Effects of Microbial Metabolites of (-)-Epigallocatechin Gallate on Glucose Uptake in L6 Skeletal Muscle Cell and Glucose Tolerance in ICR Mice.

Glucose uptake ability into L6 skeletal muscle cell was examined with eleven kinds of ring fission metabolites of (-)-epigallocatechin gallate (EGCG) produced by intestinal bacteria. The metabolites 5-(3,5-dihydroxyphenyl)-γ-valerolactone (EGC-M5), 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M6), 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (EGC-M7) and 5-(3-hydroxyphenyl)valeric acid (EGC-M11) have been found to promote uptake of glucose into L6 myotubes significantly. EGC-M5, which is one of the major ring fission metabolites of EGCG, was also found to have a promotive effect on glucose transporter 4 (GLUT4) translocation accompanied by phosphorylation of AMP-activated protein kinase (AMPK) signaling pathway in skeletal muscle both in vivo and in vitro. Furthermore, the effect of oral single dosage of EGC-M5 on glucose tolerance test with ICR mice was examined and significant suppression of hyperglycemia was observed. These data suggested that EGC-M5 has an antidiabetic effect in vivo.

Function of Green Tea Catechins in the Brain: Epigallocatechin Gallate and its Metabolites.

Over the last three decades, green tea has been studied for its beneficial effects, including anti-cancer, anti-obesity, anti-diabetes, anti-inflammatory, and neuroprotective effects. At present, a number of studies that have employed animal, human and cell cultures support the potential neuroprotective effects of green tea catechins against neurological disorders. However, the concentration of (-)-epigallocatechin gallate (EGCG) in systemic circulation is very low and EGCG disappears within several hours. EGCG undergoes microbial degradation in the small intestine and later in the large intestine, resulting in the formation of various microbial ring-fission metabolites which are detectable in the plasma and urine as free and conjugated forms. Recently, in vitro experiments suggested that EGCG and its metabolites could reach the brain parenchyma through the blood-brain barrier and induce neuritogenesis. These results suggest that metabolites of EGCG may play an important role, alongside the beneficial activities of EGCG, in reducing neurodegenerative diseases. In this review, we discuss the function of EGCG and its microbial ring-fission metabolites in the brain in suppressing brain dysfunction. Other possible actions of EGCG metabolites will also be discussed.

Inhibitory Activity of Catechin Metabolites Produced by Intestinal Microbiota on Proliferation of HeLa Cells.

Eleven kinds of catechin metabolites produced from (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCg) by intestinal microbiota were evaluated for inhibitory activity on the proliferation of HeLa cells, which are human cervical cancer cells. Among the catechin metabolites, 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M2), 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M7), and 5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M9) were found to show inhibitory activity on HeLa cell proliferation as compared with control. The results suggested that three adjacent hydroxyl groups in the phenyl moiety may play an important role in the inhibitory activity. In addition, the inhibitory activity was also examined with four (-)-epicatechin (EC) metabolites possessing two adjacent hydroxyl groups in the phenyl moiety. Only 5-(3,4-dihydroxyphenyl)valeric acid (EC-M9) showed inhibitory activity and therefore valeric acid moiety likely contributes to the inhibitory activity. EGC-M9 showed the strongest inhibitory activity with IC50 of 5.58 µM. Thus, in this study it was found for the first time that several catechin metabolites derived from EGC, EGCg, and EC inhibit the proliferation of cervical cancer cells.

Hypolipidemic effects of crude green tea polysaccharide on rats, and structural features of tea polysaccharides isolated from the crude polysaccharide.

Crude tea polysaccharide (crude TPS) was prepared from instant green tea by ethanol precipitation followed by ultrafiltration membrane treatment and its effects on blood lipid, liver lipid, and fecal lipid levels were examined with Sprague-Dawley rats fed a high-fat diet. Although crude TPS showed no effects on the serum lipid levels, it suppressed the liver lipid accumulation and increased the fecal excretion of dietary fat. Then, the structural features of crude TPS were investigated. After separation of crude TPS by DEAE-cellulose and gel-filtration column chromatography, two kinds of neutral tea polysaccharides (NTPS-LP and NTPS-HH) and an acidic polysaccharide (ATPS-MH) were obtained. According to monosaccharide composition, methylation, and NMR analyses, NTPS-LP, NPTS-HH, and ATPS-MH were presumed to be starch, arabinogalactan with ß-1,3-linked galactosyl backbone blanched at position 6 and with 1,5-linked arabinofuranosyl residues, and α-1,4-linked galacturonic acid backbone with arabinogalactan region, respectively.

Biotransformation of (-)-epicatechin, (+)-epicatechin, (-)-catechin, and (+)-catechin by intestinal bacteria involved in isoflavone metabolism.

Isoflavone-metabolizing bacteria, Adlercreutzia equolifaciens, Asaccharobacter celatus, Slackia equolifaciens, and Slackia isoflavoniconvertens catalyzed C-ring cleavage of (-)-epicatechin and (+)-catechin, (+)-epicatechin, and (-)-catechin in varying degrees. The cleaving abilities of (-)-epicatechin and (+)-catechin were enhanced by hydrogen, except (+)-catechin cleavage by S. equolifaciens, which was not accelerated. (-)-Catechin cleavage by Ad. equolifaciens was remarkably accelerated by hydrogen.

Bioconversion of (-)-epicatechin, (+)-epicatechin, (-)-catechin, and (+)-catechin by (-)-epigallocatechin-metabolizing bacteria.

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.

Biotransformation of (-)-epigallocatechin and (-)-gallocatechin by intestinal bacteria involved in isoflavone metabolism.

Four isoflavone-metabolizing bacteria were tested for their abilities to degrade (-)-epigallocatechin (EGC) and its isomer (-)-gallocatechin (GC). Biotransformation of both EGC and GC was observed with Adlercreutzia equolifaciens JCM 14793, Asaccharobacter celatus JCM 14811, and Slackia equolifaciens JCM 16059, but not Slackia isoflavoniconvertens JCM 16137. With respect to the degradation of EGC, strain JCM 14793 only catalyzed 4'-dehydroxylation to produce 4'-dehydroxylated EGC (7). Strain JCM 14811 mainly produced 7, along with a slight formation of the C ring-cleaving product 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1). Strain JCM 16059 catalyzed only C ring cleavage to form 1. Interestingly, the presence of hydrogen promoted the bioconversion of EGC by these bacteria. In addition, strain JCM 14811 revealed the ability to catalyze 4'-dehydroxylation of 1 to yield 1-(3,5-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2) in the presence of hydrogen. In the case of GC, strain JCM 14793 mainly produced C ring-cleaving product (1) with only a very small amount of 4'-dehydroxylated GC (8), while Strain JCM 14811 only catalyzed 4'-dehydroxylation to form 8. Strain JCM 16059 formed 1. The bioconversion of GC by the three strains was stimulated by hydrogen. Strain JCM 14793 showed the ability to convert 1 into 2 in the presence of hydrogen as did strain JCM 14811. Furthermore, strains JCM 14793 and JCM 14811 were found to have the ability to catalyze p-dehydroxylation of the pyrogallol moiety in the EGC metabolites 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (3) and 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (4), and this ability was enhanced by the presence of hydrogen.

Isolation and characterization of rat intestinal bacteria involved in biotransformation of (-)-epigallocatechin.

Two intestinal bacterial strains MT4s-5 and MT42 involved in the degradation of (-)-epigallocatechin (EGC) were isolated from rat feces. Strain MT4s-5 was tentatively identified as Adlercreutzia equolifaciens. This strain converted EGC into not only 1-(3, 4, 5-trihydroxyphenyl)-3-(2, 4, 6-trihydroxyphenyl)propan-2-ol (1), but also 1-(3, 5-dihydroxyphenyl)-3-(2, 4, 6-trihydroxyphenyl)propan-2-ol (2), and 4'-dehydroxylated EGC (7). Type strain (JCM 9979) of Eggerthella lenta was also found to convert EGC into 1. Strain MT42 was identified as Flavonifractor plautii and converted 1 into 4-hydroxy-5-(3, 4, 5-trihydroxyphenyl)valeric acid (3) and 5-(3, 4, 5-trihydroxyphenyl)-γ-valerolactone (4) simultaneously. Strain MT42 also converted 2 into 4-hydroxy-5-(3, 5-dihydroxyphenyl)valeric acid (5), and 5-(3, 5-dihydroxyphenyl)-γ-valerolactone (6). Furthermore, F. plautii strains ATCC 29863 and ATCC 49531 were found to catalyze the same reactions as strain MT42. Interestingly, formation of 2 from EGC by strain MT4s-5 occurred rapidly in the presence of hydrogen supplied by syntrophic bacteria. Strain JCM 9979 also formed 2 in the presence of the hydrogen or formate. Strain MT4s-5 converted 1, 3, and 4 to 2, 5, and 6, respectively, and the conversion was stimulated by hydrogen, whereas strain JCM 9979 could catalyze the conversion only in the presence of hydrogen or formate. On the basis of the above results together with previous reports, the principal metabolic pathway of EGC and EGCg by catechin-degrading bacteria in gut tract is proposed.

Antioxidative activity of microbial metabolites of (-)-epigallocatechin gallate produced in rat intestines.

The antioxidative activities of (-)-epigallocatechin gallate (EGCg) metabolites degraded by rat intestinal flora were investigated by a flow injection analysis coupled to an on-line antioxidant detection system with the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation. All of the metabolites were found to have antioxidative activity, suggesting that the EGCg metabolites may show antioxidative activity in the body.

Blood-Brain Barrier Permeability of Green Tea Catechin Metabolites and their Neuritogenic Activity in Human Neuroblastoma SH-SY5Y Cells.

SCOPE: To understand the mechanism by which green tea lowers the risk of dementia, focus was placed on the metabolites of epigallocatechin gallate (EGCG), the most abundant catechin in green tea. Much of orally ingested EGCG is hydrolyzed to epigallocatechin (EGC) and gallic acid. In rats, EGC is then metabolized mainly to 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5) and its conjugated forms, which are distributed to various tissues. Therefore, we examined the permeability of these metabolites into the blood-brain barrier (BBB) and nerve cell proliferation/differentiation in vitro. METHODS AND RESULTS: The permeability of EGC-M5, glucuronide, and the sulfate of EGC-M5, pyrogallol, as well as its glucuronide into the BBB were examined using a BBB model kit. Each brain- and blood-side sample was subjected to liquid chromatography tandem-mass spectrometry analysis. BBB permeability (%, in 0.5 h) was 1.9-3.7%. In human neuroblastoma SH-SY5Y cells, neurite length was significantly prolonged by EGC-M5, and the number of neurites was increased significantly by all metabolites examined. CONCLUSION: The permeability of EGC-M5 and its conjugated forms into the BBB suggests that they reached the brain parenchyma. In addition, the ability of EGC-M5 to affect nerve cell proliferation and neuritogenesis suggests that EGC-M5 may promote neurogenesis in the brain.

Blood brain barrier permeability of (-)-epigallocatechin gallate, its proliferation-enhancing activity of human neuroblastoma SH-SY5Y cells, and its preventive effect on age-related cognitive dysfunction in mice.

BACKGROUND: The consumption of green tea catechins (GTCs) suppresses age-related cognitive dysfunction in mice. GTCs are composed of several catechins, of which epigallocatechin gallate (EGCG) is the most abundant, followed by epigallocatechin (EGC). Orally ingested EGCG is hydrolyzed by intestinal biota to EGC and gallic acid (GA). To understand the mechanism of action of GTCs on the brain, their permeability of the blood brain barrier (BBB) as well as their effects on cognitive function in mice and on nerve cell proliferation in vitro were examined. METHODS: The BBB permeability of EGCG, EGC and GA was examined using a BBB model kit. SAMP10, a mouse model of brain senescence, was used to test cognitive function in vivo. Human neuroblastoma SH-SY5Y cells were used to test nerve cell proliferation and differentiation. RESULTS: The in vitro BBB permeability (%, in 30 min) of EGCG, EGC and GA was 2.8±0.1, 3.4±0.3 and 6.5±0.6, respectively. The permeability of EGCG into the BBB indicates that EGCG reached the brain parenchyma even at a very low concentration. The learning ability of SAMP10 mice that ingested EGCG (20 mg/kg) was significantly higher than of mice that ingested EGC or GA. However, combined ingestion of EGC and GA showed a significant improvement comparable to EGCG. SH-SY5Y cell growth was significantly enhanced by 0.05 µM EGCG, but this effect was reduced at higher concentrations. The effect of EGC and GA was lower than that of EGCG at 0.05 µM. Co-administration of EGC and GA increased neurite length more than EGC or GA alone. CONCLUSION: Cognitive dysfunction in mice is suppressed after ingesting GTCs when a low concentration of EGCG is incorporated into the brain parenchyma via the BBB. Nerve cell proliferation/differentiation was enhanced by a low concentration of EGCG. Furthermore, the additive effect of EGC and GA suggests that EGCG sustains a preventive effect after the hydrolysis to EGC and GA.

Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4(+) T Cell and Natural Killer Cell Activities.

Tea catechins, such as (-)-epigallocatechin-3-O-gallate (EGCG), have been shown to effectively enhance immune activity and prevent cancer, although the underlying mechanism is unclear. Green tea catechins are instead converted to catechin metabolites in the intestine. Here, we show that these green tea catechin metabolites enhance CD4(+) T cell activity as well as natural killer (NK) cell activity. Our data suggest that the absence of a 4'-hydroxyl on this phenyl group (B ring) is important for the effect on immune activity. In particular, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5), a major metabolite of EGCG, not only increased the activity of CD4(+) T cells but also enhanced the cytotoxic activity of NK cells in vivo. These data suggest that EGC-M5 might show immunostimulatory activity.

Effects of Metabolites Produced from (-)-Epigallocatechin Gallate by Rat Intestinal Bacteria on Angiotensin I-Converting Enzyme Activity and Blood Pressure in Spontaneously Hypertensive Rats.

Inhibitory activity of angiotensin I-converting enzyme (ACE) was examined with (-)-epigallocatechin gallate (EGCG) metabolites produced by intestinal bacteria, together with tea catechins. All of the metabolites showed ACE inhibitory activities and the order of IC50 was hydroxyphenyl valeric acids > 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (1) > trihydroxyphenyl 4-hydroxyvaleric acid ≫ dihydroxyphenyl 4-hydroxyvaleric acid ≫ 5-(3,5-dihydroxyphenyl)-γ-valerolactone (2). Among the catechins, galloylated catechins exhibited stronger ACE inhibitory activity than nongalloylated catechins. Furthermore, the effects of a single oral intake of metabolites 1 and 2 on systolic blood pressure (SBP) were examined with spontaneously hypertensive rats (SHR). Significant decreases in SBP were observed between 2 h after oral administration of 1 (150 mg/kg in SHR) and the control group (p = 0.002) and between 4 h after administration of 2 (200 mg/kg in SHR) and the control group (p = 0.044). These results suggest that the two metabolites have hypotensive effects in vivo.

Catabolism of (+)-catechin and (-)-epicatechin by rat intestinal microbiota.

Catabolism of (+)-catechin (+C) and (-)-epicatechin (EC) by rat intestinal microbiota was examined in vitro. +C and EC metabolites isolated were identified by LC-MS and NMR analyses. As a result, 4-hydroxy-5-(3-hydroxyphenyl)valeric acid (C-5 and EC-5), 4-oxo-5-(3,4-dihydorxyphenyl)valeric acid (EC-7), 4-oxo-5-(3-hydorxyphenyl)valeric acid (EC-8), and 1-(4-hydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EC-13) were identified as new metabolites of +C or EC. From the measurement of optical rotation, each of the +C and EC metabolites possessing the same chemical structure and chiral carbon was inferred to have an enantiomeric relationship to each other and to maintain the configurations at the 3-position of the original catechins. On the basis of these findings together with previous information, the proposed metabolic pathway of +C and EC by rat intestinal microbiota was updated.

Metabolism of (-)-epigallocatechin gallate by rat intestinal flora.

Anaerobic metabolism of (-)-epigallocatechin gallate (EGCg) by rat intestinal bacteria was investigated in vitro. First, intestinal bacteria which are capable of hydrolyzing EGCg to (-)-epigallocatechin (EGC) and gallic acid (2) were screened with 169 strains of enteric bacteria. As a result, Enterobacter aerogenes, Raoultella planticola, Klebsiella pneumoniae susp. pneumoniae, and Bifidobacterium longum subsp. infantis were found to hydrolyze EGCg. Subsequent steps of EGCg metabolism are degradation of EGC (1) by intestinal bacteria. Then, EGC was incubated with rat intestinal bacteria in 0.1 M phosphate buffer (pH 7.1) and the degradation products were analyzed with time by HPLC or LC-MS. Further, the products formed from EGC were isolated and identified by LC-MS and NMR analyses. The results revealed that EGC was converted first to 1-(3',4',5'-trihydroxyphenyl)-3-(2'',4'',6''-trihydroxyphenyl)propan-2-ol (3) by reductive cleavage between 1 and 2 positions of EGC, and subsequently metabolite 3 was converted to 1-(3',5'-dihydroxyphenyl)-3-(2'',4'',6''-trihydroxyphenyl)propan-2-ol (4) followed by the conversion to 5-(3,5-dihydroxyphenyl)-4-hydroxyvaleric acid (5) by decomposition of the phloroglucinol ring in metabolite 4. This degradation pathway was considered to be the major route of EGCg metabolism in the in vitro study, but two minor routes were also found. In addition to the in vitro experiments, metabolites 3, 4, 5, and 6 were detected as the metabolites after direct injection of EGC into rat cecum. When EGCg was administered orally to the rats, metabolites 4, 5, 6, 11, and 12 were found in the feces. Among the metabolites detected, metabolite 5 was dominant both in the cecal contents and feces. These findings suggested that the metabolic pathway of EGCg found in the in vitro study may be regarded as reflecting its metabolism in vivo.

Butyricimonas synergistica gen. nov., sp. nov. and Butyricimonas virosa sp. nov., butyric acid-producing bacteria in the family 'Porphyromonadaceae' isolated from rat faeces.

Two bacterial strains, designated MT01(T) and MT12(T), isolated from rat faeces were characterized by using a polyphasic taxonomic approach that included analysis of their phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogeny based on 16S rRNA gene sequences. The 16S rRNA gene sequence analysis showed that these strains were members of the family 'Porphyromonadaceae'. The strains shared 94 % 16S rRNA gene sequence similarity with each other and were related to Odoribacter splanchnicus NCTC 10825(T) (86-87 % sequence similarity). The strains consisted of obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. Growth of the strains was inhibited on medium containing 20 % bile. The two strains produced significant levels of butyric and isobutyric acids as end products from glucose. Although the major cellular fatty acid of these two strains and O. splanchnicus JCM 15291(T) was iso-C(15 : 0), strains MT01(T) and MT12(T) showed a higher level of iso-C(15 : 0) (66 and 74 %, respectively) than did O. splanchnicus JCM 15291(T) (48 %). In addition, the ratios of iso-C(15 : 0) to anteiso-C(15 : 0) in whole-cell methanolysates of the two isolates were very much higher than that of O. splanchnicus JCM 15291(T). The major menaquinone of the isolates was MK-10. This menaquinone composition was different from those of other genera of the family 'Porphyromonadaceae', such as Barnesiella (predominant menaquinones: MK-11 and MK-12), Odoribacter (MK-9), Paludibacter (MK-8), Parabacteroides (MK-9 and MK-10), Porphyromonas (MK-9 and MK-10) and Tannerella (MK-10 and MK-11). Menaquinone composition is therefore an important chemotaxonomic characteristic of these micro-organisms. Strains MT01(T) and MT12(T) have DNA G+C contents of 46 mol%. On the basis of these data, strains MT01(T) and MT12(T) represent two novel species of a novel genus, for which the names Butyricimonas synergistica gen. nov., sp. nov. and Butyricimonas virosa sp. nov., respectively, are proposed. The type strains of B. synergistica and B. virosa are MT01(T) (=JCM 15148(T) =CCUG 56610(T)) and MT12(T) (=JCM 15149(T)=CCUG 56611(T)), respectively.

Deodorizing effects of tea catechins on amines and ammonia.

Deodorizing effects of tea catechins on amines were examined under alkaline conditions to eliminate the neutralization reaction. They showed deodorizing activity on ethylamine, but none on dimethylamine or trimethylamine. Deodorizing activity on ethylamine was found to be in the order of (-)-epigallocatechin gallate > gallic acid > (-)-epigallocatechin (EGC) > (-)-epicatechin gallate > ethyl gallate >> (+)-catechin = (-)-epicatechin. Further, reaction products of EGC with methylamine, ethylamine, and ammonia were detected by HPLC, indicating that a deodorizing reaction other than neutralization occurs. From structural analysis of the reaction product with the methylamine isolated as a peracetylated derivative, the product was presumed to be methylamine substituted EGC, in which the hydroxyl group of EGC at the 4' position is replaced by the methylamino group. The same replacement reaction took place in the case of ethylamine and ammonia.