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Cancer Ther. Author manuscript; available in PMC 2008 November 17.
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Cancer Ther. 2008 ; 6(b): 495–510.
From here to eternity - the secret of Pharaohs: Therapeutic

potential of black cumin seeds and beyond
Subhash Padhye, Sanjeev Banerjee, Aamir Ahmad, Ramzi Mohammad, and Fazlul H
Sarkar*
Department of Pathology and Division of Internal Medicine, Barbara Ann Karmanos Cancer Institute,
Wayne State University, School of Medicine, Detroit, MI- 48201, USA
Summary
Over many centuries humans have been mining the bounties of nature for discovering substances
that have been used for the treatment of all human diseases; many such remedies are useful even
today as modern day medicine. Emerging evidence also suggests that the search is still continuing
for harnessing active compounds from nature in combating human illnesses although pharmaceutical
industries are equally active for synthesizing small molecule compounds as novel therapeutics. The
lesson learned over many centuries clearly suggests that further sophisticated search for finding
compounds from natural resources together with robust characterization and chemical synthesis will
lead to the discovery of novel drugs that may have high therapeutic efficacy against all human
diseases including cancer. Black cumin seed (Nigella sativa) oil extracts have been used for many
centuries for the treatment of many human illnesses, and more recently the active compound found
in black seed oil, viz. thymoquinone (TQ) has been tested for its efficacy against several diseases
including cancer. However, further research is needed in order to assess the full potential of TQ as
a chemopreventive and/or therapeutic agent against cancers. Here, we have summarized what is
known regarding the value of black seed oil and its active compound TQ, and how this knowledge
will help us to advance further research in this field by creating awareness among scientists and health
professionals in order to appreciate the medicinal value of TQ and beyond.
Keywords
Thymoquinone; Chemoprevention; Chemotherapy
I. Introduction
Out of the several accompanying articles found in the tomb of Egyptian Pharaoh Tutankhamen
were the seeds of Black cumin [Nigella sativa; (Zohary and Hopf, 2001)] not to be mistaken
with common cumin seed (Cuminum cyminum). It is a spice that grows in the Mediterranean
region and in Western Asian countries including India, Pakistan and Afghanistan. The
historical references to these seeds are also found in some of the oldest religious and medical
texts. For example, it is referred to as ‘Melanthion’ by Hippocrates and Dioscorides, while the
Bible describes it as the ‘curative black cumin’ (Isaiah 28:25, 27 NKJV). It is, therefore, no
wonder that they were thought to be worthy accompaniments in the ‘From Life here to Eternity’
by the pharaoh as described earlier.
*Correspondence: Fazlul H Sarkar, Department of Pathology and Division of Internal Medicine, Barbara Ann Karmanos Cancer
Institute, Wayne State University, School of Medicine, Room-740 HWCRC Bldg., 110 E Warren, Detroit, MI 48201, USA; Tel:
313-576-8327; Fax: 313-576-8389; E-mail: fsarkar@med.wayne.edu
Padhye et al. Page 2
The black cumin herb goes by many different names. For example, in old Latin it is called as
‘Panacea’ meaning ‘cure all’ while in Arabic it is termed as ‘Habbah Sawda’ or ‘Habbat el
Baraka’ translated as ‘Seeds of blessing’. In India it is called as Kalonji while in China it is
referred as Hak Jung Chou (Aggarwal et al, 2008). The plant belongs to the Ranunculaceae
family of flowering plants and genus of about 14 species including Nigella arvensis, Nigella
ciliaris, Nigella damascene, Nigella hispanica, Nigella integrifolia, Nigella nigellastrum,
Nigella orientalis and Nigella sativa, respectively. Among these, Nigella sativa is the species
most exhaustively investigated for therapeutic purposes although other species have also been
implicated for therapeutic uses (Aggarwal et al, 2008).
The species grow to 20-30 cm tall, with finely divided leaves wherein the leaf segments are
narrowly linear to threadlike (Figure 1A). The flowers are white, yellow, pink, pale blue or
pale purple, with 5-10 petals. The fruit is a capsule composed of several united follicles, each
containing numerous seeds while in some species (e.g. Nigella damascena), the capsule is large
and inflated. The parts of the plant most commonly used for the therapeutic purposes in the
“Alternative Medicinal” systems are the seeds (Figure 1B) which are contained in an inflated
capsule formed from the united follicles containing considerable amount of oil having pungent
and bitter taste. Commonly the seeds are used primarily as a spice and food preservative. In
folk medicinal practices they are ingested with food or mixed with honey and are primarily
used as lactogogues, carminitative and antihelmnthic agents. The seeds have also been used as
diuretics, anti-hypertensive, muscle relaxants and as immunity enhancers in immune-
compromised people. Importantly, the seeds have been reported to be safe when used orally
in moderate amount in food (DerMarderosian. et al, 2005). They have been shown to stimulate
uterine contractions when used in large amounts, leading to abortion (Aqel and Shaheen,
1996). There are reports that the oil from the seeds can be used to treat dermatitis topically
(Zedlitz et al, 2002). Several beneficial pharmacological effects have been attributed to various
crude or purified components of these seeds including antihistaminic (Chakravorty, 1993),
antihypertensive (Zaoui et al, 2000), hypoglycemic (Al-Hader et al, 1993), antifungal (Khan
et al, 2003), anti-inflammatory (Al-Ghamdi, 2001) along with significant anti-neoplastic
(Worthen et al, 1998) activities. These studies collectively provide early indication that further
development of agents derived from black cumin seeds could be useful in modern medicine.
A. Chemistry of the active compounds found in black seed extracts

Most of the earlier studies on this plant involved use of either the seeds or the oil extracted
from it. For example, the ethanol extract of Nigella sativa seeds has been shown to possess
antitumor activity as well as lifespan expanding activity in mice bearing Ehrlich ascites tumor
cells by Musa and co-workers (Musa et al, 2004). Benkaci-Ali and co-workers (Benkaci-Ali
et al, 2006) investigated different methods of extracting oil from Nigella sativa and found that
microwave extraction gave the best results in terms of reduction of extraction time and better
yield in obtaining higher amounts of oil, and this is particularly significant since microwave
extraction is a green technique reducing environmental burden.
The chemical composition of the black Nigella sativa seed is diverse and contains amino acids,
proteins, carbohydrates, fixed and volatile oils, alkaloids, saponins and many other compounds.
Thin Layer Chromatography (TLC) screening of the oil samples showed the presence of four
main components, viz. thymoquinone, carvacrol, tanethole and 4-terpineol, which
demonstrated respectable radical scavenging property. These four constituents and the essential
oil possessed variable antioxidant activity when tested in the 2,2′-diphenyl-p-picrylhydrazyl
(DPPH) assay for non-specific hydrogen atom or electron donation. The oil samples showed
variable antioxidant activity which was ascribed mainly to the variable composition of these
constituents (Abou Basha et al, 1995).

According to the common practices of ‘evidence-based herbal medicine’, the bioactive

constituents of the volatile oil of black seed (54%) were identified by El-Dakhakhany in 1963
showing that Thymoquinone (I) or, in short, TQ was the main active constituent of volatile oil
of the black seed although it is accompanied by other analogous compounds such as Thymol
(II) and Thymoquinone dimer named as Dithymoquinone (TQ2, III) (Figure 2). Ghosheh et al.
(Ghosheh et al, 1999) carried out High Performance Liquid Chromatography (HPLC) analysis
of the oil of Nigella sativa using the isocratic mobile phase of water-methanol-2-propanol
(50:45:5%v/v) which revealed that the concentration of different constituents in the oil
including TQ and Thymohydroquinone (THQ, termed ‘Nigellone’ by earlier workers) depends
upon storage conditions. This is an important reminder to the researchers using oil as a source
of test substance in various biological assays because such results may include a factor of
variability depending upon the source and storage conditions of the Black seed oil.
A HPLC method for quantifying the putative pharmacologically active constituents: TQ, DTQ
(dithymoquinone), THQ, and thymol (THY), in the oil of Nigella sativa seed has been described
earlier (Ghosheh et al, 1999) employing a reversed-phase C18 analytical column and by using
an isocratic mobile phase of water:methanol:2-propanol (50:45:5% v/v) at a flow rate of 2 ml
min-1 and UV detection at 254 nm for TQ, DTQ, and THY, and at 294 nm for THQ. This
method provides a good quality control methodology for the pharmacologically active
components in these widely used Nigella sativa extracts as a natural remedy for many human
illnesses. Although TQ has been investigated presently for determining its therapeutic
potential, the other constituents of Nigella sativa seed oil also deserve further investigation for
assessing their therapeutic value. Chemically, TQ belongs to 2, 5-di-substituted benzoquinone
class of compounds having methyl and isopropyl groups at C-2 and C-5 positions, respectively.
The compound can be readily prepared in gram quantities by oxidation of thymol as shown
previously (Dockal et al, 1955). Since many of the biological activities of TQ largely originate
from its antioxidant properties, the electrochemical characteristics are important. Michelitsch
and Rittmannsberger (Michelitsch and Rittmannsberger, 2003) have described the
polarographic behavior of TQ in Sörensen buffer: methanol (3:7, v/v; pH 8.5) and have found
that the compound exhibits a single, reversible peak at dropping mercury electrode at -0.095
V vs. Ag/AgCl electrode. This perhaps can explain its facile inter-conversion under biological
environment leading to its antioxidant nature. The polarographic method can be applied to
determine TQ in black seed oil preparations where the limit of detection has been calculated
to be 0.05μg/ml.
Khalife and Lupidi (Khalife and Lupidi, 2007) have found that TQ reacts with GSH, NADH
and NADPH chemically. Such reactions occurring under the physiological conditions clearly
indicate the formation of two products, viz. glutathione dihydrothymoquinone after rapid
reaction with GSH and dihydrothymoquinone (DHTQ) after slow reaction with NADH and
NADPH, respectively. Measurement of antioxidant activity of the reduced compounds against

organic radicals such as 2,2/-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and
DPPH revealed a potential scavenging activity for glutathionyl-dihydrothymoquinone similar
to that of DHTQ while TQ showed lower scavenging activities. It is interesting to note that
these compounds exhibit antioxidant capacity equivalent to Trolox which is considered as a
standard antioxidant compound. These results indicate a possible intracellular non-enzymatic
activation of TQ dependent upon GSH, NADH and NADPH representing perhaps the ‘cellular
switch’ for modulating cellular antioxidant defenses.
The crystal structure of TQ was determined by Pagola and colleagues in 2004 using high-
resolution powder diffraction which indicated that it belongs to the triclinic system with Z =
2 and space group P··. The thermal analysis performed on the crystals indicated that weak van
der Walls forces are present in the molecules in the solid state and that only one crystalline

system exists in the compound. Availability of this crystal structure opens up the possibility
of using it for molecular modeling in various protein ligands.
B. Biological Activities of TQ
Amongst the various bioactivities examined for TQ or the extracts of black seed oil, most are
concerned with the anticancer or the antioxidant potential although only few have shown a
direct correlation between the two. Several possible targets have been suggested for the
observed activities and yet there seems to be no unique target. In this connection recent studies
reported by Kaseb et al. have indicated effects of TQ on cell cycle regulatory and proapoptotic
proteins in prostate cancer cells (Kaseb et al, 2007), and more recent studies have documented
the cancer cell specific effects of TQ affecting multiple targets (Sethi et al, 2008; Yi et al, 2008;
Aggarwal et al, 2008), suggesting that TQ deserves further in-depth investigations for
delineating its role as an anticancer agent. Moreover, a few studies have been devoted to anti-
inflammatory properties and they obviously have connotations to the anti-cancer activities of
TQ and thus prompt further in-depth investigation as indicated below.
1. Antioxidant Activity—TQ has been shown to exhibit antioxidant property through

different mechanisms in several recent reports. For example, it inhibits the production of 5-
hydroxyeicosa-tetraenaoic as well as 5-lipoxygenase products (El-Dakhakhny et al, 2002),
both of which are required for the viability of colon cancer cells. It was shown to work as a
scavenger of various reactive oxygen species including superoxide radical anion and hydroxyl
radicals (Kruk et al, 2000; Mansour et al, 2002; Badary et al, 2003). Additionally, it was able
to produce significant reductions in hepatic antioxidant enzymes such as superoxide dismutase
(SOD), catalase and glutathione peroxidase. It has been shown that TQ could inhibit iron-
dependent microsomal lipid peroxidation efficiently in rats with doxorubicin-induced
hyperlipidemic nephropathy (Badary et al, 2000). The compound was observed to decrease
cellular oxidative stress by inducing glutathione in experimental allergic encephalomyelitis in
female Lewis rats (Mohamed et al, 2003).
Multiple epidemiological studies have shown that a high intake of antioxidant rich foods is
inversely related to cancer risk (Borek, 2004). Recent experimental and clinical studies have
implicated oxidative stress in the development and progression of different cancers (Kim et al,
2004; Pathak et al, 2005). Present findings suggest that TQ has a potent chemopreventive
potential of inhibiting the process of carcinogenesis by modulating lipid peroxidation and
cellular antioxidant milieu (Badary, et al, 1999; Badary et al, 2007). Wilson and co-workers
(Wilson-Simpson et al, 2007) have analyzed the effect of low and high doses of
Epigallocatechin-3-gallate (EGCG), Selenium, and TQ on ES-2 ovarian cells at 24, 48, and 72
h in terms of morphology, cell count, and biochemical markers. During this phase I study,
experimental groups were administered physiological doses of specific antioxidants, EGCG,

selenium, and TQ. Selenium exhibited the largest effect on biochemical assays. Analysis of
collected data showed that the antioxidants suppress metabolic activity, alter behavioral
responses, and cause molecular damage. However, it has not been shown that the use of
antioxidants results in total destruction of ES-2 ovarian cancer cells. Norwood et al. (Norwood
et al, 2007) further confirmed the effects of sustained drug delivery of TQ, EGCG and 5-
fluorouracil (5-FU) on the metabolic activity as well as structural changes in the SW-626 human
colon cancer cells. Their results indicate that the sustained drug delivery of EGCG and TQ
induces significant cellular destruction and interference of cellular metabolic functions which
are comparable with damages caused by sustained drug delivery of 5-FU. Morphological
cellular changes occurring at 24 h after exposure to these two agents are also comparable to
those caused by 5-FU. Whether these results are indicative of a safer alternative to 5-FU using
natural photochemicals remains to be seen and thus further in-depth investigations are needed.

Sayed-Ahmed and co-worker (Sayed-Ahmed and Nagi, 2007) have investigated the possible
protective effects of TQ against Gentamicin (GM)-induced nephrotoxicity. Supplementation
with TQ resulted in significant decrease in reduced glutathione (GSH), and increased levels of
glutathione peroxidase (GPx), catalase and ATP, and a complete reversal of the GM-induced
increase in blood urea nitrogen, creatinine, thiobarbituric acid-reactive substances (TBARS)
and total nitrate/nitrite (NOx), and decrease in GSH, GPx, CAT and ATP to control values.
Histopathological examination of kidney tissues confirmed the biochemical data wherein TQ
supplementation prevents GM-induced degenerative changes in kidney tissues, suggesting that
these effects, at least in part, may be related to the ability of TQ to modulate cellular oxidative
stress. The protective effects of TQ have been assessed by Khattab and Nagi in 2007 in rats
after chronic inhibition of nitric oxide (NO) synthesis with N (omega)-nitro-l-arginine methyl
esters (l-NAME). Treatment with TQ decreased the elevated creatinine and increased GSH
levels compared to normal levels and inhibited the in vitro production of superoxide radicals
in enzymatic and non-enzymatic systems, thus offering protection against l-NAME-induced
hypertension and renal damage possibly via antioxidant activity of TQ.
Both Nigella sativa oil and TQ can partly protect gastric mucosa from acute alcohol-induced
mucosal injury which is partly ascribed to their radical scavenging activity (Kanter et al,
2005). The gastroprotective activity of Nigella sativa oil and TQ against gastric mucosal injury
induced by ischemia/reperfusion in rats was also investigated by El-Abhar and co-workers
(El-Abhar et al, 2003) who found that both agents offer protection against the gastric lesions
and these might be related to the conversion of the gastric mucosal redox state. Interestingly,
Farah and colleagues in 2005 have compared the effects of the water soluble and lipid soluble
fractions of Black seed and pure TQ on A549 cells in culture for 24, 48 and 72 h wherein the
cell number was found to decrease and remained so for the duration of the study. The water
soluble fraction showed a trend similar to TQ but the ethanol fraction showed a negative shift
in cell number at 48 h when compared with the control. This is contrary to the expectation that
lipid soluble TQ shall accompany the ethanol fraction and hence needs further corroboration
from the HPLC profiles of both the fractions, which clearly suggests further in-depth
investigation.
The state of hyperhomocysteinemia (HHcy) appears to be associated with higher risks of

coronary, cerebral and peripheral vascular diseases as well as a number of other clinical
conditions and is thought to be capable of inducing a pathogenic state of oxidative stress
although its underlying molecular mechanisms are not fully elucidated. El-Saleh and co-
workers (El-Saleh et al, 2004) have shown that active antioxidant components of black seeds
of Nigella sativa plants are capable of rendering protection against the development of
methionine-induced HHcy and its associated state of oxidative stress. Pre-treatment of rats
with an oral dose of 100 mg/kg of TQ for 30 min and for one week provided complete protection
against induced HHcy after methionine load (100 mg/kg). Under the state of induced HHcy,
there were significant increases in the plasma levels of triglycerides, lipid peroxidation, and
cholesterol as well as in the activities of glutathione peroxidase and SOD although catalase
activity was not affected. The total antioxidant status was significantly depressed. All of these
effects were almost totally blocked by the prior treatment with TQ. Mahgoub has investigated
in 2003 the effects of TQ on acetic acid-induced (intracolonic injection of 3% acetic acid)
colitis in rats. The study showed that pre-treatment of rats for 3 days with TQ (10 mg/kg) was
able to give complete protection against acetic acid-induced colitis as against the control group
treated with significantly higher dose of sulfasalazine (500 mg/kg). The smaller dose of TQ (5
mg/kg) produced partial protection, which clearly suggests that the effects of TQ could be
partly due to its antioxidant activity.
Mansour and co-workers have studied in 2002 the effects of TQ on antioxidant enzymes, lipid
peroxidation and DT-diaphorase in hepatic, cardiac and kidney tissues of normal mice.

Treatment with the different doses of TQ produced significant reductions in hepatic SOD, CAT
and GSH-Px activities although neither produced any change in GST activity nor influenced
reduced glutathione content in any of the tissues studied. These differences were attributed to
varying concentrations of DT-diphorase enzyme present in different tissues; highest being in

the hepatic tissues which was responsible for the reduction of TQ to DHTQ. These results
revealed that TQ and DHTQ acted not only as superoxide anion scavengers but as general free
radical scavengers. The median inhibitory concentration (IC50) values for TQ and DHTQ in
biochemical and photochemical assays were in the nanomolar and micromolar ranges,
respectively. These authors have suggested that the reported beneficial in vivo protective effects
of TQ are likely to be through the combined antioxidant properties of both compounds, which
needs further confirmatory studies. Badary and Gamal have studied in 2001 the inhibitory
effects of TQ against 20-methylcholanthrene (MC)-induced fibrosarcoma tumorigenesis where
the compound delayed the onset of MC-induced fibrosarcoma tumors that appeared at 12 weeks
and produced less MC-induced mortality. Compound alone showed a significant induction in
the enzyme activities of hepatic GST and quinone-reductase (QR). Mice treated with TQ along
with MC showed reduction in hepatic lipid peroxides and increased GSH content and increased
enzyme activities of GST and QR compared to the control group. The IC50 value for TQ against
fibrosarcoma cells was found to be 15 μM, indicating its potential as a powerful
chemopreventive and/or therapeutic agent.
The effects of TQ on carbon tetrachloride (CCl4)-induced hepatotoxicity have been

investigated in male Swiss albino mice by Nagi and colleagues in 1999 (Nagi et al, 1999). Oral
administration of TQ in a single dose (100 mg/Kg) resulted in a significant protection against
the hepatotoxic effects of CCl4. When tested as a substrate for mice hepatic DT-diaphorase in
the presence of NADH, TQ was found to undergo reduction to DHTQ which turned out to be
more potent than TQ and butylated hydroxytoluene (BHT). The IC50 values for DHTQ, TQ
and BHT were found to be 0.34, 0.87 and 0.58 μM, respectively. The data suggests that the
protective action of TQ against CCl4-induced hepatotoxicity may be mediated through the
combined antioxidant properties of TQ and its metabolite DHTQ.
The influence of TQ on doxorubicin (DOX)-induced hyperlipidemic nephropathy and

oxidative stress in rats has been examined by al-Shabanah and colleagues in 1998. Treatment
with TQ significantly suppressed DOX-induced proteinuria, albuminuria, and urinary
excretion of N-Acetyl Glucosamine (NAG) which confirmed the involvement of free radicals
in the pathogenesis of nephropathy induced by DOX. The study suggested that TQ might be
applicable as a protective agent against proteinuria and hyperlipidemia associated with
nephrotic syndrome. The cardio toxicity of the widely used antitumor agent, Doxorubicin, has
been suggested to result from the generation of oxygen free radicals. These workers observed
that TQ offers protection against doxorubicin-induced cardio toxicity without compromising
its antitumor activity. This finding was based on significant reductions in serum lactate
dehydrogenase and creatine kinase elevated levels, and further supplemented by
histopathological examination of cardiac tissue. The compound did not alter the plasma and
heart DOX levels as monitored by fluorometric analysis.
TQ has also been tested in isolated rat hepatocytes as a hepatoprotective agent against the
toxicity inflicted by tert-butyl hydroperoxide (TBHP) by Daba and Abdel-Rahman in 1998 and
compared against the protection offered by known hepatoprotective agent, silybin. Although
both compounds prevented TBHP-induced depletion of GSH to the same extent, degree of
protection by TQ against the liver enzyme leakage was less than that by silybin. The antioxidant
and pro-oxidant effects of TQ and a synthetic structurally-related compound, viz. tert-
butylhydroquinone (TBHQ), were examined in vitro by Badary et al. (Badary et al, 2003). Both
compounds efficiently inhibited iron-dependent microsomal lipid peroxidation in a
concentration-dependent manner with IC50 values of 16.8 and 14.9 μM, respectively. TBHQ

was stronger than TQ as a scavenger of DPPH and hydroxyl radicals whereas TQ was more
active than TBHQ as a superoxide anion scavenger. Only TBHQ significantly promoted DNA
damage in the bleomycin-Fe (III) system. These results suggest that both compounds have
strong antioxidant potentials although TQ acts mainly as a potent superoxide anion scavenger.
Moreover, Al-Majed and co-workers (Al-Majed et al, 2006) have evaluated the neuroprotective
effect of TQ against transient forebrain ischemia-induced neuronal damage in the rat
hippocampus. The pre-treatment of ischemic rats with the compound decreased the elevated
levels of MDA and increased GSH, catalase and SOD activities to normal levels. TQ and its
reduced product, THQ, inhibited the in vitro non-enzymatic lipid peroxidation in hippocampal
homogenate induced by iron-ascorbate. The IC50 for TQ and THQ were found to be 12 and 3
μM respectively. This spectacular protection makes TQ a promising agent in pathologies
implicating neurodegenaration such as cerebral ischemia.
2. Anti-inflammatory and Chemopreventive Activity of TQ—Inflammation has been

known to produce pro-inflammatory cytokines and diverse reactive oxygen species (ROS) and
reactive nitrogen species (RNS) creating pre-disposition to various patho-physiological
disorders such as Cohn’s disease or ulcerative colitis (Greenstein et al, 1979; Ekbom et al,
1990; D’Haens et al, 1993; Brentnall et al, 1996; Krok and Lichtenstein, 2004), gastric
helicobacter pylori infection (Crowe, 2005), and colorectal adenocarcinoma (Ekbom et al,
1990; Nielsen et al, 2005). Intervention in the inflammatory cascade can lead to a delay in
cancer progression and may improve patient’s morbidity and mortality as well. Arachidonic
acid is another example of a major precursor of several classes of signal transduction molecules
whose metabolism is altered significantly in human carcinogenesis. For example, it has been
well established that 5-lipooxynenase (5-LOX) converts arachidonic acid to hydroxyl-
eicosaterraenoic acids or leukotrienes (LT) which, in turn, enhance proliferation and survival
and suppress apoptosis of human cells. It may, therefore, be expected that inhibition of 5-LOX
protein may lead to apoptosis (Hoque et al, 2005). As a result, potential of TQ in suppressing
inflammation through inhibition of leukotrienes constitutes a very active area of research at
the present time. The compound is reported to be a potent inhibitor of leukotrienes formation
in human blood cells (Mansour and Tornhamre, 2004). The inhibitory effect was found to be
dose as well as time-dependent, and the effect was exerted on both 5-lipooxgenase and
Leucotiriene-C4-synthase (LT4synthase) activity (Mansour and Tornhamre, 2004). In another
study (Mahgoub, 2003), the rats pre-treated with oral TQ doses showed complete protection
against acetic acid-induced colitis compared to sulfasalazine (500 mg/kg) control group
wherein TQ was found to suppress the production of NO by macrophages which is useful in
ameliorating the inflammatory and autoimmune conditions (El-Mahmoudy et al, 2002).
El-Dakhakhny and co-workers have evaluated in 2002 the effects of Nigella sativa oil, TQ and
polythymoquinone (Nigellone) on the synthesis of 5-LOX products from polymorphonuclear

leukocytes from rats. TQ inhibited the production of 5-LOX products (IC50=0.26 mg/ml) and
5-HETE production (IC50=0.36 mg/ml) which may be ascribed to its antioxidant potential.
These observations explain the traditional use of Nigella sativa oil for ameliorating
inflammatory conditions in various folk medicinal systems. El-Gazzar et al, reported in 2007
that TQ inhibits LPS-induced pro-inflammatory cytokine production in RBL-2H3 cells by
blocking GATA transcription factor expression and promoter binding, which demonstrates its
anti-inflammatory effect (El-Gazzar et al, 2007). The compound inhibits LPS-induced IL-5
and IL-13 mRNA expression as well as protein production but not the production of IL-10.
Since LTs are important mediators in asthma and inflammatory processes, the effects of TQ
on leukotriene formation were studied in human blood cells by Mansour and his co-worker in
2004 found that it provoked a significant concentration-dependent inhibition of both LTC4 and
LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50
values of 1.8 and 2.3 μM, respectively, at 15 min. Their major inhibitory effect was on the 5-

lipoxygenase activity (IC50=3μM) as evidenced by suppressed conversion of exogenous

arachidonic acid into 5-HETE in sonicated polymorphonuclear cell suspensions.
Staurosporine, which is an unselective protein kinase inhibitor, failed to prevent inhibition of
LTC4 synthase activity induced by TQ which clearly indicates that the compound indeed
inhibits the formation of leukotrienes in human blood cells (Mansour and Tornhamre, 2004).
The anti-inflammatory activity of Black cumin seed oil has also been evaluated using
carrageenan-induced paw edema in rats and croton oil-induced ear edema in mice by
Hajhashemi and colleagues in 2004. Although oral administration of the oil at doses of 100,
200 and 400 μl/kg did not exert a significant anti-inflammatory effect in the carrageenan test,
the intraperitoneal injection of the same doses significantly inhibited carrageenan-induced paw
edema (Hajhashemi et al, 2004). The oil could also reduce croton oil-induced edema at smaller
doses and was found to produce a significant analgesic effect in acetic acid-induced writhing,
formalin and light tail flick tests. It seems that mechanism other than opioid receptors is
involved in the analgesic effect of the oil since naloxone, an opioid antagonist, could not reverse
this effect. Being one of the major components of the oil (13.5%), TQ obviously has an
important role in these pharmacological effects. Experimental Allergic Encephalitis (EAE) is
a T-cell mediated autoimmune disease, which resembles the human disease of Multiple
Sclerosis (MS) in rodents. The infiltration of inflammatory cells and the induction of astrocyte
proliferation correlate with the severity of the disease. Since oxidative stress has been
postulated as the causative factor of initiation and progression of MS, the amelioration of the
inflammation by TQ was examined by El-Gouhary and colleagues in 2005 who showed potent
effects, which were thought to occur via induction of glutathione (El-Gouhary et al, 2005).
El-Gazzar and colleagues has investigated in 2005 whether TQ affects Th2 cytokine response
in vitro in lipopolysaccharide (LPS)-activated rat mast cells. TQ significantly inhibited LPS-
induced IL-5 and IL-13 mRNA expression and protein production but did not affect IL-10
production probably by blocking GATA transcription factor expression (El-Gazzar et al,
2005). The inhibitory effects of TQ on activation of the redox-sensitive transcription factor
nuclear factor kappa B (NF-··B) and interleukin-6 (IL-6) were studied in vitro (Sayed and
Morcos 2007). Human proximal tubular epithelial cells (pTECs) in vitro were cultivated and
stimulated with Advanced Glycation End Products (AGEs) and the effects of TQ were studied.
A significant reduction of AGE-induced NF-··B-activation and IL-6 expression was observed
in Human proximal tubular epithelial cells (pTECs) cultivated and stimulated with AGEs.
Sayed studied in 2008 the effect of angiotensin II (AT II) on proximal tubular epithelial cells
(pTECs) in vitro. AT II has been found to activate NF-··B and its controlled genes, IL-6, in a
time-dependent manner wherein the first point of maximum NF-··B activation occurs after 12
h and the second after 3.5 days, respectively (Sayed 2008). TQ was found to decrease NF-··B
activation in a dose-dependent manner with maximum inhibitory effect at a concentration of

500nM. Preincubation of pTECs with TQ leads to disappearance of the second peak of NF-
··B. These data suggest the therapeutic value of TQ in delaying end stage renal diseases in
diabetics. Kanter has investigated in 2008 the possible beneficial effects of Black seed oil and
TQ on neurodegeneration in hippocampus after chronic toluene exposure in rats. Treatment
with TQ caused morphologic improvement on neurodegeneration indicating necessity of
further preclinical research in this area. The author has also studied the effects of same
compounds on histopathological changes of sciatic nerves in streptozotocin (STZ)-induced
diabetic rats (Kanter, 2008a). The histopathological evaluation of the tissues in diabetic animals
treated with these compounds showed fewer morphological alterations. Myelin breakdown
decreased significantly after treatment with both. The ultra-structural features of axons also
showed remarkable improvement. These results suggest that further preclinical research may
be able to highlight the advantages of using these agents as a potential treatment on peripheral
neuropathy in STZ-induced diabetic rats.

McDermott and co-workers have assessed in 2008 the chemoprotective potential of two
antioxidants, EGCG and TQ, against n-hexane toxicity in terms of increase in ROS formation
with a corresponding decrease in Jurkat T-cell proliferation since n-Hexane is an important
industrial solvent and ambient air pollutant. Treatment of cells with EGCG, at a concentration
reached in plasma, reduced the ROS formation caused by exposure to n-hexane and inhibited
the decrease in cell proliferation. Similar effects were obtained with TQ. Both the compounds
were able to significantly reduced n-hexane-induced LDH leakage to the control levels.
As indicated earlier, NF-··B is a molecular target of TQ (Sethi et al, 2008), and further studies
by Mohamed and colleagues in 2005 showed the effects of TQ on the inhibition of activation
of NF-··B in an experimental autoimmune encephalomyelitis in the rat model of multiple
sclerosis. The encephalomyelitis was induced in Lewis rats by injecting myelin basic protein
emulsified in complete Freund’s adjuvant. Several clinical and biochemical parameters
including activation of NF-··B were determined to assess the degree of protection. TQ was able
to counter peri-vascular cuffing and infiltration of mononuclear cells in the brain and spinal
cord, increase the red blood cell glutathione, and inhibit the activation of NF-··B in the brain
and spinal cord. These results clearly provide some early indication that many of the biological
activity of TQ could in part be due to inactivation of NF-··B and its downstream genes.
Collectively, these results suggest that NF-··B is a molecular target of TQ among many other
legitimate targets.
Moreover, El-Gazzar and colleagues have investigated in 2007 the effect of TQ on LPS-
induced TNF-··production in the rat basophil cell line, RBL-2H3 (El Gazzar et al, 2007). The
administration of TQ to LPS-stimulated cells did not noticeably alter NF-··B cytosolic
activation or nuclear expression as demonstrated by western blot analysis. Instead, the
compound significantly increased the amount of the repressive NF-··B p50 homodimer and
simultaneously decreased the amount of transactivating NF-··B p65:p50 heterodimer, bound
to the TNF-··promoter as revealed by electrophoretic mobility shift and chromatin
immunoprecipitation assays. These results suggest that TQ attenuates the pro-inflammatory
response in LPS-stimulated mast cells by modulating nuclear transactivation of NF-··B and
TNF-··production. The above results collectively suggest that further in-depth research in this
area is warranted.
3. Anti-proliferative (anti-tumor) and Cell Cycle Regulatory Activity—Shoieb and

colleagues have investigated in 2003 the anti-proliferative effects of TQ in cancer and normal
cell lines, viz. canine osteocarcinoma (COS31) and its cisplatin-resistant variant (COS31/
rCDDP), human breast adenocarcinoma (MCF-7), Human ovarian adenocarcinoma (BG-1)
and Mandin-Darby canine (MDCK) cells (Shoieb et al, 2003). The compound was found to
inhibit proliferation in a concentration-dependent manner as assessed by MTT assay. MDCK
cells (normal kidney cells) were the most resistant cells to the inhibitory effects of TQ (IC50
= 101 μM). Ait and colleagues have evaluated in 2007 the anti-tumor properties of the black
seed oil and its ethyl extract against P815 cell line and both were found to be cytotoxic. The
extracts were also tested on a variety of cell lines such as ICO1, Vero cells and BSR cell line
which showed that the extent of cytotoxicity depends upon the tumor cell type. In animal model
employing DBA2/P815 (H2d) mouse model it was observed that the injection of the essential
oil into the tumor site significantly inhibited solid tumor development as well as the incidence
of liver metastasis, thus improving mouse survival. These results indicate that the anti-tumor
activity or cell growth inhibition could in part be due to the effect of TQ on cell cycle.
The cell cycle checkpoints allow the cells to correct possible defects and avoid progression to
cancer (Hartwell and Weinert, 1989; Hartwell and Kastan, 1994). There are two major
checkpoints to detect DNA damage: one at the G1-S transition that prevents the cell from
replicating damaged DNA and one at the G2-M transition that prevents chromosome

segregation, if the chromosome is not intact. The principle activity of TQ was found to be due
to its effects on the expression of cell cycle regulatory proteins. The treatment of cells with 30
μM concentration for 48 h induced G1 cell-cycle arrest in papilloma cells, which correlated
with a sharp increase in the expression of the cyclin-dependent kinase inhibitor p16 and down-
regulation of cyclin D1 protein expression (Gali-Muhtasib et al, 2004b). It would be helpful
to define further targets of TQ by studying other cell cycle regulating proteins in future studies.
In Flow cytometric studies of DNA content by propidium iodide staining it has been revealed
that TQ induces G1 cell-cycle arrest of osteosarcoma cancer cells (COS31) as well as human
colon cancer cells (HCT-116), at 100μM concentration treated for 48 h (Gali-Muhtasib et al,
2004a). The effect was observed starting after 24 h at a concentration of 50 μM for COS31
cells while for HCT-116 cells it started at 60 μM, respectively. The G1 arrest was associated
with up-regulation of p21WAF1 in HCT-116 cells which was suggested as the principal
transcriptional target of p53 in the context of the G1 checkpoint. The resulting high levels of
p21WAF1 blocks cdk2 activity and possibly cdk4 and cdk6 activities leading to G1 arrest. There
was also an up-regulation of p53 expression which plays important roles in cancer development
as documented by multiple observations. For example, the p53 knockout-mice invariably
develop spontaneous tumors within first 6 months of life highlighting the protective role played
by p53 against cancer (el-Deiry et al, 1993). Although virtually all human tumors deregulate
either the pRB or p53 pathway or sometimes both, the unique effects of TQ on p53 protein
clearly warrant further studies in determining the precise molecular targets of TQ (Yamasaki,
2003). Moreover, TQ induced growth inhibition in spindle carcinoma cells by inducing G2/M
cell-cycle arrest which was associated with an increase in p53 expression and down-regulation
of cyclin B1 protein. It is worth mentioning that p53 can regulate the G2/M transition through
either induction of p21 or 14-3-3··, a protein that normally sequesters cyclin B1-Cdc2
complexes in the cytoplasm, ultimately leading to the induction of apoptosis (Yonish-Rouach
et al, 1991; Shaw et al, 1992; Hermeking et al, 1997; Bunz et al, 1998). These limited studies
further suggest that it would be highly desirable to investigate the effects of TQ on other
proteins that are involved in G2-M transition in order to delineate the molecular mechanism
(s) by which TQ may function as an inhibitor of cell cycle progression and thus as an anti-
tumor agent.
Having shown that TQ is an anti-cellular and anti-neoplastic drug that induces p53-dependent
apoptosis in human colon cancer cells, Roepke and colleagues have evaluated in 2007 the anti-
proliferative and pro-apoptotic effects of TQ in two human osteosarcoma cell lines with
different p53 mutation status. Cell viability was reduced more selectively in MG63 tumor cells
than in normal human osteoblasts. Flow cytometric analysis showed that TQ induced a much
greater increase in the Pre-G1 (apoptotic) cell population, but no cell cycle arrest in MG63
cells. G2/M arrest in MNNG/HOS cells was associated with p21WAF1 up-regulation. Using
three DNA damage assays, the compound was confirmed to induce greater extent of apoptosis
in p53 null MG63 cells. Although the Bax/Bcl-2 ratios were not differentially modulated in
both cell lines, the mitochondrial pathway appeared to be involved in apoptosis induced by TQ
in MG63 by showing the cleavage of caspases-9 and -3, respectively. Since TQ was found to
induce p53-independent apoptosis in human osteosarcoma cells, it suggests the potential
clinical usefulness of TQ for the treatment of these malignancies.
The serine/threonine kinase Polo-like kinase 1 (Plk1) is over-expressed in many types of human
cancers, and has been implicated as an adverse prognostic marker. Plk1 localizes to its
intracellular anchoring sites via its polobox domain (PBD). Reindl and colleagues have
reported in 2008 that TQ and its synthetic C-1 imino analog, Poloxin, are good inhibitors of
Plk 1 PBD in vitro and cause deregulation of its cellular localization, chromosomal defects,
mitotic arrest and apoptosis in HeLa cells (Reindl et al, 2008). These results provide early
indication as to the value of further research into the development of synthetic analogs of TQ
as anticancer agents. Ivankovic and colleagues have investigated in 2006 the anti-tumor activity

of TQ and THQ in L929 mouse fibroblasts and two other tumor cell lines, viz. squamous cell
carcinoma (SCC VII) and fibrosarcoma (FsaR), respectively. Both compounds showed dose
dependent potent cytotoxicities, which was more pronounced in tumor cells compared to L929
normal fibroblasts. The growth inhibitory (anti-tumor) effects of TQ were further studied by
Badary and colleagues in 2001 studied fibrosarcoma induced by 20-methylcholanthrene (MC)
in male Swiss albino mice. Administration of TQ (0.01% in drinking water) one week before
and after MC treatment significantly inhibited the tumor incidence and tumor burden compared
with the results in the group receiving MC alone (Badary et al, 2001). Moreover, TQ also
delayed the onset of MC-induced fibrosarcoma tumors indicating that it could be a powerful
chemopreventive agent against MC-induced fibrosarcomas (Badary et al, 2001).
Gali-Muhtasib and colleagues have also evaluated in 2008 the therapeutic potential of TQ in
two different murine colon cancer models, viz.1, 2-dimethyl hydrazine (DMH) and xenografts.
They examined the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor
invasion in vitro and found that the tumor multiplicity was reduced from 17.8 in the DMH
group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long
lasting since tumors did not re-grow even when TQ injection was discontinued for 10 weeks.
In a xenograft model of HCT116 colon cancer cells, TQ significantly delayed the growth of
the tumor cells.
In addition to TQ, a recent review article has shown the beneficial effects of many chemical
compounds isolated from natural resources (Aggarwal et al, 2008), suggesting that exploitation
of natural compounds for therapeutic application is an active area of research. Moreover, recent
studies have also shown that NF-··B is a legitimate target of TQ which was associated with cell
growth inhibition and induction of apoptosis in cancer cells (Sethi et al, 2008). As indicated
earlier, Kaseb and colleagues have observed in 2007 the biological effects of TQ in the
inhibition of DNA synthesis, cell proliferation, and viability of prostate cancer cells (LNCaP,
C4-2B, DU145, and PC-3) but not of the non-cancerous (BPH-1) prostate epithelial cells which
was mechanistically linked with the down-regulation of AR and E2F-1. In LNCaP cells, this
was associated with a dramatic increase in p21WAF1 (Cip1), p27 (Kip1), and Bax proteins. It
also blocked the progression of synchronized LNCaP cells from G1 to S phase, with a
concomitant decrease in AR and E2F-1 as well as the E2F-1-regulated proteins necessary for
cell cycle progression. In a xenograft prostate tumor model, TQ inhibited growth of C4-2B-
derived tumors in nude mice which was associated with a dramatic decrease in AR, E2F-1,
and cyclin A as determined by Western blot analysis (Kaseb et al, 2007). These results
collectively suggest that TQ may prove to be an effective agent in treating hormone-sensitive
as well as hormone-refractory prostate cancers with reasonable degree of selectivity and other
cancers (Kaseb et al, 2007; Aggarwal et al, 2008; Sethi et al, 2008). In the following section
we will catalogue several other studies that are relevant to cancer prevention and/or therapy
although further scientific studies are needed in order to justify the application of TQ alone or
in combination with other agents as novel therapeutics.
Although 5-FU continues to be the chemotherapeutic gold-standard for the treatment of colon
cancer, the side effects of 5-FU are numerous due to its ability to attack both healthy and
cancerous cells. Hence, Norwood and colleagues have compared in 2007 the effects of 5-FU
and natural chemotherapeutic agents, EGCG and TQ on the metabolic activity as well as
structural changes in the SW-626 human colon cancer cell line. These studies indicate a
significant cellular destruction and interference of cellular metabolic functions which opens
up new possibility for sustained drug delivery of other natural agents for the safe alternative
treatment of colon cancer. Consequently the studies were extended by these workers (Richards
et al, 2007) to the ceramic drug delivery system of TQ which demonstrated the greatest
reduction in cell count as well as the most cell membrane damage according to
malondialdehyde (MDA) levels. This supports the hypothesis that sustained delivery of

antioxidants may be a means of treating cancers, both safely as well as effectively. Obviously
further studies are needed to test the mechanisms behind these reactions.
Badary and colleagues have shown in 2007 that TQ protects the mice against benzo(a)pyrene-
induced forestomach carcinogenesis and chromosomal aberrations (CAs) in mouse bone
marrow cells when supplemented in the drinking water (Badary et al, 2007). It was observed
that daily intake of the compound before and after or during exposure to benzo(a)pyrene
significantly reduced the frequencies of CAs and damaged cells compared to the highly
clastogenic activity of B(a)P alone. Womack and colleagues have evaluated in 2006 the effects
of a single dose of 5 μM of TQ which showed a 50% reduction in Hep-2 cell numbers after 24
h. After 48 h the cells exhibited a four-fold decrease in total cell number which indicates that
TQ given in a sub-therapeutic dose could alter cellular viability. Tan and colleagues have also
examined in 2006 the effects of TQ on the proliferation and viability of PANC-1 cell line. The
PANC-1 cells were treated with three pre-determined doses of TQ (5, 25, and 50 μg/ml) and
medium viability and morphology were examined microscopically after each 24 h interval.
The compound was found to be the potent inhibitor of human pancreatic carcinoma, reducing
their propagation activities.
Recently, Richards and co-workers have used the androgen-dependent LNCaP human prostate
cancer cell line as a cell model to evaluate the physiological effects to conventional treatments
with both low doses and high doses of TQ (Richards et al, 2006). All treated cells showed a
reduction in cell growth, and high doses of the compound seemed to be the most potent. The
group treated with high doses of TQ also demonstrated the greatest decrease in total protein
levels in comparison to the control. Morphologically, the cells demonstrated significant
changes, such as swelling and irregularity in appearance upon TQ exposure. These results seem
to suggest that TQ could serve as a chemopreventive agent for prostate cancer. However, further
experiments are needed to understand the mechanism involved. Because TQ is a potent agent,
it has been explored further for combination with other known chemopreventive agents; one
such study reported by Brewer and colleagues showed in 2006 that selenomethione, lycopene
and thymoquinone could be effective on SiHa cells in the presence or absence of estrogen
(Brewer et al, 2006). Their results indicate that selenomethione alone appeared to be chemo-
protective, however, when used in combination with estrogen, lycopene and TQ, it caused
cellular damage as evidenced by decreased proliferation rate, increased glutathione levels, and
increased MDA levels (Brewer et al, 2006).
It appears that TQ is a pleiotropic agent targeting multiple signaling pathways in many patho-
physiological conditions as documented earlier and thus TQ has also been tested for its optimal
delivery. Martin et al. have determined the effects of TQ on the viability and metabolic activity
of SH-SY5Y human neuroblastoma cells alone or challenged with levo-dopa (L-dopa) using
conventional and sustained drug delivery routes (Martin et al, 2006). These results suggest that
the compound is able to offer some protection although the exact mechanism for this is not yet
known. It is suggested that the compound may be preventing the quinone formation, which
has been implicated in the pathogenesis of Parkinson’s disease as a result of L-dopa auto-
oxidation and thus it could be further investigated as a neuroprotective agent.
Further studies using a crude gum, a fixed oil, and two purified components of Nigella
sativa seed, viz. TQ and DTQ were assessed for their cytotoxicity on several parental and multi-
drug resistant (MDR) human tumor cell lines (Worthen et al, 1998). Although as much as 1%
w/v of the gum or oil was devoid of cytotoxicity, both TQ and DTQ were cytotoxic for all of
the tested cell lines. The parental cell lines and their corresponding MDR variants, viz.
Doxorubicin-resistant and etopside-resistant cell lines, were equally sensitive to TQ and DTQ.
The MDR modulator, viz. quinine, reversed MDR Dx-5 cell resistance to DOX and ETP by

6- to 16-fold, but had no effect on the cytotoxicity of TQ or DTQ These results suggest further
pre-clinical in vitro and in vivo animal experiments to advance our knowledge on the subject.
4. Apoptosis Induction Activity—It has been shown that TQ triggers apoptosis in

HCT-116 cells in a dose and time-dependent manner, starting at a concentration of 100 μM
after 12 h of incubation which is associated with a 2.5 to 4.5 fold increase in p53 and
p21WAF1 mRNA expression and a significant decrease in Bcl-2 protein levels (Yamasaki,
2003). Co-incubation with pifithrin-··, a p53 inhibitor, restored the Bcl-2, p53 and p21WAF1
levels to the untreated control levels and absolved the effects of TQ. These results suggest role
of TQ in influencing cell cycle regulators involved in apoptosis as well as in down-regulating
the anti-apoptotic proteins. This was supported by similar effects on primary mouse
keratinocytes, papilloma (SP-1), and spindle carcinoma cells respectively. At longer incubation
times (48 h) the compound induced apoptosis in both cell lines by increasing the ratio of Bax/
Bcl-2 protein expression and down-regulating the Bcl-xL protein.
TQ has been shown to initiate apoptosis even via p53-independent pathways through activation
of caspase-3, 8 and 9 in p53-null myeloblastic leukemia HL-60 cells (El-Mahdy et al, 2005).
It was observed that caspase-8 activity was highest after 1 h following the treatment of TQ,
while caspase-3 activity was highest after 6 h respectively. These observations were explained
on the basis of up-regulation of pro-apoptotic Bax protein along with down-regulation of anti-
apoptotic Bcl-2 proteins resulting in enhance Bax/Bcl-2 ratio. It is thus apparent that TQ
induces apoptosis through modulation of multiple targets and hence is a promising

phytochemical that could be useful for the killing of many types of cancer cells. These results
are also supported by reports in prostate and other cancer cells (Kaseb et al, 2007; Aggarwal
et al, 2008; Sethi et al, 2008).
A very recent report has identified checkpoint kinase 1 homolog, CHEK1, a serine/threonine
kinase, as the target of TQ, leading to apoptosis in p53+/+ colon cancer cells (Gali-Muhtasib
et al, 2008b). The study compared the effect of TQ on p53+/+ as well as p53-/- HCT116 colon
cancer cells and p53+/+ cells were found to be more sensitive to TQ in terms of DNA damage
and apoptosis-induction. As a possible explanation for such sensitivity, it was observed that
CHEK1 was up-regulated upto 9 folds in p53-null HCT116 cells. Further, transfection of p53
cDNA and CHEK1 siRNA in p53 null cells resulted in restoration of apoptosis to the levels of
p53+/+ cells. The results were also corroborated in vivo and it was demonstrated that tumors
lacking p53 had higher levels of CHEK1 which was associated with poorer apoptosis, advance
tumor stages and worse prognosis.
Despite the potential interest in TQ as a skin anti-neoplastic agent, its mechanism of action
was not examined for a long time. Gali-Muhtasib and colleagues studied in 2004 the cellular
and molecular events involved in antineoplastic activity of TQ using primary mouse

keratinocytes, papilloma (SP-1) and spindle carcinoma cells. The non-cytotoxic concentrations
of TQ reduce the proliferation of neoplastic keratinocytes by 50%. The sensitivity of cells to
TQ treatment appears to be stage dependent. For example, papilloma cells are twice as sensitive
to the growth inhibitory effects of TQ as the spindle cancer cells. At longer times of incubation
the compound induced apoptosis in both cell lines by increasing the ratio of Bax/Bcl-2 protein
expression and decreasing Bcl-xL protein. Collectively, these findings support a potential role
for TQ as a chemopreventive agent, particularly at the early stages of skin tumorigenesis.
Rooney and Ryan sought to understand in 2005 the mechanisms involved in the action ··-
hederin and TQ which are the active constituents of Nigella sativa, against human laryngeal
carcinoma (HEp-2) cancer cells by using buthionine sulfoximine (BSO), a selective inhibitor
of GSH synthesis, to determine the importance of GSH in the apoptosis elicited and using
cisplatin as internal standard. Both compounds elicited necrosis and apoptosis with a higher

incidence of the latter induced by TQ. BSO significantly enhanced ··-hederin- and cisplatin-
mediated toxicity without changes in apoptosis or necrosis levels. The compound TQ and
cisplatin significantly decreased GSH levels in a dose-dependent manner, with BSO
pretreatment synergistically depleting GSH levels in only cells treated with TQ. Since the
caspase-3 inhibitor significantly decreased apoptosis induced by the latter combination, it was
concluded that GSH depletion and caspase 3-activation mediate apoptosis induced by TQ in
this cell line, suggesting that exploitation of cause and effect relationship between the biological
activities of TQ with many cellular targets are warranted.
Tumor growth and metastasis are known to be angiogenesis-dependent and several clinical
trials have suggested that anti-angiogenic therapy might provide an attractive target for
therapeutic intervention. Alteration in the balance between pro-angiogenic and anti-angiogenic
molecules in the local tissue microenvironment may provide the starting point for such strategy
using the active constituent of black seed oil. The anti-aniogenic effects of TQ have been
assessed by cell proliferation and migration assays (Bawadi et al, 2004). The TQ significantly
decreased the proliferation of human breast (MCF-7), colon (Caco-2) and prostate (DU-145)
cancer cells at concentration of 100 μM and also prevented their metastasis. It inhibited
HIF-1··expression and decreased HIF-1··DNA binding activity in all cancer cells in addition
to reducing VEGF and cathepsin D secretion in normal human lung fibroblast cells. It, however,
did not affect normal cell proliferation even at 200μM concentration. Presently, there are no
studies reported, dealing with effects of TQ on angiogenesis.
5. Other Activities—The immunomodulatory and immunotherapeutic potentials of black

seed oil and its active ingredients have been discussed by Salem in 2005. The oil and some of
its active ingredients showed beneficial immunomodulatory properties, augmenting the T cell-
and natural killer cell-mediated immune responses. Further studies are urgently required to
explore bystander effects of TQ on the professional antigen presenting cells, including
macrophages and dendritic cells, as well as its modulatory effects upon Th1- and Th2-mediated
inflammatory immune diseases which is likely to substantially improve the immunotherapeutic
application of TQ in clinical settings. El-Mahmoudy and co-workers (El-Mahmoudy et al,
2005b) have compared the macrophage-derived cytokine and NO profiles in type I and type II
diabetes mellitus (DM) in order to determine whether TQ has any modulatory effects.
Peritoneal macrophages were collected from Otsuka Long-Evans Tokushima Fatty (OLETF)
rats as a model for type II DM and its control, Long-Evans Tokushima Otsuka (LETO) rats,
as well as from STZ-injected LETO ones as a model for type I DM. The cells were cultured
and incubated with or without TQ (10 μM) in the absence or presence of lipopolysaccharide.
Nitrite, IL-1·· and TNF-·· are significantly higher in macrophage supernatants and sera of the
acutely affected STZ-LETO rats either with or without LPS stimulation compared to
corresponding controls. On the other hand, chronically diabetic OLETF rats’ macrophage
supernatants showed significant decreases of IL-1·· and TNF-·· level upon LPS stimulation. In
absence of stimulation (IL-1··) insignificant increase in nitrite concentration was observed
which turned significant upon LPS stimulation. Sera of these animals, however, showed
significant increase in TNF-··level. The compound TQ normalized the elevated nitrite and
cytokine profiles both in vitro and in vivo but had no significant effect on the already decreased
parameters in chronically affected OLETF rats. These data suggest that there is a tendency for
macrophage inflammatory products to increase in acute type I Diabetes and to decrease in
chronic type II Diabetes respectively. Collectively, TQ has been suggested to have the potential
to normalize the elevated levels of these macrophage-derived inflammatory mediators.
It has been established that NO is involved in the destruction of ··-cells during the development
of type I DM. El-Mahmoudy and colleagues demonstrated in 2005 the possibility of rescuing
··-cells by intervention with TQ using STZ rat diabetic model. The hyperglycemic and
hypoinsulinemic responses to STZ were significantly abrogated in rats treated with TQ, and

this abrogation persisted for 1 month after termination of the treatment. TQ was found to have
no effect on either I··B degradation or NF-··B activation, although it significantly inhibited both
p44/42 and p38 mitogen-activated protein kinases (MAPKs) which contribute to the
transcriptional machinery of inducible nitric oxide synthase and NO production, respectively.

These data emphasize the protective role of TQ against development of type I diabetes via NO
inhibitory pathway.
The protective effect of black seed extract and TQ has been studied on mouse cells infected
with schistosomiasis by Aboul-Ela in 2002. Bone marrow cells and spleen cells in both in vitro
and in vivo experiments were used to evaluate the potentially protective effects on the induction
of chromosomal aberrations. Karyotyping of the mice cells illustrated that the main
abnormalities were gaps, fragments and deletions especially in chromosomes 2, 6 and some in
chromosomes 13 and 14. The seed extract as well as TQ both offered protection against the
chromosomal aberrations induced by schistosomiasis.
Nigellone is the carbonyl polymer of TQ, isolated from Nigella sativa L. seeds. The polymer
is far less toxic but retains much of the pharmacologic properties of TQ, which is the active
principle of the plant. Chakravorty’s investigations (Chakravorty, 1993), carried out on rat
peritoneal mast cells in vitro, have shown that nigellone, in relatively low concentrations, is
very effective in inhibiting histamine release induced by the secretagogues antigen in sensitized
cells, viz. compound 48/80, and the calcium ionophore,viz.A23187 respectively. The
mechanism of action seems to involve decreasing intracellular calcium by inhibiting its uptake
and stimulating the efflux as well as by an inhibition of protein kinase C. El-Gazzar and
colleagues have examined in 2006 the effect of TQ on airway inflammation in a mouse model
of allergic asthma. Intraperitoneal injection of TQ before airway challenge of ovalbumin
(OVA)-sensitized mice resulted in a marked decrease in lung eosinophilia and the elevated
Th2 cytokines; both in vivo and in vitro, following stimulation of lung cells with OVA. TQ
also decreased the elevated serum levels of OVA-specific IgE and IgG1. Histological
examination of lung tissue demonstrated that the compound significantly inhibited allergen-
induced lung eosinophilic inflammation and mucus-producing goblet cells. It was concluded
that TQ attenuates allergic airway inflammation by inhibiting Th2 cytokines and eosinophil
infiltration into the airways and thus demonstrating its potential anti-inflammatory role during
the allergic response in the lung.
III. Conclusions and Future Directions

We have attempted in summarizing the state-of-knowledge on the biological activity of TQ;
however in this short article we were not able to include all the published results and thus we
hope that the authors whose results have not been included in this article will forgive us. It is
however tantalizing to speculate that because TQ has attracted significant scientific attention
in recent years, further pre-clinical and clinical research to assess the health benefits of TQ is
urgently needed. Moreover, emerging evidence suggests that synthetic analogues of treasures
from natural resources could indeed be useful for making more effective compounds than TQ
which should also be an active area of research. It is hoped that this article will promote such
research and thus novel agents could be discovered based on the chemical structure of TQ for
the prevention and/or treatment of human diseases in the near future.
Acknowledgement
The authors’ work cited in this review was partly funded by grants from the National Cancer Institute, NIH
(5R01CA083695, 5R01CA101870, and 5R01CA108535) awarded to FHS, a sub-contract award to FHS from the
University of Texas MD Anderson Cancer Center through a SPORE grant (5P20-CA101936) on pancreatic cancer
awarded to James Abbruzzese, and a grant from the Department of Defense (DOD Prostate Cancer Research Program
DAMD17-03-1-0042) awarded to FHS.

Abbreviations
(DMH), 1, 2-dimethyl hydrazine
(ABTS), 2,2/-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)

(DPPH), 2,2′-diphenyl-p-picrylhydrazyl
(MC), 20-methylcholanthrene
(5-FU), 5-fluorouracil
(5-LOX), 5-lipooxynenase
(AGEs), Advanced Glycation End Products
(AT II), angiotensin II
(BSO), buthionine sulfoximine
(BHT), butylated hydroxytoluene
(CCl4), carbon tetrachloride
(CAs), chromosomal aberrations
(DM), diabetes mellitus
(DHTQ), dihydrothymoquinone
(DTQ), dithymoquinone
(TQ2,III), Dithymoquinone
(DOX), doxorubicin
(EGCG), Epigallocatechin-3-gallate
(EAE), Experimental Allergic Encephalitis
(FsaR), fibrosarcoma
(GM), Gentamicin
(GPx), glutathione peroxidase
(GSH), glutathione
(HPLC), High Performance Liquid Chromatography
(HHcy), hyperhomocysteinemia
(IL-6), interleukin-6
(LT4synthase), Leucotiriene-C4-synthase
(LT), leukotrienes
(L-dopa), levo-dopa
(LPS), lipopolysaccharide
(LETO), Long-Evans Tokushima Otsuka
(MDA), malondialdehyde
(IC50), median inhibitory concentration
(MAPKs), mitogen-activated protein kinases
(MDR), multi-drug resistant
(MS), Multiple Sclerosis
(l-NAME), N(omega)-nitro-l-arginine methyl esters
(NAG), N-Acetyl Glucosamine

(NOx), nitrate/nitrite
(NO), nitric oxide
(NF-··B), nuclear factor kappa B
(OLETF), Otsuka Long-Evans Tokushima Fatty
(OVA), ovalbumin
(PBD), polobox domain
(Plk1), Polo-like kinase 1
(pTECs), proximal tubular epithelial cells
(pTECs), proximal tubular epithelial cells
(QR), quinone-reductase
(RNS), reactive nitrogen species
(SCC VII), squamous cell carcinoma
(STZ), streptozotocin

(SOD), superoxide dismutase

(TBHP), tert-butyl hydroperoxide
(TBHQ), tert-butylhydroquinone
(TLC), Thin Layer Chromatography

(TBARS), thiobarbituric acid-reactive substances
(THQ), Thymohydroquinone
(I), Thymoquinone
(TQ), thymoquinone
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Figure-1.
(A) Morphological features of Nigella sativa plant, and (B) black cumin seeds containing oil
having Thymoquinone (TQ) as the active principle.

Figure-2.
Chemical structures of Thymoquinone, Thymol and Dithymoquinone.

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Cancer Ther. 2008 ; 6(b): 495–510.

From here to eternity - the secret of Pharaohs: Therapeutic

A. Chemistry of the active compounds found in black seed extracts

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

According to the common practices of ‘evidence-based herbal medicine’, the bioactive

NADPH, respectively. Measurement of antioxidant activity of the reduced compounds against

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

1. Antioxidant Activity—TQ has been shown to exhibit antioxidant property through

experimental groups were administered physiological doses of specific antioxidants, EGCG,

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

The state of hyperhomocysteinemia (HHcy) appears to be associated with higher risks of

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

varying concentrations of DT-diphorase enzyme present in different tissues; highest being in

The effects of TQ on carbon tetrachloride (CCl4)-induced hepatotoxicity have been

The influence of TQ on doxorubicin (DOX)-induced hyperlipidemic nephropathy and

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

2. Anti-inflammatory and Chemopreventive Activity of TQ—Inflammation has been

polythymoquinone (Nigellone) on the synthesis of 5-LOX products from polymorphonuclear

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

lipoxygenase activity (IC50=3μM) as evidenced by suppressed conversion of exogenous

activation in a dose-dependent manner with maximum inhibitory effect at a concentration of

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

3. Anti-proliferative (anti-tumor) and Cell Cycle Regulatory Activity—Shoieb and

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

4. Apoptosis Induction Activity—It has been shown that TQ triggers apoptosis in

induces apoptosis through modulation of multiple targets and hence is a promising

and molecular events involved in antineoplastic activity of TQ using primary mouse

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

5. Other Activities—The immunomodulatory and immunotherapeutic potentials of black

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

transcriptional machinery of inducible nitric oxide synthase and NO production, respectively.

III. Conclusions and Future Directions

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

(ABTS), 2,2/-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)

(NAG), N-Acetyl Glucosamine

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

(SOD), superoxide dismutase

(TLC), Thin Layer Chromatography

doxorubicin-induced hyperlipidemic nephropathy in rats. Toxicology 2000;143:219–226. [PubMed:

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

thymoquinone inhibit synthesis of 5-lipoxygenase products in polymorphonuclear leukocytes from

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

El-Mahmoudy A, Shimizu Y, Shiina T, Matsuyama H, El-Sayed M, Takewaki T. Successful abrogation

El-Mahmoudy A, Shimizu Y, Shiina T, Matsuyama H, Nikami H, Takewaki T. Macrophage-derived

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

Cancer Ther. Author manuscript; available in PMC 2008 November 17.

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