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Antimutagenic components in Spatholobus suberectus Dunn against N-methyl-N-nitrosourea

Antimutagenic components in Spatholobus suberectus Dunn against N-methyl-N-nitrosourea Background: An extract from Spatholobus suberectus (S. suberectus) Dunn has been reported to show potent antimutagenic effects against N-alkyl-N-nitrosoureas in umu screening. The aim of this study was to identify the antimutagenic components from extracts of S. suberectus against N-methyl-N-nitrosourea (MNU) in the Ames assay with Salmonella typhimurium strain TA1535 and to elucidate the antimutagenic mechanism of the flavonoids. Results: From the ethyl acetate fraction obtained from fractionation of the methanol extract of S. suberectus Dunn, medicarpin, formononetin and isoliquiritigenin were successfully isolated through a combination of normal- and reversed-phase chromatography. Genistein and naringenin, which were already reported to be contained in S. suberectus Dunn, were also tested for their antimutagenicity towards MNU, along with formononetin, isoliquiritigenin and medicarpin. Our results demonstrated that genistein, isoliquiritigenin, medicarpin and naringenin were antimutagenic against MNU without showing cytotoxicity. MNU is reported to cause not only DNA alkylation but also induce reactive oxygen species. The hydroxyl radical scavenging capacity of the flavonoids was correlated with the antimutagenic capacity, indicating that the hydroxyl radical scavenging activity was involved in their antimutagenicity towards MNU. Conclusions: It is important to prevent DNA damage by N-nitrosamines for cancer chemoprevention. Genistein, isoliquiritigenin, medicarpin and naringenin were demonstrated to possess an antigenotoxic effects against carcinogenic MNU due to their radical scavenging activity. Keywords: Antimutagenicity, Formononetin, Genistein, Isoliquiritigenin, Medicarpin, Naringenin, Hydroxyl radical Introduction can prevent the mutagenicity and carcinogenicity of N- N-Nitroso compounds, are implicated as carcinogens in nitrosamines. the human environment such as food, tobacco smoke, N-Methyl-N-nitrosourea (MNU) is a direct-acting air, water and cosmetics [1–6]. In addition, N-nitroso mutagen that forms the corresponding methyldiazohydr- compounds have been reported to be formed endogen- oxides and methylates DNA to form DNA adducts that ously, mainly in the stomach and bowel, or in an infec- miscode during DNA replication, causing mutations that tion site [2]. The endogenous formation of N- lead to tumour formation. MNU is formed by the nitro- nitrosamines is a possible explanation for the epidemio- sation of methylurea with nitrite in guinea-pig stomachs, logical connection of gastrointestinal cancers [7, 8]. and MNU is absorbed from the stomach into the blood Therefore, for chemoprevention, it is important to dis- [9]. Additionally, MNU can be detected by the nitrosa- cover naturally occurring or synthetic compounds that tion of creatinine or fermented foods at gastric pH [10– 12]. Therefore, for cancer chemoprevention, it is import- ant to find some compounds that can inhibit the muta- genicity induced by MNU. * Correspondence: inami@rs.socu.ac.jp Faculty of Pharmaceutical Sciences, Sanyo-onoda City University, Spatholobus suberect (S. suberectus) Dunn (Legumino- Daigakudo-ri 1-1-1, Sanyo-onoda-shi, Yamaguchi 756-0884, Japan sae) is a traditional Chinese herbal medicine used to Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 treat rheumatism, anaemia, menoxenia, and other Noda-shi, Chiba 278-8510, Japan © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Inami et al. Genes and Environment (2019) 41:22 Page 2 of 9 disorders [13]. Pharmacologically, it exhibits anti- (Minnesota, USA). All reagents used were of the best inflammatory [14, 15] and antitumour activities [16, 17]. commercially available quality from Wako Pure Chem- Our previous study showed that an aqueous extract of S. ical Industries (Osaka, Japan) and were not further puri- suberectus Dunn showed the most potent antimutagenic fied unless otherwise noted. Medicarpin (mp. 194 °C) effects against MNU by umu test screening [18]. was synthesized according to the method of Goel et al The umu test is a convenient method because the data [23]. MNU [mp. 106 °C (decomp.) was prepared as de- are obtained in a short period of time; however, the scribed previously [24]. Purity of the synthesized medi- Ames test is a more precise method to evaluate the gen- carpin and MNU were determined by the H-NMR otoxic activities of a wide variety of environmental car- spectra, which all required integral values matches the cinogens and mutagens than the umu test [19, 20]. The number of protons without any other peak. Ames test using bacterial strains of Salmonella typhi- murium (S. typhimurium) is widely used by regulatory Preparation of the aqueous extract of S. suberectus Dunn agencies, academic institutions and pharmaceutical com- Dried S. suberectus Dunn was cut with a pair of scissors. panies to assess the mutagenic potential of compounds Distilled water (130 mL) was added to the cut S. suberec- [21, 22]. In the present study, we identified antimutagens tus Dunn (30 g) and refluxed for 30 min. The mixture against MNU using the Ames assay, and the inhibitory was filtered with suction, and the filtrate was concen- mechanism was investigated. trated under reduced pressure. The whole procedure was repeated twice. The crude extracts were combined, Materials and methods dried under vacuum, and finally gave a reddish solid General experimental procedures (5.30 g). The reaction progress was monitored using thin-layer chromatography (TLC) on silica gel 60 F (0.25 mm, Preparation of the methanol extract of S. suberectus Dunn Merck, Darmstadt, Germany). Column chromatography Dried S. suberectus Dunn was cut with a pair of scissors. was performed using silica gel 60 (0.04–0.063 mm, Methanol (130 mL) was added to the cut S. suberectus Merck). Melting points were determined using a Yanaco Dunn (30 g) and stirred for 30 min at room temperature. (Tokyo, Japan) micro-melting point apparatus without The mixture was filtered with suction, and the filtrate correction. The LC system used was equipped with an was concentrated under reduced pressure. The whole LC-6 AD pump (Shimadzu, Kyoto, Japan), a UVDEC- procedure was repeated twice. The crude extracts were 100 V spectrometric detector (JASCO, Tokyo, Japan), a combined, dried under vacuum, and finally a reddish YRD-880 IR detector (Shimamura Tech. Co. Ltd., Tokyo, solid (2.32 g) was obtained. Japan), or a SPD-20A (Shimadzu), and a Capcell pack RP-18 column (Shiseido, Tokyo, Japan). NMR spectra Fractionation of the methanol extract of S. suberectus were recorded with a JEOL JNM-LA400 spectrometer Dunn based on the solubility in organic solvents (Tokyo, Japan). The chemical shifts were expressed as Methanol (3.5 L) was added to the cut S. suberectus ppm downfield from TMS. The mass spectra were mea- Dunn (1 kg) and incubated overnight at room sured on a JEOL JMS-SX102A mass spectrometer. The temperature. The mixture was filtered with suction, and ESR spectra were collected on a JEOL JES-X320. the filtrate was concentrated under reduced pressure. The whole procedure was repeated 5 times. The crude Reagents extracts were combined, dried under vacuum, and finally Bacto agar and Bacto nutrient broth were obtained from a reddish solid (162 g) was obtained. Ethyl acetate (300 Becton Dickinson Microbiology Systems (Sparks, USA). mL) and water (300 mL) were added to the solid, and 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) and ethyl- the aqueous phase was separated. The aqueous phase enediamine-N,N,N′,N′-tetraacetic acid disodium salt was re-extracted with ethyl acetate (150 mL × 6), and the dihydrate (EDTA) were purchased from Dojindo Labora- combined organic phases were washed with water (100 tories (Kumamoto, Japan). Sodium ammonium hydrogen mL). The aqueous phase was extracted with water- phosphate tetrahydrate was obtained from Merck saturated n-butanol (150 mL × 6), and the combined or- (Darmstadt, Germany). S. suberectus Dunn was pur- ganic phases were washed with n-butanol-saturated chased from Matsumoto Yakugyo Co., Ltd. (Nagoya, water (100 mL). The ethyl acetate, n-butanol and aque- Japan). Genistein [mp. 304 °C (decomp.), recrystallized ous extracts were filtered, and the filtrates were removed from ethanol and H O] and isoliquiritigenin [mp. of solvent with a rotary evaporator, and the residue was 197 °C, recrystallized from ethanol and chloroform] were dried in vacuo. Finally, the ethyl acetate fraction (23.8 g), obtained from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, n-butanol fraction (48.0 g), and aqueous fraction (86.0 g) Japan). Formononetin (mp. 256 °C) and naringenin (mp. were obtained from the methanol extract of S. suberectus 251 °C) was purchased from LKT Laboratories, Inc. Dunn. The recovery of the weight was 97%. Inami et al. Genes and Environment (2019) 41:22 Page 3 of 9 Isolation of antimutagenic compounds from the ethyl 0.1 M sodium phosphate buffer (pH 7.4, 0.5 mL), each acetate fraction solution of plant extract (50 μL), and a culture of S. From the ethyl acetate fraction obtained from the typhimurium TA1535 (0.1 mL). A portion of the mixture methanol extract of S. suberectus Dunn, medicarpin, for- was diluted 10 -fold times in 1/15 M PB. The diluted so- mononetin and isoliquiritigenin were successfully iso- lution (200 μL) was supplemented with histidine-free top lated through a combination of normal and reversed agar (2 mL) and poured on a nutrient broth agar plate. systems of chromatography (see the additional file 1). The colonies were counted after incubation at 37 °C for The three purified compounds were characterized by 20 h. Experiments were performed in triplicate and re- comparing their spectroscopic properties with literature peated three times with similar results. The results are values. expressed as the mean ± SE. A substance was considered cytotoxic when the bacterial survival was less than 80% Bacterial mutation assay of that observed in the negative control [25]. The muta- The antimutagenic effect of each plant extract was tion frequency was estimated as the number of mutants assayed according to the Ames method using the plate- per 10 surviving bacterial cells. incorporation protocol [21, 22]. Dr. T. Nohmi (National Institute of Health Sciences, Tokyo, Japan) kindly pro- Reaction with MNU and isoliquiritigenin vided the S. typhimurium TA1535. A solution of MNU (1.5 μmol/50 μL of DMSO) was A solution of MNU (1.5 μmol/50 μL of DMSO) was added to a test tube and supplemented with 0.1 M so- added to a test tube and supplemented with 0.1 M so- dium phosphate buffer (pH 7.4, 0.6 mL) and a solution dium phosphate buffer (pH 7.4, 0.5 mL), a solution of isoliquiritigenin (0.5 mg/50 μL of DMSO). DMSO (50 μL) with various concentrations of each fraction, and (50 μL) was used instead of the isoliquiritigenin solution a culture of S. typhimurium TA1535 (0.1 mL), and the for the blank. The reaction conditions were similar to solution was thoroughly mixed. Then, top agar (2 mL) those of the mutation assay, although 0.1 M sodium was added, and the mixture was poured onto a minimal- phosphate buffer (pH 7.4, 0.1 mL) was used instead of a glucose agar plate. The revertant colonies were counted bacterial culture (0.1 mL). A portion of the mixture was after incubation at 37 °C for 44 h. Experiments were per- diluted 10-fold in 0.1 M sodium phosphate buffer (pH formed in triplicate and repeated three times with simi- 7.4) at specified intervals and the solution (1 μL) was lar results. The results are expressed as the mean ± SE. injected into HPLC. Plates with neither MNU nor plant extract were consid- The kinetics of the reactions were followed by moni- ered negative controls. MNU (1.5 μmol/50 μL) resulted toring of the MNU peak by HPLC. A plot of [MNU] ver- in 1826 ± 52 colonies. All of the tested plates were sus time gave a straight line with a slope of –k. The half- microscopically examined for thinning, the absence of a life was calculated from ln2/k. Experiments were re- background lawn and/or the presence of microcolonies, peated four times with similar results. The results were which are considered indicators of toxicity induced by expressed as the mean ± SD. the test material. Neither MNU nor the plant extracts MNU was determined using HPLC with a Shiseido displayed toxicity to S. typhimurium TA1535 under the capcell pack UG80 (5 μm, 250 × 4.6 mm) column with conditions of the antimutagenicity test. 8% methanol–H O as the eluent at 0.7 mL/min and 254 Mutagenic activity in the presence of the extracts is nm. expressed as the percent of mutagenicity (% = Rs/R × Isoliquiritigenin was determined using HPLC with a 100), where Rs is the number of his revertants/plate for Shiseido capcell pack UG80 (5 μm, 250 × 4.6 mm) col- plates exposed to MNU and plant extracts, and R is the umn with 60% methanol–H O as the eluent at 0.7 mL/ + 2 number of his revertants/plate of MNU. The number of min and 323 nm. spontaneous revertants was subtracted beforehand to give Rs and R. Thus, the mutagenicity of MNU in the absence of plant extracts was defined as 100% MNU Detection of DMPO-OH adducts by ESR spectroscopy mutagenicity. Each flavonoid was diluted in acetonitrile. To a test tube containing 0.9 M DMPO (10 μL, final conc. 40.9 mM), Cytotoxicity test flavonoid (each concentration in 33 μL), 0.1 M sodium Toxicity assays under the same conditions as those used phosphate buffer (pH 7.4, 147 μL), 20 mM EDTA (10 μL, for the Ames test were performed to determine the max- final conc. 0.91 mM) and 10 mM FeSO (10 μL, final imum concentrations of each plant extract that could be conc. 0.45 mM) was added 10 mM H O (10 μL, final 2 2 added without exerting toxic effects on the bacteria used conc. 0.45 mM), followed by mixing on a vortex mixer in the Ames test. A solution of MNU (1.5 μmol/50 μLof and then transferring to an aqueous flat-cell. Acetonitrile DMSO) was added to a test tube and supplemented with (33 μL) was used instead of compound solution for the Inami et al. Genes and Environment (2019) 41:22 Page 4 of 9 blank. Further increasing the dose of flavonoid resulted component, the antimutagenic activity against MNU was in precipitation in the reaction mixture. compared among aqueous and methanol extracts (Fig. After 2 min, the ESR spectrum was acquired using the 1). The aqueous and methanol extraction yields were following parameters: magnetic field of 336.0 ± 5.0 mT, 10.6 and 4.6%, respectively. The methanol extract had a microwave power of 1.0 mW, modulation frequency of lower extraction yield; however, the antimutagenic activ- 9.42 GHz, modulation width of 0.05 mT, sweep time of ity in the methanol extract (40%) was better than that in 30 s, response time of 0.03 s, and receiver gain of 250. the aqueous extract (25%) at a dose of 20 mg/plate on S. Experiments were repeated three times with similar re- typhimurium TA1535. sults. The results were expressed as the mean ± SD. The methanol fraction was sequentially partitioned The capacity of the OH scavenging activity at each into ethyl acetate, n-butanol and the residuals. Among flavonoid concentration was presented as a relative in- these fractions, the ethyl acetate fraction (60% inhib- tensity determined by calculating the peak height of the ition) showed the highest antimutagenic activity against ESR signal due to the OH adduct of DMPO (DMPO– MNU at a dose of 15 mg/plate in S. typhimurium OH). The OH scavenging activity in the presence of fla- TA1535 (Fig. 2). Therefore, the ethyl acetate fraction vonoids is expressed as the percent of OH scavenging was separated through a combination of normal and re- activity [% = (R-Rs)/R × 100], where Rs is the DMPO-OH versed phases of chromatography (Fig. 3, additional file adduct intensity in the presence of flavonoid and R is 1). In each fractionation step, the recoveries of the the DMPO-OH adduct intensity in the absence of weights were more than 85% (see the additional file 1). flavonoid. The methanol extract and its ethyl acetate fraction have appropriate polarity to yield a flavonoid-rich frac- Results and discussion tion [26]. In our study, the ethyl acetate fraction was Identification of the main component from the prepared by partitioning the methanol extract of S. sub- antimutagenic fractions erectus Dunn, which possessed the highest antimutageni- The aqueous fraction of S. suberectus Dunn inhibited city, assuming that the antimutagens were flavonoids. MNU-induced mutagenicity in preliminary screening Finally, three flavonoids were isolated and identified as using the umu test. To identify the antimutagenic formononetin, isoliquiritigenin and medicarpin, and Fig. 1 Effect of the extracts of S. suberectus Dunn on MNU-induced mutagenicity in S. typhimurium TA1535 Inami et al. Genes and Environment (2019) 41:22 Page 5 of 9 Fig. 2 Effect of the fractions from the methanolic extracts of S. suberectus Dunn on MNU-induced mutagenicity in S. typhimurium TA1535 these flavonoids have been reported to be isolated from have already been isolated from S. suberectus Dunn and S. suberectus Dunn [27, 28]. are commercially available [29, 30]. In the cytotoxicity assays, samples with values of > 80% viable cells were considered non-toxic compared Inhibitory effect of the components of S. suberectus Dunn with the viability of the negative control [25]. In this on MNU-induced mutagenicity study, no cell toxic effects were observed at a concentra- Formononetin, isoliquiritigenin, and medicarpin were tion of 1.0 mg/plate for formononetin and naringenin evaluated for their ability to inhibit MNU-induced muta- and 0.5 mg/plate for genistein, isoliquiritigenin and med- genicity in S. typhimurium TA1535 (Fig. 3). Additionally, icarpin. To assess the precise antimutagenic potency of genistein and naringenin were tested for their antimuta- the plant extracts, the mutation frequency mutagenicity genicity towards MNU (Fig. 3). Genistein and naringenin was calculated by dividing the number of mutants with Fig. 3 Structures of flavonoids from S. suberectus Dunn Inami et al. Genes and Environment (2019) 41:22 Page 6 of 9 the surviving fraction of bacteria. These data clearly [41], and the reaction between flavonoids and the meta- showed that genistein, isoliquiritigenin, medicarpin and bolic antimutagenic-activating mutagen [42]. In this naringenin possessed antimutagenic activity against study, the direct-acting MNU was used, and the activity MNU in S. typhimurium TA1535 (Fig. 4). The antimuta- was evaluated using S. typhimurium TA1535, which did genic activity of the flavonoids was of the following not contain the plasmid pKM101 [43]. Therefore, the in- order: isoliquiritigenin > genistein > medicarpin = narin- hibitory effect on the mutagenicity of direct-acting mu- genin. Formononetin did not show antimutagenicity tagens was thought to be caused by a chemical reaction against MNU. between MNU and the flavonoids. The half-lives of Flavonoids are well-known antimutagens that have MNU in the presence or absence of isoliquiritigenin been detected by Ames assays [31, 32], and there are were compared, and the results were 21.0 ± 1.2 min and several reports that use MNU as a mutagen [33]. 20.0 ± 2.3 min, respectively. Furthermore, we could not Although genistein has been reported to inhibit MNU- detect any new product from a reaction mixture of induced mutagenicity, we also tested for its antimuta- MNU and isoliquiritigenin, and no significant change in genicity towards MNU to compare with those of the the amount of isoliquiritigenin was observed by HPLC. other isolated flavonoids [34]. Naringenin has been re- These data indicated that isoliquiritigenin did not de- ported to have antimutagenic activity against the compose MNU and did not scavenge on an electrophilic indirect-mutagen aflatoxin B through inhibition of product generated from MNU in vitro. metabolic activating enzymes, but it did not show anti- MNU treatments have been reported to induce not mutagenicity against the direct-acting N-methyl-N′-ni- only DNA alkylation but also increase intracellular ROS tro-N-nitrosoguanidine [35]. In this study, naringenin levels [44, 45]. Therefore, five flavonoids were evaluated inhibited MNU-induced mutagenicity, which is the rea- for their antioxidant activities using a hydroxyl radical son why the higher concentration of naringenin was ( OH)-scavenging assay (Fig. 5). To investigate the reac- used. tion of OH with flavonoids, the electron spin resonance For the first time, isoliquiritigenin, naringenin and (ESR) spin-trapping technique was used [46]. The Fen- 2+ 3+ − • medicarpin were demonstrated to possess antimutagenic ton reaction (Fe +H O → Fe + OH + OH) was 2 2 activity against MNU. These flavonoids have been re- the source of OH [47], and 5,5-dimethyl-1-pyrroline N- ported to have anticancer activity [36–39], and the anti- oxide (DMPO) was used as the OH-trapping agent [48]. mutagenic activity was considered to contribute to their The capacity of the OH-scavenging activity is presented anticancer activity. as the inhibition percent (%) relative to the intensity of the DMPO-OH adduct (Fig. 5). Hydroxyl radical-scavenging activity of the flavonoids Genistein, isoliquiritigenin, medicarpin and narin- derived from S. suberectus Dunn genin, which possess antimutagenic activity, inhibited The antimutagenic mechanism of the flavonoids in the the formation of the DMPO-OH adduct. Formononetin, Salmonella assay was reported to be by inhibition of en- which did not have antimutagenic activity towards zymatic activation [40], induction of the SOS response MNU, showed very low OH-scavenging activities. These Fig. 4 Mutagenicity (A), survival rate (B), and mutation frequency (C) of flavonoids on MNU-induced mutagenicity in S. typhimurium TA1535. The SE bars were too small to show Inami et al. Genes and Environment (2019) 41:22 Page 7 of 9 Conclusions It is important to prevent DNA damage by N-nitrosa- mines for cancer chemoprevention. In the present study, four components with antimutagenic activity against MNU from S. suberectus Dunn were identified as genis- tein, isoliquiritigenin, medicarpin, and naringenin. Isoli- quiritigenin was the most active component of S. suberectus Dunn in inhibiting MNU-induced mutagenic- ity. This report describes the first demonstration of the antigenotoxic effects of these components against car- cinogenic MNU. Many flavonoids are known to have a bioactive effect on human health [54], and the dietary intake of flavo- noids has been reported to reduce the risk of developing cancer, such as gastric, breast, prostate, and colorectal cancers [55]. The radical scavenging potency of flavo- noids might be involved in their chemopreventive effects. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s41021-019-0137-4. Additional file 1: Antimutagenic components in Spatholobus suberectus Dunn against N-methyl-N-nitrosourea. Fig. 5 OH-scavenging activity of flavonoids Acknowledgements Not applicable. Authors’ contributions data indicated that antimutagenic activity against MNU- KI coordinated the study, analysed the data, and wrote the manuscript. YA separated the active fractions and performed the Ames assay. TH and NU induced mutagenicity had the same tendency as the evaluated the antimutagenicity of the isolated flavonoids against MNU and OH-scavenging activity. analysed the data. YO measured the radical scavenging activity and analysed Despite the fact that flavonoids scavenged OH, there the data. MM conceived of the study, participated in designing the study, and helped to draft the manuscript. All authors have read and approved the were no significant changes in the amount of isoliquiriti- final manuscript. genin in a reaction with MNU in a test tube. As a small amount of extracellular ROS was generated from MNU Funding This work was supported in part by a Grant-in-Aid from the Ministry of Edu- [44], the decrease of isoliquiritigenin was hard to detect. • cation, Culture, Sports, Science and Technology of Japan and by a Grant-in- Many studies have reported OH-scavenging activity of Aid for the Science Research Promotion Fund from the Japan Private School flavonoids [49] and the structure-activity relationship for Promotion Foundation. free radical scavenging activity [50]. Furthermore, Availability of data and materials Makhafola et al. reported a direct correlation with anti- The datasets analysed during the current study are available from the oxidant activity and antimutagenicity towards 4- corresponding author upon reasonable request. nitroquinoline N-oxide [51]. Our results demonstrated Ethics approval and consent to participate that the hydroxyl radical scavenging activity of flavo- Not applicable. noids was involved in their antimutagenicity against Consent for publication direct-acting MNU in the Ames assay with strain S. Not applicable. typhimurium TA1535. Since antimutagenicity of flavo- noids toward MNU was significantly effective (approxi- Competing interests mately 100% inhibition), there are possibilities to have The authors declare that they have no competing interests. other antimutagenic mechanism. The flavonoids may Received: 22 October 2019 Accepted: 25 November 2019 block the reaction between methyldiazonium ion and DNA due to the interaction with DNA and flavonoids References [52, 53]. Further investigation is required to quantify O - 1. Lijinsky W. Chemistry and biology of N-nitroso compounds. 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Antimutagenic components in Spatholobus suberectus Dunn against N-methyl-N-nitrosourea

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Copyright © 2019 by The Author(s)
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Biomedicine; Human Genetics; Life Sciences, general
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10.1186/s41021-019-0137-4
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Abstract

Background: An extract from Spatholobus suberectus (S. suberectus) Dunn has been reported to show potent antimutagenic effects against N-alkyl-N-nitrosoureas in umu screening. The aim of this study was to identify the antimutagenic components from extracts of S. suberectus against N-methyl-N-nitrosourea (MNU) in the Ames assay with Salmonella typhimurium strain TA1535 and to elucidate the antimutagenic mechanism of the flavonoids. Results: From the ethyl acetate fraction obtained from fractionation of the methanol extract of S. suberectus Dunn, medicarpin, formononetin and isoliquiritigenin were successfully isolated through a combination of normal- and reversed-phase chromatography. Genistein and naringenin, which were already reported to be contained in S. suberectus Dunn, were also tested for their antimutagenicity towards MNU, along with formononetin, isoliquiritigenin and medicarpin. Our results demonstrated that genistein, isoliquiritigenin, medicarpin and naringenin were antimutagenic against MNU without showing cytotoxicity. MNU is reported to cause not only DNA alkylation but also induce reactive oxygen species. The hydroxyl radical scavenging capacity of the flavonoids was correlated with the antimutagenic capacity, indicating that the hydroxyl radical scavenging activity was involved in their antimutagenicity towards MNU. Conclusions: It is important to prevent DNA damage by N-nitrosamines for cancer chemoprevention. Genistein, isoliquiritigenin, medicarpin and naringenin were demonstrated to possess an antigenotoxic effects against carcinogenic MNU due to their radical scavenging activity. Keywords: Antimutagenicity, Formononetin, Genistein, Isoliquiritigenin, Medicarpin, Naringenin, Hydroxyl radical Introduction can prevent the mutagenicity and carcinogenicity of N- N-Nitroso compounds, are implicated as carcinogens in nitrosamines. the human environment such as food, tobacco smoke, N-Methyl-N-nitrosourea (MNU) is a direct-acting air, water and cosmetics [1–6]. In addition, N-nitroso mutagen that forms the corresponding methyldiazohydr- compounds have been reported to be formed endogen- oxides and methylates DNA to form DNA adducts that ously, mainly in the stomach and bowel, or in an infec- miscode during DNA replication, causing mutations that tion site [2]. The endogenous formation of N- lead to tumour formation. MNU is formed by the nitro- nitrosamines is a possible explanation for the epidemio- sation of methylurea with nitrite in guinea-pig stomachs, logical connection of gastrointestinal cancers [7, 8]. and MNU is absorbed from the stomach into the blood Therefore, for chemoprevention, it is important to dis- [9]. Additionally, MNU can be detected by the nitrosa- cover naturally occurring or synthetic compounds that tion of creatinine or fermented foods at gastric pH [10– 12]. Therefore, for cancer chemoprevention, it is import- ant to find some compounds that can inhibit the muta- genicity induced by MNU. * Correspondence: inami@rs.socu.ac.jp Faculty of Pharmaceutical Sciences, Sanyo-onoda City University, Spatholobus suberect (S. suberectus) Dunn (Legumino- Daigakudo-ri 1-1-1, Sanyo-onoda-shi, Yamaguchi 756-0884, Japan sae) is a traditional Chinese herbal medicine used to Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 treat rheumatism, anaemia, menoxenia, and other Noda-shi, Chiba 278-8510, Japan © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Inami et al. Genes and Environment (2019) 41:22 Page 2 of 9 disorders [13]. Pharmacologically, it exhibits anti- (Minnesota, USA). All reagents used were of the best inflammatory [14, 15] and antitumour activities [16, 17]. commercially available quality from Wako Pure Chem- Our previous study showed that an aqueous extract of S. ical Industries (Osaka, Japan) and were not further puri- suberectus Dunn showed the most potent antimutagenic fied unless otherwise noted. Medicarpin (mp. 194 °C) effects against MNU by umu test screening [18]. was synthesized according to the method of Goel et al The umu test is a convenient method because the data [23]. MNU [mp. 106 °C (decomp.) was prepared as de- are obtained in a short period of time; however, the scribed previously [24]. Purity of the synthesized medi- Ames test is a more precise method to evaluate the gen- carpin and MNU were determined by the H-NMR otoxic activities of a wide variety of environmental car- spectra, which all required integral values matches the cinogens and mutagens than the umu test [19, 20]. The number of protons without any other peak. Ames test using bacterial strains of Salmonella typhi- murium (S. typhimurium) is widely used by regulatory Preparation of the aqueous extract of S. suberectus Dunn agencies, academic institutions and pharmaceutical com- Dried S. suberectus Dunn was cut with a pair of scissors. panies to assess the mutagenic potential of compounds Distilled water (130 mL) was added to the cut S. suberec- [21, 22]. In the present study, we identified antimutagens tus Dunn (30 g) and refluxed for 30 min. The mixture against MNU using the Ames assay, and the inhibitory was filtered with suction, and the filtrate was concen- mechanism was investigated. trated under reduced pressure. The whole procedure was repeated twice. The crude extracts were combined, Materials and methods dried under vacuum, and finally gave a reddish solid General experimental procedures (5.30 g). The reaction progress was monitored using thin-layer chromatography (TLC) on silica gel 60 F (0.25 mm, Preparation of the methanol extract of S. suberectus Dunn Merck, Darmstadt, Germany). Column chromatography Dried S. suberectus Dunn was cut with a pair of scissors. was performed using silica gel 60 (0.04–0.063 mm, Methanol (130 mL) was added to the cut S. suberectus Merck). Melting points were determined using a Yanaco Dunn (30 g) and stirred for 30 min at room temperature. (Tokyo, Japan) micro-melting point apparatus without The mixture was filtered with suction, and the filtrate correction. The LC system used was equipped with an was concentrated under reduced pressure. The whole LC-6 AD pump (Shimadzu, Kyoto, Japan), a UVDEC- procedure was repeated twice. The crude extracts were 100 V spectrometric detector (JASCO, Tokyo, Japan), a combined, dried under vacuum, and finally a reddish YRD-880 IR detector (Shimamura Tech. Co. Ltd., Tokyo, solid (2.32 g) was obtained. Japan), or a SPD-20A (Shimadzu), and a Capcell pack RP-18 column (Shiseido, Tokyo, Japan). NMR spectra Fractionation of the methanol extract of S. suberectus were recorded with a JEOL JNM-LA400 spectrometer Dunn based on the solubility in organic solvents (Tokyo, Japan). The chemical shifts were expressed as Methanol (3.5 L) was added to the cut S. suberectus ppm downfield from TMS. The mass spectra were mea- Dunn (1 kg) and incubated overnight at room sured on a JEOL JMS-SX102A mass spectrometer. The temperature. The mixture was filtered with suction, and ESR spectra were collected on a JEOL JES-X320. the filtrate was concentrated under reduced pressure. The whole procedure was repeated 5 times. The crude Reagents extracts were combined, dried under vacuum, and finally Bacto agar and Bacto nutrient broth were obtained from a reddish solid (162 g) was obtained. Ethyl acetate (300 Becton Dickinson Microbiology Systems (Sparks, USA). mL) and water (300 mL) were added to the solid, and 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) and ethyl- the aqueous phase was separated. The aqueous phase enediamine-N,N,N′,N′-tetraacetic acid disodium salt was re-extracted with ethyl acetate (150 mL × 6), and the dihydrate (EDTA) were purchased from Dojindo Labora- combined organic phases were washed with water (100 tories (Kumamoto, Japan). Sodium ammonium hydrogen mL). The aqueous phase was extracted with water- phosphate tetrahydrate was obtained from Merck saturated n-butanol (150 mL × 6), and the combined or- (Darmstadt, Germany). S. suberectus Dunn was pur- ganic phases were washed with n-butanol-saturated chased from Matsumoto Yakugyo Co., Ltd. (Nagoya, water (100 mL). The ethyl acetate, n-butanol and aque- Japan). Genistein [mp. 304 °C (decomp.), recrystallized ous extracts were filtered, and the filtrates were removed from ethanol and H O] and isoliquiritigenin [mp. of solvent with a rotary evaporator, and the residue was 197 °C, recrystallized from ethanol and chloroform] were dried in vacuo. Finally, the ethyl acetate fraction (23.8 g), obtained from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, n-butanol fraction (48.0 g), and aqueous fraction (86.0 g) Japan). Formononetin (mp. 256 °C) and naringenin (mp. were obtained from the methanol extract of S. suberectus 251 °C) was purchased from LKT Laboratories, Inc. Dunn. The recovery of the weight was 97%. Inami et al. Genes and Environment (2019) 41:22 Page 3 of 9 Isolation of antimutagenic compounds from the ethyl 0.1 M sodium phosphate buffer (pH 7.4, 0.5 mL), each acetate fraction solution of plant extract (50 μL), and a culture of S. From the ethyl acetate fraction obtained from the typhimurium TA1535 (0.1 mL). A portion of the mixture methanol extract of S. suberectus Dunn, medicarpin, for- was diluted 10 -fold times in 1/15 M PB. The diluted so- mononetin and isoliquiritigenin were successfully iso- lution (200 μL) was supplemented with histidine-free top lated through a combination of normal and reversed agar (2 mL) and poured on a nutrient broth agar plate. systems of chromatography (see the additional file 1). The colonies were counted after incubation at 37 °C for The three purified compounds were characterized by 20 h. Experiments were performed in triplicate and re- comparing their spectroscopic properties with literature peated three times with similar results. The results are values. expressed as the mean ± SE. A substance was considered cytotoxic when the bacterial survival was less than 80% Bacterial mutation assay of that observed in the negative control [25]. The muta- The antimutagenic effect of each plant extract was tion frequency was estimated as the number of mutants assayed according to the Ames method using the plate- per 10 surviving bacterial cells. incorporation protocol [21, 22]. Dr. T. Nohmi (National Institute of Health Sciences, Tokyo, Japan) kindly pro- Reaction with MNU and isoliquiritigenin vided the S. typhimurium TA1535. A solution of MNU (1.5 μmol/50 μL of DMSO) was A solution of MNU (1.5 μmol/50 μL of DMSO) was added to a test tube and supplemented with 0.1 M so- added to a test tube and supplemented with 0.1 M so- dium phosphate buffer (pH 7.4, 0.6 mL) and a solution dium phosphate buffer (pH 7.4, 0.5 mL), a solution of isoliquiritigenin (0.5 mg/50 μL of DMSO). DMSO (50 μL) with various concentrations of each fraction, and (50 μL) was used instead of the isoliquiritigenin solution a culture of S. typhimurium TA1535 (0.1 mL), and the for the blank. The reaction conditions were similar to solution was thoroughly mixed. Then, top agar (2 mL) those of the mutation assay, although 0.1 M sodium was added, and the mixture was poured onto a minimal- phosphate buffer (pH 7.4, 0.1 mL) was used instead of a glucose agar plate. The revertant colonies were counted bacterial culture (0.1 mL). A portion of the mixture was after incubation at 37 °C for 44 h. Experiments were per- diluted 10-fold in 0.1 M sodium phosphate buffer (pH formed in triplicate and repeated three times with simi- 7.4) at specified intervals and the solution (1 μL) was lar results. The results are expressed as the mean ± SE. injected into HPLC. Plates with neither MNU nor plant extract were consid- The kinetics of the reactions were followed by moni- ered negative controls. MNU (1.5 μmol/50 μL) resulted toring of the MNU peak by HPLC. A plot of [MNU] ver- in 1826 ± 52 colonies. All of the tested plates were sus time gave a straight line with a slope of –k. The half- microscopically examined for thinning, the absence of a life was calculated from ln2/k. Experiments were re- background lawn and/or the presence of microcolonies, peated four times with similar results. The results were which are considered indicators of toxicity induced by expressed as the mean ± SD. the test material. Neither MNU nor the plant extracts MNU was determined using HPLC with a Shiseido displayed toxicity to S. typhimurium TA1535 under the capcell pack UG80 (5 μm, 250 × 4.6 mm) column with conditions of the antimutagenicity test. 8% methanol–H O as the eluent at 0.7 mL/min and 254 Mutagenic activity in the presence of the extracts is nm. expressed as the percent of mutagenicity (% = Rs/R × Isoliquiritigenin was determined using HPLC with a 100), where Rs is the number of his revertants/plate for Shiseido capcell pack UG80 (5 μm, 250 × 4.6 mm) col- plates exposed to MNU and plant extracts, and R is the umn with 60% methanol–H O as the eluent at 0.7 mL/ + 2 number of his revertants/plate of MNU. The number of min and 323 nm. spontaneous revertants was subtracted beforehand to give Rs and R. Thus, the mutagenicity of MNU in the absence of plant extracts was defined as 100% MNU Detection of DMPO-OH adducts by ESR spectroscopy mutagenicity. Each flavonoid was diluted in acetonitrile. To a test tube containing 0.9 M DMPO (10 μL, final conc. 40.9 mM), Cytotoxicity test flavonoid (each concentration in 33 μL), 0.1 M sodium Toxicity assays under the same conditions as those used phosphate buffer (pH 7.4, 147 μL), 20 mM EDTA (10 μL, for the Ames test were performed to determine the max- final conc. 0.91 mM) and 10 mM FeSO (10 μL, final imum concentrations of each plant extract that could be conc. 0.45 mM) was added 10 mM H O (10 μL, final 2 2 added without exerting toxic effects on the bacteria used conc. 0.45 mM), followed by mixing on a vortex mixer in the Ames test. A solution of MNU (1.5 μmol/50 μLof and then transferring to an aqueous flat-cell. Acetonitrile DMSO) was added to a test tube and supplemented with (33 μL) was used instead of compound solution for the Inami et al. Genes and Environment (2019) 41:22 Page 4 of 9 blank. Further increasing the dose of flavonoid resulted component, the antimutagenic activity against MNU was in precipitation in the reaction mixture. compared among aqueous and methanol extracts (Fig. After 2 min, the ESR spectrum was acquired using the 1). The aqueous and methanol extraction yields were following parameters: magnetic field of 336.0 ± 5.0 mT, 10.6 and 4.6%, respectively. The methanol extract had a microwave power of 1.0 mW, modulation frequency of lower extraction yield; however, the antimutagenic activ- 9.42 GHz, modulation width of 0.05 mT, sweep time of ity in the methanol extract (40%) was better than that in 30 s, response time of 0.03 s, and receiver gain of 250. the aqueous extract (25%) at a dose of 20 mg/plate on S. Experiments were repeated three times with similar re- typhimurium TA1535. sults. The results were expressed as the mean ± SD. The methanol fraction was sequentially partitioned The capacity of the OH scavenging activity at each into ethyl acetate, n-butanol and the residuals. Among flavonoid concentration was presented as a relative in- these fractions, the ethyl acetate fraction (60% inhib- tensity determined by calculating the peak height of the ition) showed the highest antimutagenic activity against ESR signal due to the OH adduct of DMPO (DMPO– MNU at a dose of 15 mg/plate in S. typhimurium OH). The OH scavenging activity in the presence of fla- TA1535 (Fig. 2). Therefore, the ethyl acetate fraction vonoids is expressed as the percent of OH scavenging was separated through a combination of normal and re- activity [% = (R-Rs)/R × 100], where Rs is the DMPO-OH versed phases of chromatography (Fig. 3, additional file adduct intensity in the presence of flavonoid and R is 1). In each fractionation step, the recoveries of the the DMPO-OH adduct intensity in the absence of weights were more than 85% (see the additional file 1). flavonoid. The methanol extract and its ethyl acetate fraction have appropriate polarity to yield a flavonoid-rich frac- Results and discussion tion [26]. In our study, the ethyl acetate fraction was Identification of the main component from the prepared by partitioning the methanol extract of S. sub- antimutagenic fractions erectus Dunn, which possessed the highest antimutageni- The aqueous fraction of S. suberectus Dunn inhibited city, assuming that the antimutagens were flavonoids. MNU-induced mutagenicity in preliminary screening Finally, three flavonoids were isolated and identified as using the umu test. To identify the antimutagenic formononetin, isoliquiritigenin and medicarpin, and Fig. 1 Effect of the extracts of S. suberectus Dunn on MNU-induced mutagenicity in S. typhimurium TA1535 Inami et al. Genes and Environment (2019) 41:22 Page 5 of 9 Fig. 2 Effect of the fractions from the methanolic extracts of S. suberectus Dunn on MNU-induced mutagenicity in S. typhimurium TA1535 these flavonoids have been reported to be isolated from have already been isolated from S. suberectus Dunn and S. suberectus Dunn [27, 28]. are commercially available [29, 30]. In the cytotoxicity assays, samples with values of > 80% viable cells were considered non-toxic compared Inhibitory effect of the components of S. suberectus Dunn with the viability of the negative control [25]. In this on MNU-induced mutagenicity study, no cell toxic effects were observed at a concentra- Formononetin, isoliquiritigenin, and medicarpin were tion of 1.0 mg/plate for formononetin and naringenin evaluated for their ability to inhibit MNU-induced muta- and 0.5 mg/plate for genistein, isoliquiritigenin and med- genicity in S. typhimurium TA1535 (Fig. 3). Additionally, icarpin. To assess the precise antimutagenic potency of genistein and naringenin were tested for their antimuta- the plant extracts, the mutation frequency mutagenicity genicity towards MNU (Fig. 3). Genistein and naringenin was calculated by dividing the number of mutants with Fig. 3 Structures of flavonoids from S. suberectus Dunn Inami et al. Genes and Environment (2019) 41:22 Page 6 of 9 the surviving fraction of bacteria. These data clearly [41], and the reaction between flavonoids and the meta- showed that genistein, isoliquiritigenin, medicarpin and bolic antimutagenic-activating mutagen [42]. In this naringenin possessed antimutagenic activity against study, the direct-acting MNU was used, and the activity MNU in S. typhimurium TA1535 (Fig. 4). The antimuta- was evaluated using S. typhimurium TA1535, which did genic activity of the flavonoids was of the following not contain the plasmid pKM101 [43]. Therefore, the in- order: isoliquiritigenin > genistein > medicarpin = narin- hibitory effect on the mutagenicity of direct-acting mu- genin. Formononetin did not show antimutagenicity tagens was thought to be caused by a chemical reaction against MNU. between MNU and the flavonoids. The half-lives of Flavonoids are well-known antimutagens that have MNU in the presence or absence of isoliquiritigenin been detected by Ames assays [31, 32], and there are were compared, and the results were 21.0 ± 1.2 min and several reports that use MNU as a mutagen [33]. 20.0 ± 2.3 min, respectively. Furthermore, we could not Although genistein has been reported to inhibit MNU- detect any new product from a reaction mixture of induced mutagenicity, we also tested for its antimuta- MNU and isoliquiritigenin, and no significant change in genicity towards MNU to compare with those of the the amount of isoliquiritigenin was observed by HPLC. other isolated flavonoids [34]. Naringenin has been re- These data indicated that isoliquiritigenin did not de- ported to have antimutagenic activity against the compose MNU and did not scavenge on an electrophilic indirect-mutagen aflatoxin B through inhibition of product generated from MNU in vitro. metabolic activating enzymes, but it did not show anti- MNU treatments have been reported to induce not mutagenicity against the direct-acting N-methyl-N′-ni- only DNA alkylation but also increase intracellular ROS tro-N-nitrosoguanidine [35]. In this study, naringenin levels [44, 45]. Therefore, five flavonoids were evaluated inhibited MNU-induced mutagenicity, which is the rea- for their antioxidant activities using a hydroxyl radical son why the higher concentration of naringenin was ( OH)-scavenging assay (Fig. 5). To investigate the reac- used. tion of OH with flavonoids, the electron spin resonance For the first time, isoliquiritigenin, naringenin and (ESR) spin-trapping technique was used [46]. The Fen- 2+ 3+ − • medicarpin were demonstrated to possess antimutagenic ton reaction (Fe +H O → Fe + OH + OH) was 2 2 activity against MNU. These flavonoids have been re- the source of OH [47], and 5,5-dimethyl-1-pyrroline N- ported to have anticancer activity [36–39], and the anti- oxide (DMPO) was used as the OH-trapping agent [48]. mutagenic activity was considered to contribute to their The capacity of the OH-scavenging activity is presented anticancer activity. as the inhibition percent (%) relative to the intensity of the DMPO-OH adduct (Fig. 5). Hydroxyl radical-scavenging activity of the flavonoids Genistein, isoliquiritigenin, medicarpin and narin- derived from S. suberectus Dunn genin, which possess antimutagenic activity, inhibited The antimutagenic mechanism of the flavonoids in the the formation of the DMPO-OH adduct. Formononetin, Salmonella assay was reported to be by inhibition of en- which did not have antimutagenic activity towards zymatic activation [40], induction of the SOS response MNU, showed very low OH-scavenging activities. These Fig. 4 Mutagenicity (A), survival rate (B), and mutation frequency (C) of flavonoids on MNU-induced mutagenicity in S. typhimurium TA1535. The SE bars were too small to show Inami et al. Genes and Environment (2019) 41:22 Page 7 of 9 Conclusions It is important to prevent DNA damage by N-nitrosa- mines for cancer chemoprevention. In the present study, four components with antimutagenic activity against MNU from S. suberectus Dunn were identified as genis- tein, isoliquiritigenin, medicarpin, and naringenin. Isoli- quiritigenin was the most active component of S. suberectus Dunn in inhibiting MNU-induced mutagenic- ity. This report describes the first demonstration of the antigenotoxic effects of these components against car- cinogenic MNU. Many flavonoids are known to have a bioactive effect on human health [54], and the dietary intake of flavo- noids has been reported to reduce the risk of developing cancer, such as gastric, breast, prostate, and colorectal cancers [55]. The radical scavenging potency of flavo- noids might be involved in their chemopreventive effects. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s41021-019-0137-4. Additional file 1: Antimutagenic components in Spatholobus suberectus Dunn against N-methyl-N-nitrosourea. Fig. 5 OH-scavenging activity of flavonoids Acknowledgements Not applicable. Authors’ contributions data indicated that antimutagenic activity against MNU- KI coordinated the study, analysed the data, and wrote the manuscript. YA separated the active fractions and performed the Ames assay. TH and NU induced mutagenicity had the same tendency as the evaluated the antimutagenicity of the isolated flavonoids against MNU and OH-scavenging activity. analysed the data. YO measured the radical scavenging activity and analysed Despite the fact that flavonoids scavenged OH, there the data. MM conceived of the study, participated in designing the study, and helped to draft the manuscript. All authors have read and approved the were no significant changes in the amount of isoliquiriti- final manuscript. genin in a reaction with MNU in a test tube. As a small amount of extracellular ROS was generated from MNU Funding This work was supported in part by a Grant-in-Aid from the Ministry of Edu- [44], the decrease of isoliquiritigenin was hard to detect. • cation, Culture, Sports, Science and Technology of Japan and by a Grant-in- Many studies have reported OH-scavenging activity of Aid for the Science Research Promotion Fund from the Japan Private School flavonoids [49] and the structure-activity relationship for Promotion Foundation. free radical scavenging activity [50]. Furthermore, Availability of data and materials Makhafola et al. reported a direct correlation with anti- The datasets analysed during the current study are available from the oxidant activity and antimutagenicity towards 4- corresponding author upon reasonable request. nitroquinoline N-oxide [51]. Our results demonstrated Ethics approval and consent to participate that the hydroxyl radical scavenging activity of flavo- Not applicable. noids was involved in their antimutagenicity against Consent for publication direct-acting MNU in the Ames assay with strain S. Not applicable. typhimurium TA1535. Since antimutagenicity of flavo- noids toward MNU was significantly effective (approxi- Competing interests mately 100% inhibition), there are possibilities to have The authors declare that they have no competing interests. other antimutagenic mechanism. The flavonoids may Received: 22 October 2019 Accepted: 25 November 2019 block the reaction between methyldiazonium ion and DNA due to the interaction with DNA and flavonoids References [52, 53]. Further investigation is required to quantify O - 1. Lijinsky W. Chemistry and biology of N-nitroso compounds. 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