Powerpoint about phytochemicals

Eur Food Res Technol (2006) 224: 109–115 DOI 10.1007/s00217-006-0295-z

O R I G I N A L PA P E R

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. Ear- lier we have reported that bitter cumin (Cuminum nigrum L.) possess the most potent antioxidant activity among cumin varieties—cumin, 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 ex- tracted 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 1,1- diphenyl-2-picryl hydrazyl (DPPH) free radical scaveng- ing, soybean lipoxygenase-dependent lipid peroxidation, rat liver microsomal lipid peroxidation and superoxide an- ion (O2−) scavenging. The bitter cumin extract exhibited high antioxidant activity with IC50 values of 14.0 ± 0.5 µg, 28.0 ± 3.0 µg, 110 ± 14.0 µg and 125.4 ± 8.7 µg of the ex- tract, respectively for DPPH free radical scavenging, soy- bean lipoxygenase-dependent lipid peroxidation, rat liver microsomal lipid peroxidation and superoxide anion scav- enging. 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: [email protected] 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, phagocy- tosis, 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 muta- tion, which can further initiate or propagate the develop- ment of many diseases [1, 2]. Antioxidants are compounds that can delay, inhibit, or prevent the oxidation of oxi- dizable matters by scavenging free radicals and diminish oxidative stress. Plants contain a wide variety of antiox- idant 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 associ- ated 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

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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 pos- sess high antioxidant activity compared to other two cumin varieties [4]. In this study, we report isolation and charac- terization of bioactive polyphenolic compounds from bitter cumin and their antioxidant and antimicrobial properties.

Materials and methods

Materials

C. nigrum seeds were obtained from the local market, iden- tified and authenticated at the Department of Botany, Agri- culture 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 pur- chased from Hi Media, Mumbai, India. Nitroblue tetra- zolium (NBT) was purchased from Sisco Research Labo- ratories, Mumbai, India. pUC18 DNA was purchased from Bangalore Genei, Bangalore, India. All other chemicals and solvents used were analytical grade.

Extraction of polyphenolic compounds from C. nigrum seeds

Bitter cumin seeds were powdered in a mixer grinder. The powder was defatted with hexane in a soxhlet’s 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 ex- tracted thrice with above solvents. The combined extracts were concentrated under vacuum in a rotavapour and sub- jected 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

The total phenolic content of the above extract was esti- mated by Folin–Ciocalteau method [5]. In brief, the extract

Table 1 Phenolic compounds identified in C. nigrum seeds

Polyphenolic compounds Concentration (µg/g dry weight)

Gallic acid 173.50 Protocatechuic acid 130.50 Caffeic acid 500.90 Ellagic acid 150.10 Ferulic acid 375.50 Quercetin 154.60 Kaempferol 94.70

was dissolved in methanol and an aliquot of this solution was added to 2 ml of 2% Na2CO3 and after 2 min 100 µl of Folin-reagent (diluted 1:1) was added. After 30 min, the absorbance was measured at 750 nm using Shimadzu UV- Visible Spectrophotometer-1601. The total phenol content was expressed as gallic acid equivalents (GAE) per mg of extract calculated from standard graph of gallic acid.

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 fol- lowing 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 (µg/g dry weight) were calculated by compar- ison of peak areas (254 nm) of the samples with that of standards (Fig. 1 and Table 1). The HPLC retention times

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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

6.37 169 125 M-COO Gallic acid 8.41 153 109 M-COO− Protocatechuic acid 13.47 179 135 M-COO− Caffeic acid 17.66 300.8 170 M-125 Ellagic acid

125 Trihydroxybenzene fragment 21.24 193 178 M-O− Ferulic acid

149 M-COO−

29.21 301.1 151 M-free phenol at 2 position and a portion of the benzopyranone ring moiety

Quercetin

37.31 285 133 M-151 Kaempferol 151 Free phenol at position 2 and a portion of the

benzopyranone part

of the individual peaks of polyphenolic compounds are shown in Table 2.

An API 200 triple quadrupole mass spectrometer (Ap- plied Biosystems) was used for determining the mass of the polyphenolic compounds. Analysis were performed on a Turbo ions spray source in negative mode by using settings nebuliser gas 16 (N2) (arbitrary units), focusing potential − 400 V, entrance potential − 10, declustering potential (DP) 25–60 and collision energy (CE) 15–35. Full scan acquisition was performed scanning from m/z 150–700 u at a cycle time of 2 s. MS product ions were produced by collision-associated dissociation (CAD) of the selected precursor ions in collision cell. In all the experiments, both the quadrupoles (Q1 and Q3) were operated at unit resolu- tion. Product ion scan of selected molecules were carried out in order to confirm the structure of the compounds.

Determination of antioxidant activity

Measurement of DPPH radical scavenging activity

The DPPH free radical scavenging test was carried out as described elsewhere [6]. Different dilutions of the extract of bitter cumin were incubated with 1 ml of DPPH solution (50 × 10−5 M). The decrease in absorbance due to scavenging DPPH radicals by bitter cumin extract was determined at 517 nm using a Shimadzu UV-Visible Spectrophotometer-1601 (Shimadzu, Kyoto, Japan). BHT were used as BHA standard. The percentage of remaining DPPH after 5 min was calculated for individual experi- ments and the concentration at which 50% of the initial and remaining DPPH concentrations were calculated from standard DPPH graph.

Measurement of superoxide anion scavenging activity

The superoxide scavenging ability of the bitter cumin ex- tract was assessed by a modified method as described else- where [7]. Superoxide anions were generated in samples that contained 100 µl each of 1.0 mM NBT, 3.0 mM NADH and 0.3 mM PMS and the final volume was adjusted to 1 ml

with 0.1 M phosphate buffer (pH 7.8) at ambient tempera- ture. The reaction mixture (NBT and NADH) was incubated with or without cumin extract at ambient temperature for 2 min and the reaction was started by adding PMS. The absorbance at 560 nm was measured against blank sam- ples for 3 min. Decrease in absorbance in the presence of cumin extracts indicated superoxide anion scavenging ac- tivity. The percent inhibition was calculated by using the following formula.

Superoxide scavenging activity (%)

= Control OD − Sample OD Control OD

× 100

Measurement of rat liver microsomes lipid peroxidation activity

Rat liver was homogenized in 0.25 M Tris–HCl–Sucrose– EDTA buffer at pH 7.4. The homogenate was centrifuged at 7649 × g for 30 min to remove unbroken cells and cell debris. The supernatant was centrifuged at 100,000 × g in a refrigerated ultracentrifuge (Beckman L7-65 Ultracen- trifuge, USA) for 1 h to isolate the microsomal pellet, which is dissolved, into 2 ml of 125 mM KCl. The protein content of microsomes was estimated by Lowry method [8]. One milligram of microsomal protein was incubated with 0.2 mM FeSO4 and 0.2 mM ascorbic acid with or without cumin extract in a final volume of 1 ml at 37 ◦C for 1 h. Malondialdehyde (MDA) formed in the incubation mixture was reacted with thiobarbituric acid and thiobarbi- turic acid reactive substances (TBARS) were measured at 535 nm spectrophotometrically [9]. The antioxidant activ- 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 1.56 × 105 M/cm.

Lipoxygenase-dependent lipid peroxidation activity

Soybean lipoxygenase-dependent lipid peroxidation was measured as a decrease in absorbance of lipid hydroper-

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oxide formation at 234 nm spectrophotometrically [10]. Briefly, the reaction mixture in a final volume of 1 ml con- tained 200 µM linoleic acid and 5 nM soybean lipoxy- genase 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 spectrophotome- ter. 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 µl) contained 3 µg of calf thymus DNA in 20.0 mM phosphate buffer saline (pH 7.4) and different concentra- tions of bitter cumin extract (0.5, 1.0, 1.5 and 2.0 µg) 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 incu- bated them for 1 h at 37 ◦C. Similarly 1.0 µg of pUC18 DNA was incubated with different concentrations of cumin extract for 30 min at ambient temperature and 2.0 µl each of 100.0 µM FeSO4, 600.0 µM ascorbic acid and 60.0 mM H2O2 were added. The final volume was adjusted to 20.0 µl 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 infu- sion 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. Dif- ferent concentrations of cumin aqueous methanol–acetone extract were added to the wells and the control well re- ceived 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 concen- tration required to inhibit 50% of enzyme activity/scavenge 50% of free radicals) value was calculated from the dose- response 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 propor- tion of known natural antioxidants [11]. There is an increas- ing 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 an- tioxidant 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 high- molecular weight polyphenols. Thus, with the above sol- vent 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 µg GAE/mg of extract.

Many phenolic compounds are normally present as gly- cosides or aglycones in plants. The cumin extract was sub- jected 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). Inter- estingly, 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 char- acteristics 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 activ- ity, but each method has its own limitations [12]. Hence, we tested the effect of bitter cumin extract on different

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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 µg 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 µg/ml) (Fig. 1) but, less potent than BHA (IC50 8.25 ± 0.35 µg). 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 reac- tive 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 µg (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 as- sociated with carcinogenesis, autogenesis and ageing [15, 16]. Membrane lipids are particularly susceptible to ox- idation 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

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p er

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g in

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an io

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Fig. 2 C. nigrum seed extract scavenged superoxide anion radicals. Values mean ± SEM of three individual experiments

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n m

o le

s o

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rm ed

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o f

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te in

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 capa- ble 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 µg (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 mi- crosomal lipid peroxidation (Table 3).

The autoxidation of lipids as well as the enzymatic oxi- dation of fats, oils and fat-containing foods during storage and processing are responsible for rancidity and deteri- oration of food quality. Soybean lipoxygenase-dependent lipid peroxidation is an enzymatic lipid peroxidation as- say used to determine the antioxidant activity of test compounds. Cumin extract showed significant inhibition of lipoxygenase-dependent lipid peroxidation and the in- hibitory effect is found to be dose-dependent with an IC50 value of 28.0 ± 3.0 µg (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 [19–21]. 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 rad- ical 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-2- deoxyguanosine 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

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

Correlation coefficient between total phenol content and antioxidant activity

Antioxidant assay Free radicals IC50 value (µg)

DPPH free radical scavenging DPPH 14.0 ± 0.5 0.94 Soybean lipoxygenase-dependent lipid peroxidation

LOO 28.0 ± 3.0 0.92

Microsomal lipid peroxidation OH 110.0 ± 14.0 0.98 Superoxide anion scavenging O2− 125.4 ± 8.7 0.94Note. Values are mean ± SEMof three experiments

0.5–2.0 µg 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 abil- ity 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 food- borne 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). Enterobac- ter 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 Gram- negative bacteria. The antibacterial activity of flavonoids was attributed to inhibition of synthesis of DNA and RNA and other related macromolecules [25, 26]. Further, pheno- lic 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, protocate- chuic 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 antioxi- dant systems. Further, bitter cumin extract also showed an- tibacterial 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 µg cumin seeds extract. Lane 4: DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid + 1.0 µg cumin seeds extract. Lane 5: DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid + 1.5 µg cumin seeds extract. Lane 6: DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid + 2.0 µg cumin seeds extract

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Fig. 6. Protective effect of the extract of C. nigrum seeds on oxida- tive damage to pUC18 DNA. Lane 1: pUC18 DNA. Lane 2: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM H2O2. Lane 3: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM ascor- bic acid + 6.0 mM H2O2 + 0.5 µg of Cumin seeds extract. Lane 4: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM H2O2 + 1.0 µg of cumin seeds extract. Lane 5: pUC18 DNA + 10.0 µM FeSO4 + 60.0 µM ascorbic acid + 6.0 mM H2O2 + 2.5 µg of cumin seeds extract. Lane 6: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM H2O2 + 2.5 µg 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, Direc- tor 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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