Isolation and Characterization of a Human Intestinal Bacterium

May 11, 2017 - The metabolite of 3′-DMAG by strain AUH-JLD49s was identified as 3′-desmethyl-4′-dehydroxyarctigenin (DMDH-AG) based on electrosp...
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Isolation and Characterization of a Human Intestinal Bacterium Eggerthella sp. AUH-JLD49s for the Conversion of (−)-3′Desmethylarctigenin Ye Wang,† Fei Yu,† Ming-Yue Liu,† Yi-Kai Zhao, Dong-Ming Wang, Qing-Hong Hao, and Xiu-Ling Wang* College of Life Sciences, Agricultural University of Hebei, 289 Lingyusi Street, Baoding, Hebei 071001, People’s Republic of China ABSTRACT: Arctiin is the most abundant bioactive compound contained in the Arctium lappa plant. In our previous study, we isolated one single bacterium capable of bioconverting arctigenin, an aglycone of arctiin, to 3′-desmethylarctigenin (3′-DMAG) solely. However, to date, a specific bacterium capable of producing other arctiin metabolites has not been reported. In this study, we isolated one single bacterium, which we named Eggerthella sp. AUH-JLD49s, capable of bioconverting 3′-DMAG under anaerobic conditions. The metabolite of 3′-DMAG by strain AUH-JLD49s was identified as 3′-desmethyl-4′-dehydroxyarctigenin (DMDH-AG) based on electrospray ionization mass spectrometry (ESI−MS) and 1H and 13C nuclear magnetic resonance spectroscopy. The bioconversion kinetics and bioconversion capacity of strain AUH-JLD49s were investigated. In addition, the metabolite DMDH-AG showed an inhibitory effect on cell growth of human colon cancer cell line HCT116 and human breast cancer cell line MDA-MB-231. KEYWORDS: arctigenin, 3′-desmethylarctigenin (3′-DMAG), 3′-desmethyl-4′-dehydroxyarctigenin (DMDH-AG), human intestinal bacteria, isolation, microbial biotransformation

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INTRODUCTION

In the present study, we isolated one human intestinal bacterium, which we named Eggerthella sp. AUH-JLD49s, capable of anaerobically converting 3′-DMAG solely and efficiently. The bioconverting properties of strain AUHJLD49s and the growth inhibitory effect of the metabolite of 3′-DMAG on cancer cell lines were studied as well.

Arctium lappa L., a herbal medicine (seeds and root) listed in the Chinese National Pharmacopoeia, is popularly used as a vegetable as well. Studies have shown that consumption of A. lappa is associated with many health benefits, including dispelling pathogenic wind-heat, promoting eruption, relieving a sore throat, etc.1 Arctiin, which is the most abundant lignan in the seeds of A. lappa,2 was reported to be anti-austeric,3 antiallergic,4 anti-inflammatory,5 anti-asthmatic,6 and antidiabetic,7 to have a chemopreventive effect against cancer8 and an ameliorative effect to attenuate the deterioration of renal function,9 etc. Because most herbal medicines are administered orally, intestinal bacteria play a pivotal role in arctiin metabolism. In 1992, Nose et al. reported that the first microbial metabolite of arctiin was arctigenin (an aglycone of arctiin), and arctigenin was subsequently converted to 3′desmethylarctigenin (3′-DMAG) when being incubated with rat feces.10 At present, it is clear that arctiin is metabolized to various metabolites, including arctigenin, 3′-DMAG, dihydroxyenterolactone, enterolactone, etc. by both human11 and rat12 intestinal bacteria. In 2007, the first human intestinal bacterium named Eubacterium sp. ARC-2 (EF413640), which was capable of converting arctigenin, was isolated. Strain ARC-2 was reported to convert the substrate arctigenin to seven different metabolites.13 In 2013, we reported Blautia sp. AUH-JLD56, a human intestinal bacterium capable of converting the substrate arctiin or arctigenin to 3′-DMAG under anaerobic conditions.14 However, to date, no particular bacteria responsible for the conversion of 3′-DMAG have been isolated and identified. © 2017 American Chemical Society

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MATERIALS AND METHODS

Chemicals and Bacterial Strains. The authentic arctigenin was from Senbeijia Biological Company (Nanjing, China). Compound 3′DMAG was prepared using our previous microbial biotransformation method.14 Staphylococcus aureus (ATCC27217), Salmonella paratyphi (CMCC50001), and Escherichia coli (CICC10372) were from the Institute of Food Science and Technology, Hebei Agricultural University. The reagents, including acetonitrile, methanol, and acetic acid, were of high-performance liquid chromatography (HPLC) grade. Brain heart infusion (BHI) powder was from Difco Co. (Sparks, MD, U.S.A.). Bacterial Isolation and Identification. The fresh feces sample used in this study was from a healthy female volunteer. Bacterium isolation and identification were carried out using our previously described methods.14 The isolated bacterium was inoculated in BHI liquid medium and cultured in an anaerobic chamber (Concept 400, Ruskinn, U.K.) containing 5% CO2, 10% H2, and 85% N2 at 37 °C. The 16S rRNA gene sequence of the isolated bacterium has been submitted to the National Center for Biotechnology Information (NCBI) GenBank with the accession number KX650621. Four randomly synthesized primers, including S400 (TGGTGGACCA), S424 (GACCGACCCA), and S495 (GGGTAACGTG), were used for Received: Revised: Accepted: Published: 4051

January 12, 2017 May 3, 2017 May 4, 2017 May 11, 2017 DOI: 10.1021/acs.jafc.7b00114 J. Agric. Food Chem. 2017, 65, 4051−4056

Article

Journal of Agricultural and Food Chemistry

Figure 1. HPLC elution profiles of 3′-DMAG after 3 days of incubation with strain AUH-JLD49s (regular line) and the authentic compound 3′DMAG (dotted line). The inset shows the UV spectrum of peak 1. The concentration of the substrate 3′-DMAG was 0.5 mmol/L.

Figure 2. (A) UV spectrum of the metabolite of 3′-DMAG by human intestinal bacterium strain Eggerthella sp. JLD49s and (B) ESI−MS[+] spectrum of the metabolite of 3′-DMAG. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Assay. The in vitro antioxidant test was carried out using DPPH free radicals. The DPPH radical scavenging activity was examined using our previously reported method.15 However, we made a modification in the reaction temperature and reaction time. The temperature for reaction was 20 °C, and the reaction time was changed to 48 h. The purity of the substrate 3′-DMAG and the metabolite of 3′-DMAG by strain AUHJLD49s was 99%. Antimicrobial Activity of the Metabolite of 3′-DMAG. The authentic arctigenin, biosynthesized 3′-DMAG, and metabolite of 3′DMAG were tested in the antimicrobial activity. After the agar medium was cooled to about 50 °C, 10% of the pre-prepared bacterial culture, including S. aureus (ATCC27217), S. paratyphi (CMCC50001), and E. coli (CICC10372), was inoculated separately. The medium was then beaten with a diameter of 0.5 cm hole. Each

random amplification of polymorphic DNA (RAPD) analysis. The random primers mentioned previously were synthesized by Sangon Co. Production and Identification of the Metabolite of 3′-DMAG by Strain AUH-JLD49s. The metabolite of 3′-DMAG by strain AUH-JLD49s was detected, prepared, and chemically identified as we described previously.14 An A21171-Autopol III Rudolph Research Analytical automatic polarimeter (Hackettstown, NJ, U.S.A.) was used for detection of the optical rotation of the metabolite. Biotransformation Kinetics of 3′-DMAG by Strain AUHJLD49s. The biotransforming kinetics of strain AUH-JLD49s was studied as we described previously.14 The concentration of the substrate 3′-DMAG was 0.4 mmol/L. The cultural broth was sampled after every 3 h of incubation, and the experiments were performed in triplicate. 4052

DOI: 10.1021/acs.jafc.7b00114 J. Agric. Food Chem. 2017, 65, 4051−4056

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Journal of Agricultural and Food Chemistry

Figure 3. Proposed biosynthesis procedures of the metabolite DMDH-AG by human intestinal bacterium strain AUH-JLD56 and strain AUHJLD49s under anaerobic conditions. hole was added with 20 μL solution of arctigenin, 3′-DMAG, and the metabolite of 3′-DMAG. All of the compounds mentioned previously were dissolved in 100% methanol, and the concentration of each compound was 5 mmol/L. Streptomycin (0.1 × 104 unit) was used as a positive control. Growth Inhibitory Effect of the Metabolite of 3′-DMAG on Cancer Cells. Both human colon cancer cell line HCT116 and human breast cancer cell line MDA-MB-231 were obtained from the China Centre for Type Culture Collection, Chinese Academy of Sciences. The microbial metabolite of 3′-DMAG by bacterium strain Eggerthella sp. AUH-JLD49s was applied to HCT116 and MDA-MB-231 to study the inhibitory effect. The purity of the biosynthesized microbial metabolite of 3′-DMAG was 99%. The two cell lines were cultured as we described previously.16 The metabolite of 3′-DMAG was dissolved in 100% dimethyl sulfoxide (DMSO) at the concentration of 100 mmol/L. The cells (5 × 104/mL) were seeded in 96-well tissue culture plates and treated with the metabolite of 3′-DMAG at the concentrations of 50, 100, and 200 μmol/L and incubated for 24 h, followed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 570 nm.

(1470 bp) of strain AUH-JLD49s had 99.8% similarity to that of Eggerthella sp. strain AUH-Julong365 (accession number JN874873). However, strain AUH-JLD49s and strain AUHJulong365 showed different band patterns when primer S424 or S495 was used for RAPD analysis (data not shown). Our result indicated that, although the two Eggerthella strains showed about 99.8% 16S rRNA gene sequence similarity, an obvious difference also exists in the genomic component. Identification of the Metabolite of 3′-DMAG Produced by Strain AUH-JLD49s. One single metabolite from 3′-DMAG by bacterium strain AUH-JLD49s, which eluted at 14.3 min (peak 2 in Figure 1) in the HPLC elution profile, was detected. On the bases of the HPLC retention time and ultraviolet (UV) spectrum, we inferred that peak 1 was the residual substrate 3′-DMAG left in the cultural broth (Figure 1). The metabolite of 3′-DMAG gave absorbance maxima at 228 and 278 nm, respectively (Figure 2A). The electrospray ionization mass spectrometry (ESI−MS) spectrum showed a [M + H]+ ion at m/z 343 (Figure 2B), 16 mass units (−OH) less than that of the substrate 3′-DMAG, suggesting that the metabolite is a dehydroxylation product of 3′-DMAG. For further structural identification, we purified the metabolite and detected it using 1H and 13C nuclear magnetic resonance (NMR) analyses. Both the 1H and 13C NMR data were identical to those for compound 4 [3′-desmethyl-4′-dehydroxyarctigenin (DMDH-AG)] reported by Xie et al.11 Therefore, the metabolite of 3′-DMAG by strain AUH-JLD49s was accurately identified as DMDH-AG. The bioconversion procedures of the substrate arctiin by strain AUH-JLD56 and strain AUH-JLD49s were summarized in Figure 3.

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RESULTS Identification of Human Intestinal Bacterium Strain AUH-JLD49s. In the effort to search for a single bacterium that shows activity to metabolize 3′-DMAG, we incubated each single bacterium with the substrate 3′-DMAG under anaerobic conditions. One single bacterium with 3′-DMAG bioconversion activity was isolated by the HPLC method. The newly isolated bacterium, which we named strain AUH-JLD49s, was a rodshaped, non-spore-forming, non-motile, and Gram-negative obligately anaerobic bacterium. Strain AUH-JLD49s did not ferment the carbohydrates in API kits. Neither indole nor H2S was produced by this strain. The 16S rRNA gene sequence 4053

DOI: 10.1021/acs.jafc.7b00114 J. Agric. Food Chem. 2017, 65, 4051−4056

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Journal of Agricultural and Food Chemistry H NMR (CDCl3, 400 MHz): δ 2.41−2.55 (2H, m, H-3, Ha7″), 2.59−2.62 (2H, m, H-2, Hb-7″), 2.91 (1H, dd, J = 13.90 and 6.80 Hz, Ha-7′), 2.98 (1H, dd, J = 13.90 and 5.30 Hz, Hb7′), 3.82 (3H, s, −OCH3), 3.85 (3H, s, −OCH3), 3.90 (1H, dd, J = 9.18 and 7.72 Hz, Ha-4), 4.14 (1H, dd, J = 8.70 and 7.12 Hz, Hb-4), 6.48 (1H, d, J = 2.18 Hz, H-2″), 6.51−6.58 (1H, dd, J = 7.99 and 1.94 Hz, H-6″), 6.62 (1H, t, J = 2.18 Hz, H-2′), 6.68 (1H, brd, H-6′), 6.71 (1H, dd, H-4′), 6.82 (1H, d, J = 7.99 Hz, H-5″), 7.14 (1H, t, J = 7.72 Hz, H-5′). 13 C NMR (CDCl3, 100 MHz): δ 179.1 (C-1), 156.1 (C-3′), 149.0 (C-3″), 147.8 (C-4″), 139.4 (C-1′), 130.5 (C-1″), 129.8 (C-5′), 121.5 (C-6′), 120.7 (C-6″), 116.2 (C-2′), 114.0 (C-4′), 111.8 (C-2″), 111.4 (C-5″), 71.5 (C-4), 55.9 (−Me), 55.8 (−Me), 46.2 (C-2), 41.3 (C-3), 38.2 (C-7″), 34.7 (C-7′). Chirality Study of Biosynthesized DMDH-AG. In this study, we observed that the metabolite DMDH-AG made the plane-polarized light rotate. Our results indicated that biosynthesized DMDH-AG from 3′-DMAG by strain AUHJLD49s is an optically active substance. The specific rotation ([α]25 D ) of the metabolite DMDH-AG was −58.2° (c = 0.79 × 10−3, MeOH). Bioconversion Kinetics of 3′-DMAG by Strain AUHJLD49s. The study on the biotransformation kinetics of 3′DMAG indicated that bacterium strain AUH-JLD49s began to convert 3′-DMAG to DMDH-AG after 3 h of inoculation. During bacterial growth, production of DMDH-AG gradually increased with time until 15 h, by which the metabolite DMDH-AG reached its highest concentration in the medium at 0.31 mmol/L. The average conversion percentage of the substrate 3′-DMAG by strain AUH-JLD49s was 96.8% (Figure 4). 1

Figure 5. Bioconversion capacity of strain AUH-JLD49s after 3 days of incubation with different concentrations of the substrate 3′-DMAG (black bars) and the metabolite DMDH-AG (gray bars).

to the bacterial cells of strain AUH-JLD49s even when we used a high concentration of the substrate 3′-DMAG or that of the product DMDH-AG (data not shown). We surmised that exposure to a high concentration of 3′-DMAG may result in expression changes of particular proteins, which subsequently influenced the bioconversion capacity of bacterium strain AUHJLD49s. Antimicrobial Activity of DMDH-AG. To know whether the metabolite DMDH-AG is of any cytotoxicity, we tested the antimicrobial activity of arctigenin, 3′-DMAG, and the metabolite DMDH-AG (data not shown). However, none of the tested compounds showed any antimicrobial activity to S. aureus (ATCC27217), S. paratyphi (CMCC50001), or E. coli (CICC10372). Growth Inhibitory Effect of DMDH-AG on Different Cancer Cell Lines. In this study, we found that DMDH-AG, the metabolite of 3′-DMAG, inhibited the growth of both human colon cancer cell line HCT116 and human breast cancer cell line MDA-MB-231 (Figure 6). The inhibitory rates

Figure 4. Biotransformation kinetics of 3′-DMAG (●) by strain AUHJLD49s in BHI liquid medium, with the initial concentration of 3′DMAG of 0.4 mmol/L, and the metabolite DMDH-AG (○).

Bioconversion Capacity of 3′-DMAG by Strain AUHJLD49s. To know the conversion capacity of the newly isolated bacterium, we incubated the bacterium strain AUH-JLD49s with different concentrations of the substrate 3′-DMAG. The results showed that the average conversion percentage of 3′DMAG was 92.8% when the concentration of the substrate 3′DMAG was lower than 1.2 mmol/L. However, when the concentration of 3′-DMAG was increased to 1.6 mmol/L, the conversion capacity of 3′-DMAG by strain AUH-JLD49s sharply dropped to 18.0% (Figure 5). A possible explanation for this phenomenon may be due to the toxicity of the high concentration of the substrate or the elevated concentration of the product. However, we did not observe any inhibitory effect

Figure 6. Inhibitory effect of DMDH-AG on the growth of human colon cancer cell line HCT116 (black bars) and human breast cancer cell line MDA-MB-231 (gray bars).

of DMDH-AG on HCT116 and MDA-MB-231 cell lines were 54.3 and 45.7%, respectively, when the concentration of DMDH-AG was 200 μmol/L. In addition, we observed that exposure to DMDH-AG resulted in protein expression changes in both HCT116 and MDA-MB-231 cell lines (data not shown). The growth inhibitory effect of DMDH-AG on cancer 4054

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exert strong anticarcinogenic activity.8,11,17−19 However, to date, reports on anticarcinogenic activity of microbial metabolites of arctiin are still quite rare. Professor Hattori and his team had obtained DMDH-AG (i.e., compound 4) by anaerobic incubation of arctiin with human intestinal flora. They tested the anticarcinogenic activity of DMDH-AG at extremely low concentrations (form 10−8 to 10−5 mol/L). According to Hattori et al., DMDH-AG significantly stimulated cell growth of MCF-7 at the concentration of 10−8 mol/L (p < 0.05). However, Hattori et al. did not detect the anticarcinogenic activity of DMDH-AG at high concentrations. It was probably because Hattori et al. could not obtain enough amount of DMDH-AG by anaerobic incubation of arctiin with human intestinal flora. Here, in the present study, we applied high concentrations of DMDH-AG to different cancer cell lines. Our study provides the first evidence that DMDH-AG had an obvious inhibitory effect on the growth of both human colon cancer cell line HCT116 and human breast cancer cell line MDA-MB-231 at higher concentrations (≥50 μmol/L). However, it is worth noticing here that the concentrations of DMDH-AG applied to the cancer cell lines were quite high. Although high concentrations of DMDH-AG did not show any cytotoxicity to bacterial cells in this study, the potential toxicity of DMDH-AG to animals should be evaluated. Studies on the distribution and concentration of DMDH-AG in different tissues, in vivo metabolism after administration, and conjugation with possible conjugates are also necessary. On the other hand, our previous study has demonstrated that 3′-DMAG, the microbial bioconversion metabolite of arctigenin by bacterium strain AUH-JLD56, was of strong DPPH radical scavenging activity.14 However, the study on the antioxidative activity showed that the DPPH radical scavenging activity of DMDH-AG was significantly decreased in comparison to that of arctigenin and 3′-DMAG (Figure 7) in the present study. Our results demonstrated that the hydroxyl group of 3′-DMAG at C-4′ is a key functional group for maintaining high DPPH radical scavenging activity.

cells was due to the ability of DMDH-AG to block the cell cycle and to induce apoptosis. The anticarcinogenic mechanisms for DMDH-AG will be investigated in details in our future studies.

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DISCUSSION The metabolism of arctiin by intestinal microflora has been known for over 2 decades; however, specific bacteria responsible for the bioconversion of arctigenin or the microbial metabolites of arctigenin are still quite limited. In 2007, the first single bacterium strain Eubacterium sp. ARC-2, which is capable of bioconverting arctigen into seven different metabolites, including three monodesmethylarctigenins, three didesmethylarctigenins, and dihydroxyenterolactone, was isolated from human feces by Professor Hattori and his team.13 In 2013, we reported the second single human intestinal bacterium, which we named Blautia sp. AUH-JLD56, capable of bioconverting arctigenin to 3′-DMAG solely and efficiently.14 According to the available references, other single bacteria responsible for the bioconversion of arctigenin or the microbial metabolites of arctigenin have not been isolated. In the present study, for the first time, we isolated one single bacterium strain Eggerthella sp. AUH-JLD49s, which is capable of bioconverting 3′-DMAG to DMDH-AG solely and efficiently under anaerobic conditions. Bacterium strain Eubacterium sp. ARC-2 and strain Blautia sp. AUH-JLD56 mentioned previously showed the demethylation activity in lignans. However, in the present study, the newly isolated bacterium strain Eggerthella sp. AUH-JLD49s is capable of dehydroxylation of lignan, such as 3′-DMAG. In fact, specific bacteria capable of dehydroxylating plant lignan secoisolariciresinol diglucoside (SDG) have been reported previously. For example, Eubacterium sp. strain SDG-2 (EF413638), isolated by Wang et al. in 2000,19 was a bacterium capable of dehydroxylation in the bioconversion of SDG to enterodiol and enterolactone. It is worth noticing here that Eubacterium sp. strain SDG-2, was reclassified to Eggerthella sp. strain SDG-2 by a phylogenetic position based on 16S rRNA gene sequence analysis.20 Moreover, Jin et al. isolated a different bacterium, designated strain ARC-1 (EF413639), capable of dehydroxylating (−)-dihydroxyenterodiol to (−)-enterodiol from human feces in 2007. Strain ARC-1 showed 95% similarity with Denitrobacterium CCUG45665 (AJ518870) and 92% similarity with that of Eggerthella sp. strain SDG-2 in 16S rRNA gene sequence. Similarly, in our previous study, human intestinal bacterium strain Julong732, capable of dehydroxylating tetrahydrodaidzein to equol under anaerobic conditions, showed 92.8% similarity with Eggerthella hongkongenis HKU10 (AY288517) in 16S rRNA gene sequence.21 The microbial biotransforming studies carried out thus far seem to support that dehydroxylation is a common catalytic reaction to Eggerthella species. However, some results did not support this point of view. According to Clavel et al., although the dehydroxylation activity was observed for several strains of Eggerthella lenta, however, E. lenta SECO-Mt75m2 did not show any bioconversion activity toward secoisolariciresinol (SECO) when being incubated alone in Mt-6 medium.22 Therefore, the present evidence are still quite limited and warrant more studies to clarify whether dehydroxylation capacity is a special trait of some particular Eggerthella species or common to Eggerthella species. Numerous studies have demonstrated that arctiin or its aglycone arctigenin are of various bioactivities (see the Introduction). In recent years, more findings have revealed that arctigenin and chemically synthesized arctigenin analogues

Figure 7. DPPH radical scavenging capacity of arctigenin (black bars), 3′-DMAG (light gray bars), and DMDH-AG (dark gray bars) at different concentrations.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 86-312-7528257. Fax: 86-312-7528265. E-mail: [email protected]. ORCID

Xiu-Ling Wang: 0000-0001-8743-671X 4055

DOI: 10.1021/acs.jafc.7b00114 J. Agric. Food Chem. 2017, 65, 4051−4056

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Journal of Agricultural and Food Chemistry Author Contributions

(14) Liu, M. Y.; Li, M.; Wang, X. L.; Liu, P.; Hao, Q. H.; Yu, X. M. Study on human intestinal bacterium Blautia sp. AUH-JLD56 for the conversion of arctigenin to (−)-3′-desmethylarctigenin. J. Agric. Food Chem. 2013, 61, 12060−12065. (15) Liang, X. L.; Wang, X. L.; Li, Z.; Hao, Q. H.; Wang, S. Y. Improved in vitro assays of superoxide anion and 1,1-diphenyl-2picrylhydrazyl (DPPH) radical-scavenging activity of isoflavones and isoflavone metabolites. J. Agric. Food Chem. 2010, 58, 11548−11552. (16) Liang, X. L.; Li, M.; Li, J.; Wang, X. L. Equol induces apoptosis in human hepatocellular carcinoma SMMC-7721 cells through the intrinsic pathway and the endoplasmic reticulum stress pathway. AntiCancer Drug 2014, 25, 633−640. (17) Takasaki, M.; Konoshima, T.; Komatsu, K.; Tokuda, H.; Nishino, H. Antitumor-promoting activity of lignans from the aerial part of Saussurea medusa. Cancer Lett. 2000, 158, 53−59. (18) Kang, K.; Lee, H. J.; Kim, C. Y.; Lee, S. B.; Tunsag, J.; Batsuren, D.; Nho, C. W. The chemopreventive effects of Saussurea salicifolia through induction of apoptosis and phase II detoxification enzyme. Biol. Pharm. Bull. 2007, 30, 2352−2359. (19) Wang, L. Q.; Meselhy, M. R.; Li, Y.; Qin, G. W.; Hattori, M. Human intestinal bacteria capable of transforming secoisolariciresinol diglucoside to mammalian lignans, enterodiol and enterolactone. Chem. Pharm. Bull. 2000, 48, 1606−1610. (20) Jin, J. S.; Zhao, Y. F.; Nakamura, N.; Akao, T.; Kakiuchi, N.; Min, B. S.; Hattori, M. Enantioselective dehydroxylation of enterodiol and enterolactone precursors by human intestinal bacteria. Biol. Pharm. Bull. 2007, 30, 2113−2119. (21) Wang, X. L.; Hur, H. G.; Lee, J. H.; Kim, K. T.; Kim, S. I. Enantioselective synthesis of S-equol from dihydrodaidzein by a newly isolated anaerobic human intestinal bacterium. Appl. Environ. Microbiol. 2005, 71, 214−219. (22) Clavel, T.; Henderson, G.; Engst, W.; Doré, J.; Blaut, M. Phylogeny of human intestinal bacteria that activate the dietary lignin secoisolariciresinol diglucoside. FEMS Microbiol. Ecol. 2006, 55, 471− 478.

†

Ye Wang, Fei Yu, and Ming-Yue Liu contributed equally to this work. Funding

This work was supported by the Applied and Fundamental Research Plan in Hebei Province of China (Key Basic Research Project 16962504D) and the National Natural Science Foundation of China (NSFC, Grant 31670057). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED 3′-DMAG, 3′-desmethylarctigenin; DMDH-AG, 3′-desmethyl4′-dehydroxyarctigenin; DPPH, 1,1-diphenyl-2-picrylhydrazyl; BHI, brain heart infusion; ESI−MS, electrospray ionization mass spectrometry

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REFERENCES

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DOI: 10.1021/acs.jafc.7b00114 J. Agric. Food Chem. 2017, 65, 4051−4056