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Antimutagenicity of lemon grass (Cymbopogon citratus Stapf) to various known mutagens in salmonella mutation assay
Genetic
ELSEVIER
Toxicology
Mutation Research 341 (1994) 71-75
Antimutagenicity of lemon grass (Cymbopogon citratus Stapf) to various known mutagens in salmonella mutation assay Usanee Vinitketkumnuen ap*,Rawiwan Puatanachokchai a, Prachya Kongtawelert Nirush Lertprasertsuke b, Taijiro Matsushima ’
a,
a Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50002, Thailand b Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50002, Thailand ‘Japan Bioassay Laboratory, Hadano 257, Japan
Received 13 October 1993; revision received 21 June 1994; accepted 30 June 1994
Abstract Lemon grass (Cymbopogon citrutus Stapf) was extracted with 80% ethanol. The extract was not found to be mutagenic in the Salmonella mutation test with or without metabolic activation. However, the extract was found to possess antimutagenic properties towards chemical-induced mutation in Salmonella typhimurium strains TA98 and TAlOO. Mutagenicity of AFB,, Trp-P-l, Trp-P-2, Glu-P-1, Glu-P-2, IQ, MNNG and AF-2, was inhibited by the extract of lemon grass in a dose-dependent manner, but no effect was found on the mutagenic activity of benzo[ alpyrene. Keywords:
Antimutagenicity;
Cymbopogon
citratus;
Medicinal plant
1. Introduction
as mutagenic precursors in foods may be important factors in the occurrence of cancer (Doll and
Epidemiological studies suggest that carcinogenie contamination of the environment as well
Peto, 1981). It is likely that this environmental influence not only depends on the exposure to carcinogenic factors but is also caused by a lack of antimutagenic or anticarcinogenic agents (Ames, 1983; Hayatsu et al., 1988). Increasing reports on dietary carcinogens and anticarcinogens have given rise to the hope that antimutagens/anticarcinogens, in particular those contained naturally in our diet, are of potential value in preventing cancer and other diseases linked with mutational occurrence (Ames, 1979). Medicinal plants have played a major role in the livelihood of the Thai people with little scientific
Abbreviations: AFB,, Aflatoxin Bl; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NADH, nicotinamide adenine nucleotide (oxidised form), Trp-P-l, 3-amino-1,4-dimethyl-SH-pyrido[4,3-b]-indole; Trp-P-2, 3amino-1-methyl-SH-pyrido[4,3-b]-indole; Glu-P-1, 2-amino-6methyldipyrido-1,2-a:3’,2’-d-imidazole, Glu-P-2, 2-aminodipyrido1,2-a:3’,2’-d-imidazole, B[a]P, benzo[a]pyrene; IQ, 2amino-3-methylimidazo[4,_5-flquinoline; MNNG, N-methylN’-nitro-N-nitrosoguanidine; AF-2, 2-(2-fury&3(5-nitro-2furyl) a&amide. * Corresponding author. Fax 053-217-144.
0165-1218/94/$07.00 0 1994 Elsevier Science B.V. All rights resewed SSDIO165-1218(94)00053-O
72
U. Vinitketkumnuen et al. /Mutation Research 341 (1994) 71-75
knowledge. Realizing their importance, research has been conducted to promote the use of Thai medicinal plants. In the present study, the antimutagenic activity of lemon grass (Cymbupogon citrutus, Stapf), a Thai medicinal plant commonly used in the diet and in medicine, was investigated by Ames Salmonella mutation preincubation assay. The influence of crude extract on the induction of His+ revertants by various known mutagens was studied in Salmonella typhimurium TA98 and TAlOO.
2. Materials
were pooled and dried in a rotary evaporator under reduced pressure at 50°C. The dry residue was weighed and redissolved in known volumes of dimethylsulfoxide. The solution was passed through a Millipore filter membrane before being analysed for its mutagenicity and antimutagenicity. Antimutagenicity assay Tester strains Salmonella typhimurium TA98
and TAlOO were kindly provided by Prof. B.N. Ames, University of California, Berkeley. The bacteria were kept and regularly checked for their genetic markers (Maron and Ames, 1983). The assay was performed essentially as ‘preincubation technique’ (Matsushima et al., 1980). 50 ~1 of known mutagens, O-100 ~1 of plant extract, 500 ~1 of S9 mix (or 500 ~1 of 0.2 M phosphate buffer, pH 7.4) and 100 ~1 of stationary phase bacteria (2-3 x lo9 cells/ml) were preincubated at 30°C for 30 min. The tubes were shaken at a moderate speed in a shaking water bath. After the preincubation period, 2 ml of molten top agar supplemented with L-histidine and o-biotin at 45°C was added to the mixtures, gently mixed and poured onto a minimal glucose agar plate. The plates were inverted and incubated at 37°C for 48 h. His+ revertants colonies were counted. The experiments were repeated, and 3 plates were used for each concentration of plant extract. Results are expressed as the mean of 6 plates per point minus the mean of spontaneous revertants. In each assay triplicate plate incubations (using
and methods
Glucose-6-phosphate, D-biotin, dimethysulfoxide, B(a)P, MNNG and AFB, (A6636, Lot 38F4061) were obtained from Sigma, Chemical, St. Louis, USA and NADPH and NADH from Oriental Yeast Company, Japan, pyrolysate products, Trp-p-l, Trp-p-2, Glu-p-l, Glu-p-2 and IQ were obtained from the Nard Institute, Osaka, Japan. Sample preparation
Lemon grass was purchased from local markets in Chiang Mai, Thailand. The plant was cleaned, minced, freeze-dried and blended to a fine powder. Fifty grams of the plant powder was extracted by stirring with 500 ml of 80% ethanol for 6 h, then filtered by suction. The residue was extracted again with 500 ml of 80% ethanol in the same way for 4 h and after filtering, the filtrates
Table 1 Effect of lemon grass extract Amount extract
(mg) 0 0.5 1.0 2.5 5.0 10.0
of
on mutagenesis
Number
induced
of His+ revertants
by various
induced
in Salmonella typhimurium strain TA98
mutagens
by mutagens/plate
mu1
Trp-p-l
Glu-p-2
(0.05 /.Lg)
Trp-p-2 (0.02 /.Lg)
Glu-p-l
(0.05 /Lg)
(0.03 /Lg)
(0.5 /LB)
901 f 103 444 f 47 260 k 40 133 + 14 41 f 10 3+ 2
329 f 512 f 442 + 295 + 165 f 53*
1035 1646 1131 739 306 102
688 f 510* 358 + 243 + 89f 20*
234 ~fr12 73 f 14 39+ 6 24* 2 12* 3 0
17 22 32 37 14 7
Spontaneous revertants have been substracted already. The results Zero concentration indicates mutagens alone without any extract.
+ + f + f f
42 31 58 49 55 14
32 9 48 25 11 5
are mean f SEM of 6 plates from 2 independent
experiments.
U. Vbitketkumnuen
et al. /Mutation Research 341 (1994) 71-75
only the plant extract) were performed together with the test plates incubation to determine the mutagenic activity of the plant extract. Metabolic activation
Rat liver S9 fraction was used as the metabolic activation system in this assay. Phenobarbital and 5,6-benzoflavone were used to induce liver microsome enzymes (Matsushima et al., 1976). S9 mix was freshly prepared each day and kept in ice. 1 ml of S9 mix contained: 0.1 mmol of sodium phosphate buffer, pH 7.4, 8 pmol of MgCl,-KCl, 5 pmol of Glu-6-phosphate, 4 pmol of NADPH and NADH and 0.1 ml of S9 fraction.
73
tract was concentration dependent. At the highest dose (10.0 mg of extract per plate), the number of histidine auxotrophic revertants was reduced by approximately 100%. Lemon grass extract also showed antimutagenie activity in strain TAlOO exposed to IQ and AF-2. In the case of MNNG, no clear antimutagenie effects could be observed at low doses of the extract, whereas at a concentration of 10 mg of the extract per plate a pronounced inhibition of the MNNG-induced mutation in TAlOO was found. (Table 2) The lemon grass extract did not influence the formation of His+ revertants induced by benzo[a]pyrene in TAlOO in this study.
3. Results 4. Discussion Before testing for the antimutagenicity of the lemon grass extract, the mutagenicity of the extract was tested; it was ascertained that the amount of plant extract added to the indicator bacteria did not influence their spontaneous mutation frequencies (Data not shown). Table 1 shows the antimutagenic activity of the ethanol extract of lemon grass against various known mutagens in S. typhimurium TA98. It was found that lemon grass extract exerted significant antimutagenic action in the Salmonella mutation assay. Decrease of mutant colonies of S. typhimurium strain TA98 upon exposed to APB,, Trp-P-l, Trp-P-2, Glu-P-1 and Glu-P-2 in the presence of different amount of lemon grass ex-
The present findings clearly demonstrate that ethanol extract of lemon grass modifies mutation in S. typhimurium strains TA 98 and TAlOO which are induced by various known mutagens such as APB,, Trp-P-l, Trp-P-2, Glu-P-1, Glu-P-2, IQ, AF-2 and MNNG. But the extract did not influence the benzo[a]pyrene mutagenesis. The results suggest that some active compounds in lemon grass may either deactivate these mutagens by directly trapping or deactivate involved enzyme in S9 mix. However, lemon grass extract cannot inhibit mutagenesis induced by B[a]P, a promutagen which is activated by cytochrome P-448 dependent system. Therefore, it was suggested that
Table 2 Effects of lemon grass extract on mutagenesis induced by various mutagens in Salmonella typhimurium strain TAlOO Amount of extract
ms. 0
0.5 1.0 2.5 5.0 10.0
Number of His+ revertants induced by mutagens/plate
1015 f 261 + 121+ 100 + 16+ 13+
5PLg
MNNG 0.5 Pg
1496 f 97 1550 f 95 1389 f 62 1807 f 24 1763 f 118 1777+ 73
1545 f 1408 + 1355 + 842 + 399 + 80 +
BlalP
fQ 0.1 Pg 43 21 33 19 5 6
AF-2 0.01 /kg 74 172 202 170 108 29
810 f 46 631+ 22 653 + 23 579 f 48 440+34 280 + 19
Spontaneous revertants have been substracted already. The results are mean f SEM of 6 plates from 2 independent Zero concentration indicates mutagens alone without any extract.
experiments.
74
U. Wnitketkumnuen
et al. /Mutation
the extract might not be able to influence this activation system. Many active substances in lemon grass have been identified, such as essential oil, citral, myrcene, citral, and lignin-related compounds (Ruangrungsri, 1991). Some study has reported that many substances in lignin such as phenylpropanoid, caffeic acid inhibited mutagenicity of AFB,. (San and Chan, 1987). This investigation is a preliminary study of the antimutagenic activity of Thai medicinal plants commonly used in diet and medicine. Interesting results have prompted further studies, including those on the identification of the main active substances in lemon grass and experiments are now being performed. It might turn out that the active substance is a compound that is already known; on the other hand, it could lead to the discovery of some hitherto unrecognized chemical compond which can be used as a chemoprevention agent. Lemon grass seems to have several advantages
as a chemopreventive agent. In addition to completely inhibits mutagenesis of many mutagens in the Salmonella mutation assay, it inhibits chromosomal aberration in human lymphocytes exposed to mitomycin C (Meevatee et al., 1993), it inhibits micronucleus formation in rats exposed to cyclophosphamide (Pinsaeng, 1993), and is responsible for retardation of tumor growth and lessening the degree of tumor metastases in rats transplanted with fibrosarcoma (Puatanachokchai, 1994). Lemon grass extract enhanced glutathione-S-transferase activity in mouse intestine (Luke and Boling, 1991). It also inhibited the activity of Epstein-Barr virus induced by teleocidin (Murakami et al., 1993). Pharmacological studies have revealed of lemon grass is be nontoxic (Carlini et al., 1986; Souza et al., 1986). Apart from cancer, mutagenesis causes many degenerative diseases such as heart disease, and aging (Ames and Gold, 1991). Thus lemon grass might also be used to prevent these conditions. The prospect that development and assessment of chemoprotective agents from lemon grass seems reasonable. Therefore, chemoprotective agents from lemon grass could be administered to high-risk, but otherwise healthy individuals to improve their quality of health.
Research 341 (1994) 71-75
Acknowledgement
This work was supported by the Hitachi Research Foundation, Japan, 1992.
References Ames, B.N. (1979) Identifying environmental chemicals causing mutations and cancer. Science, 204, 587-593. Ames, B.N. (1983) Dietary carcinogens and anticarcinogens. Science 221, 1256-1264. Ames, B.N. and L.S. Gold (1991) Endogenous mutagens and the cause of aging and cancer. Mutation Res., 250. 3-16. Carlini, E.A., J.P. Contar, A.R. Silva Filho, N.G. Da SilveriaFilho, M.L. Frochtengarten and O.F.A. Bueno (1986) Pharmacology of lemon grass (Cymbopogon citratus Stapf) I. Effect of teas prepared from the leaves on laboratory animals. J. Ethanopharmacol., 17, 37-64. Doll, R. and R. P&o (1981) The cause of cancer: quantitative estimate of avoidable risks of cancer in the United States today. J. Natl. Cancer. Inst., 66, 1195-1308. Hayatsu, H., Arimoto, S. and T. Negishi (1988) Dietary inhibitors of mutagenesis and carcinogenesis. Mutation Res., 202, 429-446. Luke, K.T. and 2. Baling (1991) Effects of essential oils on glutathione-S-transferase activity in mice. J. Agric. Food Chem. 39, 660-662. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res., 113, 173-215. Matsushima, T., M. Sawamura, K. Hara and T. Sugimura (1976) A safe substitute for polychlorinated biphenyl as an inducer of metabolic activation system, in: F. de Serres et al. (Eds.), In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier/North-Holland, Amsterdam, pp. 85-88. Matsushima, T., T. Sugimura, T. Nagao, A. Shirai and M. Sawamura (1980) Factors modulating mutagenicity in microbial test, in: K.H. Norpoth and R.C. Garner (Eds.), Short-term Test System for Detecting Mutagens and Carcinogens, Springer Berlin, pp. 273-285. Meevatee, U., S. Boontim, 0. Keereeta, U. Vinitketkumnuen and N. 0-ariyakul (1993) Antimutagenic activity of lemon grass, in: Boot-in S. (Ed.), Man and Environment, Chiang Mai University Press, p. 346. Murakami, A., H. Ohigashi and K. Koshimizu (1993) Possible anti-tumor promoters from traditional plant food items of Thailand. Abstracts of the 11th Asia Pacific Cancer Conference, Bangkok, Thailand, p. 83. Pinsaeng, K. (1993) Anti-micronucleus formation of lemon grass extract, Master thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. Puatanachokchai, R. (1994) Antimutagenicity, cytotoxicity and antitumor activity from lemon grass (Cymbopogon citratus) extract. Master thesis, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
U. V5nitketkumnuen et al. /Mutation Research 341 (I 994) 71-75 Ramel, C., V.K. Alekperov, B.N. Ames, T. Kada and L.W. Wattenberg (1986) ICPEMC Publication No. 12. Inhibitors of mutagenesis and their relevance to carcinogenesis, Report by ICPEMC expert group on antimutagens and desmutagens. Mutation Res., 168, 47-65. Ruangrungsri, N. (1991) Herbs: lemon grass, Chulalongkorn Press, p. 39-40. San, R.H.C. and RIM. Chan (1987) Inhibitory effect of
75
phenolic compounds on aflatoxin B, metabolism and induced mutagenesis. Mutation Res., 177, 229-239. Souza Formigoni, M.L.O., H.M. Lodder, O.G. Filho, T.M.S. Ferreira and E.A. Carlini (1986) Pharmacology of lemon grass (Cymbopogon citrates Staph) II. Effect of daily two month administration in male and female rats and in offspring exposed “in utero”. J. Ethanopharmacol., 17, 65-74.
ELSEVIER
Toxicology
Mutation Research 341 (1994) 71-75
Antimutagenicity of lemon grass (Cymbopogon citratus Stapf) to various known mutagens in salmonella mutation assay Usanee Vinitketkumnuen ap*,Rawiwan Puatanachokchai a, Prachya Kongtawelert Nirush Lertprasertsuke b, Taijiro Matsushima ’
a,
a Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50002, Thailand b Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50002, Thailand ‘Japan Bioassay Laboratory, Hadano 257, Japan
Received 13 October 1993; revision received 21 June 1994; accepted 30 June 1994
Abstract Lemon grass (Cymbopogon citrutus Stapf) was extracted with 80% ethanol. The extract was not found to be mutagenic in the Salmonella mutation test with or without metabolic activation. However, the extract was found to possess antimutagenic properties towards chemical-induced mutation in Salmonella typhimurium strains TA98 and TAlOO. Mutagenicity of AFB,, Trp-P-l, Trp-P-2, Glu-P-1, Glu-P-2, IQ, MNNG and AF-2, was inhibited by the extract of lemon grass in a dose-dependent manner, but no effect was found on the mutagenic activity of benzo[ alpyrene. Keywords:
Antimutagenicity;
Cymbopogon
citratus;
Medicinal plant
1. Introduction
as mutagenic precursors in foods may be important factors in the occurrence of cancer (Doll and
Epidemiological studies suggest that carcinogenie contamination of the environment as well
Peto, 1981). It is likely that this environmental influence not only depends on the exposure to carcinogenic factors but is also caused by a lack of antimutagenic or anticarcinogenic agents (Ames, 1983; Hayatsu et al., 1988). Increasing reports on dietary carcinogens and anticarcinogens have given rise to the hope that antimutagens/anticarcinogens, in particular those contained naturally in our diet, are of potential value in preventing cancer and other diseases linked with mutational occurrence (Ames, 1979). Medicinal plants have played a major role in the livelihood of the Thai people with little scientific
Abbreviations: AFB,, Aflatoxin Bl; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NADH, nicotinamide adenine nucleotide (oxidised form), Trp-P-l, 3-amino-1,4-dimethyl-SH-pyrido[4,3-b]-indole; Trp-P-2, 3amino-1-methyl-SH-pyrido[4,3-b]-indole; Glu-P-1, 2-amino-6methyldipyrido-1,2-a:3’,2’-d-imidazole, Glu-P-2, 2-aminodipyrido1,2-a:3’,2’-d-imidazole, B[a]P, benzo[a]pyrene; IQ, 2amino-3-methylimidazo[4,_5-flquinoline; MNNG, N-methylN’-nitro-N-nitrosoguanidine; AF-2, 2-(2-fury&3(5-nitro-2furyl) a&amide. * Corresponding author. Fax 053-217-144.
0165-1218/94/$07.00 0 1994 Elsevier Science B.V. All rights resewed SSDIO165-1218(94)00053-O
72
U. Vinitketkumnuen et al. /Mutation Research 341 (1994) 71-75
knowledge. Realizing their importance, research has been conducted to promote the use of Thai medicinal plants. In the present study, the antimutagenic activity of lemon grass (Cymbupogon citrutus, Stapf), a Thai medicinal plant commonly used in the diet and in medicine, was investigated by Ames Salmonella mutation preincubation assay. The influence of crude extract on the induction of His+ revertants by various known mutagens was studied in Salmonella typhimurium TA98 and TAlOO.
2. Materials
were pooled and dried in a rotary evaporator under reduced pressure at 50°C. The dry residue was weighed and redissolved in known volumes of dimethylsulfoxide. The solution was passed through a Millipore filter membrane before being analysed for its mutagenicity and antimutagenicity. Antimutagenicity assay Tester strains Salmonella typhimurium TA98
and TAlOO were kindly provided by Prof. B.N. Ames, University of California, Berkeley. The bacteria were kept and regularly checked for their genetic markers (Maron and Ames, 1983). The assay was performed essentially as ‘preincubation technique’ (Matsushima et al., 1980). 50 ~1 of known mutagens, O-100 ~1 of plant extract, 500 ~1 of S9 mix (or 500 ~1 of 0.2 M phosphate buffer, pH 7.4) and 100 ~1 of stationary phase bacteria (2-3 x lo9 cells/ml) were preincubated at 30°C for 30 min. The tubes were shaken at a moderate speed in a shaking water bath. After the preincubation period, 2 ml of molten top agar supplemented with L-histidine and o-biotin at 45°C was added to the mixtures, gently mixed and poured onto a minimal glucose agar plate. The plates were inverted and incubated at 37°C for 48 h. His+ revertants colonies were counted. The experiments were repeated, and 3 plates were used for each concentration of plant extract. Results are expressed as the mean of 6 plates per point minus the mean of spontaneous revertants. In each assay triplicate plate incubations (using
and methods
Glucose-6-phosphate, D-biotin, dimethysulfoxide, B(a)P, MNNG and AFB, (A6636, Lot 38F4061) were obtained from Sigma, Chemical, St. Louis, USA and NADPH and NADH from Oriental Yeast Company, Japan, pyrolysate products, Trp-p-l, Trp-p-2, Glu-p-l, Glu-p-2 and IQ were obtained from the Nard Institute, Osaka, Japan. Sample preparation
Lemon grass was purchased from local markets in Chiang Mai, Thailand. The plant was cleaned, minced, freeze-dried and blended to a fine powder. Fifty grams of the plant powder was extracted by stirring with 500 ml of 80% ethanol for 6 h, then filtered by suction. The residue was extracted again with 500 ml of 80% ethanol in the same way for 4 h and after filtering, the filtrates
Table 1 Effect of lemon grass extract Amount extract
(mg) 0 0.5 1.0 2.5 5.0 10.0
of
on mutagenesis
Number
induced
of His+ revertants
by various
induced
in Salmonella typhimurium strain TA98
mutagens
by mutagens/plate
mu1
Trp-p-l
Glu-p-2
(0.05 /.Lg)
Trp-p-2 (0.02 /.Lg)
Glu-p-l
(0.05 /Lg)
(0.03 /Lg)
(0.5 /LB)
901 f 103 444 f 47 260 k 40 133 + 14 41 f 10 3+ 2
329 f 512 f 442 + 295 + 165 f 53*
1035 1646 1131 739 306 102
688 f 510* 358 + 243 + 89f 20*
234 ~fr12 73 f 14 39+ 6 24* 2 12* 3 0
17 22 32 37 14 7
Spontaneous revertants have been substracted already. The results Zero concentration indicates mutagens alone without any extract.
+ + f + f f
42 31 58 49 55 14
32 9 48 25 11 5
are mean f SEM of 6 plates from 2 independent
experiments.
U. Vbitketkumnuen
et al. /Mutation Research 341 (1994) 71-75
only the plant extract) were performed together with the test plates incubation to determine the mutagenic activity of the plant extract. Metabolic activation
Rat liver S9 fraction was used as the metabolic activation system in this assay. Phenobarbital and 5,6-benzoflavone were used to induce liver microsome enzymes (Matsushima et al., 1976). S9 mix was freshly prepared each day and kept in ice. 1 ml of S9 mix contained: 0.1 mmol of sodium phosphate buffer, pH 7.4, 8 pmol of MgCl,-KCl, 5 pmol of Glu-6-phosphate, 4 pmol of NADPH and NADH and 0.1 ml of S9 fraction.
73
tract was concentration dependent. At the highest dose (10.0 mg of extract per plate), the number of histidine auxotrophic revertants was reduced by approximately 100%. Lemon grass extract also showed antimutagenie activity in strain TAlOO exposed to IQ and AF-2. In the case of MNNG, no clear antimutagenie effects could be observed at low doses of the extract, whereas at a concentration of 10 mg of the extract per plate a pronounced inhibition of the MNNG-induced mutation in TAlOO was found. (Table 2) The lemon grass extract did not influence the formation of His+ revertants induced by benzo[a]pyrene in TAlOO in this study.
3. Results 4. Discussion Before testing for the antimutagenicity of the lemon grass extract, the mutagenicity of the extract was tested; it was ascertained that the amount of plant extract added to the indicator bacteria did not influence their spontaneous mutation frequencies (Data not shown). Table 1 shows the antimutagenic activity of the ethanol extract of lemon grass against various known mutagens in S. typhimurium TA98. It was found that lemon grass extract exerted significant antimutagenic action in the Salmonella mutation assay. Decrease of mutant colonies of S. typhimurium strain TA98 upon exposed to APB,, Trp-P-l, Trp-P-2, Glu-P-1 and Glu-P-2 in the presence of different amount of lemon grass ex-
The present findings clearly demonstrate that ethanol extract of lemon grass modifies mutation in S. typhimurium strains TA 98 and TAlOO which are induced by various known mutagens such as APB,, Trp-P-l, Trp-P-2, Glu-P-1, Glu-P-2, IQ, AF-2 and MNNG. But the extract did not influence the benzo[a]pyrene mutagenesis. The results suggest that some active compounds in lemon grass may either deactivate these mutagens by directly trapping or deactivate involved enzyme in S9 mix. However, lemon grass extract cannot inhibit mutagenesis induced by B[a]P, a promutagen which is activated by cytochrome P-448 dependent system. Therefore, it was suggested that
Table 2 Effects of lemon grass extract on mutagenesis induced by various mutagens in Salmonella typhimurium strain TAlOO Amount of extract
ms. 0
0.5 1.0 2.5 5.0 10.0
Number of His+ revertants induced by mutagens/plate
1015 f 261 + 121+ 100 + 16+ 13+
5PLg
MNNG 0.5 Pg
1496 f 97 1550 f 95 1389 f 62 1807 f 24 1763 f 118 1777+ 73
1545 f 1408 + 1355 + 842 + 399 + 80 +
BlalP
fQ 0.1 Pg 43 21 33 19 5 6
AF-2 0.01 /kg 74 172 202 170 108 29
810 f 46 631+ 22 653 + 23 579 f 48 440+34 280 + 19
Spontaneous revertants have been substracted already. The results are mean f SEM of 6 plates from 2 independent Zero concentration indicates mutagens alone without any extract.
experiments.
74
U. Wnitketkumnuen
et al. /Mutation
the extract might not be able to influence this activation system. Many active substances in lemon grass have been identified, such as essential oil, citral, myrcene, citral, and lignin-related compounds (Ruangrungsri, 1991). Some study has reported that many substances in lignin such as phenylpropanoid, caffeic acid inhibited mutagenicity of AFB,. (San and Chan, 1987). This investigation is a preliminary study of the antimutagenic activity of Thai medicinal plants commonly used in diet and medicine. Interesting results have prompted further studies, including those on the identification of the main active substances in lemon grass and experiments are now being performed. It might turn out that the active substance is a compound that is already known; on the other hand, it could lead to the discovery of some hitherto unrecognized chemical compond which can be used as a chemoprevention agent. Lemon grass seems to have several advantages
as a chemopreventive agent. In addition to completely inhibits mutagenesis of many mutagens in the Salmonella mutation assay, it inhibits chromosomal aberration in human lymphocytes exposed to mitomycin C (Meevatee et al., 1993), it inhibits micronucleus formation in rats exposed to cyclophosphamide (Pinsaeng, 1993), and is responsible for retardation of tumor growth and lessening the degree of tumor metastases in rats transplanted with fibrosarcoma (Puatanachokchai, 1994). Lemon grass extract enhanced glutathione-S-transferase activity in mouse intestine (Luke and Boling, 1991). It also inhibited the activity of Epstein-Barr virus induced by teleocidin (Murakami et al., 1993). Pharmacological studies have revealed of lemon grass is be nontoxic (Carlini et al., 1986; Souza et al., 1986). Apart from cancer, mutagenesis causes many degenerative diseases such as heart disease, and aging (Ames and Gold, 1991). Thus lemon grass might also be used to prevent these conditions. The prospect that development and assessment of chemoprotective agents from lemon grass seems reasonable. Therefore, chemoprotective agents from lemon grass could be administered to high-risk, but otherwise healthy individuals to improve their quality of health.
Research 341 (1994) 71-75
Acknowledgement
This work was supported by the Hitachi Research Foundation, Japan, 1992.
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