Metabolism of Kaempferia parviflora Polymethoxyflavones by Human

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Metabolism of Kaempferia parviflora Polymethoxyflavones by Human Intestinal Bacterium Bautia sp. MRG-PMF1 Mihyang Kim, Nayoung Kim, and Jaehong Han* Metalloenzyme Research Group and Department of Systems Biotechnology, Chung-Ang University, Anseong 456-756, Republic of Korea S Supporting Information *

ABSTRACT: Poylmethoxyflavones (PMFs) are major bioactive flavonoids, which exhibit various biological activities, such as anticancer effects. The biotransformation of PMFs and characterization of a PMF-metabolizing human intestinal bacterium were studied herein for the first time. Hydrolysis of aryl methyl ether functional groups by human fecal samples was observed from the bioconversion of various PMFs. Activity-guided screening for PMF-metabolizing intestinal bacteria under anaerobic conditions resulted in the isolation of a strict anaerobic bacterium, which was identified as Blautia sp. MRG-PMF1. The isolated MRGPMF1 was able to metabolize various PMFs to the corresponding demethylated flavones. The microbial conversion of bioactive 5,7-dimethoxyflavone (5,7-DMF) and 5,7,4′-trimethoxyflavone (5,7,4′-TMF) was studied in detail. 5,7-DMF and 5,7,4′-TMF were completely metabolized to 5,7-dihydroxyflavone (chrysin) and 5,7,4′-trihydroxyflavone (apigenin), respectively. From a kinetics study, the methoxy group on the flavone C-7 position was found to be preferentially hydrolyzed. 5-Methoxychrysin, the intermediate of 5,7-DMF metabolism by Blautia sp. MRG-PMF1, was isolated and characterized by nuclear magnetic resonance spectroscopy. Apigenin was produced from the sequential demethylation of 5,7,4′-TMF, via 5,4′-dimethoxy-7-hydroxyflavone and 7,4′-dihydroxy-5-methoxyflavone (thevetiaflavone). Not only demethylation activity but also deglycosylation activity was exhibited by Blautia sp. MRG-PMF1, and various flavonoids, including isoflavones, flavones, and flavanones, were found to be metabolized to the corresponding aglycones. The unprecedented PMF demethylation activity of Blautia sp. MRG-PMF1 will expand our understanding of flavonoid metabolism in the human intestine and lead to novel bioactive compounds. KEYWORDS: biotransformation, Balutia, demethylation, human intestinal bacteria, polymethoxyflavone

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INTRODUCTION

Rhizome of Kaempferia parviflora, known as krachaidum, is a functional food popularly consumed in southeast Asia. PMFs were also found to be the major active compounds in krachaidum.9 The PMFs in krachaidum were reported to be responsible for the anticancer, steroid 5α-reductase inhibition, and anti-inflammatory activities.10 In this study, a mixture of PMFs obtained by ethanol extraction of krachaidum was reacted with human fecal samples to monitor biotransformation. After evaluation of the bioconversion of the PMFs, the human intestinal bacterium with the ability to metabolize the PMFs was isolated and characterized. To obtain the detailed PMF metabolism, biotransformation kinetics of 5,7-dimethoxyflavone (5,7-DMF) and 5,7,4′-trimethoxyflavone (5,7,4′-TMF) was carried out. The ability of the isolated bacterium to participate in the biotransformation of other flavonoids was also examined, and flavonoid deglycosylation activity and demethylation activity were found.

In general, the study of biotransformation by human intestinal bacteria is essential to evaluate the actual health-promoting effects of the bioactive compounds present in foods.1 Therefore, the microbial biotransformation of secondary metabolites in herbal medicine, functional foods, and dietary supplements has drawn significant attention over the years. For example, the biotransformation of soy isoflavone daidzin by human intestinal bacteria has been extensively studied to explain the strong phytoestrogenic and anticancer effects observed in vivo.2 As a result, numerous intestinal bacteria and metabolites contributing to daidzin metabolism were isolated and characterized.3 Later, the strongly phytoestrogenic S-equol,4 produced by sequential reductions of daidzein,5 was found to be responsible for the in vivo biological activities. Poylmethoxyflavones (PMFs), mainly found in Citrus fruits, such as orange and tangerine, are known to exhibit various biological activities, including anticancer, antiviral, anti-inflammatory, antithrombogenic, and antiatherogenic effects.6 Although the biological activity of PMFs and their metabolism in the body7,8 have been extensively studied via in vitro and in vivo assays, to the best of our knowledge, the metabolism of PMFs by human intestinal bacteria has never been studied. As evident from soy isoflavone metabolism, characterization of PMF metabolism in the human intestine is important to understand the bioavailability, pharmacokinetics, and biological activity of PMFs in the body. © XXXX American Chemical Society

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MATERIALS AND METHODS

Chemicals and Culture Media. The mixture of PMFs was prepared by dissolving dried ethanol extract of krachaidum in N,Ndimethylformaide (DMF).9 In detail, air-dried krachaidum (5 g, powder), purchased from a local market in Khon Kaen, Thailand, was Received: August 25, 2014 Revised: November 24, 2014 Accepted: December 1, 2014

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Bacterial Growth and pH Changes. The bacterial culture stored in liquid nitrogen was thawed in an anaerobic chamber, and 100 μL was then transferred to 2 mL of GAM broth. When the optical density at 600 nm (OD600) reached 0.7, 400 μL of the cell culture was transferred to 20 mL of fresh GAM broth. Bacterial growth was monitored by measuring OD600 with the ultraviolet−visible (UV−vis) spectrophotometer S-3100 (Scinco, Seoul, Korea), and the pH of the medium was also measured by the Laqua pH meter F-74BW (Horiba Scientific, Kyoto, Japan) every 30 min. Biotransformation of 5,7-DMF and 5,7,4′-TMF. 5,7-DMF and 5,7,4′-TMF were used for the study of biotransformation kinetics in an anaerobic chamber and the identification of the intermediates. When the growth of the bacteria in GAM broth reached OD600 of 0.8, each substrate was added to the reaction media (1.5 mL, 100 μM) to initiate biotransformation. Aliquots of the reaction mixtures (100 μL) were taken and allocated into Eppendorf tubes every 3 h. The allocated media were extracted with 1 mL of ethyl acetate, and the supernatant (800 μL), collected after vortexing and centrifugation (10770g for 10 min), was dried under vacuum. The residue was dissolved in 100 μL of methanol and analyzed by HPLC. The solvent gradient profile for 5,7-DMF biotransformation started with 53% solvent B for 1 min and then increased from 53 to 55% solvent B over 14 min and from 55 to 70% solvent B over 3 min. The solvent gradient profile for 5,7,4′-TMF biotransformation started with 45% solvent B for 1 min and then increased from 45 to 55% solvent B over 9 min, from 55 to 61% solvent B over 3 min, and finally from 61 to 70% over 2 min. Structural Analyses of 5,7-DMF and 5,7,4′-TMF Metabolites. 5,7-DMF and 5,7,4′-TMF metabolites were analyzed by a Dionex Ultimate 3000 HPLC system (Thermo Scientific, Waltham, MA) equipped with a C18 reversed-phase column (Kinetex, 100 × 2.1 mm, 1.7 μm, Phenomenex, Torrance, CA) and a diode array detector (DAD). A Thermo Fisher Scientific LCQ fleet instrument (Thermo Scientific, Waltham, MA) was coupled to the HPLC system for electrospray ionization mass spectrometry (ESI−MS) analysis. The mobile phase was a gradient of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The initial composition of the mobile phase was 33% solvent B for 1 min. In linear gradients, the composition changed to 45% solvent B over 2 min and then 50% solvent B over 8 min, where it stayed for 10 min at the flow rate of 0.2 mL/min. ESI−MS analyses were performed in positive-ion mode within the m/z range of 50−500 and processed with Xcalibur software (Thermo Scientific, Waltham, MA). ESI conditions: spray voltage, 5.4 kV; sheath gas, 15 arbitrary units; auxiliary gas, 5 arbitrary units; heated capillary temperature, 275 °C; capillary voltage, 27 V; and tube lens, 100 V. For the isolation of intermediate A produced during the biotransformation of 5,7-DMF, 1 mL of GAM medium containing MRG-PMF1 (OD600 of 0.6) was added to 200 mL of GAM medium. When the OD600 of the GAM medium reached 0.8, 5,7-DMF (10 mg, 5 mL of 5 mM DMF) was added to the reaction medium and reacted in an anaerobic chamber. The conversion of 5,7-DMF was monitored by TLC analysis of the medium. After 58 h, the reaction medium was centrifuged (10770g for 20 min) and the supernatant was extracted with EtOAc (100 mL × 3). The combined organic layer was dried with anhydrous MgSO4, and the organic solvent was removed under reduced pressure by a rotatory evaporator. After vacuum desiccation of the residue, the fraction containing intermediate A (2 mg) was isolated by vacuum liquid chromatography.14 Intermediate A was further purified by preparative TLC (0.5 mg, isolation yield of 2.4%), and the proton nuclear magnetic resonance (1H NMR) spectrum was recorded in MeOH-d4 on a Varian VNS (600 MHz, Palo Alto, CA). Chemical shift (δ) was reported in parts per million (ppm). Intermediate A (5methylchrysin): UV (λmax, nm), 264, 314; 1H NMR (600 MHz, CD3OD) δ, 7.92 (m, 2H, H-2′, H-6′), 7.52 (m, 2H, H-3′, H-5′), 7.51 (m, H, H-4′), 6.64 (s, 1H, H-3), 6.56 (d, 1H, J = 2.2 Hz, H-6), 6.41 (d, 1H, J = 2.2 Hz, H-8), 3.96 (s, 3H, −OCH3). Reactivity Study of PMF-Metabolizing Bacterium. To study the substrate specificity of flavonoid biotransformation, the isolated bacterium was reacted with 100 μM of various substrates. In detail,

extracted with ethanol (100 mL) after soaking at room temperature for 24 h. The ethanol solution was filtered through filter paper, and then the filtrate was then evaporated under reduced pressure at 40 °C. The purple residue after vacuum desiccation resulted in a gummy product (237 mg, 4.7%). 5,7-DMF and 5,7,4′-TMF were isolated according to the published method.11 Flavonoids used for the reactivity study were purchased from Indofine Chemical Company (Hillsborough, NJ). Gifu anaerobic medium (GAM) from Nissui Pharmaceutical Co. (Tokyo, Japan) was used for isolation and growth media. GAM broth was prepared according to the instructions of the manufacturer, and GAM plates were prepared by the addition of 1.5% agar to the GAM broth. PMF Biotransformation with Fecal Samples. The experiment was performed in compliance with the Bioethics and Safety Act enforced by the Korean government and the policies of the ChungAng University Institutional Research Board (CAU-IRB). Fresh fecal samples from a healthy 34-year-old female were collected directly once in deoxygenated GAM broth (4 mL), covered with sterilized mineral oil, and placed immediately in an anaerobic chamber (5% CO2, 10% H2, and 85% N2) at 37 °C. The inoculated medium was stabilized for 2 h after filtration through sterilized gauze, and a portion of the medium was stored in liquid N 2 for further investigation. For the biotransformation of PMFs, 100 μL of the medium was inoculated in GAM broth (2 mL) and grown for 17 h at 37 °C. PMFs obtained from the ethanol extraction of krachaidum were dissolved in DMF at the concentration of 3 mg/mL, and then 2 μL was added to the 200 μL of bacterial culture and reacted for 3 days at 37 °C. After 3 days, ethyl acetate (1 mL) was added to each tube to stop the reaction and the mixture was vortexed for 20 s. After centrifugation at 10770g for 10 min, 800 μL of the organic supernatant was taken and the solvent was evaporated with a centrifugal evaporator (Micro-Cenvac NB-503CIR, N-Biotek, Gyeonggi, Korea). The residue was redissolved in 10 μL of methanol for further analysis. For thin-layer chromatography (TLC) analysis, 1 μL each of the methanol extract and the PMFs were applied on a silica gel TLC plate (Merck silica gel 60 F254). The TLC plate was developed in a mixture of hexanes/acetone (1:1, vol/vol). For HPLC analysis, the Finnigan Surveyor Plus high-performance liquid chromatography (HPLC) system (Thermo Scientific, Waltham, MA) equipped with a photodiode array detector (PDA Plus) and a C18 reverse-phase column (Hypersil GOLD 5 μm, 4.6 × 100 mm, Thermo Scientific, Waltham, MA) was employed. The mobile phase was composed of 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol (B). The solvent gradient profile started with 45% solvent B for 1 min and then increased from 45 to 55% solvent B over 14 min, from 55 to 60% solvent B over 9 min, and from 60 to 65% solvent B over 8 min, after which it decreased to 60% solvent B for 5 min at the flow rate of 1.0 mL/min. Isolation of PMF-Metabolizing Bacterium and Culture Conditions. Strain MRG-PMF1 was isolated from the fecal samples, which showed PMF bioconversion activity. The mixed cell culture (360 μL) prepared from fecal samples was diluted to 10−4 with GAM broth after the addition of 3.6 μL of the PMF solution (3.0 mg/mL in DMF). The diluted solution was spread on GAM agar plates for isolation. After incubation for 2 days at 37 °C, single colonies were picked from the plates, transferred into 500 μL of GAM broth, and incubated overnight. The PMF-metabolizing activity was checked by TLC and HPLC, as described above. Subcultures and activity checks were repeated until a pure bacterium was obtained. 16S rRNA Gene Sequencing and Phylogeny Analysis. 16S rRNA gene sequence analysis of the isolated bacterium was performed for identification of the bacterial strain. The bacterium was streaked on GAM agar plates in an anaerobic chamber and grown for 2 days at 37 °C. The 16S rRNA gene was amplified by PCR with universal bacteria primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′), and sequencing was performed via the sequencing service at Solgent, Inc. (Daejeon, Korea). The 16S rDNA sequences were analyzed using the BLASTN tool of the National Center for Biotechnology Information (NCBI) to identify bacterial strains with sequence similarity.12 Phylogenetic trees were constructed by the neighbor-joining method using the MEGA 6 program.13 B

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bacteria were grown to OD600 of 0.7 in 495 μL of GAM broth in an anaerobic chamber at 37 °C, after which 5 μL of each substrate (10 mM in DMF) was added. After incubation for 15 min and 1, 2, 3, and 7 days, 100 μL of reaction media was taken from each culture and 1 mL of ethyl acetate was added to stop the biotransformation. The mixture was then vortexed for 20 s and centrifuged (10770g for 10 min). The supernatant (800 μL) was taken and dried under reduced pressure. The residue was dissolved in 100 μL of methanol, and a 10 μL aliquot was injected for HPLC. The solvent gradient profile started with 20% solvent B for 1 min and then increased from 20 to 50% solvent B over 9 min and from 50 to 70% solvent B over 5 min at the flow rate of 1.0 mL/min. The reaction products were detected and identified from comparison to the expected standard flavonoids.

The PMF biotransformation by strain MRG-PMF1 was studied by HPLC analysis. The ethanol extract of krachaidum is known to contain 11 PMFs, for which TLC and HPLC analyses of the compositional changes were reported before.11 The mixture of PMFs used herein for the reaction with fecal samples contained 5,7-DMF (10%), 5,7,4′-TMF (12%), and 3,5,7,3′,4′pentamethoxyflavone (3,5,7,3′,4′-PMF, 20%) as the major compounds. Along with these three compounds, 3,5,7,4′tetramethoxyflavone (3,5,7,4′-TMF, 6%) and 5-hydroxy-3,7,4′trimethoxyflavone (5-OH-3,7,4′-TMF, 6%) were also abundant.9 After the 3 day reaction with MRG-PMF1, biotransformation products of the PMFs were analyzed by HPLC (Figure 2). Chrysin (5,7-dihydroxyflavone) and apigenin (5,7,4′trihydroxyflavone) were identified by comparing their retention times and UV spectra to the standard compounds. The other metabolites 3-O-methylquercetin and 3-O-methylkaempferol were assigned on the basis of the HPLC retention times and published UV spectra.15 From analysis of the biotransformation of PMFs by MRG-PMF1 (Figure 3), it was found that the methyl group at the C-3 position of flavone was not hydrolyzed even after 3 days of incubation. A growth curve of the isolated bacteria in GAM medium under anaerobic conditions was obtained by monitoring the changes in OD600. Blautia sp. MRG-PMF1 reached the stationary phase in 9 h after a lag phase of 3.5 h at 37 °C (see Figure S2 of the Supporting Information). After the stationary phase, a decrease in OD600 was observed. The growth of MRG-PMF1 showed good correlation with the pH changes of the media. The initial pH of the medium was pH 6.9, and a decrease to pH 5.4 was observed during exponential cell growth of the strain. Biotransformation of 5,7-DMF and 5,7,4′-TMF and Kinetics Study. Biotransformation of 5,7-DMF and 5,7,4′TMF by Blautia sp. MRG-PMF1 produced chrysin and apigenin, respectively, through the hydrolysis of methyl aryl ether functional groups of the substrates (Figure 2). In the time-dependent study, Blautia sp. MRG-PMF1 was found to metabolize completely 0.1 mM 5,7-DMF and 5,7,4′-TMF within 30 h (see Figure S3 of the Supporting Information). Furthermore, biotransformation intermediates were observed for both PMFs via TLC (see Figure S4 of the Supporting Information) and HPLC (Figure 4) analyses. During the biotransformation of 5,7-DMF, an intermediate A was identified on the HPLC chromatogram. The HPLC retention time and UV spectrum of intermediate A were different from 7methoxychrysin, another PMF found in krachaidum extract.11 The UV absorption at 264 and 314 nm was the same as the reported data for 5-methoxychrysin.16 The molecular ion [M + H]+ peak of intermediate A was found at m/z 269.23 from the ESI−MS spectrum, which was smaller than that of 5,7-DMF (m/z 283.24) by 14 Da and larger than that of chrysin by 14 Da (see Figure S5 of the Supporting Information). Between two possible demethylation intermediates of 5,7-DMF, intermediate A was assigned as 5-methoxychrysin by comparison of the HPLC retention times and electronic spectra. The molecular structure of intermediate A was confirmed by 1H NMR spectroscopy. Two methoxy groups of 5,7-DMF are found at δ 3.96 ppm (5-OCH3) and δ 3.92 ppm (7-OCH3),11 but the peak corresponding to the 7-methoxy group disappeared from the 1 H NMR spectrum of intermediate A. Two metabolic intermediates B and C were observed during the biotransformation of 5,7,4′-TMF, which were assigned as 7-OH-5,4′-DMF and thevetiaflavone, respectively (Figure 5).

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RESULTS PMF Biotransformation by Human Fecal Sample and the Isolation of Strain MRG-PMF1. The PMF mixture obtained by ethanol extraction of krachaidum was completely metabolized by the fecal sample obtained from a healthy 34year-old female (see Figure S1 of the Supporting Information). From the diluted fecal samples showing PMF-metabolizing activity, 75 single colonies were isolated and further tested for activity. An obligatory anaerobic Gram-positive coccobacilli, which can metabolize PMFs was isolated under anaerobic conditions (see Figure S1 of the Supporting Information) and successfully subcultured on GAM plates without losing the PMF-metabolizing activity. The 16S rRNA gene of the isolate was sequenced for identification and deposited in GenBank under accession number KJ078647. The partial 16S rRNA gene sequence (1361 bp) was most closely related with Blautia producta ATCC 27340T (X94966), showing 99.85% similarity. Other strains in the family Lachnospiraceae and genus Blautia, particularly Blautia coccoides ATCC 29236T (M59090, 99.70% similarity), Blautia stercoris GAM6-1T (HM626177, 95.39% similarity), Blautia hansenii DSM 20583T (ABYU01000028, 95.37% similarity), Blautia stercoris SJTU C 02 47 (EF403927, 95.30% similarity), Blautia schinkii BT (X94965, 95.13% similarity), Blautia faecis M25T (HM626178, 95.00% similarity), and Blautia glucerasea HFTH-1T (AB439724, 94.77% similarity), also showed high sequence similarity to the isolated bacterium (Figure 1). Therefore, the isolated PMF-metabolizing anaerobic bacterium was identified as Blautia sp. MRGPMF1.

Figure 1. Molecular phylogenetic tree analysis of Blautia sp. MRGPMF1. Numbers at the branching points indicate bootstrap values greater than 50% based on 1000 samplings. Escherichia coli was used as an out group. Bar, 0.02 nucleotide substitutions per position. C

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Figure 2. HPLC analysis of PMF metabolism by fecal sample. The solid and dotted lines represent PMFs before and after 3 days of reaction, respectively. (Inset) PDA UV spectra and the retention times of metabolites from the 3 day reaction data were used for the identification of 3-Omethylquercetin and 3-O-methylkaempferol. Abbreviations: 5,7,3′,4′-TMF, 5,7,3′,4′-tetramethoxyflavone; 3,5,7,3′,4′-PMF, 3,5,7,3′,4′-pentamethoxyflavone; 3,5,7-TMF, 3,5,7-trimethoxyflavone; 3,5,7,4′-TMF, 3,5,7,4′-tetramethoxyflavone; 5-OH-3,7,3′,4′-TMF, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone; 5-OH-7-MF, 5-hydroxy-7-methoxyflavone; 5-OH-7,4′-DMF, 5-hydroxy-7,4′-dimethoxyflavone; 5-OH-3,7-DMF, 5-hydroxy-3,7-dimethoxyflavone; and 5-OH-3,7,4′-TMF, 5-hydroxy-3,7,4′-trimethoxyflavone.

Figure 3. Bioconversion of the PMF mixture by Blautia sp. MRG-PMF1.

Blautia sp. MRG-PMF1 also showed flavone O-glucose and flavone O-rutinose hydrolysis activities. Apigetrin and rutin were converted to apigenin and quercetin, respectively. Various isoflavones, except for puerarin, were also metabolized by Blautia sp. MRG-PMF1. For example, daidzin, genistin, glycitin, ononin, and sissotrin were converted to their aglycones, daidzein, genistein, glycitein, formononetin, and biochanin A, respectively, because of the O-glucose and O-methyl hydrolysis

Biotransformation of Other Flavonoids by Blautia sp. MRG-PMF1. Blautia sp. MRG-PMF1 was tested for the ability to metabolize other flavonoids (Table 1). The metabolites, except for eriodictyol (5,7,3′,4′-tetrahydroxyflavanone), were identified by comparison to the retention times and UV spectra of the standard compounds by HPLC. The other two metabolites were confirmed by comparing the UV spectrum to the published data.17 Along with PMF-metabolizing activity, D

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anticancer and anti-inflammatory properties. To investigate PMF metabolism in the human intestine, PMFs were reacted with human fecal samples herein for the first time. Demethylated metabolites of the PMFs from the fecal samples of rats to which krachaidum extract was orally administered were reported previously.8 Although PMFs were considered to be stable bioactive compounds because of the chemical stability of the methyl aryl ether functional group, the robust biotransformation of PMFs was observed by a human fecal sample at room temperature under anaerobic conditions. The chemical cleavage of ether bonds usually requires high temperatures in the presence of strong acid or base.22 As shown in Figure 2, the PMF biotransformation by MRG-PMF1 produced chrysin, apigenin, 3-methylkeampferol, and 3methylquercetin. It is interesting to note that these compounds are reported to be present in propolis.23 Therefore, different flavonoids were produced by demethylation of the PMFs, and new biological effects of these metabolites are expected to be observed in humans. In the case of nobiletin, the metabolites 3′-demethylnobiletin, 4′-demethylnobiletin, and 3′,4′-bisdemethylnobiletin, were reported to exhibit stronger anti-inflammatory and anticarcinogenic properties.24 From the time-dependent biotransformation of 5,7-DMF and 5,7,4′-TMF, the metabolic pathways of 5,7-DMF → 5methylchrysin → chrysin and 5,7,4′-TMF → 5,4′-dimethoxy7-hydroxyflavone → thevetiaflavone → apigenin were elucidated, respectively. Demethylation was faster for the 7-methoxy group and slower for the 5-methoxy group. From biotransformation of the mixture of PMF, Blautia sp. MRG-PMF1 was found not to be able to hydrolyze the 3-methoxy group of flavone. There are surprisingly few reports on the biotransformation intermediates identified herein, such as 5-methylchrysin, 5,4′-dimethoxy-7-hydroxyflavone, and thevetiaflavone. Most PMF metabolites and their intermediates were reported from bee products.23 In addition to the PMFs, various flavonoids were also observed to be biotransformed by Blautia sp. MRGPMF1 (Table 1). Through the tested substrates, it was found that MRG-PMF1 exhibited hydrolysis activity for methyl ether, O-glucose, and O-rutinose functional groups of flavone, isoflavone, and flavanone. The only exception of this finding was naringin, but it was not clear whether or not naringin metabolites have antibacterial activity against MRG-PMF1. Although bioinformatic approaches using high-throughput sequencing contribute significantly to the areas of food and clinical microbiology, it is evident from our previous work that the biotransformation of bioactive flavonoids by human intestinal bacteria can produce new bioactive compounds that may not exist in the human diet and that cannot be biosynthesized in the body.25 For example, the new metabolic intermediates, R-DHD,26 (3S,4R)-THD,27 and R-O-DMA28 were discovered from the biosynthetic study of S-equol. Besides, the isolation of new intestinal bacteria and characterization of metabolites are of great significance to the discovery of novel enzymes and leading bioactive compounds. Identification of bioactive metabolites produced by specific intestinal bacteria is also important and is a prerequisite to investigating microbiota−host interactions at the molecular level. Furthermore, the structural characterization of various metabolites formed from dietary flavonoids can provide valuable data for the metabolomic study in human microbiome research.29 The unprecedented PMF demethylation activity of Blautia sp. MRG-PMF1 will expand our understanding of

Figure 4. HPLC chromatograms (265 nm) of Blautia sp. MRG-PMF1 biotransformation products obtained from 5,7-DMF (18 h, above) and 5,7,4′-TMF (21 h, below).

Figure 5. Metabolic pathway of 5,7,4′-TMF biotransformation.

activities. Glycitein biotransformation by human fecal flora was reported to produce the metabolite 6,7,4′-trihydroxyisoflavone.18 However, strain MRG-PMF1 did not show Cglucoside-hydrolyzing activity.19 Flavone and isoflavone Cglucosides, such as puerarin and vitexin, were not metabolized by Blautia sp. MRG-PMF1. Flavanones with methoxy groups, hesperidin and hesperetin, were metabolized, but naringin was not.

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DISCUSSION Although dietary polyphenols, including flavonoids, are known to exhibit various biological activities, there is accumulating evidence that it is their metabolites, produced by human intestinal bacteria, which contribute more significantly to human health.20,21 PMFs are a group of bioactive flavonoids that exhibit a broad spectrum of biological activities, including E

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Table 1. Flavonoid Metabolism by Blautia sp. MRG-PMF1 substrate

product

activity

time

O-glucose hydrolysis O-rutinose hydrolysis