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Antimutagenicity of hydrolyzable tannins from Terminalia chebula in Salmonella typhimurium
Mutation Research 419 Ž1998. 169–179
Antimutagenicity of hydrolyzable tannins from Terminalia chebula in Salmonella typhimurium Simran Kaur a , I.S. Grover b
a,)
, Majar Singh b, Satwinderjeet Kaur
a
a Department of Botanical Sciences, Guru Nanak DeÕ UniÕersity, Amritsar 143 005, India Department of Applied Chemical Sciences and Technology, Guru Nanak DeÕ UniÕersity, Amritsar 143 005, India
Received 21 April 1998; revised 3 September 1998; accepted 3 September 1998
Abstract A tannin fraction ŽTC-E. from the dried fruit pulp of Terminalia chebula was obtained by successfully extracting with 95% ethyl alcohol and ethyl acetate. TC-E was subjected to silica gel chromatography which yielded four fractions, viz., TC-EI, TC-EII, TC-EIII and TC-EIV. Thin layer chromatography ŽTLC. and 13C-NMR revealed that TC-EI was gallic acid ŽGA. derivative while the other fractions were tannin in nature. TC-E and its fractions were evaluated for their antimutagenic potential against two direct-acting mutagens, 4-nitro-o-phenylenediamine ŽNPD. and 4-nitroquinoline-N-oxide Ž4NQNO., and S9-dependent mutagen, 2-aminofluorene Ž2AF. in TA98 and TA100 strains of Salmonella typhimurium. The study revealed that the extract ŽTC-E. and its fractions were highly significant against S9-dependent mutagen, 2AF. The effect was found to be more or less corresponding with the nature of the fractions, as the monomeric TC-EI Ža GA derivative. was least effective as compared to other fractions which were oligomeric, and the order of their effectiveness as per their IbD50 value being TC-EIV Ž8.9 mg. ) TC-EIII Ž17.8 mg. ) TC-EII Ž45 mg. ) TC-EI Ž320 mg. in TA98; TC-EIV being 40 times more effective than TC-EI in inhibiting hisq revertants. A similar effect was noticed in TA100 too, where TC-EI was the least effective and TC-EII had the maximum effect. A similar result was noticed when the antimutagenicity of GA Ža monomeric. was compared with tannic acid ŽTA, an oligomeric.. However, chebula tannins were found to be partly effective against NPD but not at all effective against 4NQNO. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Antimutagen; Anticarcinogen; Ames’ assay; Tannin; Terminalia chebula; Salmonella typhimurium; NPD; 4NQNO; 2AF; Gallic acid; Tannic acid
1. Introduction Two-pronged strategy to counter the menace of environmental mutagens envisages eliminatingrdecreasing their input in the biosphere andror characterize the substances capable of inactivatingrneutralizing the environmental mutagens and eventually )
Corresponding author. Fax: q91-183-258820
work out their potential as therapeuticrprophylactic agents w1x. Most of the mutagenic inhibitors are basically of plant origin and have diverse chemical structure w2–7x. India, China and several other Asian countries have been traditionally relying on medicinal plants for one or another ailments since ages. Indeed, there is a sort of resurgence on the use of plant-based medicines. Even in the developed countries, like USA, 25% of prescription for pharmaceuti-
1383-5718r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 3 0 - 2
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S. Kaur et al.r Mutation Research 419 (1998) 169–179
cal drugs contain one or more substances of plant origin and the turnover of plant-based medicines being US$2.2 billion per annum w8,9x. Keeping this in view, coupled with our consistent interest in antimutagens w10–13x, prompted us to plan the programme to isolate chemical compoundrs from the medicinal plants and evaluate their antimutagenicity employing Ames’ assay. Since plant phenolics are one of the diverse chemical compounds suspected to possess antimutagenicity w4,14–17x, an attempt has been made to isolate tannins from chebula myroblan ŽTerminalia chebula Retz.. and characterize their antimutagenicity. Chebula myroblan is one of the important medicinal plants used against several ailments like piles, dropsy, diarrhoea, biliousness, headaches, dyspepsia, ophthalmia, enlarged liver and ascites. It is one of the constituents of ‘triphla’ Ža mixture of dried fruit pulps of chebula and bellerica myroblans and gooseberry. which is considered as highly efficacious cardiotonic w18x.
2. Materials and methods 2.1. Isolation and fractionation of tannins Tannins were isolated following the method of Kannan et al. w19x. A total of 40 g of finely powdered fruit pulp was extracted with 95% alcohol for 48 h. Alcoholic extract was concentrated to almost dryness in a rotary vacuum evaporator and dissolved in distilled water Ž100 ml.. Aqueous extract, duly filtered, was washed four times with 200 ml of diethyl ether in a separating funnel and then saturated with sodium chloride. The dark brown precipitates, thus formed, were removed by filtration and the filtrate was then washed 4–5 times with ethyl acetate Ž100 ml each. in a separating funnel. The ethyl acetate washings were combined and reduced to a volume of 10 ml Žin the rotary vacuum evaporator. transferred to China dish and dried to a constant weight at 508C. A buff-coloured mass Žnamed as TC-E. was obtained. This buff-coloured mass was subjected to silica gel column chromatography in a 66.5 cm long glass column having 35 mm internal diameter by suspending it in benzene. A total of 1 g of TC-E, dissolved in 4 ml of ethyl acetate, was fed onto the column. The column was eluted with sol-
vent systems: benzene, 10% ethyl acetate ŽEA., 20% EA, 40% EA, 60% EA, 80% EA and 100% EA in benzene and finally with methanol. Materials eluted in 40% EA, 60–80% EA, 100% EA and methanol were labelled as fraction TC-EI, TC-EII, TC-EIII and TC-EIV, respectively. 2.2. TentatiÕe identification The tentative chemical nature of the extracted material was inferred using thin layer chromatography ŽTLC. and 13 C-NMR Žnuclear magnetic resonance. spectroscopy. For TLC studies, glass plates Ž5 = 20 cm2 . were coated with silica gel slurry Žsilica gel:water::1:2, wrv. and duly dried. When required for use, the plates were heated at 1108C for 30 min in an oven. Solvent systems used were: solÕent system 1—toluene:ethyl acetate:formic acid Ž45:45:10. and solÕent system 2—ethyl acetate: butanol:formic acid:water Ž25:15:5:5.. The plates were air-dried and subsequently developed in an oven at 1008C. Gallic acid ŽGA. and tannic acid ŽTA. were used as the standard. 13 C-NMR spectra of the fractions were recorded on Bruker Fourier NMR spectrometer ACF ŽSwitzerland.. The samples were dissolved in acetone-d6 . The addition of CDCl 3 was required for locking of the NMR. 2.3. Antimutagenicity screening The procedures followed for the preparation of bacterial culture media and S9 mix were the same as described by Maron and Ames w20x. In order to verify the toxicity of the test sample, a simple toxicity test was performed. Equal quantities Ž0.1 ml. of bacterial culture in a nutrient broth and test samples Ždissolved in DMSO. were added to the test tubes containing histidinerbiotin-supplemented soft agar and overlaid onto minimal agar plates. The number of spontaneous revertant colonies, size of the colonies and intensity of the background lawn were assessed after the plates were incubated at 378C for 48 h and compared with that of control where no test sample was added. Non-toxic concentrations were categorized as those where there was well-developed lawn, almost similar size of colonies and no statistical difference in the number of spontaneous revertants in test and control plates. The concentrations of the test
Table 1 13 C-NMR spectral data of the column chromatography fractions of TC-E in the aromatic region Ž d : 107–175.
d : 162–175 1 cs; d : 168.21 10 cs; d : 164.87; 165.62; 166.25; 168.12; 168.75; 170.00; 171.25; 172.50; 173.12; 175.62 TC-EIII 15 cs; d : 164.48; 164.78; 165.08; 166.84; 167.43; 167.72; 168.02; 168.60; 169.19; 169.78; 172.78; 173.31; 173.60; 173.90; 174.19;
TC-EI TC-EII
X
C-8
C-9
C-9
C-10
C-11
d : 121–125
d : 109–110
d : 114–117
d : 144–146
d : 139
1 cs; d : 121.90 1 cs; d : 110.14 4 cs; d : 120.62; 3 cs; d : 108.75; 121.25; 123.75; 111.25; 111.85; 125.00;
1 cs; d : 145.96 6 cs; d : 142.50; 143.75; 145.00; 146.25; 146.50; 147.50; 5 cs; d : 119.19; 4 cs; d : 108.60; 109.19 3 cs; d : 115.49; 116.25 6 cs; d : 143.31; 119.78; 123.31; 109.78; 110.37; 116.66; 144.19; 144.48; 124.48; 125.07; 145.07; 145.66; 146.25 – 4 cs; d : 116.27; 116.87; 117.50; 118.75;
1 cs; d : 138.72 6 cs; d : 137.50; 138.75; 139.00; 139.33; 140.00; 141.25; 8 cs; d : 136.25; 137.43; 138.02; 138.31; 139.13; 139.33; 139.78; 140.48;
Unassigned cs
2 cs; d : 129.61; 132.02 4 cs; d : 130.00; 131.87; 133.75; 135.00;
2 cs; d : 107.23; 107.50;
S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fraction C-7
cs, carbon signals.
171
172
Table 2 13 C-NMR spectral data of the column chromatography fractions of TC-E in the carbohydrate region Ž d ; 5–95.
C-1
C-2
C-3 and C-5
C-4
C-6
d : 90–95
d : 70–72
d : 73–75
d : 65–69
d : 60–63
TC-EI
–
–
–
1 cs; d : 68.36
–
TC-EII
3 cs; d : 91.25; 93.75; 95.00
3 cs; d : 71.25; 71.87; 72.50
1 cs; d : 74.65
4 cs; d : 65.00; 67.50; 68.12; 68.75
4 cs; d : 60.00; 61.22; 62.50; 63.75;
TC-EIII
4 cs; d : 90.37; 91.25; 94.88; 95.05
1 cs; d : 72.13
3 cs: d : 73.31; 73.90; 74.48
5 cs; d : 68.31; 68.90; 69.49; 70.07; 70.66
5 cs; d : 60.37; 60.96; 61.54; 62.72; 63.12
cs, carbon signals.
Unassigned cs
5 cs; 3 cs; 6 cs; 6 cs; 4 cs; 6 cs; 4 cs; 2 cs;
d : 7.54–25.62 d : 32.59–39.67 d : 11.25–24.37 d : 33.75–46.25 d : 76.25;79.37 d : 33.90-44.48 d : 65.66–67.43 d : 77.43; 78.02
S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fraction
S. Kaur et al.r Mutation Research 419 (1998) 169–179
samples used for investigating the antimutagenicity were 5 = 10 3, 4 = 10 3, 3 = 10 3, 2 = 10 3, 1 = 10 3, 0.5 = 10 3 , 0.25 = 10 3, 0.10 = 10 3 and 0.01 = 10 3 mg 0.1 mly1 platey1 . These were tested against two direct-acting mutagens w4-nitro-o-phenylenediamine ŽNPD, 20 mg 0.1 mly1 platey1 . and 4-nitroquinoline-N-oxide Ž4NQNO, 0.25 mg 0.1 mly1 platey1 .x and S9-dependent mutagen w2-aminofluorene Ž2AF, 20 mg 0.1 mly1 platey1 .x in TA98 and TA100 tester strains. All the test samples and mutagensr promutagens were dissolved in DMSO. The plate incorporation assay, as described by Maron and Ames w20x, with a little modification as given by Bala and Grover w10x, was used for the present investigation. Both pre- and co-incubation
173
modes of experiments were done. Each concentration was tested in triplicate and the entire experiment was repeated twice. The dose inhibiting 50% of mutagenicity ŽIbD50 . was inferred from the dose–response curve and used as indication of antimutagenicity potency w1x.
3. Results and discussion The present gross identification of the isolates from T. chebula is based on the available NMR data on tannins, GA esters, hexahydroxyphenyl esters, etc. w21–23x. Some aspects are supported by TLC studies. Four fractions were obtained from the alco-
Fig. 1. Effect of TC-E and its column chromatography fractions on NPD induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of NPD of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
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holic extract using column chromatography. A brief description of each isolate is given below. TC-EI: It is evident from the 13 C-NMR spectral analysis that it is a non-tannin fraction, as it lacked the glucose core Žonly one carbon signal in the carbohydrate region; Table 2.. It had seven carbon signals Žcs. in the region of d 107–175 Žaromatic region., including one of GA ŽC7 at d 168.21., with five saturated carbons Žsee Table 1 for carbon signals.. It may have some ethylene group substituent indicated by carbon signals in the aromatic region at d 129.60 and 132.02. Its TLC showed a single spot at Rf 0.54 and 0.83 in solvent systems 1 and 2, respectively, which are comparable with pure GA. TC-EII: It contains three tannins as manifestable by cs at d 91.25, 93.25 and 95.00 in the carbohydrate region ŽTable 2., and numerous signals in the aromatic region. These tannins had the depside
linkage Ž d 114–119. and one glucose residue esterified with hexahydroxyphenoyl group Žcs at d 108.75.. Impregnation of TC-EI is also indicated by the 13 C-NMR and TLC analysis. TLC analysis revealed the presence of four major spots. Probably, one of the spots with Rf 0.83 corresponds to TC-EI which is expected in the column chromatography. Other three spots, as deciphered by the solvent system 1 with Rf 0.30, 0.52 and 0.70, are the characteristics of these tannins of this group. TC-EIII: Spectral analysis of TC-EIII indicated it to be a mixture of four tannins. The presence of depside linkages and hexahydroxyphenyl ester group is also suggested ŽTable 1.. In solvent system 2, it showed the presence of four spots at Rf 0.28, 0.37, 0.52 and 0.70 corresponding to four tannins. 13 C-NMR spectrum of TC-EIV could not be recorded due to technical reasons. Moreover, as it
Fig. 2. Effect of TC-E and its column chromatography fracttions on 4NQNO induced mutagenicity in strain TA100 of S. typhimurium. ŽPositive control of 4NQNO of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
S. Kaur et al.r Mutation Research 419 (1998) 169–179
did not rise in both the solvent systems of TLC. However, the preliminary chemical tests indicated that it is tannin in nature. All the above mentioned fractions were investigated for their antimutagenicity against two directacting mutagens, NPD and 4NQNO, and S9-dependent mutagen, 2AF, in TA98 and TA100 strains of Salmonella typhimurium. Besides this, antimutagenic activity of GA and TA was also determined. Results of these are presented in Figs. 1–8. The positive control of mutagen in each case was considered as 100% mutagenicity. It is evident from the results that these extractsrfractions show a strong mutagen specificity, as it did not inhibit 4NQNO-induced hisq revertants in TA100 strain. On the contrary, the extract TC-E and the fraction TC-EIV rather enhanced the frequency of hisq revertants ŽFig. 2.. These fractions though show antimutagenicity against NPD, yet the activity of only TC-III was more than
175
50%. TC-EI was toxic at concentration 3 = 10 3 mg 0.1 mly1 platey1 and at higher than this. At concentration less than it, the inhibitory effect was insignificant. Against 2AF-induced mutagenicity, the fractions as well as the tannin extract were highly potent in both the strains and the effect enhanced was more in the pre-incubation mode of experimentation ŽFigs. 3 and 4.. In strain TA98, TC-E exhibited 97.3% inhibitory activity in the pre-incubation experiment. Its fractions were also equally potent and the order of their antimutagenic potency, as determined by IbD50 , was: TC-EIV Ž8.9 mg. ) TC-EIII Ž17.8 mg. ) TC-EII and TC-E Ž45 mg. ) TC-EI Ž320 mg.. It is significant to note that though TC-EI, which is a non-tannin fraction, shows antimutagenic potential, but its IbD50 was much more than the tannin fractions. Similarly, their inhibitory activity against 2AF in TA100 was also significant and the order of their potency on the basis of IbD50 values being TC-E
Fig. 3. Effect of TC-E and its column chromatography fractions on 2AF induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
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S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fig. 4. Effect of TC-E and its column chromatography fractions on 2AF induced mutagenicity in strain TA100 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
Ž56 mg. ) TC-EII Ž90 mg. ) TC-EIV Ž160 mg. ) TC-EIII Ž215 mg. ) TC-EI Ž450 mg.. On analysis of the results obtained in the present investigation, it was observed that ethyl acetate extract ŽTC-E. and its column chromatography ŽTC-EI, TC-EII, TC-EIII and TC-EIV. fractions exhibited mutagen specificity, as they were highly effective in inhibiting the indirect-acting S9-dependent mutagen, 2AF, and its suppressing effect being ‘low’ against NPD and negligible against 4NQNO. A pronounced enhancement was observed in inhibitory activity in the pre-incubation experiments against 2AF-induced mutagenicity, implying that the added modulator interfered with the metabolic activation of the promutagen or it tended to interact directly with the ultimate mutagenic metabolite. Cytochrome P-450 enzyme system catalyzes the formation of N-hydroxy derivative, i.e., N-hydroxy-2-aminofluorene, and this metabolic intermediate interacts directly with DNA.
Thus, the alteration in the structure and function of P-450 enzymes may result in altered rates and differential pathways of metabolism of mutagens and carcinogens, and in some cases provide protection against chemically induced mutagenesis. Any such activity is expected of polyphenols, since their special biochemical property includes precipitation of large macromolecules, particularly proteins. The association of polyphenols with proteins derives from the fact that polyphenols are multidentate ligands able to bind simultaneously via different phenolic groups at more than one point on the periphery of the molecule to the protein surface. This interaction leads to the formation of a reversible complex via hydrophobic interaction w23x. The extent of the association depends upon the oligomeric nature of the polyphenol; more the oligomeric nature, more is its association. On comparison of the inhibitory potency of GA Ža monomer. and TA Ža polymer of molecules
S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fig. 5. Effect of GA and TA on 2AF induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
with a core of b-penta-O-galloyl-D-glucose to which a chain of galloyl residues is attached. against 2AF in TA98 and TA100 strains ŽFigs. 5 and 6.. It was
Fig. 6. Effect of GA and TA on 2AF induced mutagenicity in strain TA100 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
177
Fig. 7. Effect of GA and TA on NPD induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of NPD of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
observed that the low molecular weight GA was less potent than the high molecular weight TA and their IbD50 value being 275 and 65 mg, respectively, in
Fig. 8. Effect of GA and TA on 4NQNO induced mutagenicity in strain TA100 of S. typhimurium. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
178
S. Kaur et al.r Mutation Research 419 (1998) 169–179
strain TA98. Similarly, in TA100, TA exhibited 50% antimutagenicity at the dose of 62 mg whereas GA failed to suppress the mutagenicity of 2AF by more than 50%. The column chromatography fractions of TC-E behaved similarly. It is a common fact that the low molecular weight constituents are eluted first and the highest molecular weight fraction is eluted last. TLC studies also supported the point and revealed that TC-EI gave a single spot and its 13 C-NMR spectrum showed that this fraction was a GA derivative and not a tannin, since it lacked the carbohydrate core. TC-EII and TC-EIII were polyphenols as was quite evident from their TLC properties and 13 CNMR spectrum. TLC revealed TC-EIV to be a high molecular weight polymer. The antimutagenic potency of the four fractions was in the order of Žstrain TA98.: TC-EIV) TC-EIII) TC-EII) TC-EI. The order was not the same in TA100, but TC-EI was the least active. This assumption is supported by the observations of Wang et al. w24x, who reported that the inhibitory effect of TA, GA, green tea polyphenols ŽGTP. and Žy.epicatechin ŽEC. derivatives wepigallocatechin ŽEGC. and epigallocatechin3-gallate ŽEGCG.x on cytochrome P-450-dependent monooxygenases, NADPH cytochrome-C-reductase and epoxide hydrolase activities was in the order of: TA ) EGCG ) GTP ) EGC ) EC ) GA. They stated that the structure–activity relationship indicated that the potency of inhibition is dependent on the number of hydroxyl groups present on these substituents. Gali et al. w25x reported that hydrolyzable tannins, like TA, ellagic acid and GA, which had decreasing molecular weights, also had decreasing inhibitory effects on 12-O-tetradecanoylphorbol13-acetate ŽTPA.-induced tumor promotion in mouse skin, suggesting that oligomeric hydrolyzable tannins have more antitumor promotion activities than monomeric hydrolyzable tannins. TC-EIII was the only fraction successful in inhibiting the mutagenicity of NPD by more than 50% ŽFig. 1.. Similarly, GA had no inhibitory effect on NPD or 4NQNO, but TA inhibited the revertants by 58.14% ŽFig. 7.. Since NPD being a direct-acting mutagen, it is suggested that the antimutagen acts by interacting with the active groups on the mutagens. The present results are in accordance with the observations made by a few workers, where they noticed that plant phenolics, like ellagic acid, quercitin,
myrecitin and anthraflavic acid, interact with the active electrophiles w15,16,26,27x. Imanishi et al. w28x reported that under certain experimental conditions, tannin components extracted from green tea and black tea increased UV-induced sister chromatid exchange ŽSCE. frequencies in human cells. They concluded that the modifying action of these tannin-related compounds is due to the inhibition of excision repair. The work done by other workers on antimutagenic, antitumor and anticarcinogenic activity of plant phenolics revealed that these biochemical constituents do not act via a single mechanism w15,16,26x. They interfere with the metabolic activity of promutagen, act as blocking agents, and form adducts with ultimate metabolite and scavenging of free radicals or by DNA adduct formation as well as other mechanisms which are still to be defined.
Acknowledgements The research fellowship awarded to Simran Kaur by University Grants Commission, New Delhi is duly acknowledged. We are also grateful to Prof. B.N. Ames, Department of Biochemistry, University of California, California for supplying TA98 and TA100 strains of S. typhimurium for the present studies.
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carcinogen N-nitrosomethylbenzylamine, Biomed. Environ. Sci. 4 Ž1991. 225–231. L. Awasthi, B. Nath, Chemical examination of Terminalia bellerica Roxb: Part I. A new cardiac glycoside, Ind. Chem. Soc. 45 Ž1968. 913–917. L.V. Kannan, J.P.S. Sarin, G.K. Ray, Estimation of tannins in myroblan ŽTerminalia chebula Retz.., Ind. J. Pharmacol. 22 Ž1960. 314–315. D.M. Maron, B.N. Ames, Revised methods for Salmonella mutagenicity test, Mutation Res. 113 Ž1983. 173–215. E. Haslam, M. Uddin, Gallotanins: Part XV. Some observations on the structure of chebulinic acid and its derivatives, J. Chem. Soc. Ž1967. 2381–2384. E. Haslam, The metabolism of gallic acid and hexahydroxydiphenic acid in higher plants, in: W. Herz, H. Grisebach, G.W. Kirby ŽEds.., Progress in the Chemistry of Organic Natural Products, Springer-Verlag, New York, 1982, pp. 1–42. M.P. Spencer, Y. Cal, R. Martin, S.H. Gaffney, P.N. Goulding, D. Magnolato, T.H. Lilley, E. Haslam, Polyphenol complexation: some thoughts and observations, Phytochemistry 27 Ž1988. 2397–2409. Z.Y. Wang, M. Das, D.R. Bickers, H. Mukhtar, Interaction of epicatechins derived from green tea with rat hepatic cytochrome P-450, Drug Metab. Dispos. 16 Ž1988. 98–103. H.U. Gali, E.M. Perchellet, D.S. Klish, J.M. Johnson, J.-P. Perchellet, Hydrolyzable tannins: potent inhibitors of hydroperoxide production and tumor promotion in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate in vivo, Int. J. Cancer 51 Ž1992. 425–432. M.-T. Huang, A.W. Wood, H.L. Newmark, J.M. Sayer, H. Yagi, D.M. Jerina, A.H. Conney, Inhibition of the mutagenicity of bay region diol epoxides of polycyclic aromatic hydrocarbons by phenolic plant flavonoids, Carcinogenesis 4 Ž1983. 1631–1637. H.L. Newmark, Plant phenolics as inhibitors of mutational and pre-carcinogenic events, Can. J. Physiol. Pharmacol. 65 Ž1987. 461–466. H. Imanishi, Y.F. Sasaki, T. Ohta, M. Watanabe, T. Kato, Y. Shirasu, Tea tannin components modify the induction of sister chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells in mice, Mutation Res. 259 Ž1991. 79–87.
Antimutagenicity of hydrolyzable tannins from Terminalia chebula in Salmonella typhimurium Simran Kaur a , I.S. Grover b
a,)
, Majar Singh b, Satwinderjeet Kaur
a
a Department of Botanical Sciences, Guru Nanak DeÕ UniÕersity, Amritsar 143 005, India Department of Applied Chemical Sciences and Technology, Guru Nanak DeÕ UniÕersity, Amritsar 143 005, India
Received 21 April 1998; revised 3 September 1998; accepted 3 September 1998
Abstract A tannin fraction ŽTC-E. from the dried fruit pulp of Terminalia chebula was obtained by successfully extracting with 95% ethyl alcohol and ethyl acetate. TC-E was subjected to silica gel chromatography which yielded four fractions, viz., TC-EI, TC-EII, TC-EIII and TC-EIV. Thin layer chromatography ŽTLC. and 13C-NMR revealed that TC-EI was gallic acid ŽGA. derivative while the other fractions were tannin in nature. TC-E and its fractions were evaluated for their antimutagenic potential against two direct-acting mutagens, 4-nitro-o-phenylenediamine ŽNPD. and 4-nitroquinoline-N-oxide Ž4NQNO., and S9-dependent mutagen, 2-aminofluorene Ž2AF. in TA98 and TA100 strains of Salmonella typhimurium. The study revealed that the extract ŽTC-E. and its fractions were highly significant against S9-dependent mutagen, 2AF. The effect was found to be more or less corresponding with the nature of the fractions, as the monomeric TC-EI Ža GA derivative. was least effective as compared to other fractions which were oligomeric, and the order of their effectiveness as per their IbD50 value being TC-EIV Ž8.9 mg. ) TC-EIII Ž17.8 mg. ) TC-EII Ž45 mg. ) TC-EI Ž320 mg. in TA98; TC-EIV being 40 times more effective than TC-EI in inhibiting hisq revertants. A similar effect was noticed in TA100 too, where TC-EI was the least effective and TC-EII had the maximum effect. A similar result was noticed when the antimutagenicity of GA Ža monomeric. was compared with tannic acid ŽTA, an oligomeric.. However, chebula tannins were found to be partly effective against NPD but not at all effective against 4NQNO. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Antimutagen; Anticarcinogen; Ames’ assay; Tannin; Terminalia chebula; Salmonella typhimurium; NPD; 4NQNO; 2AF; Gallic acid; Tannic acid
1. Introduction Two-pronged strategy to counter the menace of environmental mutagens envisages eliminatingrdecreasing their input in the biosphere andror characterize the substances capable of inactivatingrneutralizing the environmental mutagens and eventually )
Corresponding author. Fax: q91-183-258820
work out their potential as therapeuticrprophylactic agents w1x. Most of the mutagenic inhibitors are basically of plant origin and have diverse chemical structure w2–7x. India, China and several other Asian countries have been traditionally relying on medicinal plants for one or another ailments since ages. Indeed, there is a sort of resurgence on the use of plant-based medicines. Even in the developed countries, like USA, 25% of prescription for pharmaceuti-
1383-5718r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 3 0 - 2
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S. Kaur et al.r Mutation Research 419 (1998) 169–179
cal drugs contain one or more substances of plant origin and the turnover of plant-based medicines being US$2.2 billion per annum w8,9x. Keeping this in view, coupled with our consistent interest in antimutagens w10–13x, prompted us to plan the programme to isolate chemical compoundrs from the medicinal plants and evaluate their antimutagenicity employing Ames’ assay. Since plant phenolics are one of the diverse chemical compounds suspected to possess antimutagenicity w4,14–17x, an attempt has been made to isolate tannins from chebula myroblan ŽTerminalia chebula Retz.. and characterize their antimutagenicity. Chebula myroblan is one of the important medicinal plants used against several ailments like piles, dropsy, diarrhoea, biliousness, headaches, dyspepsia, ophthalmia, enlarged liver and ascites. It is one of the constituents of ‘triphla’ Ža mixture of dried fruit pulps of chebula and bellerica myroblans and gooseberry. which is considered as highly efficacious cardiotonic w18x.
2. Materials and methods 2.1. Isolation and fractionation of tannins Tannins were isolated following the method of Kannan et al. w19x. A total of 40 g of finely powdered fruit pulp was extracted with 95% alcohol for 48 h. Alcoholic extract was concentrated to almost dryness in a rotary vacuum evaporator and dissolved in distilled water Ž100 ml.. Aqueous extract, duly filtered, was washed four times with 200 ml of diethyl ether in a separating funnel and then saturated with sodium chloride. The dark brown precipitates, thus formed, were removed by filtration and the filtrate was then washed 4–5 times with ethyl acetate Ž100 ml each. in a separating funnel. The ethyl acetate washings were combined and reduced to a volume of 10 ml Žin the rotary vacuum evaporator. transferred to China dish and dried to a constant weight at 508C. A buff-coloured mass Žnamed as TC-E. was obtained. This buff-coloured mass was subjected to silica gel column chromatography in a 66.5 cm long glass column having 35 mm internal diameter by suspending it in benzene. A total of 1 g of TC-E, dissolved in 4 ml of ethyl acetate, was fed onto the column. The column was eluted with sol-
vent systems: benzene, 10% ethyl acetate ŽEA., 20% EA, 40% EA, 60% EA, 80% EA and 100% EA in benzene and finally with methanol. Materials eluted in 40% EA, 60–80% EA, 100% EA and methanol were labelled as fraction TC-EI, TC-EII, TC-EIII and TC-EIV, respectively. 2.2. TentatiÕe identification The tentative chemical nature of the extracted material was inferred using thin layer chromatography ŽTLC. and 13 C-NMR Žnuclear magnetic resonance. spectroscopy. For TLC studies, glass plates Ž5 = 20 cm2 . were coated with silica gel slurry Žsilica gel:water::1:2, wrv. and duly dried. When required for use, the plates were heated at 1108C for 30 min in an oven. Solvent systems used were: solÕent system 1—toluene:ethyl acetate:formic acid Ž45:45:10. and solÕent system 2—ethyl acetate: butanol:formic acid:water Ž25:15:5:5.. The plates were air-dried and subsequently developed in an oven at 1008C. Gallic acid ŽGA. and tannic acid ŽTA. were used as the standard. 13 C-NMR spectra of the fractions were recorded on Bruker Fourier NMR spectrometer ACF ŽSwitzerland.. The samples were dissolved in acetone-d6 . The addition of CDCl 3 was required for locking of the NMR. 2.3. Antimutagenicity screening The procedures followed for the preparation of bacterial culture media and S9 mix were the same as described by Maron and Ames w20x. In order to verify the toxicity of the test sample, a simple toxicity test was performed. Equal quantities Ž0.1 ml. of bacterial culture in a nutrient broth and test samples Ždissolved in DMSO. were added to the test tubes containing histidinerbiotin-supplemented soft agar and overlaid onto minimal agar plates. The number of spontaneous revertant colonies, size of the colonies and intensity of the background lawn were assessed after the plates were incubated at 378C for 48 h and compared with that of control where no test sample was added. Non-toxic concentrations were categorized as those where there was well-developed lawn, almost similar size of colonies and no statistical difference in the number of spontaneous revertants in test and control plates. The concentrations of the test
Table 1 13 C-NMR spectral data of the column chromatography fractions of TC-E in the aromatic region Ž d : 107–175.
d : 162–175 1 cs; d : 168.21 10 cs; d : 164.87; 165.62; 166.25; 168.12; 168.75; 170.00; 171.25; 172.50; 173.12; 175.62 TC-EIII 15 cs; d : 164.48; 164.78; 165.08; 166.84; 167.43; 167.72; 168.02; 168.60; 169.19; 169.78; 172.78; 173.31; 173.60; 173.90; 174.19;
TC-EI TC-EII
X
C-8
C-9
C-9
C-10
C-11
d : 121–125
d : 109–110
d : 114–117
d : 144–146
d : 139
1 cs; d : 121.90 1 cs; d : 110.14 4 cs; d : 120.62; 3 cs; d : 108.75; 121.25; 123.75; 111.25; 111.85; 125.00;
1 cs; d : 145.96 6 cs; d : 142.50; 143.75; 145.00; 146.25; 146.50; 147.50; 5 cs; d : 119.19; 4 cs; d : 108.60; 109.19 3 cs; d : 115.49; 116.25 6 cs; d : 143.31; 119.78; 123.31; 109.78; 110.37; 116.66; 144.19; 144.48; 124.48; 125.07; 145.07; 145.66; 146.25 – 4 cs; d : 116.27; 116.87; 117.50; 118.75;
1 cs; d : 138.72 6 cs; d : 137.50; 138.75; 139.00; 139.33; 140.00; 141.25; 8 cs; d : 136.25; 137.43; 138.02; 138.31; 139.13; 139.33; 139.78; 140.48;
Unassigned cs
2 cs; d : 129.61; 132.02 4 cs; d : 130.00; 131.87; 133.75; 135.00;
2 cs; d : 107.23; 107.50;
S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fraction C-7
cs, carbon signals.
171
172
Table 2 13 C-NMR spectral data of the column chromatography fractions of TC-E in the carbohydrate region Ž d ; 5–95.
C-1
C-2
C-3 and C-5
C-4
C-6
d : 90–95
d : 70–72
d : 73–75
d : 65–69
d : 60–63
TC-EI
–
–
–
1 cs; d : 68.36
–
TC-EII
3 cs; d : 91.25; 93.75; 95.00
3 cs; d : 71.25; 71.87; 72.50
1 cs; d : 74.65
4 cs; d : 65.00; 67.50; 68.12; 68.75
4 cs; d : 60.00; 61.22; 62.50; 63.75;
TC-EIII
4 cs; d : 90.37; 91.25; 94.88; 95.05
1 cs; d : 72.13
3 cs: d : 73.31; 73.90; 74.48
5 cs; d : 68.31; 68.90; 69.49; 70.07; 70.66
5 cs; d : 60.37; 60.96; 61.54; 62.72; 63.12
cs, carbon signals.
Unassigned cs
5 cs; 3 cs; 6 cs; 6 cs; 4 cs; 6 cs; 4 cs; 2 cs;
d : 7.54–25.62 d : 32.59–39.67 d : 11.25–24.37 d : 33.75–46.25 d : 76.25;79.37 d : 33.90-44.48 d : 65.66–67.43 d : 77.43; 78.02
S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fraction
S. Kaur et al.r Mutation Research 419 (1998) 169–179
samples used for investigating the antimutagenicity were 5 = 10 3, 4 = 10 3, 3 = 10 3, 2 = 10 3, 1 = 10 3, 0.5 = 10 3 , 0.25 = 10 3, 0.10 = 10 3 and 0.01 = 10 3 mg 0.1 mly1 platey1 . These were tested against two direct-acting mutagens w4-nitro-o-phenylenediamine ŽNPD, 20 mg 0.1 mly1 platey1 . and 4-nitroquinoline-N-oxide Ž4NQNO, 0.25 mg 0.1 mly1 platey1 .x and S9-dependent mutagen w2-aminofluorene Ž2AF, 20 mg 0.1 mly1 platey1 .x in TA98 and TA100 tester strains. All the test samples and mutagensr promutagens were dissolved in DMSO. The plate incorporation assay, as described by Maron and Ames w20x, with a little modification as given by Bala and Grover w10x, was used for the present investigation. Both pre- and co-incubation
173
modes of experiments were done. Each concentration was tested in triplicate and the entire experiment was repeated twice. The dose inhibiting 50% of mutagenicity ŽIbD50 . was inferred from the dose–response curve and used as indication of antimutagenicity potency w1x.
3. Results and discussion The present gross identification of the isolates from T. chebula is based on the available NMR data on tannins, GA esters, hexahydroxyphenyl esters, etc. w21–23x. Some aspects are supported by TLC studies. Four fractions were obtained from the alco-
Fig. 1. Effect of TC-E and its column chromatography fractions on NPD induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of NPD of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
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holic extract using column chromatography. A brief description of each isolate is given below. TC-EI: It is evident from the 13 C-NMR spectral analysis that it is a non-tannin fraction, as it lacked the glucose core Žonly one carbon signal in the carbohydrate region; Table 2.. It had seven carbon signals Žcs. in the region of d 107–175 Žaromatic region., including one of GA ŽC7 at d 168.21., with five saturated carbons Žsee Table 1 for carbon signals.. It may have some ethylene group substituent indicated by carbon signals in the aromatic region at d 129.60 and 132.02. Its TLC showed a single spot at Rf 0.54 and 0.83 in solvent systems 1 and 2, respectively, which are comparable with pure GA. TC-EII: It contains three tannins as manifestable by cs at d 91.25, 93.25 and 95.00 in the carbohydrate region ŽTable 2., and numerous signals in the aromatic region. These tannins had the depside
linkage Ž d 114–119. and one glucose residue esterified with hexahydroxyphenoyl group Žcs at d 108.75.. Impregnation of TC-EI is also indicated by the 13 C-NMR and TLC analysis. TLC analysis revealed the presence of four major spots. Probably, one of the spots with Rf 0.83 corresponds to TC-EI which is expected in the column chromatography. Other three spots, as deciphered by the solvent system 1 with Rf 0.30, 0.52 and 0.70, are the characteristics of these tannins of this group. TC-EIII: Spectral analysis of TC-EIII indicated it to be a mixture of four tannins. The presence of depside linkages and hexahydroxyphenyl ester group is also suggested ŽTable 1.. In solvent system 2, it showed the presence of four spots at Rf 0.28, 0.37, 0.52 and 0.70 corresponding to four tannins. 13 C-NMR spectrum of TC-EIV could not be recorded due to technical reasons. Moreover, as it
Fig. 2. Effect of TC-E and its column chromatography fracttions on 4NQNO induced mutagenicity in strain TA100 of S. typhimurium. ŽPositive control of 4NQNO of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
S. Kaur et al.r Mutation Research 419 (1998) 169–179
did not rise in both the solvent systems of TLC. However, the preliminary chemical tests indicated that it is tannin in nature. All the above mentioned fractions were investigated for their antimutagenicity against two directacting mutagens, NPD and 4NQNO, and S9-dependent mutagen, 2AF, in TA98 and TA100 strains of Salmonella typhimurium. Besides this, antimutagenic activity of GA and TA was also determined. Results of these are presented in Figs. 1–8. The positive control of mutagen in each case was considered as 100% mutagenicity. It is evident from the results that these extractsrfractions show a strong mutagen specificity, as it did not inhibit 4NQNO-induced hisq revertants in TA100 strain. On the contrary, the extract TC-E and the fraction TC-EIV rather enhanced the frequency of hisq revertants ŽFig. 2.. These fractions though show antimutagenicity against NPD, yet the activity of only TC-III was more than
175
50%. TC-EI was toxic at concentration 3 = 10 3 mg 0.1 mly1 platey1 and at higher than this. At concentration less than it, the inhibitory effect was insignificant. Against 2AF-induced mutagenicity, the fractions as well as the tannin extract were highly potent in both the strains and the effect enhanced was more in the pre-incubation mode of experimentation ŽFigs. 3 and 4.. In strain TA98, TC-E exhibited 97.3% inhibitory activity in the pre-incubation experiment. Its fractions were also equally potent and the order of their antimutagenic potency, as determined by IbD50 , was: TC-EIV Ž8.9 mg. ) TC-EIII Ž17.8 mg. ) TC-EII and TC-E Ž45 mg. ) TC-EI Ž320 mg.. It is significant to note that though TC-EI, which is a non-tannin fraction, shows antimutagenic potential, but its IbD50 was much more than the tannin fractions. Similarly, their inhibitory activity against 2AF in TA100 was also significant and the order of their potency on the basis of IbD50 values being TC-E
Fig. 3. Effect of TC-E and its column chromatography fractions on 2AF induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
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S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fig. 4. Effect of TC-E and its column chromatography fractions on 2AF induced mutagenicity in strain TA100 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone. = 100.
Ž56 mg. ) TC-EII Ž90 mg. ) TC-EIV Ž160 mg. ) TC-EIII Ž215 mg. ) TC-EI Ž450 mg.. On analysis of the results obtained in the present investigation, it was observed that ethyl acetate extract ŽTC-E. and its column chromatography ŽTC-EI, TC-EII, TC-EIII and TC-EIV. fractions exhibited mutagen specificity, as they were highly effective in inhibiting the indirect-acting S9-dependent mutagen, 2AF, and its suppressing effect being ‘low’ against NPD and negligible against 4NQNO. A pronounced enhancement was observed in inhibitory activity in the pre-incubation experiments against 2AF-induced mutagenicity, implying that the added modulator interfered with the metabolic activation of the promutagen or it tended to interact directly with the ultimate mutagenic metabolite. Cytochrome P-450 enzyme system catalyzes the formation of N-hydroxy derivative, i.e., N-hydroxy-2-aminofluorene, and this metabolic intermediate interacts directly with DNA.
Thus, the alteration in the structure and function of P-450 enzymes may result in altered rates and differential pathways of metabolism of mutagens and carcinogens, and in some cases provide protection against chemically induced mutagenesis. Any such activity is expected of polyphenols, since their special biochemical property includes precipitation of large macromolecules, particularly proteins. The association of polyphenols with proteins derives from the fact that polyphenols are multidentate ligands able to bind simultaneously via different phenolic groups at more than one point on the periphery of the molecule to the protein surface. This interaction leads to the formation of a reversible complex via hydrophobic interaction w23x. The extent of the association depends upon the oligomeric nature of the polyphenol; more the oligomeric nature, more is its association. On comparison of the inhibitory potency of GA Ža monomer. and TA Ža polymer of molecules
S. Kaur et al.r Mutation Research 419 (1998) 169–179
Fig. 5. Effect of GA and TA on 2AF induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
with a core of b-penta-O-galloyl-D-glucose to which a chain of galloyl residues is attached. against 2AF in TA98 and TA100 strains ŽFigs. 5 and 6.. It was
Fig. 6. Effect of GA and TA on 2AF induced mutagenicity in strain TA100 of S. typhimurium. ŽPositive control of 2AF of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
177
Fig. 7. Effect of GA and TA on NPD induced mutagenicity in strain TA98 of S. typhimurium. ŽPositive control of NPD of each experiment is indicated in each graph.. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
observed that the low molecular weight GA was less potent than the high molecular weight TA and their IbD50 value being 275 and 65 mg, respectively, in
Fig. 8. Effect of GA and TA on 4NQNO induced mutagenicity in strain TA100 of S. typhimurium. Percent of controls Žhisq revertantsrplate with mutagen and antimutagen.rŽhisq revertantsrplate with mutagen alone.=100.
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S. Kaur et al.r Mutation Research 419 (1998) 169–179
strain TA98. Similarly, in TA100, TA exhibited 50% antimutagenicity at the dose of 62 mg whereas GA failed to suppress the mutagenicity of 2AF by more than 50%. The column chromatography fractions of TC-E behaved similarly. It is a common fact that the low molecular weight constituents are eluted first and the highest molecular weight fraction is eluted last. TLC studies also supported the point and revealed that TC-EI gave a single spot and its 13 C-NMR spectrum showed that this fraction was a GA derivative and not a tannin, since it lacked the carbohydrate core. TC-EII and TC-EIII were polyphenols as was quite evident from their TLC properties and 13 CNMR spectrum. TLC revealed TC-EIV to be a high molecular weight polymer. The antimutagenic potency of the four fractions was in the order of Žstrain TA98.: TC-EIV) TC-EIII) TC-EII) TC-EI. The order was not the same in TA100, but TC-EI was the least active. This assumption is supported by the observations of Wang et al. w24x, who reported that the inhibitory effect of TA, GA, green tea polyphenols ŽGTP. and Žy.epicatechin ŽEC. derivatives wepigallocatechin ŽEGC. and epigallocatechin3-gallate ŽEGCG.x on cytochrome P-450-dependent monooxygenases, NADPH cytochrome-C-reductase and epoxide hydrolase activities was in the order of: TA ) EGCG ) GTP ) EGC ) EC ) GA. They stated that the structure–activity relationship indicated that the potency of inhibition is dependent on the number of hydroxyl groups present on these substituents. Gali et al. w25x reported that hydrolyzable tannins, like TA, ellagic acid and GA, which had decreasing molecular weights, also had decreasing inhibitory effects on 12-O-tetradecanoylphorbol13-acetate ŽTPA.-induced tumor promotion in mouse skin, suggesting that oligomeric hydrolyzable tannins have more antitumor promotion activities than monomeric hydrolyzable tannins. TC-EIII was the only fraction successful in inhibiting the mutagenicity of NPD by more than 50% ŽFig. 1.. Similarly, GA had no inhibitory effect on NPD or 4NQNO, but TA inhibited the revertants by 58.14% ŽFig. 7.. Since NPD being a direct-acting mutagen, it is suggested that the antimutagen acts by interacting with the active groups on the mutagens. The present results are in accordance with the observations made by a few workers, where they noticed that plant phenolics, like ellagic acid, quercitin,
myrecitin and anthraflavic acid, interact with the active electrophiles w15,16,26,27x. Imanishi et al. w28x reported that under certain experimental conditions, tannin components extracted from green tea and black tea increased UV-induced sister chromatid exchange ŽSCE. frequencies in human cells. They concluded that the modifying action of these tannin-related compounds is due to the inhibition of excision repair. The work done by other workers on antimutagenic, antitumor and anticarcinogenic activity of plant phenolics revealed that these biochemical constituents do not act via a single mechanism w15,16,26x. They interfere with the metabolic activity of promutagen, act as blocking agents, and form adducts with ultimate metabolite and scavenging of free radicals or by DNA adduct formation as well as other mechanisms which are still to be defined.
Acknowledgements The research fellowship awarded to Simran Kaur by University Grants Commission, New Delhi is duly acknowledged. We are also grateful to Prof. B.N. Ames, Department of Biochemistry, University of California, California for supplying TA98 and TA100 strains of S. typhimurium for the present studies.
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