Umu test applied for screening natural antimutagenic agents

Food Chemistry 124 (2011) 1699–1707

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Analytical Methods

Umu test applied for screening natural antimutagenic agents Stéphane Caillet, Stéphan Lessard, Gilles Lamoureux, Monique Lacroix * Canadian Irradiation Center (CIC), 531 Blvd. des Prairies, Laval, Québec, Canada H7V 1B7 Research Laboratory in Sciences Applied to Food, INRS-Institut Armand-Frappier, Université du Québec, 531 Blvd. des Prairies, Laval, Québec, Canada H7V 1B7

a r t i c l e

i n f o

Article history: Received 25 March 2010 Received in revised form 27 May 2010 Accepted 26 July 2010

Keywords: Umu test Antimutagenic activity Food additives Food supplements Herbs

a b s t r a c t Antimutagenic activities of pure commercial chemicals and food supplements were compared with that of herb extracts using a method based on the umu test system for screening natural antimutagens. Of the 55 products investigated, 22 were commercial products, 12 were food supplements and 21 were aqueous and ethylic herb infusions. All commercial products showed antimtagenic properties. BHT was shown to be a very strong antimutagen. All food supplements showed medium or neutral antimutagenic properties, except for echinaforce and coenzyme-Q10 which behaved as mutagenic products. All herbs showed antimutagenic properties except Italian parsley that had mutagenic activity. With regard to the metabolites, those from commercial products showed antimutagenic properties, except those from BHA and especially Biochanin A that showed high mutagenic activity. All metabolites from food supplements showed antimutagenic properties, except those from Fluxarola that showed antimutagenic properties. Metabolites from most herb extracts showed antimtagenic properties and those from thyme showed very strong antimutagenic activities, while those from camomile, rosemary and tarragon showed mutagenic activities, and those from celeriac and sage showed very strong mutagenic activities. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction Mutagenic and carcinogenic agents are omnipresent in the human environment, and it seems impossible to eliminate all of them. Moreover, several well-known mutagenic risk factors are closely connected with a modern lifestyle, and their entire eradication appears to be very burdensome, even unattainable. Therefore, there exists a need to reduce genotoxic effects of mutagenic and carcinogenic factors by the regular intake of antimutagenic agents (Hertog, Hollman, Katan, & Kromhout, 1993). Human epidemiology and animal studies have indicated that cancer risk may be modified by changes in dietary habits or dietary components (Trichopoulou & Vasilopoulou, 2000). Humans ingest large numbers of naturally occurring antimutagens and anticarcinogens like the phytochemicals, in our food. These antimutagens and anticarcinogens may inhibit one or more stages of the carcinogenic process and prevent or delay the formation of cancer. Thus, studies on antimutagens and anticarcinogens in food are important to research in the physiological functionality of food. Recently, it has been realised that several natural occurring compounds have potent anticarcinogenic or antimutagenic activity against environmental carcinogens and mutagens (Verschaeve & Van Staden,

* Corresponding author at: Research Laboratory in Sciences Applied to Food, INRS-Institut Armand-Frappier, Université du Québec, 531 Blvd. des Prairies, Laval, Québec, Canada H7V 1B7. Tel.: +1 450 687 5010x4489; fax: +1 450 687 5792. E-mail address: [email protected] (M. Lacroix).

2008). Through techniques for detecting possible environmental carcinogens and mutagens (Ames, McCann, & Yamasaki, 1975), it has been shown that ordinary diets contain many kinds of mutagens and antimutagens. Kada (1981) has studied the antimutagenic activity of foodstuffs using microbial mutation assay systems. The umu test system was developed to evaluate the genotoxic activities of a wide variety of environmental carcinogens and mutagens, using the expression of the SOS genes to detect DNAdamaging agents in Salmonella Typhimurium (Oda, Nakamura, Oki, Kato, & Shinagawa, 1985). This strain, S. Typhimurium TA1535/pSK1002, carries the plasmid pSK1002 in which the umuC’ gene is fused inframe to the lacZ’ gene. The SOS regulatory system is controlled in part by the interplay of 2 proteins, the lexA protein, which represses a set of unlinked genes during normal cell growth, and the recA protein, which is required in vivo for inactivation of lexA protein after treatments that derepress the system by DNA damaging (Little & Mount, 1982). Natural occurring compounds, such as culinary herbs and traditional medicinal plants form a potential pool for the screening of new anti-tumour compounds. Such screening may include the screening for inhibitors of mutagenesis. Screening culinary herbs and traditional medicinal plants that have been used for ages against several illnesses, including cancer, and that proved to be efficient in curing people may be one way to narrow the search (Van Wyk, Van Oudtshoorn, & Gericke, 1997). It is also important to note that most of the culinary herbs and traditional medicinal plants have never been the subject of exhaustive toxicological tests such as is required for modern pharmaceutical

0308-8146/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.082

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compounds. Based on their traditional use for long periods of time they are often assumed to be safe. However, research has shown that a lot of plants which are used as food ingredients or in traditional medicine have in vitro mutagenic (Deciga-Campos et al., 2007; Mohd-Fuat, Kofi, & Allan, 2007) or toxic and carcinogenic (De Sa Ferreira & Ferrão Vargas, 1999) properties. Mutagenic and antimutagenic compounds have been found in several plants, and some of these structures have been elucidated (Kim, Kim, & Lee, 1991; Zheng, Kenney, & Lam, 1992). For this reason it is also important to screen culinary herbs and medicinal plants for their mutagenic potency. Plants exhibiting clear mutagenic properties should be considered as potentially unsafe and certainly require further testing before their continued use can be recommended. Plants with obvious antimutagenic potential can, on the other hand, be considered interesting for therapeutic use and merit further in depth investigations of their pharmacological properties. In a previous study, we evaluated the antioxidant properties of pure commercial products and of herb extracts (Caillet et al., 2007). Now in our search for new naturally occurring antimutagenic plants, we compared the same herb extracts and commercial compounds. Twenty-two pure commercial products, 12 naturally food supplements and 20 herb extracts were tested for their antimutagenic properties.

Table 1 List of pure commercial product, food supplements and herbs studied. Pure commercial products

Chemical family

Cadmium chloride Hydroquinone Rutin Phytic acid Glutathione N-acetyl-L-cysteine Dithiothreitol Phenidone Catechol Epicatechin Morin BHA BHT Gossypol Indol-3-acetonitrile Vitamin A Vitamin C Vitamin E Ajmalicine Biochanin A Sempervirine Bathophenanthroline

– Phenoid Flavonoid Sugar phosphate Aminothiol Aminothiol Thiol Phenylamine derivative Flavonoid Flavonoid Flavonoid Phenol derivative Phenol derivative Naphthyl derivative Indol Retinoid Ascorbic acid Tocopherol Alkaloid Isoflavone Alkaloid Aza-phenanthrene

Food supplements (trade name)

Main plant extracts or chemical compound

2. Materials and methods

Fluxarola

2.1. Tested products

Chaparral Echinaforce Red Clover Citrus bioflavonoid Garlic powder Coenzyme-Q10 Gotu Kola Lapacho Pycnogenol Red Reishi Spirulina

Corylus avellana, Cupressus sempervirens, Vitis vinifera, Aesculus hippocastanum Larrea divaricata, L. Mexicana, L. tridentate Echinacea angustifolia, E. pallida, E. purpurea Trifolium pratense Flavonoid Allium sativum L. Benzoquinone Centella asiatica Tabebuia impetiginosa Proanthocyanidins Ganoderma Lucidum Aphanizomenon flos-aquae, Sprirulina maxima, Spirulina platensis

Fifty-five pure chemical products, food supplements and herbs were used in our study (Table 1). Among twenty-two pure chemical products, BHA, BHT, biochanin A, bathophenanthroline, catechol, gossypol, hydroquinone, indole-3-acetonitrile, morin, phytic acid, phenidone, rutin, vitamins A, C and E, glutathione, cadmium chloride, N-acetyl-L-cysteine and dithiothreitol were purchased from Sigma (St. Louis, MO, USA); ajmalicine, epicatechin and sempervirine were purchased from Indofine (Somerville, NJ, USA). Twelve food supplements were purchased from Optima (Montréal, QC, Canada) while twenty-one herbs were purchased from a local farmers market (Jean–Talon Market, Montreal, QC, Canada). 2.2. Pure commercial product preparation The hydrophilic products (namely hydroquinone, phytic acid, rutin, glutathione, N-acetyl-L-cysteine, vitamin C, Cadmium chloride and dithiothreitol) were dissolved in distilled water at the concentration of 3 mg/ml while the lipophilic products (i.e., 13 other products) were dissolved in chloromethane at the same concentration. These solutions were used for the determination of antimutagenic activity after suitable dilution with the same solvents. 2.3. Food supplements preparation Fluxarola was dissolved in distilled water at a concentration of 3 mg/ml while the other products (i.e., 11) were dissolved in chloromethane at the same concentration. These solutions were used for the determination of antimutagenic activity after suitable dilution with the same solvents. 2.4. Herb extraction Herbs were dried at room temperature and extracted through infusion with distilled water or ethanol at a concentration of 1 g of herb/10 ml. We compared extraction efficacies: an extraction of short duration (1 h for water and 4 h for ethanol) vs. an extrac-

Herbs (Common Name)

Botanical name

Anise Basil Camomile Chives Celeriac Common rue Coriander Garlic Hibiscus Italian parsley Marjoram Mint Parsley Rosemary Sage Savory Tarragon Thyme Vervain Mild oregano, Strong oregano

Anethum graveolens L. Ocimum basilicum L. Chamaemelum nobile L. Allium schoenoprasum L. Vallisneria alternifolia Ruta graveolens L. Coriandrum sativum L. Allium sativum Hibiscus abelmoschus L. Anthriscus cerefolium L. Origanum marjorana L. Mentha officinalis L. Petroselinum crispum Rosemarinus officinalis L. Salvia officinalis L. Satureja hortensis Artemisia dracunculus L. Thymus vulgaris L. Verbena officinalis L. Origanum vulgare L. sp.

tion of long duration (24 h for both water and ethanol). A 24 h period should allow extraction of compounds not easily extractable and, in particular, more phenolic compounds than when using an extraction of short duration. In the case of extraction of short duration, it was necessary to carry out an extraction for 4 h in ethanol, whereas an extraction of 1 h was sufficient, in water, to obtain similar quantities of extracts, sufficient for all experiments. The mixture was homogenised for 5 min under nitrogen (Sorvall Omni-Mixer, Mandel Scientific Co. Ltd., Guelph, ON, Canada) and

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macerated, according to the herb, during 1 or 24 h in water, and 4 or 24 h in ethanol, in order to obtain the highest yield of extraction for each herb (Caillet et al., 2007). The infusion was then filtered under vacuum, and the liquid was placed in a tube sealed with a screw-cap under nitrogen, and kept at 20 °C until used. For the determination of antimutagenic activity, complete evaporation of water or ethanol extracts was done using the SpeedVac Automatic Evaporation system (Savant System, Holbrook, NY, USA). Each dried extract was weighed, then redissolved in distilled water or ethanol, to obtain known concentrations. 2.5. Determination of antimutagenic activity The antimutagenicity of herb extracts and commercial products was investigated against potassium dichromate (antimutagenicity per se) and quercetin metabolites in the presence of S9 fraction using a modified umu test. The antimutagenicity per se and of the metabolites were tested on commercial products (pure chemical products and food supplements) at 19.5, 78.1 and 313 lg/ml, respectively (Caillet et al., 2007), and herbs extract at 210, 840 and 3360 lg/ml, respectively. Bacterial strain: Salmonella Typhimurium strain TA1535/pSK1002 containing the fusion gene umuC’–‘lacZ that produces a hybrid protein with b-galactosidase activity and whose expression is controlled by the umu regulatory region (Oda et al., 1985) was purchased from DSMZ (Braunschweig, Germany). Chemicals: Magnesium chloride (MgCl2) and potassium chloride (KCl) were purchased from Fisher Scientific (Nepean, ON, Canada). Sodium dodecyl sulfate (SDS), mercaptoethanol, potassium dichromate, quercetin, the enzyme substrate o-nitrophenyl-b-D-galactopyranoside (ONPG), glucose-6-phosphate and b-nicotinamide adenine (b-NAD) were purchased from Sigma–Aldrich (Oakville, ON, Canada). Sodium phosphate (NaH2PO4) and disodium phosphate (Na2HPO4) were purchased from Laboratoires Mat (Beauport, QC, Canada). S9 fraction prepared from livers of male Wistar rats pretreated with Aroclor 1254 was purchased from Invitro Technologies (Baltimore, MD, USA). Activation mixture: The activation mixture containing 1 ml of S9 fraction, 10 ml of sterile phosphate buffer (0.2 M, pH 7.4), 0.4 ml of 0.4 M MgCl2 + 1.65 M KCl sterile solution, 7.7 ml of sterile distillated water, 0.1 ml of 1 M glucose-6-phosphate sterile and 0.8 ml of 1 M sterile b-NAD was prepared. This solution must be kept at 4 °C and the S9 fraction and NAD are added last. Umu test: TGA medium containing 1% Bacto tryptone, 0.5% NaCl, 0.2% glucose and 20 lg/ml ampicilin was inoculated with 1 ml of the tester strain Salmonella Typhimurium TA1535/pSK1002 and was incubated at 37 °C under moderate agitation during 16 h. The culture was then diluted 10 times with TGA medium and incubated for 2 h or until the bacterial density reached OD600 of 0.25– 0.3 at 37 °C, resulting in log-phase cells. One ml of the log-phase culture was further added to a test tube containing the test mixture or 1 ml of TGA medium for the control. Two hundred and eight microliter of phosphate buffer (0.1 M) for the antimutagenicity per se or 208 ll of the S9 mixture was added for the antimutagenicity evaluation of the tested product metabolites. Forty-two microliter of tested product preparations at three final concentrations (19.5, 78.1 or 313 lg/ml for commercial products and 210, 840 or 3360 lg/ml for herbs extract) or the solvent (water or ethanol for herbs extract and water or chloromethane for commercial products) in which was dissolved the compound (control) was added to each tube. Finally, 42 ll of potassium dichromate (336 lg/ml) or quercetin (313 lg/ml) in the presence of the S9 fraction was added to the test tube (Thériault, Caillet, Kermasha, & Lacroix, 2006). The metabolite compounds of quercetin in the presence of the S9 fraction or the potassium dichromate per se are mutagenic and are used as control. The test mixture and control in test tubes

were incubated for 2 h at 37 °C under moderate agitation. At the end of incubation, the cell density from each tube was measured at 600 nm using a spectrophotometer (Unicam model UV4, Cambridge, UK). The b-galactosidase activity was also assayed according to others (Whong, Wen, Stewart, & Ong, 1986). In order to evaluate the b-galactosidase activity, 50 ll of treated cells and 100 ll of O-nitrophenyl-b-D-galactopyranoside (ONPG) solution (4 mg/ml in 0.1 M phosphate buffer, pH 7.0) were added to a test tube containing 450 ll of B buffer prepared according to others (Whong et al., 1986) with 16.1 g of Na2HPO4, 5.5 g of NaH2PO4, 0.75 g of KCl, 0.25 g of MgSO4–7H2O, 1 g of SDS, 2.7 ml of b-mercaptoethanol and 1 l of distilled water at pH 7. Finally, the tubes were incubated at 28 °C during 25 min. The enzymatic reaction was stopped by adding 400 ll of 1 M sodium carbonate (Na2CO3). The OD420 nm and OD550 nm were determined with a spectrophotometer (Unicam, UV4 model, Cambridge, UK). Inhibition of SOS response or antimutagenicity was calculated as follows:

Antimutagenicity ð%Þ ¼ ðb-gal unit control-b-gal unit sample= b-gal unit controlÞ  100 b-galactosidase activity was presented as units according to the following formula: Unit = 1000  (OD420 nm–1.75 OD550 nm)/(T  V  OD600 nm) where T represented the time of reaction (min) and V the volume of cells (ml). Enzyme units with a one time dose response were considered as positive results (Miller, 1972). The following scale already used for the antioxidant activity percentages (Laughton, Evans, Moroney, Hoult, & Halliwell, 1991) was proposed for the antimutagenic activity percentages: a product having an antimutagenic activity above 70% was considered as a strong antimutagen; an antimutagen with an activity between 40% and 70% was considered as a medium antimutagen; an antimutagen with an activity less than 40% was considered as a neutral compound; a negative result indicated that the compound was mutagenic. 2.6. Statistical analysis This experiment was done in replicate and three samples of each replicate were analysed. Data were analysed using SPSS for Windows. Analyses of variance were performed by ANOVA procedures. Significant differences between means were determined by Duncan’s multiple range test (p 6 0.05). 3. Results and discussion Results of the antimutagenic properties per se of commercial products at three concentrations (19.5, 78.1, 313 lg/ml) and antimutagenic strength at 313 lg/ml are presented in Table 2. The b-galactosidase units, represent the specific enzymatic activity of the b-galactosidase produced by Salmonella Typhimurium TA1535/pSK1002 due to induction of the SOS response in the presence of mutagen agents (McDaniels, Reyes, Wymer, Rankin, & Stelma, 1990). The SOS-inducing potency of test compounds is estimated by the measurement of the induction of the level of umu operon in terms of intracellular b-galactosidase activity (Ong, Stewart, Wen, & Whong, 1987). The percentage of antimutagenicity is calculated to illustrate the inhibition of the SOS response. The results showed that the antimutagenic activity of most commercial products increased with the concentration. Among hydrophylic compounds, rutin (at three concentrations) and cadmium chloride were strong antimutagens with activities above 70%, while other hydrophylic compounds were neutral as antimutagens with activities below 40%. With regard to the lipophilic compounds, BHT was shown to be very strong antimutagen

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Table 2 Antimutagenicity per se of pure commercial products. Products

Solubility

a

b-galactosidase Units (mean ± SD)b at

Antimutagenicity (%, mean ± SD)c at

19.5 lg/ml

78.1 lg/ml

313 lg/ml

19.5 lg/ml

78.1 lg/ml

313 lg/ml

Antimutagenic strength at 313 lg/ ml

Control Cadmium chloride Dithiothreitol Glutathione Hydroquinone N-acetyl-L-cysteine Phytic acid Rutin Vitamin C

Hydrophylic

120.90 ± 4.81 37.60 ± 2.93 86.08 ± 4.35 87.92 ± 4.25 112.52 ± 4.52 103.48 ± 4.24 115.32 ± 4.88 3.96 ± 2.62 119.92 ± 3.33

120.90 ± 4.81 27.36 ± 2.30 79.60 ± 5.30 70.61 ± 3.19 113.23 ± 4.30 99.99 ± 3.85 115.32 ± 5.39 4.11 ± 2.49 109.10 ± 5.23

120.90 ± 4.81 25.80 ± 2.12 75.12 ± 4.44 76.08 ± 3.70 96.04 ± 3.82 101.15 ± 5.21 114.96 ± 4.36 4.90 ± 2.71 105.41 ± 5.08

N/A 68.66 ± 3.34Ae 28.26 ± 2.61Ad 27.27 ± 1.31Ad 6.93 ± 1.81Ab 14.40 ± 2.86Ac 3.90 ± 1.23Aab 96.72 ± 4.14Af 0.8 ± 1.02Aa

N/A 77.20 ± 3.48Be 33.66 ± 3.24ABc 41.59 ± 2.51Bd 6.34 ± 1.40Aa 17.29 ± 1.01Ab 4.10 ± 2.20Aa 96.60 ± 4.51Af 9.76 ± 2.55Ba

N/A 78.50 ± 3.45Be 37.40 ± 2.21Bd 36.60 ± 2.78Bd 19.92 ± 2.41Bc 16.33 ± 2.84Abc 4.20 ± 1.20Aa 95.94 ± 4.42Af 12.81 ± 2.33Bb

N/A Strong Neutral Neutral Neutral Neutral Neutral Strong Neutral

Control Ajmalicine Bathophenanthroline Biochanin A BHA BHT Catechol Epicatechin Gossypol Indol-3-acetonitrile Morin Phenidone Sempervirine Vitamin A Vitamin E

Lipophilic

128.28 ± 5.52 86.47 ± 4.36 132.95 ± 4.51 96.90 ± 4.18 59.06 ± 4.25 107.04 ± 4.06 95.28 ± 3.83 6.69 ± 2.61 50.50 ± 3.62 58.54 ± 2.29 16.39 ± 2.69 132.62 ± 5.64 33.01 ± 2.36 41.81 ± 2.69 8.22 ± 1.75

128.28 ± 5.52 70.70 ± 4.12 120.82 ± 4.69 105.97 ± 3.01 65.71 ± 4.59 112.16 ± 4.09 71.32 ± 4.32 4.97 ± 2.50 52.75 ± 2.99 59.57 ± 3.73 21.71 ± 2.82 126.09 ± 5.41 22.11 ± 2.56 28.74 ± 2.30 4.86 ± 1.41

128.28 ± 5.52 66.64 ± 3.56 126.89 ± 4.03 119.11 ± 3.28 184.72 ± 6.17 179.46 ± 6.96 52.14 ± 4.20 4.77 ± 2.46 58.20 ± 3.21 53.60 ± 2.54 54.81 ± 3.87 108.01 ± 4.63 7.61 ± 1.52 41.10 ± 2.87 4.77 ± 1.35

N/A 32.56 ± 2.01Ad 3.64 ± 2.26Aa 24.42 ± 2.25Cc 53.96 ± 2.88Be 16.55 ± 1.09Ab 25.72 ± 2.57Ac 94.78 ± 3.64Aj 60.62 ± 3.34Af 54.28 ± 2.12Ae 87.21 ± 3.56Bi 3.38 ± 3.22Aa 74.26 ± 2.30Ah 67.40 ± 2.33Ag 93.59 ± 2.53Aj

N/A 44.88 ± 2.61Bd 5.8 ± 2.42Ba 17.39 ± 1.98Bc 48.77 ± 2.40Bd 12.56 ± 1.91Ab 44.40 ± 2.62Bd 96.12 ± 3.47Ah 58.87 ± 3.22Ae 53.55 ± 2.81Ae 83.07 ± 2.87Bg 1.70 ± 2.11Aa 82.76 ± 2.83Bg 77.59 ± 2.20Bf 103.78 ± 3.75Bi

N/A 48.05 ± 2.56Be 1.08 ± 2.07ABb 7.52 ± 2.16Ac 44.06 ± 2.39Aa 239.90 ± 11.97Bj 59.35 ± 3.78Cf 96.28 ± 3.28Ah 54.63 ± 3.01Af 58.21 ± 2.75Af 57.27 ± 3.04Af 15.80 ± 2.11Bd 105.94 ± 4.23 Ci 67.96 ± 2.74Ag 103.72 ± 3.21Bi

N/A Medium Neutral Neutral Mutagenic Very strong Medium Strong Medium Medium Medium Neutral Strong Medium Strong

a

Control is the mixture containing only the mutagen agent and the cells. b-galactosidase units represents the enzymatic specific activity. Percentage of antimutagenicity is the percentage of inhibition of the SOS response. Means in the same row bearing the same uppercase letter are not significantly different (P > 0.05). Means within a column for each solubility group that have the same lowercase letter are not significantly different (P > 0.05). b

c

with activity at almost 240%. Sempervirine, vitamin E, epicatechin were shown to be strong antimutagens. Vitamin A, catechol, indole-3-acetonitrile, morin, gossypol and ajmalicine showed medium antimutagenic properties. Phenidone, biochanin A and bathophenanthroline were considered neutral, while BHA behaved as mutagenic product. Results of the antimutagenic properties per se of food supplements at three concentrations (19.5, 78.1, 313 lg/ml) and antimutagenic strength at 313 lg/ml are presented in Table 3. The antimutagenic activity of most food supplements did not seem to be affected by the concentration compared with the antimutagenic

activity of most commercial products and herb extracts. The only hydrophilic compound, Fluxarola was considered neutral. With regard to the lipophilic compounds, citrus bioflavonoid, lapacho and chaparral showed medium antimutagenic properties. The other compounds were considered neutral, except echinaforce and Coenzyme-Q10 showing mutagenic activity. Table 4 presents the antimutagenic properties per se of herb extracts at three concentrations (210, 840, 3360 lg/ml) and antimutagenic strength at 3360 lg/ml. The antimutagenic activity of most herb extracts increased with the concentration. In the case of extraction in water, the extracts from tarragon and basil showed

Table 3 Antimutagenicity per se of food supplements.

a

Products

Solubility

b-galactosidase units (mean ± SD)b at

Antimutagenicity (%, mean ± SD)c at

19.5 lg/ml

78.1 lg/ml

313 lg/ml

19.5 lg/ml

78.1 lg/ml

313 lg/ml

Controla Fluxarola

Hydrophylic

119.85 ± 4.23 115.35 ± 4.15

119.85 ± 4.23 93.78 ± 3.29

119.85 ± 4.23 95.88 ± 3.96

N/A 3.75 ± 2.24A

N/A 21.75 ± 2.08A

N/A 20.00 ± 2.16A

N/A Neutral

Control Chaparral Echinaforce Red Clover Citrus 60.10 ± 3.89 Garlic powder CoenzymeQ10 Gotu Kola Lapacho Pycnogenol Red Reishi Spirulina

Lipophilic

127.83 ± 5.35 69.99 ± 2.85 143.36 ± 5.63 93.60 ± 3.45

127.83 ± 5.35 70.82 ± 2.98 175.91 ± 6.10 95.71 ± 3.78

127.83 ± 5.35 71.78 ± 2.92 169.55 ± 5.18 85.50 ± 3.38

N/A 44.59 ± 2.67Ag 37.61 ± 2.56Aa 25.12 ± 1.01Ae

86.51 ± 3.82 121.87 ± 4.13

104.40 ± 4.68 133.21 ± 4.19

115.81 ± 4.71 131.61 ± 3.80

N/A 45.24 ± 2.11Afg 12.14 ± 1.04Ba 26.77 ± 1.00Ad bioflavonoid 53.69 ± 2.67Ah 32.32 ± 1.43Ce 4.66 ± 1.43Bb

54.14 ± 2.33Ah 18.32 ± 0.89Bd 4.20 ± 1.32Ab

N/A 43.92 ± 2.66Afg 32.64 ± 2.52Aa 33.11 ± 2.46Be 59.19 ± 2.95 52.98 ± 3.05Ah 9.40 ± 0.84Ac 2.96 ± 1.90Ab

N/A Medium Mutagenic Neutral 58.62 ± 2.53 Medium Neutral Mutagenic

112.30 ± 4.23 68.32 ± 3.72 76.36 ± 3.79 111.53 ± 4.23 76.49 ± 3.95

108.70 ± 4.69 70.93 ± 3.54 79.89 ± 3.21 111.93 ± 4.52 79.25 ± 4.04

109.23 ± 4.52 69.57 ± 3.72 78.89 ± 3.33 123.30 ± 4.36 77.64 ± 3.59

12.22 ± 1.42Ac 46.55 ± 2.54Ag 40.26 ± 2.6Af 12.75 ± 0.50Bc 40.16 ± 2.07Af

14.96 ± 1.64Ac 44.51 ± 2.25Ag 37.50 ± 2.50Af 12.43 ± 0.55Bc 39.26 ± 2.83Af

14.55 ± 1.78Ad 45.57 ± 2.75Ag 38.28 ± 2.27Ae 5.54 ± 0.68Ac 39.26 ± 2.37Aef

Neutral Medium Neutral Neutral Neutral

Antimutagenic strength at 313 lg/ml

Control is the mixture containing only the mutagen agent and the cells. b-galactosidase Units represents the enzymatic specific activity. c Percentage of antimutagenicity is the percentage of inhibition of the SOS response. Means in the same row bearing the same uppercase letter are not significantly different (P > 0.05). Means within a column for each solubility group that have the same lowercase letter are not significantly different (P > 0.05). b

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Solvent

b-galactosidase units (mean ± SD)b at

Antimutagenicity (%, mean ± SD)c at

210 lg/ml

840 lg/ml

3360 lg/ml

210 lg/ml

840 lg/ml

3360 lg/ml

– 1 1 24 1 1 1 1 1

122.80 ± 4.98 99.80 ± 4.44 53.72 ± 3.09 90.75 ± 3.33 91.04 ± 3.25 108.82 ± 3.69 116.33 ± 4.17 150.91 ± 5.28 100.06 ± 4.53

122.80 ± 4.98 93.49 ± 4.02 57.43 ± 4.82 85.13 ± 3.61 91.44 ± 3.51 103.56 ± 4.13 102.31 ± 4.48 137.76 ± 4.69 89.43 ± 4.34 1

122.80 ± 4.98 88.19 ± 3.80 61.14 ± 3.27 79.40 ± 3.56 101.87 ± 4.19 98.70 ± 4.12 92.87 ± 4.28 95.48 ± 3.12 82.27 ± 3.37 127.42 ± 4.83

N/A 23.86 ± 2.58ABd 53.23 ± 3.12ABg 30.67 ± 3.30ABed 25.53 ± 2.09Bd 15.66 ± 2.31ABc 16.68 ± 2.09Bc 12.18 ± 2.06Ba 27.17 ± 2.88Bde 145.19 ± 5.87 Mutagenic 30.48 ± 2.57Be 75.24 ± 3.52 39.07 ± 2.25Bh 31.97 ± 2.66ABe 14.07 ± 2.38Bc 0.90 ± 1.38Bb 113.38 ± 6.68Ah 32.48 ± 2.78Ae

N/A 28.18 ± 2.73Bef 50.21 ± 2.68Ai 35.34 ± 2.58Bgh 17.60 ± 2.41Ac 19.62 ± 2.21Bcd 24.37 ± 2.38Cde 22.24 ± 2.19Cde 33.00 ± 3.15Bfg 3.76 ± 2.92Bc

N/A Neutral Medium Neutral Neutral Neutral Neutral Neutral Neutral 9.25 ± 2.88Ba

31.63 ± 2.60Bfg 74.85 ± 3.33 Neutral 26.18 ± 2.73Ae 10.92 ± 2.18Bb 8.15 ± 1.73Cb 113.78 ± 6.88Aj 39.36 ± 2.47Bh

Neutral 33.71 ± 2.40Ah Neutral Neutral Neutral Strong Neutral

Antimutagenic strength at 3360 lg/ ml

Marjoram Mild

24

97.49 ± 4.07 oregano

85.36 ± 3.41

83.95 ± 4.25 1

Parsley Rosemary Savory Tarragon Vervain

1 1 24 24 1

78.95 ± 3.21 119.71 ± 4.22 135.92 ± 3.91 14.75 ± 1.72 88.21 ± 3.76

83.54 ± 3.89 105.51 ± 4.19 121.68 ± 4.49 16.44 ± 1.56 82.90 ± 4.26

90.65 ± 3.02 109.39 ± 4.13 112.79 ± 4.07 16.92 ± 1.87 74.46 ± 3.69

N/A 18.72 ± 2.62Af 56.25 ± 2.23Bi 26.09 ± 2.24Ag 25.86 ± 2.50Bg 11.38 ± 2.96Ae 5.26 ± 2.62Ad 22.89 ± 2.25Aa 18.51 ± 1.22Af 134.16 ± 5.28 18.24 ± 1.98Aa 20.60 ± 2.07Af 81.40 ± 3.04 38.72 ± 2.37Bf 35.70 ± 2.90Bh 2.51 ± 2.77Acd 10.68 ± 2.73Ab 112.01 ± 6.59Aj 28.16 ± 2.51Ag

– 24

117.72 ± 4.35 36.72 ± 2.35

117.72 ± 4.35 47.71 ± 2.61

117.72 ± 4.35 60.34 ± 3.87

N/A 68.80 ± 3.07Cd

N/A 59.46 ± 2.12Bc

N/A 48.74 ± 3.12Ac

N/A Medium

4 4

51.02 ± 2.88 20.61 ± 2.66 oregano

47.61 ± 2.87 19.68 ± 2.17

34.46 ± 1.22 16.06 ± 2.01 4

120.10 ± 6.64

115.87 ± 5.39

88.76 ± 4.29

59.55 ± 2.71Ac 83.28 ± 2.04Ad 55.56 ± 3.19 34.93 ± 2.34Ab 1.57 ± 2.89Aa

70.72 ± 2.50Bd 86.35 ± 4.43Ae 76.60 ± 4.14 Neutral 24.60 ± 1.74Ba

Strong Strong 50.67 ± 2.30Bb

4

56.65 ± 2.63Ac 82.48 ± 2.23Ae 58.07 ± 3.53 52.80 ± 2.87Bb 2.02 ± 2.67Aa

Control Anise Basil Camomile Chives Celeriac Coriander Garlic Hibiscus Italian

Control Common rue Mint Sage Strong Thyme

Water

Extraction time (h)

parsley

Ethanol

Neutral

a

Control is the mixture containing only the mutagen agent and the cells. b-galactosidase units represents the enzymatic specific activity. c Percentage of antimutagenicity is the percentage of inhibition of the SOS response. Means in the same row bearing the same uppercase letter are not significantly different (P > 0.05). Means within a column for each solubility group that have the same lowercase letter are not significantly different (P > 0.05). b

strong and medium antimutagenic properties, respectively. The other aqueous extracts were neutral with respect to antimutagenic activity, except Italian parsley that had mutagenic activity. Among the ethylic extracts, sage and mint were strong antimutagens, while common rue showed a medium antimutagenic activity. Strong oregano and thyme were considered neutral. Results of the antimutagenic properties of metabolites from commercial products at three concentrations (19.5, 78.1, 313 lg/ ml) and antimutagenic strength at 313 lg/ml are presented in Table 5. The relationship between the concentrations of metabolites from commercial products and the observed antimutagenic or mutagenic activity was less obvious as compared with the activity per se of the same compounds. Indeed, the antimutagenic activity of metabolites from several compounds did not seem to be affected by the concentration. Among hydrophylic compounds, metabolites from cadmium chloride were strong antimutagens, while those from hydroquinone rutin and glutathione showed medium antimutagenic properties. The metabolites from other compounds were considered neutral. In the case of lipophilic compounds, metabolites from morin and gossypol were shown to be strong antimutagens, while metabolites from catechol, ajmalicine, indole-3-acetonitrile, epicatechin and vitamin E showed medium antimutagenic properties. The metabolites from other compounds were considered neutral, while those from BHA behaved as mutagenic products and those from biochanin A showed high mutagenic activity. Results of the antimutagenic properties of metabolites from food supplements at three concentrations (19.5, 78.1, 313 lg/ml) and antimutagenic strength at 313 lg/ml are presented in Table 6. The antimutagenic activity of metabolites from most food supplements seems to be affected by the concentration as in antimutagenic activity per se of the same compounds. Metabolites from

Fluxarola showed mutagenic properties. Among lipophilic products, metabolites from pycnogenol, garlic powder, lapacho, coenzyme-Q10, red clover and chaparral were strong antimutagens while metabolites from citrus bioflavonoid and spirula showed medium antimutagenic properties. Metabolites from other products were considered neutral. Table 7 presents the antimutagenic properties of metabolites from herb extracts at three concentrations (210, 840, 3360 lg/ ml) and antimutagenic strength at 3360 lg/ml. The antimutagenic activity of metabolites from most herb extracts increased with the concentration as in antimutagenic activity per se of the same compounds. In the case of extraction in water, metabolites from parsley, basil, chives, mild oregano and garlic showed medium antimutagenic properties. Metabolites from Tarragon, camomile and rosemary behaved as mutagenic products while those from celeriac were shown to be very strong mutagen with activity of 380%. The metabolites from other compounds were considered neutral. In the case of extraction in ethanol, metabolites from thyme showed very strong antimutagenic activity while those of oregano were strong antimutagens. Metabolites from mint and common rue showed medium antimutagenic properties while those from sage showed very strong mutagenic activity. Among 55 products investigated, nine yielded strong antimutagenic activity and eleven products showed medium antimutagenic properties. These products were chosen from the literature as having antioxidant or other biological properties beneficial to cells susceptible to carcinogenic transformation (Appel, Roverts, & Woutersen, 1991; Laughton et al., 1991; Stahelin et al., 1991; Zheng & Wang, 2001). Pure commercial compounds and food supplements allowed comparison of their antimutagenic properties within the same family of chemical compounds (Tables 1, 2 and 4). However, there

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S. Caillet et al. / Food Chemistry 124 (2011) 1699–1707

Table 5 Antimutagenicity of metabolites from pure commercial products. Products

Solubility

a

b-galactosidase units (mean ± SD)b at

Antimutagenicity (%, mean ± SD)c at

19.5 lg/ml

78.1 lg/ml

313 lg/ml

19.5 lg/ml

78.1 lg/ml

313 lg/ml

Antimutagenic strength at 313 lg/ml

Control Cadmium chloride Dithiothreitol Glutathione Hydroquinone N-acetyl-L-cysteine Phytic acid Rutin Vitamin C

Hydrophylic

124.19 ± 4.78 41.62 ± 2.85 66.61 ± 3.51 53.81 ± 2.89 44.56 ± 2.16 121.48 ± 5.02 98.68 ± 4.64 66.29 ± 3.36 78.64 ± 4.12

124.19 ± 4.78 16.83 ± 1.07 78.56 ± 4.24 53.48 ± 2.79 46.28 ± 2.08 125.23 ± 5.44 93.89 ± 4.20 68.40 ± 3.47 85.68 ± 4.52

124.19 ± 4.78 17.75 ± 0.96 83.15 ± 5.29 65.47 ± 3.58 45.52 ± 1.89 123.68 ± 5.69 93.58 ± 4.38 65.34 ± 3.28 96.21 ± 4.66

N/A 65.68 ± 2.91Af 46.36 ± 2.46Bd 56.67 ± 2.50Be 64.09 ± 3.14Af 2.18 ± 1.72Aa 20.54 ± 1.27Ab 46.62 ± 2.34Ad 36.67 ± 1.90Cc

N/A 86.44 ± 3.87Bh 36.74 ± 2.19Ad 56.93 ± 2.47Bf 62.73 ± 2.79Ag 0.83 ± 1.03Aa 24.39 ± 1.75Bb 44.92 ± 2.29Ae 31.00 ± 1.64Bc

N/A 85.70 ± 3.26Bf 33.04 ± 2.12Ac 47.28 ± 2.59Ad 63.34 ± 2.65Ae 0.41 ± 1.05Aa 24.64 ± 1.85Bb 47.38 ± 2.95Ad 22.53 ± 1.56Ab

N/A Strong Neutral Medium Medium Neutral Neutral Medium Neutral

Control Ajmalicine Bathophenanthroline Biochanin A BHA BHT Catechol Epicatechin Gossypol Indol-3-acetonitrile Morin Phenidone Sempervirine Vitamin A Vitamin E

Lipophilic

130.22 ± 5.69 59.40 ± 3.31 95.09 ± 4.07 226.03 ± 11.23 48.50 ± 2.57 87.89 ± 4.01 65.96 ± 3.81 72.39 ± 3.21 36.34 ± 1.63 76.22 ± 3.48 63.00 ± 2.74 128.07 ± 5.88 115.23 ± 4.69 165.24 ± 7.20 65.67 ± 3.85

130.22 ± 5.69 62.88 ± 3.49 109.27 ± 4.75 270.14 ± 13.02 57.93 ± 2.44 92.18 ± 3.85 68.08 ± 3.62 74.45 ± 3.41 37.29 ± 1.85 73.98 ± 3.56 48.44 ± 1.89 125.56 ± 5.67 105.27 ± 4.36 161.22 ± 6.86 80.48 ± 4.25

130.22 ± 5.69 66.86 ± 3.67 121.36 ± 5.27 278.97 ± 12.38 132.99 ± 4.96 88.43 ± 3.82 66.07 ± 3.84 71.08 ± 3.92 35.18 ± 1.71 75.90 ± 3.37 2.08 ± 0.83 104.09 ± 4.89 89.42 ± 3.65 118.05 ± 4.20 77.78 ± 4.30

N/A 54.38 ± 3.02Ai 26.97 ± 1.36Ce 73.57 ± 3.62Ba 62.74 ± 3.35Cj 32.50 ± 1.87Af 49.34 ± 2.84Ahi 44.41 ± 1.98Agh 72.09 ± 3.26Ak 41.46 ± 2.27Ag 148.38 ± 6.43Bl 1.65 ± 0.75Ac 11.51 ± 1.14Ad 26.89 ± 2.15Ab 49.56 ± 2.96Bhi

N/A 51.71 ± 2.86Aij 16.08 ± 1.39Bd 107.44 ± 5.15Aa 55.51 ± 2.33Bj 29.21 ± 1.51Ae 47.71 ± 2.54Ahi 42.82 ± 2.00Ag 71.36 ± 3.55Ak 43.18 ± 2.48Agh 137.20 ± 5.39Bl 3.57 ± 1.61Ac 19.16 ± 1.45Bd 23.80 ± 1.72Ab 38.19 ± 2.01Af

N/A 48.65 ± 2.63Ah 6.80 ± 0.98Ac 114.23 ± 5.06Aa 2.13 ± 1.00Ab 32.09 ± 1.88Ae 49.26 ± 2.75Ah 45.41 ± 2.68Agh 72.98 ± 3.57Ai 41.71 ± 2.36Afg 98.40 ± 3.95Aj 20.06 ± 1.48Bd 31.33 ± 2.10Ce 9.34 ± 1.77Bc 40.27 ± 2.24Af

N/A Medium Neutral Mutagenic Mutagenic Neutral Medium Medium Strong Medium Strong Neutral Neutral Neutral Medium

a

Control is the mixture containing only the mutagen agent and the cells. b-galactosidase units represents the enzymatic specific activity. Percentage of antimutagenicity is the percentage of inhibition of the SOS response. Means in the same row bearing the same uppercase letter are not significantly different (P > 0.05). Means within a column for each solubility group that have the same lowercase letter are not significantly different (P > 0.05). b

c

Table 6 Antimutagenicity of metabolites from food supplements. Products

Solubility

b-galactosidase units (mean ± SD)b at

Antimutagenicity (%, mean ± SD)c at

19.5 lg/ml

78.1 lg/ml

313 lg/ml

19.5 lg/ml

78.1 lg/ml

313 lg/ml

Antimutagenic strength at 313 lg/ml

Controla Fluxarola

Hydrophylic

117.26 ± 4.18 164.09 ± 6.80

117.26 ± 4.18 166.81 ± 6.32

117.26 ± 4.18 147.67 ± 5.75

N/A 39.93 ± 2.23B

N/A 42.25 ± 2.05B

N/A 25.94 ± 1.21A

N/A Mutagenic

Control Chaparral Echinaforce Red Clover Citrus 54.03 ± 2.95 Garlic powder CoenzymeQ10 Gotu Kola Lapacho Pycnogenol Red Reishi Spirulina

Lipophilic

123.84 ± 5.49 37.15 ± 2.07 81.38 ± 3.86 31.21 ± 1.26

123.84 ± 5.49 35.87 ± 1.86 89.25 ± 4.36 31.47 ± 1.44

123.84 ± 5.49 35.23 ± 1.74 90.42 ± 4.63 32.17 ± 1.59

N/A 70.97 ± 3.68Ad 27.93 ± 1.37Aa 74.58 ± 3.41Ad

0.58 ± 1.22 33.16 ± 1.69

1.26 ± 1.17 31.81 ± 1.35

0.94 ± 0.83 32.03 ± 1.54

N/A 70.00 ± 3.91Ae 34.28 ± 1.62Bb 74.79 ± 3.02Ae bioflavonoid 50.81 ± 3.16Ad 100.46 ± 4.88Af 73.22 ± 3.73Ae

55.45 ± 2.88Ac 98.98 ± 4.84Af 74.31 ± 3.22Ad

N/A 71.55 ± 3.52Ad 26.98 ± 2.28Aa 74.02 ± 3.66Ade 60.91 ± 3.79 56.37 ± 2.81Ac 99.24 ± 4.61Af 74.13 ± 3.57Ade

N/A Strong Neutral Strong 55.17 ± 2.79 Medium Strong Strong

74.87 ± 4.05 27.04 ± 2.26 36.97 ± 1.89 107.43 ± 5.96 62.98 ± 3.11

76.52 ± 4.37 26.81 ± 2.19 5.47 ± 1.17 79.70 ± 4.65 59.23 ± 2.87

77.79 ± 4.66 23.91 ± 1.23 14.57 ± 1.72 78.32 ± 4.54 54.34 ± 2.64

39.54 ± 2.13Ac 78.16 ± 5.52Ae 70.14 ± 3.38Ae 13.25 ± 1.15Aa 49.85 ± 2.45Ad

38.21 ± 2.18Ab 78.35 ± 5.35Ad 95.58 ± 4.53Be 35.64 ± 2.07Bb 52.17 ± 2.51Ac

37.18 ± 2.72Ab 80.69 ± 4.12Ae 111.77 ± 5.38Cg 36.75 ± 2.56Bb 56.12 ± 2.74Bc

Neutral Strong Srong Neutral Medium

a

Control is the mixture containing only the mutagen agent and the cells. b-galactosidase Units represents the enzymatic specific activity. c Percentage of antimutagenicity is the percentage of inhibition of the SOS response. Means in the same row bearing the same uppercase letter are not significantly different (P > 0.05). Means within a column for each solubility group that have the same lowercase letter are not significantly different (P > 0.05). b

seems to be no significant correlation between the antimutagenic properties of compounds and their chemical family as has been noted for antioxidant properties (Caillet et al., 2007). For instance, the two commonly used synthetic food additives, BHA and BHT, showed different antimutagenic potential, although they are chemically similar (phenol derivative). Although BHA is very efficient in preventing autoxidation (Caillet et al., 2007), this compound behaved as a mutagenic product. In contrast, BHT was shown to be very strong antimutagen. BHA is currently used less and less in the food industry, with the profit of natural antioxidant

compounds. BHA and BHT have been suspected to cause or promote negative health effects (Namiki, 1990). All studied flavonoids (i.e., catechol, epicatechine, rutin, morin, and citrus bioflavonoid) showed strong or medium antimutagenic activities. Flavonoids are a group of natural benzo-c-pyran derivatives and occur as aglycones, glycosides and methylated derivatives. Flavonoids have been reported to have a wide variety of biological activities (Cody, Middleton, & Harborne, 1986). Certain flavonoids have been shown to have mutagenic and carcinogenic effects (Cody et al., 1986) whereas other flavonoids have been reported to exert inhibition

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S. Caillet et al. / Food Chemistry 124 (2011) 1699–1707 Table 7 Antimutagenicity of metabolites from herb extracts. Herb extracts a

Control Anise Basil Camomile Chives Celeriac Coriander Garlic Hibiscus Italian

Solvent

Water

Extraction time (h)

b-galactosidase units (mean ± SD)b at

Antimutagenicity (%, mean ± SD)c at

210 lg/ml

840 lg/ml

3360 lg/ml

210 lg/ml

840 lg/ml

3360 lg/ml

– 1 1 24 1 1 1 1 1

122.85 ± 4.98 118.59 ± 5.36 57.20 ± 5.63 173.49 ± 7.22 76.28 ± 3.35 550.41 ± 14.38 104.05 ± 4.81 96.42 ± 3.80 87.87 ± 3.56

122.85 ± 4.98 117.01 ± 5.30 61.15 ± 4.82 168.71 ± 7.68 73.39 ± 3.28 585.36 ± 14.96 89.62 ± 3.51 85.84 ± 3.45 89.54 ± 3.61 1

122.85 ± 4.98 110.12 ± 5.24 65.10 ± 4.21 172.04 ± 7.72 63.09 ± 2.78 589.44 ± 15.26 78.07 ± 3.26 70.59 ± 3.21 101.58 ± 4.19 90.93 ± 3.60

N/A

N/A

3.42 ± 0.15Af 53.42 ± 5.25Ak 41.22 ± 2.47Ac 37.90 ± 1.67Aj 348.03 ± 12.68Aa 15.30 ± 1.80Ag 21.51 ± 0.94Ah 28.47 ± 1.16Bi 90.20 ± 3.45

4.71 ± 0.21Bf 50.19 ± 3.95Ak 37.33 ± 2.48Ac 40.26 ± 1.78Aj 376.48 ± 11.62Ba 27.04 ± 2.05Bhi 30.11 ± 1.21Bi 27.11 ± 1.09Bhi 102.96 ± 4.39

N/A 10.32 ± 0.49Ce 46.98 ± 3.03Ajk 40.10 ± 2.56Ac 48.62 ± 1.68Bk 380.10 ± 12.84Ba 36.42 ± 2.18 Ci 42.51 ± 2.34Cj 17.28 ± 1.66Af 25.98 ± 1.14Bi

parsley

Antimutagenic strength at 3360 lg/ml N/A Neutral Medium Mutagenic Medium Very Mutagenic Neutral Medium Neutral 26.57 ± 1.01Bh

Marjoram Mild

24

116.23 ± 4.38 oregano

118.13 ± 4.72

94.11 ± 3.86 1

16.15 ± 1.02Af 5.38 ± 1.89Af 74.23 ± 3.64

Neutral 3.84 ± 1.60Af 64.12 ± 3.15

23.36 ± 2.15Bg 63.70 ± 3.04

48.12 ± 2.29Bk 69.71 ± 4.08Al 7.00 ± 2.61Ad 12.43 ± 1.01Bg 109.72 ± 5.22Ab 2.31 ± 0.88Be

Medium 65.17 ± 3.52Al 1.41 ± 1.40Bd 30.39 ± 2.85Ch 78.44 ± 4.88Bb 10.76 ± 0.95Ce

Medium Mutagenic Neutral Mutagenic Neutral

Neutral 39.57 ± 1.94Aj

Parsley Rosemary Savory Tarragon Vervain Control Common rue Mint Sage Strong

Ethanol

1 1 24 24 1

37.77 ± 2.35 130.32 ± 4.26 132.12 ± 7.06 250.97 ± 11.56 137.45 ± 4.55

37.21 ± 2.19 131.45 ± 4.88 107.57 ± 4.29 257.65 ± 12.31 125.69 ± 4.36

42.77 ± 2.63 124.61 ± 4.79 85.51 ± 3.65 219.12 ± 10.21 109.58 ± 4.32

47.80 ± 2.36Bk 69.25 ± 4.39Al 6.08 ± 1.90Ae 7.54 ± 1.62Ae 104.37 ± 4.78Ab 11.88 ± 1.08Ad

– 24

120.32 ± 4.22 76.10 ± 3.39

120.32 ± 4.22 78.67 ± 3.23

120.32 ± 4.22 51.31 ± 3.62

N/A 36.75 ± 2.50Ab

N/A 34.61 ± 2.08Ab

N/A 57.35 ± 2.86Bb

N/A Medium

4 4

55.59 ± 2.38 320.24 ± 11.52 oregano

53.87 ± 2.43 308.88 ± 10.86

37.41 ± 2.27 284.83 ± 9.59 4

53.79 ± 2.46Ac 166.15 ± 5.97Ba 37.46 ± 2.53

55.22 ± 2.49Ac 156.71 ± 5.50Ba 33.80 ± 2.22

68.90 ± 3.08Bc 136.73 ± 5.89Aa 31.06 ± 1.86

Medium Very Mutagenic

49.01 ± 2.78

71.90 ± 3.67Ad 140.13 ± 6.70Ae

74.18 ± 3.51Ac 137.74 ± 6.76Ae

Strong 140.74 ± 6.56Ad

68.86 ± 3.65Ad Thyme

4

48.29 ± 2.71

45.42 ± 2.54

Very Strong

a

Control is the mixture containing only the mutagen agent and the cells. b-galactosidase units represents the enzymatic specific activity. Percentage of antimutagenicity is the percentage of inhibition of the SOS response. Means in the same row bearing the same uppercase letter are not significantly different (P > 0.05). Means within a column for each solubility group that have the same lowercase letter are not significantly different (P > 0.05). b

c

of tumour promotion (Wattenberg, 1983). Other research has confirmed that several plant flavonoids inhibit the mutagenicity induced by chemical mutagens (Choi, Park, Moon, Rhee, & Young, 1994; Wall et al., 1988), but they are not necessarily able to prevent all kind of mutations that can be induced by mutagen agents (Laughton et al., 1991). It has been reported that the phenolic compounds and especially flavonoids can serve as screens against UV radiation in plants (Escarpa & Gonzalez, 2001). Flavonoids have potent suppressive effects of SOS-inducing activity by chemical mutagen and UV irradiation, and these compounds may be potent as bio-antimutagens (Miyazawa & Hisama, 2003a). Cadmium salt and tocopherol were also considered as strong antimutagens. Tocopherol, b-carotene and ascorbic acid promote inhibition of oxidation products of the cell and prevent the metabolism of oncogenic molecules that need to be metabolised to exert their mutagenic activity (Stahelin et al., 1991; Thomson, 1991; Trizna, Schantz, & Hsut, 1991). Studies showed that cadmium exposure caused DNA damage in rats (Latinwo, Ikediobi, Sponholtz, Fasanya, & Riley 1997) and cultured cell lines (Coogan, Bare, & Waalkes, 1992). The mutagenic effect of cadmium chloride is believed to be related to generation of free radicals, which may interact with DNA (Meliksetova, Vasil’eva, & Zasukhina, 1997). However, it was observed that cadmium alone did not cause DNA damage (Badisa et al., 2007). O’Riordan, Hughes, and Evans (1978) found no statistically significant increase in the number of chromosome aberrations in 40 men exposed to cadmium only and speculated that effects reported by other authors might be due to combined effects of cadmium and other metals. Cadmium may also reduce the incidence of liver and lung cancers induced by other carcinogenic compounds without causing testicular damages (Waalkes et al., 1991).

According to these authors, the cadmium action probably induces a general slowing of cell division. Unger and Clausen (1973) demonstrated that injected cadmium salt gave rise to a diminished P-450 activity in the liver of mice. This effect does not occur in long-term repeated exposure due to the protective effect of induced metallothionein (Schnell, Means, Roberts, & Pence, 1979) and, although an effect of cadmium on mixed-function oxygenase would be of interest in relation to the carcinogenicity of many organic compounds, the fact that it occurs only in acute exposures makes it of limited practical importance with regard to the situation in liver tissue (Nordberg & Andersen, 1981). All thiols (aminothiols and thiols) studied, which belong to the same family of chemicals showed a low antimutagenic potential, while Caillet et al. (2007) showed that the same compounds were pro-oxidant. However, De Flora, Izzotti, D’Agostini, and Cesarone (1991) reported that thiols such as glutathione, N-acetyl-L-cysteine and dithiothreitol possess anti-cancer potential activity. These compounds apparently reduce the damage caused to DNA by X-rays or 2-acetylaminofluorene. This suggests that some compounds may also contribute to the prevention of cancer through other mechanisms than their antioxidant properties and their ability to inhibit the SOS response. The SOS-inducing potency is thus one among several mechanisms that prevent cancer. Concentrations used to study the antimutagenic activity of herb extracts were approximately 10 times higher than those of commercial products. These high concentrations are justified because the filtrates collected from the herbs were crude extracts without purification, while commercial products were highly purified. Once purified, micronutrients with antimutagenic properties present in these extracts will likely be equal or superior to the positive

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controls when used at similar concentrations. Our study showed that, of the 21 tested herb extracts, tarragon, mint and sage were the most efficient. Many reports on antimutagens in natural products including medicinal plants have been published (Cherdshewasart, Sutjit, Pulcharoen, Panriansaen, & Chulasiri, 2008; Verschaeve & Van Staden, 2008), but few have reported antimutagenic properties of spices and culinary herbs (Lvova & Zasukhina, 2002; Miyazawa & Hisama, 2003b). The results of antimutagenic activity obtained for herb extracts are comparable with those presented in the literature (Samejima, Kanazawa, Ashida, & Danno, 1995) which indicate that mint, sage and thyme showed a strong antimutagenic effect against Trp-P-2-induced SOS response. Wong, Lau, Yamasaki, and Teel (1993) showed that several herbs inhibited mutagenesis, DNA binding and metabolism of benzo(a)pyrene (BaP) in Salmonella Typhimurium TA100. Also, these authors suggest that herb extracts inhibited the mutagenicity of BaP metabolites by inhibiting cytochrome P-450IA1 activity of hepatic S9. As in the case of antioxidant activity (Caillet et al., 2007), the antimutagenic activity of herbs and spices is caused mainly by phenylpropanoids and phenolic compounds, such as flavonoids, phenolic acids and phenolic monoterpenes (Miyazawa & Hisama, 2003b; Samejima et al., 1995; Zheng et al., 1992).The authors reported that luteolin, myristicin, dehydrodieugenol and trans-coniferyl aldehyde which is contained in mint, sage, thyme, parsley and clove showed a strong antimutagenic effect. Kaur and Saini (2000) have associated antimutagenicity with antioxidant properties, due to the capacity of the phenolic compounds to inhibit the DNA damage caused by the presence of free radicals. In fact, inhibition of mutagenesis is generally not based on one specific mechanism. Indeed, the herb extracts containing phenolic compounds were complex mixtures, meaning that different phenomenons such as synergy or co inhibition can interfere with the antimutagenic activity. Hence, compounds and complex mixtures with antimutagenic activity have different modes of action and act in parallel at different levels. As inhibitors, they may prevent the formation of mutagens. According to Krul et al. (2001), as blocking agents, they can prevent the biotransformation of premutagens into reactive metabolites by inhibiting metabolic activation or by scavenging reactive molecules. As suppressing agents they may modulate intracellular processes, which are involved in DNA repair mechanisms. The antimutagenic activity of the metabolites of products investigated showed an overall higher antimutagenic potential than the potential obtained for the antimutagenicity per se of the same compounds. This means that the compounds are getting their antimutagenic activity after being metabolised in the liver in the presence of S9 fraction. Among metabolites of 55 products investigated, eleven yielded strong antimutagenic activity and sixteen products showed medium antimutagenic properties. However, some commercial products, such as BHT, Biochanin A, sampervirine or fluarola lost their antimutagenic potency after being metabolised by S9. With regard to metabolites of herb extracts, five showed mutagenic activities. It is interesting to note that the sage showed a high antimutagenic potency, but behaved as a mutagenic product after being metabolised by S9. In contrast, some commercial products (coenzyme-Q10, pycnogenol, red clover, garlic powder) or herb (thyme and strong oregano) can be efficient in preventing mutations but they need to be metabolised by S9 to produce an antimutagenic activity. 4. Conclusion The discovery and exploration of phytochemical compounds with antimutagenic and anticarcinogenic potency is at the present time of great importance because of the undesirable consequences of an increased rate of mutations and the related possible risks of cancer in humans. Recently, the global demands for plant products

and their accompanying health benefits are increasing. These raw materials are composed of a vast variety of dietary supplements and food ingredients. The umu test system showed that the majority of products tested in this study possessed antimutagenic potency. However, a strong antimutagenic activity was measured for metabolites of compounds such as morin, gossypol, pycnogenol, cadmium chloride, garlic powder, lapacho, coenzyme-Q10, red clover chaparral and some herbs (strong oregano, thyme). The results from the present study provide evidence to support the safe consumption of products with clear antimutagenic properties at doses studied. Also, the rationale for screening for antimutagenicity was that antimutagenic compounds can possibly also be anticarcinogens. However, it should be kept in mind that some phytochemicals can contribute to the carcinogenic process without inducing mutations. Such chemicals can induce intracellular signalling, alter gap junctional intercellular communication and alter patterns of gene expression, for example by modifications of methylation and acetylation of DNA and histones. They may contribute to cancer by a ‘epigenetic mechanism’ rather than by mutation (Trosko & Upham, 2005). Hence, screening for antimutagenicity is certainly important but it does not cover all aspects of relevance in the cancer prevention. References Ames, B. N., McCann, J., & Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research, 31(6), 347–363. Appel, M. J., Roverts, G., & Woutersen, R. A. (1991). Inhibitory effects of micronutrients on pancreatic carcinogenesis in azaserine-treated rats. Carcinogenesis, 12, 2157–2161. Badisa, V. L. D., Latinwo, L. M., Odewumi, C. O., Ikediobi, C. O., Badisa, R. B., AyukTakem, L. T., et al. (2007). Mechanism of DNA damage by cadmium and interplay of antioxidant enzymes and agents. Environmental Toxicology, 22(2), 144–151. Caillet, S., Yu, H., Lessard, S., Lamoureux, G., Ajdukovic, D., & Lacroix, M. (2007). Fenton reaction applied for screening natural antioxidants. Food Chemistry, 100(1), 542–552. Cherdshewasart, W., Sutjit, W., Pulcharoen, K., Panriansaen, R., & Chulasiri, M. (2008). Antimutagenic potential of the Thai herb, Mucuna collettii Lace. Journal of Ethnopharmacology, 115, 96–103. Choi, J. S., Park, K. Y., Moon, S. H., Rhee, S. H., & Young, H. S. (1994). Antimutagenic effect of plant flavonoids in the Salmonella assay system. Archives of Pharmacal Research, 17, 71–75. Cody, E., Middleton Jr., & Harborne, J. B. (1986). Plant flavonoids in biology and medicine. New York: Allen R. Liss. Coogan, T. P., Bare, R. M., & Waalkes, M. P. (1992). Cadmium-induced DNA strand damage in cultured liver cells: Reduction cadmium genotoxicity following zinc pretreatment. Toxicology and Applied Pharmacology, 113(2), 227–233. Deciga-Campos, M., Rivero-Cruz, I., Arriaga-Alba, M., Castaneda-Corral, G., AngelesLopez, G. E., Navarrete, A., et al. (2007). Acute toxicity and mutagenic activity of Mexican plants used in traditional medicine. Journal of Ethnopharmacology, 110(2), 334–342. De Flora, S., Izzotti, A., D’Agostini, F., & Cesarone, C. F. (1991). Antioxidant activity and other mechanisms of thiols involved in chemoprevention of mutation and cancer. American Journal of Medicine, 91(3/3), S122–S130. De Sa Ferreira, I. C. F., & Ferrão Vargas, V. M. (1999). Mutagenicity of medicinal plant extracts in Salmonella/microsome assay. Phytotherapy Research, 13(5), 397–400. Escarpa, A., & Gonzalez, M. C. (2001). An overview of analytical chemistry of phenolic compounds in foods. Critical Reviews in Analytical Chemistry, 31(2), 57–139. Hertog, M. G. L., Hollman, P. C. H., Katan, M. B., & Kromhout, D. (1993). Intake of potentially anticarcinogenic flavonoids and their determinants in adults in the Netherlands. Nutrition and Cancer, 20(1), 21–29. Kada, T. (1981). Recent research on environment mutagens. Nippon Nogeikagaku Kaisi, 55, 597–605. Kaur, I. P., & Saini, A. (2000). Sesamol exhibits antimutagenic activity against oxygen species mediated mutagenicity. Mutation Research – Genetic Toxicology and Environmental Mutagenesis, 470(1), 71–76. Kim, S., Kim, J., & Lee, S. (1991). Antimutagenic compounds identified from the chloroform fraction of garlic (Allium sativum). Journal of the Korean Society of Food Science and Nutrition, 20(3), 253–259. Krul, C., Luiten-Schuite, A., Tenfelde, A., van Ommen, B., Verhagen, H., & Havenaar, R. (2001). Antimutagenic activity of green tea and black tea extracts studied in a dynamic in Vitro gastrointestinal model. Mutation Research – Fundamental and Molecular Mechanisms of Mutagenesis, 474(1–2), 71–85. Latinwo, L. M., Ikediobi, C. O., Sponholtz, G., Fasanya, C., & Riley, L. (1997). Comparative studies of in vivo genotoxic effects of cadmium on brain, kidney, and liver cells. Cellular and Molecular Biology, 43(2), 203–210.

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