Demethylation of Polymethoxyflavones by Human Gut Bacterium

Using an anaerobic in vitro study, 12 PMFs, 5,7-dimethoxyflavone (5,7-DMF), 5-hydroxy-7-methoxyflavone (5-OH-7-MF), 3,5,7-trimethoxyflavone (3,5,7-TMF...
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Demethylation of Polymethoxyflavones by Human Gut Bacterium, Blautia sp. MRG-PMF1 Supawadee Burapan, Mihyang Kim, and Jaehong Han* Metalloenzyme Research Group and Department of Integrative Plant Science, Chung-Ang University, Anseong 17546, Korea S Supporting Information *

ABSTRACT: Polymethoxyflavones (PMFs) were biotransformed to various demethylated metabolites in the human intestine by the PMF-metabolizing bacterium, Blautia sp. MRG-PMF1. Because the newly formed metabolites can have different biological activities, the pathways and regioselectivity of PMF bioconversion were investigated. Using an anaerobic in vitro study, 12 PMFs, 5,7-dimethoxyflavone (5,7-DMF), 5-hydroxy-7-methoxyflavone (5-OH-7-MF), 3,5,7-trimethoxyflavone (3,5,7-TMF), 5-hydroxy3,7-dimethoxyflavone (5-OH-3,7-DMF), 5,7,4′-trimethoxyflavone (5,7,4′-TMF), 5-hydroxy-7,4′-dimethoxyflavone (5-OH-7,4′DMF), 3,5,7,4′-tetramethoxyflavone (3,5,7,4′-TMF), 5-hydroxy-3,7,4′-trimethoxyflavone (5-OH-3,7,4′-TMF), 5,7,3′,4′-tetramethoxyflavone (5,7,3′,4′-TMF), 3,5,7,3′,4′-pentamethoxyflavone (3,5,7,3′,4′-PMF), 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (5OH-3,7,3′,4′-TMF), and 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (5,3′-diOH-3,7,4′-TMF), were converted to chrysin, apigenin, galangin, kaempferol, luteolin, and quercetin after complete demethylation. The time-course monitoring of PMF biotransformations elucidated bioconversion pathways, including the identification of metabolic intermediates. As a robust flavonoid demethylase, regioselectivity of PMF demethylation generally followed the order C-7 > C-4′ ≈ C-3′ > C-5 > C-3. PMF demethylase in the MRG-PMF1 strain was suggested as a Co-corrinoid methyltransferase system, and this was supported by the experiments utilizing other methyl aryl ether substrates and inhibitors. KEYWORDS: biotransformation, Blautia, Co-corrinoid, flavonoid, human intestinal bacteria, polymethoxyflavone, regioselectivity

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INTRODUCTION

Biochemically, cleavage of a methyl aryl ether bond is catalyzed by the corrinoid-dependent O-demethylase.23 To test whether demethylation of PMFs is also performed by this Coenzyme, a few preliminary experiments utilizing substrate analogues and inhibitors were also performed.

Intestinal metabolism of plant secondary metabolites by human gut microbiota is of a great importance to human health.1,2 In particular, biotransformation of polyphenolics by human microbiota has extensively been studied.3,4 In the case of flavonoids, chemical reactions of deglycosylation of C- and Oglycosides, reduction and cleavage of the C-ring, hydrolysis of esters, and demethylation occur during the gut metabolism.5−10 These reactions of flavonoid metabolism are well exemplified by S-equol formation from the soy isoflavone.11−13 Recently, demethylation of methoxyflavones drew our attention, because chemical cleavage of the methyl aryl ether bond requires very rigorous reaction conditions. Until now, only two gut bacteria, Eubacterium limosum and Blautia sp. MRG-PMF1, were reported to metabolize flavonoids with a methoxy group, such as isoxanthohumol and icaritin, respectively.14−16 Polymethoxyflavones (PMFs), a unique class of flavonoids isolated from the rhizome of Kaempferia parvif lora and the epicarp of citrus fruits, have shown various biological functions such as anticancer,17,18 antiinflammation,19 antiallergic disorder,20 antimutagenicity,21 and neuroprotection.22 As PMFs become new compounds leading to the development of new drugs and functional foods, it is important to understand their interactions with human gut microbiota. Furthermore, the newly produced metabolites and the metabolic intermediates could exhibit different biological activities. In this report, biotransformation of structurally diverse PMFs (Figure 1) was carried out with Blautia sp. MRG-PMF1 to identify the metabolic intermediates and elucidate the bioconversion pathway. © XXXX American Chemical Society

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MATERIALS AND METHODS

The experimental protocol was evaluated and approved by the Institutional Review Board of Chung-Ang University (Approval Number: 1041078-201502-BR-029-01). Chemicals and Bacterium. Twelve PMFs, 5,7-dimethoxyflavone (5,7-DMF), 3,5,7-trimethoxyflavone (3,5,7-TMF), 5,7,4′-trimethoxyflavone (5,7,4′-TMF), 3,5,7,4′-tetramethoxyflavone (3,5,7,4′-TMF), 5,7,3′,4′-tetramethoxyflavone (5,7,3′,4′-TMF), 3,5,7,3′,4′-pentamethoxyflavone (3,5,7,3′,4′-PMF), 5-hydroxy-7-methoxyflavone (5-OH7-MF), 5-hydroxy-3,7-dimethoxyflavone (5-OH-3,7-DMF), 5-hydroxy7,4′-dimethoxyflavone (5-OH-7,4′-DMF), 5-hydroxy-3,7,4′-trimethoxyflavone (5-OH-3,7,4′-TMF), 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (5,3′-diOH-3,7,4′-TMF), and 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (5-OH-3,7,3′,4′-TMF), were isolated and purified from K. parvif lora.24 Alkyl derivatives of PMF, 5,3′-dihydroxy-3,7,4′-triethylquercetin (5,3′diOH-3,7,4′-TEF), 5-hydroxy-3,7,3′,4′-tetrapropylquercetin (5-OH3,7,3′,4′-TPF), and 3,5,7,3′,4′-pentaethoxyflavone (3,5,7,3′,4′-PEF), were synthesized from quercetin, according to a published method (Figure 1).25 Syringic acid and vanillic acid were purchased from Sigma-Aldrich Korea (Seoul, Korea). HPLC-grade methanol (MeOH) and acetonitrile (MeCN) were purchased from Burdick & Jackson Laboratories, Inc. (Muskegon, MI, USA). Formic acid for mass Received: Revised: Accepted: Published: A

January 25, 2017 February 8, 2017 February 13, 2017 February 13, 2017 DOI: 10.1021/acs.jafc.7b00408 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Molecular structures of PMFs and other substrates used in the study. Abbreviations: 5,7-dimethoxyflavone (5,7-DMF), 3,5,7trimethoxyflavone (3,5,7-TMF), 5,7,4′-trimethoxyflavone (5,7,4′-TMF), 3,5,7,4′-tetramethoxyflavone (3,5,7,4′-TMF), 5,7,3′,4′-tetramethoxyflavone (5,7,3′,4′-TMF), 3,5,7,3′,4′-pentamethoxyflavone (3,5,7,3′,4′-PMF), 5-hydroxy-7-methoxyflavone (5-OH-7-MF), 5-hydroxy-3,7-dimethoxyflavone (5-OH-3,7-DMF), 5-hydroxy-7,4′-dimethoxyflavone (5-OH-7,4′-DMF), 5-hydroxy-3,7,4′-trimethoxyflavone (5-OH-3,7,4′-TMF), 5,3′-dihydroxy3,7,4′-trimethoxyflavone (5,3′-diOH-3,7,4′-TMF), 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (5-OH-3,7,3′,4′-TMF), 5-hydroxy-3,7,3′,4′-tetraethylflavone (5-OH-3,7,3′,4′-TEF), 5-hydroxy-3,7,3′,4′-tetrapropylflavone (5-OH-3,7,3′,4′-TPF), and 5,3′-dihydroxy-3,7,4′-tributylflavone (5,3′-diOH3,7,4′-TBF). USA), was used for HPLC analysis. The injection volume was 10 μL, and the flow rate was 1.0 mL/min. The mobile phase for the analysis of PMF biotransformation was composed of 0.1% acetic acid in deionized water (solvent A) and 0.1% acetic acid in MeCN (solvent B). For the eluent gradient system, solution B was started at 20% and increased to 40% in 8 min, to 50% in 12 min, to 55% in 15 min, to 70% in 20 min, to 80% in 22 min, and finally to 20% in 30 min. Preliminary Study on MRG-PMF1 Demethylase. For the biotransformation of PMF analogues and other substrates, 5,3′dihydroxy-3,7,4′-triethylquercetin (5,3′-diOH-3,7,4′-TEF), 5-hydroxy3,7,3′,4′-tetrapropylquercetin (5-OH-3,7,3′,4′-TPF), 3,5,7,3′,4′-pentaethoxyflavone (3,5,7,3′,4′-PEF), syringic acid, and vanillic acid were used (Figure 1). Under anaerobic conditions (CO2 5%, H2 10%, N2 85%), 10 μL of substrate (10 mM in DMF) was added to 1 mL of GAM broth medium containing Blautia sp. MRG-PMF1 (OD600 0.9), and the culture was incubated at 37 °C. Biotransformation of quercetin alkyl derivatives was monitored for 6 days, and that of syringic acid and vanillic acid was monitored for 3 days. Aliquots of the reaction mixtures (100 μL) were allocated into Eppendorf tubes at fixed times to monitor the biotransformation. The allocated reaction mixtures were extracted by vortexing with 1 mL of EtOAc and centrifuged (10770g for 10 min). The supernatant EtOAc extract (800 μL) was collected after centrifugation and dried under vacuum. The dried residue was dissolved in DMF (100 μL) and filtered through a 0.2 μm filter (Grace, USA) for HPLC analysis. For the inhibition study, 5,7-DMF was biotransformed in the presence of MeI, EtI, and KCN. The Blautia sp. MRG-PMF1 was grown in GAM broth medium (2 mL) until OD600 reached 0.9. Each inhibitor was added at different concentrations (0.1, 1, and 5 mM) with 5,7-DMF (0.1 mM) to the culture medium. After 1, 2, 3, and 4 days of incubation at 37 °C, an aliquot of the culture medium (100 μL) was transferred to an Eppendorf tube and extracted with EtOAc (1 mL). The HPLC analytes were prepared and analyzed as described above.

spectrometry (98%) was purchased from Fluka (Buchs, Switzerland). Ethyl acetate (99.5%, EtOAc), acetic acid (99.5%), N,N-dimethylformamide (99.5%, DMF), methyl iodide (99%, MeI), and potassium cyanide (95.5%, KCN) were purchased from Samchun Pure Chemicals (Gyeonggi-do, Korea). Ethyl iodide (99%, EtI) was purchased from Junsei (Tokyo, Japan). Gifu anaerobic medium (GAM) was from Nissui Pharmaceutical Co. (Tokyo, Japan). The GAM broth was prepared following the manufacturer’s instructions, and GAM plates were prepared by adding 1.5% (w/v) agar in GAM broth. Human intestinal bacterium, Blautia sp. MRG-PMF1, isolated from our laboratory (GenBank accession number: KJ078647), was used in these experiments. Bacterial growth and substrate conversion experiments were performed under anaerobic conditions, according to published methods.5,15 In detail, the stock culture of Blautia sp. MRGPMF1 (50% glycerol in GAM) stored in liquid nitrogen was thawed in an anaerobic chamber (CO2 5%, H2 10%, N2 85%) at 37 °C. It was subcultured on a GAM agar plate, and after incubation for 2 days at 37 °C, a single colony was picked from the plate, transferred into 500 μL of GAM broth, and incubated overnight in the presence of 5,7-DMF (0.1 mM). The PMF-metabolizing activity was checked by TLC and HPLC. A 100 μL aliquot of the broth cultured MRG-PMF1 strain was then transferred to 2 mL of GAM broth. When the optical density at 600 nm (OD600) reached 0.7, 200 μL of the cell culture was transferred to 10 mL of fresh GAM broth. Bacterial growth was monitored by measuring the OD600 with the UV-vis spectrophotometer S-3100 (Scinco, Seoul, Korea) Biotransformation of PMFs. The following experimental method was generally used for the study of the biotransformation of 12 PMFs by the MRG-PMF1 strain. Under anaerobic conditions (CO2 5%, H2 10%, N2 85%) at 37 °C, 100 μL of a PMF substrate (10 mM in DMF) was added to 10 mL of GAM broth medium containing Blautia sp. MRG-PMF1 (OD600 0.9). Aliquots of the reaction mixtures (100 μL) were allocated into Eppendorf tubes to monitor the biotransformation. The allocated reaction mixtures were extracted by vortexing with 1 mL of EtOAc and centrifuged (10770g for 10 min). The supernatant EtOAc extract (800 μL) was collected after centrifugation and dried under vacuum. The dried residue was dissolved in DMF (100 μL) and filtered through a 0.2 μm filter (Grace, USA) before chromatographic analysis. General HPLC Method. A Finnigan Surveyor Plus HPLC with a Thermo PDA Plus detector, equipped with a C18 Hypersil GOLD column (4.6 × 100 nm, 5 μm, Thermo Scientific, Waltham, MA,

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RESULTS Biotransformation of Chrysin Methyl Ethers. Biotransformation of 5,7-DMF by the MRG-PMF1 strain was reported previously, and the metabolism of 5,7-DMF to chrysin, via 7OH-5-MF, was elucidated.15 When 5-OH-7-MF was added to the reaction medium as a substrate, chrysin was produced in 2 d B

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Figure 2. HPLC chromatogram changes of 5-OH-7-MF (5-hydroxy-7-methoxyflavone, A, 310 nm) and 5-OH-7,4′-DMF (5-hydroxy-7,4′dimethoxyflavone, B, 330 nm) metabolisms by Blautia sp. MRG-PMF1, and metabolic pathways of chrysin methyl ethers (C) and apigenin methyl ether, 5-OH-7,4′-DMF (D). The insets show UV spectra of the compounds.

decreased. Hence, the metabolic pathway of 3,5,7-TMF was established as follows: 3,5,7-TMF → A → B → C. The final metabolite identified as peak C was galangin, and the two intermediates, peaks A and B, were assigned as 7-hydroxy-3,5dimethoxyflavone (7-OH-3,5-DMF) and 5,7-dihydroxy-3-methoxyflavone (5,7-diOH-3-MF), respectively, based on UV and MS spectra (see Figure S1). If the metabolite B was 3,7dihydroxy-5-methoxyflavone (3,7-diOH-5-MF), its strong characteristic UV absorptions at 260 and 350 nm should have been detected.26 Biotransformation of 5-OH-3,7-DMF was expected to produce only one metabolic intermediate before galangin formation. As shown in Figure 3B, a new peak at the retention time of 13.99 min formed in 24 h, and the final product at the retention time of 13.32 min was produced in 48 h. These two peaks appeared to be the same as peaks B and C produced by the 3,5,7-TMF biotransformation (Figure 3A), based on their retention times and UV spectra. Therefore, the metabolism of 5-OH-3,7-DMF by MRG-PMF1 strain was found to produce 5,7-diOH-3-MF as a metabolic intermediate before the formation of galangin (Figure 3C). Biotransformation of Kaempferol Methyl Ethers. The metabolism of 3,5,7,4′-TMF by Blautia sp. MRG-PMF1 was very slow due to an unknown reason. The reaction mixture was biotransformed for 13 d, and 4 metabolites including kaempferol were detected on HPLC chromatograms (Figure 4A). In 5 d, metabolic intermediates A (14.43 min), B (10.76 min), and C (7.31 min) were detected. The intermediates B and C reached maximum concentrations at 7 d, whereas the peaks corresponding to the original 3,5,7,4′-TMF and intermediate A decreased and completely disappeared in 8 d. The final product, kaempferol, identified by comparison with an authentic reference, appeared at the retention time of 9.83 min in 8 d too. The intermediate B was then identified as 3-Omethylkaempferol (5,7,4′-trihydroxy-3-methoxyflavone, 5,7,4′trOH-3-MF) based on the UV and MS spectroscopic data.15 Therefore, the metabolic pathway of 3,5,7,4′-TMF was

without any metabolic intermediates (Figure 2A). Therefore, the metabolic pathway of two chrysin methyl ethers by Blautia sp. MRG-PMF1 was established as shown at Figure 2C. Biotransformation of Apigenin Methyl Ethers. Sequential demethylation of 5,7,4′-TMF was reported previously to produce 7-hydroxy-5,4′-dimethoxyflavone (7-OH-5,4′-DMF), 7,4′-dihydroxy-5-methoxyflavone (7,4′-diOH-5-MF), and apigenin.15 When the other apigenin methyl ether, 5-OH-7,4′DMF, was reacted, apigenin was produced in a day and the conversion was completed within 2 days (Figure 2B). Due to the fast conversion, only two small peaks of metabolic intermediates were found near the retention time of 13.00 min on the chromatogram. The first demethylation of 5-OH7,4′-DMF could produce acacetin (5,7-dihydroxy-4′-methoxyflavone, 5,7-diOH-4′-MF) and genkwanin (5,4′-dihydroxy-7methoxyflavone, 5,4′-diOH-7-MF), and both metabolic intermediates were confirmed by MS analysis (see Figure S1). The peak at the retention time of 13.00 min was identified as acacetin by comparison of HPLC retention time and UV absorption with an authentic reference. Similarly, the peak at the retention time of 13.28 min was assigned as genkwanin (5,4′-diOH-7-MF) by comparison with an authentic reference. Whereas demethylation of 5,7,4′-TMF was regioselective,15 demethylation of 5-OH-7,4′-DMF was not regioselective. Namely, the methyl groups at C-7 and C-4′ positions were equally reactive and two metabolic intermediates were produced (Figure 2D). Biotransformation of Galangin Methyl Ethers. Two galangin derivatives of 3,5,7-TMF and 5-OH-3,7-DMF were reacted with the MRG-PMF1 strain. The bioconversion of both of these flavonoids was relatively slow, and the metabolic intermediates were identified on the HPLC chromatograms (Figure 3). The substrate, 3,5,7-TMF, at the retention time of 13.35 min decreased with the occurrence of new peaks at retention times of 10.33 min (A) and 13.99 min (B) in 12 h. Peak A disappeared in 48 h, as peak B increased. In 72 h, a new peak (C) at the retention time of 13.32 min increased as peak B C

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Figure 3. HPLC chromatogram changes (310 nm) of 3,5,7-TMF (3,5,7-trimethoxyflavone, A) and 5-OH-3,7-DMF (5-hydroxy-3,7dimethoxyflavone, B) metabolisms by Blautia sp. MRG-PMF1, and metabolic pathway of galangin methyl ethers (C). The insets show UV spectra of the compounds.

The biotransformation of 5-OH-3,7,4′-TMF was relatively faster than that of 3,5,7,4′-TMF. After 1 d, two metabolic intermediates, A and B, were found at the retention times of 14.41 and 10.78 min, respectively (Figure 4B). While the peak height of intermediate A was reduced, that of intermediate B reached a maximum in 2 d. The final metabolite of 5-OH3,7,4′-TMF biotransformation appeared in 48 h at the retention time of 10.32 min, and the UV and MS spectra were identical to

established, based on the chromatographic changes and MS spectra of the peaks, as follows: 3,5,7,4′-TMF → A → C → 5,7,4′-trOH-3-MF → kaempferol. The metabolic intermediates A and C were assigned as 7-hydroxy-3,5,4′-trimethoxyflavone (7-OH-3,5,4′-TMF) and 7,4′-dihydroxy-3,5-dimethoxyflavone (7,4′-diOH-3,5-DMF), respectively, based on MS and UV spectroscopic data (see Figure S2). D

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Figure 4. HPLC chromatogram changes of 3,5,7,4′-TMF (3,5,7,4′-tetramethoxyflavone, A, 340 nm) and 5-OH-3,7,4′-TMF (5-hydroxy-3,7,4′trimethoxyflavone, B, 350 nm) metabolisms by Blautia sp. MRG-PMF1, and metabolic pathway of kaempferol methyl ethers (C). The insets show UV spectra of the compounds.

3,7,4′-TMF was established as follows: 5-OH-3,7,4′-TMF → 5,7-diOH-3,4′-DMF → 5,7,4′-trOH-3-MF → kaempferol (Figure 4C). Biotransformation of Luteolin Methyl Ether. The biotransformation of 5,7,3′,4′-TMF was faster than that of the other PMFs, and the final metabolite was produced in 48 h (Figure 5A). During the bioconversion, a metabolic intermediate was observed at the retention time of 7.18 min (A) in 3 h. The peak height of A was maximum in 9 h, and new peaks at retention times of 3.33 min (B) and 6.43 min (final metabolite)

those of kaempferol. Additionally, the intermediate B at the retention time of 10.76 min was identified as 5,7,4′-trOH-3MF, as in the case of 3,5,7,4′-TMF. Accordingly, the intermediate A was assigned as 5,7-dihydroxy-3,4′-dimethoxyflavone (5,7-diOH-3,4′-DMF) based on the MS spectrum (see Figure S2). Even though peak A in Figure 4B has the same MS molecular ion peak as the intermediate C (7,4′-diOH-3,5DMF) in Figure 4A, the UV absorption of intermediate A (5,7diOH-3,4′-DMF) in Figure 4B suggested the presence of a 4′methoxy group. Therefore, the metabolic pathway of 5-OHE

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Figure 5. HPLC chromatogram changes (335 nm) of 5,7,3′,4′-TMF (5,7,3′,4′-tetramethoxyflavone) metabolism by Blautia sp. MRG-PMF1 (A), and metabolic pathway of luteolin methyl ethers (B). The insets show UV spectra of the compounds.

Figure 6. HPLC chromatogram changes of 5,3′-diOH-3,7,4′-TMF (5,3′-dihydroxy-3,7,4′-trimethoxyflavone, A, 355 nm), 5-OH-3,7,3′,4′-TMF (5hydroxy-3,7,3′,4′-tetramethoxyflavone, B, 355 nm), and 3,5,7,3′,4′-PMF (3,5,7,3′,4′-pentamethoxyflavone, C, 350 nm) metabolisms by Blautia sp. MRG-PMF1, and metabolic pathway of quercetin methyl ethers (D). The insets show UV spectra of the compounds.

5,7,3′,4′-TMF was established as follows: 5,7,3′4′-TMF → 7,4′diOH-5,3′-DMF → 7,3′,4′-trOH-5-MF → luteolin (Figure 5B). Biotransformation of Quercetin Methyl Ethers. Three quercetin derivatives, 3,5,7,3′,4′-PMF, 5-OH-3,7,3′,4′-TMF, and 5,3′-diOH-3,7,4′-TMF, were isolated from K. parvif lora (Figure 1). The biotransformation of 5,3′-diOH-3,7,4′-TMF by the MRG-PMF1 strain was very slow. The major products, observed in 13 d, were 3-O-methylquercetin (5,7,3′,4′tetrahydroxy-3-methoxyflavone, 5,7,3′,4′-teOH-3-MF) at the retention time of 8.84 min and quercetin at the retention time

were observed. After 48 h, the final metabolite was the major product, which was identified as luteolin by comparison with the standard compound. Although four consecutive demethylations were expected for 5,7,3′,4′-TMF to produce luteolin, only two metabolic intermediates, A and B, were observed by HPLC analysis. Based on MS spectral analysis, intermediate A was assigned as 7,4′-dihydroxy-5,3′-dimethoxyflavone (7,4′diOH-5,3′-DMF), and intermediate B was assigned as 5-Omethylluetolin (7,3′,4,′-trihydroxy-5-methoxyflavone, 7,3′,4′trOH-5-MF) (see Figure S3). The metabolic pathway of F

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Figure 7. HPLC chromatogram changes of syringic acid (top, 275 nm) and vanillic acid (bottom, 260 nm) metabolisms by Blautia sp. MRG-PMF1 (A) and the metabolic pathways of syringic acid and vanillic acid (B). The insets show UV spectra of the compounds.

8.54 min and was observed until 24 h. This compound was assigned as 7-hydroxy-3,5,3′,4′-tetramethoxyflavone (7-OH3,5,3′,4′-TMF) based on its MS spectrum. A peak appeared in 24 h at the retention time of 8.84 min, and another peak appeared in 36 h at the retention time of 8.26 min. These peaks were 3-O-methylquercetin and quercetin, respectively. During the biotransformation of 3,5,7,3′,4′-PMF, many small peaks were observed. For instance, more than 5 minor peaks were observed in the chromatogram obtained in 12 h. Some of these corresponded to the metabolites, 5,7,3′-trOH-3,4′-DMF and 5,3′,4′-trOH-3,7-DMF, observed from the biotransformation of 5,3′-diOH-3,7,4′-TMF (Figure 6A). It appears that biotransformation of 3,5,7,3′,4′-PMF is very complicated because of low regioselectivity of demethylation. Preliminary Study of Blautia sp. MRG-PMF1 Demethylase. When quercetin alkyl ethers, 5-OH-3,7,3′,4′-TEF, 5OH-3,7,3′,4′-TPF, and 3,5,7,3′,4′-PEF (Figure 1), were introduced to the bacterial culture, the quercetin derivatives were recovered because no reaction product was formed (see Figure S4). Therefore, the putative demethylase of Blautia sp. MRG-PMF1 seemed to catalyze the cleavage of methyl aryl ether functional groups only. Next, syringic acid and vanillic acid were used as substrates to test whether the enzyme is reactive to other methyl aryl ether compounds. As shown in Figure 7A, both substrates were converted very rapidly. Vanillic acid was metabolized to protocatechuic acid, and syringic acid was metabolized to gallic acid via 3-O-methylgallic acid (Figure 7B).

of 8.26 min (Figure 6A). There were two metabolic intermediates formed before the appearance of 5,7,3′,4′teOH-3-MF, and their peaks, at retention times of 12.10 min (A) and 10.58 min (B), appeared in 3 and 6 d, respectively. The intermediates, A and B, were assigned as 5,7,3′-trihydroxy-3,4′dimethoxyflavone (5,7,3′-trOH-3,4′-DMF) and 5,3′,4′-trihydroxy-3,7-dimethoxyflavone (5,3′,4′-trOH-3,7-DMF), respectively. No regioselectivity of PMF demethylation on the C-7 and C-4′ positions was found for the biotransformation of 5,3′diOH-3,7,4′-TMF, as was observed for the demethylation of 5OH-7,4′-DMF. Therefore, the biotransformation of 5,3′-diOH3,7,4′-TMF was established as shown in Figure 6D. When 5-OH-3,7,3′,4′-TMF was reacted, only two intermediates were detected (Figure 6B). The peak at the retention time of 8.84 min was 5,7,3′,4′-teOH-3-MF, and the peak at the retention time of 12.16 min was assigned as 5,7,3′-trOH-3,4′DMF from comparison with the previous results (Figure 6A). The expected metabolic intermediate of 5,7-diOH-3,3′,4′-TMF was not detected in the HPLC chromatograms. It appeared that demethylation of the methyl group at C-7 was very fast. The production of quercetin did not increase from 3 d to the next 10 d. The biotransformation pathway of 5-OH-3,7,3′,4′-TMF was established as follows: 5-OH-3,7,3′,4′-TMF → [5,7-diOH3,3′,4′-TMF] → 5,7,3′-trOH-3,4′-DMF → 5,7,3′,4′-teOH-3MF → quercetin. When 3,5,7,3′,4′-PMF was reacted with the MRG-PMF1 strain, chromatographic changes were observed for the first 2 d, after which no changes were observed for 10 d (Figure 6C). The first intermediate appeared in 6 h at the retention time of G

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Journal of Agricultural and Food Chemistry

Figure 8. Inhibition of 5,7-DMF demethylation by Blautia sp. MRG-PMF1 in the presence of inhibitors. Chrysin production was measured by HPLC (310 nm), and the concentration of inhibitors was 0.1 mM.

reactivity with other substrates has never been published.14 A group of acetogenic bacteria was also reported to demethylate secoisolariciresinol lignan, but their ability for PMF-demethylation was also not known.29 Due to the increasing number of publications on PMFs in the area of health and food science, this study will provide additional resources for further research. For example, the extracts of K. parvif lora rich in PMFs are used for the production of various foods,30 and the metabolism of the extracts by human intestinal microbiota produces new PMFs metabolites (see Figure S6). Therefore, the biotransformation study was expected to identify the flavonoid metabolic intermediates, the biological function of which needs to be investigated further. The selected substrates for PMF biotransformation were 12 methyl ethers of chrysin, apigenin, galangin, kaempferol, luteolin, and quercetin. Both chrysin methyl ethers, 5,7-DMF and 5-OH-7-MF, were completely metabolized to chrysin by the MRG-PMF1 strain in 2 d. Previously, it was reported that 5,7,4′-TMF (apigenin trimethyl ether) was regioselectively metabolized to apigenin via 7-OH-5,4′-DMF and 7,4′-diOH-5MF.15 However, such selectivity was not observed for the demethylation of 5-OH-7,4′-DMF. Both metabolic intermediates, 5,7-diOH-4′-MF (acacetin) and 5,4′-diOH-7-MF, were produced at the same time, and the C-7 and C-4′ positions appeared to have similar reactivity. Lack of regioselectivity was also observed during the biotransformation of 3,5,7,3′,4′-PMF. After a selective first demethylation on the C-5 position, the metabolic intermediate 7-OH-3,5,3′,4′-TMF was metabolized further to various demethylated metabolites. Two galangin methyl ethers of 3,5,7-TMF and 5-OH-3,7DMF showed sequential demethylation, and the regioselectivity of C-7 > C-5 > C-3 in the flavone was found. During the biotransformation of two kaempferol methyl ethers, 3,5,7,4′TMF and 5-OH-3,7,4′-TMF, a similar trend was also observed and regioselectivity of demethylation was found as C-7 > C-4′ > C-5 > C-3. When a luteolin derivative of 5,7,3′,4′-TMF was reacted, only three metabolites, including luteolin, were observed on the HPLC chromatograms. Two metabolic

For the inhibition study, 5,7-DMF was biotransformed in the presence of MeI, EtI, and KCN. All the inhibitors inhibited biotransformation of 5,7-DMF, and the inhibition was concentration dependent. The three inhibitors completely inhibited demethylase activity of the MRG-PMF1 strain at the concentration of 1 mM and higher. Therefore, timedependent inhibition of 5,7-DMF demethylation was studied at a concentration of 0.1 mM (see Figure S5). The inhibitory effect appeared to follow the order KCN > MeI > EtI, as shown in Figure 8.

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DISCUSSION Human gut microbiota plays an important role in human health. Changes in gut microbiota are closely associated with obesity, immune response, allergy, and various diseases including cancer and diabetes.27 Along with the genomics study, investigation of the gut microbial metabolites has become a necessary component of molecular-level research.28 Flavonoids, an important class of plant polyphenolics, undergo various chemical modifications of hydrolysis, reduction, and other less defined reactions during the human gut microbiota metabolism.5−13 Recently, we reported that the anaerobic bacterium Blautia sp. MRG-PMF1 can metabolize 5,7-DMF and 5,7,4′-TMF to the corresponding demethylated flavones chrysin and apigenin, respectively.15 During the biotransformation, rare flavonoids of 7-OH-5-MF and thevetiaflavone were also isolated and characterized as metabolic intermediates. For example, 7-OH5-MF was only identified from propolis. Furthermore, it was found that the flavone demethylation was not the hydrolysis reaction, because O-18 labeled water was not incorporated into the metabolic products. From the standpoint of microbial biotechnology and nutraceuticals, further study of this unique biotransformation system is important. In this report, various PMFs were reacted under anaerobic conditions to investigate the metabolic reactivity of the MRGPMF1 strain. The previously reported E. limosum was the only other gut bacterium with flavonoid demethylase activity, but its H

DOI: 10.1021/acs.jafc.7b00408 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry intermediates were identified as 7,4′-diOH-5,3′-DMF and 7,3′,4′-trOH-5-MF, implying that demethylation of B-ring methoxy groups was faster than that of C5-methoxy groups on PMFs. The biotransformation of PMFs demonstrated a general regioselectivity of demethylation as C-7 > C-4′ ≈ C-3′ > C-5 > C-3. In addition, bioconversions were found for the production of rare flavonoids, such as 7-OH-3,5-DMF, 7-OH3,5,4′-TMF, 7,4′-diOH-3,5-DMF, and 7-OH-3,5,3′,4′-TMF (see Table S1). Quercetin methyl ethers were the largest flavone among the 12 PMF substrates that can be metabolized by the MRG-PMF1 strain. Biotransformation of three quercetin methyl ethers was not complete, even after 13 d incubation. Demethylation on the C-7 position of three quercetin derivatives was generally preferred, and 3-O-methylquercetin (5,7,3′,4′-teOH-3-MF) was the distinct metabolic intermediate commonly observed. We suspect that 3-O-methylquercetin might have an inhibitory effect on the bioconversion. Metabolism of 5-OH-3,7,3′,4′TMF and 3,5,7,3′,4′-PMF to 3-O-methylquercetin was relatively fast, but that of 5,3′-diOH-3,7,4′-TMF was very slow. All 12 PMFs studied were metabolized by the MRGPMF1 strain within 3 days, but the metabolisms of 5,3′-diOH3,7,4′-TMF and 3,5,7,4′-TMF were very slow. Metabolism of PMFs in rats was reported recently, and a few demethylated PMFs were identified as major metabolites in blood and feces.31 From our experimental results, it was possible to delineate the metabolism of PMFs in human intestine. For example, 3-O-methylquercetin, apigenin, 3-Omethylkaempferol, and chrysin were the major metabolites when the ethanol extract of K. parvif lora was reacted with the MRG-PMF1 strain for 2 days (see Figure S6). The result was in good agreement with the results obtained from the metabolism of each individual PMF. Additionally, these results can provide detailed structural information to the pharmacokinetic data obtained from the rat model.31 In environmental microbiology, it has been known for a long time that methyl aryl ether cleavage by methyltransferase is vital for one-carbon (C1) metabolism of certain types of bacteria.32 For example, simple phenolics with methoxy groups, including syringate, vanillate, and veratrole, were demethylated by methylotropic bacteria.33 Analogous to lignin degradation, biochemical reactions of PMF demethylation were thought to be due to the corrinoid-dependent O-demethylase.23 If PMF Odemethylation is catalyzed by the Co-corrinoid system, it would be a demethylase with the largest substrate binding site, because it can metabolize 3,5,7,3′,4′-PMF. Because the Co-corrinoid system is inhibited by cyanide and alkyl iodide,34,35 KCN and alkyl iodide were added to the cell culture metabolizing 5,7DMF (Figure 8). All three compounds inhibited the demethylation of 5,7-DMF in a concentration-dependent manner, which strongly suggested that the demethylation of PMFs is catalyzed by a Co-corrinoid enzyme. This was further supported by the fact not only that other alkyl (R = ethyl, propyl) derivatives of quercetin were not metabolized but also that syringic acid and vanillic acid were metabolized very rapidly by Blautia sp. MRG-PMF1.

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General HPLC-MS method, ESI-MS spectra, and biotransformation details (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +82 31 670 4830. Fax: +82 31 675 1381. E-mail: [email protected]. ORCID

Jaehong Han: 0000-0002-5328-3927 Funding

This work was supported under the framework of international cooperation program managed by the National Research Foundation of Korea (NRF-2015K2A1A2068137) and by the Korean government (MSIP) (NRF-2015R1A1A3A04001198). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.7b00408 J. Agric. Food Chem. XXXX, XXX, XXX−XXX