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Background: A powdered ethanolic extract of Glycyrrhiza aspera root exhibits antimutagenic activity against N-methyl-N-nitrosourea (MNU) based on the Ames assay with Salmonella typhimurium TA1535. The aim of this study was to identify the antimutagenic components of the powdered ethanolic extract of G. aspera root. Results: The powdered ethanolic extract of G. aspera root was sequentially suspended in n-hexane, carbon tetrachloride, dichloromethane, ethyl acetate, and ethanol, and each solvent soluble fraction and the residue were assayed for antimutagenic activity against MNU in S. typhimurium TA1535. The dichloromethane soluble fraction exhibited the highest antimutagenicity and was fractionated several times by silica gel chromatography. The fraction with the highest antimutagenic activity was further purified using HPLC, and the fractions were assayed for antimutagenicity against MNU in S. typhimurium TA1535. Finally, five components with antimutagenic activity against MNU were identified as glyurallin A, glyasperin B, licoricidin, 1-methoxyphaseollin, and licoisoflavone B. Conclusions: The five components were demonstrated to possess an antigenotoxic effect against carcinogenic MNU for the first time. It is important to prevent DNA damage by N-nitrosamines for cancer chemoprevention. Keywords: Glyurallin A, Glyasperin B, Licoricidin, 1-Methoxyphaseollin, Licoisoflavone B Background be caused by N-nitroso compounds that form during Humans are exposed to endogenous and exogenous N- meat processing or cooking [10]. nitroso compounds [1]. Approximately 45–75% of the N-Methyl-N-nitrosourea (MNU) is a DNA alkylating total human exposure to N-nitroso compounds is esti- carcinogen that induces cancer in various organs, mated to be due to in vivo synthesis [2]. Almost all particularly the forestomach, brain and nervous system, tested N-nitroso compounds have carcinogenic activity in rodents [11]. MNU is produced by the nitrosation of in experimental animals [1]. Therefore, exposure to N- creatinine or fermented foods at the gastric pH [12–15]. nitroso compounds is suspected to induce human can- Additionally, MNU is formed by the nitrosation of cer. Several epidemiological studies have demonstrated methylurea with nitrite in guinea pig stomachs [16]. that the endogenous formation of N-nitroso compounds Therefore, for cancer chemoprevention, it is important is correlated with the cancer incidence in humans [3–9]. to identify compounds that can inhibit mutagenicity Recently, the International Agency for Research on Can- induced by MNU. cer (IARC) has reported that the consumption of red Short-term bacterial mutation assays, such as the meat and processed meat is carcinogenic, and this may Ames assay, are an effective screening tool for the identification of various mutagenic or antimutagenic compounds in complex materials [17]. The assay has * Correspondence: inami@rs.noda.tus.ac.jp advantages as an inexpensive and flexible screening Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki method that provides preliminary information related to 2641, Noda, Chiba 278-8510, Japan antimutagenesis. There are many reports about the Full list of author information is available at the end of the article © The Author(s). 2017 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 (2017) 39:5 Page 2 of 7 antimutagenicity of edible plants; however, the inhibitory ethanolic extract of G. aspera (China) root was kindly effects against MNU mutagenesis are less well studied provided by Tokiwa Phytochemical Co. Ltd. (Chiba, [18, 19]. Japan). Glycyrrhiza root has long been used worldwide as an herbal medicine and natural sweetener [20–22]. The Preparation of a powdered ethanolic extracts of genus Glycyrrhiza (Leguminosae) consists of about 30 Glycyrrhiza aspera root species including G. glabra, G. uralensis, G. inflata, G. Arootof G. aspera (100 g) was refluxed with 95% aspera, G. korshinskyi and G. eurycarpa [23]. In Japanese ethanolic aqueous solution (1000 mL) for 1 h, and the pharmacopeia, only G. glabra and G. uralensis are mixture was filtered with suction. The residue was permitted to be used as licorice and licorice powder, and refluxed again with 95% ethanolic aqueous solution the other Glycyrrhiza species can be used as raw mate- (1000 mL) for 1 h, and the mixture was filtered with rials of licorice extract [23]. Glycyrrhiza has a reported suction. The combined filtrates were concentrated under chemopreventive effect based on its anticarcinogenesis reduced pressure and vacuum dried to a constant and antimutagenesis toward both indirect-acting and weight, and finally a brown powder was obtained. direct-acting mutagens [24–29]; however, the inhibitory effects against MNU mutagenesis have not been studied Fractionation of the powdered ethanolic extract of in detail. Glycyrrhiza aspera root based on solubility in organic In our previous study, a powdered ethanolic extract of solvents G. aspera root decreased MNU-induced mutagenicity in The powered ethanolic extract of G. aspera root (10 g) a preliminary antimutagenic screen using the Ames was added to hexane (100 mL) and stirred for 10 min. assay [30]. The aim of this study was to identify the anti- The supernatant was filtered with suction. The stirring mutagenic components of the powdered ethanolic and filtration of the residue was repeated twice. Sequen- extract of G. aspera root. tially, the residue was suspended in carbon tetrachloride (100 mL × 3), dichloromethane (100 mL × 3), ethyl Methods acetate (100 mL × 3), and ethanol (100 mL × 3) following General experimental procedures the same procedure. The organic solvent portions were The reaction progress was monitored using thin-layer removed organic solvent by rotary evaporator and the chromatography (TLC) on silica gel 60 F (0.25 mm, residue was dried in vacuo. The whole extraction Merck, Darmstadt, Germany). Column chromatography procedure was repeated twice; the organic portions and was performed using silica gel 60 (0.04–0.063 mm, residue were combined. Finally, hexane soluble fraction Merck). Melting points were determined using a Yanaco (62 mg), carbon tetrachloride soluble fraction (880 mg), (Tokyo, Japan) micro-melting-point apparatus without dichloromethane soluble fraction (15.6 g), ethyl acetate correction. HPLC was performed using an EYELA Pre- soluble fraction (11.4 g), ethanol soluble fraction parative LC system [VSP-3050 pump, UV-9000 spectro- (700 mg) and the residue (1.7 g) were obtained from the metric detector, LiChrosorb RP-18 column (10 μm, powdered ethanolic extract of G. aspera root (30 g). 25 mm × 300 mm)] (Tokyo Rikakikai Co. Ltd., Tokyo, Recovery of the weight was 101%. Japan) and a Shimadzu LC system [LC-6 AD pump, SPD-20A UV spectrometric detector, Mightysil RP-18 Isolation of antimutagenic compounds from the column (5 μm, 20 mm × 250 mm)] (Kyoto, Japan). The dichloromethane soluble fraction NMR spectra were recorded with a JEOL JNM-LA400 The dichloromethane soluble fraction was chromato- spectrometer (Tokyo, Japan). The chemical shifts were graphed on a silica gel, eluted with 5% methanol-CH Cl , 2 2 expressed in ppm, downfield from TMS. The mass spec- 3% methanol-CH Cl ,1%methanol-CH Cl , 10% ethyl 2 2 2 2 tra were collected using a JEOL JMS-SX102A mass spec- acetate-CH Cl , and later separated on an RP-18 column 2 2 trometer (Tokyo, Japan). by preparative HPLC and eluted with 80% methanol in water (see the Additional file 1). Five peaks representing active components were purified using HPLC and charac- Reagents terized by comparing their spectroscopic (NMR and MS) Sodium ammonium hydrogen phosphate tetrahydrate properties with literature values. was purchased from Merck (Darmstadt, Germany). Bacto agar and Bacto nutrient broth were obtained from Becton Dickinson Microbiology Systems (Sparks, USA). Bacterial mutation assay MNU were obtained from Toshin Gousei (Tokyo, The antimutagenic effect of each plant extract was Japan). Other reagents were purchased from Wako Pure assayed according to the Ames method using the plate- Chemical Industries (Osaka, Japan). A powdered incorporation protocol [31, 32]. Dr. T. Nohmi (National Inami et al. Genes and Environment (2017) 39:5 Page 3 of 7 Institute of Health Sciences, Tokyo, Japan) kindly DMSO) was added to a test tube, and supplemented provided the S. typhimurium TA1535. with 0.1 M sodium phosphate buffer (pH 7.4, 0.5 mL), A solution of MNU (1.5 μmol/50 μL DMSO) was each solution of plant extract (50 μL), and a culture of S. added to a test tube and supplemented with 0.1 M so- typhimurium TA1535 (0.1 mL). A portion of the mixture dium phosphate buffer (pH 7.4, 0.5 mL), a solution was diluted 10 times in 1/15 M PB. The diluted (50 μL) with various concentrations of fraction, and a solution (200 μL) was supplemented with histidine-free culture of the S. typhimurium TA1535 (0.1 mL), and the Top Agar (2 mL) and poured on a Nutrient Broth agar solution was thoroughly mixed. Then, Top Agar (2 mL) plate. The colonies were counted after incubation at 37 ° was added, and the mixture was poured onto a minimal- C for 20 h. Each sample was assayed using duplicate glucose agar plate. The revertant colonies were counted plates. A substance was considered cytotoxic when the after incubation at 37 °C for 44 h. Each sample was bacterial survival was less than 80% of that observed in assayed using duplicate plates. The results are expressed the negative control. The mutation frequency was esti- as the mean number of revertant colonies per plate. mated as the number of mutants per 10 surviving bac- Plates with neither MNU nor plant extract were consid- terial cells [31, 32]. ered negative controls. MNU (1.5 μmol/50 μL) resulted in 1470 ± 70 colonies. All of the tested plates were Results and discussion microscopically examined for thinning or the absence of Identification of antimutagenic components from a a background lawn and/or presence of microcolonies, powdered ethanolic extract of Glycyrrhiza aspera root which are considered indicators of toxicity induced by A powdered ethanolic extract of G. aspera root was the test material. Neither MNU nor plant extracts dis- sequentially suspended in n-hexane, carbon tetrachlor- played toxicity to S. typhimurium TA1535 under the ide, dichloromethane, ethyl acetate, and ethanol. Each conditions of the antimutagenicity test. soluble fraction and the residue were assayed for inhibi- Mutagenic activity in the presence of extracts is tory effects against MNU mutagenesis in Salmonella expressed as the percentage of mutagenicity (% = Rs/R × typhimurium TA1535 (Fig. 1). 100), where Rs is the number of his revertants/plate for The dichloromethane soluble fraction showed the plates exposed to MNU and plants extracts, and R is the highest antimutagenic activity, and was fractionated number of his revertants/plate of MNU. The number using a silica gel column and preparative high- of spontaneous revertants was subtracted beforehand to performance liquid chromatography (HPLC), and its give Rs and R. Thus, the mutagenicity of MNU in the antimutagenic components were identified. The fraction- absence of plant extracts was defined as 100% MNU ation and mutagenicity assay were conducted repeatedly mutagenicity. (Fig. 2 and the Additional file 1). In each fractionation step, the recoveries of weights were more than 89%. Cytotoxicity test Finally, the fraction (Fr.3-2-2-3-2) with the highest Toxicity assays under the same conditions as those used antimutagenic activity was separated into fractions 1–9 for the Ames test were performed to determine the using HPLC (Fig. 3a). Those fractions were tested for maximum concentrations of plant extracts that could be mutagenicity against MNU, and the compounds from added without exerting toxic effects on the bacteria used peaks 4–8 each inhibited MNU-induced mutagenicity in the Ames test. A solution of MNU (1.5 μmol/50 μL (Fig. 3b) Fig. 1 Effect of each soluble fraction on MNU-induced mutagenicity in S. typhimurium TA1535 Inami et al. Genes and Environment (2017) 39:5 Page 4 of 7 and pterocarpans are not well-known for their antimutagenicity. Inhibitory effect of licoricidin from the powdered ethanolic extract of Glycyrrhiza aspera root on MNU- induced mutagenicity Licoricidin (peak 6) did not show any revertant colonies by cytotoxicity (Fig. 3b). For the antimutagenicity assay, it was necessary to determine the concentration at which the viability of the tester strain did not decrease. Licori- cidin reduced revertant colonies induced by MNU in S. typhimurium TA1535 (Fig. 5a) without cytotoxicity at the maximum concentration of 100 μg/plate (Fig. 5b). To assess the precise antimutagenic potency of licorici- din, mutation frequency was calculated by dividing the number of mutants with the surviving fraction of bac- Fig. 2 Diagram of the separation procedure for the dichloromethane teria (Fig. 5c). These data clearly showed that licoricidin soluble fraction possessed antimutagenic activity against MNU in S. typhimurium TA1535. The compounds from fractions 4–8wereeach analysed The antimutagenic activity of the G. glabra extract using mass spectrometry and H nuclear magnetic reson- against ethyl methanesulfonate (EMS) is reportedly ance spectroscopy, and five antimutagenic compounds attributed to glabrene [41]. In our study, glabrene was were identified, i.e., glyurallin A [33], glyasperin B [34], not isolated from the fraction with the highest muta- licoricidin [35, 36], 1-methoxyphaseollin [37], and licoiso- genic activity. The difference between the isolated com- flavone B [38], by comparing their spectroscopic proper- pounds in antimutagenic activity for the powdered ties with literature values (Fig. 4). ethanolic extract of G. aspera root probably reflects dif- Glyurallin A was a pterocarpene and 1-methoxyphaseollin ferences in the mechanism of action between MNU and was a pterocarpan. Glyasperin B, licoricidin, and licoisofla- EMS. MNU reacts mainly via an S 1 mechanism and ef- vone B were an isoflavanone, an isoflavan, and an isoflavone, ficiently alkylated both nitrogens and oxygens in DNA. respectively. A phenolic hydroxyl group is a common in five EMS, which reacts predominantly via an S 2 mechan- isolated compounds with antimutagenicity. ism, alkylates the nitrogens at the DNA bases and Flavonoids are well-known antimutagens based on the produced little alkylation of the oxygens in DNA bases results of Ames assays [39, 40], whereas pterocarpenes [42, 43]. Thus, the MNU is far more mutagenic than Fig. 3 a HPLC diagram of Fr. 3-2-2-3-2; b Effect of each fraction from Fr.3-2-2-3-2 on MNU-induced mutagenicity in S. typhimurium TA1535 Inami et al. Genes and Environment (2017) 39:5 Page 5 of 7 Fig. 4 Structures of antimutagenic agents from the powdered ethanolic extract of Glycyrrhiza aspera root EMS. Additionally, the main bioactive components were epoxide [48]. Therefore, we assumed that the antimuta- different between G. glabra and G. aspera [23]. genic mechanisms of the isolated compounds were simi- In vitro,N-nitrosamine formation is inhibited by phen- lar to that of phenolic acid or hydroxylated flavonoids. olic compounds, ascorbic acid, thiols, and metals [18, Furthermore, MNU treatments are reported to induce 19]. Mutagenesis by direct-acting mutagens can be not only DNA alkylation but also increase intracellular reduced or prevented in several ways; MNU can be ROS level [49–51], and then the antimutagenicity was decomposed to non-mutagenic products via antimuta- partly attributed to its radical scavenging potency of fla- gens, or reactive mutagenic products from MNU can vonoids [52–54]. We are working to elucidate the anti- react with antimutagens before reaching DNA. It is also mutagenic mechanisms for the isolated compounds possible to induce repair enzymes in the Salmonella against MNU. strain [44]. Antimutagens and anticarcinogens in the diet are A number of known flavonoids (such as genistein etc) suggested to be highly effective for cancer prevention possess significant antimutagenic activity [40, 45, 46]; [44, 55, 56]. The intake of medicinal and edible plants however, the detailed mechanism for the antimutageni- that include antimutagens may play a role in improving city has not been completely established. The inhibitory human health. effect on the mutagenicity of direct-acting mutagens is probably caused by a chemical reaction between the Conclusions mutagen and antimutagen. The inhibitory effect of It is important to prevent DNA damage by N-nitrosa- phenolic acid results from the scavenging action on an mines for cancer chemoprevention. In this study, five electrophilic decomposition product of MNU [47]. Hung components with antimutagenic activity against MNU et al have reported that hydroxylated flavonoids showed from a powdered ethanolic extract of G. aspera root antimutagenic activity toward benzo[a]pyrene 7,8-diol- were identified as glyurallin A, glyasperin B, licoricidin, 9,10-epoxide by direct interaction with the 7,8-diol-9,10- 1-methoxyphaseollin, and licoisoflavone B. To the best ab c Fig. 5 Mutagenicity (a), survival rate (b), and mutation frequency (c) of licoricidin on MNU-induced mutagenicity in S. typhimurium TA1535 Inami et al. Genes and Environment (2017) 39:5 Page 6 of 7 of our knowledge, this report describes the first demon- 9. Jakszyn P, Gonzalez CA. Nitrosamine and related food intake and gastric and oesophageal cancer risk: a systematic review of the epidemiological stration of the antigenotoxic effects of these components evidence. World J Gastroenterol. 2006;12:4296–303. against carcinogenic MNU. 10. International agency for research on cancer (IARC). 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Genes and Environment – Springer Journals
Published: Jan 6, 2017
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