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Recent studies by us have identified anticancer efficacy of grape seed extract (GSE) against prostate cancer (PCA) via its antiproliferative, pro-apoptotic and antiangiogenic activities in both cell culture and animal models. GSE is a complex mixture containing gallic acid (GA), catechin (C), epicatechin (EC) and several oligomers (procyanidins) of C and/or EC, some of which are esterified to GA. To determine which components are most active against PCA, GSE was dissolved in water and ethyl acetate-extractable material separated by semi-preparative C18 HPLC. Ten prominent peaks were collected and each analyzed by HPLC-negative ion electrospray mass spectrometry (LC-MS). Based on HPLC retention times, UV and mass spectra, and comparisons with authentic standards, we identified major components in six fractions as GA, C, EC, and procyanidins B1 (EC-C dimer), B2 (EC-EC dimer), and B3 (C-C dimer). Although not completely identified, another dimer, four trimers, and two dimer-gallate conjugates also were detected in later fractions. Efficacy studies for GA, C, EC and dimmers B1-B3 showed that GA is far more effective in causing growth inhibition and apoptotic death of human PCA DU145 cells, while C, EC and dimmers were almost ineffective. Next, GSE was fractionated using a molecular sizing column (Toyopearl HW-40S). With methanol as eluant, eight fractions (Fr) were collected and each analyzed by LC-MS to characterize multiple components. GA was the first and only component eluted in Fr#1, and both C and EC eluted in Fr#2. Frs #3 & #4 contained four procyanidin dimers including B1 and B2 and a dimer-gallate conjugate. Fr#5 contained three trimers of C and/or EC and one dimer-gallate conjugate; Fr#6 contained three different trimers of C and/or EC and one dimer-gallate; Fr#7 contained three additional trimers of C and/or EC, a trimer-gallate, and two tetramers of C and/or EC; and Fr#8 contained one additional trimer-gallate, one additional tetramer, and one pentamer. Biological testing of these fractions showed that other than GA, Frs #5-8 were considerably more active than #2-4 in causing growth inhibition and apoptosis in DU145 cells; in general, biological activity increased with the molecular size of the polyphenol. High activity of GA in GSE was further confirmed by preparing a sample of GSE with GA removed resulting in a substantial loss of activity. Next, studies were done using pure GA that showed a very strong dose- and time-dependent growth inhibition, S-phase arrest and apoptotic death of DU145 cells. Mechanistic studies showed a strong induction in Cip1/p21, and caspase-9, -3 and PARP cleavages by GA in DU145 cells. Together, these observations identify GA as one of the major active constituents in GSE; however, high-molecular size procyanidins and their gallate esters also seem promising in GSE efficacy against PCA.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]