Abstract
A rapid and inexpensive method has been developed for preparation of gram quantities of (-)-epigallocatechin gallate, the major flavanol of green tea. Under optimum conditions (pH 4, 30 mM caffeine), the formation of insoluble complexes between caffeine and the gallate esters of green tea brew is maximised. Separation of these precipitates and decaffeination (by partition against chloroform) produces a gallate-rich fraction, which, on partition against ethyl hexanoate, then propyl acetate, yields (-)-epigallocatechin gallate (purity 81 %).The enriched (-)-epigallocatechin gallate fraction, together with (-)-epicatechin, (+)-catechin and gallic add were supplied to rats maintained on a purified C10 diet. Collection and analysis of urine samples by HPLC enabled detection of metabolites of the fed test substances. Glucuronide and sulphate conjugates (most probably 3-0-methylated forms of the parent compounds and a glucuronide conjugate of the ring-fission product, 3-hydroxyphenylpropionic add) accounted for the major proportion of metabolites for (-)-epicatechin and (+)-catechin, whilst gallic add was predominantly metabolised to 4-O-methyl gallic add. (-)-Epigallocatechin gallate did not appear to be metabolised in a similar way to (-)-epicatechin and (+)-catechin — novel peaks on chromatograms of urine from rats fed this compound corresponded to gallic add and unchanged (-)-epigallocatechin gallate. Quantification of certain urinary metabolites by HPLC and 1H NMR when (-)-epicatechin was fed to rats at a range of concentrations, enabled the formulation of dose-response curves linking output to intake. These methods were extended to a volunteer study with encapsulated green tea. Using these calibrations, estimated intake values were in close agreement with actual values of intake. Results indicate that HPLC and/or 1H NMR profiling of urine has considerable potential as a biomarker of exposure and merits further development.