will show varying levels of self-compatibility rather than a complete breakdown of the
self-incompatibility system (Hauck et al., 2002; Yamane et al., 2003; Mable, 2003; Ushijima
et al., 2004). These findings are consistent with sexual reproduction low efficiency observed
in GHG (Hirschegger et al., 2006) and wild Leek.
Given the clonal pattern of spatial distribution into the wild environment the most likely
matings between related individuals will happen on short distances. It follows that true seeds
from wild Leek will produce phenotypes affected by inbreeding depression. Inbreeding
depression of wild Leek would be even more severe if individuals were originated from
selfing because of their autopolyploid nature. Clonal propagation (by cloves and bulblets)
ensures adaptive advantage to a wide array of citotypes composing the species-complex.
Thus, GHGs actually reported as a rare specimen at world level, must be unique clones
originated from very rare natural crosses. Domestication, by allowing the GHG introduction
into crop field, was important to increase population size above the minimum threshold size
necessary to avoid extinction due to drift.
Both the plasticity of morpho-physiological traits and the variable genome size make
difficult the precise classification of plants and bulbs of A. ampeloprasum species-complex.
Plant material from this species-complex, as already pointed out, can appear very variable for
size, organ colour, number of reproduction units (bulblets, bulbils and cloves), reproductive
strategies, ability to produce true seeds and ploidy level. Given the controversial
morphological classification, it follows that the molecular markers revealing naturally
occurring variation at diagnostic loci can be properly used for distinctness studies (Avise,
1994). The M13 marker (universal primer: 5’-GAGGGTGGCGGTTCT-3’) (Latouche et al.,
1997) revealed to be the most efficient in distinguishing A. ampeloprasum from A. sativum
gene-pool. This marker showed gene-pool specific fragments (Figure 7). Overall, considering
the major bands, two different patterns were detected within A. ampeloprasum group and only
one specific to A. sativum. Upon a diagnostic point of view, the M13 marker could be
proposed as a perfect tool to differentiate market cultivars of bulb producing garlic in order to
label them as A. sativum or A. ampeloprasum complex in a more efficient, fast, cheap and
achievable way.
ALLIUM AMPELOPRASUM COMPLEX ALLIUM SATIVUM
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 S1 S2 S3 S4 S5 S6 S7
Figure 7. M13 minisatellite diagnostic DNA marker distinguishing Ampeloprasum species-complex

from Sativum types. The 1000 bp band is specific to A. sativum gene-pool. Legend: A1 and A2 = wild
Leek from Stintino (Sardinia); A3 and A4 = wild Leek from Capocaccia (Sardinia); A5 and A6 = wild
Leek from Tremiti; A7, A8 and A9 = wild Leek from Basilicata (south Italy); A10 = Leek commercial
cultivar; A11, A12 and A13 = Great headed garlic landraces from Basilicata (south Italy); S1 = Hard
neck cultivar; S2, S3 and S4 = single bulb Chinese garlic; S5, S6 and S7 = soft neck garlic landrace
from Basilicata (south Italy).
CONCLUSION
The wild gene-pool of A. ampeloprasum species-complex is widely distributed at
geographic level and reflects a broad range of environmental adaptation. From our studies it
results that the wild Leek would be better defined as a species-complex rather than a group of
different separate species. Furthermore, Island garlic (A. commutatum), based on the
evaluation in a common environment either at morphological or molecular level, was
classified as A. ampeloprasum. The variable ploidy level associated with both high
polymorphism and a variable level of heterozygosity can determine striking morphological
variation and the establishment of different polyploid lineages at landscape level. Ecological
consequences of the establishment of different cytotypes can be linked to a differential pattern
of adaptation (Mable, 2003). Nonetheless, in A. ampeloprasum species-complex genome size
alone should not be interpreted as the unique factor explaining adaptation. The contribution of
gene interaction, heterozygosity and the genetic basis of sexuality on plant establishment and
adaptation in different ecological niches must be better assessed together with ploidy level.
For instance, the diploid wild Leek growing on the harsh and sandy dunes of northern
Sardinia is tolerant to high salt soil content and water deficits while the hexaploid GHG
prefers fertile, well drained and input rich soils.
Great headed garlic is a cultigen derived from the wild Leek gene-pool following rare
events of cross pollination between genetically distant parents. Once established, GHG has
been propagated by cloves. Heterozygosity is probably associated to the chromosomal genetic
distance of the parental ancestors. It is well recognized that within the genus Allium hybrid
vigour is present (McCollum, 1987), that also in A. sativum, the degree of heterozygosis for
isozymes is high (Maaβ & Klaas, 1995) and selfing determines inbreeding depression in Leek
cultivars (De Clercq et al., 2004). The clonal reproduction of the bulb-producing garlic (either
sativum or ampeloprasum) limits the efficient cultivar propagation as a crop. By using bulbils
as propagules it is possible to produce only plantlets in the first cultivation year whereas the
propagation by cloves significantly reduces the yield of the previous year. The best solution
would be the reproduction through seeds (easily treated for virus) following the selection of
male-sterile hybrids with high level of heterozygosity. The identification of cytoplasmic male
sterile genetic factors within the A. ampeloprasum species-complex along with the selection
of fertile parental clones will be the next frontier for garlic breeding (Bozzini and De Luca,
1991; Etoh 1985 and 1997; Hirschegger et al., 2006).
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Chapter 6
INHIBITORY EFFECTS OF DIALLYL SULFIDES

FROM GARLIC (ALLIUM SATIVUM L.) ON FAMILY
X DNA POLYMERASE ACTIVITY, AND HUMAN
CANCER CELL GROWTH
Yoshiyuki Mizushina1,2*, Masayuki Nishida1, Yuko Kumamoto-

Yonezawa1, Isoko Kuriyama1 and Hiromi Yoshida1,2
1
Laboratory of Food & Nutritional Sciences, Department of Nutritional Science,
Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
2
Cooperative Research Center of Life Sciences, Kobe-Gakuin University,
Nishi-ku, Kobe, Hyogo 651-2180, Japan
ABSTRACT
Diallyl sulfides are characteristic flavor components obtained from garlic. These
compounds are thought to be responsible for their epidemiologically proven anti-cancer
effect on garlic eaters. Diallyl sulfides are selectively inhibitors of mammalian family X
DNA polymerases (pols) such as pol β, pol λ and terminal deoxynucleotidyl transferase
(TdT), in vitro, and the order of their effect is as follows: Sample-A (the purified fraction
from garlic consisted of diallyl trisulfide, diallyl tetrasulfide and diallyl pentasulfide
[molecular ratio: 5.3 : 3 : 1]) > diallyl trisulfide > diallyl disulfide > diallyl monosulfide,
suggesting that the number of sulfur atoms in the compounds may play an important
structural role in enzyme inhibition. The inhibitory effect of Sample-A on rat pol β,
human pol λ and calf TdT is dose-dependent, and 50 % inhibition was observed at a
concentration of 9.7, 34.5 and 37.1 μM, respectively. The compound influences neither
the activities of other eukaryotic pols such as α, γ, δ, ε, η, ι and κ, prokaryotic pols, nor
*
Corresponding author: Yoshiyuki Mizushina, Ph.D.; Laboratory of Food & Nutritional Sciences, Department of
Nutritional Science, Kobe-Gakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe, Hyogo 651-2180,
Japan; Tel.: +81-78-974-1551 (ext. 3232); fax: +81 78 974 5689; E-mail address:
mizushin@nutr.kobegakuin.ac.jp (Y. Mizushina).
200 Yoshiyuki Mizushina, Masayuki Nishida, Yuko Kumamoto-Yonezawa et al.
the other DNA metabolic enzymes tested. The inhibitory effect of N-terminal truncated
pol λ lacking a BRCA1 C-terminus (BRCT) domain and a proline-rich region (residues
245–575, Del-2 pol λ) is stronger than that of intact pol λ (i.e., residues 1–575, Full-
length pol λ) and truncated pol λ lacking a BRCT domain (residues 133–575, Del-1 pol
λ). Sample-A-induced inhibition of pol X activities is competitive with respect to both
the DNA template-primer and the dNTP substrate. The suppression of human cancer cell
(promyelocytic leukemia cell line, HL-60) growth has the same tendency as the inhibition
of pol X family among compounds. Allicin and alliin, which are organosulfur
compounds containing garlic, hardly affect their activities. In conclusion, diallyl sulfides
are suggested to bind to the pol β-like region of family X pols, and have anti-cancer
activity.
Keywords: Diallyl sulfides; Garlic; DNA polymerase β (pol β); DNA polymerase λ (pol
λ); Terminal deoxynucleotidyl transferase (TdT); Family X of DNA polymerases (pol
X); Pol β-like region; Enzyme inhibitor; Cytotoxicity; Anti-cancer activity
Abbreviations: pol, DNA-directed DNA polymerase (E.C. 2.7.7.7); TdT, terminal

deoxynucleotidyl transferase (E.C. 2.7.7.31); dsDNA, double-stranded DNA; BRCT,
BRCA1 C-terminus; NLS, nuclear localization signal; HhH, helix-hairpin-helix; dTTP,
2’-deoxythymidine 5’-triphosphate; dNTP, 2’-deoxyribonucleoside 5’-triphosphate;
IC50, 50 % Inhibitory concentration; LD50, 50 % lethal dose; NP-40, Nonidet P-40.
1. INTRODUCTION
Garlic (Allium sativum L.), fresh garlic extract, garlic compounds or synthetically
prepared substitutes may be food items that significantly affect human health. Egyptian
records dating to about 1,550 BC mention garlic as a remedy for a variety of diseases [1], and
garlic has been demonstrated to possess antibiotic, fungicidic, anti-helmentic, anti-thrombotic
as well as anti-cancer and anti-carcinogenic properties [2, 3]. Epidemiologic investigations
have found that the risk of developing stomach, colon, and prostate cancers seems to be
inversely related to garlic consumption [4, 5]. Experimental animal and laboratory studies
provide convincing evidence that garlic and some of its organosulfur components are
effective inhibitors of a variety of cancers and cancer cells in culture, including breast, colon,
skin, uterine, esophagus, and lung [5, 6]; however, the specific components of garlic that
underline specific cellular and molecular events, which govern anti-cancer properties, have
not been clarified. Depending on its cultivation conditions, garlic can contain at least 33
different organosulfur compounds in addition to amino acids, vitamins, and micronutrients.
The allyl sulfur compounds formed by enzymatic activity when garlic is minced or crushed,
such as allicin, water-soluble S-allylmercaptocysteine (SAMC) and S-allylcysteine (SAC),
and oil-soluble diallyl disulfide and diallyl sulfide, probably account for the majority of these
anti-cancer effects [7, 8].
We established an assay to detect DNA polymerase (pol) inhibitors for the development of
anti-cancer functional foods and drugs, and have searched for natural materials, including foods,
for more than 10 years. Pol (DNA-dependent DNA polymerase, E.C.2.7.7.7) catalyzes the
addition of deoxyribonucleotides to the 3'-hydroxyl terminus of a primed double-stranded DNA
Inhibition of Selective DNA Polymerases by Diallyl Sulfides 201
molecule [9]. The human genome encodes 14 pols to conduct cellular DNA synthesis [10].
Eukaryotic cells reportedly contain three replicative types: pols α, δ, and ε, mitochondrial pol γ,
and at least twelve repair types: pols β, δ, ε, ζ, η, θ, ι, κ, λ, μ, σ and REV1 [11]. Eukaryotic
pols are classified based on amino acid sequence homology into four families, A, B, X and Y
[12, 13]. Family X pols (pol X) include pols β, λ, μ, and σ and terminal deoxynucleotidyl
transferase (TdT). The pols of this family have evolved as nucleotidyl transferases to catalyze
DNA polymerization in a distributive manner [14]. The assay method for pol activity was
described previously [15, 16]. The substrates of the pols were poly(dA)/oligo(dT)12-18 and 2'-
deoxythymidine 5'-triphosphate (dTTP) as the DNA template-primer and nucleotide substrate
(2'-deoxynucleoside 5'-triphosphate (dNTP)), respectively.
We screened the natural products of garlic extract, which inhibit eukaryotic pol activity,
and the newly-found compounds were found to selectively inhibit the activities of family X
pols, such as pols β, λ and TdT. The natural components were diallyl sulfides, which are
organosulfur compounds.
Here, we consider the structure-activity relationship in the inhibition by diallyl sulfides of
the pol X family and human cancer cell growth, and discuss the inhibitory action of the
compounds and its relation to the enzyme structure of the pols.
2. ISOLATION OF POL INHIBITORS FROM GARLIC

We screened food materials for eukaryotic pol inhibitors and found that ethanol extract
from garlic (Allium sativum L.) inhibited activity. Fresh garlic (1 kg) was homogenized and
extracted with 2,000 ml of hot water. The tissue cake was freeze-dried (100 g), and extracted
with 1,000 ml of ethanol. The extracted solution was subjected to Diaion HP-20 column
chromatography (5.0 X 40 cm), a hydrophobic type of chromatography. The passed solution
of ethanol was evaporated and dissolved with n-hexane, and the solution was applied to 1st
silica gel column chromatography (PSQ 60B, Fuji Silysia, 5.0 X 15 cm). After the column
was washed with n-hexane, the eluate with n-hexane : diethyl ether (v/v 48 : 2) was collected.
The active eluate was purified by 2nd silica gel column chromatography (PSQ 60B, Fuji
Silysia, 2.5 X 20 cm) using n-hexane : diethyl ether (v/v 49 : 1). The active fractions were
evaporated, and the purified material (Sample-A) was obtained (35 mg).
The chemical structure of the purified fraction from garlic (Sample-A) was characterized
by NMR, IR and MS. Sample-A was identified as three diallyl sulfides (1: diallyl trisulfide,
2: diallyl tetrasulfide, and 3: diallyl pentasulfide) as an inseparable mixture, and are the major
organosulfur compounds and flavor components of garlic [17–19], and the ratio of
compounds 1 to 3 was estimated to be 5.3 : 3 : 1 by the 1H NMR spectrum. The chemical
structures of compounds 1–3 are shown in Figure 1A.
3. EFFECTS OF SAMPLE-A AND ORGANOSULFUR COMPOUNDS ON THE

ACTIVITIES OF MAMMALIAN DNA POLYMERASES α, β AND λ
The commercially available diallyl sulfides, diallyl monosulfide (compound 4), diallyl
disulfide (compound 5) and diallyl trisulfide (compound 1), and other organosulfur
compounds containing garlic, such as allicin (compound 6) and alliin (compound 7) were
prepared (Figure 1B). Diallyl tetrasulfide (compound 2) and diallyl pentasulfide (compound
3), which consisted of Sample-A, could not be commercially obtained as reagents. The
inhibitory activity of calf pol α, rat pol β and human pol λ against each compound was
investigated. In mammalian pols, pol α and pols β and λ were used as representative
replicative pol and repair / recombination-related pols, respectively. One unit of each pol
activity was defined as the amount of enzyme that catalyzed the incorporation of 1 nmol of
2’-deoxyribonucleotide 5’-triphosphates (i.e., dTTP) into the synthetic template-primers (i.e.,
poly(dA)/oligo(dT)12-18, A/T = 2/1) in 60 min at 37 °C under the normal reaction conditions
for each enzyme [10, 11]. The molecular concentration of Sample-A was calculated by the
ratio of compounds 1 to 3 (5.3 : 3 : 1, respectively). As shown in Table 1, Sample-A had the
strongest inhibitory effect on pols β and λ of the tested compounds, diallyl trisulfide was the
second strongest, diallyl disulfide and allicin showed moderate inhibition, and diallyl
monosulfide and alliin had no influence. These results suggested that the effect of the diallyl
sulfides ranked as follows: diallyl pentasulfide > diallyl tetrasulfide > diallyl trisulfide >
diallyl disulfide > diallyl monosulfide. The inhibitory effects of allicin and alliin had the
same tendency as that of diallyl disulfide, suggesting that the number of sulfur atoms in the
compound may be important for the inhibition of pols β and λ. On the other hand, no
organosulfur compounds affected the activity of pol α (Table 1). When activated DNA (i.e.,
DNA digested by bovine deoxyribonuclease I) was used as the DNA template-primer instead
of poly(dA)/oligo(dT)12-18 (A/T = 2/1), the mode of inhibition by these compounds did not
change (data not shown).
4. EFFECTS OF SAMPLE-A AND ORGANOSULFUR COMPOUNDS ON

CULTURED HUMAN CANCER CELLS
Pols have recently emerged as important cellular targets for chemical intervention in the
development of anti-cancer agents. Organosulfur compounds could therefore be useful in
chemotherapy and we investigated the cytotoxic effect of Sample-A and the five purchased
compounds (i.e., compounds 1 and 4–7) against a human promyelocytic leukemia cell line,
HL-60.
Sample-A had the strongest growth inhibitory effect on human cancer cells
(promyelocytic leukemia cell line, HL-60) of the tested compounds, diallyl trisulfide, diallyl
disulfide were the second and third strongest, and diallyl monosulfide had no effect (Table 2).
The suppression of cell growth had the same tendency as the inhibition of mammalian pols β
and λ among the compounds, suggesting that the influence on cancer cells may be the activity
of pols, especially repair- and recombination-related pols. The cytotoxic dose was
approximately 1.4 to 5.1-fold higher than the enzyme inhibitory concentrations (LD50 and
IC50 values of Sample-A were 49.1 μM and 9.7–34.5 μM, respectively) (Tables 1 and 2).
We therefore concentrated our efforts on Sample-A (mixture of compounds 1–3), which
is the purified mixture of diallyl sulfides from garlic, and diallyl trisulfide (compound 1), a
commercially purchased fine chemical reagent, in the latter part of this study.
A
(1)
S S
S
(2)
S S
S S
(3)
S S S
S S
B
(4)
S
(5)
S
S
(6)
O
S
S
(7)
O NH2
S
COOH
Figure 1.Structures of organosulfur compounds from garlic. (A) The three diallyl sulfides comprising
Sample-A. Compound 1: diallyl trisulfide, compound 2: diallyl tetrasulfide, and compound 3: diallyl
pentasulfide. (B) Commercially purchased reagents of Sample-A (i.e., diallyl sulfides)-related
compounds. Compound 4: diallyl monosulfide, compound 5: diallyl disulfide, compound 6: allicin
(diallyl disulfide-oxide), and compound 7: L(+)-alliin (S-allyl-L-cysteine sulfoxide).
Table 1. IC50 values of organosulfur compounds from garlic on the activities of

mammalian DNA polymerases α, β and λ
Compound IC50 value (μM)

Calf pol α Rat pol β Human pol λ
Diallyl monosulfide >1000 >1000 >1000
Diallyl disulfide >1000 292 ± 16 397 ± 23
Diallyl trisulfide >1000 115 ± 7.5 146 ± 10
Sample-A >1000 9.7 ± 0.8 34.5 ± 2.1
Allicin >1000 478 ± 27 544 ± 31
Alliin >1000 >1000 >1000
Each compound was incubated with mammalian pol (0.05 units each). Pol activity was measured as
described in Mizushina et al. [15, 16]. Enzyme activity in the absence of the compound was taken
as 100 %. Data are shown as the mean ± SEM of three independent experiments.
A
Diallyl tris
Family
Sample-A
A Human DNA polymerase γ
Calf DNA polymerase α
B Human DNA polymerase δ
Human DNA polymerase ε
Rat DNA polymerase β
Human DNA polymerase λ (Full-length)
X Human DNA polymerase λ (Del-1)
Human DNA polymerase λ (Del-2)
Calf terminal deoxynucleotidyl transferase
Human DNA polymerase η
Y Human DNA polymerase ι
Human DNA polymerase κ
0 20 40 60 80
DNA polymerase activity (%)
Cherry salmon DNA polymerase δ
Fruit fly DNA polymerase α
Fruit fly DNA polymerase δ
Fruit fly DNA polymerase ε
Cauliflower DNA polymerase α
E. coli DNA polymerase I
T4 DNA polymerase
Taq DNA polymerase
0 20 40 60 80 100
DNA polymerase activity (%)
Calf primase of DNA polymerase α
HIV-1 reverse transcriptase
Human telomerase
T7 RNA polymerase
T4 Polynucleotide kinase
Bovine deoxyribonuclease I
0 20 40 60 80 100
Relative activity (%)
Figure 2. Effect of organosulfur compounds from garlic on the activities of various DNA polymerases
and other DNA metabolic enzymes. (A) Mammalian pols, (B) plant, fish and prokaryotic pols, and (C)
other DNA metabolic enzymes. Diallyl trisulfide (gray bars) and Sample-A (black bars) (200 M
each) were incubated with each enzyme (0.05 units). % of relative activity. Enzymatic activity was
measured as described previously [15, 16, 27]. Enzyme activity in the absence of the compounds was
taken as 100 %. Data are shown as the means ± SEM of four independent experiments.
5. EFFECTS OF SAMPLE-A ON THE ACTIVITIES OF DNA

POLYMERASES AND OTHER DNA METABOLIC ENZYMES
As shown in Figure 2A, 200 μM of Sample-A and diallyl trisulfide inhibited the activities
of rat pol β, human pol λ and calf TdT, and the inhibitory effect of pol β was the strongest of
these enzymes. These compounds had no influence on the activities of nuclear replicative
pols, such as calf pol α, human pol δ and human pol ε, human mitochondrial replicative pol γ,
or repair-related pols, such as human pols η, ι and κ. Given that the pol A family includes
pol γ, the pol B family includes pols α, δ and ε, the pol X family includes pol β, pol λ and
TdT, and the pol Y family includes pols η, ι and κ [12-14], diallyl sulfides did not inhibit the
activities of family A, B and Y pols.
In human pol λ, “Full-length” (residues 1–575) and N-terminal-deleted versions, “Del-1”
(133–575) and “Del-2” (245–575), were prepared. Full-length and fragments of pol λ were
dose-dependently inhibited by Sample-A. The inhibitory effect of Sample-A on truncated
versions of pol λ, such as Del-1 and Del-2, was slightly stronger than that on the full-length
enzyme, with 50 % inhibition observed at 34.5, 16.3 and 9.5 μM, respectively. The inhibition
of Del-2 pol λ was the strongest, and this was the same value as for pol β inhibition. The effects
seem to be relatively selective between pol β and Del-2 pol λ lacking nuclear localization signal
(NLS), the BRCA1 C-terminus (BRCT) domain and proline-rich region [20].
Sample-A and diallyl trisulfide had no inhibitory effect on fish (i.e., cherry salmon) pol δ,
insect (i.e., fruit fly) pols α, δ and ε, plant (i.e., cauliflower) pol α, or prokaryotic pols such as
the Klenow fragment of E. coli pol I, Taq pol and T4 pol (Figure 2B). The compounds also
did not inhibit the activities of other DNA-metabolic enzymes such as primase of calf pol α,
HIV-1 reverse transcriptase, human telomerase, T7 RNA polymerase, T4 polynucleotide

kinase or bovine deoxyribonuclease I (Figure 2C).
Table 2. LD50 values of organosulfur compounds from garlic on human cancer cell growth
Compound LD50 value (μM)

Diallyl monosulfide >1000
Diallyl disulfide 405 ± 27
Diallyl trisulfide 153 ± 11
Sample-A 49.1 ± 4.0
Allicin 587 ± 41
Alliin >1000
Each compound was added to the culture of a promyelocytic leukemia cell line HL-60, and incubated
for 24 hours. The rate of viability was determined by MTT assay [28]. Cell viability of cancer
cells in the absence of the compound was taken as 100 %. Data are shown as the mean ± SEM of
five independent experiments.
These results suggest that diallyl sulfides could selectively inhibit the activity of
eukaryotic family X pols, such as pols β and λ and TdT, and the inhibitory effect of Sample-
A was stronger than that of diallyl trisulfide.
6. EFFECT OF INTERACTION OF NUCLEIC ACID, PROTEIN AND

SAMPLE-A
To determine whether the inhibitor resulted in binding to DNA or enzymes, the
interaction of Sample-A with double-stranded DNA (dsDNA) was investigated based on the
thermal transition of dsDNA with or without Sample-A. The Tm of dsDNA with an excess
amount of Sample-A (200 μM) was measured using a spectrophotometer equipped with a
thermoelectric cell holder. In the concentration range used, no thermal transition of Tm was
observed, whereas ethidium bromide used as a positive control, a typical intercalating
compound, produced clear thermal transition (data not shown). These results indicate that the
diallyl sulfides of Sample-A do not intercalate to DNA as a template-primer, and the
compound may directly bind to the enzyme and inhibit its activity.
To determine the effects of a non-ionic detergent on the binding of Sample-A to X family
pols, such as pols β, λ and TdT, Nonidet P-40 (NP-40) was added to the reaction mixture at a
concentration of 0.05 or 0.1 %. In the absence of Sample-A, the activities of these enzymes
were not affected by the addition of NP-40, and we designated the activities in these cases as
100 %. The inhibitory effect of Sample-A at 20 μM was not reversed by the addition of 0.1
% NP-40 to the reaction mixture (data not shown). These results suggest that Sample-A can
bind to and interact with the hydrophilic region of the enzyme protein. We also tested
whether an excess amount of a substrate DNA analog, poly(rC) (50 μM), or a protein, BSA
(200 μg/ml), could prevent the inhibitory effects of Sample-A. If the compound binds to the
enzymes by non-specific adhesion, the addition of the nucleic acid and/or protein will be
expected to reduce inhibitory activity. Neither poly(rC) nor BSA influenced the inhibitory
effects on Sample-A, suggesting that the compound can occur selectively or bind to a specific
site on the enzymes and not to the nucleic acid.
Diallyl sulfide binding region
Pol β-like region Diallyl sulfide

inhibitory
NLS HhH HhH Pol X motif activity
1 36 133 245
Pol λ
(Full-length) BRCT Proline-rich +
15 132 244 575
133
Pol λ
(Del-1) ++
575
245
Pol λ
(Del-2) +++
575
Pol β +++
335
1 27 118 154
TdT +
26 117 153 509
Figure 3. Schematic representation of pols β, λ and TdT of family X pols. NLS (nuclear localization
signal), BRCT (BRCA1 C-terminus) domain, proline-rich region, HhH (helix-hairpin-helix) and pol X
motif are indicated. The pol β-like region includes two HhHs and a pol X motif. The inhibitory
activity of diallyl sulfide (i.e., Sample-A) against these enzymes is indicated below, “+++” is an IC50
value of < 10 μM, “++” is an IC50 value of 10 to 20 μM, and “+” is an IC50 value of 20 to 40 μM
7. MODE OF INHIBITION OF POL X FAMILY BY SAMPLE-A

Next, to elucidate the mechanism by which Sample-A inhibited family X pols, such as pols β,
λ and TdT, the extent of inhibition as a function of substrate concentration was studied. In kinetic
analysis of pols β and λ, poly(dA)/oligo(dT)12-18 and dTTP were used as the DNA template-
primer and dNTP substrate, respectively. Double reciprocal plots of the results showed that the
Sample-A-induced inhibition of rat pol β activity was competitive with respect to both the DNA
template-primer and the dNTP substrate (Table 3). In the case of the DNA template-primer, the
apparent maximum velocity (Vmax) was unchanged at 111 pmol/h, whereas 48.7 pmol/h of the
Michaelis constant (Km) increased in the presence of 9 μM of Sample-A. The Vmax for the
dNTP substrate was unchanged at 62.5 pmol/h, and the Km for the dNTP substrate increased from
3.05 to 16.7 μM in the presence of 9 μM of Sample-A. The inhibition constant (Ki) values,
obtained from Dixon plots, were found to be 2.61 μM and 3.68 μM for the DNA template-primer
and dNTP substrate, respectively. As shown in Table 3, the inhibition of human full-length-pol λ
activity was also competitive with respect to both the DNA template-primer (Vmax was
unchanged at 83.3 pmol/h) and the dNTP substrate (Vmax was unchanged at 52.6 pmol/h).
Table 3. Kinetic analysis of the inhibitory effects of Sample-A on the activities of

mammalian DNA polymerases β, λ (Full length, residues 1–575), and terminal
deoxynucleotidyl transferase as a function of the DNA template-primer dose and the
nucleotide substrate concentration
Enzyme DNA Sample-A Kma) Vmaxa) Kib) Inhibitory

modea)
Substrate (μM) (μM) (pmol / h) (μM)
Rat Template 0 6.74 111 2.61 Competitive
pol β -primerc) 3 12.5
6 21.1
9 48.7
Nucleotided) 0 3.05 62.5 3.68 Competitive
substrate 3 4.76
6 8.33
9 16.7
Human Template 0 2.38 83.3 8.00 Competitive
pol λ -primerc) 10 4.55
20 7.69
30 14.3
substrate 10 1.92
20 3.33
30 5.56
Calf Primere) 0 2.15 90.9 8.62 Competitive
TdT 10 4.88
20 9.09
30 18.2
substrate 10 2.00
20 2.23
30 5.26
a) Data obtained from the Lineweaver Burk plot.
b) Data obtained from the Dixon plot.
c) poly(dA)/oligo(dT)
12-18.
d) dTTP.
e) oligo(dT)
12-18.
In kinetic analysis of TdT, oligo(dT)12-18 and dTTP were used as the DNA primer and
dNTP substrate, respectively. Double reciprocal plots of the results showed that the Sample-A-
induced inhibition of calf TdT activity was competitive with respect to both the DNA primer
and the dNTP substrate (Table 3). In the case of the DNA primer, the Vmax was unchanged at
90.9 pmol/h, whereas 8.47-fold increases in the Km were observed in the presence of 30 μM of
Sample-A. The Vmax for the dNTP substrate was unchanged at 55.6 pmol/h, and the Km for
the dNTP substrate increased from 1.02 to 5.26 μM in the presence of 0 to 30 μM of Sample-A.
The inhibition constant (Ki) values, obtained from Dixon plots, were found to be 8.62 μM and
12.1 μM for the DNA primer and dNTP substrate, respectively.
The inhibition of pols β and λ by Sample-A had the same kinetic mode as that of TdT,
i.e., competitive with respect to both the DNA primer and the dNTP substrate, suggesting that
the compound can bind directly to both the DNA primer-binding site and the dNTP substrate-
binding site, and then may directly inhibit the DNA polymerization process. As the Ki values
for nucleic acid were smaller than those for the dNTP substrate, the affinity of the diallyl
sulfides of Sample-A might be greater for the enzyme-nucleic acid binary complex than for
the enzyme-nucleotide substrate complex.
8. DIALLYL SULFIDE BINDING REGION OF FAMILY X POLS

Pols β, λ and TdT belong to the pol X family, which includes pol μ, yeast pol IV,
mitochondrial pol β, nuclear pol β from protozoans and 20-kDa African swine fever virus pol X
[14]. Family X pols are composed of an NLS, a BRCT domain, a proline-rich region, and a pol β-
like region containing two helix-hairpin-helixes (HhHs) and a pol X motif (Figure 3). Human pol
λ shares 54, 47 and 30 % homology to human pols β and μ, and yeast pol IV, respectively.
Sample-A inhibited the activities of pol β, intact and truncated pol λ and TdT among the
eukaryotic pols and other DNA metabolic enzymes tested, with the strongest inhibitory effect on
both pol β and Del-2 pol λ (residues 245–575) which mainly consisted of a pol β-like region. The
compound did not intercalate to dsDNA, and did not non-specifically interact with proteins and
nucleic acids. The Sample-A-induced inhibition of pols β, λ and TdT was competitive with
respect to both the DNA template-primer and the dNTP substrate (Table 3), indicating that
Sample-A directly binds to the DNA template-primer-binding site and the dNTP substrate-binding
site of the enzymes. Since both of those sites are present in the pol β-like region among pols β, λ
and TdT [20], Sample-A may directly bind to the pol β-like region, which has the activity of DNA
polymerization. There was no reversibility in the inhibition of pols β, λ and TdT by NP-40,
indicating that Sample-A may bind to or interact with the hydrophilic region (maybe DNA primer-
binding and dNTP-binding regions) of the pol β-like core on enzyme molecules. Since the
inhibitory effect of pol β and Del-2 pol λ, consisting only of the pol β-like core region, was
approximately 3.6-fold stronger than that of Full-length pol λ, an N-terminal part such as the NLS,
BRCT domain or proline-rich region of pol λ may prevent binding to the pol β-like region by
Sample-A. The inhibitory activity of Sample-A against Del-2 pol λ was as strong as that for pol β.
This result suggests that the three-dimensional structure of pol β and Del-2 pol λ may be quite
similar. We are trying to co-crystallize diallyl sulfide and pol β / Del-2 pol λ for further study.
9. DISCUSSION
Garlic (Allium sativum L.), a widely used herbal vegetable, has been suggested as an
anti-cancer agent for several decades in epidemiological studies [21]. The most convincing
evidence comes from studies on digestive tract tumors (i.e., esophageal, gastric and colorectal
cancers) and prostate cancer. For example, a study on the association of gastric cancer and
consumption of Allium vegetables showed that persons with high intake of total Allium
vegetables (>24 kg/year) had 60 % reduced risk of this cancer compared to those with low
consumption (<11.5 kg/year) on 564 patients with stomach cancer and 1,131 normal controls
[22]. A more significant population-based, case control investigation on 238 histologically
confirmed prostate cancer patients and 471 normal male controls conducted in China reported
that men with high intake of garlic (>2.14 g/day) had a significantly lower risk of prostate
cancer than those with low or no garlic consumption [23]. These results suggested that garlic
may play a positive role in the prevention of certain human cancers.
An ethanol extract from garlic inhibited the activity of mammalian pols, and the active
fraction (Sample-A) was characterized and identified as a mixture of three diallyl sulfides
(i.e., diallyl trisulfide, diallyl tetrasulfide and diallyl pentasulfide) by chromatographic and
spectroscopic means. It is reported that diallyl sulfides suppress cancer cell growth, and have
effective action against cancer cell lines. Diallyl sulfides, especially diallyl trisulfide from
garlic, arrest the G2/M phase in the cell cycle [24], increase the sub-G1 DNA content [24],
induce caspase-3 activity and apoptosis through downregulation of Bcl-2 protein and
activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase
(JNK) pathways [25], and inhibit tublin polymerization [24, 26]. Here, we add a novel
activity (inhibition of pol X family) of diallyl sulfides to the anti-cancer cell growth effects.
10. CONCLUSION
Diallyl sulfides could selectively inhibit the activities of family X pols and human cancer
cells, a promyelocytic leukemia cell line (HL-60), growth, and in order of their effects, diallyl
sulfides might be ranked as follows: diallyl pentasulfide > diallyl tetrasulfide > diallyl
trisulfide > diallyl disulfide > diallyl monosulfide. These results suggest that the number of
sulfur atoms in the compounds may play an important structural role in inhibition.
Furthermore, the inhibition of repair / recombination-related pols, such as the pol X family,
not replicative pols, may be involved in the cytotoxicity of cancer cells.
In this review, diallyl sulfides, which are the major organosulfur compounds in garlic
(Allium sativum L.), are indicated as potent anti-cancer agents based on the inhibitory activity
of family X pols.
ACKNOWLEDGMENTS
We are grateful for the donations of calf pol α, rat pol β, human pol γ, human pols δ and
ε, human pols η and ι, human pol κ, and human pol λ by Dr. M. Takemura of Tokyo
University of Science (Tokyo, Japan), Dr. A. Matsukage of Japan Women's University
(Tokyo, Japan), Dr. M. Suzuki of Nagoya University (Nagoya, Japan), Dr. K. Sakaguchi of
Tokyo University of Science (Chiba, Japan), Dr. F. Hanaoka and Dr. C. Masutani of Osaka
University (Osaka, Japan), Dr. H. Ohmori of Kyoto University (Kyoto, Japan), and Dr. O.
Koiwai of Tokyo University of Science (Chiba, Japan), respectively.
This work was supported in part by a Grant-in-Aid for Kobe-Gakuin University Joint
Research (A), and the “Academic Frontier” Project for Private Universities: matching fund
subsidy from the Ministry of Education, Science, Sports, and Culture of Japan (MEXT),
2006–2010, (H. Y. and Y. M.). Y. M. acknowledges a Grant-in-Aid for Young Scientists (A)
(No. 19680031) from MEXT, Grants-in-Aid from the Nakashima Foundation (Japan),
Foundation of Oil & Fat Industry Kaikan (Japan), and The Salt Science Research Foundation,
No. 08S3 (Japan).
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& Fraumeni, J. F. Jr. (2002). Allium vegetables and risk of prostate cancer: a
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Chapter 7
GARLIC: A PROMISING ANTIDOTE

TO HEAVY METAL TOXICITY
Rajdeep Chowdhury and Keya Chaudhuri*

Molecular and Human Genetics Division, Indian Institute of Chemical Biology,
4 Raja S C Mullick Road, Kolkata-700032, India
ABSTRACT
Garlic for centuries has been well known for its medicinal attributes in addition to its
other virtues. Garlic in different forms has antioxidant properties. These properties are
shown to be due to the existence of compounds such as water soluble organosulfur
compounds, S-allylcysteine and lipid soluble compounds like diallyl sulfide. It shows
phenomenal ameliorating properties against heavy metal poisoning due to its possession
of chemicals containing organo-sulfur groups, volatile oils, enzymes, carbohydrates and
amino acids. With the threat of heavy metal poisoning increasing every day and lead,
mercury, cadmium, arsenic, and copper poisoning gradually attaining alarming
proportions, garlic was extensively exploited to treat the metal-induced toxicities. Recent
supportive evidences indicate that garlic contain compounds capable of detoxifying lead,
cadmium, methlymercury, phenylmercury and arsenic. The restorative property of garlic
was attributed to its antioxidant activity and/or chelating efficacy. The clastogenic effects
of the heavy metals were also pronouncedly reduced by dietary administration of garlic.
Fatal effects with respect to body metal burden, oxidative stress and mitochondrial injury
were effectively reduced by garlic. The curative effect of garlic was superior to those of
2,3-dimercapto-1-propanol (BAL) and D-penicillamine(PEN), 2,3-dimercaptosuccinic
acid (DMSA) and N-acetyl-DL-penicillamine (APEN), and the current remedies. In this
commentary, the research advances on the chemistry and pharmacology of garlic and the
potential and molecular mechanism of garlic mediated attenuation of heavy metal toxicity
are discussed.
*
Corresponding author: Dr. Keya Chaudhuri Scientist F; Molecular & Human Genetics Division Indian Institute of
Chemical Biology (A CSIR Organization) 4, Raja S C Mullick Road, Kolkata-700032, India, Tel: +91-33-
2473-3491, Fax: +91-33-2473-5197, Email: keya.chaudhuri@gmail.com, kchaudhuri@iicb.res.in
216 Rajdeep Chowdhury and Keya Chaudhuri
HEAVY METAL TOXICITY

There are 35 metals that concern us because of occupational or residential exposure; 23
of these are the heavy elements or "heavy metals": antimony, arsenic, bismuth, cadmium,
cerium, chromium, cobalt, copper, gallium, gold, iron, lead, manganese, mercury, nickel,
platinum, silver, tellurium, thallium, tin, uranium, vanadium, and zinc (Wasserman et al.
2008, Jarup 2003). Interestingly, small amounts of these elements are common in our
environment and diet and are actually necessary for good health, but large amounts of any of
them may cause acute or chronic toxicity (poisoning). Heavy metals become toxic when they
are not metabolized by the body, accumulate in the soft tissues and cause nutritional
deficiencies, hormonal imbalances, neurological disorders, and even autoimmune disorders
and other debilitating chronic conditions. Our bodies have evolved in a less polluted world
and are not prepared to handle this heavy load of environmental pollutants and would soon
become walking toxic time bombs if there does not exist defenses to disarm these many, and
virtually unavoidable, invaders (Ostrowski et al. 1999).
Therefore, it is important for us to inform ourselves about the heavy metals and to pursue
conventional and natural medical procedures or protective measures against excessive
exposure. If unrecognized or inappropriately treated, toxicity can result in significant illness
and reduced quality of life.
Synthetic Therapies to Heavy Metal Overload
Heavy metals combine preferentially with -SH radicals of proteins and enzymes and
inhibit their functions. Active research on the protective effects of chelating compounds
containing -SH radicals is being carried out for prevention and treatment of heavy metal
poisoning in the fields of biochemistry, pharmacology, medicine and public health. The
current approved clinical intervention method is to give chelating agents that form an
insoluble, stable complex with heavy metals and remove it from burdened tissues (Blanusa et
al. 2005). EDTA is the main chelating component that has been used for ages. EDTA,
removes toxic metals, improves circulaton, enhances the immune system and inhibits the
creation of "free radicals". Subsequently -SH compounds like, British Anti Lewisite (BAL)
and penicillamine were developed as a remedy to heavy metal toxicity (Snider et al. 1990,
Muckter et al. 1997). But due to adverse side effects their use was very much compromised
(Vilensky and Redman 2003, Wang et al. 2007). Moreover, although many doctors still
prescribe penicillamine for heavy metal poisoning, this use of the drug has not been approved
by the Food and Drug Administration. Later this was followed by the use of compounds with
less toxicity and side effects like, meso 2, 3-dimercaptosuccinic acid (DMSA) and
Dimercaptopropanesulfonate (DMPS) (Kreppel et al. 1990, Blanusa et al. 2005, Kalia and
Flora 2005, Flora et al. 2007). The chemical structures of the conventional antidotes are
represented in Figure 1. These compounds could preferentially chelate mercury, copper and
zinc but their application to patients were not devoid of side effects. This makes finding a
simple and perhaps natural approach to reducing the toxic effects of heavy metals even more
poignant. A summary of major chelating agents used to reduce heavy metal load and theirs
shortcomings are enlisted in Table 1.
Garlic: A Promising Antidote to Heavy Metal Toxicity 217
Figure 1. Chemical structures of synthetic chelating agents used against heavy metal toxicity.
Emergence of Garlic, the Wonder Condiment as an Antidote
Allium sativum, commonly known as garlic, a species of the onion family Alliaceae,
contains an abundant quantity of S-S and -SH compounds. Garlic and its organosulfur
components therefore have recently been utilized as a potential antidote to heavy metal
toxicity (Jacob and Schwandt 1992, Isensee et al. 1993, Torok et al. 1994). The organosulfur
compounds, with potential anti-heavy metal activity, extracted from garlic under various
conditions are represented in Figure 2. The chemopreventive effects of garlic constituents are
based on the following mechanisms: (i) chelation of heavy metals by organosulfur
components (ii) induction of phase II enzymes enhancing detoxification and increasing the
rate of excretion of the polar heavy-metal-chelate complex, (iii) increased synthesis of GSH,
an endogenous tripeptide thiol that directly protects cells from damage by free radicals, and
(iv) scavenging free radicals thus inhibiting heavy metal induced oxidative stress (Cha 1987).
The various modes of heavy metal detoxification by organosulfur components with respect to
selective heavy metals are discussed in details below. The chemical structures of some key
garlic organosulfur components reactive with heavy metals are diagrammatically represented
in Figure 3.
As noted earlier, there are 35 metals of concern, with 23 of them called the heavy metals.
Toxicity can result from any of these metals. This commentary will address the effect of
garlic on metals that are most likely encountered in our daily environment. Briefly covered
are the four metals that are included in the ATSDR's "Top 20 Hazardous Substances" list.
Copper will also be discussed even though it does not appear among the top 20 on the
ATSDR's list. The toxicity and symptom profiles of the above mentioned five metals are
represented in Table 2.
Figure 2. The major organosulfur compounds of garlic present in different preparations.
Figure 3. Chemical structures of garlic organosulfur components reactive with heavy metals
Table 1. Properties of synthetic chelating agents used against heavy metal toxicity
Sl. Chelating Agent Systemic IUPAC Name (Molecular Metals Chelated Route of Administration Side Effects
No. Formula)
1. British anti-Lewisite 2,3-Dimercaptopropanol As, Pb, Hg, Cd Intramuscular Tachycardia, anxiety, nausea, vomiting,
(BAL) / Dimercaprol (C3H8OS2) abdominal pain, dental and muscle pain,
burning sensation, conjunctivitis,
rhinorrhoea
2. Dimercaptosuccinic 2,3-bis-sulfanylbutanedioic acid As, Pb, Hg Oral Nausea, vomiting, diarrhea and anorexia
Acid (DMSA) (C4H6O4S2 )
3. Dimercaptopropane 2,3-Dimercapto-1-propanesulfonic Hg, Cu Intramuscular Agitation, chest constriction,
sulfonate (DMPS) acid Oral tachycardia, allergic reactions, skin
(C3H8O3S3) rashes and diarrhea
4. D-Penicillamine (2S)-2-amino-3-methyl-3-sulfanyl- As, Hg, Pb Oral Acute allergy-like reactions
butanoic acid (C5H11NO2S)
5. Ethylenediamintetra- 2-[2- Pb Intravenous Proximal nephron degeneration, fever,
acetic acid (EDTA) (Bis(carboxymethyl)amino)ethyl- nasal congestion and dermatitis
(carboxymethyl)amino]acetic acid
(C10H16N2O8)
Table 2. The Toxicity Profile of Arsenic, Lead, Cadmium, Mercury and Copper
No. Heavy Atomic Number Sources Toxicity Status* Chronic Toxicity Symptoms#
Metal
1. Arsenic 33 Drinking water, food or 1 Hyperkeratosis, mottled brown skin, cutis edema, limb
(As) breathing sawdust or burning paralysis and reduced deep tendon reflexes, nausea or
smoke from wood treated with vomiting, abdominal pain, diarrhea, fatigue, paresthesia,
arsenic paralysis, kidney failure, progressive blindness, dementia and
anorexia
Sl. Chelating Agent Systemic IUPAC Name Metals Chelated Route of Administration Side Effects
No. (Molecular Formula)
2. Lead (Pb) 82 Food, drinking water or 2 Gastrointestinal complaints, anemia,
paints nausea, peripheral neuropathy, depression,
insomnia, delusions, cognitive dysfunction,
impotence, depression of thyroid and
adrenal function, chronic renal failure and
gout
3. Mercury (Hg) 80 Mining ore deposits, 3 Excess salivation, gingivitis, tremors,

burning coal and waste, and stomach and kidney troubles, shyness,
from manufacturing plants, irritability, apathy and depression,
release of mercury from psychosis, mental deterioration, and
dental work anorexia
4. Cadmium (Cd) 48 Mining, industry, burning 7 Kidney dysfunction, increased proteinurea,

coal, household wastes, proximal renal tubular dysfunction,
cigarette smoke hypophosphatemia, muscle weaknesses,
coma, gout, hyperuricemia, hyperchloremia
and liver poisoning
5. Copper (Cu) 29 Air, drinking water, foods, 128 Irritation of the nose, mouth and eyes,
or skin contact with copper vomiting, diarrhea, stomach cramps,
or copper compounds nausea, and even death
*
According to ASTDR (Agency for Toxic Substances & Disease Registry) Priority List for hazardous substances (2007) available at
http://www.atsdr.cdc.gov/cercla/07list.html. This priority list is a prioritization of substances based on a combination of the frequency, toxicity, and potential
for human exposure. # References
The Protective Mechanism of Garlic against Cadmium (Cd)
Exposure to cadmium happens mostly in the workplace where cadmium products are
made. The general population is also exposed from breathing cigarette smoke or eating
cadmium contaminated foods. Cadmium mediates its lethal effects through ROS generation
or by direct toxic bio-accumulation causing damage to lungs, kidney and digestive tract. The
present therapy includes anti-oxidants like L-ascorbic acid, chelators like EDTA and
progesterone treatment; but according to Agency for Toxic Substances and Disease Registry
(ATSDR) there is no specific antidote for acute cadmium poisoning while prevention of
further exposure is the most important step in management of patients with symptoms
suggestive of chronic cadmium intoxication (ATSDR-Cadmium 1999).
Garlic compounds have added a new dimension to the use of antidotes to cadmium
poisoning. Garlic compounds due to presence of sulfhydryl groups, possess the capability to
reduce Cd accumulation in tissues and associated poisoning without showing any side effects
(Murugavel and Pari 2004). It has been reported that alkaline phosphatase, a kind of enzyme
in the cell membrane, activity drops on prolonged cadmium exposure. The inhibition of
alkaline phosphatase activity showed a trend of recovery on garlic administration. In
considering cadmium accumulation and the histopathological changes in liver, kidneys, bone
and testes which are target organs of cadmium poisoning, a significant decrease of cadmium
accumulation was apparent on garlic treatment. Tissue damages like edematous changes or
necrosis of liver recovered to show normal architecture on garlic supplementation.
Garlic compounds are characterized by their higher antioxidant activity and health
protective potential (Horie et al. 1992, Yamasaki and Lau 1997). Antioxidants can inhibit or
delay oxidative stress induced apoptosis. Garlic was found to have a protective effect on Cd
induced apoptosis, and this seems to be directed against the cytotoxicity associated with ROS
generation, mitochondrial injury and DNA damage induced by Cd. Hence, garlic could be a
potential therapeutic agent for Cd and other related heavy metal induced toxicity associated
with mitochondrial injury (Vasil'eva and Zasukhina 2002, Zhang et al. 2005, Massadeh et al.
2007, Murugavel and Pari 2007, Pari and Murugavel 2007, Suru 2008).
Garlic was also found to facilitate the discharge of cadmium mainly through the faeces as
its main route of excretion. An extremely small amount of cadmium was excreted via urine
upon garlic administration. Thus based on present research reports we can conclude that
garlic can essentially chelate and effectively eliminate cadmium thus attenuating its toxicity
(Vasil'eva and Zasukhina 2002, Zhang et al. 2005, Unyayar et al. 2006, Murugavel et al.
2007, Pari and Murugavel 2007, Suru 2008).
The Protective Mechanism of Garlic against Mercury (Hg)
Exposure to mercury occurs mainly from breathing contaminated air, ingesting

contaminated water and food, and having dental and medical treatments. Mercury, at high
levels, may damage the brain, kidneys, and can lead to thyroid diseases and anaemia
(ATSDR-Mercury 1999). EDTA, thiol chelators like BAL, DMSA, DMPS or selenium that
grabs or binds up heavy metal ions in the blood stream and anti-oxidants like N-acetyl-
Cysteine are presently the most preferred antidotes to mercury poisoning.
Figure 4. A schematic representation of the possible mechanism of garlic mediated arsenic

detoxification.
However many individual's sulfur stores are greatly depleted which impairs sulfur
containing chelating or complexing agents effectiveness as they are metabolized and utilized
as a source of sulfur. Here comes through the utility of sulfur containing natural substances,
like garlic that serves as an effective agent to supply organic sulfur for detoxification. Garlic
with its thiosulfinate components has significant protective effect over mercury poisoning.
Garlic was found to be more effective than BAL, DMPS or selenium (Cha 1987, el-Sabban
and Radwan 1997, Lee et al. 1999, Wu et al. 2001). Garlic supplementation with Hg protected
liver and kidney damage. Garlic's anti-toxic effects were related, in part, to its ability to
increase activity of several liver enzymes, such as glutathione-S-transferase and cytochrome
P450. Garlic plus Hg-treated test animals had fewer diploid and aneuploid cells and higher
proliferation index compared to Hg-treated group. Garlic is not reported to have any effect on
mercury accumulation. Hence the results were attributed to the antioxidant activity or the free
radical scavenging effect of garlic and its organosulfur constituents and/or their enhancing
effect on the antioxidant capacity of the body (El-Shenawy and Hassan 2008).
Some contradictory reports however does not nullify the protective effect of garlic -SH
and -S-S- radicals by forming sulfur compounds with mercury in the body and promoting
their excretion through bile juice in faeces. Thus the protective effect of garlic can be
attributed to the synergistic anti-oxidant effects of garlic components and the enhanced
elimination of mercury compounds from the body by garlic (Cha 1987, el-Sabban and
Radwan 1997, Lee et al. 1999, Wu et al. 2001).
The Protective Mechanism of Garlic against Lead (Pb)
Lead poisoning occurs when a person swallows or inhales lead in any form. The result
can be damage to the brain, nerves, and many other parts of the body. Acute lead poisoning,
which is relatively rare, occurs when a large amount of lead is taken into the body over a
short period of time. Chronic lead poisoning, which is a common problem in children, occurs
when small amounts of lead are taken in over a longer period. The body excretes lead very
slowly; consequently, it can accumulate in the body to toxic levels. Mostly lead is stored in
the bones and liver, where it reacts with cell membranes to alter their permeability or destroy
them completely (ATSDR-Lead 2007).
Figure 5. A schematic representation of the probable mechanism of garlic action against heavy metals.
Historically, one method for treating lead poisoning has been "chelation," which involves
using a compound that binds with the metal, making it easier to remove from the body.
However, the synthetic compounds like, EDTA, DMSA, DMPS that have been used in the
past, for this purpose, had severe side effects; synthetic compounds did also bind to essential
minerals in the body, and did more harm than good by redistributing the toxins, making them
a risk for use in children (Snider et al. 1990, Vilensky and Redman 2003, Wang et al. 2007).
Garlic was proven to be a safe, effective alternative for intoxicated people. Several
studies showed that it selectively binds to heavy metals, rather than essential minerals, and
can be administered orally. Adnan Massadeh at Jordan University of Science and
Technology, have demonstrated the potential of garlic as an antidote to lead toxicity
(Massadeh et al. 2007). The team analyzed the effect of different aqueous concentrations of
garlic at 12.5 to 100 mg per litre. They found that lead concentrations were reduced by up to
half in liver, kidney, heart, and spleen by almost two thirds in blood. Animals exposed to high
levels of lead have significantly lower levels of lead in their livers after also being fed garlic
cloves. Another group of researchers found that garlic significantly diminished symptoms of
lead poisoning in lead mine workers. Lead levels in different organs of exposed mice and the
effect of garlic on its distribution in these organs was observed by different research groups.
For the exposed Pb group of animals without treatment of garlic the distribution was in the
following order: Liver > kidney > spleen > heart > blood. Results showed a reduction in Pb
concentrations in all organs after treatment with garlic. The percentage removal of Pb
concentrations in liver ranged 29.1–44.6%; in kidney ranged 23.9–42.1%; in heart ranged
20.24–38.4%; in blood ranged 41.09– 66.62% at 100, 50, 25, and 12.5 mg/ml of garlic. The
weekly blood Pb profiles revealed a significant and constant dose-dependent decreasing trend
in the animals receiving garlic along with Pb. The highest dose (400mg per kg body weight)
decreased blood Pb concentration most efficiently, almost to the values recorded in healthy
rats (Senapati et al. 2001). Garlic further had the ability to reduce residues of Pb not only
from soft tissues (like; liver, kidneys and brain) but as well as from the bone sink in the body.
The efficiency of garlic in attenuating lead toxicity was accredited to the presence of the
sulfur-containing amino acids and compounds having free carboxyl (C=0) and amino (NH2)
groups in their structures. These biologically active compounds might have chelated Pb and
enhanced its excretion from the body resulting in reduced Pb accumulation in tissues and
blood. Further published results also show that garlic extracts increases the Pb concentration
in the urine as well as the faeces (Senapati et al. 2001) lending credence to this hypothesis.
Besides chelation, other components of garlic extracts (S-allyl cystein and S-allyl
mercaptocystein and some micronutrients) also prevent absorption of Pb from the gastro-
intestinal tract (Crowe and Morgan 1996a,b,c). It can be suggested, therefore, that the
ameliorative potential of garlic juice was perhaps due to combined effects both on metal
absorption and on excretion from the body. Therefore, it can be concluded that garlic can be
used for amelioration of chronic lead toxicity or reducing body lead residues.
The Protective Mechanism of Garlic against Copper (Cu)
Copper is a metal that occurs naturally in the environment, and also in plants and
animals. Low levels of copper are essential for maintaining good health. High levels can
cause harmful effects such as irritation of the nose, mouth and eyes, vomiting, diarrhea,
stomach cramps, nausea, and even death (ATSDR-Copper 2004).
Garlic compounds such as S-allylcysteine, N-acetyl-S-allylcysteine, S-
allylmercaptocysteine, and allin, are able to prevent Cu induced low density lipoprotein
(LDL) oxidation that can lead to atherosclerosis (Fields et al. 1992, Ide et al. 1997, Lau
2006). Dr Rahman and his colleagues of School of Biomolecular Sciences, Liverpool John
Moores University, Liverpool, UK found that garlic not only has the capability to chelate
copper ions but also negate copper mediated toxicity by its components’ (DAS and DADS)
anti-oxidant effects (Dillon et al. 2003). The protective effect of garlic on Cu induced LDL
oxidation may be explained, at least in part, for their ability to chelate Cu. Interestingly,
however the diethyl ether extract of garlic, which also inhibits Cu induced LDL oxidation, is
unable to chelate Cu indicating that its ability to prevent LDL oxidation is secondary to Cu
Chelation (Perez-Severiano et al. 2004). The precise mechanism by which garlic extracts
inhibit Cu induced lipoprotein oxidation remains to be studied.
The Protective Mechanism of Garlic against Arsenic (As)
Exposure to higher than average levels of arsenic occur mostly in the workplace, near
hazardous waste sites, or in areas with high natural levels. At high levels, inorganic arsenic
can cause death. Exposure to lower levels for a long time can cause a discoloration of the skin
and the appearance of small corns or warts (ATSDR-Arsenic 2007). Though there is no
effective treatment for arsenic toxicity the use of chelation therapy is imperative in all
symptomatic arsenic poisoning. However, the efficacy of chelation therapy in providing

either laboratory or clinical improvement in intoxicated patients is lacking.
Garlic may provide some relief for millions of Bangladeshis and Indians whose drinking
water is contaminated with arsenic (Chowdhury et al. 2008). Toxic effects of arsenic are
mediated primarily by triggering the production of reactive oxygen species (ROS), inhibiting
the activity of enzymes like superoxide dismutase and catalase, leading to alterations in cells’
intrinsic antioxidant defenses; and resulting in oxidative stress or disturbed antioxidant/pro-
oxidant ratio (Liu et al. 2001, Nordenson and Beckman 1991).
Garlic was found to have an overwhelming inhibitory activity over arsenic induced
toxicity. Garlic reduced arsenic induced cytotoxicity and ROS production in vitro. Moreover,
animals which were fed garlic extracts had 40 per cent less arsenic in their blood and liver,
and passed 45 per cent more arsenic in their urine. Furthermore, sulphur-containing
substances in garlic scavenged arsenic from tissues and blood reducing body arsenic burden
(Chowdhury et al. 2008).
The authors postulate that the diverse components of garlic, participate in a chemical
reaction with NaAsO2 (Chowdhury et al. 2008). The sulfur moieties of garlic can be
documented as active Lewis acids with electron affinity and high electro-negativity and
therefore have a tendency to form compounds with positively charged ions (Block 1985). In
contrast, arsenic is a highly electropositive metalloid exhibiting ionic states of +3 and +5 and
is an active Lewis base. It thus possesses an affinity for negative ions forming stable
compounds. In the reaction, major organosulphur compounds, which act as oxidants (like,
allicin) (Amagase et al. 2001, Lawson et al. 1992) are probably reduced while oxidizing
arsenic (AsIII) to its less toxic pentavalent form (AsV) (Barrett et al. 1989). Other major
thiosulfinate components (like, DADS, DAS, etc.) thereafter precipitate AsV formed and
remaining AsIII as arsenic sulphide (As2S3 and As2S5). A high amount of arsenic (both
trivalent and pentavalent) containing precipitate obtained upon mixing of AGE and NaAsO2
also impresses upon such a presumption. Therefore, the likely inference following the
restorative potential of AGE can be that, when applied simultaneously, arsenic and sulfur-
bearing components, which as elements is antithesis of each other, might react forming stable
and soluble salts (As2S3 and As2S5) that precipitate out, thus essentially chelating arsenic
(Chowdhury et al. 2008). A schematic representation of the possible mechanism of garlic
mediated arsenic detoxification is represented in Figure 4.
Comparison of the Protective Effect of Garlic to Current Remedies
According to the chelating mechanism of metal in the living body, BAL, penicillamine
and other conventional therapeutic drugs form metal chelates in the blood or outside cell and
excrete metals through urine. But they are considered inappropriate as remedies because the
toxicity of the metal is increased when they are reabsorbed by the kidney or redistributed in
the brain cell during the excretion process (Snider et al. 1990, Vilensky and Redman 2003,
Wang et al. 2007). 2,3-dimercaptosuccinic acid (DMSA), a derivative of BAL, discharges
metal through urine after forming metal-DMSA chelate compounds in the blood without
reabsorption by the kidney(Snider et al. 1990, Vilensky and Redman 2003, Wang et al. 2007).
Moreover, mild or severe side effects are associated with the conventional synthetic remedies
(Aposhian et al. 1997, Aposhian et al. 1984).
For over three decades, researchers have suspected that garlic may be helpful in cases of
heavy metal poisoning. Indeed, recent research advances suggests that garlic acts not only as
an effective antioxidant or chelator but also as a sulfur donor. A schematic representation of
the probable mechanism of garlic action against heavy metals is depicted in Figure 5. Garlic
therapy has several advantages over the market available heavy metal antidotes. Research
studies suggest that there is no reabsorption of garlic chelates in kidney like the instance of
BAL; furthermore garlic treated animals had a higher rate of heavy metal discharge when
compared to BAL, penicillamine or other conventional therapies. Thus the protective effect of
the natural supplement garlic against heavy metal poisoning is superior to that of BAL,
DMSA or penicillamine. Moreover, because of lipophobic properties of current remedial
drugs (Bosque et al. 1994, Ding and Liang 1991) they neither cross the cell membrane nor
capture intracellular heavy metals. In order to address this problem more effectively, garlic
constituting lipophilic compounds can be a potential remedy.
Lipophilic sulfur-bearing components of garlic (e.g., DADS, DATS) can permeate freely
through phospholipid membranes in human cells(Bosque et al. 1994, Ding and Liang 1991,
Miron et al. 1998, 2000); which is well justified by the considerable reduction of intracellular
heavy metal burden on garlic application in heavy metal exposed rats along with increased
elimination through urine. Therefore, it is a reasonable proposition that garlic components,
because of their ease of membrane penetration may serve as a prospective natural remedy
accounting for curative effects from chronic heavy metal poisoning. The identification of
such a regimen is essential, since garlic can be given as a dietary supplement to human
population exposed to environmental toxicants and would provide maximum protection
against toxic effects without being appreciably harmful itself.
Trends and Future Directions
The main threats to human health from heavy metals are associated with exposure to
lead, cadmium, mercury and arsenic. These metals have been extensively studied and their
effects on human health regularly reviewed by international bodies such as the WHO.
Although several adverse health effects of heavy metals have been known for a long time,
exposure to heavy metals continues, and is even increasing in some parts of the world. In
spite of reaching alarming proportions there is presently no specific remedy or drug that can
ameliorate heavy metal toxic symptoms effectively without any harmful side effects.
Moreover, we are still in the dark about the molecular mechanisms of action and also about
the intracellular and extracellular chelation status of clinically important chelators in relation
to mobilization of aged metal deposits and minimization of the mobilization of essential trace
elements during long-term chelation.
The emergence of use of garlic marks adds a new dimension to the use of antidotes to
heavy metal toxicity which ahs been demonstrated by in vitro and in vivo studies. However,
there is a need for well-designed clinical studies in order to better determine the therapeutic
value, dosage levels, duration of treatment, and other parameters. The benefits provided by
garlic must be viewed as part of the entire diet; since several dietary constituents can
influence the degree of protection. Future research should standardize the dosage, duration of
treatment of garlic and type, i.e., whether it should be taken fresh, cooked, or aged. The
identity of the specific compound or compounds from garlic responsible for heavy metal
ameliorating activity would be another promising area of research. The identification of such
a regimen is essential, since garlic can be given as a dietary supplement to human population
exposed to environmental toxicants and would provide maximum protection against toxic
effects without being appreciably harmful itself.
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Chapter 8
PHASE PROPERTIES AND LIPID COMPOSITION OF

MICROSOMAL MEMBRANES FROM STORAGE LEAF
OF GARLIC: MODIFICATIONS INDUCED BY
SPROUTING RADIOINHIBITION
Mónica B. Pérez and Clara A. Croci

Laboratorio de Radioisótopos, Dpto. de Química, Universidad Nacional del Sur.
Avda. Alem 1253, B8000FWB Bahia Blanca, Argentina
ABSTRACT
The aim of the present work was to evaluate the effect of gamma rays on the storage
leaf of garlic bulbs in terms of phase properties of microsomal membranes and their lipid
and fatty acid composition in order to correlate these features with sprouting inhibition
induced by gamma irradiation.
Garlic bulbs were irradiated 30 days after harvest with an average dose of 60 Gy of
Co-60 gamma rays. Rough and smooth microsomal membranes were isolated by
ultracentrifugation from tissues of irradiated and non-irradiated storage leaves. The
integrity of the microsomes was corroborated by transmission electron microscopy.
Wide-angle X-ray diffractograms of both fractions were recorded along 240 days of
storage using PW 1700 diffractometer. Lipids were separated by thin layer
chromatography. The fatty acid composition of major lipid fractions was studied by gas-
liquid chromatography.
The diffractograms featured peaks at Bragg spacing of 4.15 Å and 3.75 Å, revealing
the presence of a gel (crystalline) phase, while the characteristic peak of the liquid-
crystalline phase (4.6 Å) was not observed in both sorts of membranes. Irradiation was
found to bring about modifications in the intensity of 4.15 Å and 3.75 Å peaks from
smooth microsomal membranes, but not in the behavior along the period studied. Data
from the rough microsomal fraction were erratic and unreliable. Parallel to these changes,
radiation induced significant modifications in the level of smooth microsomal membrane
triacyglycerols in relation to phospholipids and their fatty acids.
These findings indicate that the storage leaf tissues of garlic are radiosensitive in
terms of physical and chemical properties of their smooth microsomal membranes. The
232 Mónica B. Pérez and Clara A. Croci
significance of the results in relation to proving the application of the radioinhibition

process in garlic bulbs is presented.
INTRODUCTION
Garlic (Allium sativum L.) is a monocotyledonous species currently included in the
Alliaceae family (Hanelt, 1990) covering a total of more than 600 Allium species. Garlic has
played an important dietary and medicinal role for centuries, and even today the medicinal
use of garlic is widespread and continues to grow. Among the ample array of therapeutic
properties ascribed to garlic, it is reported to be hypolipidemic, antiatherosclerotic,
hypoglycemic, anticoagulant, antihypertensive, antimicrobial, anticancer, hepatoprotective,
and to be an antidote for heavy metal poisoning and an immunomodulator (Hanley and
Fenwick, 1985; Augusti, 1990).
A mature garlic bulb is made up of bulblets, commonly known as cloves, which develop
from axillary buds on the younger foliage leaves (Rahim and Fordham, 1988). At harvest,
each clove is considered a dormant bud whose edible part is composed of a differentiated
fleshy leaf (the storage leaf) protecting the inner sprout. The latter consists of a sprout leaf
containing an apical meristem encircled by three to four functional leaf primordia (Shah and
Kothari, 1973). During post-harvest storage, the clove slowly emerges from dormancy via a
process regulated by hormonal factors, and the sprout begins to grow, normally entering the
sprouting stage after 60 days of storage (Argüello et al., 1991).
From the physiological point of view, sprouting is one of the main causes of quality loss
during the post-harvest storage period. With an increasing number of countries banning the
use of chemical products in the food industry, irradiation presents a viable alternative for
sprouting inhibition. The radioinhibition process been approved by the FAO/IAEA/WHO
Joint Committee on the Wholesomeness of Irradiated Foods (Diehl, 1990). Accepted as a safe
method free of adverse nutritional effects, sprouting inhibition by irradiation is now permitted
in around 40 countries, including Argentina (IAEA, 2006).
In order ensure that consumers are adequately informed and able to clearly identify
irradiated foods, the respective regulations and codes of practice in force in the food industry
need to be closely monitored. Though the methods used to detect foods subjected to high
doses of irradiation have improved considerably (Mc Murray et al., 1996), it is still not
possible to detect the application of low doses used to inhibit sprouting in fresh vegetable
products like bulbs, tubers and roots. Part of the difficulty in detecting low doses resides in
the high water content in the irradiated tissues.
In our laboratory, low doses of gamma irradiation have been successfully used to inhibit
sprouting and to extend the shelf life of c.v. Colorado garlic bulbs (Croci and Curzio, 1983).
In studies seeking to identify irradiated bulbs, we have shown that gamma rays affect several
chemical components of the sprout tissue of garlic, including growth regulators, total DNA,
RNA, proteins, soluble carbohydrates and lipids (Croci et al., 1990; Croci et al., 1994; Pérez
et al., 1998; Pérez et al., 2007). Earlier, we also reported histological and anatomical changes
in garlic sprouts associated with gamma irradiation of the bulbs (Orioli et al., 2004).
However, information on the effects of the sprouting radioinhibition process on the garlic
storage leaf is lacking.
Phase Properties and Lipid Composition of Microsomal Membranes from… 233
Biomembranes are known to be one of the most radiosensitive sites in cells (Kondoh et
al., 1998). Most effects of radiation on membranes are considered to result from the action of
the oxygen-derived free radicals generated by ionizing radiation (Kovács and Keresztes,
2002). One strategy for determining the modifications induced in membranes by free radicals
is to study the phase properties and lipid composition of microsomal membranes (Pauls and
Thompson, 1981; Senaratna et al., 1984; Voisine et al., 1991). The aim of the present work
was, therefore, to evaluate the effect of gamma rays on the storage leaf of garlic bulbs by
studying the phase properties and lipid and fatty acid composition of microsomal membranes
from storage leaf and correlating these features with the sprouting inhibition induced by
gamma irradiation.
MATERIAL AND METHODS

Sound garlic bulbs c.v. Colorado, harvested in the southwest of Buenos Aires province,
were used in these studies. Bulbs were treated 30 days after harvest with a dose of 60.0 Gy
using 60Co γ-rays (Croci et al., 1994) and stored in commercial warehouse conditions up to
and after treatment.
At selected intervals after irradiation, rough and smooth microsomal fractions from
storage leaf were isolated by ultracentrifugation as described by Thompson (1974).
Membrane pellets were stored in N2 until required for assays. The integrity of microsomes
was corroborated by transmission electron microscopy (TEM) (Robinson et al., 1987).
The phase properties of the microsomal membranes were analyzed by X-ray diffraction.
Concentrated membrane samples for wide-angle X-ray diffraction were prepared by mounting
pellets on a glass holder. Diffractograms were obtained at room temperature using a PW 1700
diffractometer (anode Cu, 45 kV, 30 mA, graphite monochromator with Philips APD 1710
analysis software). The scan used was 0.035 2θ.s-1.
Lipids were extracted from microsomes using mixtures of CHCl3–MeOH (2:1, v/v)
(Folch et al., 1957). Phospholipids (PL), triacyglycerols (TG) and free sterols (FS) were
separated by thin-layer chromatography (TLC) on plates of silica gel G, using hexane–Et2O–
HOAc (70:30:1, v/v/v). The PL were resolved into classes on commercial TLC plates using
CHCl3–MeOH–HOAc–H2O (50:37.5:3.5:2, v/v/v/v) (Holub and Skeaff, 1987).
PL were quantified by measuring lipid phosphorus (Rouser et al., 1970). TG and FS were
quantified by a standard colorimetric method used in clinical settings (Boehringer Mannheim
Gmbh, Mannheim, Germany).
Fatty acids were analyzed after differentiation in methyl esters by heating the lipid
samples overnight at 45ºC with 14% BF3 in MeOH (Morrison and Smith, 1964) under N2 in
screw cap tubes. Fatty acid analysis was performed using a Varian 3700 gas chromatograph
equipped with two (2 m × 2 mm) glass columns packed with 15% SP 2330 on 100/120
Chromosorb WAW (Supelco Inc., Bellefonte, PA) and two flame ionization detectors. The
column oven temperature was programmed from 155ºC to 230ºC at a rate of 5ºC min−1.
Injector and detector temperatures were 220 and 230ºC, respectively, with N2 (30 mL/min) as
the carrier gas. The fatty acids were identified with the aid of commercial standards.
Figure 1. Transmission electron micrograph of rough (A) and smooth (B) microsomal fraction.
Magnification: 50,000x
RESULTS
Electron Microscopy
The morphological differences between smooth and rough microsomal fractions can be
appreciated in the electron micrograph in Figure 1. The rough fraction exhibits granules
corresponding to ribosomes.
Phase Properties
The diffraction patterns of the membranes generally present characteristic peaks at Bragg
spaces of 4.15 Å, 3.75 Å and 4.6 Å (McKersie et al., 1976). The former two indicate the
presence of lipids in a crystalline or gel phase, with hexagonal or orthorhombic packing of the
lipid hydrocarbonate chains. The peak at 4.6 Å corresponds to membrane lipids in the fluid
state, known as the liquid-crystalline phase. The representative diffraction profile generated
from smooth and rough microsomal membranes from storage leaf is shown in Figure 2, where
peaks at Bragg spaces of 4.15 Å (2θ = 21.39°) and 3.75 Å (2θ = 23.71°) can be observed,
revealing the presence of gel-phase portions of lipid. There was no sign of the characteristic
peak of the liquid-crystalline phase (4.6 Å) in any of the microsomal fractions analyzed,
though this does not necessarily imply that the liquid-crystalline phase was not present in the
membranes. Certain limitations associated with the X-ray diffraction technique, particularly
when more than one phase is present in a given lipid system, make it difficult to detect the
less dominant phase (Cullis and de Kruijff, 1979).
Figure 3 shows the changes in intensity of the peaks corresponding to the smooth gel-
phase microsomal fraction of control and irradiated (60 Gy) samples. In control samples the
peak at 4.15 Å (Figure 3A) reached its maximum intensity at 30 days post harvest and then
gradually diminished to a minimum at 150 days. The intensity then increased again up to 210
days, thereafter remaining constant. The peak at 3.75 Å (Figure 3B) behaved in a similar
manner during storage, though its intensity was consistently lower than that corresponding to
the peak at 4.15 Å.
In the irradiated samples both peaks showed lower intensity than their respective controls
at 60 days post harvest. However, after 90 days the intensity was higher in the irradiated
samples (Figure 3).
In the case of the rough microsomal fraction, the variations in intensity at peaks 4.15 Å
and 3.75 Å were erratic and unreliable (data not shown).
Lipids and Fatty Acids
- Phospholipids (PL)
The analysis of PL in smooth microsomes (Table 1) showed phosphatidylcholine (PC) to

be the major component, making up over 50% of the total, followed by
phosphatidylethanolamine (PE). Phosphatidylinositol (PI) and phosphatidylserine (PS) were
minor components, and lysophosphatylcholine, phosphatidylglycerol, diphosphatidylglycerol
and phosphatidic acid were not detected at all. The relative amount of each of these PL did
not vary significantly during storage, nor was it affected by irradiation treatment.
Figure 2. A typical X-ray diffractogram of microsomal membranes from storage leaf of garlic.
350 350
A B
300
⇓ 300
Intensity [cps]
Intensity [cps]
250 250
200 200 ⇓
150 150
100 100
50 50
0 0
30 60 90 120 150 180 210 240 30 60 90 120 150 180 210 240
Days post-harvest Days post-harvest
Control Irradiated
Figure 3. Changes in the intensity of phase gel peaks in smooth microsomal membranes. A) 4.15 Å
peak, B) 3.75 Å peak. The arrow points to the day on which the samples were irradiated.
Table 1. Content of major phospholipids in smooth microsomes from storage leaf of garlic
Control Irradiated
Days post-harvest 30 150 240 30 150 240
% %
PC 54.3 ± 3.7 53.8 ± 2.7 52.7 ± 3.1 56.0 ± 2.7 56.0 ± 2.2 56.2 ± 3.1
PE 26.9 ± 1.5 27.2 ± 1.9 28.1 ± 2.0 25.8 ± 1.4 27.2 ± 1.0 24.7 ± 1.2
PI 15.3 ± 0.7 16.0 ± 1.2 15.4 ± 1.3 15.9 ± 0.5 13.4 ± 0.7 15.6 ± 0.5
PS 3.5 ± 0.2 3.0 ± 0.3 3.8 ± 0.4 3.3 ± 0.2 3.5 ± 0.2 3.6 ± 0.2
Results are expressed as mean values of three determinations ± S.D.
- Neutral lipids (NL)
The NL of smooth microsomes from storage leaf were analyzed in terms of TG and FS,
expressed as a fraction of PL content (Figure 4). In the control samples a significant increase
was observed in the relative amount of TG at 150 days post harvest and a significant decrease
at 240 days. The results for smooth microsomes from irradiated storage leaves showed the
relative amount of TG to be initially higher than in the control and then to decrease linearly as
storage continued (Figure 4A).
Relative FS content in smooth microsomes (Figure 4B) was significantly lower than that
of TG and did not vary significantly during storage or as a result of irradiation.
- Fatty acids (FA)
The FA composition of total lipids (TL), PL and TG from smooth microsomes is given in
Table 2. The major FA in the samples analyzed were found to be palmitic (16:0), palmitoleic
(16:1), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3). Significant changes
were observed during storage in the percentage of some FA in the TG of controls, with a
particularly marked increase of 78% in 18:3. These changes, which were also observed in
irradiated samples, did not modify the degree of unsaturation (unsaturated:saturated ratio) of
the FA in TL, which was not affected by storage duration or by irradiation (Figure 5). Similar
results were obtained with the FA of PL (Table 2, Figure 5). However, the behavior of the FA
of TG was different: in the control samples the most significant change was observed in 18:0,
which tripled its initial value at 150 days post harvest (Table 2). The percentage changes in
these and other FA of control sample TG produced significant variations in the
unsaturated:saturated ratio during storage (Figure 5). In the irradiated samples the changes
were of a lower magnitude, giving rise to significant differences in the degree of unsaturation
between the treated and non-treated samples.
Long-chain fatty acids (more than 20 carbons) were also detected in smooth microsomal
FA, constituting a significant proportion in the TG fraction (Figure 6).
DISCUSSION
Based on analysis of the phase properties and lipid composition of smooth microsomal
membranes, the results of the present paper clearly show the tissue of garlic storage leaves to be
radiosensitive; this could not be corroborated in the case of rough microsomes. The rough
microsomal fraction is made up mostly of rough endoplasmic reticulum in which the amount of
non-membrane proteins (ribosomes) is variable (Alberts et al., 1994). The erratic results obtained
in the phase behavior of the lipids in this fraction could reflect variations in its composition.
A change in the behavior of smooth microsomal membranes was observed during post-
harvest storage: at 30 days post harvest and very shortly after irradiation treatment, a dose of 60
Gy induced a reduction in gel-phase lipids (Figure 3), indicating that radiation caused
membrane fluidity to increase, as reported by Edwards et al. (1984) for other biological
membranes treated with ionizing radiation. At the same time, the irradiated membranes showed
a higher relative content of TG than the respective control (Figure 4). These results could be
interpreted as a physiological response of garlic storage leaf tissue to the stress caused by the
application of irradiation to inhibit the sprouting of the bulbs (Kikuchi et al., 1998).
Variations in the amount of polyunsaturated FA and lipid peroxidation have been observed
in artificial membranes subjected to low doses of irradiation (Edwards et al., 1984; Stark, 1991).
However, our results show that at 30 days post harvest the relative proportions of FA in TL and
PL of smooth microsomal membranes were not modified by the 60 Gy dose of irradiation
(Table 2). This is in agreement with the findings of Voisine et al. (1991) for microsomal
membranes of cauliflower irradiated with even higher doses (2 and 4 kGy). These authors
attributed their results to the hydrophilic nature of the radio-induced free radicals, which is not
conducive to interaction with the lipids at the hydrophobic core of the membrane.
Changes in the phase properties of smooth microsomal membranes from storage leaf
were observed over the long-term as a result of irradiation treatment. Control samples showed
a gradual decrease in the proportion of gel-phase lipids up to 150 days post harvest (Figure 3)
concomitant with the growth of the sprout observed in earlier research (Pérez et al., 2007;
Croci et al., 1994). This decrease reflects an increase in membrane fluidity to enable the
transfer of nutrients from the storage leaf to the sprout, essential for the sprout’s development
(Argüello, 1991). In smooth microsomes from storage leaf irradiated with 60 Gy, the increase
in gel-phase lipids as of 90 days post harvest provoked a reduction in fluidity, resulting in
limited nutrient transfer. This explains the meager growth of the sprout, as recently reported
for garlic irradiated with 60 Gy (Pérez et al., 2007).
A number of post-irradiation changes were also observed over the long term in lipid
composition. A significant increase in the TG:PL ratio (Figure 4) and significant changes in
the percentage of FA in TG (Table 2) were observed in the controls at 150 days of storage,
coinciding with the sprouting of the garlic clove. This increase could be attributed to an
increase in TG content, since the higher energy requirements of the sprout during foliar
neoformation calls for the rapid mobilization of reserve lipids such as TG (Li & Ross, 1990).
This explanation is consistent with the fact that such increase does not occur in smooth
microsomes of irradiated storage leaves, where sprouting is inhibited by the irradiation
treatment.
In microsomal membranes of vegetables subjected to conditions that induce the
formation of free radicals such as dehydration or ozone treatment, the formation of gel-phase
lipids has been associated with chemical changes in the lipids like alterations (decreases or
increases) in sterol content relative to PL or the reduction in the degree of unsaturation of FA
in PL (Senaratna et al., 1984; Pauls and Thompson, 1981). This is not the case with garlic
storage leaf treated with ionizing radiation, since the phase changes observed were not
accompanied by changes in the ratio of FS:PL ratio or in the ratio of unsaurated:saturated
(Figures 4 and 5). The loss of fluidity of the irradiated membranes after 90 days of storage
can therefore be attributed to radio-induced alterations in the membrane proteins.
0.8 0.16
A B
0.6 0.12
µg FS / µg PL
µg TG / µg PL
0.4 0.08
0.2 0.04
0 0
0 30 60 90 120 150 180 210 240 270 0 30 60 90 120 150 180 210 240 270
Days post-harvest Days post-harvest
Control Irradiated
Figure 4. Content of triacyglycerols (A) and free sterols (B) in smooth microsomes. Results are
expressed as mean values of three determinations ± S.D.
5
TL
unsaturat : saturat
4
0
30 150 240
Days post-harvest
5
PL
unsaturat : saturat
0
30 150 240
Days post-harvest
10
TG
unsaturat : saturat
0
30 150 240
Days post-harvest
Control Irradiated
Figure 5. Unsaturation degree of fatty acids (unsaturated:saturated ratio) of total lipids (TL),
phospholipids (PL) and triacyglycerols (TG) of smooth microsomes. Results are expressed as mean
values of three determinations ± S.D.
IRRADIATED
Figure 6. Fatty acid profiles of triacyglycerols in smooth microsomes. Different letters indicate the
presence of long chain fatty acids. The asterisks denote an increase in detector sensitivity.
Table 2. Changes in fatty acid composition of total lipids (TL), phospholipids (PL) and
triacyglycerols (TG) from smooth microsomes
Control Irradiated
Days post-harvest 30 150 240 30 150 240

% %
TL
16:0 22.4 ± 0.2 23.2 ± 3.5 21.3 ± 1.8 25.1 ± 1.9 21.3 ± 2.5 22.2 ± 0.7
16:1 1.1 ± 0.2 2.1 ± 0.4 1.2 ± 0.4 1.2 ± 0.4 1.5 ± 0.3 1.1 ± 0.4
18:0 1.2 ± 0.4 1.3 ± 0.3 0.7 ± 0.2 1.1 ± 0.1 0.9 ± 0.2 0.8 ± 0.1
18:1 12.8 ± 2.4 10.8 ± 0.7 5.6 ± 0.1 10.7 ± 0.3 8.6 ± 0.6 5.5 ± 0.3
18:2 58.0 ± 2.7 56.8 ± 4.1 63.0 ± 1.3 57.3 ± 1.9 60.5 ± 2.2 61.9 ± 0.4
18:3 4.6 ± 0.5 5.9 ± 0.8 8.2 ± 0.1 4.6 ± 0.1 7.3 ± 0.8 8.5 ± 0.3
PL
16:0 30.2 ± 1.4 25.7 ± 2.1 25.6 ± 2.5 29.6 ± 1.9 29.2 ± 1.8 27.7 ± 1.8
16:1 2.0 ± 0.3 2.7 ± 0.3 0.8 ± 0.3 1.1 ± 0.4 1.2 ± 0.4 1.1 ± 0.2
18:0 1.6 ± 0.3 5.5 ± 0.9 1.7 ± 0.4 1.8 ± 0.4 1.5 ± 0.3 1.0 ± 0.2
18:1 11.9 ± 1.0 20.1 ± 1.5 6.2 ± 1.0 11.4 ± 8.5 ± 0.4 5.8 ± 0.3
18:2 51.8 ± 2.1 42.5 ± 2.0 59.0 ± 3.1 52.8 ±1.9 54.5 ± 2.3 57.8 ± 2.9
18:3 2.6 ± 0.3 3.5 ± 0.5 6.7 ± 0.7 3.3 ± 0.3 5.2 ± 0.4 6.6 ± 0.4
TG
16:0 12.0 ± 1.7 21.1 ± 2.4 9.4 ± 0.9 10.4 ± 0.9 15.8 ± 0.8 8.9 ± 0.6
16:1 4.4 ± 0.4 4.4 ± 0.5 2.7 ± 0.4 4.0 ± 0.4 5.6 ± 0.4 2.6 ± 0.2
18:0 2.7 ± 0.3 8.6 ± 0.8 1.4 ± 0.3 2.6 ± 0.3 4.4 ± 0.4 2.8 ± 0.4
18:1 11.1 ± 0.8 13.7 ± 1.1 6.8 ± 0.7 12.2 ± 1.0 12.7 ± 1.0 7.3 ± 0.5
18:2 60.8 ± 3.3 41.5 ± 2.7 64.7 ± 4.8 60.4 ± 3.1 50.0 ± 2.4 62.8 ± 2.5
18:3 7.3 ± 0.7 5.3 ± 0.8 13.5 ± 1.6 7.6 ± 0.9 8.0 ± 0.5 12.6 ± 0.8
Results are expressed as mean values of three determinations ± S.D.
CONCLUSIONS
This study provides the first comprehensive characterization of changes in phase
properties and lipid composition of microsomal membranes over the entire storage life of
garlic bulbs after treatment with gamma irradiation. The analysis of phase properties and lipid
composition of smooth microsomal membranes clearly shows that garlic storage leaf tissue is
radiosensitive. It may, therefore, be concluded that membrane lipids of the storage leaf are
involved in the radio-induced sprouting inhibition of garlic bulbs, though on the basis of the
present results it is not possible to determine whether the observed changes in the lipids were
a cause or an effect of this phenomenon.
The findings of the present chapter have important practical implications for
distinguishing irradiated from non-irradiated garlic bulbs on the basis of the characteristics of
smooth microsomes from the storage leaf. In particular, the phase properties of membrane
lipids and the TG:PL ratio serve as indicators of the application of the radioinhibition process
in garlic bulbs.
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Chapter 9
HEALTH EFFECTS OF GARLIC AND

PROSTATE CANCER
J. Arunakaran*
Department of Endocrinology, ALM PG Institute of Basic Medical Sciences.
University of Madras, Taramani, Chennai,Tamil Nadu 600 113, India
Garlic (Allium sativum) has been used for thousands of years for medicinal purposes.
Garlic ranks highly among foods that help to prevent disease, largely due to its high content
of organosulfur compounds and antioxidant activity. Aged garlic extract (AGE) has been
found to prevent atherosclerosis and protect against cardiovascular disease, increase
circulation and immunity. AGE has been shown to prevent various kinds of cancer,
neurodegenerative disease and has antiaging effects in improving memory, endurance and
learning [1].
Garlic traditionally has been used as a natural antibiotic thought to protect against
infection, to lower blood pressure and to treat atherosclerosis, asthma, arthritis, cancer and
circulatory problems.
It grows in the form of bulb. Sanskrit records show its medicinal use about 5000 year
ago. It has been used for at least 3000 years in Chinese medicine. The Egyptians,
Babylonians, Greeks and Romans used garlic for healing purposes. In 1859, Pasteur noted
garlic’s antibacterial activity and it was used as an antiseptic to prevent gangrene during
World War I and World War II.
Historically garlic has been used around the World to treat hypertension, infections and
snake bites etc. Currently, garlic is used for reducing cholesterol levels and cardiovascular
risk as well as for its antineoplastic and antimicrobial properties.
Garlic has a high concentration of sulfur containing compounds. The thiosulfinates,
including allicin, appear to be the active substances in garlic. Allicin is formed when alliin a
sulphur containing amino acid comes into contact with the enzyme alliinase when raw garlic
is chopped, crushed or chewed. Dried garlic preparations containing alliin and alliinase must
be enteric coated to be effective because stomach acid inhibits alliinase. Because alliinase
*
Corresponding author: E.mail: j_arunakaran@hotmail.com
246 J. Arunakaran
also is deactivated by heat, cooked garlic is less powerful medicinally. The antimicrobial,
hypolipidemic, antioxidant and anti-thrombotic effects that have been attributed to garlic are
thought to be related to allicin and other breakdown products. The antineoplastic effects may
be related to the sulfur compounds or to other unknown components [1].
Allicin, S- Allyl cysteine (SAC) and Ajoene are the three active compounds found in
varying amounts in flesh garlic. Allicin a chemical formed when garlic is crushed appears to
have antibacterial properties. SAC has been shown to be effective against the initiation of
tumors in animals. Ajoene appears to be an anti-blood clotting agent.
Garlic supplementation contains allicin, an odorless precursor of the garlic smell in the
active compounds of allicin and ajoene. The enzyme alliinase is needed to convert alliin to
allicin and ajoene. Fresh garlic in large quantities can lower cholesterol levels. Garlic thins
the blood; it may lower blood pressure [2].
Garlic has been used throughout the world to treat coughs, tooth ache, earache, dandruff,
hypertension, hysteria, diarrhoea, dysentery, diphtheria, vaginitis and many other conditions.
Garlic contains a complex mixture of oil and water soluble organosulfur compounds. Oil
soluble compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl
trisulfide (DATS) are effective as antiproliferative against various tumors. The water soluble
compounds S-allyl cysteine, S-ethyl cysteine and S-propyl cysteine have little effect.
High levels of garlic may prevent development of cancer by stimulating immune system
and hindering growth of cancer cells. Scientific and medical research continues world wide
on the health properties of various forms of garlic and garlic supplements [3]. Health benefits
ascribed to garlic and garlic supplements include:
Ä Antibiotic/antifungal effects
Ä Antioxidant effects, protecting cells from free radical damage and cancer
Ä Antiseptic properties useful in fighting infections and dysentery causing amoebas
Ä Cholesterol reduction, lowering LDL and increasing HDL
Ä Natural anticoagulant properties, preventing blood clots, strokes and antihypertensive
effects, reducing blood pressure.
Garlic and their active principles such as allicin, Diallyl sulfide, Diallyl disulfide and
diallyl trisulfide give diverse biological activities including anti-tumorigenesis
antiatherosclerosis, blood sugar modulation and antibiotics [4-7]. DADS, increases the
activities of different phase II enzyme such as glutathions S transferase, UDP glucuronyl
transferase and epoxide hydrolase [8]. DADS has been shown to inhibit the growth of human
tumor cells from colon, lung and breast origins [9-11].
The antiproliferative effect of DADS was attributed to suppression of the rate of cell
division and induction of apoptosis in human tumor cells. Recent literature indicates that
pleiotropic biological effects of DADS may involve the modulation of gene expression. With
regard to the dry metabolizing enzymes for example DADS enhances the expression of CYP
1A1, 2B1 and 3A1 genes at the mRNA and protein levels [12]. Moreover, a study using
cDNA array technology in HCT-15 human colon tumor cells provides further evidence that
DADS up or down regulates the expression of a wide range of genes [13] suggesting that
DADS may modulate the expression of specific genes through a modification of histone
acetylation. Acetylation of histones is a key process in activating transcription [14] and has
Health Effects of Garlic and Prostate Cancer 247
been reported to induce selectively the expression of specific genes such as the p21
Waf1/Cip1 cyclin dependent kinase inhibitor to effect cell cycle arrest [15]
PROSTATE CANCER
Prostate cancer is the most frequently diagnosed cancer in men. Incident rates of prostate
cancer have changed substantially over the last 20 years. Prostate cancer is a leading cause of
cancer death in men. Early prostate cancer usually has no symptoms. With more advanced
disease, individuals may experience weak or interrupted urine flow, in ability to urinate or
difficulty starting or stopping the urine flow, the need to urinate frequently especially at night,
blood in urine or pain or burning with urination.
Advanced prostate cancer commonly spreads to the bones, which can cause pain in the
hips, spine, ribs or other areas. Many of these symptoms are not specific to prostate cancers.
Risk factors for prostate cancer are age, ethnicity and family history of the disease. 64% of all
prostate cancer cases are diagnosed in men aged 65 and above. In the World, African
American men and Jamaican men of African desert have the highest prostate cancer incident
rates. The disease is common in North American and North Western Europe. Strong familial
pre disposition may be responsible for 5-10% of prostate cancers. International studies
suggest that a diet high in saturated fat may also be a risk factor. The risk of dying from
prostate cancer may increase with obesity.
Early detection is the best for therapy. PSA (prostate specific antigen) blood test and the
digital rectal examination should be offered to men at average risk beginning at age 50.
Individuals at high risk of developing prostate cancer showed begin screening at age 45.
Treatment options vary depending on age, stage of the cancer, and other medical conditions
and should be discussed with the individuals physician surgery, external beam radiation, or
radioactive seed implants (brachytherapy) may be used to treat early stage disease, hormonal
therapy may be added in some cases.
Hormonal therapy, chemotherapy, radiation, or a combination of these treatments is used
to treat more advanced disease. Hormonal treatment may control prostate cancer for long
periods by shrinking the size or limiting the growth of the cancer, thus helping to relieve pain
and other symptoms.
ENDOCRINE AGENTS
Finasteride: Androgens play an important role in prostate cancer. Androgen suppression
therapy can cause regression of established prostate cancer and enuchs do not develop
prostate cancer [16,17]
Dihydrotestosterone, 5-alpha reduced metabolite of testosterone, is the major effector
hormone on prostatic epithelium. Finasteride a compound that blocks the type II isoenzyme of
5 alpha reductase, induces prostatic volume reduction and has been used to treat symptomatic
benign prostatic hyperplasia (BPH) for years [18]
Side effects noted were marginal decreases in libido (6%) and erectile function among
men assigned finasteride. A small proportion of men also developed breast associated
248 J. Arunakaran
symptoms such as gynecomastia. No cases male breast cancers were noted in the literature.
There was a benefit with respect to urinary complaints such as prostatitis development,
urinary retention, and lower urinary tract symptoms (LUTS) among men who received
finasteride [19]
Dutasteride is a newer 5-alpha reductase inhibitor (5 AR1). Like finasteriode, it inhibits
the type II receptor, but unlike finasteride for 4 months caused a 40% reduction in tumor
volume compared with control. Dutasteride is currently being assessed in a large phase 3 trial
called REDUCE (reduction by dutasteride of prostate cancer events) [20]
Toremifene: the third studied agent for cancer prevention. Tormifene is a selective
estrogen receptor modifier (SERM) approved for the treatment of breast cancer (60 mg/day
dosage). Pre clinical work using this compared in a transgenic mouse model demonstrated an
ability to prevent prostate cancer and high grade prostatic intraepithelial neoplasia (HGPIN)
[21].
Total and free testosterone levels were increased when men consumed higher fat diet.
Postprandial androgen levels do change in response to diet. Alpha linolenic acid which is
primarily derived from plant sources such as flax, corn and perilla oil demonstrated a positive
association on prostate cancer.
Reactive oxygen species can increase oncogene expression. Prostatic tissue markers of
oxidative stress are associated with prostate cancer. Dietary fat has been shown to increase
known markers of oxidative stress. Other factors that can increase oxidative stress in prostate
tissue include smoking, sedentary life style, aging and androgens [24]. In addition, in
laboratory models of high fat diet induced prostate cancer, vitamin E and other antioxidants
including lycopene and selenium can eliminate or blunt this effect.
Chemoprevention of MNU and testosterone induced prostate carcinogenesis by vitamin
D in adult male albino rats were studied in our laboratory [25]. Recent studies have
demonstrated in vivo and in vitro effects of vitamin E on apoptosis, cell cycle arrest, and
proliferation arrest in prostate cancer tumor model systems [26].
Similar to the vitamin E history, the results of this phase 3 trial led to additional studies
suggesting that selenium possessed significant anticancer properties in vivo and in vitro
including induction of apoptosis, DNA repair augmentation and cell cycle arrest in human
prostate cancer cell lines [27].
Green tea contains catechins, including epigallocatechin (EGCG), the best studied
constituent. In vitro studies suggest that this agent can induce cell cycle arrest, inhibit insulin
like growth factor-I synthesis and induce apoptosis in a variety of prostate cancer cell lines
[28].
Soy is rich in isoflavones which induce cell cycle arrest and inhibit proliferation in a
variety of prostate cancer tumor model systems [13]. Soy is also rich in alpha subtype of the
estrogen receptor when has been linked to prostate carcinogenesis [29].
Curcumin has been shown to induce apoptosis in both androgen dependent and androgen
independent prostate cancer cells; this was accomplished by downregulating apoptosis
suppressor proteins and other crucial proteins such as androgen receptor [30]. Curcumin was
also shown to induce a marked reduction of tumor volume and MMP-2 and MMP-9 activity
in the tumor bearing site in xenograft model. The metastatic nodules in vivo were
significantly fewer in the curcumin treated group than untreated group [31].
Resveratrol (3,4-5'-trihydroxy-trans-stilbene) a phytoalexin found in grape skin has been
shown to induce apoptosis in LNCaP and DU 145 prostate cancer cell lines through different
PKC mediated and MAPK-dependent pathways [32]. Resveratrol caused modulation of a

number of important genes in the androgen pathway, including PSA and AR in LNCaP cells.
Resveratrol caused modulation of a number of important genes in the androgen pathway
including PSA and AR in LNCaP cells. Resveratrol decreased cyclin B1 and cdk1 expression
and cyclin B/cdk1 kinase activity were decreased in both cell lines [33].
Lycopene which is a natural pigment synthesized by plants is a carotenoid, an acyclic
isomer of β carotene which is a highly unsaturated, straight chain hydrocarbon containing 11
conjugated and two non-conjugated double bonds. Several studies have indicated that
lycopene is an effective antioxidant and free radical scavenger. Lycopene because of its high
number of conjugated double bonds, exhibits higher singlet oxygen quenching ability
compared to β carotene or α-tocopherol [34].
Lycopene more potently inhibited the growth of the androgen independent (DU145) and
PC-3 cells than androgen dependent LNCaP cells. Our previous study demonstrated that
lycopene has been shown to decrease the levels of IGF-1 and increase the levels of IGFBP-3
in prostate cancer PC-3 cells [35]. In recent phase II study evaluating 46 patients with
androgen independent prostate cancer, lycopene did not appear effective [36].
So far various laboratory studies both in cell culture system and in animal models
convincingly argue for a definitive role of selected dietary natural occurring products for
prevention and treatment of prostate cancer. Many of these agents are antioxidants in nature.
A flavonoid, quercetin commonly present in many vegetables like onion, tea, apple has
been known to possess remarkable spectrum of biochemical and pharmacological activities
suggesting that it induces apoptosis by activity of caspases and suppressing Bcl-2 in various
tumor cell lines including human leukemia, human colon cells etc. Our earlier studies proved
that quercetin induced growth inhibition and cell death in prostatic carcinoma cells which are
associated with increase in p21 and hypophosphorylated retinoblastoma protein expression
[37]. Insulin like growth factor binding protein (IGFBP-3) is the positive regulator of
quercetin induced apoptosis in prostate cancer cells. The role of IGFBP-3 as an effector of
p53 independent apoptotic pathways has particular relevance in the treatment of prostate
cancer cell [38].
Quercetin induces p53 independent apoptosis in human prostate cancer cells by
modulating Bcl-2 related proteins a possible mediation by IGFBP-3 [39]. Quercetin
downregulates matrix metalloproteinase’s 2 and 9 proteins expression which are enzymes
known to involve in tumor invasion and metastases in prostate cancer cells [40]. Quercetin
has chemopreventive activity in prostate carcinogenesis of albino rats in vivo [41].
Our earlier studies proved that DADS induced apoptosis in PC-3 cells in a dose
dependent manner. DADS increased the number of both early and late apoptotic cells.
Histone acetylation was also observed in DADS treated prostate cancer cells. Thus DADS
induces apoptosis by influencing histone acetylation in prostate cancer cells [42].
The chemopreventive effect of diallyl disulfide on prostate carcinogenesis in Sprague
Dawley rats has been proved [43]. Growth suppressing effect of garlic compound diallyl
disulfide on prostate cancer cell line in vitro was studied through induction of apoptosis in our
laboratory [44]. DADS induces cell cycle arrest in prostate cancer cells [45]. Effect of DADS
on expression of apoptosis associated proteins in androgen independent human prostate
cancer cells have also been studied in our laboratory [46].
AKT or protein kinase B is a serine/threonine kinase that plays an important role in
intracellular signaling cascades. A variety of neoplasia shows perturbations in the
250 J. Arunakaran
biochemical pathways affected by Akt. Prostate cancer specifically shows biochemically

abnormalities related to Akt that may be of importance in sustaining tumor growth by
preventing apoptosis and promoting proliferation and angiogenesis [47]. Akt over expression
has been demonstrated in prostate cancer [48]. Three central regulation of the cell cycle
affected by Akt are cyclin D, p21 and p27 which oppose cell cycle progression.
Complete activation of the catalytic activity of Akt requires phosphorylation of a
threonine residue at 473. It is possible that Akt shows partial activation with phosphorylation
at the threonine 308 position [49]. Hence, phosphorylation at ser 473 is the key for the
complete activation of Akt. In our present study, the 100 µM DADS treatment caused a
significant decrease in the expression of ser 473 phosphorylated Akt. The total Akt was also
decreased significantly indicating the inhibition of Akt at 50 and 100µM concentration of
DADS in LNCaP cells [50].
These Akt is a cell survival protein that acts to promote survival through inhibition of
proapoptotic factors and activation of antiapoptotic factors. DADS reduced the expression of
Akt and p-Akt and it is evidenced that Akt inhibition is a good target for prostate cancer
therapy.
To conclude, Garlic (Allium Sativum) belongs to Lily family, called the bulb which is
made up of separate cloves. Native to central Asia by the 6th century BC, garlic was known in
both India and China. Currently China, South Korea, India, Spain and United States are
among the top commercial producers of garlic.
Garlic is an excellent source of manganese. It is also a very good source of vitamin B6
and vitamin C. Garlic is a good source of protein and thiamin (B1) as well as minerals such as
phosphorous, selenium, calcium, potassium, iron and copper.
Garlic appears to have no effect on drug metabolism but patients taking anticoagulants
should be cautious. It seems prudent to stop taking high doses of garlic seven to 10 days
before surgery because garlic can prolong bleeding time. The adult dosage raw garlic is
4g/per day; dried powder means 300 mg 2 to 3 times per day; Aged garlic 7.2g/per day.
Thus, garlic has several health benefits. Fresh garlic is the best. It is a good diet. Garlic
has proven itself as a popular food and nutrition item. It is gaining scientific credibility as a
significant contributor to good health.
FUTURE DIRECTION
It is extremely important to consider seriously the issue of bioavailability and metabolism
of the dietary garlic since the biological properties of agents depend on their presence at the
time of damage. This review presents an extensive analysis of the key findings from studies
on the effect of dietary antioxidants such as tea polyphenols, curcumin, resveratrol, lycopene,
pomegranate and organosulfur compounds such as diallyl disulfide (garlic extract) on various
cancer particular reference to prostate cancer.
This research is leading to the identification of novel cancer drug targets. The approach
can be explored in clinical studies in the future.
ACKNOWLEDGMENTS
The financial assistance provided by University of Madras as URF to one of the scholars
Mr. M. R. Vijayababu, as UWPFEP fellowship to Mr. A. Arunkumar and Mr. K.
Senthilkumar. CSIR (Council for Scientific and Industrial Research, India) as SRF, to Mr. P.
Kanagaraj is gratefully acknowledged and Ms. S. Banudevi, Ms. Gunadharani DN are the
research scholars working on prostate cancer.
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Chapter 10
THE INFLUENCE OF GARLIC (ALLIUM SATIVUM L.,

ALLIACEAE) EXTRACTS ON THE
PHARMACODYNAMIC EFFECTS OF DRUGS
Biljana Bozin1, Neda Mimica-Dukic2 and Isidora Samojlik3

1
Department of Pharmacy, Faculty of Medicine, Hajduk Veljkova 3
2
Department of Chemistry, Faculty of Sciences, Trg D. Obradovica 3
3
Department of Pharmacology and Toxicology, Faculty of Medicine, Hajduk Veljkova 3
University of Novi Sad, 21 000 Novi Sad, Republic of Serbia
ABSTRACT
Garlic (Allium sativum L., Alliaceae) has played one of the most important dietary
and medicinal roles in many cultures for centuries. It has been used as a spice and
condiment and, due to its potential benefits, in preventive and curative treatments.
Epidemiological, clinical and preclinical studies have shown the close relationship
between dietary habits, including garlic intake, and the occurrence of diseases. A wide
array of therapeutic effects such as hypolipidaemic, antiatherosclerotic, hypoglycaemic,
anticoagulant, antihypertensive, antimicrobial and hepatoprotective action have been
reported. However, the most common complication of garlic use is prolonged bleeding
and side effects including halitosis, nausea, hypotension, headache and bloating. Drug-
herb interactions with hypoglycaemics, cardiovascular medications and monoamine
oxidize inhibitors also have been reported. With respect to this, the aim of the study was
to examine the influence of polar extracts derived from garlic on pentobarbitone- and
thiopentone-induced sleeping time, midazolam-induced impairment in motor
coordination and analgesic effect of codeine in mice. The examined extracts were
obtained from the immature garlic plant, ground and air-dried garlic bulbs (prepared as an
aged garlic extract) and fresh garlic bulbs, with determined content of total phenolics,
flavonoid glycosides and aglycones. The animals were divided into four groups (three
extracts’ groups and a control) according to peroral pretreatment regime with a particular
extract during five consecutive days. Tested drugs were administered 2 h after the
application of particular extract on the fifth day of experimental procedure, and the
measurements were done according to survey protocol. Pretreatment with all extracts
produced changes in both pentobarbitone- and thiopentone-induced sleeping time. Also,
256 Biljana Bozin, Neda Mimica-Dukic and Isidora Samojlik
examined extracts exhibited notable changes in induction time after the application of
both hypnotic drugs. All three extracts reduced motor impairment caused by midazolam
and decreased the analgesic effect of codeine. Because garlic alone does not induce sleep,
motor discoordination or analgesic effect, and because its use produces changes in tested
drug effects, the interaction between drugs and phytopreparations containing garlic
should be additionally examined/confirmed in humans.
INTRODUCTION
Garlic (Allium sativum L., Alliaceae) has been used for centuries in folk medicine.
Sanskrit records show its medicinal use about 5.000 years ago, and it has been used for at
least 3.000 years in Chinese traditional medicine. Drawings and carvings picturing garlic
were uncovered in Egyptian tombs dating from 3.700 BC. Furthermore, its use as a remedy
for heart disease, tumors and headaches is documented in the Codex Ebers dating from 1.550
years ago. The Babylonians, Indians, Greeks and Romans used this plant for healing purposes
as well. Garlic is also mentioned in the Bible, and virtually every early civilization known had
been using this plant for a variety of diseases and to preserve good health [1, 2]. Even today,
the medical use of garlic is widespread and growing. Thus, garlic is investigated extensively
for health benefits, resulitng in more than 1.000 publications over the last decade.
Epidemiological, preclinical and clinical studies have shown the close relation between
dietary habits including garlic intake and the occurrence of diseases. It is considered to be one
of the best disease-preventive foods based on its potential and varied effects [3]. A wide array
of therapeutic effects such as hypolipidaemic, antiatherosclerotic, hypoglycaemic,
anticoagulant, antihypertensive, antimicrobial, antidote (for heavy metal poisoning) and
hepatoprotective action have been reported [1, 2, 4]. Furthermore, it prevents cold and flu
symptoms through immune enhancement and exhibits anticancer and chemopreventive
activities [3-6]. The antioxidant properties of garlic and different garlic preparations are also
well documented [4, 5, 7-10].
Generally, these health-related functions are mostly attributed to the rich content of γ-
glutamyl cysteine in fresh garlic and many sulfur-containing compounds, which produce a
characteristic flavour formed during storage and processing [3-5]. However, additional
compounds in garlic present a wide range of primary and secondary nonsulfur biomolecules
such as prostaglandins; fructan; pectin; adenosine; vitamins B1, B2, B6, C and E; biotin,;
nicotinic acid; fatty acids; glycolipids; phospholipids; essential amino acids [12]; lectins [13];
steroidal glycosides [14]; essential oils [15, 16]; flavonoids [10, 17] and anthocyanins [18].
The majority of these components work synergistically to provide various health benefits [3,
5, 10].
Although many biological and pharmacological activities have been confirmed, in
official medicine garlic is used only for the reduction both of cholesterol levels and risk of
cardiovascular disease, as well as for its antineoplastic and antimicrobial properties [1, 11].
Aside from an unpleasant odor of the skin, side effects recorded after garlic intake include
halitosis, nausea, hypotension, headache and bloating [19]. The most common complication
of garlic use is increased bleeding secondary to its antiplatelet and antithrombotic effects [20-
22]. Bleeding after transurethral resection of the prostate has been documented as well [23].
The Influence of Garlic (Allium sativum L., Alliaceae) Extracts… 257
In rare instances, gastrointestinal symptoms, changes in gut flora or allergic reactions are
noted [24]. Chemical burns as a result of topical application of crushed garlic cloves also have
been reported [25]. Drug-herb interactions that may occur with garlic include those with
hypoglycemics, anticoagulants, cardiovascular medications, protease and monoamine oxidize
inhibitors and anticancer drugs [19, 26-29]. Although other possible garlic-drug interactions
are not known, further investigations are necessary. Furthermore, there are no data regarding
the pharmacodynamic effects of immature garlic plants widely used in salads and as spices in
most of the countries of the Balkan peninsula.
With respect to this, this study investigated potential influence of different garlic extracts
on some CNS responses (hypnotic, anaesthetic, antinociceptive and analgesic actions) caused
by penthobarbitone, thiopentone, midazolam and codeine.
MATERIALS AND METHODS

Plant Material and Extract Preparation
The whole plants of cultivated immature garlic-bulbs with steam and leaves (A. sativum
L., Alliaceae) were collected in April 2006, while the bulbs of mature garlic plants were
collected in July 2006, both in Padej, Province of Vojvodina, Republic of Serbia. Voucher
specimens of collected plants (immature garlic No G-12/06 and mature garlic No G-19/06)
were confirmed and deposited at the Herbarium of the Laboratory of Pharmacognosy,
Department of Pharmacy, Faculty of Medicine (HLPhM), The University of Novi Sad,
Serbia.
Ground and air-dried immature garlic plants (I), garlic bulbs (II; prepared as Aged Garlic
Extract) and fresh garlic bulbs (III) were extracted, as described previously [10]. The
quantities of dry extracts were determined gravimetrically and for extracts I, II and III they
were 3.32%, 9.17% and 5.99%, respectively.
Phytochemical Analyses
Evaluation of total phenolic content
The amount of total phenolic compounds in the investigated extracts was determined
spectrophotometrically with the Folin-Ciocalteu (FC) reagent, as described before [10]. The
concentration of total phenolics was expressed as mg of gallic acid equivalents (GAE) per g
of dried extract (DE), by using a standard curve of gallic acid. All measurements were carried
out in five replicates.
Estimation of total flavonoid glycosides and aglycones content
The amount of total flavonoids in the extracts was determined spectrophotometrically

[10], using a method based on the formation of flavonoid-aluminium complex with the
maximum of absorbtivity at 430 nm. Hydrolysis of flavonoid glycosides in the investigated
extracts was performed with 2 M HCl for 30–40 min. at 100oC. After filtration, flavonoids
were extracted with ethyl acetate [30]. The flavonoid glycosides content was expressed as μg
of rutin equivalents per g of DE while the total flavonoid aglycones content was expressed as
μg of quercetin equivalents per g of DE both by using standard graph. All measurements were
carried out in five repetitions.
Animals
Adult male NMRI-Albino mice, body weight 25–30 g, were obtained from Louis Pasteur
Institute (Novi Sad, Republic of Serbia). The animals were housed in individual cages (8
mice/cage) one week prior to experimental procedures. They were allowed free access to
pelleted food and water. Temperature of the environment was 24±3ºC and the laboratory was
maintained on a 12 h day/night cycle. All experiments were carried out during the light phase.
The experimental procedures were approved by Ethical Committee for Animal Use in
Experiments, The University of Novi Sad.
Study Design
All animals were divided into four groups according to pretreatment regime: three groups
were pretreated with daily dose (DD) of tested garlic extracts (I, II and III) during 5
consecutive days before the examination and the control group (C) which was pretreated with
saline for five days. The last dose of particular extract was applied 2 h before testing. Human
DD of garlic recommended by German Commission E [24] (4 g of fresh clove or equivalent
dose of garlic preparations) was adapted for the experimentation on mice by using the
following conversion equation for Human Equivalent Dose (HDE):
HDE (mg/kg or mL/kg) = animal dose (mg/kg or mL/kg) x (animal weight [kg]/human weight [kg])0.33
Calculated DD of garlic extract I, II and III were 0.11 mL/kg, 0.44 mL/kg and 0.20
mL/kg, respectively. Each animal received appropriate DD of garlic extracts in a form of
solution in amount of 10 mL/kg by per os gavage. All experiments were carried out on eight
animals per experimental group.
Pharmacological Assays
Potentiation of pentobarbitone and thiopenthone induced sleeping time
Pentobarbitone sodium (40 mg/kg) or thiopentone sodium (25 mg/kg) (Sigma-Aldrich,

Germany) were administered intraperitoneally (i.p.). The interval between the drug
application and the moment at which the animals have lost righting reflex was registered as
sleeping induction time (IT), while the period of loss and regaining of righting reflex was
considered as sleeping time (ST). The duration of sleeping was considered completed when
mice did not accept the dorsal position for three consecutive trials [31].
Motor coordination test
The impairment of motor coordination caused by midazolam (AD Galenika,

Belgrade, Republic of Serbia) was assayed by the fixed speed rotarod method [31]. The
animals were placed on iron rod (3.5 cm in diameter) covered with sandpaper to prevent
slippage, elevated for about 60 cm above bedding, turning at the speed of 12 rpm and
divided into two compartments by plexiglass disks. In this test, coordination insufficiency
produced by midazolam was indicated by the inability of the animals to maintain their
equilibrium for at least 180 s on the rotating rod. Before the midazolam application (5
mg/kg i.p.) the mice were given one training trial during which they were allowed to
remain on the rotarod for up to 180 s. After the injection of the drug, latencies before fall
prior to reaching maximum 180 s were recorded in regular time intervals of 1, 5, 10, 15
and 20 min.
Hot plate test
The temperature of metal plate enclosed by plexiglas walls was maintained at

55±0.5ºC. The end point, i.e., response, was the time measured in seconds at which the
animal licked or flinched one of the hind paws or jumped off the plate [31]. To prevent
tissue damage, a cut-off time was used as double value of latencies measured before drug
application (i.e., double pre-drug responses). Response latencies were determined three
times before the application of tested compound in order to determine a pre-drug
response of each mouse, and 10, 30, 50, 70, 90, 110 and 130 min. following drug
administration. After responding or reaching cut-off time, mice were removed from the
plate. The tested compound - codeine hydrochloride (Fampharm, Kruševac, Republic of
Serbia) - was applied intraperitoneally a dose of 25 mg/kg.
To show drug effect as a value between the baseline value and the value at cut-off we
expressed hot-plate response latencies as a percentage of the maximum possible effect
(%MPE) = (post-drug time – pre-drug time)/(cut-off time – pre-drug time)x100.
Statistical Analysis
The values are presented as means. Statistical significance in both hot plate and
motor coordination tests was analyzed using one-way analysis of variance (ANOVA)
followed by Dunnett’s multiple comparison test. Data groups gained in pentobarbital and
thiopenthone induced sleeping time tests were compared using two tailed t-test for
independent samples. Statistical significance was considered at P<0.05.
RESULTS AND DISCUSSION

Total Phenolic and Flavonoid Content
Although many of pharmacological activities of phytomedicines are related to the

phenolic-type compounds [10, 32], these effects do not always correlate with the presence of
large quantities of phenolics. Therefore, both sets of data need to be examined together.
The amount of total phenolics in investigated garlic extracts varied broadly, ranging from
0.07 to 0.70 mg GAE/g of DE (Figure 1). Furthermore, the results obtained from evaluation
of total flavonoid glycosides and aglycones content showed great variations, too (Figure 2),
especially regarding the plant phenophasis. A notably higher content of rutin (RE) and
quercetin equivalents (QE) was confirmed in extract obtained from fresh garlic bulbs (7.37
and 7.46 µg/g of DE, respectively) than in extract prepared as aged garlic extract (0.38 and
2.84 µg/g of DE) and extract of immature garlic plant (1.08 and 1.45 µg/g of DE).
Pharmacodynamic Effects
The application of pentobarbitone and thiopentone resulted in hypnosis in all tested

animals regardless of the pretreatment regime (Figure 3). The differences in IT between
groups after pentobarbitone-induced hypnosis were noted, particularly in groups pretreated
with extract I and II. There were no significant changes in IT in groups treated with
thiopentone compared to control. The prolongation of ST after the application of
pentobarbitone was noted in all groups treated with garlic extracts, but the statistically
significant increase in ST was observed only in groups treated with extracts I and III.
Pretreatment with garlic extracts II and III shortened ST after the thiopentone application,
while extract of immature garlic plant (III) caused statistically significant prolongation of ST.
The impairment of motor coordination caused by midazolam was observed in all tested
groups (Figure 4). Pretreatment with garlic extract brought an increase of the interval spent on
rotating rod in the course of time, as a result of reduction in midazolam action. Statistically
significant difference comparing to control was observed in all groups treated with garlic
extracts in all measured intervals up to 15 min.
The analgesic effect of codeine in all tested groups was noticed as a typical biphasic
curve (Figure 5). Pretreatment with garlic extracts produced changes in the intensity of
analgesia resulting in decrease of overall effect. Significantly the lowest analgesic effect in all
garlic treated groups was noted in 90 and 110 min. compared with control, while the most
depleted values were gained in the group pretreated with extract III.
The effects of tested drugs depend not only on their pharmacodynamic properties but on
their pharmacokinetics, too, especially metabolic pathways. The hypnotic effect of
pentobarbitone and thiopentone, motor impairment caused by midazolam, and the analgesic
effect of codeine are subject to the pharmacokinetic properties of drugs; thus, these effects
can be affected by changes in their liver metabolism. Pentobarbitone and thiopentone are
inactivated by liver microsomal oxidative enzymes, including cytochrome P450 (CYP) [33].
Metabolism of midazolam is mostly dependent on isoenzyme CYP3A4 activity and it is
usually used as a probe of CYP3A4 induction/inhibition [34], while the analgesic effect of
codeine is dependent on its biotransformation into morphine by CYP2D6 action in liver [35].
GAE (mg/g DE) 0.7
0.62
0.7
0.6
I
0.5 II
0.4 III
0.3 0.07
0.2
0.1
0
Phenolics
Figure 1. Total phenolic content for the studied extracts of garlic (I, II and III).
μg/g DE 7.37 7.46
7.5
7
6.5
6
5.5
5
4.5 I
2.84
4
II
3.5
III
3
2.5 1.08 1.45
2
1.5 0.38
1
0.5
0
Flavonoid glycosides (RE) Flavonoid aglycones (QE)
Figure 2. Total flavonoid content for the studied extracts of garlic (I, II and III).
Data obtained in studies on animals showed that garlic influences various phase I and
phase II enzymes, which are involved in the metabolism of drugs and other xenobiotics [36-
39]. The influence of fresh and various kinds of processed garlic on CYP isoenzymes of
human liver microsomes in vitro was also demonstrated [38, 40]. However, clinical studies,
which investigated the influence of garlic on the pharmacokinetics and metabolism of drugs,
came to opposing results. While one study’s results suggest that multiple-dose treatment with
garlic powder preparations activates saquinavir (HIV protease inhibitors) metabolism via
CYP3A4, others find no effect on a single dose of alprazolam (CYP3A4 substrate) or

metabolism of dextrometorphan (CYP2D6 substrate) [41, 42]. Results of clinical studies
indicate that the influence of garlic on human drug metabolism varies with its processing and
formulation [43]. Our study confirmed that the maturation stadium of the garlic plant can also
play role in its effects and interactions.
% of change 342.62
350
300
250
200 110.24
98.75
I
150 43.13
II
III
100
50 28.44 4.17 4.17

-6.58
0 -8.33 -38.52
-48.77
-30.01
-50
penthobarbitone thiopentone penthobarbitone thiopentone
Induction time Sleeping time
Figure 3. Potentiation of pentobarbitone and thiopenthane induced sleeping time by garlic extracts (I, II
and III) in mice.
time (s)
200
180
160
140
Control
120
I
100 II
80 III
60
40
20
0
1' 5' 10' 15' 20' 25'
time (min)
Figure 4. Time intervals spent on rotating rod in rotarod test in mice after the application of midazolam
(5 mg/kg i.p.) in control group and groups pretreated with garlic extracts I, II and III.
% MPE 70
60
50
Control
40
I
II
30
III
20
*
10
0
10 20 30 40 50 60 70 80 90 100 110 120 130 *
time points (min)
Figure 5. Response latencies over time points in hot plate test in mice after the application of codeine
(25 mg/kg i.p.) in control group and groups pretreated with garlic extracts I, II and III.
Thus, the prolongation of sleeping time, decrease in motor impairment and analgesia can
be assigned to compounds from our garlic extracts that affect liver metabolic enzymes
involved in the biotransformation of tested drugs. Although pharmacological properties of
garlic are usually attributed to its sulfur-containing compounds [3, 4], they cannot be
excluded from involvement in possible interactions with drugs, especially when it is known
that they have an effect on various detoxifying phase II metabolizing enzymes [44].
Moreover, additional components like flavonoids and other phenolics present in tested
extracts should not be rejected from the possible contribution to observed changes of drug
effects.
Despite the widespread use of garlic-based phytopreparations, well-documented herb-
drug interactions are rare and many observed herb-drug interactions are based on individual
case reports and case series [19, 26-29, 41, 42]. The clinical importance of herb-drug
interactions depends on various factors that are related to coadministered drugs (dose,
administration route, pharmacokinetic properties), herbs (species, maturation, dose,
administration route) and patients (genetic polymorphism, age, pathological conditions) [45].
Although some herb-drug interactions may be beneficial, in many cases garlic-drug
interactions may increase drug toxicity [26-29, 41]. Further work relating to the isolation and
characterization of the active constituent(s) of tested garlic extracts involved in those
biological activities and interactions as well as the evaluation of mechanism(s) of those
effects are underway in our laboratory.
ACKNOWLEDGMENTS
The Ministry of Sciences and Environmental Protection, Republic of Serbia (Grant No.
142036) supported this research work.
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INDEX
104, 106, 107, 109, 116, 117, 124, 125, 126,

129, 132, 133, 134, 135, 136, 137, 138, 139,
4 140, 141, 149, 151, 152, 167, 168, 169, 177,
178, 179, 210, 250
4-hydroxynonenal, 101
active oxygen, 107, 108
activity level, 68
6 acute, 18, 41, 42, 69, 74, 81, 83, 85, 90, 92, 98,
103, 104, 108, 110, 116, 184, 216, 221, 228
6-OHDA, 86, 87, 88 acute renal failure, 69, 83
adaptability, 186
adaptation, 32, 189, 193
A adducts, 95, 105, 144
adenocarcinoma, 134, 252
AAS, 156, 160, 161, 162, 163, 164, 169, 172, adenoma, 126
174, 178, 179 adenosine, 3, 16, 136, 256
aberrant crypt foci, 126 adhesion, 16, 90, 109, 136, 142, 146, 153, 206
abnormalities, 21, 250 adjunctive therapy, 254
absorption, 11, 14, 32, 79, 156, 158, 160, 162, administration, x, 12, 16, 20, 21, 26, 30, 33, 34,
163, 164, 165, 170, 171, 173, 174, 176, 177, 35, 39, 42, 43, 44, 67, 69, 70, 76, 79, 82, 84,
178, 224 86, 93, 95, 96, 99, 100, 101, 104, 123, 124,
absorption spectroscopy, 170, 177 126, 136, 139, 140, 215, 221, 229, 259, 263,
accounting, 81, 85, 86, 226 264
accuracy, 164, 166, 170, 171, 174 ADP, 16, 83, 133, 134
ACE, 13 adrenal gland, 136
acetaminophen, 68, 76, 78, 82, 87, 91, 106, 109, adrenaline, 15
115, 127, 145, 146, 152 adriamycin, 67, 110, 115
acetate, 8, 21, 65, 165, 173, 258 ADS, xii, 40, 62, 99, 131, 137, 147, 149, 150,
acetic acid, 219 246
acetone, 29, 109, 125 adult, 29, 124, 126, 177, 248, 250, 252
acetylation, 147, 246, 249, 252 adults, 12, 35, 44, 153
acetylcholinesterase, 229 advanced glycation end products, 81
ACI, 105 AEA, 232
acidic, 62, 122 Ag, 159, 161, 167, 169
acidity, 3 age, 8, 9, 24, 26, 60, 125, 247, 263
acquired immunodeficiency syndrome, 27 ageing, 81
ACS, 49, 109 agent, viii, xii, 2, 6, 19, 23, 30, 34, 36, 44, 49, 55,
activated carbon, 164 57, 59, 61, 69, 71, 80, 82, 84, 85, 86, 87, 88,
activation, 20, 21, 27, 43, 45, 46, 47, 48, 51, 59, 92, 93, 95, 119, 127, 136, 150, 210, 212, 221,
74, 75, 81, 82, 89, 90, 92, 93, 95, 96, 100, 103, 222, 246, 248
268 Index
agents, 14, 15, 19, 24, 27, 29, 31, 49, 50, 54, 56, AMS, 89, 123
75, 85, 86, 102, 120, 135, 202, 210, 216, 217, AMT, 122, 124, 126
219, 222, 249, 250, 254 amyloid, 80, 86, 97, 107, 108, 112
aggrecan, 24 amyloid beta, 80, 107, 108
aggregation, 15, 16, 39, 40, 42, 45, 49, 51, 53, 56, anaemia, 25, 35, 221
152, 228 analgesia, 260, 263
aging, 24, 26, 44, 51, 64, 85, 113, 248 analgesic, xii, 255, 257, 260
aging process, 24 analog, 206
agricultural, viii, ix, 119, 181, 188, 189, 195 analysis of variance, 259
agriculture, 183 analytical techniques, 23, 156, 157, 158, 160
aid, 233 analytical tools, 157
AIDS, 27 androgen, 79, 81, 84, 103, 142, 150, 248, 249,
air, xii, 24, 178, 221, 255, 257 252, 253, 254
air-dried, xii, 255, 257 androgens, 248
AKT, 134, 249 anemia, 220
alanine, 68, 127 aneuploid, 183, 188, 222
alanine aminotransferase, 127 angiogenesis, 23, 128, 135, 136, 137, 142, 146,
Albino, 258 150, 250
albumin, 17 angiogenic, 136, 149, 150
alcohol, 6, 28, 53, 90, 121, 125 angiotensin II, 12, 13, 54
alkaline, 91, 96, 221 angiotensin-converting enzyme, 13
alkaline phosphatase, 91, 96, 221 angiotensin-converting enzyme (ACE), 13
alkaloids, 120, 132 animal models, viii, 11, 14, 26, 37, 61, 76, 85,
alkane, 5 123, 136, 249
allele, 197 animal studies, 60
alleles, 192 animal tissues, 242
allergens, 33 animals, xii, 9, 11, 14, 36, 52, 83, 88, 92, 93, 98,
allergic reaction, 219, 257 120, 123, 126, 127, 139, 222, 223, 224, 225,
allergy, 33, 39, 219 226, 229, 246, 255, 258, 259, 260, 261, 265
allies, 182, 196 anode, 233
Allium cepa, 38, 40, 45, 105, 175, 176 anorexia, 219, 220
alpha, 48, 54, 56, 77, 103, 110, 113, 141, 146, ANOVA, 259
229, 247, 248, 253 anther, 184
ALT, 127 anthracene, 70, 73, 91, 98, 101, 102, 114, 125,
alternative, 12, 26, 27, 31, 36, 44, 223, 232 144
alternative medicine, 44 anthropogenic, 156
alternatives, 82, 166 anti-angiogenic, 136
alters, 47, 95, 142 antiapoptotic, 91, 250
aluminium, 164, 257 anti-apoptotic, 84
aluminum, 121 anti-apoptotic, 135
Alzheimer’s disease, 24, 26, 42, 54 antibacterial, xi, xii, 3, 28, 41, 245, 246
Amadori, 25, 81, 143 antibacterial properties, xii, 3, 246
amelioration, viii, 62, 68, 70, 76, 82, 224 antibiotic, viii, xi, 29, 119, 200, 245, 251
amine, 95, 126 antibiotics, 29, 246
amino, x, xi, 3, 13, 17, 34, 35, 95, 98, 101, 108, anti-cancer, vii, ix, 19, 22, 23, 57, 81, 82, 97,
140, 200, 201, 215, 219, 224, 245, 256 116, 119, 120, 123, 127, 132, 133, 152, 199,
amino acid, x, xi, 3, 13, 17, 35, 101, 108, 140, 200, 202, 210, 212
200, 201, 215, 224, 245, 256 anticancer drug, 82, 257
amino acids, x, 3, 13, 35, 101, 200, 215, 224, 256 anti-carcinogenic, 151
AML, 150 anticoagulant, xii, 34, 232, 246, 255, 256, 265
ammonium, 165, 173 anticoagulants, 250, 257
amniotic, 92 antidiabetic, 56, 142
amphetamine, 88 antigen, 84, 134, 247
Index 269
antimicrobial protein, 53 aspirin, 228

antimony, 178, 216 assessment, 54, 112, 160, 187
antineoplastic, viii, xi, 23, 34, 92, 119, 245, 246, asthma, xi, 2, 33, 39, 50, 245
256 astrocytoma, 22
antioxidative, 105, 106, 110, 212 atherogenesis, 74
antioxidative activity, 106 atherosclerosis, xi, 9, 12, 14, 18, 38, 41, 44, 58,
antiparasitic, 31, 50 105, 224, 245
antithesis, 225 atherosclerotic plaque, 71, 103, 104
antitumor, viii, 3, 22, 26, 27, 34, 46, 53, 61, 148 athletes, 2, 120
anti-tumor, 130, 149, 246 atmospheric deposition, 156
antiviral, 27, 34 atmospheric pressure, 264
anxiety, 219 atomic absorption spectrometry, 156, 160, 162,
aorta, 41, 54, 67, 70, 71, 74, 103 163, 164, 173, 174, 176, 177, 178
apathy, 220 atoms, x, 199, 202, 210
apoptosis, ix, 21, 23, 25, 41, 43, 48, 49, 52, 53, ATP, 132
55, 59, 69, 75, 80, 95, 96, 98, 103, 107, 108, atrophy, 51, 85
109, 112, 113, 114, 116, 119, 128, 131, 132, authentication, 156
133, 134, 135, 137, 138, 142, 143, 146, 147, autoimmune disorders, 216
148, 149, 150, 151, 152, 210, 211, 212, 213, automation, 166
221, 229, 246, 248, 249, 250, 252, 253, 254 autonomy, 26
apoptosis proteins, 133 autopsy, 41
apoptotic, 21, 76, 84, 91, 95, 96, 130, 132, 133, availability, 85, 108, 171
134, 135, 146, 249 avoidance, 70, 73, 113, 211
apoptotic cells, 22, 249 axonal, 85
apoptotic effect, 76, 133 azoxymethane, 71, 95, 108
apoptotic rate, 84
APP, 42
application, xi, xii, 33, 54, 60, 128, 146, 176, B
177, 213, 216, 226, 232, 237, 241, 251, 255,
B cells, 127
257, 258, 259, 260, 262, 263, 264
B. subtilis, 29
arachidonic acid, 16, 56
Bacillus, 29
arginine, 25, 54, 72, 77, 107, 113
back, 120, 188
arid, 185
bacteria, 19, 28, 29, 32, 39, 197
aromatics, 176, 177
bacterial, 27, 28, 55, 150
arrest, 47, 53, 76, 96, 128, 129, 130, 131, 132,
bacterial infection, 55, 150
142, 146, 147, 148, 149, 210, 211, 213, 247,
bacteriostatic, 29, 41
248, 249, 253
bacterium, 29
arrhythmias, 66
BAL, x, 215, 216, 219, 221, 222, 225, 226, 229
arsenic, x, 21, 42, 178, 215, 216, 219, 222, 224,
Bangladesh, 164, 175
225, 226, 227, 228, 229
barium, 121
arsenic poisoning, 225
barrier, 84, 188
arsenite, 42, 70, 73, 103, 227
barriers, 196
arteries, 9
basic research, 120
arteriosclerosis, 11, 18, 25
Bax, 46, 59, 91, 95, 108, 116, 132, 133, 134, 135,
artery, 11, 16, 40, 43, 48, 68, 74, 85, 265
149, 150, 251, 252
arthritis, xi, 25, 245
bcl-2, 134
asbestos, 89, 109
Bcl-2, 46, 84, 91, 95, 133, 134, 135, 149, 150,
ascites, 52, 96, 106, 136, 150
210, 212, 249
ascorbic, 25, 81, 89, 93, 95, 133, 138, 221
Bcl-2 proteins, 134
ascorbic acid, 25, 81, 89, 93, 95, 133, 138, 221
Bcl-xL, 95, 127, 133, 134
ash, 3
beam radiation, 247
aspartate, 68
beef, 89
Aspergillus niger, 30
behavior, viii, xi, 62, 86, 87, 88, 231, 237, 243
270 Index
behavioral change, 108 boils, 28

behaviours, 60 bomb, 160
beneficial effect, vii, viii, 1, 11, 61, 84, 187 bonds, 249
benefits, vii, xii, 1, 4, 19, 37, 38, 62, 122, 226, bone marrow, 71
246, 250, 255, 256 bounds, 17
benign, 247 bovine, 74, 202, 206
benign prostatic hyperplasia, 247 bowel, 26
benzo(a)pyrene, 20, 21, 56, 124, 151 brachytherapy, 247
bile, 8, 11, 222 brain, 26, 51, 70, 78, 84, 85, 86, 87, 88, 93, 99,
bile acids, 8, 11 103, 107, 111, 112, 221, 222, 224, 225, 227,
biliary obstruction, 68, 104, 264 229
bilirubin, 68 brain damage, 78, 87, 111
binding, 20, 21, 57, 59, 86, 90, 94, 95, 103, 116, branching, 85
125, 126, 137, 144, 206, 209, 249, 253 breakdown, xi, 30, 31, 43, 56, 128, 192, 246
bioactive compounds, 5, 36, 37, 104 breast cancer, 21, 42, 49, 52, 79, 95, 98, 105, 132,
bioassay, 151 149, 248, 251
bioavailability, 6, 12, 13, 36, 79, 109, 142, 156, breathing, 219, 221
250 breeding, ix, 181, 182, 187, 188, 192, 194, 197
biochemistry, 216 broad spectrum, 30, 31, 86
biological activity, 51, 156, 229 buffer, 171
biologically active compounds, 224 bulbs, vii, ix, x, xi, xii, 3, 31, 44, 50, 58, 105,
biomarker, 100 160, 164, 179, 181, 186, 189, 190, 192, 231,
biomarkers, 24, 125, 252 232, 233, 237, 241, 242, 255, 257, 260, 264
biometals, 115, 229 Burkitt lymphoma, 127
biomolecules, 256 burn, 70
biosynthesis, 8, 9, 10, 11, 12, 25, 44, 45, 48, 49, burning, 219, 220, 247
75, 89, 126 burns, 44, 53, 257, 265
biotin, 3, 256
biotransformation, 94, 261, 263
bismuth, 216 C
bladder, 19, 22, 35, 48, 70, 73, 113, 117, 123,
Ca2+, 95, 133, 146
133, 141, 149
cabbage, 160
bladder cancer, 48, 133, 149
CAD, 16, 133
bleeding, xii, 15, 34, 41, 250, 255, 256, 265
cadherin, 24, 81, 84, 142, 152
bleeding time, 250
cadmium, x, 92, 110, 111, 115, 215, 216, 221,
blindness, 219
226, 227, 228, 229
blocks, 247
calcification, 41
blood, xi, xii, 3, 6, 8, 9, 11, 12, 13, 14, 15, 25, 26,
calcium, 3, 16, 32, 86, 112, 121, 133, 250
34, 35, 36, 38, 39, 40, 42, 44, 45, 47, 48, 49,
calf, 125, 199, 202, 205, 209, 210
51, 54, 56, 69, 71, 83, 84, 93, 100, 101, 103,
cAMP, 16, 90
104, 107, 108, 115, 116, 135, 153, 221, 223,
cancer cells, ix, 21, 49, 52, 53, 95, 96, 98, 108,
224, 225, 228, 230, 245, 246, 247, 265
114, 119, 120, 128, 129, 130, 131, 132, 133,
blood clot, xii, 246
134, 136, 137, 140, 141, 143, 146, 147, 148,
blood flow, 69, 101
149, 150, 151, 200, 202, 206, 210, 211, 212,
blood pressure, xi, xii, 12, 13, 39, 42, 45, 51, 54,
213, 246, 248, 249, 253, 254
100, 245, 246
cancer progression, 52
blood stream, 221
cancer treatment, 23, 253
blood urea nitrogen, 83
cancerous cells, 127, 137
blood vessels, 15
Candida, 30, 31, 38, 49, 196
body fluid, 158
capillary, 136
body weight, 68, 69, 70, 71, 74, 92, 94, 96, 97,
capsule, 6, 7, 95
124, 136, 224, 258
carbazole, 132
boiling, 176
carbohydrate, 27, 32
Index 271
carbohydrates, x, 3, 32, 120, 215, 232 cell growth, 27, 57, 127, 128, 137, 138, 141, 143,
carbon, 8, 76, 104, 164 150, 201, 202, 206, 210, 251
carbon tetrachloride, 76, 104 cell invasion, 142, 152
carboxyl, 224 cell line, x, 21, 23, 46, 49, 52, 55, 75, 79, 84, 127,
carboxylic, 72 128, 129, 130, 131, 132, 133, 134, 146, 147,
carcinogen, ix, 20, 34, 58, 70, 71, 95, 116, 119, 149, 200, 202, 206, 210, 248, 249, 251, 253
123, 124, 125, 139, 140, 144, 145, 251 cell lines, 21, 23, 46, 52, 55, 75, 79, 84, 128, 129,
carcinogenesis, 21, 22, 23, 59, 71, 73, 76, 83, 87, 130, 131, 132, 133, 146, 147, 149, 210, 248,
94, 95, 101, 114, 115, 123, 124, 126, 138, 139, 249, 251, 253
140, 142, 144, 145, 229, 248, 249, 251, 252, cell membranes, 35, 223
253, 266 cell metabolism, 36
carcinogenic, 23, 34, 46, 55, 76, 125, 138, 141, cell signaling, 137
144, 151, 200 cerebellar granule cells, 75, 101
carcinogens, 20, 49, 76, 97, 123, 124, 125, 126, cerebral ischemia, 73, 84, 85, 92, 113
139 cerebrospinal fluid, 43
carcinoma, 19, 22, 48, 75, 113, 126, 128, 133, cerebrovascular, 25
136, 138, 140, 144, 146, 147, 149, 150, 151, cerebrovascular disease, 25
152, 249, 253 cerium, 216
carcinomas, 95 cervical cancer, 134
cardiac muscle, 39 cervix, 124, 144
cardiovascular disease, vii, viii, xi, 1, 3, 8, 9, 12, chelates, 173, 225, 226
15, 17, 18, 25, 38, 50, 51, 53, 58, 82, 88, 113, chelating agents, 216, 217, 219
119, 123, 245, 253, 256 chelators, 221, 226
cardiovascular risk, xi, 17, 38, 50, 51, 53, 245 chemical properties, xi, 52, 231, 242
cardiovascular system, 12 chemical structures, 201, 216, 217
carotene, 66, 249 chemicals, x, 32, 34, 120, 215
carotenoids, 24 chemokines, 48
carrier, 233 chemoprevention, vii, ix, 19, 75, 76, 95, 115,
caspase, 48, 52, 74, 80, 84, 90, 95, 96, 107, 115, 120, 125, 141, 143, 148, 251
132, 133, 134, 135, 143, 146, 148, 149, 150, chemotherapeutic drugs, 134
151, 210 chemotherapy, 27, 38, 43, 45, 47, 58, 82, 96, 109,
caspase-12, 80, 107 150, 202, 247, 251
caspase-dependent, 81, 150 chest, 219
caspases, 133, 134, 249 chicken, 93
CAT, 67, 68, 71, 74, 75, 78, 80, 81, 84, 89, 90, children, 222, 223
92, 93, 96, 98, 99 210, 250
catalase, 25, 67, 102, 112, 133, 139, 140, 225 Chinese medicine, xi, 245
catalyst, 162, 173 chitosan, 161, 174, 176
catalytic activity, 250 chloride, 87, 170, 228
catalytic effect, 171 chloroplast, 195
cataract, 26 chloroquine, 229
catechins, 51, 120, 132, 141, 248 cholera, 2
CDK2, 131 cholesterol, xi, xii, 3, 8, 9, 11, 12, 13, 14, 31, 32,
cDNA, 24, 148, 246 34, 40, 44, 45, 49, 50, 55, 56, 59, 60, 71, 72,
cell adhesion, 90, 109, 142, 153 100, 103, 104, 105, 153, 245, 246, 251, 256
cell culture, 21, 65, 76, 80, 123, 130, 142, 249 cholesterol-lowering drugs, 34
cell cycle, 22, 53, 75, 76, 96, 128, 129, 130, 131, chondrocytes, 141
132, 135, 142, 147, 148, 149, 210, 211, 247, chromatographic technique, 157
248, 249, 250, 253 chromatography, xi, 156, 165, 166, 167, 175,
cell death, 21, 25, 75, 80, 89, 93, 95, 101, 107, 201, 231, 233, 264
131, 132, 133, 134, 136, 137, 138, 146, 147, chromium, 107, 121, 139, 152, 164, 175,176,
149, 150, 249, 253 216
cell division, 21, 75, 135, 246 chromosome, 183, 188, 191, 197
272 Index
chromosomes, 28, 55, 183, 184 community, 156

chronic disease, 26, 64 compatibility, 192, 195
chronic diseases, 26, 64 compilation, 65, 79, 80, 88
chronic renal failure, 83, 220 compliance, 7
chrysanthemum, 242 complications, 14, 100
cigarette smoke, 220, 221 components, ix, xi, 4, 6, 9, 11, 14, 15, 16, 21, 23,
cigarette smoking, 9 24, 26, 28, 32, 34, 37, 39, 40, 48, 51, 52, 78,
circulation, xi, 8, 15, 26, 245 100, 104, 110, 123, 125, 126, 129, 130, 132,
cis, 53, 54, 63 133, 136, 137, 138, 139, 140, 142, 166, 169,
cisplatin, 69, 72, 113 199, 200, 201, 217, 218, 222, 224, 225, 226,
citrus, 122 227, 232, 235, 246, 256, 263, 264, 266
classes, 233 composites, 178
classical, 132 composition, vii, x, xi, 5, 6, 30, 34, 53, 56, 64,
classification, 156, 182, 184, 189, 190, 192, 196, 109, 110, 120, 121, 156, 157, 160, 163, 164,
212 169, 175, 176, 231, 233, 236, 237, 238, 240,
clay, 2 241, 243, 252
cleavage, 133, 134, 135, 150 concentration, x, xi, 16, 17, 18, 19, 28, 29, 30, 36,
climatic factors, 187 67, 75, 113, 126, 130, 131, 152, 156, 161, 163,
clinical trial, 13, 18, 34, 37, 109 164, 168, 169, 170, 172, 174, 178, 199, 200,
clinical trials, 13, 18, 34, 37 202, 206, 207, 208, 224, 245, 250, 257
clone, 139, 184, 187, 189, 190 condensation, 74, 134
cloning, 58 confidence, 123
closure, 142 confidence interval, 123
CMV, 22 confidence intervals, 123
cNOS, 12 conjugated dienes, 96
CNS, 257 conjugation, 127
Co, xi, 51, 119, 142, 157, 158, 159, 160, 161, conjunctivitis, 219
162, 164, 165, 167, 168, 169, 174, 231 constipation, 2
coagulation, 15 consumers, 6, 18, 32, 156, 232
coal, 220 consumption, vii, viii, 2, 7, 17, 19, 32, 36, 37,
cobalt, 216 103, 122, 123, 158, 173, 187, 200, 210
codes, 232 contact dermatitis, 33
coenzyme, 76, 140 contaminated food, 221
cognitive disorders, 85 contamination, 156, 160, 171, 172, 173, 177, 188,
cognitive dysfunction, 220 228
cognitive function, 26 control, xii, 11, 14, 21, 26, 27, 42, 48, 58, 68,
cohort, 122 109, 112, 122, 124, 127, 128, 131, 132, 137,
collagen, 15, 16, 58, 69, 70, 136 142, 143, 144, 156, 160, 164, 197, 206, 210,
colon, vii, 1, 19, 20, 21, 22, 32, 47, 52, 56, 57, 235, 236, 237, 247, 248, 251, 252, 255, 258,
58, 75, 76, 79, 84, 95, 96, 114, 122, 123, 125, 260, 262, 263
126, 130, 131, 133, 137, 141, 143, 145, 146, control group, 124, 127, 131, 258, 262, 263
147, 148, 149, 151, 200, 212, 213, 246, 249, convective, 173
251 conversion, 21, 75, 258
colon cancer, 22, 52, 56, 75, 76, 96, 114, 122, cooking, vii, 1, 2, 15, 34, 36, 41, 62, 66
131, 133, 137, 141, 143, 145, 146, 147, 148, copper, x, 36, 121, 175, 176, 179, 215, 216, 220,
151, 212, 213 224, 227, 228, 250
colon carcinogenesis, 21, 95, 114 corn, 125, 248
colorectal cancer, 22, 128, 136, 210 corolla, 184
coma, 220 coronary arteries, 9
combined effect, 102, 224 coronary artery disease, 11, 16, 40, 43, 48, 265
combustion, 171 coronary heart disease, 53, 155
communication, 109, 136 correlation, 23, 139, 189, 190
communities, 189 correlations, 169
Index 273
Corynebacterium, 48 cytotoxicity, 114, 136, 149, 210, 213, 221, 225,

costs, 26, 171 229
cough, 3
country of origin, 178
coupling, 188 D
covalent, 163
dandruff, xii, 246
covering, 182, 192, 232
dating, 120, 200, 256
COX-2, 141
DBP, 12
COX-2 enzyme, 141
de novo, 8
CRC, 44, 195, 241, 242
death, 8, 9, 21, 25, 46, 75, 80, 84, 89, 93, 95, 101,
creatinine, 69, 83
107, 108, 131, 132, 133, 134, 136, 137, 138,
CREB, 90
142, 147, 149, 150, 220, 224, 247, 249, 253
credibility, 37, 250
deaths, 146
crops, 160, 178, 186, 195, 241, 242
decomposition, 178
cross-linking, 58
defects, 17
crown, 116
defence, 25
crude oil, 3
defense, 69, 92, 115, 229
Cryptococcus, 30, 43, 55
defenses, 216, 225
crystalline, xi, 231, 234
deficiency, 17, 18, 43, 55, 57, 60, 227, 228
C-terminus, x, 200, 205, 207
deficit, 85, 86, 110
cultivation, 2, 182, 188, 194, 200
deficits, 85, 104, 111, 113, 193, 229
cultivation conditions, 200
degenerative conditions, 24
cultural practices, 189
degradation, 66, 132, 156
culture, 21, 76, 80, 113, 115, 120, 123, 130, 142,
dehydration, 238, 243
146, 152, 200, 206, 249
dehydrogenase, 31, 68, 71, 81, 86, 92, 93, 96, 127
cumin, 163
delivery, 142
curcumin, 47, 55, 85, 90, 103, 108, 248, 250, 252
delusions, 220
cyclooxygenase, 25, 51, 54, 95, 114, 152
dementia, 26, 102, 219
cyclooxygenase-2, 51, 54, 95, 114, 152
density, 56, 65, 103, 105, 106, 107, 109, 212,
cyclosporin, 73
224, 228
cyclosporine, 103, 134
dental caries, 29
cystathionine, 82
deoxyribonucleotides, 140, 200
cysteine, vii, ix, xii, 1, 4, 13, 14, 17, 18, 45, 48,
Department of Health and Human Services, 227
50, 53, 54, 55, 57, 62, 63, 64, 68, 74, 75, 77,
depolarization, 134
82, 100, 101, 103, 104, 105, 106, 107, 108,
depolymerization, 59, 116
109, 110, 114, 120, 127, 128, 129, 134, 139,
deposition, 8, 9, 156, 160, 227
146, 149, 203, 221, 246, 256
deposits, 220, 226
cysteine proteases, 103, 134, 149
depressed, 126
cytochrome, 20, 43, 51, 74, 81, 82, 90, 93, 94, 95,
depression, 135, 188, 192, 194, 220
100, 109, 115, 124, 126, 132, 133, 134, 141,
deprivation, 80, 254
144, 152, 222, 260, 266
derivatives, 5, 13, 14, 20, 24, 31, 36, 37, 46, 47,
cytokine, 26, 102
75, 76, 77, 102, 106, 113, 121, 152
cytokines, 48, 89, 92, 140
dermatitis, 3, 33, 219
cytology, 182, 190
dermatophytes, 30
cytomegalovirus, 22, 28
desert, 247
cytometry, 130, 189
desorption, 50
cytoplasm, 8, 196
destruction, 148, 171
cytoplasmic membrane, 243
detection, 42, 164, 165, 166, 170, 171, 247
cytosol, 133, 134, 137
detoxification, 21, 23, 25, 75, 94, 104, 139, 217,
cytosolic, 4, 95, 133
222, 225, 266
cytotoxic, 9, 22, 53, 92, 96, 126, 146, 202
detoxifying, ix, x, 20, 25, 82, 93, 97, 102, 108,
cytotoxic action, 22
119, 143, 215, 263
developed countries, 8, 9
274 Index
developmental deficits, 229 diversity, 5, 188, 189, 196

deviation, 163, 164, 171 division, 21, 75, 135, 246
diabetes, viii, 9, 14, 18, 25, 32, 41, 48, 50, 56, 57, DNA, ix, x, 20, 22, 23, 24, 25, 28, 34, 57, 59, 66,
59, 78, 81, 87, 89, 96, 97, 119, 123, 137, 153, 74, 76, 92, 94, 95, 96, 100, 103, 105, 114, 116,
155 119, 125, 126, 127, 129, 130, 132, 133, 134,
diabetes mellitus, 41, 48, 56, 59, 153 137, 140, 144, 145, 147, 148, 152, 181, 190,
diabetic patients, 14, 17, 52, 56, 75, 80 191, 193, 196, 199, 200, 201, 202, 203, 205,
diarrhea, 32, 33, 34, 219, 220, 224 206, 207, 208, 209, 210, 211, 212, 213, 221,
diarrhoea, xii, 246 232, 248
diastolic blood pressure, 12, 100 DNA damage, 23, 25, 66, 92, 100, 103, 127, 145,
dienes, 96 221
diet, 2, 8, 12, 17, 18, 25, 32, 53, 66, 67, 69, 71, DNA polymerase, x, 28, 140, 152, 199, 200, 203,
72, 84, 87, 94, 95, 98, 100, 109, 126, 127, 143, 205, 208, 211, 212, 213
169, 178, 216, 226, 247, 248, 250 DNA repair, ix, 22, 119, 125, 248
dietary, vii, x, xii, 1, 6, 8, 9, 12, 14, 36, 37, 39, DNA strand breaks, 95, 116
47, 50, 53, 85, 93, 102, 105, 108, 122, 126, doctors, 216
141, 144, 157, 176, 177, 178, 215, 226, 227, dogs, 15, 42
232, 249, 250, 255, 256 domestication, ix, 181, 195
dietary habits, xii, 255, 256 donations, 210
dietary intake, 177, 178 donor, 226
dietary supplementation, 9, 12, 39 donors, 22
diets, 8, 24, 60, 67, 114, 179, 266 dopamine, 86
differentiation, ix, 49, 75, 128, 149, 150, 181, dopaminergic, 86, 93
196, 233 dosage, 14, 34, 35, 37, 72, 73, 85, 87, 97, 226,
diffraction, 233, 234, 242 248, 250
digestion, 120, 159, 160, 161, 162, 163, 164, 165, double bonds, 249
166, 168, 169, 171, 172, 173, 174, 177, 179 downregulating, 248
digestive tract, 210, 221 down-regulation, 95, 132, 134, 149
dimer, 138 D-penicillamine, x, 215
diphtheria, xii, 246 drinking, 69, 76, 82, 84, 99, 178, 220, 225
diploid, 183, 188, 189, 193, 222 drinking water, 69, 76, 82, 84, 99, 178, 220, 225
direct action, 75 drug interaction, 58, 257, 263
discharges, 225 drug metabolism, 250, 262
discriminant analysis, 163 drug targets, 250
discrimination, 264 drug toxicity, 263
disease model, 87, 105 drug-induced, 46
diseases, vii, viii, 8, 9, 15, 18, 25, 29, 64, 100, drug-resistant, 29
119, 123, 137, 197, 200, 221, 256 drugs, xii, 3, 14, 17, 20, 30, 32, 34, 43, 81, 120,
disorder, viii, 119 123, 130, 134, 200, 225, 226, 255, 257, 260,
dispersion, 178 261, 263, 265
disposition, 247 drying, 49
distillation, 6, 62, 121 DSM, 149
distress, viii, 2 duodenum, 123, 141
distribution, 79, 116, 157, 182, 187, 192, 223, duplication, 128
228, 229 duration, 13, 16, 37, 226, 237, 259
disulfide, ix, x, xii, 18, 20, 33, 34, 39, 40, 47, 48, dust, 39
52, 55, 57, 62, 63, 65, 79, 91, 98, 102, 103, dysregulation, 132
104, 106, 109, 111, 115, 120, 121, 123, 126,
137, 143, 147, 148, 149, 150, 151, 199, 200,
201, 202, 203, 206, 210, 246, 249, 250, 251, E
253, 254
E. coli, 29, 205
diuretic, 2
eating, 11, 221
divergence, 186, 189
E-cadherin, 81, 84, 142, 152
Index 275
ecological, 193 estrogen, 22, 248

ecology, 182, 190 ethanol, 6, 29, 66, 72, 77, 90, 92, 96, 97, 106,
edema, 84, 85, 219 108, 110, 114, 116, 201, 210
eicosanoid, 38 ethnicity, 247
eicosanoids, 52 ethyl acetate, 258
elaboration, 11 etiology, viii, 119, 127
elderly, 41 evolution, ix, 36, 39, 156, 181, 194, 195, 242
electrodes, 170 excitotoxic, 85, 112
electromagnetic, 170 excitotoxicity, 86
electron, 225, 231, 233, 234 exclusion, 166
electron microscopy, xi, 231, 233 excretion, 11, 20, 69, 100, 151, 217, 221, 222,
elephant, 196, 197 224, 225, 227
embryo, 188 exercise, 12, 14, 24
embryonic development, 132 exposure, 24, 71, 75, 83, 125, 127, 128, 130, 132,
emission, 156, 160, 162, 166, 175, 176 139, 140, 146, 160, 216, 220, 221, 224, 226
encoding, 137 extinction, 192
endonuclease, 132 extracellular matrix, 58, 142
endoplasmic reticulum, 237 extraction, 24, 29, 30, 34, 35, 41, 121, 164, 165,
endothelial cell, 18, 72, 74, 75, 80, 81, 90, 104, 166, 173, 174, 177, 178
106, 108, 116, 136, 138, 143, 149, 150, 230 extraction process, 34
endothelial cells, 18, 72, 74, 75, 81, 90, 104, 106, eyes, 220, 224
108, 116, 136, 138, 143, 149, 150, 230
endothelial dysfunction, 13, 54, 59
endothelium, 18 F
endurance, xi, 245
failure, 35, 83, 87, 219
energy, 3, 14, 26, 86, 166, 238
familial, 247
enterococci, 29, 47
family, x, 2, 22, 62, 140, 152, 199, 201, 205, 206,
environment, 16, 137, 183, 184, 190, 192, 193,
207, 209, 210, 212, 217, 232, 247, 250
216, 217, 224, 258
family history, 247
environmental change, 137
FAO, 6, 155, 176, 232
environmental chemicals, 34
farming, 121
environmental conditions, 189, 190
farms, 191
environmental factors, 14
fast food, 37
enzymatic, 20, 21, 45, 69, 93, 99, 140, 200
fat, 3, 9, 25, 47, 62, 71, 72, 108, 109, 114, 247,
enzymatic activity, 200
248, 252
epidemiologic studies, 16, 19
fatigue, 219
epidermal growth factor, 131, 253
fatty acids, xi, 3, 9, 93, 212, 231, 233, 237, 239,
epidermal growth factor receptor, 131
240, 243, 256
epinephrine, 16
FDP, 165, 173
epithelial cell, 20, 48, 58, 59, 95, 109, 116, 130,
feeding, 35, 39, 66, 70, 71, 95, 108
131, 134
females, 19, 94, 122
epithelial cells, 20, 48, 58, 59, 95, 109, 116, 134
fermentation, 53
epithelium, 247
fertility, 187, 189, 196
equilibrium, 259
fertilization, 187
ERK1, 136, 138, 210
fever, 2, 209, 219
erosion, 33
fibers, 89
erythrocytes, 25, 67, 71, 84, 97, 99, 116, 212
fibrinogen, 40, 265
Escherichia coli (E. coli), 29, 47, 205
fibroblast growth factor, 136
esophageal cancer, 19, 59, 116, 125, 144
fibroblasts, 127
esophagus, 123, 141, 200
fibrosarcoma, 27
essential oils, 256
fibrosis, 18, 68, 73, 82, 87, 92, 104, 107, 110, 264
ester, 28, 114, 126, 251
filament, 184
esters, 8, 233, 242
filtration, 258
276 Index
first generation, 192 gamete, 191

fish, 102, 205 gametes, 183, 191
fish oil, 102 gamma, 123
flame, 160, 162, 163, 164, 167, 175, 176, 177, gamma rays, vii, x, xi, 166, 231, 232, 233, 242,
179, 233 243
flame ionization detector, 233 gamma-ray, 242
flatulence, 33 gamma-tocopherol, 110
flavonoid, xii, 11, 101, 249, 255, 257, 260, 261, ganglion, 133, 146
265 gangrene, xi, 3, 245
flavonoids, viii, 2, 14, 24, 49, 51, 54, 156, 256, gas, xi, 231, 233, 264
257, 263 gas chromatograph, 233, 264
flavor, ix, 48, 62, 126, 142, 145, 199, 201 gastric, 7, 19, 22, 29, 49, 51, 53, 57, 71, 73, 75,
flora, 33, 257 83, 87, 96, 97, 101, 108, 115, 122, 125, 128,
flow, 15, 69, 101, 170, 176, 177, 178, 179, 189, 131, 133, 134, 143, 146, 148, 149, 210
247 gastric ulcer, 96, 108
fludarabine, 134 gastritis, 29
fluid, 7, 43, 234 gastrointestinal, vii, viii, 1, 2, 11, 32, 34, 36, 46,
flumazenil, 265 79, 119, 120, 257
fluorescence, 158, 160, 162, 165, 166, 177 gastrointestinal tract, vii, 1, 11, 79
fluoride, 242 gastroscopy, 128, 146
folate, 17, 18, 51 gel, xi, 6, 7, 201, 231, 233, 234, 235, 236, 237,
folic acid, 17, 18, 43, 57, 60 238
food, viii, ix, 2, 14, 29, 36, 53, 56, 92, 119, 120, gender, 8, 9
155, 157, 160, 166, 169, 170, 174, 176, 177, gene, ix, 8, 22, 24, 26, 40, 47, 58, 90, 94, 137,
178, 179, 197, 200, 201, 219, 221, 232, 242, 141, 147, 181, 182, 185, 186, 187, 188, 190,
250, 258 191, 192, 193, 194, 195, 197, 246, 252
Food and Drug Administration, 216 gene expression, 8, 24, 47, 58, 141, 147, 246
food industry, 232 gene transfer, 187
food poisoning, 29 generation, 18, 21, 42, 43, 74, 80, 82, 92, 93, 95,
food safety, 53 98, 108, 116, 122, 134, 149, 161, 162, 163,
foodstuffs, 37, 175 164, 165, 166, 176, 177, 178, 192, 221
forensic, 166 genes, 8, 34, 57, 117, 137, 141, 148, 152, 246,
forestry, 52 249
fragmentation, 74, 96, 127, 132, 133, 134 genetic defect, 17
free radical, 23, 25, 34, 46, 76, 81, 82, 91, 111, genetic factors, 194
116, 140, 216, 217, 222, 229, 233, 237, 238, genetics, 14, 184
243, 246, 249 genome, 140, 182, 187, 188, 191, 192, 193, 195,
free radical scavenger, 116, 249 201
free radicals, 23, 34, 76, 81, 91, 140, 216, 217, genomes, 183, 191
229, 233, 237, 238, 243 genotoxic, 70, 229
freeze-dried, 201 genotype, 16
freezing, 32 genotypes, 183
fructooligosaccharides, 50 genre, 194
fruits, 3, 8, 24, 122, 176, 178 gentamicin, 69, 78, 83, 93, 110, 111, 112
fungal, 27, 30, 31, 57 genus Candida, 196
fungi, 30, 31, 57 geochemistry, 166
fungicidal, 30, 52 geothermal, 160
fungus, 30 geothermal field, 160
fusion, 188 germanium, 3, 23, 121, 176, 177, 178, 179
germination, 4, 30, 38, 243
GHG, ix, 181, 184, 185, 186, 187, 189, 190, 191,
G 192, 193, 194
gift, 142
gallium, 216
Index 277
ginger, 56, 153 haplotypes, 197

gingivitis, 220 harm, 223
gland, 123, 136, 141 harmful effects, 224
glass, 166, 233 harvest, xi, 53, 231, 232, 233, 235, 236, 237, 240
glioblastoma, 22, 95, 103, 133, 134, 138, 149 harvesting, 156, 187, 189
glucose, 8, 14, 40, 58, 71, 75, 80, 81, 89, 92, 93, hazardous substances, 220, 229
99 HDL, 9, 11, 246
glucose metabolism, 8 headache, xii, 2, 255, 256
glutamate, 25, 91 healing, xi, 25, 135, 245, 256
glutathione, 4, 20, 21, 25, 34, 36, 49, 50, 56, 67, health, vii, viii, 1, 4, 6, 12, 18, 32, 35, 37, 38, 40,
68, 104, 109, 113, 115, 126, 127, 128, 133, 41, 42, 45, 48, 50, 56, 59, 62, 65, 102, 119,
139, 140, 145, 151, 152, 222 121, 142, 143, 156, 157, 160, 175, 200, 216,
glutathione peroxidase, 25, 67 221, 224, 226, 228, 246, 250, 256
glycation, 14, 75, 81, 89, 100, 111 Health and Human Services, 227
glycolipids, 3, 256 health effects, 40, 42, 56, 102, 226
glycoprotein, 33, 266 heart, 2, 8, 9, 17, 25, 35, 39, 41, 42, 51, 52, 53,
glycoside, 50, 264 58, 66, 67, 70, 71, 76, 78, 81, 82, 87, 89, 90,
glycosides, xii, 255, 256, 257, 260 96, 99, 101, 102, 103, 110, 120, 155, 223, 256
glycosylated, 14 heart disease, 9, 17, 53, 58, 120, 155, 256
gold, 170, 177, 216 heat, xi, 15, 36, 246
gout, 220 heating, 36, 62, 66, 112, 173, 233
GPx, 67, 68, 69, 71, 74, 75, 76, 78, 80, 81, 82, heavy metal, vii, x, 156, 160, 174, 178, 215, 216,
83, 84, 89, 90, 92, 93, 95, 96, 98, 99 217, 218, 219, 221, 223, 226, 227, 228, 230,
grains, 19 232, 256
gram-positive bacteria, 29 heavy metals, x, 156, 174, 178, 215, 216, 217,
granule cells, 75, 101 218, 223, 226, 230
granules, 234 height, ix, 181, 189, 190
graph, 258 Helicobacter pylori, 29, 41, 42, 52, 55
graphite, 160, 170, 177, 178, 233 helix, 200, 207, 209
grasslands, 182 heme oxygenase, 90, 104
green tea, 51, 120, 132, 141, 252 hemorrhoids, 3
grounding, 6 hemostasis, 53
groups, x, xii, 6, 20, 21, 28, 29, 35, 66, 75, 79, hepatic injury, 68, 82, 102, 111, 114, 145
85, 90, 127, 139, 174, 215, 221, 223, 224, 255, hepatitis, 68, 73, 74, 104, 153
258, 259, 260, 262, 263 hepatitis a, 68, 104, 153
growth factor, 107, 131, 136, 138, 140, 248, 249, hepatocarcinogenesis, 126
253 hepatocellular, 22, 134, 150
growth factors, 136, 140 hepatocellular carcinoma, 134, 150
growth inhibition, 128, 130, 131, 132, 133, 134, hepatocytes, 9, 11, 44, 49, 50, 69, 90, 94, 96, 107,
140, 146, 249, 253 114, 115, 139, 152
GST, 20, 21, 25, 68, 69, 70, 71, 76, 81, 90, 92, hepatoma, 128, 132, 138, 139, 143, 148
93, 94, 96, 97, 99, 126, 139 hepatotoxicity, 68, 73, 74, 76, 82, 90, 91, 98, 106,
GSTP, 139 109, 115, 127, 145, 146
gut, 257 herbal, 37, 47, 58, 136, 150, 177, 210, 265
gynecomastia, 248 herbs, 48, 148, 176, 177, 263
heredity, 8
heritability, ix, 181, 189, 190
H herpes, 28, 44, 57
herpes simplex, 28, 57
H2, 94, 138, 147, 161, 162, 172
herpes simplex virus type 1, 28
half-life, 79
herpes zoster, 44
halitosis, xii, 255, 256
heterozygosity, 192, 193, 194
handling, 97
heterozygote, 183, 190, 191
haplotype, 197
278 Index
hexane, 122, 201, 233 hydrogen sulfide, 82, 103

high blood cholesterol, 100, 103 hydrolysis, 32
high blood pressure, 42 hydroperoxides, 94, 96
high fat, 9, 248 hydrophilic, 72, 75, 77, 78, 206, 209, 237
high risk, 60, 247, 252 hydrophobic, 201, 237
high temperature, 25, 34 hydroxyl, viii, 53, 62, 65, 111, 113, 140, 200
high-fat, 25, 47, 72, 108 hyperactivity, 105
high-performance liquid chromatography, 175, hypercholesterolemia, 12, 18, 44, 71, 155
264 hyperglycemia, 14
hippocampal, 80, 85, 107, 108, 110 hyperhomocysteinemia, 17, 18, 40, 60
hippocampus, 113 hyperlipidemia, 52
hippocrates, 2, 19 hyperphosphorylation, 148
hips, 247 hyperplasia, 247
histochemical, 125 hypersensitivity, 54
histological, 69, 82, 83, 232 hypertension, xi, xii, 8, 9, 12, 13, 18, 38, 43, 44,
histone, 24, 131, 134, 147, 246, 249, 252, 253 51, 58, 83, 100, 103, 155, 245, 246
histone deacetylase, 252 hypertensive, vii, 1, 12, 13, 45, 47, 51, 55, 93, 98,
histopathology, 127 108, 116, 153
HIV, 28, 34, 57, 206, 261 hyperuricemia, 220
HIV-1, 206 hypnosis, 260
HO-1, 90, 92 hypnotic, xii, 256, 257, 260
homeostasis, 21, 26, 132, 137 hypocholesterolemic, vii, 1, 3, 11, 36
homocysteine, 8, 17, 18, 38, 43, 46, 50, 51, 52, hypophosphatemia, 220
53, 56, 57, 58, 59, 60, 107 hypotension, xii, 255, 256
homogenized, 173, 201 hypotensive, 13
homology, 53, 140, 201, 209 hypothesis, 26, 137, 191, 224
hormonal therapy, 247 hypoxic, 44
hormone, 14, 247, 254 hysteria, xii, 246
hormones, 8, 252
horticulture, 195
host, 32, 175 I
household, 220
IAEA, 232, 242
household waste, 220
IAP, 133
HPLC, 43, 165, 166, 167, 173, 177
ICAM, 143
HSB, 41
ice, 131
human exposure, 220
id, 20, 190, 259
human genome, 140, 201
identification, 23, 24, 32, 194, 196, 212, 226,
human leukemia cells, 148
227, 250
human subjects, 40
identity, 226
humans, ix, xii, 9, 11, 12, 15, 31, 32, 39, 40, 42,
idiopathic, 92
79, 100, 103, 120, 121, 122, 142, 155, 181,
IgE, 33
187, 227, 229, 256
IGF, 249, 253
hybrid, 133, 146, 184, 187, 188, 191, 192, 194,
IGF-1, 249
195, 197
IL-1, 89, 92, 107, 141, 152
hybridization, 196
IL-10, 89
hybrids, 194
IL-6, 89
hydride, 162, 163, 164, 165, 176, 177, 178
IL-8, 136
hydrides, 163
imbalances, 216
hydro, 75, 77, 78, 206, 209, 237
immune disorders, 137
hydrocarbon, 249
immune function, 26, 41
hydrogen, viii, 12, 13, 42, 62, 65, 70, 82, 100,
immune response, ix, 22, 27, 60, 119
103, 171
immune system, 32, 216, 246
hydrogen peroxide, viii, 62, 65, 100
immunity, xi, 27, 137, 197, 245
Index 279
immunocompetence, 48 inflammatory response, 26

immunodeficiency, 27 influenza, 27, 52, 57
immunomodulator, 232 influenza vaccine, 27
immunomodulatory, vii, 1, 26, 27 information systems, 137
immunosuppression, 145 ingestion, 7, 50, 98, 99, 100
immunosuppressive, 43 inhalation, 50
immunotherapy, 48, 144 inherited, 51
implants, 247 inhibitor, 21, 23, 26, 40, 51, 81, 95, 128, 133,
impotence, 220 134, 136, 138, 141, 200, 206, 213, 247, 248,
in situ, 92, 177 252
in vitro, viii, x, 6, 9, 13, 15, 19, 21, 23, 27, 29, 30, inhibitor protein, 95
31, 36, 37, 38, 41, 50, 54, 55, 56, 57, 58, 61, inhibitors, x, xii, 15, 21, 23, 34, 42, 47, 128, 138,
64, 65, 66, 79, 80, 81, 84, 85, 86, 90, 93, 95, 143, 151, 152, 199, 200, 201, 212, 242, 255,
99, 103, 105, 109, 111, 112, 127, 141, 142, 257, 261, 266
146, 147, 148, 188, 197, 199, 212, 225, 226, inhibitory, x, 11, 15, 16, 23, 29, 35, 44, 45, 52,
228, 242, 248, 249, 252, 253, 261, 266 75, 81, 89, 128, 133, 134, 136, 137, 149, 199,
in vivo, viii, 3, 6, 15, 19, 20, 21, 22, 27, 28, 29, 201, 202, 205, 206, 207, 208, 209, 210, 212,
30, 36, 37, 42, 44, 50, 55, 58, 59, 61, 62, 65, 225, 251
71, 72, 73, 74, 78, 85, 86, 87, 93, 95, 97, 98, inhibitory effect, x, 11, 15, 16, 35, 44, 45, 52, 75,
99, 100, 102, 103, 109, 111, 125, 126, 127, 81, 89, 128, 134, 136, 149, 199, 202, 205, 206,
141, 142, 144, 147, 149, 150, 226, 227, 228, 208, 209, 251
248, 249, 252 initiation, xii, 14, 22, 25, 59, 76, 116, 125, 142,
inactivation, 124, 134, 136, 143, 149 144, 243, 246
inactive, 26, 137 injection, 21, 30, 68, 82, 87, 88, 91, 136, 176,
inbreeding, 192, 194, 195 178, 179, 259
incidence, viii, 19, 23, 93, 108, 119, 124, 126, injections, 35, 127
127, 140 injuries, 34
incompatibility, 192, 195, 197 injury, x, 15, 25, 66, 68, 69, 70, 73, 74, 76, 78,
incubation, 64, 72, 74 80, 82, 83, 84, 87, 88, 90, 92, 93, 97, 98, 100,
incurable, viii, 119 101, 102, 104, 105, 106, 107, 108, 111, 114,
indication, 2 115, 116, 125, 127, 144, 145, 152, 215, 221,
indicators, 241 229, 230, 243
indices, 125 innate immunity, 137
inducer, 86 inorganic, 224
induction, xii, 20, 21, 39, 50, 53, 55, 59, 68, 75, iNOS, 12, 68, 81, 83, 89, 92, 95, 96, 107, 109,
76, 81, 83, 93, 94, 95, 96, 101, 104, 125, 132, 141
133, 134, 135, 136, 137, 138, 139, 141, 142, insight, 23
143, 146, 149, 150, 211, 213, 217, 246, 248, insomnia, 220
249, 253, 256, 258, 261 instability, 6, 28, 36
induction time, xii, 256, 258 insulin, 14, 89, 101, 248, 253
inductor, 92 insulin like growth factor, 249
industrial, 178 integrin, 28, 57, 131, 146
industry, 220, 232 integrity, xi, 15, 188, 231, 233
infarction, 8, 18, 42, 56, 78, 81, 82, 87, 90, 97, interaction, xii, 9, 34, 36, 49, 90, 146, 193, 206,
103, 114 237, 256, 265
infection, xi, 22, 24, 55, 58, 150, 245 interactions, xii, 14, 32, 58, 140, 255, 257, 262,
infections, xi, 3, 27, 28, 31, 32, 245, 246 263, 265
infectious diseases, 28 interface, 166, 175
inflammation, 24, 25, 26, 82, 140, 152 interference, 164
inflammatory, 3, 23, 24, 25, 59, 82, 92, 141, 152 interferon, 74
inflammatory bowel disease, 26 interleukin, 136
inflammatory cells, 82 interleukin-2, 136
inflammatory disease, 25, 82 interstitial, 82
280 Index
interval, 258, 260

intervention, 24, 202, 216, 253
K
intestinal flora, 33
kappa, 26, 45, 81, 89, 104, 106, 137
intestinal tract, 33, 224
kappa B, 26, 45, 81, 89, 104, 137
intestine, 20, 34, 46, 70, 74, 123
karyotype, ix, 181, 183, 189, 190
intoxication, 221, 228
kidney, 7, 25, 36, 38, 39, 69, 70, 78, 79, 84, 93,
intracellular signaling, 249
94, 96, 97, 98, 99, 101, 127, 219, 220, 221,
intraperitoneal, 26, 35
222, 223, 225, 226, 228
intravenous, 21, 30, 43, 265
kidney failure, 219
intrinsic, 95, 107, 132, 134, 149, 225
kidneys, 35, 221, 224
inulin, 32
kinase, 47, 59, 84, 88, 90, 94, 108, 109, 116, 130,
invasive, 81, 128, 136, 142
131, 133, 135, 138, 147, 148, 206, 210, 212,
investigations, 103
247, 249, 253, 254
iodine, 4
kinase activity, 47, 84, 131, 133, 147, 249
ionic, 156, 206, 225
kinases, 103, 134, 138, 149
ionization, 50, 233, 264
Kirchhoff, 212
ionizing radiation, 233, 237, 238, 242, 243
knockout, 71
ions, 21, 30, 140, 173, 174, 221, 224, 225
iron, 3, 46, 89, 93, 103, 107, 121, 175, 176, 216,
227, 250, 259 L
iron deficiency, 227
irradiated foods, 232 labeling, 129
irradiation, x, 27, 66, 92, 138, 166, 169, 177, 231, laboratory studies, 19, 200, 249
232, 233, 235, 236, 237, 238, 241, 242, 243 lactate dehydrogenase, 68, 96, 127
irradiations, 169 Lactobacillus, 29
irrigation, 156 laryngeal cancer, 122, 143
irritability, 220 laser, 50
irritation, 224 LDL, 9, 12, 14, 25, 48, 65, 66, 71, 72, 74, 75, 80,
ischaemia, 105, 114 81, 89, 90, 100, 105, 109, 111, 212, 224, 228,
ischaemic heart disease, 17 246
ischemia, 66, 68, 69, 72, 73, 74, 78, 83, 84, 85, leaf sheaths, 184, 187
92, 98, 101, 107, 111, 113, 114 leakage, 127
ischemic, 9, 52, 66, 81, 84, 85, 87, 88, 101, 108, learning, xi, 26, 27, 60, 85, 86, 110, 111, 113,
111 245
ischemic heart disease (IHD), 9 17 left ventricular, 82
isoenzymes, 140, 261 legislation, 39
isoflavones, 120, 248 lending, 224
isolation, 242, 263 lesions, 18, 41, 120, 125, 140
isoniazid, 68, 111 leucine, 138
isorhamnetin, 13, 54 leukaemia, 22, 134, 150
isotope, 174 leukemia, x, 41, 48, 75, 79, 132, 133, 134, 148,
isozyme, 126, 141, 196 149, 151, 200, 202, 206, 210, 249
isozymes, 34, 194 leukemia cells, 41, 134, 148
leukemic, 22, 148, 149
leukocytes, 42, 66, 83, 89
J
leukotrienes, 89
Lewis acids, 225
jejunum, 94
libido, 247
JNK, 94, 138, 143, 210
life cycle, 187, 189
Jun, 59, 94, 115, 116, 138, 147, 149, 152, 153,
life span, 110, 136
210, 212
lifestyle, 8, 12
Jung, 264
ligands, 137
limitations, 171, 235
Index 281
links, 32 lymphocytes, 26, 53

linolenic acid, 248 lymphoid, 127
lipid, x, xi, 1, 9, 12, 18, 19, 21, 22, 24, 25, 31, 38, lymphoma, 22, 127
39, 42, 43, 44, 53, 55, 65, 66, 68, 71, 84, 87, lysine, 25
89, 92, 94, 95, 100, 103, 104, 105, 106, 107, lysis, 35
111, 112, 114, 115, 116, 140, 153, 215, 228, lysosomal enzymes, 81, 111
229, 231, 233, 234, 237, 238, 241, 242, 243,
264
lipid metabolism, 42, 53, 105, 153 M
lipid oxidation, 25
mace, 55
lipid peroxidation, 25, 39, 43, 55, 65, 103, 104,
macronutrients, 24
105, 106, 107, 111, 112, 114, 115, 116, 140,
macrophage, 54
228, 229, 237, 264
macrophages, 46, 52, 66, 72, 81, 89, 102, 108,
lipid peroxides, 68, 92, 95, 111
109, 141
lipid profile, 18, 31, 100
magnesium, 3, 32, 121, 175
lipids, 9, 11, 24, 25, 31, 39, 40, 48, 49, 50, 71,
Maillard reaction, 25, 36, 41, 107
232, 234, 236, 237, 238, 239, 240, 241, 242,
maintenance, 132
243, 264, 265
malate dehydrogenase, 81
lipophilic, 226
males, 19, 94, 122
lipopolysaccharide, 68, 72, 73, 89, 104
malignancy, 254
lipoprotein, 8, 9, 56, 65, 103, 105, 106, 107, 111,
malignant, 95, 107, 125, 132, 133, 137, 140, 149,
212, 224, 228
211
lipoproteins, 9, 14, 40, 56, 103, 109
malignant growth, 132
lipoxygenase, 23, 25, 40, 47, 53, 74, 89, 113,
malondialdehyde, 67
141, 152
malondialdehyde (MDA), 67, 68, 69, 70, 71, 82,
liquid chromatography, xi, 156, 165, 166, 167,
84, 90, 92, 94, 100, 127, 132, 142
175, 231, 264
mammal, 23
liver cells, 139
mammalian, 201, 205
liver damage, 34, 52, 78, 82, 87, 90, 97, 101, 110
mammalian cells, 135, 138, 140
liver enzymes, 34, 222
management, 12, 56, 221
livestock, 31
manganese, 121, 175, 176, 216, 250
loading, 50, 227
manufacturing, 5, 6, 37, 220
localization, 200, 205, 207
MAPK, ix, 90, 95, 104, 120, 133, 138, 249
location, 37, 136
MAPKs, 138, 139, 143, 151
locus, 197
market, vii, 1, 6, 7, 37, 186, 193, 226
LOD, 159, 161, 162, 165, 168, 169
marrow, 71
long period, 107, 247
masking, 142
longevity, 2, 26, 110
mass spectrometry, 50, 156, 160, 176, 178, 264
losses, 171, 173
mast cells, 26
low temperatures, 187
matrix, 50, 58, 136, 142, 189, 249
low-density, 105, 106, 212
matrix metalloproteinase, 249
low-density lipoprotein, 105, 106, 212
maturation, 262, 263
LPS, 68, 73, 89, 109, 141
MCA, 26
lumen, 11
meals, 9, 252
lung, 19, 20, 21, 22, 44, 46, 49, 54, 69, 70, 78,
measurement, ix, 17, 155, 166, 170, 177
79, 82, 84, 87, 92, 94, 99, 123, 124, 125, 128,
measures, 18, 189, 216, 228
131, 132, 136, 138, 139, 141, 142, 144, 146,
meat, 34
147, 149, 200, 246, 251
media, 36
lung cancer, 22, 44, 46, 49, 131, 149, 251
mediation, 38, 249, 253
lungs, 19, 96, 221
medication, 11
lutein, 141
medications, viii, xii, 2, 255, 257
lycopene, 114, 115, 248, 249, 250, 252, 253
medicinal plants, 120, 264, 265
lymphocyte, 27, 134
282 Index
medicine, xi, 2, 3, 27, 44, 105, 120, 147, 150, microcirculation, 59

211, 216, 245, 256 microencapsulation, 36, 49
meiosis, 183, 184, 212 microflora, 29
melanoma, 26, 128, 136, 146, 151 micronutrients, 24, 200, 224
melatonin, 101 micro-organisms, 54
membranes, vii, x, xi, 25, 35, 51, 106, 223, 226, microscopy, 231, 233
228, 229, 231, 233, 234, 235, 236, 237, 238, microsomes, xi, 8, 66, 89, 95, 124, 125, 126, 231,
241, 242, 243 233, 235, 236, 237, 238, 239, 240, 241, 261
memory, xi, 26, 85, 92, 110, 245 microtubule, 49, 59, 116, 146
memory deficits, 85 microtubules, 75
men, 16, 50, 60, 103, 210, 247, 248, 252 microwave, 49, 62, 66, 160, 163, 166, 171, 174,
meningitis, 30 178, 179
mercury, x, 170, 215, 216, 219, 220, 221, 222, microwave heating, 62
226, 227, 228 migration, 136, 142, 150
meristem, 232 mimicking, 44
mesophyll, 62, 64 mineralization, 164, 173
mesothelial cells, 89 minerals, 3, 120, 223, 250
messengers, 25 Ministry of Education, 38, 211
meta-analysis, 12, 52, 54, 59 mitochondria, 39, 92, 95, 132, 133, 134, 148
metabolic, vii, x, 1, 5, 14, 17, 36, 51, 82, 124, mitochondrial, x, 81, 86, 92, 95, 96, 105, 111,
125, 126, 156, 200, 205, 209, 260, 263 112, 132, 133, 134, 135, 143, 150, 187, 188,
metabolic pathways, vii, 1, 5, 260 201, 205, 209, 215, 221, 229
metabolism, ix, 8, 17, 20, 23, 28, 36, 38, 39, 40, mitochondrial membrane, 95, 133, 134
42, 43, 50, 53, 56, 57, 58, 59, 75, 83, 86, 102, mitogen, 138
105, 117, 119, 125, 126, 141, 144, 145, 146, mitogen-activated protein kinase, 138
147, 153, 250, 260, 261 mitosis, 22
metabolite, 13, 36, 82, 99, 247 mitotic, 146, 148, 213
metabolites, viii, 19, 21, 36, 45, 54, 79, 119, 125, mixing, 6, 225
127, 144, 176 MMP, 142, 248
metabolizing, 21, 50, 59, 93, 94, 101, 102, 104, MMP-2, 142, 248
114, 246, 263, 266 MMP-9, 248
metabolomics, 24 model system, 248
metal content, 176 models, viii, 2, 11, 13, 14, 21, 26, 37, 61, 65, 72,
metal extraction, 173 73, 74, 76, 78, 79, 80, 83, 84, 85, 86, 87, 88,
metal ions, 140, 173, 221 97, 98, 99, 104, 105, 123, 127, 136, 141, 248,
metalloproteinase, 136, 249 249
metalloproteinases, 253 modulation, ix, 18, 21, 70, 89, 97, 99, 109, 114,
metals, x, 55, 156, 166, 170, 174, 177, 178, 215, 119, 138, 146, 153, 246, 249
216, 217, 218, 223, 225, 226, 227, 230 moieties, 225
metastases, 142, 249 moisture, 3, 120
metastasis, 136, 137, 142, 151 molecular markers, 190, 192
metastatic, 84, 248, 252 molecular mechanisms, 226
metastatic cancer, 84 molecular medicine, 150
methane, 5 molecular structure, 84
methanol, 165, 173 molecules, ix, 24, 25, 45, 83, 85, 120, 137, 139,
methicillin-resistant, 58 140, 142, 209
methionine, 17, 18, 43, 50, 63, 167 monoamine, xii, 255, 257
methylation, 24, 125 monochromator, 233
methylene, 44 mononuclear cells, 22
methylenetetrahydrofolate reductase, 17, 57 monosaccharides, 32
metric, 155 monoterpenes, 120, 145
microbes, 27, 29 morbidity, 75
microbial, 3, 27, 52, 54, 60, 177 morphine, 261
Index 283
morphological, ix, 132, 134, 181, 182, 183, 186, natural, xi, 8, 14, 23, 24, 26, 27, 36, 38, 43, 45,
187, 189, 192, 193, 194, 196, 234 48, 49, 115, 120, 146, 148, 150, 152, 182, 183,
morphology, ix, 39, 101, 181, 182, 184, 186, 190 185, 192, 194, 200, 201, 216, 222, 224, 226,
mortality, 14, 19, 75, 81, 82, 93, 108, 122, 127 245, 249
mortality rate, 82 natural environment, 185
motor activity, 86 nausea, xii, 219, 220, 224, 255, 256
motor coordination, xii, 92, 255, 259, 260 neck, ix, 141, 181, 188, 189, 190, 193
mouse, 20, 21, 22, 35, 39, 40, 48, 51, 53, 56, 66, necrosis, 67, 74, 81, 89, 93, 101, 221
78, 96, 98, 102, 108, 110, 115, 125, 133, 142, neem, 101
144, 145, 146, 147, 151, 187, 228, 248, 252, negativity, 225
259 neoformation, 238
mouse model, 51, 115, 142, 248 neoplasia, 29, 56, 126, 151, 248, 249
mouth, 220, 224 neoplasms, 82
MPS, 222 neoplastic, 22, 47, 128, 132, 150
MPTP, 86, 87 neovascularization, 136
mRNA, 90, 93, 94, 137, 139, 141, 246 nephrectomy, 83, 87
MSC, 130, 131, 132 nephron, 219
mucoid, 134 nephropathy, 111, 153
mucosa, 33, 46, 96, 124, 141, 144 nephrotoxicity, 69, 72, 73, 78, 87, 93, 97, 98,
multidrug resistance, 102 103, 112, 113, 115, 229
multiple alleles, 192 nerve, 107
multiple factors, 8 nerve growth factor, 107
multivariate, 163, 189 nerves, 222
murine model, 151 nervous system, 84
muscle, 57, 75, 148, 219, 220 network, 135, 137
muscle cells, 75 neuroblastoma, 22, 95, 107, 128, 133, 137, 146,
muscle weakness, 220 147, 149
muscles, 13, 39 neurodegenerative, xi, 24, 26, 86, 88, 245
muscular contraction, 13 neurodegenerative diseases, 24, 26
musculoskeletal, 120 neurodegenerative disorders, 86
mustard oil, 21 neurological disorder, 216
mutagen, 66, 139, 151 neuronal cells, 26
mutagenesis, 21, 57, 59, 125, 126, 144, 145, 211 neuronal death, 108
mutations, 25 neurons, 57, 85, 108, 110
mycobacterium, 29 neuropathy, 220
myeloid, 41, 134, 150 neuroprotection, 113
myeloperoxidase, 68 neuroprotective, 80, 85, 86, 88, 92, 108
myocardial infarction, 8, 18, 42, 56, 78, 81, 87, neurotoxic, 84, 85, 86, 104
90, 97, 103, 114 neurotoxicity, 86, 87, 112, 229
myocardial necrosis, 67, 74, 81, 101 neurotrophic, 26, 85
NFkB, 81, 89
NF-kB, 26
N NF-κB, 89, 92, 107
Ni, 157, 158, 159, 160, 161, 163, 164, 166, 167,
NAA, 169, 177
168, 169, 174, 199
N-acety, x, 36, 68, 69, 75, 77, 81, 82, 101, 134,
niacin, 4
215, 221, 224
nickel, 175, 216
NaCl, 84
nicotine, 70, 73, 78, 96, 97, 105, 114
NAD, 21, 55, 93, 94, 151
nicotinic acid, 3, 121, 256
NADH, 81
nitrate, 19, 51, 96
nanoparticles, 134, 149
nitric acid, 172, 173
naphthalene, 70, 111
nasopharyngeal carcinoma, 138, 151
National Academy of Sciences, 58
284 Index
nitric oxide (NO), viii, 9, 12, 13, 16, 18, 25, 43, oils, vii, x, 1, 28, 37, 40, 58, 64, 105, 141, 143,
54, 62, 66, 68, 81, 83, 85, 89, 102, 107, 108, 152, 215, 256
109, 110, 114, 136, 150 oilseed, 242
nitric oxide synthase, viii, 12, 43, 54, 62, 68, 107, olefins, 212
114 oligosaccharide, 41
nitrogen, 65, 69, 83, 141, 189 oligosaccharides, 32, 44, 51
nitrosamines, 46, 144 olive oil, 122
nitrosative stress, 69, 78, 83, 93, 111 Olympic Games, 2
NMDA, 107 oncogene, 27, 55, 134, 148, 212, 248
NMR, 201 Oncogene
NO synthases, 12 oncology, 148
nodules, 248 Oncology, 49, 53, 105, 116, 146, 149, 251, 253,
non-destructive, 166 265
non-enzymatic, 140 onion, 2, 23, 31, 38, 39, 40, 41, 42, 44, 45, 46,
non-small cell lung cancer, 44 47, 48, 51, 53, 56, 62, 105, 126, 141, 152, 157,
normal, 11, 12, 16, 17, 18, 22, 26, 35, 39, 57, 68, 158, 163, 169, 173, 175, 176, 177, 182, 183,
69, 95, 98, 111, 116, 126, 128, 131, 132, 135, 186, 196, 217, 249, 264, 265
202, 210, 221, 227 online, 45, 47, 57, 123, 166, 177, 252
normal conditions, 26 on-line, 165
North America, 122, 247, 252 operator, 172
NOS, 12, 25 optical, 156, 160
nose, 220, 224 oral, 7, 12, 14, 18, 20, 21, 29, 36, 39, 42, 45, 67,
Nrf2, 69, 90, 94, 102, 107, 139, 143, 152 68, 79, 81, 95, 101, 124, 125, 126
NS, 65 organ, 82, 114, 125, 127, 139, 192
N-terminal, x, 138, 200, 205, 209, 210, 212 organic, 15, 40, 102, 121, 131, 156, 166, 171,
nuclear, 20, 26, 43, 45, 46, 47, 58, 81, 89, 90, 94, 172, 173, 211, 222
104, 106, 116, 125, 133, 134, 139, 144, 149, organic compounds, 121, 156
158, 167, 174, 190, 191, 200, 205, 207, 209 organic matter, 171, 172
nuclear factor kappa, 137 organic solvent, 173
nuclear genome, 191 organic solvents, 173
nuclear reactor, 167 organism, 25, 31
nuclei, 95, 129 organoselenium, 44
nucleic acid, 24, 31, 206, 209 ornithine, 75, 95, 125, 144
nucleic acid synthesis, 31 orthorhombic, 234
nucleoli, 92 oxidants, 172, 221, 225
nucleotides, 192 oxidation, 17, 24, 25, 48, 56, 65, 66, 70, 71, 72,
nucleus, 133, 137 74, 75, 80, 89, 105, 109, 111, 166, 212, 224,
nutraceuticals, 56, 127 228, 252
nutrient, 120, 136, 238 oxidative damage, viii, 26, 61, 68, 70, 71, 72, 73,
nutrient transfer, 238 74, 78, 83, 85, 86, 87, 92, 93, 94, 96, 98, 104,
nutrients, 37, 121, 237 109, 110, 112, 229, 264
nutrition, 38, 44, 59, 250 oxidative stress, viii, x, 25, 46, 61, 65, 66, 68, 69,
nutritional deficiencies, 37, 216 70, 73, 75, 76, 78, 79, 80, 82, 85, 88, 91, 92,
nuts, 178 93, 96, 97, 98, 100, 101, 102, 103, 104, 106,
107, 108, 109, 110, 111, 112, 113, 133, 137,
140, 146, 149, 152, 153, 215, 217, 221, 225,
O 228, 229, 248
oxide, viii, 12, 25, 38, 39, 43, 54, 62, 66, 68, 75,
obesity, viii, 8, 14, 119, 137, 155, 247
102, 107, 110, 114, 121, 125, 127, 136, 150,
observations, 86, 88, 131, 139
203
obstruction, 68, 73, 104, 264
oxygen, viii, 21, 24, 31, 43, 58, 62, 65, 80, 103,
occlusion, 68, 85
106, 107, 108, 110, 111, 112, 131, 135, 138,
occupational, 33, 216
147, 148, 149, 225, 229, 233, 248, 249
odors, 6, 32
Index 285
oxyradicals, 228 peroxidation, 25, 39, 43, 52, 55, 65, 103, 104,
ozone, 238, 242 105, 106, 107, 111, 112, 114, 115, 116, 140,
228, 229, 237, 264
peroxide, 21, 62, 65, 100
P peroxynitrite, viii, 62, 65, 108, 110, 112
perturbation, 16
p38, 90, 94, 95, 133, 138, 151
perturbations, 249
p53, 40, 46, 132, 133, 136, 149, 150, 249, 251,
PGE, 136
252, 253
P-glycoprotein, 266
PAA, 168, 177
pH, 3, 4, 5, 36, 62, 122, 171
pain, 3, 140, 219, 247
pH values, 62
paints, 220
phagocyte, 48
pairing, 166, 173, 183, 188, 195
pharmaceutical, ix, 13, 181
pancreas, 14, 123, 141
pharmaceuticals, 31
parainfluenza, 28
pharmacokinetic, 142, 260, 263
paralysis, 219
pharmacokinetics, 44, 53, 79, 260, 261, 266
parameter, 82, 156
pharmacological, 3, 50, 54, 80, 120, 249, 256,
parasites, 31, 120
260, 263
parasitic infection, 27
pharmacology, vii, x, 109, 215, 216
parenteral, 125
phenolic, 23, 24, 27, 43, 59, 89, 257, 260, 261,
parents, 189, 194
265
Parkinson’s disease, 86, 87, 115
phenolic compounds, 24, 27, 257, 265
particles, 8
phenotypes, 192
particulate matter, 178
phenotypic, 186, 196
pasta, 187
phenotypic plasticity, 186
pathogenic, 9, 50, 57, 197
phorbol, 65, 114
pathogens, 29
phosphate, 71, 92, 93, 171
pathophysiology, 58
phosphatidic acid, 235
pathways, vii, ix, 1, 5, 23, 81, 90, 94, 95, 97, 102,
phosphatidylcholine, 235
109, 114, 120, 137, 142, 150, 151, 210, 211,
phosphatidylethanolamine, 235
249, 250, 260
phosphatidylserine, 235
patients, 11, 14, 16, 17, 18, 25, 30, 31, 34, 37, 40,
phospholipase C, 16
41, 50, 52, 56, 75, 80, 89, 100, 103, 122, 210,
phospholipids, xi, 3, 11, 32, 231, 236, 239, 240,
216, 221, 225, 249, 250, 252, 263, 265
243, 256
Pb, 157, 158, 159, 160, 161, 162, 163, 164, 166,
phosphorous, 3, 121, 243, 250
167, 168, 169, 170, 174, 219, 220, 222, 223,
phosphorus, 233
224
phosphorylates, 137
PC12 cells, 80, 107
phosphorylation, 42, 47, 94, 95, 131, 133, 138,
PCR, 190
212, 250
PDGF, 138
photocatalysis, 165, 177
pearls, 100
photolysis, 166, 177
pectin, 3, 256
photon, 168, 169
penicillin, 3, 29
photosynthesis, 32
peptide, 31, 80, 86, 87, 107, 112
phylogenetic, 182, 190, 191
peptides, 4, 31, 48
phylogeny, 190, 191
perianth, 184
physical exercise, 24
perilla, 248
physical properties, 41
periodic, 158
physicians, 19, 40, 52, 55, 251
periodic table, 158
physicochemical, 16
periodontitis, 29
physicochemical properties, 16
peripheral neuropathy, 220
physiological, 12, 25, 44, 58, 121, 135, 169, 192,
permeability, 30, 51, 134, 223, 229
232, 237
permeabilization, 134
physiology, 103
286 Index
phytochemicals, viii, 8, 23, 24, 53, 119, 120, 123, polypeptides, 102
127, 132, 146, 211 polyphenols, 250
phytoestrogens, 252 polyploid, 189, 192, 193
phytotherapy, 251 polyploidization, 191
PI3K, 90, 138 polyploidy, 192, 196
pigments, 24 pomegranate, 250
pistil, 197 pools, 190, 191
PKC, 249 poor, 6, 122, 163
placebo, 12, 14, 16, 18, 51, 56 population, 46, 96, 122, 128, 131, 187, 189, 192,
placental, 94, 109 210, 212, 221, 226, 227
plants, viii, 2, 19, 44, 49, 61, 120, 156, 160, 177, population size, 187, 192
178, 183, 185, 187, 188, 189, 190, 192, 197, pore, 134
220, 224, 249, 257, 264, 265 positive correlation, 139
plaque, 44, 71, 103, 104 postoperative, 41, 265
plaques, 8, 9 potassium, 3, 69, 110, 112, 121, 174, 250
plasma, 7, 12, 16, 17, 18, 30, 36, 40, 43, 44, 50, powder, 6, 7, 8, 9, 11, 12, 14, 28, 33, 34, 35, 36,
52, 56, 60, 67, 71, 75, 79, 80, 81, 84, 89, 98, 49, 52, 55, 56, 62, 63, 64, 65, 67, 69, 71, 72,
99, 103, 105, 106, 153, 156, 158, 159, 160, 108, 112, 113, 126, 127, 128, 129, 131, 144,
175, 178, 264 250, 251, 261
plasma membrane, 16, 30, 52 powders, 145
plasma proteins, 17 power, 140, 160
plasticity, 186, 192 power plant, 160
platelet, 8, 12, 15, 16, 39, 40, 42, 45, 49, 51, 53, power plants, 160
56, 142, 152, 153, 228 prebiotics, 37, 42
platelet activating factor, 16 precedents, 165
platelet aggregation, 15, 16, 39, 40, 42, 45, 49, preclinical, xii, 255, 256
51, 53, 56, 152 pregnant, 228
platelets, 15, 16 preservatives, 27
platinum, 216 press, 104, 105, 113, 115, 116
play, x, 14, 18, 20, 24, 25, 26, 75, 122, 138, 139, pressure, xi, xii, 12, 13, 38, 39, 42, 45, 51, 54,
186, 190, 199, 210, 247, 262 169, 245, 246, 264
PLC, 165 prevention, viii, 3, 8, 18, 23, 26, 37, 42, 46, 48,
ploidy, ix, 181, 185, 186, 188, 192, 193 56, 59, 61, 68, 69, 70, 71, 80, 82, 83, 85, 86,
PMA, 124 93, 100, 105, 114, 119, 125, 137, 138, 139,
pneumonia, 28 140, 143, 144, 147, 151, 155, 210, 211, 216,
poisoning, x, 29, 215, 216, 220, 221, 222, 223, 221, 248, 249, 252
225, 226, 227, 228, 232, 256 preventive, xii, 9, 104, 120, 255, 256
polarity, 5, 20 primary tumor, 142, 151
pollen, 188, 197 primates, 85
pollination, 188, 191, 192, 194 pro-apoptotic protein, 22, 135
pollinators, 194 probe, 140, 261
pollutants, 216 probiotics, 42
poly(ADP-ribose) polymerase (PARP), 133, 134 producers, 156, 250
polyamine, 75, 113, 152 production, 6, 8, 12, 13, 14, 16, 18, 25, 27, 28,
polymerase, 95, 116, 133, 134, 137, 140, 141, 34, 54, 55, 65, 66, 72, 74, 81, 82, 83, 89, 90,
200, 206, 212, 213 95, 102, 109, 110, 111, 114, 133, 134, 138,
polymerization, 140, 201, 209, 210 147, 155, 156, 174, 183, 185, 187, 188, 189,
polymerization process, 209 196, 225
polymorphism, ix, 181, 184, 186, 193, 242, 263 productivity, 156
polymorphisms, 190, 191 progesterone, 221
polymorphonuclear, 89 prognosis, 130
polynucleotide, 206 pro-inflammatory, 23, 25, 48
polynucleotide kinase, 206 prokaryotic, x, 199, 205
Index 287
proliferation, viii, 18, 21, 46, 47, 54, 75, 81, 84, PSQ, 201
115, 116, 119, 126, 127, 128, 130, 131, 132, psychological stress, 27, 48, 58
134, 135, 136, 137, 138, 140, 142, 146, 147, psychosis, 220
148, 150, 188, 196, 212, 213, 222, 248, 250 public, 216
promoter, 23, 51 public health, 216
promyelocytic, x, 75, 132, 134, 200, 202, 206, Public Health Service, 227
210 pulmonary hypertension, 44
pro-oxidant, 85, 152, 225 pulmonary vascular resistance, 69
propagation, 188, 192, 194 pulse, 168, 177
prophylactic, 43, 56, 84, 88, 107 pulses, 177
proposition, 226 purification, 30, 242
prostaglandin, 89, 100, 102, 103, 140 pus, 28
prostaglandins, 3, 140, 256 putrescine, 75
prostanoids, 140 pyrene, 20, 21, 56, 124, 151
prostate cancer, vii, 22, 23, 46, 47, 52, 53, 59, 79, pyridoxamine, 81
81, 84, 95, 103, 108, 131, 134, 142, 143, 148, pyruvate, 91
149, 150, 200, 210, 212, 247, 248, 249, 250, pyruvic, 127
251, 252, 253, 254
prostate carcinoma, 75, 113, 140, 152
prostate specific antigen, 247 Q
prostatitis, 248
quality control, 160
protease inhibitors, 34, 261
quality loss, 232
proteases, 28, 103, 134, 149
quality of life, 216
protection, 13, 25, 26, 32, 75, 83, 85, 86, 94, 107,
quartile, 122
115, 120, 152, 226
quartz, 179
protective factors, 42
quercetin, 11, 13, 14, 16, 41, 45, 54, 90, 249, 253,
protective mechanisms, 88
258, 260
protein, 3, 8, 17, 18, 27, 30, 31, 36, 46, 48, 50,
quinine, 90
53, 58, 65, 69, 70, 75, 76, 80, 81, 83, 84, 90,
quinolinic acid, 85, 86, 112, 229
92, 93, 94, 95, 96, 102, 107, 108, 109, 120,
quinone, 21, 55, 93, 94, 139
127, 131, 132, 134, 135, 136, 137, 138, 141,
142, 147, 148, 150, 197, 206, 210, 246, 249,
250, 251, 253, 254 R
protein kinase C, 253
protein kinases, 138 radiation, xi, 25, 54, 125, 144, 171, 231, 233,
protein oxidation, 70, 80 237, 238, 242, 243, 247
protein synthesis, 76, 90 radical reactions, 26
proteins, 3, 17, 22, 24, 27, 31, 33, 36, 53, 91, 94, radio, 237, 238, 241
96, 132, 133, 134, 136, 137, 209, 216, 232, rain, 84
237, 238, 248, 249, 253, 254 Raman, 159, 163, 177
proteinuria, 69, 83 random, 190
proteolysis, 22 range, 2, 24, 34, 37, 82, 142, 164, 166, 167, 170,
proteolytic enzyme, 21 182, 186, 188, 190, 193, 197, 206, 246, 256
proteomics, 33 RAPD, 196
protocol, xii, 130, 255 ras, 55
protons, 169 RAS, 55
proto-oncogene, 27, 134 RAW, 109, 141
protozoa, 50 reactive oxygen species (ROS), 21, 24, 25, 43,
protozoan, 31 61, 65, 68, 69, 80, 82, 83, 84, 85, 88, 89, 90,
protozoan parasites, 31 92, 95, 96, 103, 106, 108, 129, 131, 133, 134,
PSA, 247, 249 137, 138, 139, 140, 147, 148, 149, 221, 225
pseudo, 187 reactivity, 7, 28, 66, 84, 153
pseudomonas, 29 reagent, 164, 165, 168, 170, 177, 178, 202, 257
288 Index
reagents, 173, 202, 203 reverse transcriptase, 206

receptors, 137 rhinitis, 33
recombination, 202, 210 Rho, 48, 149
recovery, 86, 173, 221 riboflavin, 3, 121
rectal examination, 247 ribose, 83, 133, 134
red blood cells, 25, 35, 47, 107, 108, 115, 116, ribosomal, ix, 181
230 ribosomes, 234, 237
redness, 33 rice, 179
redox, 21, 47, 50, 58, 95, 104, 110, 111, 115, ringworm, 30
135, 138 risk, vii, xi, 1, 8, 9, 12, 14, 15, 17, 18, 19, 25, 29,
reductases, 139 32, 38, 40, 41, 42, 44, 46, 50, 52, 53, 56, 60,
reflexes, 219 102, 120, 122, 143, 160, 188, 200, 210, 212,
regenerate, 188 223, 245, 247, 252, 256, 265
regeneration, 196 risk factors, 8, 9, 12, 14, 18, 38, 44, 50, 53, 143
regression, 247 risks, 51, 171
regular, 25, 46, 144, 259 RNA, 28, 95, 116, 137, 141, 206, 232
regulation, ix, 24, 38, 94, 95, 108, 119, 132, 134, rodents, 85, 126
136, 137, 142, 148, 149, 241, 242, 250 room temperature, 6, 28, 121, 233
regulations, 156, 232 rotavirus, 28
regulators, 232 rural, ix, 181, 189
regulatory bodies, 18 rural communities, 189
relationship, vii, xii, 14, 19, 23, 38, 52, 59, 108, rutin, 258, 260
201, 211, 255
relationships, ix, 181, 188, 195
relatives, ix, 181, 190, 191, 195, 196 S
relevance, 38, 85, 86, 95, 97, 194, 249
S phase, 129, 130, 131
remethylation, 18
SAC, xii, 4, 5, 6, 7, 8, 9, 11, 13, 21, 24, 27, 30,
renal, 50, 69, 78, 83, 87, 93, 107, 110, 111, 114,
36, 64, 68, 73, 74, 75, 76, 78, 79, 80, 81, 82,
220, 229
83, 84, 85, 86, 87, 88, 93, 99, 122, 123, 127,
renal failure, 69, 83, 87, 220
128, 139, 140, 142, 200, 246
repair, ix, 22, 119, 125, 201, 202, 205, 210, 248
safety, 6, 7, 34, 53, 167
repair system, ix, 119
saline, 258
reparation, 40, 45, 119, 136, 155, 167, 174
salmon, 205
reperfusion, 66, 68, 69, 72, 73, 74, 83, 85, 87, 92,
salmonella, 29, 47
97, 98, 101, 105, 107, 113, 114
salt, 12, 193
repetitions, 258
salts, 225
replication, 28, 190, 211
sample, ix, 33, 155, 156, 158, 160, 163, 164, 166,
repressor, 81
167, 170, 171, 173, 174, 177, 237
reproduction, 128, 183, 188, 192, 194
saponin, 23
resection, 256
saponins, viii, 1, 3, 7, 11, 24, 27, 31, 120, 156
residential, 216
satellite, 184
residues, x, 171, 176, 200, 205, 208, 209, 224
saturated fat, 109, 247
resin, 174
sawdust, 219
resistance, 29, 31, 69, 102, 125, 188, 196
SBP, 12
resources, 188, 194
scavenger, 88, 90, 116, 249
respiration, 57, 120
scientific community, 156
respiratory, 81
SCs, 142
restriction enzyme, 195
seafood, 92
resveratrol, 248
search, 37, 120, 188
retention, 248
secretion, 14, 48, 102, 136
reticulum, 237
sedentary, 248
retina, 133, 146
seed, 185, 187, 188, 195, 242, 247
retinoblastoma, 249
seedlings, 188, 196
Index 289
seeds, 31, 53, 183, 187, 188, 192, 194, 195 skin, 19, 21, 22, 26, 39, 40, 52, 53, 73, 77, 79, 84,
segregation, 188 106, 123, 124, 138, 141, 151, 196, 200, 219,
selectivity, 163, 165, 170 220, 224, 248, 256
selenium, viii, 1, 3, 23, 24, 36, 46, 121, 126, 130, skin cancer, 124, 138
144, 147, 163, 164, 165, 166, 170, 175, 176, sleep, xii, 256
177, 178, 179, 221, 222, 228, 248, 250, 252 Sm, 157, 167, 168, 174
SEM, 88, 203, 205, 206 small intestine, 20, 34, 123
semi-natural, 182 smoke, 24, 219, 220, 221
senescence, 25, 85, 87, 110, 111, 243 smoking, 8, 9, 18, 19, 103, 122, 248
sensation, 219 smooth muscle, 13, 57, 148
sensitivity, 29, 128, 163, 164, 165, 170, 171, 174, social costs, 26
240 SOD, 25, 67, 68, 69, 71, 74, 78, 81, 83, 84, 90,
separation, 6, 167, 170, 243 92, 93, 94, 96, 97, 98, 99
sepsis, 24 sodium, 3, 15, 21, 42, 70, 87, 103, 121, 171, 227,
series, 87, 109, 128, 146, 183, 213, 263 258
serine, 249 sodium arsenate, 21
serum, 9, 11, 13, 14, 16, 18, 32, 39, 40, 49, 50, software, 233
56, 59, 65, 68, 69, 83, 84, 92, 93, 111, 127, soil, 3, 121, 163, 178, 193
265 soils, 156, 178, 185, 193
serum glutamic pyruvic transaminase, 127 solid phase, 164, 178
severity, 11, 125, 128, 228 solvent, 5, 41, 173
sex, 252 solvents, 44, 173
sex hormones, 252 soy, 50, 252
sexual reproduction, 192 soybean, 23, 40, 141, 152, 243
sexuality, 193 spatial, 110, 192
shape, 182, 184, 185, 186 spatial memory, 110
shares, 209 speciation, 156, 157, 160, 164, 165, 166, 174,
sheep, 141 175, 176, 177
shigella, 29 specificity, 171
shoot, 188, 196 spectrophotometric, 229
short period, 4, 222 spectrophotometry, 176, 178, 179
short-term, 44, 92, 153 spectroscopy, 158, 161, 166, 169, 170, 173, 175,
short-term memory, 92 177
shyness, 220 spectrum, 22, 30, 31, 86, 125, 170, 201, 249
sickle cell, 25, 39 speed, 164, 259
side effects, vii, viii, xii, 2, 31, 34, 36, 120, 216, spermatogenesis, 35
221, 223, 225, 226, 255, 256 spermine, 75
sign, 234 SPF, 139
signaling, ix, 21, 81, 90, 94, 97, 109, 119, 136, S-phase, 129, 130, 131, 132
137, 138, 142, 143, 147, 151, 249, 254 spices, viii, 8, 48, 56, 107, 119, 122, 158, 164,
signaling pathway, ix, 81, 90, 94, 97, 109, 119, 169, 173, 175, 176, 177, 178, 179, 257
137, 143, 151, 254 spinach, 242
signaling pathways, ix, 81, 94, 97, 109, 120, 137, spine, 247
151 spleen, 223
signalling, 21 spore, 30
signals, 34, 102, 136, 138, 143 Sprague-Dawley rats, 125
silica, 201, 233 sprouting, x, 231, 232, 233, 237, 238, 241, 242
silver, 216 stability, 37, 44, 109, 122
similarity, 16, 190 stages, 41, 53, 121
single nucleotide polymorphism, 190 stamens, 184
sites, 33, 184, 190, 191, 192, 209, 224, 233 standard deviation, 163, 164, 166, 170, 171
skills, 85 standardization, 6
standards, 233
290 Index
Staphylococcus aureus, 28, 58 supply, 135, 222

starches, 14 suppression, x, 27, 48, 54, 76, 89, 96, 134, 136,
statin, 41 140, 141, 200, 202, 246, 247
statins, 34 suppressor, 248
statistical analysis, 163 surface area, 71
sterile, 194 surgery, 34, 41, 57, 101, 145, 247, 250, 265
steroid, viii, 1, 3, 8, 11, 23, 31 survival, 26, 80, 85, 134, 135, 138, 213, 250
steroid hormones, 8 susceptibility, 29, 43, 71
sterols, 233, 238 symptom, 217
stigma, 188 symptoms, 33, 140, 221, 223, 226, 247, 248, 252,
stimulant, 2 256, 257
stings, 2 syndrome, 27
stock, 186 synergistic, 25, 30, 52, 222
stomach, vii, xi, 1, 19, 22, 29, 33, 60, 70, 79, 84, synovitis, 141
122, 123, 125, 141, 143, 144, 200, 210, 212, synthesis, 8, 13, 14, 15, 26, 31, 38, 42, 43, 44, 49,
220, 224, 245 50, 55, 75, 76, 90, 95, 125, 128, 130, 140, 201,
stomatitis, 34 217, 248
storage, vii, x, xi, 4, 6, 16, 25, 36, 37, 41, 51, 62, systems, ix, 20, 25, 26, 71, 115, 119, 127, 137,
64, 231, 232, 233, 234, 235, 236, 237, 238, 167, 173, 177, 229, 248
241, 242, 243, 256 systolic blood pressure, 12
strain, 28
strains, 29, 30, 85
S-transferase (GST), 20, 25, 68, 126 T
strategies, 24, 86, 163, 176, 177, 192, 211
T cells, 45, 81, 104
streptococci, 45
T lymphocyte, 134
streptomyces, 55
T-2 toxin, 196
striatum, 86, 105
tachycardia, 219
stroke, 9, 88, 93, 108, 116
target organs, 221
strokes, 246
targets, 44, 120, 148, 202, 250
subsidy, 211
taste, 37
substances, xi, 11, 34, 67, 75, 137, 139, 220, 222,
taxa, 182, 184, 192
225, 229, 242, 245
taxonomic, ix, 2, 181, 184, 196
substitutes, 200
taxonomy, 190, 195
substitution, 23
tea, 2, 51, 120, 132, 141, 148, 179, 248, 249, 250,
substrates, 201
252
sucrose, 32, 60
tellurium, 176, 177, 216
sugar, 14, 52, 246, 265
telomerase, 148, 206
sulfa drugs, 3
TEM, 233
sulfate, 70, 73, 117, 127
temperature, 5, 6, 28, 32, 36, 121, 172, 173, 233,
sulfonylurea, 14
243, 259
sulfur, vii, x, xi, 1, 27, 34, 35, 36, 40, 52, 96, 101,
temperature gradient, 172
105, 106, 110, 121, 124, 131, 135, 143, 149,
temporal, 24
156, 199, 200, 202, 210, 211, 212, 215, 222,
tenascin, 24
224, 225, 226, 245, 251, 256, 263
tendon, 219
sulfur dioxide, 121
testes, 221
sulphur, vii, viii, xi, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 16,
testicular torsion, 70, 73, 115
17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31,
testosterone, 96, 98, 113, 247, 248, 252
36, 45, 52, 158, 167, 175, 225, 245
testosterone levels, 248
request104, 105, 114, 139, 140, 225
thallium, 216
superoxide dismutase, 25, 34, 67, 104, 105, 139,
theaflavin, 114
140, 225
therapeutic agents, 29, 54
supplements, vii, 1, 6, 7, 8, 11, 12, 24, 32, 33, 34,
therapy, 9, 12, 27, 34, 36, 41, 50, 92, 138, 150,
37, 44, 49, 53, 65, 157, 163, 177, 246, 266
221, 224, 226, 247, 250, 252, 254
Index 291
thiamin, 250 transcription, 22, 26, 81, 90, 102, 137, 139, 141,
thiobarbituric acid, 66, 127 142, 143, 246
thioredoxin, 28, 139 transcription factor, 26, 102, 137, 138, 139, 143
threat, x, 215 transcription factors, 26
threats, 31, 226 transcriptional, 8, 52, 112, 141, 251
three-dimensional, 136, 209 transcriptomics, 24
threonine, 249, 250 transduction, 211
threshold, 192 transfer, 53, 96, 187, 188, 237, 251
thrombosis, 15, 53 transformation, 27, 227
thrombotic, xi, 200, 246 transformation product, 28
thromboxanes, 15 transformations, 5
thrombus, 15, 26, 39 transgenic, 42, 248, 252
thymidine, 128 transgenic mouse, 248
thymocytes, 90, 92 transition, 81, 134, 190, 206
thymus, 125 transitional cell carcinoma, 48, 144
thyroid, 220, 221 translocation, 90, 133, 134, 143
tin, 216 transmembrane, 96, 133
TiO2, 165, 166, 177 transmission, xi, 183, 231, 233
tissue, vii, 1, 21, 68, 86, 92, 94, 105, 114, 115, transmission electron microscopy, xi, 231, 233
116, 125, 126, 132, 136, 137, 166, 201, 227, transplant, 50
229, 232, 237, 241, 248, 252, 259 transplant recipients, 50
tissue engineering, 150 transplantation, 149
tissue homeostasis, 132, 137 transposon, 29
TNF, 48, 89, 92, 107, 110, 132, 136 transsulfuration, 18, 51
TNF-alpha (TNF-α), 48, 89, 92, 107,110 transurethral resection, 256
tobacco, 24, 55, 125, 144 trauma, 24
tobacco smoke, 24 trial, 40, 52, 109, 189, 248, 252, 259
tolerance, 187 triglyceride, 49
tomato, 70, 108, 253 triglycerides, 8, 9, 11, 13, 14, 25, 40
total plasma, 17 tripeptide, 76, 217
toxic, 34, 35, 76, 79, 80, 84, 85, 90, 99, 139, 156, trypsin, 28
169, 216, 221, 222, 225, 226, 227 tuberculosis, 30
toxic effect, 35, 84, 99, 216, 222, 226, 227 tubers, 232
toxic metals, 216 tubular, 93, 100, 220
toxic substances, 139 tumor, 21, 23, 27, 40, 47, 52, 53, 54, 55, 57, 76,
toxicities, x, 106, 145, 215 84, 94, 95, 96, 100, 106, 115, 124, 125, 127,
toxicity, vii, x, 6, 21, 27, 32, 34, 35, 37, 42, 49, 128, 130, 131, 136, 137, 138, 140, 142, 143,
58, 69, 70, 72, 73, 74, 79, 80, 81, 82, 85, 86, 146, 147, 148, 149, 150, 151, 153, 211, 246,
87, 90, 92, 97, 98, 100, 103, 107, 110, 111, 248, 249, 250, 251
114, 115, 120, 127, 142, 215, 216, 217, 219, tumor cells, 21, 22, 47, 54, 106, 115, 124, 128,
220, 221, 223, 224, 225, 226, 227, 263, 266 130, 131, 142, 143, 146, 147, 148, 149, 211,
toxicological, 27 246
toxicology, 107 tumor growth, 21, 55, 124, 136, 142, 151, 250
toxin, 28, 29, 86, 92, 196 tumor invasion, 249
toxins, 86, 139, 223 tumorigenesis, 53, 126, 137, 145, 246
trace elements, ix, 36, 155, 156, 159, 160, 161, tumors, xii, 2, 20, 22, 23, 25, 39, 47, 52, 120,
163, 166, 167, 168, 169, 171, 173, 174, 175, 123, 124, 126, 142, 144, 145, 151, 210, 246,
176, 177, 178, 226 256
trade, 6 tumour, 19, 35, 57, 89, 150
training, 259 tumour growth, 35, 150
traits, ix, 181, 183, 186, 188, 189, 190, 192 tumours, 19, 23, 35, 84
transcriptase, 206 turnover, 11, 132
TXA2, 16
292 Index
type 2 diabetes mellitus, 56 vasoconstriction, 15

type II diabetes, 14 vasodilator, 13
vegetable oil, 6, 7
vegetables, viii, 3, 8, 14, 19, 45, 46, 60, 119, 122,
U 123, 126, 143, 148, 151, 152, 155, 158, 160,
169, 170, 176, 177, 178, 187, 210, 212, 238,
UGT, 139
249, 251, 266
ulcer, 33
vegetative reproduction, 183
ulceration, 34
vein, 90, 138, 149, 150
ultrastructure, 39
velocity, 207
ultraviolet B, 54
vero, 229
undernutrition, 230
versatility, 164
uranium, 216
vessels, 15, 172
urea, 69, 83
Vibrio cholerae, 29, 197
urea nitrogen, 83
Vicia faba, 229
urinary, 28, 69, 70, 73, 100, 113, 151, 227, 248
virulence, 28
urinary bladder, 70, 73, 113
virus, 22, 27, 55, 57, 188, 194, 209
urinary retention, 248
viruses, 57
urinary tract, 28, 248
vitamin B1, 57
urinary tract infection, 28
vitamin B12 deficiency, 57
urine, 7, 36, 98, 103, 221, 224, 225, 226, 247
vitamin B6, 17, 58, 250
urokinase, 47
vitamin C, 3, 71, 92, 120, 121, 250
urticaria, 33
vitamin D, 248, 252
UV, 24, 25, 27, 138, 165, 166, 169, 170, 171,
vitamin E, 3, 92, 93, 101, 105, 248
177, 242
vitamins, viii, 2, 3, 17, 24, 76, 78, 84, 121, 200,
UV irradiation, 27
256
UV light, 24, 25
VLDL, 9
UV-irradiation, 166, 177
voltammetric, 170, 177
vomiting, 219, 220, 224
V
W
vaccine, 27
vacuum, 29, 49
walking, 92, 216
vaginitis, xii, 30, 246
Warfarin, 34, 50
values, 17, 23, 38, 62, 68, 88, 132, 160, 164, 169,
warts, 224
175, 202, 203, 206, 207, 209, 224, 236, 238,
wastewaters, 170
239, 241, 259, 260
water, x, xii, 5, 6, 7, 9, 11, 19, 24, 29, 32, 34, 36,
vanadium, 175, 216
64, 69, 70, 76, 82, 84, 85, 99, 103, 113, 125,
vancomycin, 29, 47
128, 130, 131, 135, 148, 152, 165, 170, 178,
vapor, 162, 165, 177
189, 193, 200, 201, 215, 219, 220, 221, 225,
variability, 188
232, 246, 258
variables, 11, 24
water-soluble, 5, 6, 7, 24, 32, 34, 36, 64, 103,
variance, 259
125, 128, 135, 152, 200
variation, ix, 5, 122, 181, 184, 186, 187, 188,
weight loss, 12, 35, 95
189, 191, 192, 193, 195, 196, 197
winter, 187
vascular bundle, 62, 64
Wistar rats, 69, 107, 114, 126, 139, 152
vascular cell adhesion molecule, 90, 109
women, 14, 47, 103, 122, 143
vascular disease, 18, 40, 42, 43, 59, 89
woodland, 182
vascular diseases, 89
workers, 18, 33, 223
vascular endothelial growth factor (VEGF), 136,
workplace, 221, 224
149
World Health Organization (WHO), 226, 232,
vascular system, 56
264
vascular wall, 9
World War II, xi, 245
Index 293
worms, 2 yield, ix, 6, 122, 124, 181, 187, 188, 189, 190,
wound healing, 25, 135 194
yuan, 147
X
Z
xenobiotic, 75, 117, 141
xenobiotics, 261 zinc, 4, 121, 175, 176, 177, 179, 216
xenograft, 59, 84, 96, 103, 149, 248, 251 Zn, 157, 158, 159, 160, 161, 162, 163, 164, 166,
xenografts, 57, 84, 95 167, 168, 169, 170, 171, 172, 174, 176, 177
X-ray diffraction, 233, 235, 242
X-rays, 166
Β
Y β-amyloid, 80, 86
yeast, 31, 196, 209

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FOOD AND BEVERAGE CONSUMPTION AND HEALTH SERIES

GARLIC CONSUMPTION AND HEALTH

Marketing Food to Children and Milk Consumption and Health

Red Wine and Health Alcoholic Beverage Consumption

Handbook of Green Tea and Health Fruit and Vegetable Consumption

GARLIC CONSUMPTION AND HEALTH

MIHAIL PĂCURAR AND GAVRIL KREJCI

Nova Science Publishers, Inc.

NOTICE TO THE READER

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Available upon request.

ISBN: 978-1-61324-623-8 (eBook)

Chapter 9 Health Effects of Garlic and Prostate Cancer 245

A COMPREHENSIVE SURVEY OF GARLIC

Alejandra Cardelle-Cobas, Ana Cristina Soria,

THE CHEMISTRY OF GARLIC

Properties Values Minerals Values Vitamins Values

Energy 119 kcal Potassium 446 mg Thiamin (Vit. B1) 0.16 mg

Moisture 70 % Phosphorus 134 mg Riboflavin (Vit. B2) 0.02 mg

Protein 4.3 g Magnesium 24.1 mg Niacin (Vit. B3) 1.02 mg

Carbohydrate 24.3 g Sodium 19 mg Piiridoxin (Vit. B6) 0.32 mg

Fiber 1.2 g Calcium 17.8 mg Folic acid 4.8 μg

GARLIC CONSUMPTION AND GARLIC SUPPLEMENTS

Table 2. Garlic supplements on the market.

Garlic supplement Main compounds and characteristics

Garlic powder Commercially available in capsule and tablet forms.

Garlic essential oil Commercially available as oil capsules.

Garlic oil macerate Commercially available as soft gel capsules.

EFFECTS RELATED TO CARDIOVASCULAR DISEASE

Effects on Levels of Serum Lipids (Cholesterol and Triglycerides)

Cholesterol is an extremely important biological molecule that has roles in membrane

Of particular clinical importance is the abnormal deposition of cholesterol and

Figure 2. Cholesterol biosynthesis pathway.

Active Compounds and Anti-Cholesterolemic Pathway by Garlic Derivatives

Active Compounds and Anti-Hypertensive Pathway by Garlic Derivatives

Anti-Hyperglycaemic or Anti-Diabetic Potential

Active Compounds and Anti- Hyperglycaemic Pathway by Garlic Derivatives

Anti-Platelet or Anti-Thrombotic Effect

Active Compounds and Anti- Platelet Pathway by Garlic Derivatives

Antiplatelet activity is substantially affected by genotype, environment and storage

Homocysteine (Hcy) is a sulphur-containing amino acid formed during metabolism of

Figure 5. Homocysteine: a risk factor for cardiovascular disease.

EFFECTS ON CANCER AND MUTAGENESIS

methylbenzylamine), by inhibiting the conversion of procarcinogens to ultimate carcinogens

Large-scale gene expression analysis in combination with functional assays yields a

The bacterial strain Staphylococcus aureus causes pus-producing infections, such as

as well as Streptococcus (including S. viridans and S. haematyticus), Vibrio cholerae,

Literature on the antiparasitic capacity of garlic focuses mainly on protozoan parasites.

Drug and Chemical Interactions

Dosage, Administration Route and Formulation Type

Figure 9. Summary of the multiple health-promoting effects of garlic.

Basksh R. & Chughatai, M. I. D. (1984). Influence of garlic on serum cholesterol, serum

Higdon J. & Lawson L. (2005). Garlic and organosulfur compounds. Micronutrient

transcriptional activity in colon cancer cells: structure-activity relationship. Japanese

Sharifi, A. M., Darabi, R. & Akbarloo, N. (2003). Investigation of antihypertensive

atherosclerosis. Proceedings of the National Academy of Sciences of the United States of

MEDICINAL PROPERTIES OF GARLIC: IMPORTANCE

Perla D. Maldonado1, Daniel Limón2, Sonia Galván-Arzate3, Abel

Table 1. Main organosulfur compounds found in garlic presentations

Garlic Presentation Organosulfur compound

1.1. Garlic Powder, Garlic Extracts, Garlic Homogenates, and Allicin

The following is a compilation of studies evidencing the therapeutic potential of garlic

1.1.1. In vitro studies