Genotoxic and antimutagenic activities of extracts from pseudocereals in the Salmonella mutagenicity assay

Food and Chemical Toxicology 48 (2010) 1483–1487

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Genotoxic and antimutagenic activities of extracts from pseudocereals in the Salmonella mutagenicity assay S. Mošovská a,*, M. Mikulášová b, L. Brindzová a, L´. Valík a, L. Mikušová a a b

Department of Nutrition and Food Evaluation, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic Institute of Cell Biology, Comenius University, Mlynská Dolina, 842 15 Bratislava, Slovak Republic

a r t i c l e

i n f o

Article history: Received 8 October 2009 Accepted 11 March 2010

Keywords: Pseudocereals Phenolic compounds Genotoxicity Antimutagenicity

a b s t r a c t Extracts of amaranth (Amaranthus L.), sorghum (Sorghum bicolor L.) and Japanese millet (Echinochloa frumentacea L.) were evaluated for mutagenicity in Salmonella typhimurium strains TA98, TA100 and TA102. All three pseudocereal extracts were also assessed for their antimutagenic properties against the direct mutagens 2-nitrofluorene (2NF) for strain TA98, 3-(5-nitro-2-furyl)acrylic acid (5NFAA) for TA100 and H2O2 for TA102 strain and against the indirect mutagen aflatoxin B1 (AFB1). No mutagenicity was induced by any of the pseudocereal extracts when tested at concentrations as high as 50 mg/ml. All three extracts showed similar antimutagenicity against 5NFAA and no antimutagenicity against 2NF. The number of revertants induced by H2O2 extract was inhibited in order amaranth > Japanese milet > sorghum. All extracts were effective in the inhibition of mutagenic activity of aflatoxin B1. The total polyphenol content as well as the amount of the flavonoids and phenolic acids as main component of polyphenolics were also determined. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The consumption of plant products is associated with a lowering risk of number of chronic diseases, including atherosclerosis and cancer. These beneficial effects have been partly attributed to antioxidants, which may play important roles in inhibition of free radicals and oxidative chain-reactions within tissues and membranes (Podsedek, 2007; Nsimba et al., 2008; Chatterjee et al., 2005). Phenolic compounds, including simple phenolics, flavonoids, phenolic acids, coumarins, tannins, stilbens, lignans or lignins, (Peyrat-Maillard et al., 2001; Amarowicz et al., 2004; Naczk and Shahidi, 2006) are secondary plant metabolites, and are an important part of both human and animal diets (Gülçin, 2006; Naczk and Shahidi, 2006). They protect plants against tissue injuries, high levels of oxygen, free radicals and reactive oxygen species formed by the photosynthesis (Peyrat-Maillard et al., 2001). They belong to antioxidants, which may act as both radical scavengers and metal chelators (Sikwese and Duodu, 2007). The antioxidant activity of these compounds is mainly due to their redox properties, which allow them to act as reducing agents or hydrogen-atom donors (Amarowicz et al., 2004). Some epidemiologic studies have also reported protective associations between phenolics and cardiovascular diseases risk factors and in animal and cell culture models of * Corresponding author. Tel.: +421 259325761. E-mail address: [email protected] (S. Mošovská). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.03.015

cancer (Kris-Etherton et al., 2002). They are found mainly in fruits, vegetables and legumes (Thériault et al., 2006; Cardador-Martínez et al., 2002). Besides of fruits and vegetables, the important sources of antioxidants are also pseudocereals. They are plants, which are grown as a crop to produce starchy grain suitable for human food, excluding pulses, oilseeds or nuts. The best-known representative pseudocereal seeds are amaranth (Amaranthus spp.), quinoa (Chenopodium quinoa) or buckwheat (Fagopyrum spp.) (Fletcher, 2004; Cai et al., 2004). Pseudocereals can grow and give higher and more stable grain yields in regions characterized by low rainfall or drought, high temperature and low soil fertility (Ragaee et al., 2006). As pseudocereals do not contain gluten, the causative agent for celiac disease, they are suitable in diet of people with allergy to typical cereals. They have high nutritional and functional values, are rich in proteins containing essential amino acids such as lysine, fats and antioxidant potential (Vogelmann et al., 2009; Pas´ko et al., 2009). Amaranth grain has also high content of arginine and histidine, important amino acids for child nutrition (Gimplinger et al., 2008). Amaranth oil contains significant levels of squalene, an important precursor for all steroids (Barba de la Rosa et al., 2009). Starch, the main component of amaranth grain, is important in its food applications. It is used as fat replacers, gravies sauces, in breakfast cereals, cookies, health food and in cosmetics too (Kong et al., 2009). Sorghum grain has higher protein content than other cereals although its nutritional quality is lower (Salinas et al., 2006). In spite of it, sorghum is important source of

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phytochemicals, including polyphenols, plant sterols which have positive influence to human health (Kamath et al., 2004). Japanese millet, belonging to millet family, is the main crop in the region of India. It has higher content of dietary fiber than traditional Indian crop such as rice and wheat (Hegde et al., 2005). According to Watanabe (1999), ethanolic extract from this type of millet contains antioxidants such as serotonin, luteolin and tricin. The aim of the study was to investigate the mutagenic and antimutagenic effects of extracts from amaranth, sorghum, and Japanese millet, in the Salmonella/microsome assay. The amount of total phenols, flavonoids and phenolic acids, potential contributors to antimutagenic activities of used extracts was also determined.

2. Materials and methods 2.1. Bacterial strains Histidine-dependent strain of Salmonella typhimurium TA98, TA100 and TA102 were purchased from the Collection of Microorganisms, Masaryk University, Brno (Czech Republic).

2.2. Metabolic activation system Liver S9 homogenate was prepared from male Wistar rats induced by Delor 103 (500 mg/kg) 5 days before killing as described by Maron and Ames (1983).

2.3. Plant materials The amaranth (Amarathus L.) seeds, variety PI 604671, were obtained from the gene bank of the Research Institute of Plant Production, Pieštˇany (Slovakia). The sorghum seeds (Sorghum bicolor L.) variety Zsófia and Japanese millet (Echinochloa frumentacea L.) variety Udalaja were acquired from Plant Production Station in Uhrˇíneˇves (Czech Republic).

2.4. Preparation of extract Defatted flour (Kim et al., 2006) was hydrolyzed with 2 M NaOH for 4 h at 50 °C, then acidified with 6 M HCl to pH 2. The free phenolic acids were extracted with ethyl acetate at ratio 1:1 (v/v). Ethyl acetate extracts were evaporated to dryness in a rotary evaporator at temperature lower than 40 °C (Krygier et al., 1982). The residue was dissolved in methanol. Methanolic extracts was used for determination of phenolic acids. For Ames test, methanol was evaporated and then dissolved in DMSO to need concentration or in 96% ethanol for total phenolic and flavonoid content.

2.7. Determination of total phenolic compounds, flavonoid and phenolic acids The total polyphenol content of the ethanolic pseudocereal extracts was measured by Folin–Ciocalteu-reagent according to Yu et al. (2004). The concentration of total phenolics was expressed in mg of gallic acid equivalents (GAE) per 100 g dry weight of flour. The flavonoid content was determined in ethanolic pseudocereal extracts according to Kreft et al. (2002). The results were expressed as mg of rutin equivalents per 100 g dry weight of flour. The amount of phenolic acids (gallic, vanilic, caffeic, syringic, p-coumaric and ferulic acid) was performed in methanolic extracts by RP-HPLC on column Eclipse XDB-C18 4.6 mm ID  250 mm with DAD detector. Pumps, autosampler and detector were conducted by program CHEMSTATION. The mobile phase consisted of water solution of acetic acid (A) and acetonitrile (B). The flow rate was maintained at 1 ml/min and the injection volume was 10 ll. Detection of phenolic acids was carried out at 272 and 320 nm (ferulic acid). 2.8. Statistical analysis The statistical significances of all calculated values were determined by paired Student’s t-test (pt) and variance analysis ANOVA (F-test) (pA). The values represent the means ± standard deviation (SD) of three separate experiments with triplicate plates/dose/experiment. The regression analysis was carried out using Microsoft Excel 2000. Mean values and standard errors of determination of total phenolic compounds, flavonoids and phenolic acids were calculated from the data obtained from two experiments.

3. Results In the plate incorporation assay with S. typhimurium TA98, TA100 and TA102 strains without metabolic activation no significant increase in the number of His+ revertant colonies was observed in the range of tested concentrations (from 3.125 to 50 mg/ml) of tested extracts (Table 1). The pseudocereal extracts were tested for their antimutagenic activity against 2-nitrofluoren (2NF) induced mutagenicity in the tester strain TA98, against 3-(5-nitro-2-furyl)acrylic acid (5NFAA) in TA100 strain and against H2O2 in TA 102 strain, while activity Table 1 Genotoxic activity of pseudocereal extracts in bacterial strains Salmonella typhimurium TA98, TA100 and TA102 by the Ames test. The data are a mean of three independent experiment each done in triplicates. Sample

2.6. Assessment of cytotoxicity In order to examine the cytotoxicity of extracts or of the combination mutagenextract against the tested strains, 0.1 ml of the bacterial suspension was diluted 10 5 fold with phosphate buffer and 0.1 ml of extract or 0.1 ml of extract and 0.1 ml of reference mutagen was added to 2 ml of molten top agar. Then the mixed solution was poured onto agar plate with complete medium. After incubation at 37 °C for 3 days, the number of surviving cells was counted.

TA98 Ma

TA100 ±SDb

Ma

TA102 ±SDb

Ma

±SDb

Amaranth

0 3.12 6.25 12.5 25 50

25 27 25 28 26 23

2.52 3.00 1.53 4.36 2.00 2.08

126 114 118 129 148 133

16.65 6.86 19.66 16.29 19.65 19.74

296 196 333 228 320 226

15.10 23.12 28.84 24.06 16.70 25.13

Sorghum

0 3.12 6.25 12.5 25 50

25 24 22 26 23 22

2.52 3.00 3.61 4.00 3.61 2.65

126 179* 175* 152* 159* 111

1665 19.00 11.53 11.24 12.12 15.13

296 343* 277 276 242 274

15.10 22.30 11.37 11.68 27.50 15.18

Japanese millet

0 3.12 6.25 12.5 25 50 PC

25 22 24 23 23 22 421***

2.52 6.11 4.58 3.61 5.86 2.00 27.5

126 176* 179* 190** 123 123 3284***

16.65 17.78 16.82 14.29 5.20 5.69 39.5

296 213 138 227 283 278 962***

15.10 28.05 24.04 33.13 10.07 41.63 18.7

2.5. Assay for mutagenic and antimutagenic effect of extracts The plate-incorporation mutagenicity test was performed as described Maron and Ames (1983) using tester strains S. typhimurium TA98, TA100 and TA102. To 2 ml of melted top agar containing 50 lM of L-histidine–biotin, 0.1 ml of a cell suspension (cultivation for 16 h overnight, approximate cell density 2–5  108 cells/ ml), 0.1 ml of DMSO extract of pseudocereals, for assay antimutagenic effect 0.1 ml positive mutagens and in the case of metabolic activation 0.5 ml the mixture of S9 were added. The mixture was poured onto a minimal glucose agar plate and incubated for 48 h at 37 °C and then the number of histidine-independent revertants was counted. The results represent the mean of three separate experiments, each run in triplicate. Antimutagenicity was expressed as percentage of inhibition of mutagenicity following the formula %Inhibition = 100 [(X1/X2)100] where X1 performances the number of revertants/plate in the presence of extract and X2 is the number of revertants/plate in the absence of extract. According to Negi et al. (2003) the antimutagenic effect was considered weak or absent (inhibition up to 25%), moderate (25–40% inhibition) or strong (inhibition higher than 40%).

Dose (mg/ml)

PC-positive control: 3-(5-nitro-2-furyl)acrylic acid (5 lg/ml for each strain). pA < 0.001 (statistical significance for individual samples compared to those of the control, pt-Student´s t-test, pA-ANOVA). a Mean number of revertants/plate (M). b Standard error (SD). * pt < 0.05. ** pt < 0.01. *** pt < 0.001.

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against aflatoxin B1 induced mutagenicity was tested in all three tested strains (Fig. 1). Suitable concentrations of positive mutagens for the three strains tested were chosen from the linear part of concentration–response curve: 5.0 lg/plate of 2NF, 5.0 lg/plate of 5NFAA, 1.87 mg/plate of H2O2 and 10.0 lg/plate of AFB1. In order to discriminate cytotoxicity from true antimutagenesis we have always investigated the frequency of revertants per number of surviving cells and the effect of dose on cell viability (that was found to be above 90%) was taken into account. Extracts from amaranth, sorghum and Japanese millet displayed a moderate but no significant antimutagenic effect against 2NF and strong protective effect (37–57%) against 5NFAA at all concentrations tested with no clear dose–response effect. Amaranth displayed marked dose–response effect against H2O2 at TA 102 strain only in higher concentrations and sorghum displayed typical, but weaker protective dose–response effect in all concentrations. Whereas sorghum inhibited number of revertants induced by H2O2 about 29–51%, amaranth at the highest tested concentration inhibited at 72%. Extract from Japanese millet showed a significantly stronger protective effect against H2O2 at all concentrations tested, the percent inhibition of mutagenicity was ranged from 56% to 68% (P < 0.001). An antimutagenic effect against AFB1 was noticed with all three extracts in all strains and was found to be concentration dependent (with the exception of sorghum in strain TA100). Linear relationship between extract dose and antimutagenic response in the case of amaranth is strong in the strain TA98 (r2 = 0.914), followed by

Table 2 Comparison of total phenolics and flavonoids in pseudocereal extracts. Sample

Total phenolics (mg GAE/100 g dry sample)

Flavonoids (mg Rutin/100 g dry sample)

Amaranth Sorghum Japanese millet

104.08 ± 2.297 86.07 ± 0.773 132.32 ± 0.013

37.43 ± 0.210 52.76 ± 2.029 22.05 ± 0.192

Results are means ± SD (n = 2); P < 0.05.

TA98, 2NF (-S9) % mutagenic activity

120 % mutagenic activity

TA100 (r2 = 0.786) and TA102 (r2 = 0.891). The linear regression analysis between sorghum extract dose and antimutagenic activity showed strong correlation in TA98 (r2 = 0.810) and TA102 (r2 = 0.918), but not in TA100 and a similar relationship was recorded for Japanese millet TA98 (r2 = 0.925), TA100 (r2 = 0.263), TA102 (r2 = 0.877). The variations in the antimutagenic activity between amaranth, sorghum and Japanese millet might be due to the differences in their active constituents. Therefore their total phenolic and flavonoids content was estimated (Table 2). The total phenolic content in descending was Japanese millet > amaranth > sorghum. On the other hand, flavonoid content decreased as follows: sorghum > amaranth > Japanese millet. Tested extracts also contained different amount of phenolic acids. As shown in Table 3, ferulic acid is majority phenolic acid in the all tested extracts, order was Japanese

100 80 60 40 20

TA98, AFB1 (+S9)

120 100 80 60 40 20

0

0 0

3.125

6.25

12.5

25

50

0

3.125

concentration (mg/ml)

TA100, 5NFAA (-S9)

100 80 60 40 20

25

50

TA100, AFB1 (+S9)

120 100 80 60 40 20 0

0 0

3.125

6.25

12.5

25

0

50

3.125

120

6.25

12.5

25

50

concentration (mg/ml)

concentration (mg/ml)

TA102, H2O2 (-S9)

TA102, AFB1 (+S9)

120

% mutagenic activity

% mutagenic activity

12.5

concentration (mg/ml)

% mutagenic activity

% mutagenic activity

120

6.25

100 80 60 40 20 0

100 80 60 40 20 0

0

3.125

6.25

12.5

25

50

0

3.125

concentration (mg/ml)

amaranth

japanese millet

6.25

12.5

25

50

concentration (mg/ml)

sorghum

Fig. 1. Antimutagenic effect of amaranth, sorghum and Japanese millet extracts against mutagens 2NF with TA98, 5NAFAA with TA100, H2O2 with TA102 ( S9) and promutagen aflatoxin B1 (+S9).

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Table 3 Content of phenolic acids in amaranth, sorghum and Japanese millet extracts. Sample

Amaranth Sorghum Japanese millet

Phenolic acids (mg/100 g dry sample) Gallic

Vanilic

Caffeic

Syringic

p-coumaric

Ferulic

0.55 ± 0.065 0.37 ± 0.016 4.89 ± 0.059

0.33 ± 0020 1.04 ± 0.041 1.15 ± 0.025

nd 7.07 ± 0.133 3.03 ± 0.058

0.49 ± 0.028 0.29 ± 0.027 0.64 ± 0.032

0.27 ± 0.002 3.81 ± 0.182 33.83 ± 0.081

0.56 ± 0.054 20.78 ± 0.275 35.78 ± 0.522

Results are means ± SD (n = 2); P < 0.01.

millet > sorghum > amaranth. According to Awika and Rooney (2004), Dykes and Rooney (2006), ferulic acid is the major bound phenolic acid in sorghum. Japanese millet extract also contained high amount of p-coumaric acid. On the other hand this phenolic acid was found at lowest content in extract from amaranth. 4. Discussion There are many publications dealing with favorable properties of plant polyphenolic compounds. In our previous paper (Brindzová et al. 2009) we observed antimutagenic effect of extracts from oat, buckwheat and wheat bran. In the present paper we used the S. typhimurium assay to examine potential mutagenic and antimutagenic effect of pseudocereals amaranth, sorghum and Japanese millet. It is known, that not all polyphenols as well as not all actions of individual polyphenols are necessarily beneficial. Some of them have mutagenic and/or pro-oxidant effect, and they may interfere with essential biochemical pathways (Ferguson, 2001). Therefore before initiating experiments to evaluate the antimutagenic activity of extracts, we verified their potential mutagenic effect and we found that extracts from amaranth, sorghum and Japanese millet did not exhibit mutagenic activity in any of Salmonella strains used. We revealed marked inhibitory effect of these extracts on the number of revertants induced by selected mutagens. The inhibition of mutagenesis is often complex, acting through multiple mechanisms (Edenharder and Grünhage, 2003). The significant antimutagenic activity of three extracts against direct acting 5NFAA suggests that these extracts may directly protect DNA damage from mutagen, and activity against H2O2 confirmed their antioxidant potential. The highest protection exhibited by the extracts against promutagen AFB1 induced mutagenesis indicate, that besides of direct interaction between phenolic compounds from extracts and AFB1, these compounds may also affect the metabolic activation of AFB1. This toxin is biotransformed in the liver by cytochrome P 450 system (Nakai et al., 2008) and converted into its toxic metabolite exo-8,9-epoxide (Gomes-Carneiro et al., 2006; Nakai et al., 2008). Covalent binding of this metabolite to DNA results in the formation of adducts and mutations (Nakai et al., 2008; Wu et al., 2007). Edenharder and Grünhage (2003) reported that antimutagenesis of flavonoids and structurally related compounds may be dependent or independent of concentration. Our results confirmed concentration dependent antimutagenic activity in the case of H2O2 and AFB1 (with the exception of sorghum in TA100) and concentration independent effect on mutagenesis induced by 2NF and 5NFAA. Slightly variations in the antimutagenic activity in three tested plant extracts might be due to the difference in the active constituents and their combinations in the extracts. There are many papers describing positive antimutagenic and antioxidant effects of polyphenolic compounds, phenolic acids and flavonoids present in many types of plants. As may be seen in Table 2, the highest phenolic content was determined in Japanese millet extract, which contained the lowest

amount of flavonoids. On the contrary, the extract from sorghum had the highest content of flavonoids which made more than 50% of total phenolics content. According to Awika and Rooney (2004), phenolic acids of sorghum and other cereals are mostly concentrated in the bran and exist mostly in bound forms with ferulic acid, being the most abundant bound phenolic acid in these foodstuffs. According to these authors, the bound phenolic acids are an important source of antioxidant activity in white sorghum varieties and most other cereals which normally have very low levels of flavonoids. As shown in Table 3, ferulic acid is the major phenolic acid in tested pseudocereals. Coumaric acid was also predominate phenolic acid in extract from Japanese millet. Our results are similar to published work by Dykes and Rooney (2006) whereby ferulic, p-coumaric and cinnamic acids are the major PA in millets. Although extracts of pseudocereals have high content of polyphenolic compounds, as we stated in the paper, at this moment we cannot say, which are the effective compounds responsible for their antimutagenic ability. 5. Conclusion Data presented in this paper indicated that extracts from amaranth, sorghum and Japanese millet are good source of polyphenolic compounds and have potential antimutagenic effect on directacting mutagens, NFAA and H2O2 and on indirect-acting mutagen, AFB1. These obtained results suggest, that pseudocereals not only provide an alternative crop for people with celiac disease, but also has potential for exploration of application as antimutagenic and antioxidants agents. Conflict of interest None declared. Acknowledgments This work was supported by the Slovak Grant Agencies Vega (Grant No. 1/0845/08) APVV (Grant No. 0310-06) and AV (Grant No. 4/0013/07). The authors thank to VURV Pieštˇany for providing the amaranth seeds and plant production station in Uhrˇíneˇves for obtaining the sorghum and Japanese millet seeds for this research project. References Amarowicz, R., Pegg, R.B., Rahimi-Moghaddam, P., Barl, B., Weil, J.A., 2004. Freeradical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem. 84, 551–562. Awika, J.M., Rooney, L.W., 2004. Sorghum phytochemicals and their potential impact on human health. Phytochemistry 65, 1195–1221. Barba de la Rosa, A.P., Fomsgaard, I.S., Laursen, B., Mortensen, A.G., Olvera-Martínez, L., Silva-Sánchez, C., Mendoza-Herrera, A., González-Castañeda, J., De LeónRodríguez, A., 2009. Amaranth (Amaranthus hypochondriacus) as an alternative crop for sustainable food production: phenolic acids and flavonoids with potential impact on its nutraceutical quality. J. Cereal Sci. 49, 117–121. Brindzová, L., Mikulášová, M., Takácsová, M., Mošovská, S., Opattová, A., 2009. Evaluation of the mutagenicity and antimutagenicity of extracts from oat,

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