Environmental and Molecular Mutagenesis 44:387–393 (2004)
Antimutagenic Activity of Spearmint Tian-Wei Yu,1 Meirong Xu,1 and Roderick H. Dashwood1,2* 2
1 Cancer Chemoprotection Program, Linus Pauling Institute, Corvallis, Oregon Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon
The antimutagenic activity of spearmint (Mentha spicata), a popular food flavoring agent, was studied in the Salmonella assay. Spearmint leaves were brewed in hot water for 5 min at concentrations up to 5% (w/v), and the water extracts were tested against the direct-acting mutagens 4-nitro1,2-phenylenediamine (NPD) and 2-hydroxy amino-3-methyl-3H-imidazo[4,5-f]quinoline (N-OHIQ) using Salmonella typhimurium strain TA98. Nontoxic concentrations of spearmint extract inhibited the mutagenic activity of N-OH-IQ in a concentration-dependent fashion, but had no effect against NPD. These experiments by design focused on the water extract consumed commonly as an herbal tea, but chloroform and methanol extracts of spearmint also possessed antimutagenic activity against N-OH-IQ. Water extract of spearmint inhibited the mutagenic activity of the parent compound, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), in the presence of rat liver S9; however,
the concentration for 50% inhibition (IC50) against IQ was approximately 10-fold higher than in assays with N-OH-IQ minus S9. At concentrations similar to those used in the Salmonella assays, spearmint extract inhibited two of the major enzymes that play a role in the metabolic activation of IQ, namely, cytochromes P4501A1 and 1A2, based on ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase assays in vitro. In vivo, rats were given spearmint water extract (2%; w/v) as the sole source of drinking fluid before, during, and after 2-week treatment with IQ; colonic aberrant crypt foci were inhibited significantly at 8 weeks (P ⬍ 0.05, compared with rats given IQ alone). Collectively, these findings suggest that spearmint tea protects against IQ and possibly other heterocyclic amines through inhibition of carcinogen activation and via direct effects on the activated metabolite(s). Environ. Mol. Mutagen. 44:387–393, 2004. © 2004 Wiley-Liss, Inc.
Key words: IQ; PhIP; Trp-P-2; heterocyclic amines; colonic aberrant crypt foci; mint; Salmonella mutagenicity assay
INTRODUCTION There continues to be significant interest in the various antimutagens and anticarcinogens present in the human diet, including those found in beverages such as tea [Dashwood, 2002; Surh and Ferguson, 2003]. Tea is strictly defined as the beverage obtained from brewing the leaves of Camellia sinensis, which contains high levels of polyphenols such as catechins, theaflavins, and thearubigins [Lambert and Yang, 2003]. The promising results obtained in antimutagenicity and anticarcinogenicity assays with tea have given impetus to studies on the possible health benefits, and possible risks, of other beverages, including so-called herbal teas [Winn, 2003; Huang et al., 2004; Sparber et al., 2004; Strandell et al., 2004]. We became interested in evaluating spearmint (Mentha spicata) for antimutagenic activity. Spearmint essential oil is a common constituent in hygiene and cosmetic products, and substantial amounts are used in the food and beverage industries [Spirling and Daniels, 2001]. In countries of the Middle East and Africa, the dry or fresh leaf of spearmint is added during the brewing of tea, where it provides a pleasant aroma and refreshing taste. The ancient Romans believed that the smell of mint “stirred the mind and the taste © 2004 Wiley-Liss, Inc.
to a greedy desire of meat” [Fabre, 2003], a historical perspective that is interesting in light of the present-day understanding of the mutagens formed during the cooking of meat [Sugimura et al., 2004]. Accepting perhaps that indeed “everything old is new again” [Israelsen, 1995], we sought to examine the possible antimutagenic effects of spearmint toward 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The present investigation confirmed that spearmint had significant inhibitory effects against the cooked meat heterocyclic amine mutagen both in vitro and in vivo.
Grant sponsor: the Linus Pauling Institute; Grant sponsor: the Service Core of Program Project; Grant number: CA90890. *Correspondence to: Roderick H. Dashwood, Cancer Chemoprotection Program, Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331. E-mail:
[email protected] Received 7 May 2004; provisionally accepted 1 June 2004; and in final form 21 June 2004 DOI 10.1002/em.20063 Published online 4 November 2004 in Wiley InterScience (www. interscience.wiley.com).
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MATERIALS AND METHODS Cut and sifted dry spearmint leaves were purchased from Frontier Natural Products (Norway, IA). Heterocyclic amines were obtained from Toronto Research Chemicals(North York, Canada). The mutagen 4-nitro1,2-phenylenediamine (NPD), as well as 7-ethoxyresorufin and resorufin, were from Sigma (St. Louis, MO), whereas methoxyresorufin was from Molecular Probes (Eugene, OR). Agar number 1 and nutrient broth number 2 were from Oxoid (Ogdensburg, NY). Aroclor 1254-induced rat liver S9 was purchased from Moltox (Boone, NC). Monosodium NADP was from Calbiochem (La Jolla, CA), and disodium D-glucose 6-phosphate was from MP Biomedicals (Irvine, CA). Dimethylsulfoxide (DMSO) was from Alfa Aesar (Ward Hill, MA). All other chemicals and reagents were from Fisher Scientific (Hampton, NH).
Spearmint Extraction and Fractionation Spearmint leaves were brewed in boiling Milli-Q water (95–100°C) for 5 min, and after brief centrifugation the supernatant was filter sterilized. Initial concentrations during brewing were in the range 0.1–5% (w/v), as indicated. In the fractionation studies, spearmint leaves were ground to powder and extracted with 10 volumes of chloroform at room temperature, followed by 10 volumes of methanol at 60°C, and finally 10 volumes of water at 90 –95°C. Each sequential extraction step was conducted for 5 min. Chloroform and methanol extracts were dried under nitrogen, dissolved in DMSO, and tested directly in the Salmonella assay.
photometer, with excitation and emission wavelengths of ex ⫽ 530 nm and em ⫽ 585 nm, respectively. Enzyme activities were quantified by comparison with resorufin standard curves [Burke et al., 1985] after pilot studies had ruled out any possible nonspecific quenching by the mint extracts (data not shown).
Colonic Aberrant Crypt Assay Aberrant crypt assays with the carcinogen IQ were conducted as reported previously [Yu et al., 2001], except that F344 rats were killed at 8 weeks. Positive controls in group 1 were given IQ by oral gavage during experiment week 3 and 4 and drinking water throughout the study; group 2 received the same dosing regimen of IQ plus 2% (w/v) spearmint beverage as the sole source of drinking fluid for 8 weeks; negative controls in group 3 were given vehicle alone (no IQ) and 2% spearmint beverage for 8 weeks. Colons were fixed mucosa side up in 10% phosphate-buffered formalin, stained with methylene blue, and the putative preneoplastic lesions termed “aberrant crypt foci” (ACF) were scored as described elsewhere [Santana-Rios et al., 2001b; Yu et al., 2001].
Data and Statistical Analysis Data presented in each figure represents the mean ⫾ SD of triplicates (n ⫽ 3) except for the in vivo studies (n ⫽ 4). In the latter experiments, data were analyzed by ANOVA (Waller-Duncan K-ratio t-test) using SAS statistical software, with a significant difference considered at the P ⬍ 0.05 level.
Salmonella Mutagenicity Assay Salmonella assays were based on the methodologies described previously [Maron and Ames, 1983]. In the majority of experiments, the order of addition to the assay was as follows: 2 ml molten top agar, mutagen (or 0.01 mL DMSO solvent alone), spearmint extract (ⱕ 0.1 mL), 0.5 ml phosphate-buffered saline or 10% rat liver S9 mix, and finally 0.1 ml of an overnight culture of Salmonella typhimurium strain TA98. The mixture was vortexed and poured onto minimal glucose plates. Alternatively, the mutagen, spearmint extract, and bacteria were preincubated for up to 30 min prior to the addition of top agar and plating. Petri plates were incubated at 37°C in the dark for 48 hr, and the histidine revertant (his⫹) colonies were counted with a Sorcerer Colony Counter (Perceptive Instruments, Suffolk, U.K.). All of the experiments were repeated at least twice, and triplicate plates were used for each data point.
Toxicity Tests The toxicity of spearmint extracts toward Salmonella strain TA98 was determined as described in detail elsewhere [Santana-Rios et al., 2001a; Yu et al., 2001]. These tests confirmed that there was normal growth of the background lawn, spontaneous counts within the normal range, and no significant reduction in cell survival. Thus, for the concentrations and conditions reported here, no toxicity or other adverse effects were observed.
Enzyme Assays Ethoxyresorufin O-deethylase (EROD) and methoxyresorufin Odemethylase (MROD) assays were conducted at 37°C and contained 1 mM MgCl2, 100 mM potassium phosphate buffer (pH 7.8), 1 M substrate, 0.2 mg/ml rat liver S9 protein, and 0.1 mL spearmint extract. The enzymatic reaction was initiated by addition of 0.1 mM NADPH in a final reaction volume of 1 ml. Formation of the resorufin product was monitored continuously for up to 3 min using a Hitachi F2500 Fluorescence spectro-
RESULTS In initial experiments, spearmint was brewed at a concentration of 5% (w/v) for 5 min and increasing volumes of the water extract (herbal tea) were added to Salmonella assays containing the direct-acting mutagen NPD. Revertant counts on the order of ⬃ 2,500/plate were obtained with 20 g NPD alone, and spearmint had no significant effect on mutagenicity at nontoxic concentrations in the assay (Fig. 1a). Certain compounds, such as chlorophyllin, act as molecular complexing agents, and the ratio of mutagen to inhibitor can influence antimutagenic potency [Dashwood, 2002]. To increase this ratio in the present study, the volume of mint extract was held constant (0.1 ml/plate) and the dose of NPD was reduced from 20 g to 0.1–2.5 g/plate; under these conditions spearmint also lacked antimutagenic activity (Fig. 1b). Because some antimutagens stabilize or scavenge direct-acting mutagens and prevent DNA damage [Dashwood, 2002], spearmint extract was preincubated for up to 30 min with NPD prior to plating, but no significant inhibition of mutagenicity was detected (Fig. 1c). Collectively, these results indicated that spearmint extract had little or no effect against the direct-acting mutagen NPD. Initial experiments used NPD, a diagnostic control for Salmonella strain TA98 [Maron and Ames, 1983], but we were interested in testing spearmint against heterocyclic amine mutagens that might be ingested at the same time via the diet (e.g., mint sauce with meat). Thus, N-OH-IQ (10 ng/plate) was used in subsequent studies in which spearmint was brewed at different concen-
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Fig. 2. Spearmint inhibits the mutagenic activity of 2-hydroxyamino-3methyl-3H-imidazo[4,5-f]quinoline (N-OH-IQ). Spearmint leaves were brewed in hot water for 5 min at concentrations of 0%, 0.1%, 0.5%, or 1% (w/v) and 50 l of the aqueous extract were tested against the direct-acting mutagen N-OH-IQ (gray bars) or DMSO vehicle (white bars) in the direct-plate assay with Salmonella strain TA98. Results are given as mean ⫾ SD (n ⫽ 3).
Fig. 1. No effect of spearmint on the mutagenicity of 4-nitro-1,2-phenylenediamine (NPD). Spearmint leaves were brewed in hot water for 5 min at a concentration of 5% (w/v), and the aqueous extract was tested against the direct-acting mutagen NPD in the direct-plate assay (a and b) or in the preincubation assay (c). In the latter case, spearmint, NPD, and Salmonella strain TA98 were coincubated for 0, 15, or 30 min before plating. Results are given as mean ⫾ SD (n ⫽ 3).
trations (0%, 0.1%, 0.5%, or 1%; w/v) for 5 min. Spontaneous revertant counts with DMSO alone were on the order of ⬃ 20/plate, and 50 l of each mint extract had no toxic effect (Fig. 2, open bars). The positive control gave ⬃ 2,000 revertants per plate and, in marked contrast to the findings with NPD, spearmint inhibited the mutagenic activity of N-OH-IQ in a concentration-dependent manner (Fig 2, gray bars). Each of the spearmint concentrations shown in Figure 2 inhibited the mutagenic activity of N-OH-IQ significantly (P ⬍ 0.05). Spearmint leaf next was extracted in a sequential fashion with chloroform, methanol, and water, and each fraction was tested for antimutagenic activity in the Salmonella assay. There was a concentration-dependent inhibition of mutagenicity for all three fractions (Fig. 3), suggesting that spearmint leaf contains both lipophilic and hydrophilic compounds with antimutagenic activity against N-OH-IQ. No toxic effect was seen under these conditions, including in assays with vehicle alone (data not shown). The results in Figure 3 suggest that additional studies may be warranted on the antimutagens present in chloroform and methanol extracts of spearmint. Using similar conditions to those described in Figure 2 for spearmint vs. N-OH-IQ, the Salmonella assays were
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repeated with the parent compound, IQ, in the presence of rat liver S9. The positive control gave ⬃ 2,500 revertants at a dose of 6 ng/plate (Fig. 4). No significant antimutagenic effect was seen with the low and intermediate concentrations of spearmint in the assay, whereas 50 l of 1% spearmint extract inhibited the mutagenic activity of IQ significantly (P ⬍ 0.05). By design, the experiment in Figure 4 sought to recapitulate the previous conditions with spearmint against N-OH-IQ (Fig. 2); however, higher concentrations of spearmint were tested against IQ and inhibited mutagenicity by ⬎ 95% without producing toxicity (results not presented). By interpolation from the plot of percent inhibition vs. spearmint concentration (not shown), the concentration for 50% inhibition (IC50) was 0.15% vs. 0.014%, respectively, in assays with IQ ⫹ S9 and N-OH-IQ minus S9. Thus, lower concentrations of spearmint were highly effective against N-OH-IQ, whereas higher concentrations strongly inhibited the mutagenic activity of IQ, possibly through effects on the enzymes in liver S9. In liver S9, two of the major enzymes that metabolically activate IQ are cytochrome P450 1A1 (CYP1A1) and CYP1A2 [Turesky, 2004], and these enzymes were studied in EROD and MROD assays, respectively (Fig. 5). Concentration-dependent enzyme inhibition was seen in each assay, and the IC50 for spearmint extract was similar in each case, being 0.013% for EROD inhibition (Fig. 5a) and 0.018% for MROD inhibition (Fig. 5b). These values were comparable to the IC50 for inhibition of N-OH-IQ mutagenicity in the Salmonella assay, but somewhat lower than the IC50 for inhibition of IQ mutagenicity in the presence of S9. Based on these findings, a pilot ACF study was undertaken in rats treated with IQ and spearmint extract (Fig. 6). Male F344 rats were given IQ by oral gavage according to our standard 2-week protocol [Yu et al., 2001], or they were given IQ plus 2% (w/v) spearmint extract throughout the study. At the end of 8 weeks, none of the controls given vehicle alone plus spearmint had colonic ACF. However, animals given IQ alone or IQ plus 2% spearmint extract had 8.1 ⫾ 1.4 vs. 2.8 ⫾ 2.6 ACF per colon, respectively (mean ⫾ SD; P ⬍ 0.05; Fig. 6).
Fig. 3. Chloroform, methanol, and water fractions of spearmint inhibit the mutagenic activity of N-OH-IQ. Spearmint leaves were extracted with 10 volumes of chloroform at room temperature, followed by 10 volumes of methanol at 60°C, and finally 10 volumes of water at 90 –95°C. Fractions were tested at doses equivalent to 1, 10, 100, and 1,000 g per plate of the starting material, as indicated by the wedge-shaped symbol. The directplate assay was used with Salmonella strain TA98; vehicle controls had no toxic effect (data not shown). Results are given as mean ⫾ SD (n ⫽ 3).
DISCUSSION Despite its long history and widespread popularity, information is surprisingly scarce on the antimutagenic and antigenotoxic effects of mint. Early studies reported desmutagenic effects against mutagenic pyrolysis products [Morita et al., 1978; Natake et al., 1989]; more recently, mint was shown to inhibit carcinogenesis in the hamster cheek pouch [Samman et al., 1998]. Luteolin, from peppermint, was identified as a strong antimutagen against the heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]in-
Fig. 4. Spearmint inhibits the mutagenic activity of 2-amino-3-methyl3H-imidazo[4,5-f]quinoline (IQ). Assays were conducted exactly as described in Figure 2 except that the direct-acting mutagen N-OH-IQ was replaced with parent compound, IQ, in the presence of a rat liver metabolic activation system.
dole (Trp-P-2) [Samejima et al., 1995], whereas another constituent of peppermint, pulegone, has been recognized as a potential hepatotoxin [Nair, 2001].
Antimutagenic Activity of Spearmint
Fig. 5. Inhibition of 7-ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities by spearmint. Spearmint leaves were brewed in hot water for 5 min at a concentration of 1% (w/v) and tested in the EROD assay (a) or MROD assay (b) at the final concentrations shown. Inset: Percent inhibition vs. concentration of spearmint in the assay, showing the concentration for 50% inhibition (IC50). Percent inhibition was calculated from the initial slopes, in which each spearmint addition was compared with the 0% control. Data points and error bars indicate mean ⫾ SD (n ⫽ 3).
The volatile components of spearmint are well known, including the terpenoids carvone, dihydrocarvenone, limonene, menthone, and menthol, and compounds in this class exhibit cancer chemopreventive and therapeutic activities [Satomi et al., 1999]. Such compounds may have accounted in part for the antimutagenic activity of the chloroform and methanol extracts (Fig. 3), but volatile terpenoids have lower aqueous solubility in the water extract of spearmint. In the latter case, the more likely candidates would appear to be the nonvolatile aqueous constituents, such as glycosylated terpenes, flavonoids, anthacyanins, and chlorophylls [Zheng et al., 2003]. Members of these chemical classes have been shown to
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exhibit antimutagenic activity, as well as promutagenic activity in some cases [Hardigree and Epler, 1978; Samejima et al., 1995; Calomme et al., 1996; Dashwood, 2002]. Interestingly, a recent study identified the compound 6,7-bis-(2,2-dimethoxyethene)-2,11-dimethoxy-2Z, 4E,8E,10Z-dodecatetraendioic acid in the chloroform extract of spearmint as having inhibitory activity against tetracycline in the mouse micronucleus assay [Villasenor et al., 2002]. Collectively, these findings suggested the need for further studies on the antimutagenic and antigenotoxic properties of spearmint. In the present investigation, the water extract of spearmint acted in vitro as a blocking agent or desmutagen, that is, via inhibitory effects on carcinogen metabolism and/or DNA adduct formation [Dashwood, 2002]. The inhibition was mutagen-specific, since spearmint had no effect on NPD, but lowered the mutagenic activity of both N-OH-IQ and IQ in the Salmonella assay. Inhibition of the mutagenicity of N-OH-IQ (but not of NPD) might indicate a mechanism involving scavenging of the aryl nitrenium ion ultimate carcinogen, whereas antimutagenic activity against IQ might occur via the inhibition of carcinogen-activating enzymes. EROD and MROD activities were inhibited by spearmint in vitro, supporting interference in the metabolic activation of IQ by CYP1A1 and CYP1A2. However, since mint was more effective against N-OH-IQ than against IQ in the Salmonella assay, our interpretation is that the antimutagens in spearmint effectively scavenge the activated metabolite(s) of heterocyclic amines at low doses, but also inhibit hepatic CYP1A1 and CYP1A2 at higher doses. Based on these findings, a pilot study was conducted in vivo, and we report here for the first time that 2% spearmint given to rats before, during, and after carcinogen exposure inhibited IQ-induced ACF significantly (Fig. 6). A more extensive investigation in the future should include separate exposure protocols for blocking and suppressing activities, to distinguish inhibitory effects during the carcinogen exposure period from those acting postinitiation [Dashwood, 2002]. Nonetheless, the results support a chemopreventive role for spearmint against the earliest putative preneoplastic lesions in the rat colon [Pretlow and Bird, 2001], with a level of protection similar to that seen with white, green, and black teas against IQ and the related heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [Xu et al., 1996; Santana-Rios et al., 2001a]. It is interesting to speculate on the possible complementary mechanisms afforded by tea polyphenols acting in concert with the antimutagens in spearmint, and whether this contributes in any way to reducing the risks for colon and other cancers in the Middle East and Africa, where black tea brewed with mint is the beverage of choice.
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Fig. 6. Spearmint inhibits IQ-induced colonic ACF in the rat. Rats were treated with IQ according to a published protocol [Yu et al., 2001], namely, by oral gavage at a dose of 133 mg/kg body wt on alternating days for 2 weeks. Spearmint leaves were brewed in hot water for 5 min at a concentration of 2% (w/v), and the aqueous extract was given as the sole source of drinking fluid for 2 weeks before, 2 weeks during, and 4 weeks
after the carcinogen or vehicle treatment period (as shown). Total ACF/ colon were scored as reported previously [Yu et al., 2001]. Results are given as mean ⫾ SD (n ⫽ 4); compared with animals given IQ alone, spearmint inhibited IQ-induced ACF significantly at 8 weeks (asterisk, P ⬍ 0.05, by ANOVA).
ACKNOWLEDGMENTS
Nair B. 2001. Final report on the safety assessment of mentha piperita (peppermint) oil, mentha piperita (peppermint) leaf extract, mentha piperita (peppermint) leaf, and mentha piperita (peppermint) leaf water. Int J Toxicol 20(Suppl 3):61–73. Natake M, Kanazawa K, Mizuno M, Ueno N, Kobayashi T, Danno G, Minamoto S. 1989. Herb-water extracts markedly suppress the mutagenicity of Trp-P-2. Ag Biol Chem 53:1423–1425. Pretlow TP, Bird RP. 2001. Correspondence re: Y. Yamada et al. Cancer Res 61:7699 –7701. Samejima K, Kanazawa K, Ashida H, Danno G. 1995. Luteolin: a strong antimutagen against dietary carcinogen, Trp-P-2, in peppermint, sage, and thyme. J Ag Food Chem 43:410 – 414. Samman MA, Bowen ID, Taiba K, Antonius J, Hannan MA. 1998. Mint prevents shamma-induced carcinogenesis in hamster cheek pouch. Carcinogenesis 19:1795–1801. Santana-Rios G, Orner GA, Amantana A, Provost C, Wu SY, Dashwood RH. 2001a. Potent antimutagenic activity of white tea in comparison with green tea in the Salmonella assay. Mutat Res 495:61–74. Santana-Rios G, Orner GA, Xu M, Izquierdo-Pulido M, Dashwood RH. 2001b. Inhibition by white tea of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced colonic aberrant crypts in the F344 rat. Nutr Cancer 41:98 –103. Satomi Y, Miyamoto S, Gould MN. 1999. Induction of AP-1 activity by perillyl alcohol in breast cancer cells. Carcinogenesis 20:1957– 1961. Sparber A, Ford D, Kvochak PA. 2004. National Institutes of Health’s Clinical Center sets new policy on use of herbal and other alternative supplements by patients enrolled in clinical trials. Cancer Invest 22:132–137. Spirling LI, Daniels IR 2001. Botanical perspectives on health peppermint: more than just an after-dinner mint. J R Soc Health 121:62– 63. Strandell J, Neil A, Carlin G. 2004. An approach to the in vitro evaluation of potential for cytochrome P450 enzyme inhibition from herbals and other natural remedies. Phytomedicine 11:98 –104.
This work was conducted in the Cancer Chemoprotection Program Core Laboratory. REFERENCES Burke MD, Thompson S, Elcombe CR, Halpert J, Haaparanta T, Mayer RT. 1985. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem Pharmacol 34:3337–3345. Calomme M, Pieters L, Vlietinck A, Vanden Berghe D. 1996. Inhibition of bacterial mutagenesis by citrus flavonoids. Planta Med 62:222–226. Dashwood RH. 2002. Modulation of heterocyclic amine-induced mutagenicity and carcinogenicity: an “A-to-Z” guide to chemopreventive agents, promoters, and transgenic models. Mutat Res 511:89 –112. Fabre A. 2003. Use of ancient texts in modern therapeutic research. Rev Hist Pharm (Paris) 51:239 –250. Hardigree AA, Epler JL. 1978. Comparative mutagenesis of plant flavonoids in microbial systems. Mutat Res 58:231–239. Huang SM, Hall SD, Watkins P, Love LA, Serabjit-Singh C, Betz JM, Hoffman FA, Honig P, Coates PM, Bull J, Chen ST, Kearns GL, Murray MD. 2004. Drug interactions with herbal products and grapefruit juice: a conference report. Clin Pharmacol Ther 75:1–12. Israelsen LD. 1995. Phytomedicines: the greening of modern medicine. J Altern Complement Med 1:245–248. Lambert JD, Yang CS. 2003. Mechanisms of cancer prevention by tea constituents. J Nutr 133:3262S–3267S. Maron DM, Ames BN. 1983. Revised methods for the Salmonella mutagenicity test. Mutat Res 113:173–215. Morita K, Hara M, Kada T. 1978. Studies on natural desmutagens: screening for vegetable and fruit factors active in inactivation of mutagenic pyrolysis products from amino acids. Ag Biol Chem 42:1235–1238.
Antimutagenic Activity of Spearmint Sugimura T, Wakabayashi K, Nakagama H, Nagao M. 2004. Heterocyclic amines: mutagens/carcinogens produced during cooking of meat and fish. Cancer Sci 95:290 –299. Surh YJ, Ferguson LR. 2003. Dietary and medicinal antimutagens and anticarcinogens: molecular mechanisms and chemopreventive potential. Mutat Res 523–524:1– 8. Turesky RJ. 2004. The role of genetic polymorphisms in metabolism of carcinogenic heterocyclic aromatic amines. Curr Drug Metab 5:169 –180. Villasenor IM, Echegoyen DE, Angelada JS. 2002. A new antimutagen from Mentha cordifolia opiz. Mutat Res 515:141–146. Winn RL. 2003. Safety concerns of herbal medicines. Gen Dent 51: 10 –16.
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Xu M, Bailey AC, Hernaez JF, Taoka CR, Schut HA, Dashwood RH. 1996. Protection by green tea, black tea, and indole-3-carbinol against 2-amino-3-methylimidazo[4,5-f]quinoline-induced DNA adducts and colonic aberrant crypts in the F344 rat. Carcinogenesis 17: 1429 –1434. Yu Z, Xu M, Santana-Rios G, Shen R, Izquierdo-Pulido M, Williams DE, Dashwood RH. 2001. A comparison of whole wheat, refined wheat and wheat bran as inhibitors of heterocyclic amines in the Salmonella mutagenicity assay and in the rat colonic aberrant crypt focus assay. Food Chem Toxicol 39:655– 665. Zheng J, Wu LJ, Zheng L, Wu B, Song AH. 2003. Two new monoterpenoid glycosides from Mentha spicata L. J Asian Nat Prod Res 5:69 –73.
