Research from India reported in Phytomedicine explored the anti-mutagenic activity of green tea. Using standard mutagenicity assays, a team of research demonstrated that green tea polyphenols could inhibit the mutagenicity of tobacco in a concentration-dependent manner. The polyphenols were also found to inhibit urinary mutagenicity in rats induced by tobacco extract. Thus “green tea has a dual action to bring about a reduction in the mutagenic and carcinogenic potential of tobacco.”
Phytomedicine: International Journal of Phytotherapy &Phytopharmacology
. March 1, 2005. Ramadasan, K.; Santhosh, K.T.; Swarnam, J.
Antimutagenic activity of green tea (Camellia sinensis) was studied using Salmonella typhimurium strains (TA 102) (Ames test). Aqueous tobacco extract was found to be mutagenic to S. typhimurium TA 102 at concentration of 50 mg/plate. Green tea polyphenols was found to inhibit the mutagenicity of tobacco in a concentration-dependent manner. Concentrations needed for 50% inhibition of mutagen-induced revertant formation was found to be 5 mg/plate. Green tea polyphenols was also found to inhibit the urinary mutagenicity in rats induced by tobacco extract. Moreover green tea polyphenols were found to inhibit in vitro nitrosation reaction produced by reaction sodium nitrite and methyl urea and further inhibition of mutagenicity indicating that green tea has dual action to bring out a reduction in the mutagenic and carcinogenic potential of tobacco.
[c] 2004 Elsevier GmbH. All rights reserved.
Keywords: Antimutagenicity; Green tea; Polyphenols; Tobacco; Ames test
Carcinogenicity of tobacco is well known. Tobacco in the form of cigarettes or chewing or nasal snuff has been shown to induce cancers of oral cavity, larynx, oesophagus and lungs and is one of most potent agents of habit induced cancers (Fraumeni et al., 1993). Tobacco contains hundreds of chemicals of which nicotin, nornicotin, polyaromatic hydrocarbons, as well as their metabolites predominate (Hoffmann and Hecht, 1985). While nitrosation and subsequent oxidation to N-oxides are the major pathway for the aromatic amines, diol epoxide formation is the major pathway for polyaromatic hydrocarbons (Autrup and Harris, 1983).
Chemoprevention is being tested as a major means of inhibition of carcinogenesis. Plants have immense number of compounds with chemopreventive potential of which polyphenolic compounds forms a major class (Ferguson, 2001). Green tea which is being taken in many Asian countries contain catechin, epicatechin, catechin gallate and epigallocatechin gallate as the major polyphenols (Ho et al., 1994). Although the anti-carcinogenicity of green tea polyphenols is well-established (Wang et al., 1989), its effect on tobacco induced mutation has not been reported. In the present study we report the antimutagenicity of green tea extract on tobacco-induced mutagenicity in vitro and in vivo. We also report the inhibition of nitrosation by green tea extract is a major mechanism of action of green tea.
Materials and methods
Green tea was supplied by Kancore Extracts, Angamali, Kerala, and tobacco was purchased from local market. Nutrient broth, L. histidine and biotin were purchased from Hi-Media Laboratories, Bombay. Agar-agar, dimethyl sulphoxide and sodium azide were obtained from Sisco Research Laboratories, Bombay. All other reagents used were of analytical grade. Salmonella typhimurium strains TA 102, were kindly supplied by Prof. B.N. Ames, University of California, Berkley, USA.
Isolation of green tea polyphenols
Dried green tea (100 g) was extracted with 500 ml (75%) methanol by overnight stirring. Supernatant was collected and the residue was extracted twice with 250 ml 75% methanol. Supernatant was pooled and evaporated in a water bath at 65[degrees]C. It was resuspended in 10% methanol. This extract was taken in separating funnel and extracted with solvents of different polarity in the order petroleum ether, chloroform, ether and ethyl acetate. Ethyl acetate extract containing most of the polyphenols was evaporated and it was resuspended in water (Ho et al., 1994) and was used in the experiments. The content of polyphenols in the preparation as analysed by high-pressure liquid chromatography ([mu][C.sub.18]column, solvent system 89.5% water, 0.5% phosphoric acid, 0.5% ethyl acetate, 12% acetone nitrile). Epigallocatechin 10.23%; Epicatechin 1.91%; Epigallocatechin gallate 31.91%, Gallocatechin gallate 0.86%, Epicatechin gallate 8.31%, Total catechins 54.82%.
Preparation of aqueous extract of tobacco
Tobacco (100 g) was cut into small pieces and boiled in 500 ml of distilled water for 1 h. It was evaporated to dryness and made up to 250 mg/ml.
Mutagenicity of tobacco extract was tested in S. typhimurium strain TA 102 by Ames test (Maron and Ames, 1983). Plate incorporation method was done for the mutagenicity assay. 2 ml of molten agar (0.6 g agar and 0.5 g NaCl in 100 ml distilled water) containing 0.045 mM histidine/biotin solution held at 45[degrees]C, was inoculated with fresh 0.1 ml of Salmonella culture (1-2 X [10.sup.8] cells/ml) and different concentrations of tobacco was overlaid on minimal glucose agar plates. The plates were incubated for 48 h at 37[degrees]C and the revertant colonies were counted using a colony counter. Experiments were done in triplicate and repeated 3 times and average of nine plates were statistically analyzed using student ‘t’ test.
Antimutagenicity of tobacco extract was tested in S. typhimurium strain TA 102 by plate incorporation method. 0.1 ml of fresh bacterial culture (1-2 X [10.sup.8] cells/ml) was mixed with 2 ml of top agar containing histidine and biotin, various concentrations of green tea polyphenols and tobacco extracts (50 mg/plate). Further it was poured onto minimal glucose-agar plate and incubated for 48 h at 37[degrees]C. After incubation, revertant colonies were counted using colony counter. The percentage of inhibition of mutagenicity was calculated using the formula [([R.sub.1]-SR)-([R.sub.2]-SR)]/([R.sub.1]-SR) X 100 where [R.sub.1] is the number of revertants in presence of mutagen alone, [R.sub.2] is the number of revertants in presence of drug and SR is the spontaneous revertants. Experiments were done in triplicate and repeated three times. Average of nine plates were taken for analysis.
Determination of urinary mutagenicity in rats
Male Wistar rats were divided into five groups (four rats/group). Group 1 animals were control group which were given only distilled water (p.o). Group 2 animals were given aqueous extract of tobacco (500 mg/rat) by i.p. injection as a single dose. Group III, IV, V, animals were fed with green tea polyphenols at concentrations of 50 mg, 200 mg, 500 mg per body weight, respectively, for 2 days. On the 2nd day, tobacco extract was administered to the Group III, IV, V, rats by i.p injection. All the rats were put into metabolic cages and 24 h urine samples were collected. The urine samples were frozen and kept at -70[degrees]C until analysis. 20 ml of pooled urines from each group was passed through XAD column to concentrate weakly anionic compounds (Yamasaki and Ames, 1977).
Urinary mutagenicity was determined by plating 0.1 ml of fresh Salmonella (1-2 X [10.sup.8] cells/ml), mixed with 0.1 ml urine concentrate and 2 ml top agar containing histidine and biotin and poured on minimal glucose agar plate. The revertants were counted after incubation at 37[degrees]C for 48 h. Experiments were done in duplicate and average of six plates were analyzed statistically.
Determination of inhibition of nitrosation reaction
The reaction between methylurea and nitrite was used as a model to study the inhibition of nitrosation reaction. The method of Stich et al. (1982) as modified by Nagabhushan et al. (1988) was employed. The reaction mixture included methyl urea (25 mM) and sodium nitrate (100 mM) in standard buffer pH 3.6 (0.068 citric acid/0.64 M disodium phosphate) to give a final volume of 2 ml. Different concentration of green tea extracts were added just prior to the addition of sodium nitrite and the mixture was incubated at room temperature (30[degrees]C for 20 min). Thereafter solution was neutralized by adding 0.7 ml of 7.5% sodium bicarbonate solution. The final volume was adjusted to 3 ml.
Overnight grown cultures of Salmonella (2.5 X [10.sup.8] cells per ml) were added to the mixture containing nitrosation product and incubated for 30 min at 37[degrees]C. Bacteria were then pelleted and washed in PBS and centrifuged. Finally bacteria were resuspended in PBS at the original cell concentration and counted. For mutation assay 0.1 ml of 1 X [10.sup.8] cells per ml was spread over minimal glucose agar medium containing minimal histidine and biotin. The plates were incubated for 48 h at 37[degrees]C and the number of revertants was counted. The percentage of inhibition of nitrosation in presence of test materials was calculated considering methyl urea and nitrite alone produced 100% mutagenicity. Experiments were done in triplicate and repeated twice and average of six plates were statistically analyzed.
Mutagenicity of tobacco
Mutagenicity of tobacco was studied using S. typhimurium strains TA 102. At concentrations studied in this experiment the tobacco extract was found to be mutagenic to S. typhimurium TA 102 and maximum mutagenicity was seen at 50 mg/plate (Table 1).
Antimutagenicity of green tea polyphenols against tobacco as a mutagen
Green tea polyphenols was found to produce inhibition of mutagenicity induced by tobacco in a concentration dependent manner. At concentration of 10.0 mg of green tea polyphenols per plate, 100% of inhibition in the revertant colony formation was observed. Addition of green tea polyphenols at concentrations 1.0 mg and 5.0 mg per plate have produced 27.8% and 45.6% of inhibition, respectively (Table 2).
Effect of green tea polyphenols on urinary mutagenicity induced by tobacco
Green tea polyphenols was found to inhibit urinary mutagenicity induced by tobacco in rats. Urine collected from rats treated with tobacco was found to be significantly mutagenic to S. typhimurium strain TA 102 as seen from the number of revertant formation. Administration of green tea polyphenols could significantly inhibit urinary mutagenicity induced by tobacco in rats (Table 3).
Effect of green tea polyphenols on nitrosation reaction
Nitrosation of methyl urea with sodium nitrite at acidic condition was found to increase the mutagenicity as seen from the increased revertant formation. Incubation of bacteria with the reaction mixture produced 632.0 [+ or -] 14.5 revertant colonies. Addition of green tea polyphenols to the medium at concentrations of 0.1 mg, 0.2 mg, 0.5 mg, 1.0 mg produced 11%, 27.7%, 55.4%, 81.1% inhibition in revertant formation (Table 4).
In the present study we report the antimutagenicity of green tea polyphenols against tobacco extract in S. typhimurium. We have used the strain TA 102, which is succeptable to oxidant mutants. We have reported earlier that tobacco extract produce significant mutagenicity to this strain compared with other strains (Kuttan and Sukumaran, 1995). Maximum mutagenicity was produced at 50 mg/plate and thereafter decreased. Antimutagenicity studies using green tea polyphenols indicated that mutagenicity induced by tobacco was significantly inhibited by the addition of green tea extract. Concentration needed for 50% inhibition was found to be approximately 5 mg/plate. Green tea extract was also found to inhibit the mutagenicity induced by tobacco extract in vivo. At a concentration of 500 mg/kg body weight, green tea extract could inhibit the mutagenicity of tobacco extract completely.
Tobacco contain several potent mutagenic substances of which nicotin and nor-nicotin are the major ingredients. Conversion of nicotin to the nitroso compound is a major reaction leading to the ultimate carcinogen formation (Barrett, 1991). This has been shown to be accomplished in the mouth as well as in the acidic condition in the stomach. Present studies indicate that green tea could inhibit the nitrosation reaction and there by inhibit the causation to the ultimate carcinogen (Xu et al., 1993).
Tobacco is a ubiquitous carcinogen throughout the world. It is probably the most dangerous of the habit related carcinogen and was found to produce the cancer at various sites (Saracci, 1987). The present result indicating that green tea can inhibit the mutagenicity of tobacco is highly relevant in the present scenerio of increased cancer incidence throughout the world. Green tea is taken in many Asian countries especially in China and Japan. Green tea is known as a major chemopreventive strategy to reduce the cancer incidence as shown by several workers (Ho et al., 1994). Major ingredient in green tea is catechin and its analog epigallocatechingallate which is one of major chemopreventive agents presently known (Yoshizawa et al., 1987). Green tea also has ellagitannins which have also significant cancer preventive properties.
Mechanism of action of green tea extract seems to be due to antioxidant activity of the extract and subsequent scavenging of reactive oxygen radicals by the polyphenols present in the extract (Kada et al., 1985). It may also inhibit conversion to the ultimate carcinogen which can bind to DNA producing mutation (Jackson et al., 1987). In the case of tobacco, nitrosamines are the active compounds. Tobacco smoke contains polyaromatic hydrocarbons and its activation was inhibited by green tea. Other than these mechanisms present studies indicate that green tea could also inhibit the nitrosation reaction and thereby reduce the mutagenic potential of amines present in tobacco.
Table 1. Mutagenicity of tobacco extract to S. typhimurium TA 102
Tobacco extract (mg/plate) No. of revertants per plate
SR 124.0 [+ or -] 7.0
25 497.3 [+ or -] 8.1**
50 755.3 [+ or -] 7.0**
75 773.0 [+ or -] 5.0**
Values are mean [+ or -] SD; n = 9.
** p < 0.001. Table 2. Antimutagenicity of polyphenolic fraction of Green tea extract to S. typhimutiurm (TA 102) against tobacco as mutagen Average Concentration of number ofPercent extracts mg/plate revertants/plate inhibition (%) Tobacco alone (50 774.4 [+ or -] 14.1 mg/plate) Tobacco + 1.0 mg 594.0 [+ or -] 11.3 27.8** Green tea extract Tobacco + 5.0 mg 354.5 [+ or -] 10.6 45.6** Green tea extract Tobacco + 10.0 mg 124.0 [+ or -] 8.5 100.0** Green tea extract Values are mean [+ or -] SD; n = 9; spontaneous revertants = 124 [+ or -] 7.0. ** p < 0.001. Table 3. Effect of polyphenolic fraction of green tea on urinary mutagenicity induced by tobacco extract S. typhimueium (TA 102) Percent Average number inhibition Group number Treatment of revertants/plate(%) I Normal rat urine244.7 [+ or -] 10.7 IIControl (Tobacco) 500 495.3 [+ or -] 9.7 mg per animal III Tobacco extract and 458.6 [+ or -] 16.2** 10.3 Green tea extract (50 mg per kg) IVTobacco extract and 307.0 [+ or -] 14.1** 52.7 Green tea extract (200 mg per kg) V Tobacco extract and 203.0 [+ or -] 12.5** 81.9 Green tea extract (500 mg per kg) Values are mean [+ or -] SD; n = 6; spontaneous revertants = 128 [+ or -] 6.1. ** p < 0.001. Table 4. Inhibition of nitrosation reaction by polyphenolic fraction from Green tea extract Average Number of Percent Concentration of extracts revertants/inhibition (mg/plate) plate (%) Nitrosation product alone 632.0 [+ or -] 14.5 + 0.1 mg Green tea extract 577.3 [+ or -] 11.9* 11.0 + 0.2 mg Green tea extract 494.3 [+ or -] 7.1** 27.7 + 0.5 mg Green tea extract 356.3 [+ or -] 11.9** 55.4 + 1.0 mg Green tea extract 228.0 [+ or -] 9.9** 81.1 Values are mean [+ or -] SD; n = 6; spontaneous revertants = 134 [+ or -] 6.1. * p < 0.005; ** p < 0.001. Acknowledgement This work was funded by a project received from Newchapter, USA. Received 14 April 2003; accepted 25 September 2003 References Autrup, H., Harris, C.C., 1983. Metabolism of chemical carcinogen by human tissue. In: Harris, C.C., Autrup, H. (Eds.), Human Carcinogenesis. Academic Press, New York, pp. 169-194. Barrett, J.C., 1991. The relationship between carcinogenesis and mutagenesis. 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Phytother. Res. 1, 44-47. K.T. Santhosh, J. Swarnam, K. Ramadasan* Amala Cancer Research Centre, Amala Nagar P.O, Thrissur, Kerala 680553, India *Corresponding author. Tel.: +91-487-230-7950. E-mail address: email@example.com (K. Ramadasan).