The antioxidant components of cacao liquor, which is a major ingredient of chocolate, were isolated with column chromatography and high-performance liquid chromatography. Quercetin and its glucoside were identified by spectrometric methods. Clovamide and deoxyclovamide were characterized by (1)H and (13)C NMR and MS spectrometry. Their antioxidative activity was measured by peroxide value of linoleic acid and thiobarbituric acid reactive-substance value of erythrocyte ghost membranes and microsomes. In the bulk oil system, clovamide had the strongest antioxidative activity but was less active in the other experiments. In the case of the two hydrophilic systems, flavans such as quercetin and epicatechin were more potently effective than the glucosides. It is considered that chocolate is stable against oxidative deterioration due to the presence of these polyphenolic compounds.

We compared levels of (ϩ)-catechin, (Ϫ)-epicatechin, and their metabolites in rat plasma and urine after oral administration. Rats were divided into four groups and given (ϩ)-catechin (CA group), (Ϫ)-epicatechin (EC group), a mixture of the two (MIX group) or deionized water. Blood samples were collected before administration and at designated time intervals thereafter. Urine samples were collected 0 -24 h postadministration. (ϩ)-Catechin, (Ϫ)-epicatechin and their metabolites in plasma and urine were analyzed by HPLC-mass spectrometry after treatment with ␤-glucuronidase and/or sulfatase. After administration, absorbed (ϩ)-catechin and (Ϫ)-epicatechin were mainly present in plasma as metabolites, such as nonmethylated or 3Ј-O-methylated conjugates. In the CA and MIX groups, the primary metabolite of (ϩ)-catechin in plasma was glucuronide in the nonmethylated form. In the EC and MIX groups, in contrast, the primary metabolites of (Ϫ)-epicatechin in plasma were glucuronide and sulfoglucuronide in nonmethylated forms, and sulfate in the 3Ј-O-methylated forms. Urinary excretion of the total amount of (Ϫ)-epicatechin metabolites in the EC group was significantly higher than the amount of (ϩ)-catechin metabolites in the CA group. The sum of (ϩ)-catechin metabolites in the urine was significantly lower in the MIX group than in the CA group, and the sum of (Ϫ)-epicatechin metabolites in the MIX group was also significantly lower than in the EC group. These results suggest that the bioavailability of (Ϫ)-epicatechin is higher than that of (ϩ)-catechin in rats, and that, in combination, (ϩ)-catechin and (Ϫ)-epicatechin might be absorbed competitively in the gastrointestinal tract of rats.

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8+/-5.3%'; and 25.3+/-8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.

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