Metabolic fate of (-)-[4-(3)H]epigallocatechin gallate in rats after oral administration.

After oral administration of [4-(3)H]EGCg to rats, the radioactivity in blood, major tissues, urine, and feces was measured over time. The radioactivity in blood and most tissues remained low for 4 h postdose, began to increase after 8 h, peaked at 24 h, and then decreased. Major urinary excretion of radioactivity occurred in the 8-24 h period, and the cumulative radioactivity excreted by 72 h was 32.1% of the dose. The radioactivity in the feces was 35.2% of the dose within 72 h postdose. In the case of rats pretreated with antibiotics (antibiotic-pretreated rats), the radioactivity levels of the blood and urine were definitely lower than those in rats not pretreated with antibiotics (normal rats). The radioactivity recovered in the antibiotic-pretreated rat urine was estimated to be only (1)/(100) of that in the normal rat urine. These results clearly demonstrated that the radioactivity detected in the blood and urine of normal rats mostly originated from degradation products of EGCg produced by intestinal bacteria. Furthermore, a main metabolite in the normal rats was purified and identified as 5-(5'-hydroxyphenyl)-gamma-valerolactone 3'-O-beta-glucuronide (M-2). In feces of the normal rats, EGC (40.8% of the fecal radioactivity) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M-1, 16.8%) were detected. These results suggested that M-1 was absorbed in the body after degradation of EGCg by intestinal bacteria, yielding M-1 with EGC as an intermediate. Furthermore, M-2 was thought to be formed from M-1 in the intestinal mucosa and/or liver, then to enter the systemic circulation, and finally to be excreted in the urine. Taking into account all of the above findings, a possible metabolic route of EGCg orally administered to rats is proposed.

Synthesis of (-)-[4-3H]epigallocatechin gallate and its metabolic fate in rats after intravenous administration.

Because a great deal of attention has been focused on the metabolism of (-)-epigallocatechin gallate (EGCg), quantitative analysis of this compound is required. For this purpose we developed a method of chemical synthesis of [4-(3)H]EGCg. Synthesized [4-(3)H]EGCg showed 99.5% radiochemical purity and a specific activity of 13 Ci/mmol. To clarify the excretion route of EGCg, the radioactivity levels of bile and urine were quantified after intravenous administration of [4-(3)H]EGCg to bile-duct-cannulated rats. Results showed that the radioactivity of the bile sample excreted within 48 h accounted for 77.0% of the dose, whereas only 2.0% of the dose was recovered in the urine. The excretion ratio of bile to urine was calculated to be about 97:3. These results clearly showed that bile was the major excretion route of EGCg. Time-course analysis of the radioactivity in blood was also performed to estimate the pharmacokinetic parameters following intravenous administration of [4-(3)H]EGCg. In addition, EGCg metabolites excreted in the bile within 4 h after the intravenous dose of [4-(3)H]EGCg were analyzed by HPLC. The results showed that 4',4"-di-O-methyl-EGCg was present in the conjugated form and made up about 14.7% of the administered radioactivity.

Identification of biliary metabolites of (-)-epigallocatechin gallate in rats.

After oral administration of (-)-epigallocatechin gallate (EGCg) to rats, its biliary metabolites were examined. Although a large part of the biliary metabolites was found to exist in conjugated forms, it was difficult to separate the conjugated forms. Thus the free form of biliary metabolites was prepared by beta-glucuronidase/sulfatase treatment and was purified by HPLC. Six compounds purified were subjected to FABeta-MS and NMR analyses. The six metabolites thus obtained were shown to be EGCg, 3'-O-methyl-EGCg, 4'-O-methyl-EGCg, 3' '-O-methyl-EGCg, 4' '-O-methyl-EGCg, and 4',4' '-di-O-methyl-EGCg, respectively. The six EGCg metabolites and their conjugates excreted during a 4-h period were estimated to be roughly 0.1% and 3.3% of the administered EGCg, respectively. In addition, 4' '-O-methyl-EGCg and 4',4' '-di-O-methyl-EGCg were estimated to exist only in the sulfate form, but the other four metabolites existed in both glucuronide (and/or sulfoglucuronide) and sulfate forms.

Antifungal activity of plant extracts against Arthrinium sacchari and Chaetomium funicola.

Various plant extracts were examined for antifungal activity with the objective of improving the commercial sterility of aseptically filled tea beverage products in PET bottles. When the hot water extract and the methanol extract of 29 samples were measured for their antifungal activity against Arthrinium sacchari M001 and Chaetomium funicola M002 strains, five samples, Acer nikoense, Glycyrrhiza glabra, Lagerstroemia speciosa, Psidium guajava and Thea sinensis, showed high activity. Of these, the extracts from A. nikoense, G. glabra and T. sinensis were fractionated by extraction with CHCl3, and the CHCl3-soluble fractions from G. glabra showed antifungal activity with minimum inhibitory concentrations (MICs) between 62.5 and 125 microg/ml against the above-mentioned two fungi. When the EtOAc-soluble fraction of A. nikoense was used, the MIC against A. sacchari M001 was 62.5 microg/ml. However, none of the fractions from A. nikoense or T. sinensis showed high activity against C. funicola M002 and their MICs were greater than 500 microg/ml. A licorice preparation made from the commercially available oil-based extract of G. glabra showed a low MIC of 25 microg/ml against five tested strains of filamentous fungi, but not against Aspergillus fumigatus M008, in a blended tea. Consequently, the possibility of adding a licorice preparation made from the oil-based extract of G. glabra to tea beverages (aseptically filled into PET bottles) was suggested.

Radical scavenging activity of tea catechins and their related compounds.

(-)-Epigallocatechin gallate was found to be the most effective scavenger among tea catechins for the superoxide anion, hydroxyl radical, and 1,1-diphenyl-3-picrylhydrazyl radical. Examination of the scavenging effects of tea catechins and their glucosides on superoxide anion showed that the presence of at least an ortho-dihydroxyl group in the B ring and a galloyl moiety at the 3 position was important in maintaining the effectiveness of the radical scavenging ability. Stoichiometric factors of tea catechins were estimated to be 2 for (+)-catechin and (-)-epicatechin, 5 for (-)-epigallocatechin, 7 for (-)-epicatechin gallate, and 10 for (-)-epigallocatechin gallate.

Methylation of tea catechins by rat liver homogenates.

Methylation of (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) was carried out with a rat liver homogenate and S-adenosyl-L-methionine. A structural analysis of the reaction products by MS and NMR showed that 4'-O-methyl EGC, 4"-O-methyl ECg, and 4"-O-methyl EGCg had been formed from EGC, ECg, and EGCg, respectively. These results suggest that methylation may be one of the metabolic pathways to the catechins.

Identification of (-)-epicatechin metabolites and their metabolic fate in the rat.

After oral administration of (-)-epicatechin to rats, three kinds of metabolites (M-1, M-2, and M-3) were detected in the urine. After isolation of the compounds by preparative high-performance liquid chromatography, structural analysis was carried out by mass spectrometry and NMR. As a result, two compounds, M-1 and M-2, were identified as (-)-epicatechin and 3'-O-methyl-(-)-epicatechin, respectively. M-3 was suggested to be a monomethylated (-)-epicatechin, but definitive elucidation was not possible because of its small quantity. Methylation of (-)-epicatechin with rat liver homogenates and subsequent structural analysis showed that M-3 was 4'-O-methyl-(-)-epicatechin. Identification of conjugated forms of the urinary metabolites also was attempted. Two conjugates in the urine were purified and analyzed by mass spectrometry and NMR. These conjugates were shown to be (-)-epicatechin-5-O-beta-glucuronide and 3'-O-methyl-(-)-epicatechin-5-O-beta-glucuronide, respectively. Metabolism and excretion of (-)-epicatechin were examined. (-)-Epicatechin and its methylated derivatives in the free forms were detected in plasma and urine, but not in bile. Significant differences in the excretion ratio of the conjugated forms of (-)-epicatechin and 3'-O-methyl-(-)-epicatechin were observed between urine and bile. Time-course analysis of (-)-epicatechin metabolites showed that the most predominant metabolites in plasma and urine were the conjugates of (-)-epicatechin and 3'-O-methyl-(-)-epicatechin, respectively, and the cumulative amount of the urinary metabolites excreted during the 24-h period was about 8% of the administered (-)-epicatechin.

Interaction of tea catechins with lipid bilayers investigated with liposome systems.

Interaction of tea catechins with lipid bilayers was investigated with liposome systems, which enabled us to separate liposomes from the external medium by centrifugation. We found that epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.

Absorption of tea catechins into rat portal vein.

Following the oral administration of tea catechins, (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate, respectively, to rats, the presence of these catechins in the portal blood was examined. It was confirmed by HPLC and mass spectrometry analysis that each of the administered catechins was present in the blood. These results clearly indicate that four predominant catechins in fresh tea leaves are absorbed, at least in part, into the rat portal vein.

Scavenging effects of tea catechins and their derivatives on 1,1-diphenyl-2-picrylhydrazyl radical.

The scavenging effects of tea catechins and their epimerized, acylated, and glucostylated derivatives on 1,1-diphenyl-2-picrythydrazyl (DPPH) radical were evaluated by electron spin resonance spectrometry. Tea catechins and their epimers were shown to have 50% radical scavenging ability in the concentration range of 1 to 3 microM. No significant differences were observed between the scavenging activities of tea catechins and their epimers, and, hence, the scavenging effects of catechins are not dependent on their sterical structure. The relationship between scavenging ability and the structure of tea catechins was also examined with acylated and glucosylated catechin derivatives. It is suggested that the galloyl moiety attached to flavan-3-ol at 3 position has a strong scavenging ability on the DPPH radical as well as the ortho-trihydroxyl group in the B ring, which elevates the radical scavenging efficiency above that of the ortho-dihydroxyl group; as has been recognized in other flavonoids such as flavones. The results obtained from the reactivity of tea catechins with the DPPH radical at different pHs suggest not only that the ortho-trihydroxyl group and the galloyl moiety contribute to maintaining the DPPH radical scavenging ability more effectively in a wide range of conditions from acidic to alkaline, but also that the radical scavenging efficiency of the ortho-dihydroxyls in the B ring is limited in neutral to alkaline regions. The difference between the scavenging abilities of the trihydroxyls (probably in the galloyl moiety) and the dihydroxyls can be explained in terms of redox potentials. It is concluded that the ortho-trihydroxyl group in the B ring and the galloyl moiety at 3 position of flavan-3-ol skeleton are the most important structural features for displaying an excellent scavenging ability on the DPPH radical.

Absorption of (-)-epigallocatechin gallate into rat portal vein.

Following oral administration of (-)-epigallocatechin gallate to rats, the presence of (-)-epigallocatechin gallate was examined in the portal blood. A compound present in the blood was identified as (-)-epigallocatechin gallate by HPLC and mass spectrometry analysis. The results clearly demonstrate that (-)-epigallocatechin gallate is absorbed, at least in part, into rat portal blood.

Effects of dietary tea catechins on alpha-tocopherol levels, lipid peroxidation, and erythrocyte deformability in rats fed on high palm oil and perilla oil diets.

The effects of dietary tea catechins on the levels of alpha-tocopherol and lipid peroxidation in both plasma and erythrocytes, as well as their effects on erythrocyte deformability, were examined in rats fed on high palm and perilla oil diets. The decrease in alpha-tocopherol concentration and the increase in lipid peroxidation level were much more pronounced in the perilla oil group than in the palm oil group. The addition of tea catechins to these diets significantly prevented the alpha-tocopherol concentration from decreasing. These results suggest that tea catechins may counteract a decrease in alpha-tocopherol by acting as an antioxidant in vivo. Furthermore, the lipid peroxidation in the plasma of rats fed perilla oil was slightly but significantly reduced by the supplemented tea catechins. However, no measurable differences were observed in the deformability of the erythrocytes in any of the groups. It is therefore likely that the erythrocytes are not severely enough affected by the lipid peroxidation to influence their deformability.

Purification and hydrolytic action of a chitosanase from Nocardia orientalis.

Chitosanase from the culture filtrate of Nocardia orientalis was purified to apparent homogeneity by precipitation with ammonium sulfate followed by CM-Sephadex chromatography, biospecific affinity chromatography on a Sepharose CL-4B with immobilized chitotriose and by gel filtration on Sephadex G-75. The enzyme specifically acted on chitooligosaccharides and chitosan to yield chitobiose and chitotriose as final products. The mode of action of the chitosanase on chitooligosaccharides and their corresponding alcohols suggests that the enzyme requires substrates with four or more glucosamine residues for the expression of activity and its shows maximum activity on chitohexaose and chitoheptaose. In the hydrolysis of chitosans of varying N-acetyl content, the enzyme cleaved about 30% acetylated chitosan with maximum activity and the enzyme activity decreased with increasing the degree of deacetylation of chitosans tested. The analysis of products formed from 33% acetylated chitosan shows the chitosanase is capable of cleaving between glucosamine and glucosamine or N-acetylglucosamine, but not cleaving between N-acetylglucosamine and glucosamine. On the basis of the results, the whole pathway of enymatic degradation of partially acetylated chitosan by a combination of chitosanase, exo-beta-D-glucosaminidase and beta-N-acetylhexosaminidase is proposed.

Enzymatic method for determination of the degree of deacetylation of chitosan.

A new method was developed for the accurate determination of the degree of deacetylation of chitosan. The method involves the complete hydrolysis of chitosan to glucosamine and N-acetylglucosamine by a cooperative action of chitosanolytic enzymes exo-beta-D-glucosaminidase, beta-N-acetylhexosaminidase, and chitosanase, and subsequent determination of the monosaccharides by specific colorimetric assays or HPLC. The conditions required for the complete hydrolysis of chitosan were examined and the degree of deacetylation of several chitosan samples was determined.

Purification and characterization of an exo-beta-D-glucosaminidase, a novel type of enzyme, from Nocardia orientalis.

A new enzyme capable of hydrolyzing chitobiose, which is an induced enzyme, was purified to apparent homogeneity from the culture filtrate of Nocardia orientalis IFO 12806. Biospecific affinity chromatography on chitotriitol-Sepharose CL-4B was effective for purification of this enzyme. It is clearly demonstrated that the enzyme is an exo-hydrolase, removing single glucosamine residues from the nonreducing terminal of a sequence of beta-(1----4)-linked glucosamine chain, such as chitosan and chitooligosaccharides, and therefore characterized as an exo-beta-D-glucosaminidase. The enzyme was found to show maximum activity on chitotetraose, chitopentaose, and their corresponding alcohols and a slight decrease in rate on longer chain lengths of substrates. A significant decrease in rate was observed using p-nitrophenyl beta-D-glucosaminide and chitobiitol as substrates. In the hydrolysis of partially acetylated chitosans, the enzyme appeared to be effective in cleaving glucosamine from the GlcN beta 1----4GlcNAc beta 1----sequence as well as the GlcN beta 1----4GlcN beta 1----sequence. These observations suggest that the second residue from the terminal plays an important role in enzyme activity, but the enzyme permits the replacement of glucosamine at the second residue by N-acetylglucosamine.

p-nitrophenyl penta-N-acetyl-beta-chitopentaoside as a novel synthetic substrate for the colorimetric assay of lysozyme.

p-Nitrophenyl beta-glycosides of N-acetylchitooligosaccharides (PNP-(GlcNAc)n n = 3-5) were examined as substrates for lysozyme [EC 3.2.1.17]. The enzyme released predominantly p-nitrophenyl N-acetyl-beta-D-glucosaminide (PNP-GlcNAc) from each substrate. Furthermore, the initial rate of PNP-GlcNAc formation in lysozyme-catalyzed hydrolysis of p-nitrophenyl penta-N-acetyl-beta-chitopentaoside (PNP-(GlcNAc)5) was about 350 and 25 times faster than those of p-nitrophenyl tri-N-acetyl-beta-chitotrioside (PNP-(GlcNAc)3) and p-nitrophenyl tetra-N-acetyl-beta-chitotetraoside (PNP-(GlcNAc)4), respectively. From these results, a new colorimetric assay method of lysozyme using PNP-(GlcNAc)5 as a substrate was developed on the basis of the determination of p-nitrophenol liberated from the substrate by lysozyme through a coupled reaction involving beta-N-acetylhexosaminidase (NAHase). The assay system gave a linear dose-response curve in the range of 2-120 micrograms of lysozyme in a 15-60 min incubation. The present assay was not significantly influenced by the ionic strength of the medium and was reproducible. This method using PNP-(GlcNAc)5 as a substrate was shown to be useful for lysozyme assay.

Enzymatic synthesis of p-nitrophenyl N,N',N'',N'',N''''-pentaacetyl-beta-chitopentaoside in water-methanol system; significance as a substrate for lysozyme assay.

Transchitooligosylation from (GlcNAc)5 to the 4-position of PNP-GlcNAc was efficiently induced through lysozyme catalysis in an aqueous solution containing methanol with a high concentration. Use of the aqueous methanol system in this reaction not only guaranteed solubility of PNP-GlcNAc substrate, but also resulted in a remarkable increase in PNP-(GlcNAc)5 production. PNP-(GlcNAc)5 was substrate for lysozyme assay compared with PNP-(GlcNAc)4.

Transglycosylation reaction of a chitinase purified from Nocardia orientalis.

Chitinase from the culture filtrates of Nocardia orientalis IFO 12806 was purified to apparent homogeneity by precipitation with ammonium sulfate followed by successive chromatography on CM-Sephadex and Bio-Gel P-60, and finally by affinity chromatography on a phenyl-Sepharose CL-4B column. The enzyme, which is essentially a hydrolase, also catalyzed a transglycosylation reaction on tetra-N-acetyl-chitotetraose (GlcNAc)4 and penta-N-acetyl-chitopentaose (GlcNAc)5. The enzyme converted the tetrasaccharide into hexa-N-acetyl-chitohexaose (GlcNAc)6 (21%) and di-N-acetyl-chitobiose (GlcNAc)2 (63%) as the major products. The corresponding values for penta-N-acetyl-chitopentaose (GlcNAc)5 were hepta-N-acetyl-chitoheptaose (GlcNAc)7 23% and tri-N-acetyl-chitotriose (GlcNAc)3 59%. The rate of the transglycosylation depended on the temperature, the concentration of substrate and the pH.