Study On Depolymerization And Transformation Of Grape Seed Proanthocyanidins

Grape seed proanthocyanidins（GSPE）are composed of oligomeric and polymeric forms of flavan-3-ol monomer units.Their biological activities appear to be closely related to their degree of polymerization（DP）.Polymeric procyanidins（PPCs）are difficult to be absorbed into the blood and the bioavailability is very low,because of their high molecular size.In the product development of oligomeric procyanidn（OPCs）,polymeric procyanidins are ofen discarded,being the waste of procyanidin resoureces.Thus,it is an emerging trend for scientists to develop effective methods of converting PCs into bioavailable small molecules.In order to expand the application range of grape seed proanthocyanidins,a prodrug strategy was proposed in this paper.The proanthocyanidin depolymerisation derivatives were used as antihypertensive prodrug to improve the clinical defects of captopril.In this paper,the agricultural byproducts grape seed proanthocyanidins are taken as the research object,the chemical components in vitro,depolymerisation of GSPE by captopril and optimization of the depolymerisation conditions,the metabolites of depolymerisation derivative ECC in rat plasma,urine and feces,the pharmacokinetics of ECC were investigatedA sensitive and rapid ultra high performance liquid chromatography quadrupole time-offlight mass spectrometry was established to identify the chemical components of compound Grape seed extract in vitro.Multiple data processing menthods like mass defect filtering,product ion filtering and neutral loss filtering could improve the accuracy and efficiency of analytic process.71 compounds were separated and identified.The above components could also divided into several groups according the type of compounds:43 compounds from proanthocyanidins,8 compounds from phenolic acids,14 compounds from flavones,2 compounds from stilbenes,3 compounds from other kinds of compounds.The fragmentation patterns of different kinds of components were also illustrated.Based on the idea of prodrug design,recently we developed a new proanthocyanidin depolymerisation method.Two novel depolymerisation derivatives（-）-epicatechin-4β-Scaptopril methyl ester（ECC）and（-）-epicatechin gallate-4β-S-captopril methyl ester（ECGC）derived from the reaction of grape seed proanthocyanidin extract with captopril in the presence of acidified methanol.The depolymerisation derivatives of GSPE were separated and purified by silica gel column chromatography and RP-HPLC,and confirmed by 1D and 2D NMR spectroscopy and mass spectrometry.In addition,the fragmentation rules of depolymerisation derivatives were summarized,respectively.From preliminary single factor experiments,the two significant independent variables,including the depolymerisation temperature and time,were considered as key factors.To achieve the best depolymerisation yield,CCD was used to optimize depolymerisation factor.The depolymerisation prediction model equations have a good reproducibility.The optimum conditions for the high yield of ECC were as follows:temperature,44.88℃;time,63.35 min.The experimental yield of ECC was 366.2 mg/g GSPE.The optimum conditions for the high yield of ECGC were as follows:temperature,45.35℃;time,106.8 min.The experimental yield of ECGC was 73.14 mg/g GSPE.The results verified the validation of the response model and indicated that the model designed in the study was adequate and accurate for the prediction of the yield of ECC and ECGC.A sensitive and rapid ultra high performance liquid chromatography quadrupole time-offlight mass spectrometry coupled with multiple data processing methods were developed to analyse the plasma,urine and feces components and the relevant metabolites after administration of compound ECC.Under the optimized conditions,16 metabolites were detected in the rat plasma,urine and feces.Among these metabolites,9 metabolites（M0-M4,M6,M11,M12,M14）were detected in plasma,14 metabolites（M0-M3,M5-M10,M12-M15）in urine and 10 metabolites（M0-M6,M12-M14）in feces.The ECC mainly underwent the phase I metabolic pathways including hydrolysis,reduction,oxidation and phase II metabolic pathways including sulfate conjugation,methylation,cysteine conjugation,GSH conjugation and cysteinylglycine conjugation.The results showed that ECC underwent extensive metabolism in vivo to release captopril methyl ester and epicatechin followed by the generation further metabolites from captopril and epicatechin.The results show that EEC has a potential for becoming a prodrug with synergistic or additive hypotensive effects.And it provides reference for the ECC pharmacokinetic study.A rapid and sensitive ultra-fast liquid chromatography-tandem mass spectrometry method was first developed and fully validated for the quantification of captopril component in rat plasma.The separation was achieved on a Shim-pack XR-ODS column and the detection was conducted by the multiple reaction monitoring mode using positive ion mode.The mobile phase consisted with water（containing 0.1%formic acid）and methanol at a flow rate of 0.2 ml/min.The analyte and internal standard were separated using the validated method.The validated method was also successfully applied to compare the pharmacokinetics of captopril after oral administration of compound captopril and compound ECC.The results indicated that the analyte had different mean concentration-time curves trend between two groups.The ECC group delayed the captopril t_max and extended the t1/2 which compared with captopril group.It helps to maintain the effective blood concentration and prolong action time.ECC improved the pharmacokinetic properties of active ingredient captopril.Consequently,EEC has a potential for becoming a prodrug with synergistic or additive hypotensive effects.