Eur Food Res Technol (2006) 224: 109115

DOI 10.1007/s00217-006-0295-z

ORIGINAL PAPER

V. Ani M. C. Varadaraj K. Akhilender Naidu

Antioxidant and antibacterial activities of polyphenolic compounds

from bitter cumin (Cuminum nigrum L.)

Received: 16 November 2005 / Revised: 8 February 2006 / Accepted: 13 February 2006 / Published online: 9 March 2006
C Springer-Verlag 2006

Abstract Cumin is one of the commonly used spices in

food preparations. It is also used in traditional ayurvedic
medicine as a stimulant, carminative and astringent. Earlier we have reported that bitter cumin (Cuminum nigrum
L.) possess the most potent antioxidant activity among
cumin varietiescumin, black cumin and bitter cumin. In
this study, we have further characterized the polyphenolic
compounds of bitter cumin and also their antioxidant and
antibacterial activity using different model systems. The
major polyphenolic compounds of cumin seeds were extracted with 70% methanol, 70% acetone, water, separated
by HPLC and their structures were elucidated by LC-MS.
The profile of phenolic acids/flavonols in bitter cumin were
found to be gallic acid, protocatechuic acid, caffeic acid,
ellagic acid, ferulic acid, quercetin and kaempferol. The
antioxidant activity of the cumin extract was tested on 1,1diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging, soybean lipoxygenase-dependent lipid peroxidation,
rat liver microsomal lipid peroxidation and superoxide anion (O2 ) scavenging. The bitter cumin extract exhibited
high antioxidant activity with IC50 values of 14.0 0.5 g,
28.0 3.0 g, 110 14.0 g and 125.4 8.7 g of the extract, respectively for DPPH free radical scavenging, soybean lipoxygenase-dependent lipid peroxidation, rat liver
microsomal lipid peroxidation and superoxide anion scavenging. Further, the extract offered a significant protection
against DNA damage induced by hydroxyl radicals. Among
a spectrum of food-borne pathogenic and spoilage bacteria
tested, the cumin extract significantly inhibited the growth
of Bacillus subtilis, Bacillus cereus and Staphylococcus
aureus. Thus, bitter cumin with an array of polyphenolic
V. Ani K. A. Naidu (
)
Biochemistry and Nutrition, Central Food Technological
Research Institute,
Mysore 570 020, India
e-mail: kanaidu@mailcity.com
Fax: +91-821-2517233
M. C. Varadaraj
Human Resource Development, Central Food Technological
Research Institute,
Mysore 570 020, India

compounds possesses potent antioxidant and antibacterial

activities.
Keywords Bitter cumin . Phenolic acids . DPPH
radicals . Superoxide anion scavenging . Lipid
peroxidation . DNA damage . Antibacterial activity
Introduction
A number of physiological processes in human body lead to
the generation of a series of oxygen-centered free radicals
and other reactive oxygen species (ROS) as by-products.
ROS play a positive role in energy production, phagocytosis, regulation of cell growth, intercellular signaling and
synthesis of biologically important compounds. However,
overproduction of ROS is also harmful to the body because
the oxidation induced by ROS can result in cell membrane
disintegration, membrane protein damage and DNA mutation, which can further initiate or propagate the development of many diseases [1, 2]. Antioxidants are compounds
that can delay, inhibit, or prevent the oxidation of oxidizable matters by scavenging free radicals and diminish
oxidative stress. Plants contain a wide variety of antioxidant phytochemicals or bioactive molecules, which can
neutralize the free radicals and thus retard the progress of
many chronic diseases associated with oxidative stress and
ROS. The intake of natural antioxidants has been associated with reduced risk of cancer, cardiovascular disease,
diabetes and diseases associated with ageing. Studies on
dietary free radical scavenging molecules have attracted
the attention to characterize phenolic compounds and other
naturally occurring phytochemicals as antioxidants.
Spices and condiments have become an integral part of
human diet to impart flavour, taste and colour to the food.
Spices are also considered as nutraceuticals in view of their
nutritional, medicinal and therapeutical properties. Cumin
is one of the most popular spices used in food preparations
in India and South East Asia. Cumin (Cuminum cyminum)
and black cumin (Nigella sativa) are widely as spice
condiment in vegetarian and non-vegetarian preparations

110

along with other spices in India and Arabia. Bitter cumin

(Cuminum nigrum L.) locally known as Shahi jeera or
Kashmiri cumin belongs to the family Apiaceae and
grows mainly in Central Asia and India. It is used in spice
mix or garam masala, pickles, wheat and rice dishes. It is
bitter in taste compared to other two varieties of cumin viz.,
normal cumin and black cumin. In traditional ayurvedic
medicine, it is used as a stimulant, carminative, astringent
and useful in dyspepsia and diarrhea [3]. Earlier we
reported a comparative study on the antioxidant potency of
the three cumin varieties and showed that bitter cumin possess high antioxidant activity compared to other two cumin
varieties [4]. In this study, we report isolation and characterization of bioactive polyphenolic compounds from bitter
cumin and their antioxidant and antimicrobial properties.
Materials and methods

Table 1

Phenolic compounds identified in C. nigrum seeds

Polyphenolic compounds

Concentration (g/g dry weight)

Gallic acid
Protocatechuic acid
Caffeic acid
Ellagic acid
Ferulic acid
Quercetin
Kaempferol

173.50
130.50
500.90
150.10
375.50
154.60
94.70

was dissolved in methanol and an aliquot of this solution

was added to 2 ml of 2% Na2 CO3 and after 2 min 100 l
of Folin-reagent (diluted 1:1) was added. After 30 min, the
absorbance was measured at 750 nm using Shimadzu UVVisible Spectrophotometer-1601. The total phenol content
was expressed as gallic acid equivalents (GAE) per mg of
extract calculated from standard graph of gallic acid.

Materials
C. nigrum seeds were obtained from the local market, identified and authenticated at the Department of Botany, Agriculture University, Bangalore. Diphenyl-picryl hydrazyl
(DPPH), linoleic acid, soybean type IV lipoxygenase,
Tween 80, Tris, calf thymus DNA, reference standards of
phenolic acids viz., gallic acid, protocatechuic acid, caffeic
acid, ellagic acid, ferulic acid, quercetin and kaempferol,
thiobarbituric acid were purchased from Sigma Chemical
Co., MO, USA. Nictinamide adenine dinucleotide-reduced
(NADH) and phenazine methosuphate (PMS) were purchased from Hi Media, Mumbai, India. Nitroblue tetrazolium (NBT) was purchased from Sisco Research Laboratories, Mumbai, India. pUC18 DNA was purchased from
Bangalore Genei, Bangalore, India. All other chemicals
and solvents used were analytical grade.

Estimation of polyphenolic compounds from bitter

by HPLC and LC-MS
The hydrolyzed cumin extract was dissolved in methanol
and subjected to HPLC for the qualitative and quantitative
analysis of phenolic contents. The HPLC system Shimadzu
LC-10 A (Japan) was equipped with dual pump LC-10AT
binary system, UV detector SPD-10A, Phenomenex Luna
RP, C18 column (i.d. 4.6 mm 250 mm) and data was
integrated by Shimadzu Class VP series software. The following gradient programme was employed (A) acetic acid
(1%) and (B) acetonitrile; 18% B at 0 min, 32% at 15.0 min
and finally to 50% at 40.0 min. The amount of phenolic
compounds (g/g dry weight) were calculated by comparison of peak areas (254 nm) of the samples with that of
standards (Fig. 1 and Table 1). The HPLC retention times
120

Extraction of polyphenolic compounds

from C. nigrum seeds

BHT

Bitter cumin seeds were powdered in a mixer grinder. The

powder was defatted with hexane in a soxhlets apparatus
for 6 h. Five grams of defatted cumin powder was extracted
with 100 ml of 70% aqueous methanol and 70% aqueous
acetone in 1:1 ratio by stirring for 2 h. The residue was extracted thrice with above solvents. The combined extracts
were concentrated under vacuum in a rotavapour and subjected to hydrolysis with 2N HCl to facilitate the breakage
of glycosides. It was then phase-separated with hexane to
remove any traces of fatty acids and subsequently with
ethyl acetate to extract polyphenolic compounds. The ethyl
acetate phase was concentrated under vacuum. The residue
was weighed and kept at 4 C until use.
Estimation of total phenolic compounds

Remaining DPPH (%)

100

C.nigrum
BHA

80

60

40

20

0
0

2.5

10

15

20

30

40

60

100 140 200

Bitter cumin extract (g)

The total phenolic content of the above extract was estimated by FolinCiocalteau method [5]. In brief, the extract

Fig. 1 C. nigrum seed extract BHA and BHT-scavenged DPPH free

radicals. Values mean SEM of three individual experiments

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Table 2

HPLC and MS characteristics of C. nigrum seeds

Retention time (min)

[M-H]

Fragmented ion

Corresponding fragment

Identified compound

6.37
8.41
13.47
17.66

169
153
179
300.8
193

29.21

301.1

37.31

285

M-COO
M-COO
M-COO
M-125
Trihydroxybenzene fragment
M-O
M-COO
M-free phenol at 2 position and a portion of the
benzopyranone ring moiety
M-151
Free phenol at position 2 and a portion of the
benzopyranone part

Gallic acid
Protocatechuic acid
Caffeic acid
Ellagic acid

21.24

125
109
135
170
125
178
149
151
133
151

Ferulic acid
Quercetin
Kaempferol

of the individual peaks of polyphenolic compounds are with 0.1 M phosphate buffer (pH 7.8) at ambient temperature. The reaction mixture (NBT and NADH) was incubated
shown in Table 2.
An API 200 triple quadrupole mass spectrometer (Ap- with or without cumin extract at ambient temperature for
plied Biosystems) was used for determining the mass of the 2 min and the reaction was started by adding PMS. The
polyphenolic compounds. Analysis were performed on a absorbance at 560 nm was measured against blank samTurbo ions spray source in negative mode by using settings ples for 3 min. Decrease in absorbance in the presence of
nebuliser gas 16 (N2) (arbitrary units), focusing potential cumin extracts indicated superoxide anion scavenging ac 400 V, entrance potential 10, declustering potential tivity. The percent inhibition was calculated by using the
(DP) 2560 and collision energy (CE) 1535. Full scan following formula.
acquisition was performed scanning from m/z 150700 u
at a cycle time of 2 s. MS product ions were produced Superoxide scavenging activity (%)
by collision-associated dissociation (CAD) of the selected
Control OD Sample OD
precursor ions in collision cell. In all the experiments, both
=
100
Control OD
the quadrupoles (Q1 and Q3) were operated at unit resolution. Product ion scan of selected molecules were carried
out in order to confirm the structure of the compounds.
Measurement of rat liver microsomes lipid
peroxidation activity
Determination of antioxidant activity
Rat liver was homogenized in 0.25 M TrisHClSucrose
EDTA buffer at pH 7.4. The homogenate was centrifuged
Measurement of DPPH radical scavenging activity
at 7649 g for 30 min to remove unbroken cells and cell
The DPPH free radical scavenging test was carried out debris. The supernatant was centrifuged at 100,000 g in
as described elsewhere [6]. Different dilutions of the a refrigerated ultracentrifuge (Beckman L7-65 Ultracenextract of bitter cumin were incubated with 1 ml of DPPH trifuge, USA) for 1 h to isolate the microsomal pellet,
solution (50 105 M). The decrease in absorbance due which is dissolved, into 2 ml of 125 mM KCl. The protein
to scavenging DPPH radicals by bitter cumin extract content of microsomes was estimated by Lowry method
was determined at 517 nm using a Shimadzu UV-Visible [8]. One milligram of microsomal protein was incubated
Spectrophotometer-1601 (Shimadzu, Kyoto, Japan). BHT with 0.2 mM FeSO4 and 0.2 mM ascorbic acid with or
were used as BHA standard. The percentage of remaining without cumin extract in a final volume of 1 ml at 37 C
DPPH after 5 min was calculated for individual experi- for 1 h. Malondialdehyde (MDA) formed in the incubation
ments and the concentration at which 50% of the initial mixture was reacted with thiobarbituric acid and thiobarbiand remaining DPPH concentrations were calculated from turic acid reactive substances (TBARS) were measured at
535 nm spectrophotometrically [9]. The antioxidant activstandard DPPH graph.
ity was expressed as decrease in oxidation of microsomal
lipids measured as n moles of MDA formed per minute per
milligram protein using a molar extinction coefficient of
Measurement of superoxide anion scavenging activity
1.56 105 M/cm.
The superoxide scavenging ability of the bitter cumin extract was assessed by a modified method as described else- Lipoxygenase-dependent lipid peroxidation activity
where [7]. Superoxide anions were generated in samples
that contained 100 l each of 1.0 mM NBT, 3.0 mM NADH Soybean lipoxygenase-dependent lipid peroxidation was
and 0.3 mM PMS and the final volume was adjusted to 1 ml measured as a decrease in absorbance of lipid hydroper-

112

oxide formation at 234 nm spectrophotometrically [10].

Briefly, the reaction mixture in a final volume of 1 ml contained 200 M linoleic acid and 5 nM soybean lipoxygenase in 50 mM Tris buffer, pH 7.4. The absorption
due to the formation lipid hydroperoxide was monitored
at 234 nm in a Shimadzu UV-Visible spectrophotometer. Different concentrations of bitter cumin extract were
incubated for 2 min with soybean lipoxygenase prior to
initiation of reaction with linoleic acid. The decrease in
lipid hydroperoxide formation in the presence of cumin
extract was calculated using an extinction coefficient
of 25 mM/cm. The enzyme activity was expressed as
moles of hydroperoxide formed per minute per nM of
enzyme.
Antioxidant activity against oxidative damage to DNA
Hydroxyl radicals generated by Fenton reaction was used
to induce oxidative damage to DNA. The reaction mixture
(9 l) contained 3 g of calf thymus DNA in 20.0 mM
phosphate buffer saline (pH 7.4) and different concentrations of bitter cumin extract (0.5, 1.0, 1.5 and 2.0 g) were
added and preincubated with DNA for 15 min at ambient
temperature. The oxidation was induced by treating DNA
with 1.0 mM FeSO4 and 10.0 mM ascorbic acid and incubated them for 1 h at 37 C. Similarly 1.0 g of pUC18
DNA was incubated with different concentrations of cumin
extract for 30 min at ambient temperature and 2.0 l each
of 100.0 M FeSO4 , 600.0 M ascorbic acid and 60.0 mM
H2 O2 were added. The final volume was adjusted to 20.0 l
with 20.0 mM phosphate buffer (pH 7.4) and incubated for
1 h at 37 C. The reaction was terminated by the addition of
loading buffer (xylene cyanol, 0.25%; bromophenol blue,
0.25% and glycerol 30%) and the mixture was subjected to
gel electrophoresis in 1% agarose/TAE buffer run at 60 V
for 3 h. DNA was visualized and photographed by a digital
imaging system (Hero Lab, GMBH, Germany).
Determination of antibacterial activity
Antibacterial activity of cumin extract was tested against
food-borne pathogenic and spoilage bacteria viz., Bacillus
subtilis, Bacillus cereus, Enterobacter spp., Escherichia
coli, Listeria monocytogenes, Staphylococcus aureus and
Yersinia enterocolitica by agar diffusion method. Plate
count agar plates were prepared using 1% brain heart infusion broth inoculum of individual bacterial cultures. Five
equidistant wells of 5.0 mm each were made in the solidified
agar medium using sterile stainless steel cork borer. Different concentrations of cumin aqueous methanolacetone
extract were added to the wells and the control well received the same volume of methanol. Initially, the plates
were kept at 6 C for 3 h and then incubated for 22 h at
37 C. The inhibition zones formed around the well were
measured to an accuracy of 0.1 mm and the activity was
calculated as a mean of triplicate for each of the indicated
bacterial species.

Statistical analysis
All the experimental data are presented as mean SEM of
three individual samples. Data are presented as percentage
of free radical scavenging/inhibition lipid peroxidation on
different concentration of cumin extracts. IC50 (the concentration required to inhibit 50% of enzyme activity/scavenge
50% of free radicals) value was calculated from the doseresponse curves. Antibacterial effect was measured in terms
of zone of inhibition to an accuracy of 0.1 mm and the effect
was calculated as a mean of triplicate tests.
Results and discussion
Spices occupy an important place in our food as taste
and aroma enhancers since ancient times. A variety of
molecules derived from spice possess bioactive properties.
Of these phenolic compounds constitute the largest proportion of known natural antioxidants [11]. There is an increasing interest in natural antioxidant molecules from food to
prevent the deleterious effects of free radicals in biological
systems and also prevent the deterioration of foods due to
oxidation of lipids and microbial spoilage. Cumin is one of
the commonly used spice condiment in both vegetarian and
non-vegetarian food preparations in Asia and Arabia. Our
earlier study showed that bitter cumin was most potent antioxidant among the three cumin varieties [4]. In this study,
we have further characterized the polyphenolic compounds
and also evaluated the antioxidant and antibacterial activity
of bitter cumin.
The polyphenolic compounds from bitter cumin seeds
were extracted with aqueous 70% methanol, 70% acetone
and ethyl acetate to facilitate extraction of low- and highmolecular weight polyphenols. Thus, with the above solvent system we could extract a number of phenolic acids
and flavonols from bitter cumin (Table 1). The total phenol
content of this extract was estimated to be 551.8 0.82 g
GAE/mg of extract.
Many phenolic compounds are normally present as glycosides or aglycones in plants. The cumin extract was subjected to acid hydrolysis (2N HCl) to break the glycoside
linkages and the polyphenolic compounds in hydrolysate
were separated, identified and quantified by LC-MS. The
identification of the individual phenolic compounds was
achieved by comparing retention time and the peak area
of cumin extract compounds with that of standards). Interestingly, bitter cumin contained a number of polyphenolic
compounds including gallic acid, protocatechuic acid,
caffeic acid, ellagic acid, ferulic acid and also flavonols
such as quercetin and kaempferol (Table 1). The MS characteristics of identified polyphenolic compounds are given
in Table 2. Caffeic and ferulic acids were found to be most
abundant among phenolic acids present in bitter cumin
(Table 1). This is the first report showing the presence of
an array of polyphenolic compounds in bitter cumin seeds.
There are many methods to assess the antioxidant activity, but each method has its own limitations [12]. Hence,
we tested the effect of bitter cumin extract on different

113

percentage scavenging of superoxide anion

120

100

80

60

40

20

0
0

25

50

100

200

400

Bitter cumin extract (g)

Fig. 2 C. nigrum seed extract scavenged superoxide anion radicals.

Values mean SEM of three individual experiments

1000

nmoles of MDA formed/mg of protein

antioxidant assays including DPPH free radical scavenging,

superoxide anion radical scavenging, rat liver microsomal
lipid peroxidation, soybean lipoxygenase-dependent lipid
peroxidation and oxidative damage to DNA to understand
its antioxidant potential. Further, we have also tested its
effect on selected food-borne pathogenic and spoilage
bacteria to assess the antibacterial activity of bitter cumin.
The DPPH radical scavenging is a sensitive antioxidant
assay and is independent of substrate polarity [13].
DPPH is a stable free radical that can accept an electron
or hydrogen radical to become a stable diamagnetic
molecule. Cumin extract exhibited a dose-dependent
scavenging of DPPH radicals and 14.0 0.5 g of extract
was sufficient to scavenge 50% of DPPH radicals/ml. The
radical scavenging effect of bitter cumin was found to
be 2.6 times more potent than the standard BHT (IC50
36.4 1.2 g/ml) (Fig. 1) but, less potent than BHA (IC50
8.25 0.35 g). This suggests that bitter cumin is a good
free radical scavenger or hydrogen donor and contributes
significantly to the antioxidant capacity of bitter cumin.
Superoxide anion is a reduced form of molecular oxygen
and plays an important role in the formation of other reactive oxygen species such as hydrogen peroxide, hydroxyl
radical or singlet oxygen [14]. The bitter cumin extract
was found to be an effective scavenger of superoxide anion
radicals in a dose-dependent manner with an IC50 value of
125.4 8.7 g (Fig. 2) and thus can prevent the formation
of ROS.
Lipid oxidation of fats and fatty foods not only brings
about chemical spoilage in foods, but also produces free
radicals such as peroxy radicals, which are purportedly associated with carcinogenesis, autogenesis and ageing [15,
16]. Membrane lipids are particularly susceptible to oxidation because of their high polyunsaturated fatty acid
content but also of their association in the cell membrane.
Lipid peroxidation is a free-radical chain reaction and the

800

600

400

200

0
0

50

100

150

200

250

300

350

400

Bitter cumin extract (g)

Fig. 3 C. nigrum seeds extract inhibited the peroxidation of rat

liver microsomal membrane lipids. Values mean SEM of three
individual experiments

reactive oxygen species can accelerate lipid oxidation [17].

Hydroxyl radicals are the most reactive free radicals capable of reacting with lipids, polypeptides, proteins and DNA
[18]. Bitter cumin extract significantly inhibited hydroxyl
radical induced oxidation of rat microsomal lipids with an
IC50 value of 110.0 14.0 g (Fig. 3). The polyphenols
present in bitter cumin extract might react with hydroxyl
radical by donating hydrogen atom and convert them to
more stable no radical products and thus prevent the microsomal lipid peroxidation (Table 3).
The autoxidation of lipids as well as the enzymatic oxidation of fats, oils and fat-containing foods during storage
and processing are responsible for rancidity and deterioration of food quality. Soybean lipoxygenase-dependent
lipid peroxidation is an enzymatic lipid peroxidation assay used to determine the antioxidant activity of test
compounds. Cumin extract showed significant inhibition
of lipoxygenase-dependent lipid peroxidation and the inhibitory effect is found to be dose-dependent with an IC50
value of 28.0 3.0 g (Fig. 4). However, bitter cumin is
found to be less potent compared to synthetic antioxidant
BHA. Plant phenols and flavonoids are known to inhibit
lipid peroxidation by quenching lipid peroxy radicals and
reduce or chelate iron in lipoxygenase enzyme and thus
prevent initiation of lipid peroxidation reaction [1921].
Similarly, the polyphenolic compounds such as quercetin,
caffeic acid, ferulic acid, ellagic acid present in bitter cumin
being free radical scavengers could react with peroxy radical before the fatty acid reacted with peroxy radicals and
thus inhibited lipid oxidation.
DNA is susceptible to oxidative damage and hydroxyl
radicals oxidize guanosine or thymine to 8-hydroxyl-2deoxyguanosine and thymine glycol which change DNA
and lead to mutagenesis and carcinogenesis [22]. In this
study, hydroxyl radicals generated by Fenton reaction were
found to induce DNA strand breaks in calf thymus DNA
and uncoiling of supercoiled DNA. Bitter cumin extract at

114
Table 3 Antioxidant activity
of extract of C. nigrum seed in
different model systems

Note. Values are mean SEM

of three experiments

Correlation coefficient between

total phenol content and
antioxidant activity
Antioxidant assay

Free radicals

IC50 value (g)

DPPH free radical scavenging

Soybean lipoxygenase-dependent
lipid peroxidation
Microsomal lipid peroxidation
Superoxide anion scavenging

DPPH
LOO

14.0 0.5
28.0 3.0

0.94
0.92

OH
O2

110.0 14.0
125.4 8.7

0.98
0.94

0.52.0 g offered complete protection to DNA damage

induced by hydroxyl radicals in calf thymus DNA and also
reduced uncoiling or open circular form in pUC18 DNA
(Figs. 5 and 6). Thus, the hydroxyl radical quenching ability of polyphenolic compounds of bitter cumin could be
responsible for the protection against oxidative damage to
DNA.
Spices and their essential oils are reported to possess
antimicrobial activity [23, 24]. The antibacterial effect of
bitter cumin extract was tested against a number of foodborne pathogenic and spoilage bacteria by agar diffusion
method. As shown in Table 4, the bacterial species namely
B. subtilis, B. cereus and S. aureus were found to be highly
sensitive and showed significant inhibition of the growth in
the presence of bitter cumin extract (Table 3). Enterobacter spp. and L. monocytogenes were moderately inhibited,
while E. coli and Y. enterocolitica were resistant to bitter
cumin extract. Thus, Gram-positive bacteria were found
to be more sensitive to bitter cumin extracts than Gramnegative bacteria. The antibacterial activity of flavonoids
was attributed to inhibition of synthesis of DNA and RNA
and other related macromolecules [25, 26]. Further, phenolic compounds with more than three 3-OH were found to
possess antibacterial activity [27]. Thus, antibacterial ac-

Fig. 4 C. nigrum seeds extract and BHA inhibited soybean

lipoxygenase-dependent oxidation of linoleic acid. Values mean
SEM of three individual experiments

Table 4

Antibacterial activity of the extract of C. nigrum seeds

Organism

Inhibition

B. subtilis
B. cereus
Enterobacter sp
E. coli
Listeria monocytogens
S. aureus
Y. enterocolitica

++
++
+

+
++

Note. , Low/no inhibition (<5 mm); + , moderate inhibition (5

20 mm); + + , High inhibition (>20 mm)

tivity of bitter cumin could be attributed to the polyphenolic

compounds present in the bitter cumin extract.
In conclusion, our studies have demonstrated for the first
time the presence of a mixture of bioactive polyphenolic
compounds such as caffeic acid, ferulic acid, protocatechuic acid, ellagic acid, quercetin and kampeferol in bitter
cumin seeds. Bitter cumin seed extract also exhibited
significant antioxidant activity at microgram quantities as
quencher of DPPH radicals, lipid peroxy radicals, hydroxyl
radicals and superoxide anion radicals in different antioxidant systems. Further, bitter cumin extract also showed antibacterial activity by suppressing the growth of pathogenic
bacteria namely B. cereus and S. aureus. Thus, bitter
cumin with a mixture of polyphenolic compounds possess

Fig. 5 Protective effect of the extract of C. nigrum seeds on oxidative

damage to Calf Thymus DNA. Lane 1: Native Calf Thymus DNA.
Lane 2: DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid. Lane 3:
DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid + 0.5 g cumin
seeds extract. Lane 4: DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic
acid + 1.0 g cumin seeds extract. Lane 5: DNA + 1.0 mM FeSO4
+ 10.0 mM ascorbic acid + 1.5 g cumin seeds extract. Lane 6:
DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid + 2.0 g cumin
seeds extract

115

Fig. 6. Protective effect of the extract of C. nigrum seeds on oxidative damage to pUC18 DNA. Lane 1: pUC18 DNA. Lane 2: pUC18
DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM
H2 O2 . Lane 3: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM H2 O2 + 0.5 g of Cumin seeds extract. Lane
4: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid +
6.0 mM H2 O2 + 1.0 g of cumin seeds extract. Lane 5: pUC18
DNA + 10.0 M FeSO4 + 60.0 M ascorbic acid + 6.0 mM
H2 O2 + 2.5 g of cumin seeds extract. Lane 6: pUC18 DNA +
10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM H2 O2 +
2.5 g of BHA (S, supercoiled DNA; N, nicked DNA)

potential antioxidant and antibacterial activities and this

spice can be further exploited for nutraceutical properties.
Acknowledgements Authors are thankful to Dr. V. Prakash, Director and Dr. S.G. Bhat, Head of the Department of Biochemistry and
Nutrition, Central Food Technological Research Institute, Mysore
for their constant encouragement and support. Ani. V is thankful to
the Council of Scientific and Industrial Research (CSIR), New Delhi,
for the award of Junior and Senior Research Fellowship. This work
was partly supported by a project awarded to KAN by Department
of Science and Technology (DST), New Delhi, India.

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