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.     

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.     

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.     

Absorption and urinary excretion of (-)-epicatechin after administration of different levels of cocoa powder or (-)-epicatechin in rats.

(-)-Epicatechin is a major polyphenol component of cocoa powder. The absorption and urinary excretion of (-)-epicatechin following administration of different levels of either cocoa powder (150, 750, and 1500 mg/kg) or (-)-epicatechin (1, 5, and 10 mg/kg) were evaluated in rats. Both the sum of plasma (-)-epicatechin metabolites at 1 h postadministration and peak plasma concentrations increased in a dose-dependent fashion. The sum of (-)-epicatechin metabolites in urine, excreted within 18 h postadministration, also increased with dose. Moreover, the sum of (-)-epicatechin metabolites excreted in urine reached the same level in both (-)-epicatechin and cocoa powder administration groups for equivalent amounts of (-)-epicatechin. These results suggest that, in the dose range examined in this study, bioavailability of (-)-epicatechin following administration of either (-)-epicatechin or cocoa powder shows dose dependence and that the various compounds present in cocoa powder have little effect on the bioavailability of (-)-epicatechin in cocoa powder.     

Identification of oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin with H(2)O(2)

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are two important antioxidants in tea. They also display some antitumor activities, and these activities are believed to be mainly due to their antioxidative effects. However, the specific mechanisms of antioxidant action of tea catechins remain unclear. In this study are isolated and identified two novel reaction products of EGCG and one product of EGC when they were reacted separately with H(2)O(2). These products are formed by the oxidation and decarboxylation of the A ring in the catechin molecule. This study provides unequivocal proof that the A ring of EGCG and EGC may also be an antioxidant site. This study also indicates an additional reaction pathway for the oxidation chemistry of tea catechins.     

Yeast-induced inhibition of (+)-catechin and (-)-epicatechin degradation in model solutions

(+)-catechin and (-)-epicatechin degradation in water-alcohol solutions containing Fe2+ and tartaric acid was studied in the presence and absence of yeasts. On the basis of the results, yeast partially inhibited the degradation of both flavans, with much slower formation of browning products absorbing at 420 and 520 nm. In comparative terms, yeast was found to be more efficient toward the degradation products of (+)-catechin absorbing at the latter wavelength. Likewise, the presence of yeast decreased the yield of a group of colored compounds eluting at high retention times in HPLC and indicated these as important contributors to color darkening in white wines. This inhibitory effect may in part account for the resistance to browning observed over periods of several years in sherry wines subjected to biological aging under flor yeast.     

Identification of metabolites of [1,2,3-(13)C]Propargyl alcohol in rat urine by (13)C NMR and mass spectrometry

Little is known about the metabolism of acetylenic (C&tbd1;C) compounds commonly used in the formulation of pesticides. To better understand the in vivo reactivity of this bond, we examined the metabolism of propargyl alcohol (PA), 2-propyn-1-ol, used extensively in the chemical industry. [1,2,3-(13)C, 2,3-(14)C]PA was administered orally to male Sprague-Dawley rats. Approximately 56% of the dose was excreted in urine by 96 h. Major metabolites were characterized, directly, in the whole urine by one- and two-dimensional (13)C NMR. To determine the complete structures of metabolites of PA, rat urine was also subjected to TLC followed by purification of separated TLC bands on HPLC. The purified metabolites were identified by (13)C NMR and mass spectrometry and by comparison with available synthetic standards. The proposed metabolic pathway involves oxidation of propargyl alcohol to 2-propynoic acid and further detoxification via glutathione conjugation to yield as final products: 3, 3-bis[(2-(acetylamino)-2-carboxyethyl)thio]-1-propanol, 3-(carboxymethylthio)-2-propenoic acid, 3-(methylsulfinyl)-2-(methylthio)-2-propenoic acid, 3-[[2-(acetylamino)-2-carboxyethyl]thio]-3-[(2-amino-2-carboxyethyl)t hio]-1-propanol and 3-[[2-(acetylamino)-2-carboxyethyl]sulfinyl]-3-[2-(acetylamino)-2-car boxyethyl]thio]-1-propanol. These unique metabolites have not been reported previously and represent the first example of multiple glutathione additions to the carbon-carbon triple bond.     

Competition between (+)-catechin and (-)-epicatechin in acetaldehyde-induced polymerization of flavanols.

The reactions of (+)-catechin and (-)-epicatechin in the presence of acetaldehyde were studied in model solution systems. When incubated separately with acetaldehyde and at pH values varying from 2.2 to 4. 0, reactions were faster with (-)-epicatechin than with (+)-catechin. In mixtures containing both (+)-catechin and (-)-epicatechin with acetaldehyde, new compounds besides the homogeneous bridged derivatives were detected. These compounds were concluded to be hetero-oligomers consisting of (+)-catechin and (-)-epicatechin linked with an ethyl bridge. In this case, the reaction of (-)-epicatechin was faster than that of (+)-catechin. This was also observed in solutions containing the two flavanols and the (+)-catechin-ethanol intermediate. Under these conditions, the homogeneous (+)-catechin bridged dimers and heterogeneous dimers were obtained by action of the intermediate on (+)-catechin and (-)-epicatechin, respectively. In addition, the homogeneous (-)-epicatechin ethyl-bridged dimers were also detected, showing that ethyl linkages underwent depolymerization and recombination reactions.     

Degradation of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase

The degradation mechanism of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase (PPO) was investigated using several model systems. The enzymically generated (-)-epicatechin o-quinone could induce cyanidin 3-rutinoside degradation. The results obtained in this study allowed us to propose a pathway for cyanidin 3-rutinoside degradation in the presence of (-)-epicatechin and litchi pericarp PPO. First, enzymatic oxidation of (-)-epicatechin produced the corresponding o-quinone, and then cyanidin 3-rutinoside and (-)-epicatechin competed for (-)-epicatechin o-quinone, resulting in degradation of cyanidin 3-rutinoside and regeneration of (-)-epicatechin. Moreover, the results of kinetic studies indicated this competition was influenced by both (-)-epicatechin concentration and cyanidin 3-rutinoside concentration in the model system.     

Metabolism of 7-fluoro-6-(3,4,5,6-tetrahydrophthalimido)-4- (2-propynyl)-2H-1,4-benzoxazin-3(4H)-one (S-53482, flumioxazin) in the rat: II. Identification of reduced metabolites

On single oral administration of (14)C-S-53482 [7-fluoro-6-(3,4,5, 6-tetrahydrophthalimido)-4-(2-propynyl)-2H-1,4-benzoxazin-3( 4H)-one, Flumioxazin] labeled at the 1- and 2-positions of tetrahydrophthaloyl group to rats at 1 (low dose) or 100 (high dose) mg/kg, the radiocarbon was almost completely eliminated within 7 days after administration in both groups with generally very low residual (14)C tissue levels. The predominant excretion route was via the feces. The major fecal and urinary metabolites involved reduction or sulfonic acid addition reactions at the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety and hydroxylation of the cyclohexene or cyclohexane ring. One urinary and four fecal metabolites were identified using chromatographic techniques and spectroanalyses (NMR and MS). Three of five identified metabolites were unique forms, reduced at the 1,2-double bond of the 3,4,5, 6-tetrahydrophthalimide moiety. On the basis of the metabolites identified in this study, the metabolic pathways of S-53482 in rats are proposed. To specify tissues forming reduced metabolites, an in vitro study was conducted. Reduction was found to take place in red blood cells.     

Rapid liquid chromatography tandem mass spectrometry assay to quantify plasma (-)-epicatechin metabolites after ingestion of a standard portion of cocoa beverage in humans

A rapid liquid chromatography electrospray ionization tandem mass spectrometry with negative ion detection method was developed and validated to determine cocoa flavonoid metabolites in human plasma and urine after the intake of a standard portion of a cocoa beverage. A chromatographic run time of only 9 min provided clear separation of all metabolites and internal standards. Samples were analyzed in a product-ion scan of m/z 289, 369, and 465 to identify the metabolites and in multiple reaction monitoring acquisition mode to quantify (-)-epicatechin ((-)-Ec) (289/ 245), (-)-epicatechin-glucuronide ((-)-EcG) (465/289), and (-)-epicatechin-sulfate ((-)-EcS) (369/289). One (-)-Ec-G and three (-)-Ec-S were identified and confirmed in urine as the major metabolites, and one (-)-Ec-G was the only metabolite present in plasma volunteers (n = 5) at a mean concentration of 625.7 +/- 198.3 nmol/L at 2 h after consumption of a cocoa beverage containing 54.4 mg of (-)-Ec.     

Characterization of polyphenol oxidase from litchi pericarp using (-)-epicatechin as substrate

Polyphenol oxidase (PPO) from litchi (Litchi chinensis Sonn.) pericarp was characterized using (-)-epicatechin, which was the major endogenous polyphenol in litchi pericarp as a substrate. The optimum pH for PPO activity with (-)-epicatechin was 7.5, and the enzyme was unstable below pH 4.5 and stable in the pH range of 6.0-8.0. Residual activities of PPO were 86.25, 86.31, and 80.17% after 67 days of incubation at 4 degrees C at pH 6.0, 7.5, and 8.0, respectively. From thermostability studies, the Ki value increased with temperature and the results suggested that the enzyme was unstable above 45 degrees C. Moreover, the results also provided strong evidence that the denaturalization temperature of PPO was near 70 degrees C. The inhibition studies indicated that l-cysteine and glutathione were strong inhibitors even at low concentrations while NaF inhibited moderately. In addition, the results also indicated that the inhibition mechanisms of thiol groups were different from those of halide salts.     

Identification of metabolites of [1,2,3-(13)C]Propargyl alcohol in mouse urine by (13)C NMR and mass spectrometry and comparison to rat

Species differences in the metabolism of acetylenic compounds commonly used in the formulation of pharmaceuticals and pesticides have not been investigated. To better understand the in vivo reactivity of this bond, the metabolism of propargyl alcohol (PA), 2-propyn-1-ol, was examined in rats and mice. An earlier study (Banijamali, A. R.; Xu, Y.; Strunk, R. J.; Gay, M. H.; Ellis, M. C.; Putterman, G. J. J. Agric. Food Chem. 1999, 47, 1717-1729) in rats revealed that PA undergoes extensive metabolism primarily via glutathione conjugation. The current research describes the metabolism of PA in CD-1 mice and compares results for the mice to those obtained for rats. [1,2,3-(13)C;2,3-(14)C]PA was administered orally to the mice. Approximately 60% of the dose was excreted in urine by 96 h. Metabolites were identified, directly, in whole urine by 1- and 2-D (13)C NMR and HPLC/MS and by comparison with the available reference compounds. The proposed metabolic pathway involves glucuronide conjugation of PA to form 2-propyn-1-ol-glucuronide as well as oxidation of PA to the proposed intermediate 2-propynal. The aldehyde undergoes conjugation with glutathione followed by further metabolism to yield as final products 3,3-bis[(2-acetylamino-2-carboxyethyl)thio]-1-propanol, 3-[(2-acetylamino-2-carboxyethyl)thio]-3-[(2-amino-2-carboxyethyl)thi o]-1-propanol, 3,3-bis[(2-amino-2-carboxyethyl)thio]-1-propanol, 3-[(2-amino-2-carboxyethyl)thio]-2-propenoic acid, and 3-[(2-formylamino-2-carboxyethyl)thio]-2-propenoic acid. A small portion of 2-propynal is also oxidized to result in the excretion of 2-propynoic acid. On the basis of urinary metabolite data, qualitative and quantitative differences are noted between rats and mice in the formation of the glucuronide conjugate of PA and in the formation of 2-propynoic acid and metabolites derived from glutathione. These metabolites represent further variation on glutathione metabolism following its addition to the carbon-carbon triple bond compared to those described for the rat.     

Studies on the acetaldehyde-induced condensation of (-)-epicatechin and malvidin 3-O-glucoside in a model solution system.

The reaction between (-)-epicatechin, malvidin 3-O-glucoside, and acetaldehyde was studied in a model solution system. Ethyl-linked flavanol oligomers and anthocyanin-flavanol derivatives were observed, showing that the two polyphenols competed in the condensation process. Among the anthocyanin-ethyl-flavanol adducts, dimeric compounds in which the flavanol was linked to the anthocyanin with CH(3)-CH bridges were observed. In addition, trimeric and tetrameric products containing one anthocyanin and one, two, or three flavanols units were detected. A tetrameric product containing two anthocyanin and two flavanol units was also found as a doubly charged ion. No compound containing more than two malvidin 3-O-glucosides was detected, suggesting that only one anthocyanin A ring summit can be included in the polymerization process, which thus stops when both ends are occupied by an anthocyanin moiety. Thioacidolysis of the two isolated anthocyanin-ethyl-flavanol dimeric derivatives showed that anthocyanin-ethyl linkage was not sensitive to such reactants, whereas the flavanol-ethyl one was. In addition, flavanol-ethyl linkages involved in anthocyanin-ethyl-flavanol adducts were found to be less sensitive to those involved in flavan-ethyl dimers.     

Inhibition of gap junctional intercellular communication by the green tea polyphenol (-)-epigallocatechin gallate in normal rat liver epithelial cells

(-)-Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, is a promising chemopreventive agent against cancer due to its strong antiproliferative effects on cancer cells; however, its possible toxicity and carcinogenicity must be investigated before EGCG can be used as a dietary supplement for chemoprevention. The inhibition of gap junctional intercellular communication (GJIC) is strongly associated with carcinogenesis, particularly the tumor promotion process; thus, we investigated the effects of EGCG on GJIC in WB-F344 normal rat liver epithelial (RLE) cells. EGCG, but not (-)-epicatechin (EC), another polyphenol found in green tea, inhibited GJIC in a dose-dependent and reversible manner in RLE cells. EGCG also induced the phosphorylation of connexin 43 (Cx43), a major regulator of GJIC. The phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) was also observed in EGCG-treated RLE cells. The inhibition of GJIC and phosphorylation of Cx43 and ERK1/2 by EGCG were completely blocked by U0126, a pharmacological inhibitor of mitogen-activated protein kinase/ERK kinase. EGCG generated a larger amount of hydrogen peroxide than EC in a dose-dependent manner. Furthermore, catalase partially inhibited the EGCG-induced inhibition of GJIC and the phosphorylation of Cx43 and ERK1/2. These results indicated that EGCG inhibited GJIC mainly due to its prooxidant activity.     

Identification of pinnatoxins and discovery of their fatty acid ester metabolites in mussels ( Mytilus edulis ) from eastern Canada

Pinnatoxins are a group of fast-acting cyclic imine toxins previously identified in shellfish from Asia, the southern Pacific, and northern Europe. In this work pinnatoxins were detected in mussels from locations across the eastern coast of Canada. Pinnatoxin G (6) was the major structural variant present, sometimes at levels >80 μg/kg, whereas much lower levels of pinnatoxin A (1) were detected in some samples. Increased concentrations were observed following base hydrolysis of extracts, leading to the discovery by LC-MS of a range of fatty acid esters of 6. Information on the structures of these acylated derivatives was provided through a series of mass spectrometric experiments, supported by partial synthesis, and it is proposed that the compounds are 28-O-acyl esters of 6. Although acyl esters of a range of other phycotoxins are known to form as metabolites in shellfish, this is the first report of their existence for this particular toxin class. The occurrence of pinnatoxins in North American shellfish further highlights the international distribution of these toxins.     

Uptake and metabolic fate of [14C]-2,4-dichlorophenol and [14C]-2,4-dichloroaniline in wheat (Triticum aestivum) and soybean (Glycine max)

The uptake and metabolism of [14C]-2,4-dichlorophenol (DCP) and [14C]-2,4-dichloroaniline (DCA) were investigated in wheat and soybean. Seeds were exposed to a nutrient solution containing 50 microM of one of two radiolabeled compounds, and plant organs were harvested separately after 18 days of growth. In wheat, uptake of [14C]-2,4-DCP was 16.67 +/- 2.65 and 15.50 +/- 2.60% of [14C]-2,4-DCA. In soybean, uptake of [14C]-2,4-DCP was significantly higher than [14C]-2,4-DCA uptake, 38.39 +/- 2.56 and 18.98 +/- 1.64%, respectively. In the case of [14C]-2,4-DCP, the radioactivity absorbed by both species was found mainly associated with roots, whereas [14C]-2,4-DCA and related metabolites were associated with aerial parts, especially in soybean. In wheat, nonextractable residues represented 7.8 and 8.7% of the applied radioactivity in the case of [14C]-2,4-DCP and [14C]-2,4-DCA, respectively. In soybean, nonextractable residues amounted to 11.8 and 5.8% of the total radioactivity for [14C]-2,4-DCP and [14C]-2,4-DCA, respectively. In wheat, nonextractable residues were nearly equivalent to extractable residues for [14C]-2,4-DCP, whereas they were greater for [14C]-2,4-DCA. In soybean, the amount of extractable residues was significantly greater for both chemicals. However, in both species, nonextractable residues were mainly associated with roots. Isolation of soluble residues was next undertaken using excised shoots (wheat) or excised fully expanded leaves including petioles (soybean). Identification of metabolite structures was made by comparison with authentic standards, by enzymatic hydrolyses, and by electrospray ionization-mass spectrometric analyses. Both plant species shared a common metabolism for [14C]-2,4-DCP and [14C]-2,4-DCA since the malonylated glucoside conjugates were found as the final major metabolites.