Broad spectrum antimutagenic activity of antioxidant active fraction of Punica granatum L. peel extracts

Mutation Research 703 (2010) 99–107

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Broad spectrum antimutagenic activity of antioxidant active fraction of Punica granatum L. peel extracts Maryam Zahin, Farrukh Aqil 1 , Iqbal Ahmad ∗ Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, India

a r t i c l e

i n f o

Article history: Received 28 April 2010 Received in revised form 11 July 2010 Accepted 4 August 2010 Available online 11 August 2010 Keywords: Punica granatum Antioxidant activity Antimutagenic activity Methanol fraction

a b s t r a c t Over the past few decades, scientific research has indicated a credible basis for some of the traditional ethnomedicinal uses of pomegranate. This study aims to evaluate the broad spectrum antioxidant and antimutagenic activities of peel extracts of pomegranate. The sequentially extracted Punica granatum peel fractions were tested for their antioxidant activity by DPPH free radical scavenging, phosphomolybdenum, FRAP (Fe3+ reducing power) and CUPRAC (cupric ions (Cu2+ ) reducing ability) assays. The methanol fraction showed highest antioxidant activity by all the four in vitro assays comparable to ascorbic acid and butylated hydroxy toluene (BHT) followed by activity in ethanol, acetone, and ethyl acetate fractions. Based on the promising antioxidant activities, the methanol fraction was evaluated for antimutagenic activity by Ames Salmonella/microsome assay against sodium azide (NaN3 ), methyl methane sulphonate (MMS), 2-aminofluorene (2-AF) and benzo(a)pyrene (B(a)P) induced mutagenicity in Salmonella typhimurium (TA97a, TA98, TA100 and TA102) tester strains. The methanol fraction showed no sign of mutagenicity at tested concentration of 10–80 ␮g/mL. This fraction showed antimutagenic activity against NaN3 and MMS with percent inhibition of mutagenicity ranging from 66.76% to 91.86% in a concentration-dependent manner. Similar trend of inhibition of mutagenicity (81.2–88.58%) against indirect mutagens (2-AF and B(a)P) was also recorded. Phytochemical analysis by HPLC, LC–MS and total phenolic content revealed high content of ellagitannins which might be responsible for promising antioxidant and antimutagenic activities of P. granatum peel extract. Further, contribution of bioactive compounds detected in this study is to be explored to understand the exact mechanism of action as well as their therapeutic efficacy. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Molecular oxygen, besides being a terminal oxidant indispensable for the production of metabolic energy can also yield (5% of total oxygen) reactive oxygen species (ROS). These ROS and free radicals which are formed in the body as a consequence of normal metabolic reactions, by exposure to ionizing radiation and by the influence of many xenobiotics are indicated in the causation of several diseases [1]. ROS include superoxide radical, hydrogen peroxide and hydroxyl free radical, all of which have one or more unpaired electrons that potentially cause damage to the respiring cells. All these reactive species are highly toxic and mutagenic [2]. They can nick DNA, damage essential enzymes and proteins or

∗ Corresponding author. Tel.: +91 9412371170/571 2703516; fax: +91 571 2703516. E-mail addresses: f [email protected] (F. Aqil), [email protected], [email protected] (I. Ahmad). 1 Present address: 580 S. Preston Street, Brown Cancer Center, Baxter Research Building II, University of Louisville, Louisville, KY 40202, USA. Tel.: +1 502 762 8481; fax: +1 502 852 3842. 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.08.001

provoke uncontrolled lipid peroxidation and autooxidation reactions leading to cancer, degenerative disease, and other diseases [3]. There are endogenous as well as exogenous systems to protect cells in an animal body against such damage. However, in many conditions, exogenous supply of antioxidants is essential. Therefore, antioxidants which can scavenge free radicals have an important role in biological system and may exert their effects by different mechanisms, such as suppressing the formation of active species by reducing hydroperoxides (ROO• ) and H2 O2 and also by sequestering metal ions, scavenging free radicals, repairing or clearing damage [4]. Similarly, many instances of mutation-related carcinogenesis have been found and this has resulted in much detailed research on mutagenesis [5]. Mechanism of mutagenesis is complex however many mutagens and carcinogens may act through the generation of reactive oxygen species. ROS may play a major role as endogenous initiators of degenerative processes, such as DNA damage and mutation, which may be related to cancer, heart disease and aging [6]. Therefore, the discovery and the exploration of compounds possessing antioxidant, antimutagenic and anticancer properties are of great practical and therapeutic significance. The substances

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with antioxidant and antimutagenic activities have been found in many medicinal and food plants. Plants rich in flavonoids and phenolic compounds are known for their anticancer, antioxidant and other biological activities [7,8]. Natural antioxidants may reduce or inhibit the mutagenic potential of mutagens and carcinogens [9]. The cellular mutability control by natural antimutagens can provide ways for preventing mutations that conceivably result in cancer as well as diseases caused by genotoxic agents [10]. Pomegranate (Punica granatum) is native to the Mediterranean region and has been used extensively in the form of juice concentrate, canned beverage, jam and jelly, etc. [11]. For centuries, the fruits, leaves, flowers and barks of this plant have been used in various formulations in traditional Indian system of medicine to ameliorate diseases ranging from conjunctivitis to hematuria [12]. The phytochemistry of pomegranate has also been widely studied. The pomegranates are found to be a rich source of polyphenolic compounds that include flavonoids (anthocyanins, catechins and other complex flavonoids) and hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallagic acid and ellagic acid esters of glucose) which account for 92% of their antioxidant activities [13,14]. Various biological activities of P. granatum have been reported including antimicrobial and antimalarial [15] and antioxidant activities [16]. A fair amount of scientific literature is available on cancer chemoprevention by P. granatum extracts and active compounds in vitro as well as in experimental animal models [17,18]. However, limited information is available on broad spectrum antioxidant and antimutagenic activities of peel extracts of this plant [19]. An antimicrobial activity of peel crude extracts has been reported previously from our laboratory against drug resistant microbial pathogens [20]. On the other hand, a correlation between total phenolics with antioxidant and antimutagenic activities has also been found in other Indian medicinal plants [21,22]. Thus, considering the chemical diversity of biologically active compounds of pomegranate, we envisage that peel extracts exhibit broad spectrum antioxidant and antimutagenic activities which might have promising therapeutic potential. Therefore, sequentially extracted P. granatum peel extracts were evaluated for their antioxidant activity using different in vitro assays. Further, the most active fraction is examined for its antimutagenic properties against both direct and indirect acting mutagens. An attempt has also been made to detect major active compounds responsible for demonstrated broad spectrum activities. 2. Materials and methods 2.1. Bacterial strains and chemicals The Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 were kindly provided by Prof. B.N. Ames, University of California, Berkeley, USA. Sodium azide (NaN3 ), nicotinamide adenine dinucleotide phosphate sodium salt, d-glucose-6-phosphate disodium salt, sodium phosphate, ammonium molybdate, neocuproine, ferric chloride, l-histidine monohydrate, d-biotin and antioxidant standards (butylated hydroxy toluene (BHT), l-ascorbic acid and gallic acid) were purchased from Hi-Media Lab. Ltd., Mumbai, India. Potassium ferricyanide, cupric chloride and ammonium acetate were purchased from Qualigens Fine Chemicals, Mumbai, India. Methyl methane sulphonate (MMS) and trichloroacetic acid were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India; while 1,1diphenyl-2-picrylhydrazyl (DPPH) radicals, 2-aminofluorene and benzo(a)pyrene were purchased from Sigma Chemical Co, St. Louis, MO, USA. All the other reagents used to prepare buffers and media were of analytical grade. 2.2. Plant material and preparation of extracts P. granatum (L.) (Pomegranate), a plant of Punicaceae family was purchased from a local market of Aligarh, India. The peels of the fruits were removed manually, shade-dried and ground finely. The voucher specimen (MBD-02/06) was deposited in the Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India. The plant extract was prepared as described earlier by Zahin et al. [22]. Briefly, five hundred (500) grams of dry peel powder was soaked in 2.5 L of petrol ether for 5 days with intermittent shaking and

at the end of extraction the extract was filtered through Whatman filter paper no.1 (Whatman Ltd., England) to make a petrol ether fraction. The same dried powder of peel was further taken for fractionation with the same above procedure with benzene. After extraction, the same material was successively extracted with ethyl acetate, acetone, methanol and ethanol. The filtered fractions were concentrated to dryness under reduced pressure on rotary evaporator at 40 ◦ C and stored at 4 ◦ C for future use. Moreover, the yield of solvent dried fractions was calculated and finally reconstituted in minimum amount of DMSO to perform experiments. 2.3. Antioxidant assays Antioxidant potential of pomegranate fractions was determined by DPPH free radical scavenging assay, the reducing power by FRAP and cupric ions (Cu2+ ) reducing ability (CUPRAC) assays and total antioxidant capacity was evaluated by phosphomolybdenum method as described below. 2.3.1. Determination of total antioxidant capacity by phosphomolybdenum method The total antioxidant capacity of different fractions was evaluated by the method of Prieto et al. [23]. An aliquot of 0.1 mL of sample solution (containing 10, 20, 40 and 80 ␮g/mL of respective fraction) was combined with 1 mL of reagent (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). Methanol (0.1 mL) was used as blank in place of the sample. The tubes were capped and incubated in a boiling water bath at 95 ◦ C for 90 min, and then cooled at room temperature. The absorbance of each solution was then measured at 695 nm against a blank in a double beam UV–vis spectrophotometer UV570455 (EC, Electronic Corporation of India Limited). For samples of unknown composition, water-soluble antioxidant capacity of the extract was expressed as equivalents of ascorbic acid (␮mol/g). 2.3.2. DPPH radical scavenging assay Free radical scavenging activity of different plant fractions against stable DPPH was determined spectrophotometrically by the slightly modified method of Gyamfi et al. [24] as described below. When DPPH reacts with an antioxidant, which can donate hydrogen, it is reduced. The changes in color (from deep-violet to lightyellow) were measured at 517 nm on a UV/vis light spectrophotometer (Spectronic 20 D+ , USA). Fifty microliters of the solvent dried peel fractions in methanol, yielding 10, 20, 40 and 80 ␮g/mL, respectively, in each reaction was mixed with 1 mL of 0.1 mM DPPH in methanol solution and 450 ␮L of 50 mM Tris–HCl buffer (pH 7.4). Methanol (50 ␮L) was used as a vehicle control in the experiment. After 30 min of incubation at room temperature the reduction of the DPPH free radical was measured spectrophotometrically. Ascorbic acid and butylated hydroxyl toluene were used as controls. Percent inhibition was calculated from the following equation:



%Inhibition =

Absorbance of control − Absorbance of test sample Absorbance of control



× 100

2.3.3. FRAP assay (Fe3+ reducing power assay) Reducing power was measured by the direct reduction of Fe3+ (CN− )6 to Fe2+ (CN− )6 , and was determined by measuring absorbance resulted from the formation of the Perl’s Prussian Blue complex following the addition of excess ferric ions (Fe3+ ). Hence, the ferric reducing antioxidant power (FRAP) method of Oyaizu [25] with little modification was adopted to measure the reducing capacity. This method is based on the reduction of (Fe3+ ) ferricyanide in stoichiometric excess relative to the antioxidants [26]. Different concentrations of extracts (10–80 ␮g/mL) in 0.75 mL of distilled water were mixed with 1.25 mL of 0.2 M (pH 6.6) sodium phosphate buffer and 1.25 mL of 1% potassium ferricyanide [K3 Fe(CN6 )]. After 20 min of incubation at 50 ◦ C for 20 min, the reaction mixture was acidified with 1.25 mL of trichloroacetic acid (10%). Finally, 0.5 mL of FeCl3 (0.1%) was added to this solution, and the absorbance was measured at 700 nm. The increased absorbance of the reaction mixture indicates greater reduction capability. 2.3.4. CUPRAC assay In order to determine the cupric ions (Cu2+ ) reducing ability of P. granatum peel extracts, the method of Apak et al. [27], was used with a little modification as described by Gulcin [26]. For the assay, 0.25 mL CuCl2 solution (0.01 M), 0.25 mL ethanolic neocuproine solution (7.5 × 10−3 M) and 0.25 mL CH3 COONH4 buffer solution (1 M) were added to a test tube, followed by mixing with different concentrations of extracts (10–80 ␮g/mL). Then, total volume was adjusted to 2 mL with distilled water, and the solution was mixed well. The tubes were stoppered and kept at room temperature. Absorbance was measured at 450 nm against a reagent blank 30 min later. Increased absorbance of the reaction mixture indicates increased reduction capability. 2.4. Antimutagenicity assay The Salmonella histidine point mutation assay described by Maron and Ames in 1983 was used to test the antimutagenic activity of the extracts, with some modifications as described earlier [21]. In the pre-incubation experiment, a mixture of solvent dried P. granatum peel extract and mutagen, each having a volume

M. Zahin et al. / Mutation Research 703 (2010) 99–107 of 0.1 mL of varying concentrations, was pre-incubated at 37 ◦ C for 30 min and then 0.1 mL of 1 × 107 CFU/mL density of the bacterial culture was added, followed by the addition of 2.5 mL of top agar at 45 ◦ C (containing 0.5% NaCl and 0.6% agar) supplemented with 0.5 mM histidine–biotin. The influence of metabolic activation of indirect acting mutagens, B(a)P (benzo(a)pyrene) and 2-AF (2-aminofluorene), was tested using 500 ␮L of S9 mixture (S9 at a concentration of 0.04 mg proteins/mL of mix). The S9 microsome fraction was prepared from the livers of rats treated with Aroclor 1254 using standard protocols [6]. The combined solutions were vortexed and poured onto minimal glucose plates (having 40% glucose solution and Vogel Bonner medium). The plates were incubated at 37 ◦ C for 48 h, after which the numbers of histidine-independent revertant colonies were scored. Survival of the bacteria was routinely monitored for each experiment. To check the toxicity of the test sample, parallel controls were run with extracts alone at all concentrations tested with mutagens. The concentrations of the test sample for investigating the antimutagenicity were 10, 20, 40 and 80 ␮g solvent dried extract/0.1 mL/plate. These were tested against direct acting mutagens sodium azide (1.5 ␮g/0.1 mL/plate) and MMS (1 ␮g/0.1 mL/plate) in TA97a, TA98, TA100 and TA102 tester strains as well as against indirect acting mutagens B(a)P and 2-AF. All the test samples and mutagens were dissolved in DMSO. In each case, no toxicity was observed and the numbers of spontaneous revertants were identical to the DMSO vehicle control. Non-toxic concentrations were categorized as those where there was a well-developed lawn, almost similar size of colonies and no statistical difference in the number of spontaneous revertants in test and control plates. Triplicate plates were set up with each concentration and the entire experiment was repeated twice. Inhibition of mutagenicity was expressed as percentage decrease of reverse mutation and calculated as:



Percent inhibition =

a−b a−c



× 100

where a = number of histidine revertants induced by mutagen, b = number of histidine revertants induced by mutagen in the presence of plant extract and c = number of revertants induced in negative control. 2.5. Phytochemical analysis

Fig. 1. Radical scavenging activity of different concentrations (10–80 ␮g/mL) of Punica granatum peel fractions (ethyl acetate, acetone, methanol and ethanol) by DPPH method. The activity is compared with ascorbic acid and BHT. Each data ) point represents the mean of three experiments with standard deviation. ( ) acetone; ( ) ethanol; ( ) ascorbic acid; (––) Ethyl acetate; ( methanol and ( ) BHT. molecular ions (M−H+) obtained by ES/MS and tandem MS with the expected theoretical molecular weights from literature data [31] as punicalagin (m/z 1083); punicalin (m/z 781); gallagic acid (m/z 601) and EA (m/z 301) and many others as shown in the Fig. 4.

2.5.1. Total phenolic content of plant fractions The total phenolic content of the plant fractions was determined with the Folin–Ciocalteau reagent by the method of Spanos and Wrolstad [28], as modified by Lister and Wilson [29]. To 0.5 mL of each sample (containing 10, 20, 40 and 80 ␮g/mL of the extract), 2.5 mL of 1/10 dilution of Folin–Ciocalteau’s reagent and 2 mL of Na2 CO3 (7.5%, w/v) were added and incubated at 45 ◦ C for 15 min. Each experiment was performed in triplicates. The absorbance of all samples was measured at 765 nm using a UV/vis spectrophotometer. Results were expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g dw).

2.6. Statistical analysis

2.5.2. HPLC analysis All samples were filtered (0.22 ␮m) and analyzed (20 ␮L injection volume) on Diode Array detector (Shimadzu) in the absorbance range between 220 and 400 nm on a Premier C-18 column (250 mm × 4.6 mm, 5 ␮m). The mobile phase, solvent A (3.5% phosphoric acid in water) and solvent B (acetonitrile) were used in a gradient where initial A, 95% for 10 min; 90%, 20 min; 80%, 30 min; 62%, 35 min; 55%, 40 min; 40%, 51–56 min; 95%, 61 min; run time 61 min at flow rate 0.75 mL/min. Pure standard punicalagins (Chromadex Corporation, Irvine, CA, USA) and ellagic acid (LKT Laboratories, St. Paul, MN, USA) were accurately weighed and dissolved in H2 O:MeOH (1:1, v/v) and DMSO, respectively, and injected in duplicate. Dried pomegranate acetone and methanol extracts (1 mg each) were dissolved in H2 O:MeOH (1:1, v/v), and tR of punicalagins (two individual peaks corresponding to ␣- and ␤-anomers) and EA peaks were matched with standards for identification.

3. Results

2.5.3. LC–MS analysis The method of Seeram et al. [30] was used for the LC–MS analysis of the pomegranate acetone and methanol fractions using Accela LC from Thermo Scientific (San Jose, CA, USA) with Hypersil gold C-18 column (50 mm × 2.1 i.d.). Solvent: A 2% HCOOH/H2 O, B 2% HCOOH/MeOH; gradient% A in B: initial: 99%, 30 min: 80%, 45 min: 60%, 60 min: 5%; run time 60 min; flow rate 0.15 mL/min; injection volume 10 ␮L. MS parameters: ionization mode, electron spray (ES) negative mode; scan range: 150–1200 amu; scan rate: 1 scan/s. Peak identities were obtained by matching their

101

The results are presented as the average and standard error or standard deviation of three experiments with triplicate plates/dose/experiment. The data were further analyzed for statistical significance using analysis of variance (one way ANOVA) by Tukey test where treatment groups were compared with their respective positive controls. The regression analysis was carried out in Microsoft Excel 2003 between percent inhibition of mutagenicity and log values of concentrations of the plant extract.

The percent yields of sequentially extracted fractions of P. granatum in different solvents viz petrol ether, benzene, ethyl acetate, acetone, methanol and ethanol were 0.2, 0.16, 1.2, 5.2, 10.2 and 6.1, respectively. These fractions were tested in order to locate the fraction with broad spectrum antioxidant activity. Total antioxidant activity by phosphomolybdenum method exhibited concentration-dependent antioxidant capacity with respect to ascorbic acid equivalents (Table 1). At highest tested concentration (80 ␮g/mL), the methanol fraction showed maximum antioxidant capacity (5067.7 ␮mol) followed by ethanol (3323.0 ␮mol), and acetone (2481.6 ␮mol) fractions. However, ethyl acetate fraction displayed relatively less total antioxidant capacity at the tested concentrations of 10–80 ␮g/mL. The free radical scavenging activity of the peel extracts of P. granatum was measured as decolorizing activity following the trapping of the unpaired electron of DPPH as shown in Fig. 1. A concentration-dependent response is evident in all the frac-

Table 1 Antioxidant capacity of Punica granatum peel extracts as ascorbic acid equivalents (␮mol/g of dry extract) by phosphomolybdenum method. Concentration (␮g/mL)

Ethyl acetate

10 20 40 80

458.2 607.6 758.9 862.8

± ± ± ±

The above data are the mean of three experiments ± SE.

2.1 5.4 8.7 2.4

Acetone 754.5 1173.3 1867.7 2481.6

± ± ± ±

Methanol 13.5 12.4 14.6 17.6

1916.9 2884.5 4202.0 5067.7

± ± ± ±

Ethanol 12.9 15.3 43.7 34.6

1041.9 1692.3 2496.9 3323.0

± ± ± ±

17.8 15.0 10.2 42.0

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tions (methanol, acetone and ethanol) at tested concentrations of 10–80 ␮g/mL. These fractions almost completely inhibited DPPH absorption; methanol fraction: 90.53%, acetone: 86.4% and ethanol fraction inhibited 83.2% DPPH absorption. Positive controls (ascorbic acid and BHT) inhibited 91.1% and 85.6% DPPH absorption, respectively. Moreover, ethyl acetate fraction was relatively less efficient as radical scavengers with an inhibition of 16.2%. Petroleum ether and benzene fractions showed remarkably lower degrees of radical scavenging activity (data not shown). As can be seen from Fig. 2, these fractions had effective reducing power using the potassium ferricyanide reduction method when compared to the standards. Similarly, the antioxidant activity by CUPRAC assays indicated the highest reducing power potential in methanol fraction followed by ethanol, acetone and ethyl acetate fractions (Fig. 2A and B). The results are comparable to ascorbic acid and BHT. Based on promising broad spectrum antioxidant activity, methanol fraction of P. granatum peels was selected and evaluated for its antimutagenic activity by Ames test against direct acting (NaN3 and MMS) and indirect acting (2-AF and B(a)P) mutagens with metabolic activation of S9. In a series of experiments preceding the antimutagenicity studies, it was ascertained that the different concentrations of methanol fraction added to the Ames

Fig. 2. Reducing power of different concentrations (10–80 ␮g/mL) of Punica granatum peel fractions (ethyl acetate, acetone, methanol and ethanol) and compared with ascorbic acid and BHT. (A) FRAP assay and (B) CUPRAC assay. Each data ) point represents the mean of three experiments with standard deviation. ( ) acetone; ( ) ethanol; ( ) ascorbic acid; (––) Ethyl acetate; ( ) BHT. methanol and (

tester strains do not influence their viability. In the Ames test, it was shown that in the presence of the different doses of the extract (10, 20, 40 and 80 ␮g/plate), the mutation frequencies did not change significantly when compared to spontaneous ones indicating that there is no sign of mutagenicity of the plant extracts. The data of P. granatum methanol fraction on direct acting mutagens NaN3 and MMS are presented in Tables 2 and 3. At the concentration of 80 ␮g/plate, the extracts exhibited maximum percent antimutagenicity in TA100 (84.5) followed by TA97a (80.4), TA98 (76.8) and TA102 (66.8) tester strains against NaN3 induced mutagenicity (Table 2). The results were statistically significant while linear relationship between extract dose and antimutagenic response was strong in the strain TA97a (R2 = 0.925), TA98 (R2 = 0.946), TA100 (R2 = 0.992) and TA102 (R2 = 0.881). Likewise, the percent inhibition of MMS induced mutagenicity was recorded as 91.9% in TA100, 90.5% in TA102, 86.6% in TA97a and 76.6% in TA98. The antimutagenic effect of methanol fraction was found to be concentration-dependent as evident from the regression analysis between extract dose and antimutagenic response against respective test mutagen in TA97a (R2 = 0.995) followed by TA98 (R2 = 0.984), TA100 (R2 = 0.983) and TA102 (R2 = 0.981). The P. granatum methanol fraction was also evaluated for its antimutagenic behaviour against benzo(a)pyrene and 2aminoflourene that infers mutagenicity by microsomal activation as shown in Tables 4 and 5. The dose-dependent antimutagenic response was highly significant with percent inhibition of mutagenicity ranging from 81.2% to 87.2% (Table 4). All the strains demonstrated reduction in the revertants in a dose-dependent manner with the regression values ranging from 0.97 to 0.98. Similar trend of antimutagenic activity against 2-AF was shown by P. granatum methanol fraction. The significant reduction (p ≤ 0.05) in a number of revertants was recorded by TA100 (88.9%) followed by TA102 (86.0%), TA97a (83.8%) and TA98 (82.3%) (Table 5). Further, the linear regression analysis between extract dose and antimutagenic response showed strong correlation in TA100 (R2 = 0.989) followed by TA98 (R2 = 0.988), TA97a (R2 = 0.987) and TA102 (R2 = 0.962). Phytochemical analysis of fractions revealed the presence of phenolics as major group of compounds. The total phenolic content equivalent to gallic acid of various fractions (mg/g of dry extract) determined by the Folin–Ciocalteau method showed highest polyphenolic content (468.3 ± 5.5) in methanol fraction followed by the ethanol (414.6 ± 5.9), acetone (219.3 ± 1.1) and ethyl acetate (20.3 ± 0.7) fractions. The plant fractions which displayed fair to good antioxidant activity were subjected to HPLC followed by LC–MS analysis. LC–MS spectra by direct infusion of all punica fractions show the presence of punicalagins (M−H m/z 1083), punicalin (M−H m/z 781), corilagin (M−H m/z 633), gallagic acid (M−H m/z 601), 2.3-(S)HHDP-d-glucose (M−H m/z 433) and ellagic acid (M−H m/z 301). The other peaks of major compounds identified are presented in Table 6. Interestingly, HPLC analysis of acetone and methanol fractions confirmed the presence of punicalagins A and B as well as ellagic acid; however, the relative abundance of ellagic acid was more in methanol fraction as compared to acetone fraction (Fig. 3). The retention times of punicalagins A and B and ellagic acid in aqueous phosphoric system were found to be 28.5, 30.5 and 37.5 min, respectively, when compared with the respective standards. HPLC chromatograms of crude and ethyl acetate fractions showed the presence of EA but either very low or no punicalagins (Fig. 3E and F). The presence of various polyphenols can be seen in all the fractions by MS analysis. In addition to punicalagins and ellagic acid, punicalin, gallagic acid and few other phenolics are present in appreciable amount (Fig. 4 and Table 6).

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Table 2 Effect of methanolic fraction of Punica granatum on the mutagenicity induced by sodium azide (NaN3 ) using Salmonella typhimurium strains. Treatment

Spontaneous Positive control NaN3

Dose (␮g/plate)

1.5

Number of His+ revertants colonies/plate TA97a

TA98

TA100

TA102

140.0 ± 2.3 246.3 ± 9.2a

31.0 ± 2.1 52.7 ± 1.2a

128.7 ± 5.7 352.0 ± 19.1a

245.7 ± 13.5 343.3 ± 19.2a

Punica granatum*

10 20 40 80

185.7 ± 6.9 165.7 ± 2.3 136.3 ± 5.2 117.0 ± 2.7

46.6 ± 0.9 36.0 ± 1.5 32.7 ± 0.7 29.7 ± 0.9

180.7 ± 3.5 153.0 ± 5.3 125.3 ± 2.2 113.3 ± 1.9

296.0 ± 7.1 262.7 ± 4.9 241.7 ± 2.9 226.0 ± 4.6

Punica granatum + NaN3 **

10 20 40 80

231.0 ± 9.9ab (25.3) 201.7 ± 10.5bc (55.4) 165.3 ± 12.8cd (73.6) 142.3 ± 10.1de (80.4)

50.7 ± 2.6a (33.3) 46.0 ± 1.2ab (40.0) 39.3 ± 2.0bc (66.7) 35.0 ± 2.1cd (76.8)

326.0 ± 11.4ab (15.17) 287.0 ± 16.2b (32.66) 220.7 ± 7.5c (57.94) 150.3 ± 10.7de (84.50)

325.0 ± 11.0ab (38.7) 311.7 ± 6.9abc (39.3) 281. 3 ± 15.0bcde (61.0) 265.0 ± 10.8cde (66.8)

F value treatment

25.27

26.57

77.36

12.77

The data represented in the table are the mean ± SE values of three replicates. Mean values followed by different letters are significantly different at p ≤ 0.05 when compared to positive control. The values in parenthesis are the inhibition rates (%) of mutagenicity. Positive control: NaN3 , sodium azide. *Negative control; **pre-incubation test. Table 3 Effect of methanolic fraction of Punica granatum on the mutagenicity induced by methyl methane sulphonate (MMS) using Salmonella typhimurium strains. Treatment

Spontaneous Positive control MMS

Dose (␮g/plate)

1.0

Number of His+ revertants colonies/plate TA97a

TA98

TA100

TA102

140.0 ± 2.3 443.7 ± 11.5a

31.0 ± 2.1 51.0 ± 1.5a

128.7 ± 5.6 952.7 ± 26.6a

245.7 ± 13.5 1195.3 ± 45.6a

Punica granatum*

10 20 40 80

185.7 ± 6.9 165.7 ± 2.3 136.3 ± 5.2 117.0 ± 2.7

46.7 ± 0.9 36.0 ± 1.5 32.7 ± 0.7 29.7 ± 0.9

180.7 ± 3.5 153.0 ± 5.3 125.3 ± 2.2 113.3 ± 1.9

296.0 ± 7.1 262.7 ± 4.9 241.7 ± 2.9 226.0 ± 4.6

Punica granatum + MMS**

10 20 40 80

392.0 ± 20.0a (20.0) 315.3 ± 10.9b (46.2) 235.7 ± 13.6c (67.8) 160.7 ± 8.4de (86.6)

50.0 ± 1.2a (23.0) 45.3 ± 1.5ab (37.8) 41.3 ± 1.5bc (52.7) 34.7 ± 1.5cd (76.6)

603.0 ± 17.4b (45.3) 492.3 ± 13.0c (57.6) 365.7 ± 14.1d (71.0) 181.7 ± 14.7e (91.9)

855.3 ± 26.3b (37.8) 621.0 ± 21.4c (61.6) 457.7 ± 14.9d (77.4) 318.0 ± 15.1e (90.5)

34.61

462.63

268.22

F value treatment

91.80

The data represented in the table are the mean ± SE values of three replicates. Mean values followed by different letters are significantly different at p ≤ 0.05 when compared to positive control. The values in parenthesis are the inhibition rates (%) of mutagenicity. Positive control: MMS, methyl methane sulphonate. *Negative control; **pre-incubation.

Table 4 Effect of methanolic fraction of Punica granatum on the mutagenicity induced by benzo(a)pyrene with metabolic activation using Salmonella typhimurium strains. Treatment

Spontaneous Positive control B(a)P

Dose (␮g/plate)

1.0

Number of His+ revertants colonies/plate TA97a

TA98

TA100

TA102

143.7 ± 7.62 720.3 ± 16.4a

37.0 ± 1.7 155.0 ± 4.4a

135.3 ± 5.5 695.7 ± 9.8a

316.0 ± 8.4 648.0 ± 9.9a

34.0 ± 1.2 38. 3 ± 2.0 45.7 ± 0.9 48.0 ± 2.0

138.3 ± 4.4 151.0 ± 4.7 164.7 ± 3.8 178.0 ± 5.1

305.7 ± 9.6 320.0 ± 10.7 328.3 ± 4.3 337.3 ± 6.3

553.7 ± 16.6b (25.5) 450.0 ± 12.2c (45.1) 333.3 ± 10.8d (68.2) 275.3 ± 11.1e (81.2)

570.0 ± 15.3b (22.8) 482.3 ± 12.4c (50.5) 432.7 ± 11.8c (67.4) 377.0 ± 13.6d (87.2)

447.43

125.6

Punica granatum*

10 20 40 80

141.3 ± 2.9 150.7 ± 1.8 161.0 ± 4.7 175.3 ± 6.1

Punica granatum + B(a)P**

10 20 40 80

615.0 ± 14.2b (18.2) 530.3 ± 10.7c (33.4) 402.7 ± 14.5d (56.8) 264.0 ± 10.0e (83.7)

F value treatment

474.93

135.0 ± 10.4ab (16.5) 110.3 ± 10.7b (38.3) 75.0 ± 2.1c (73.2) 63.7 ± 2.6cd (85.4) 72.87

The data represented in the table are the mean ± SE values of three replicates. Mean values followed by different letters are significantly different at p ≤ 0.05 when compared to positive control. The values in parenthesis are the inhibition rates (%) of mutagenicity. Positive control: B(a)P, benzo(a)pyrene.*Negative control; **pre-incubation test.

4. Discussion Antioxidants have attracted much interest with respect to their protective effect against free radical damage that may be the cause of many diseases including cancer. Over the past few

decades, scientific research has indicated a credible basis for some of the traditional ethnomedicinal uses of pomegranate. Therefore, we have focused our study to elucidate the broad spectrum antioxidant potential of P. granatum peel fractions by means of four different in vitro tests including total antioxidant capacity

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Table 5 Effect of methanolic fraction of Punica granatum on the mutagenicity induced by 2-aminofluorene with metabolic activation using Salmonella typhimurium strains. Treatment

Dose (␮g/plate)

Spontaneous Positive control (2-AF)

5.0

Number of His+ revertants colonies/plate TA97a

TA98

TA100

TA102

143.7 ± 7.6 328.0 ± 6.9a

37.0 ± 1.73 262.3 ± 9.9a

135.3 ± 5.5 502.0 ± 11.0a

316.0 ± 8.4 1430.3 ± 18.7a

Punica granatum*

10 20 40 80

141.3 ± 2.9 150.7 ± 1.8 161.0 ± 4.7 175.3 ± 6.1

Punica granatum + 2-AF**

10 20 40 80

280.0 ± 6.4b (25.7) 253.3 ± 7.2b (42.1) 212.7 ± 10.1c (69.1) 200.0 ± 10.6cd (83.8)

F value treatment

84.32

34.0 ± 1.2 38.33 ± 2.03 45.7 ± 0.9 48.0 ± 2.0

138.3 ± 4.4 151.0 ± 4.7 164.7 ± 3.8 178.0 ± 5.1

220.3 ± 7.8b (18.4) 181.3 ± 9.5c (36.2) 120.7 ± 5.8d (65.4) 86.0 ± 4.6e (82.3)

446.0 ± 11.0b (15.4) 362.3 ± 9.3c (39.8) 262.7 ± 8.4d (70.1) 215.0 ± 6.3e (88.9)

227.96

329.78

305.7 ± 9.6 320.0 ± 10.7 328.3 ± 4.3 337.3 ± 6.3 1063.3 ± 13.6b (32.6) 947.0 ± 11.0c (43.5) 772.7 ± 11.0d (59.7) 490.3 ± 14.1e (86.0) 622.22

The data represented in the table are the mean ± SE of three replicates. Mean values followed by different letters are significantly different at p ≤ 0.05 when compared to positive control. The values in parenthesis are the inhibition rates (%) of mutagenicity. Positive control: 2-AF, 2-aminoflourene. *Negative control; **pre-incubation test.

by phosphomolybdenum method, DPPH free radical scavenging assay, and reducing power by two methods (FRAP Fe3+ –Fe2+ transformation and CUPRAC assay). The tested fractions showed varying levels of antioxidant potential when compared with positive controls (BHT and ascorbic acid) and they may be of interest because of their multiple biological activities. The present study demonstrated that the sequential fractionation of pomegranate peel in various solvents resulted in the extraction of antioxidant bioactive compounds mainly in acetone, methanol and ethanol fractions. These fractions were used to determine their antioxidant capacities by the formation of phosphomolybdenum complexes. This method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compounds with the formation of a green Mo(V) complex which shows maximum absorption at 695 nm [19]. Total antioxidant activity by phosphomolybdenum method revealed methanol fraction as a potential source of antioxidant compounds responsible for the demonstrated activity. The DPPH method was used to assess the determination of potential radical scavenging activities of P. granatum fractions. The results revealed that methanol fraction has the highest free radical scavenging activity probably due to the presence of high polyphenolic content. Similar correlation of antioxidant activity and phenolic contents has also been shown in other studies [27]. These fractions act via their hydrogen donating ability, intercept the free radical chain of oxidation by giving hydrogen from the phenolic hydroxyl groups, thereby forming a stable end product that blocks the oxidation of lipid [32]. On the other hand, the reducing power reflects the electron donating capacity of bioactive compounds that

is associated with antioxidant activity [33]. Antioxidants can be reductants and inactivate the oxidants. The reducing capacity of a compound can be measured by the direct reduction of Fe[(CN)6 ]3 to Fe[(CN)6 ]2 . The addition of free Fe3+ to the reduced product leads to the formation of the intense Perl’s Prussian Blue complex, Fe4 [Fe(CN− )6 ]3 , which has a strong absorbance at 700 nm. An increase in absorbance of the reaction mixture would indicate an increase in the reducing capacity due to an increase in the formation of the complex [26]. Hence, the data presented indicated that the high antioxidant activity of pomegranate peel extracts seems to be the result of their reducing power capability. Similarly, another reducing power method (CUPRAC assay) is based on the reduction of Cu2+ to Cu1+ by antioxidants. This method is simultaneously cost-effective, rapid, stable, selective and suitable for a variety of antioxidants regardless of chemical type or hydrophilicity. Besides, it was reported that the results obtained from in vitro cupric ions (Cu2+ ) reducing measurements might be more efficiently extended to the possible in vivo reactions of antioxidants. The observed antioxidant potential for these extracts can be related to the presence of various functional groups, such as hydroxyl and carbonyl groups [34,35]. The highest total polyphenolic content was revealed in methanol fraction followed by other fractions which are in agreement with the reports of other workers [19,36]. On the other hand, the ethyl acetate fraction has the lowest antioxidant potential contrary to the earlier report [37], which showed second highest activity in ethyl acetate fraction. These variations in studies are mainly due to the different parts of the plant used, source of plant material as well as methods of extraction employed for experiments.

Table 6 Major compounds detected by LC–MS analysis. S. no.

Name

Molecular formula

Mol wt.

Major group

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Gallic acid 3,4,8,9,10-Pentahydroxy-dibenzo[b,d]pyran-6-one Ellagic acid Glucogallin Eschweilenol C 2,3-(S)-HHDP-d-glucose Gallagic acid Corilagin Punicalin Pedunculagin Granatin A Granatin B Castalagin Punicalagin

C7 H6 O5 C13 H18 O7 C14 H6 O8 C13 H16 O10 C20 H16 O12 C13 H18 O14 C28 H10 O16 C27 H22 O18 C34 H22 O22 C34 H24 O22 C34 H24 O23 C34 H28 O27 C41 H26 O26 C48 H28 O30

170.12 276.20 302.19 332.26 448.33 482.32 602 634.45 782.53 784.52 800.54 952.64 934.63 1084.70

Flavonol Gallyol derivative Ellagic acid derivative Ellagic acid derivative Gallotannins Ellagitannin Ellagitannin/gallotannin Ellagitannin/gallotannin Ellagitannin/gallotannin Ellagitannin/gallotannin Ellagitannin/gallotannin Ellagitannin/gallotannin Ellagitannin/gallotannin

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Fig. 3. HPLC chromatograms of Punica granatum (peel) fractions, standard ellagic acid (EA), and punicalagin anomers (PC). (A) Chromatogram of ellagic acid standard at tR 37.6 min, (B) chromatogram of standard punicalagin anomers at tR 28 and 31 min, respectively, (C) chromatogram of pomegranate acetone fraction showing the presence of PC and EA, (D) chromatogram of pomegranate (peel) methanol fraction showing the presence of PC and EA and (E and F) chromatogram of crude (ethanol) extract and ethyl acetate fraction.

The findings of the present study indicated that the antioxidant activities of P. granatum are related to the extraction of both low and high molecular weight phenolic compounds present in one or more fractions [38]. An array of bioactive compounds is reported so far from different parts of the plant [14]. Both flavonoids and tannins are more abundant in the peels of wild and cultivated fruits. Moreover, antioxidative efficacy of polyphenolic components of pomegranate peels has been reported in vitro as well as in vivo [11]. Therefore it could be suggested that this higher polyphenol extraction yield corresponds with the higher antioxidant activity, probably due to the combined action of the substances present in variable concentrations and their high hydrogen atom donating abilities. In addition, the additive or synergistic effects of these polyphenols could produce higher antioxidant activity of the crude extracts than that of the isolated compounds [39]. Mutations are the cause of inborn errors of metabolism leading to morbidity and mortality in living organisms. Besides inherited metabolic disorders, a spectrum of age related human diseases, including cancer, are caused by mutations. Mutagenic agents may be synthetic or natural toxic substances. Since cancer has become the number one cause of death, much attention has been focused on the chemoprevention of cancer, with little success [18]. However, less attention has been given to the substances in medicinal plants and herbal medicines that may serve to protect against chemical

mutagens or carcinogens acting as initiators in the carcinogenic process. Since, antimutagenic activity of pomegranate peel extract is less explored and acetone and ethanol fraction revealed comparatively less antioxidant activity than methanol fraction. This has formed the basis for selection of methanol fraction to study its antimutagenic behaviour against the respective mutagens. Any agent which showed a two fold increase in the number of revert ants over spontaneous in the Ames Salmonella test, may be designated as mutagenic. Mutagenicity of the natural compounds has been commonly assessed on this basis [45]. Based on the above criterion, methanol extract of P. granatum is found to be non-mutagenic. In general, the ways by which inhibitors of mutagenesis can act include the inhibition of interaction between genes and biochemically reactive mutagens; the inhibition of metabolic activation of indirectly acting mutagens by inactivation of metabolizing enzymes or interaction with the pro-mutagens making them unavailable for the enzymatic process. Thus by using the Ames test, the methanol fraction exhibited concentration-dependent antimutagenicity against direct and indirect acting mutagens in the presence of S9. Our findings are in agreement with literature, which reported concentration-dependent antimutagenic activity in other natural products [21,40]. On the other hand, Edenharder et al. [41] reported that antimutagenesis of flavonoids and struc-

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Fig. 4. LC–MS spectra (A–C) of Punica granatum (peel) fractions; (A) acetone fraction, (B) ethanol fraction and (C) methanol fraction. LC–MS spectra by direct infusion of all Punica fractions show the presence of punicalagins (M−H m/z 1083), punicalin (M−H m/z 781), corilagin (M−H m/z 633), gallagic acid (M−H m/z 601), 2.3-(S)-HHDP-d-glucose (M−H m/z 433), ellagic acid (M−H m/z 301). Other compounds present in acetone fractions are defined in chromatogram and in Table 6.

turally related compounds may be dependent or independent of concentration. A concentration-dependent antimutagenicity is evident in all the tester strains. The studies on antimutagenic activity of plants have indicated the involvement of chemical constituents which could act as non-specific redox agents, free radical scavengers, or ligands for binding metals or toxic principles [42]. However, it has been found that polyphenolic molecules undergo redox reactions by donating hydrogen to reducing agents. Previous studies have shown that the total phenolic content, as determined by Folin–Ciocalteau, is highly correlated with antioxidant assays [27]. Similarly, we have also found the higher total phenolic content in P. granatum methanol fraction which is due to the solubility of ellagitannins and ellagic acid. Hence, the possible mechanism of the demonstrated antimutagenic behaviour could be due to the bioactive phenolic antioxidant compounds present in methanol fraction which might interact with the reactive intermediates (B(a)P) or interfered with the metabolic activation of the pro-mutagen (2-AF) or tends to interact directly with the ultimate mutagenic metabolite. Thus, the alteration in the structure and function of P-450 enzyme may result in altered rates and differential pathways of metabolism of mutagens and carcinogens, and provide protection against chemically induced mutagenesis [40]. This could possibly

be a reason for its potential antimutagenic behaviour which might be helpful in exploring its mechanism of action for unfolding the cure of various diseases associated with the mutagenesis. In this study, HPLC followed by LC–MS analysis showed the presence of various phenolic compounds and ellagitannins specifically punicalagins, punicalin and gallagic acid are predominant with free ellagic acid. These compounds have been reported to have high antioxidant activity. Ellagic acid has also been reported to have antimutagenic activity against aflatoxin B induced mutagenicity in Salmonella tester strains [43]. The significant antimutagenic activity of some of these fractions against direct and indirect acting mutagens suggests that these compounds may directly protect DNA damage from mutagen. However, the inhibition of mutagenesis is often complex and goes through multiple mechanisms [8,44]. Therefore, it is concluded that the most active fraction of P. granatum peel extracts could be obtained by sequential extraction using appropriate solvents. Interestingly, the methanol fraction is rich in ellagitannins, a group of phenolics that could be responsible for demonstrated broad spectrum antioxidant and antimutagenic properties. Thus, further studies should be conducted to isolate the active principles. Alternatively, methanolic fraction may be standardized by its HPTLC markers and could be selected as such to evaluate its efficacy and safety in animal model system.

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