Biological and Pharmaceutical Bulletin
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Bioconversion of (−)-Epicatechin, (+)-Epicatechin, (−)-Catechin, and (+)-Catechin by (−)-Epigallocatechin-Metabolizing Bacteria
Akiko TakagakiFumio Nanjo
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2015 Volume 38 Issue 5 Pages 789-794

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Abstract

Bioconversion of (−)-epicatechin (−EC), (+)-epicatechin (+EC), (−)-catechin (−C), and (+)-catechin (+C) by (−)-epigallocatechin (−EGC)-metabolizing bacteria, Adlercreutzia equolifaciens MT4s-5, Eggerthella lenta JCM 9979, and Flavonifractor plautii MT42, was investigated. A. equolifaciens MT4s-5 could catalyze C ring cleavage to form (2S)-1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1S) from −EC and −C, and (2R)-1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1R) from +C. The C ring cleavage by A. equolifaciens MT4s-5 was accelerated in the presence of hydrogen. E. lenta JCM 9979 also catalyzed C ring cleavage of −EC and +C to produce 1S and 1R, respectively. In the presence of hydrogen or formate, strain JCM 9979 showed not only stimulation of C ring cleavage but also subsequent 4′-dehydroxylation of 1S and 1R to produce (2S)-1-(3-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2S) and (2R)-1-(3-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2R), respectively. On the other hand, A. equolifaciens MT4s-5 did not show any 4′-dehydroxylation ability even in the presence of hydrogen. F. plautii MT42 could convert 1S, 1R, 2S, and 2R into their corresponding 4-hydroxy-5-hydroxyphenylvaleric acids and 5-hydroxyphenyl-γ-valerolactones simultaneously. Similar bioconversion was observed by F. plautii ATCC 29863 and F. plautii ATCC 49531.

Catechins such as (+)-catechin (+C), (−)-epicatechin (−EC), (−)-epigallocatechin (−EGC), (−)-epicatechin gallate (−ECG), and (−)-epigallocatechin gallate (−EGCG) are major constituents of green tea infusion. And their isomers, (+)-epicatechin (+EC), (−)-catechin (−C), (−)-gallocatechin (−GC), (−)-catechin gallate (−CG), and (−)-gallocatechin gallate (−GCG) are known to form during heat sterilization in the production of bottled green tea.1,2) It is well recognized that tea catechins have preventive effects against cancer, heart disease, diabetes, age-related diseases.35) Accordingly, study on the metabolism of tea catechins has recently received increased attention in the explanation of their physiological functions. Particularly, in the last 5 years clarification on the catabolism of tea catechins by intestinal microbiota has been the focus of intense research interest because of poor absorbability of the intact catechins in the body relative to catechin catabolites.68)

We reported previously on the catabolism of +C and −EC by rat intestinal microbiota and proposed metabolic pathway of these catechins.9) In addition to the above report, we have recently isolated and identified rat intestinal bacteria as −EGC-metabolizing bacteria and have reported their degradation abilities.10) In this study, Adlercreutzia equolifaciens MT4s-5 and Eggerthella lenta JCM 9979 were found not only to convert −EGC into 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol, but also to catalyze the subsequent 4′-dehydroxylation of the above metabolite to yield 1-(3,5-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol in the presence of hydrogen or formate, supplied by symbiotic bacteria. Flavonifractor plautii MT42 then degraded the phloroglucinol moiety of the above propan-2-ols and simultaneously produced their corresponding 4-hydroxy-5-hydroxyphenylvaleric acids and 5-hydroxyphenyl-γ-valerolactones. However, their bioconversion abilities of catechol-type catechins still remain to be investigated.

In this report, we describe bioconversion of −EC, +EC, −C, and +C by −EGC-metabolizing bacteria, A. equolifaciens MT4s-5, E. lenta JCM 9979, and F. plautii MT42.

MATERIALS AND METHODS

Chemicals, Bacteria, and Medium

(−)-Epicatechin (−EC), (+)-epicatechin (+EC), (−)-catechin (−C), and (+)-catechin (+C) were obtained from Sigma-Aldrich Japan (Tokyo). Metabolites, (2S)-1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1S), (2R)-1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1R), (2S)-1-(3-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2S), (2R)-1-(3-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2R), (4R)-4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid from −EC (3R), (4S)-4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid from +C (3S), (4R)-5-(3,4-dihydroxyphenyl)-γ-valerolactone (4R), (4S)-5-(3,4-dihydroxyphenyl)-γ-valerolactone (4S), (4R)-4-hydroxy-5-(3-hydroxyphenyl)valeric acid from −EC (5R), (4S)-4-hydroxy-5-(3-hydroxyphenyl)valeric acid from +C (5S), (4R)-5-(3-hydroxyphenyl)-γ-valerolactone (6R), and (4S)-5-(3-hydroxyphenyl)-γ-valerolactone (6S) were prepared according to the methods previously reported9) and were used as reference standards. All other chemicals were available products of analytical or HPLC grade. Adlercreutzia equolifaciens MT4s-5 and Flavonifractor plautii MT42 were isolated from rat feces as reported in our previous paper.10) Eggerthella lenta JCM 9979 was purchased from RIKEN BioResource Center (Ibaraki, Japan). Flavonifractor plautii ATC C 29863 (formerly Eubacterium plautii) and Flavonifractor plautii ATC C 49531 (formerly Clostridium orbisciendens) were obtained from American Type Culture Collection (ATC C, Manassas, VA, U.S.A.). General Anaerobic Medium (GAM) broth was obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). All cultures in this study were carried out under anaerobic condition with an AnaeroPack (anaerobic cultivation) system (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan) unless otherwise stated.

LC/MS Analysis for Structural Identification

LC/MS analysis was performed by a Surveyor HPLC and an LCQ Deca XPplus system (Thermo Fisher Scientific K. K., Yokohama, Japan) as described in our previous paper.9) Identification of the metabolites formed from −EC, +EC, −C, and +C was carried out by comparison of their HPLC retention times, UV spectra, and MS and MS/MS data to those of the reference standards.

LC/MS/MS Analysis for Quantitation

LC/MS/MS analysis was performed using a model Agilent 1100 series LC system (Agilent Technologies, Tokyo, Japan) coupled with a 3200QTRAP LC/MS/MS system (AB SCIEX, MA, U.S.A.) as reported in our previous paper.10) The mass detector was equipped with turbo-ion spray (electrospray ionization; ESI) source and operated in multiple reactions monitoring mode (MRM) under negative ion mode. For all the mass scan modes, ion spray voltage was maintained at −4500 V, curtain gas was set to 30 (arbitrary units), the collision gas was 3 (arbitrary units), and capillary temperature was set at 600°C. For MRM scans, the transition pairs of the precursor ion to the product ion were as follows: m/z 289.04 to m/z 109.10 for −EC, +EC, −C, and +C, m/z 291.03 to m/z 123.20 for 1S and 1R, and m/z 275.02 to m/z 107.20 for 2S and 2R. The transition pairs were simultaneously monitored. The optimized collision energy (CE), declustering potential (DP), and collision cell exit potential (CXP) were optimized for each metabolite, and set at −34 to −38 for CE, −50 to −55 for DP, and 0 to −4 for CXP. The LC/MS/MS system was controlled, and the data were acquired and processed by Analyst® version 1.6.1 software.

Incubation of Catechins with A. equolifaciens MT4s-5 and E. lenta JCM 9979

Two −EGC-metabolizing bacteria (A. equolifaciens MT4s-5 and E. lenta JCM 9979) were separately precultured in GAM broth (5 mL) at 37°C for 48 h. Each preculture (0.5 mL) was then inoculated into 5 mL of fresh GAM broth containing 1 mM −EC, +EC, −C, or +C and was incubated at 37°C. GAM broth containing each catechin was also incubated without bacterium under the same condition as a control. After incubation for 24, 48, and 72 h, aliquots (0.5 mL) of the incubation mixture were withdrawn in an anaerobic glovebox under CO2 atmosphere. After adding 50 µL of 2 M HCl to each sample, bacterial cells were removed by centrifugation at 12000×g for 10 min at 4°C. A portion (0.2 mL) of each resulting supernatant was then diluted with 0.8 mL of 0.5% aqueous acetic acid, and the sample solution was analyzed by LC/MS system for identification of metabolites and by LC/MS/MS system for quantitation of metabolites as described above.

Incubation of Catechins with A. equolifaciens MT4s-5 and E. lenta JCM 9979 in the Presence of Hydrogen

Each preculture (0.5 mL) of A. equolifaciens MT4s-5 and E. lenta JCM 9979 was inoculated into GAM broth (5 mL) containing 1 mM −EC, +EC, −C, or +C and then hydrogen gas was aseptically bubbled into the incubation mixture for 10 s at a flow rate of about 50 mL/min. The resultant culture in test tube was packed in an Anaero Pouch (800 mL) with AnaeroPack system (Mitsubishi Gas Chemical Company, Inc.). Air in the pouch was roughly removed by hand and hydrogen gas was injected to the pouch for 30 s at a flow rate of 800 mL/min. The resultant culture broth each was incubated anaerobically at 37°C. After sampling of the incubation mixture as mentioned below, hydrogen gas was injected into the pouch again and the culture was continued. After the 24, 48, and 72 h incubation, aliquots (0.5 mL) of the incubation mixture were withdrawn in the anaerobic glovebox. Samples for the LC/MS and LC/MS/MS analyses were prepared in the same manner as above.

Incubation of Metabolites 1S, 1R, 2S, and 2R with F. plautii Strains MT42, ATCC 29863, and ATCC 49531

After pre-incubation of each strain of F. plautii in GAM broth (5 mL) at 37°C for 24 h, aliquots (0.5 mL) of each preculture were inoculated into 5 mL of fresh GAM broth containing 1 mM of either metabolite 1S, 1R, 2S, or 2R and the resultant culture broth was incubated at 37°C. After 24 h of incubation, a portion (0.5 mL) of the culture broth was taken out. Sample preparation for LC/MS analysis was the same as above.

RESULTS

Bioconversion of −EC, +EC, −C, and +C by A. equolifaciens MT4s-5

We examined the ability of A. equolifaciens MT4s-5 to biotransform −EC, +EC, −C, and +C. The results are shown in Fig. 1. Strain MT4s-5 readily converted −EC and −C into (2S)-1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1S) and +C into (2R)-1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1R). The bioconversion was observed to be accelerated in the presence of hydrogen. However, A. equolifaciens MT4s-5 showed only limited conversion of +EC and did not stimulate this conversion in the presence of hydrogen. In addition, strain MT4s-5 showed no 4′-dehydroxylation ability of 1S and 1R in the B ring regardless of the presence of hydrogen.

Fig. 1. Bioconversion of −EC, +EC, −C, and +C by A. equolifaciens MT4s-5

−EC (○), +EC (●), −C (△), +C (▲), metabolites 1S (□), and 1R (■). Data points represent mean±standard deviation (S.D.) of three experiments.

Bioconversion of −EC, +EC, −C, and +C by E. lenta JCM 9979

Bioconversion ability of E. lenta JCM 9979 was also examined (Fig. 2). Strain JCM 9979 easily converted −EC and +C into 1S and 1R, respectively. On the other hand, little bioconversion of −C and +EC was observed. In the presence of hydrogen, E. lenta JCM 9979 not only promoted the conversion to 1S from −EC and 1R from +C, but also catalyzed 4′-dehydroxylation of 1S and 1R to produce (2S)-1-(3-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2S) and (2R)-1-(3-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2R), respectively. Similar results were also obtained in the presence of formate (data not shown).

Fig. 2. Bioconversion of −EC, +EC, −C, and +C by E. lenta JCM 9979

−EC (○), +EC (●), −C (△), +C (▲), metabolites 1S (□), 1R (■), 2S (♢), and 2R (♦). Data points represent mean±S.D. of three experiments.

Bioconversion of Metabolites 1S, 1R, 2S, and 2R by F. plautii Strains

F. plautii MT42 was found to produce (4R)-4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid (3R) and (4R)-5-(3,4-dihydroxyphenyl)-γ-valerolactone (4R) from 1S and (4S)-4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid (3S) and (4S)-5-(3,4-dihydroxyphenyl)-γ-valerolactone (4S) from 1R, as shown in Fig. 3. Similarly, strain MT42 could convert 2S and 2R to their corresponding 4-hydroxy-5-(3-hydroxyphenyl)valeric acids (5R from 2S, 5S from 2R) and 5-(3-hydroxyphenyl)-γ-valerolactones (6R from 2S, 6S from 2R). Similar results as above were also obtained with F. plautii strains ATC C 29863 and ATC C 49531 (data not shown).

Fig. 3. HPLC Profiles and Mass Spectra Showing Bioconversion of Metabolites 1S, 1R, 2S, and 2R by F. plautii MT42

DISCUSSION

In this study, we examined bioconversion of catechol-type catechins (four diastereoisomers; −EC, +EC, −C, and +C) by A. equolifaciens MT4s-5 and E. lenta JCM 9979, which have been reported to catalyze C ring cleavage of (−)-epigallocatechin (−EGC).10) Strain MT4s-5 converted −EC, −C, and +C into their corresponding 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ols. Strain JCM 9979 catalyzed C ring cleavage of −EC and +C. Thus, substrate specificity was different between A. equolifaciens MT4s-5 and E. lenta JCM 9979. In addition, only slight conversion of +EC by strain MT4s-5 and of +EC and −C by strain JCM 9979 was observed. However, it is unclear at present whether this phenomena are due to the ability of each strain to convert +EC and −C because it remains a possibility that slight isomerization of +EC to +C and −C to −EC takes place in the incubation mixture.

It was reported that A. equolifaciens MT4s-5 catalyzed not only C ring cleavage of −EGC but also subsequent 4′-dehydroxylation and the catalytic reactions were accelerated in the presence of hydrogen.10) However, in the case of catechol-type catechins strain MT4s-5 could catalyze the C ring cleavage but could not catalyze either 4′-dehydroxylation in the B ring of (2S)- (1S) or (2R)-1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1R), even in the presence of hydrogen. These observations seemed to indicate that the presence of three vicinal hydroxyl groups in the B ring is important for the dehydroxylation by A. equolifaciens MT4s-5. Wang et al.11) reported that Eggerthella sp. SDG-2 (formerly Eubacterium sp. SDG-2) showed 4′-dehydroxylation ability of 1,3-dihydroxyphenylpropan-2-ol derivatives from (3R)-flavan-3-ols such as −EC, −C, −EGC, and (−)-gallocatechin (−GC), but not from (3S)-flavan-3-nols (+C and +EC). From these results, they stated that R configuration at C-3 position seemed to be essential for the dehydroxylation. Thus, the structural feature required for the dehydroxylation by A. equolifaciens MT4s-5 was different from that by Eggerthella sp. SDG-2.

E. lenta JCM 9979 catalyzed C ring cleavage of −EC and +C but hardly catalyzed the cleavage of +EC and −C whereas Eggerthella sp. SDG-2 has been shown to have the ability to cleave C ring of all the above catechins.11) Eggerthella sp. CAT-112) and Eggerthella lenta rk313) were reported to convert −EC into 1S and +C into 1R as well as E. lenta JCM 9979, but the conversion ability of +EC and −C by these strains is not determined. E. lenta strains JCM 9979 and rk313) could not catalyze the dehydroxylation of 1S or 1R whereas 1S and 1R have been found to undergo dehydroxylation by strain CAT-112) and 1R by strain SDG-2.11) Thus, the substrate specificity and dehydroxylation ability varied among the bacterial strains. However, we revealed in this study that E. lenta JCM 9979 had the ability to catalyze 4′-dehydroxylation of 1S and 1R only in the presence of hydrogen or formate. Accordingly, it is interesting to examine the dehydroxylation ability of strain rk3 and the bioconversion ability of strains CAT-1 and SDG-2 in the presence of hydrogen or formate.

F. plautii strains MT42, ATC C 29863, and ATC C 49531 were reported to degrade the phloroglucinol moiety of 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol and 1-(3,5-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol from −EGC.10) In this study, the F. plautii strains were found to have the ability to degrade 1S, 1R, 2S, and 2R. The observations suggested that F. plautii strains degrade the phloroglucinol moiety of 1-hydroxyphenyl-3-(2,4,6-trihydroxyphenyl)propan-2-ols regardless of not only the number and attached positions of their hydroxyl group in the B ring but also their configurations at 2 position. Furthermore, the above consideration led us to examine whether or not the F. plautii strains degrade phloroglucinol. Results revealed that phloroglucinol disappeared during the incubation with each of the strains (data not shown) and hence the F. plautii strains had the ability to degrade phloroglucinol.

We demonstrated that −EGC-metabolizing bacteria, A. equolifaciens MT4s-5, E. lenta JCM 9979, and F. plautii strains MT42, ATC C 29863, and ATC C 49531 were also involved in the metabolism of catechol-type catechins. On the basis of these results together with previous reports,1113) we propose the bioconversion of catechol-type catechins by intestinal bacteria as illustrated in Fig. 4. However, since catechol-type catechins were shown to undergo further degradation in the gut tract,9) much more research is needed to clarify the intestinal bacteria involved in degradation of 4-hydroxy-5-hydroxyphenylvaleric acids (3S, 3R, 5S, and 5R) and 5-hydroxyphenyl-γ-valerolactones (4S, 4R, 6S, and 6R).

Fig. 4. Possible Bioconversion Scheme of −EC, +EC, −C, and +C by A. equolifaciens MT4s-5, E. lenta JCM 9979, and F. plautii Strains

a)Quotation from ref. 11, b)Quotation from ref. 12, c)Quotation from ref. 13.

Acknowledgment

We acknowledge the assistance of Andrea K. Suzuki (Mitsui Norin Co., Ltd.) in the proofreading of the manuscript.

Conflict of Interest

The authors are employees of Mitsui Norin Co., Ltd.

REFERENCES
 
© 2015 The Pharmaceutical Society of Japan
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