Extraction And Purification Of Proanthocyanidin From Grape Seed, Its Structure And Functionality

Grape is a fruit with a high economic value in the world and belongs to Vitaceae Visit. There are a lot of phenolic compounds found in the grape; 30-40% is found in the grape skin and 60-70% in the grape seed. The major constituent of phenolic compound is proanthocyanidin (PC) which has a highly hydroxyl structure. It can act as an antioxidant, removes free radicals, and reduces risk of chronic diseases such as cardiovascular disease and cancer.The precision and accuracy of the measurement of PC content had been improved by the modifidied Vanillin-HCL assay, the coloring stability has been increased greatly, and the analysis time had been shortened. It is an instant and convenient assay, and can be applied to the measurement of free PC content in the grape skin, seed, stem and its extracts. The PC can be separated from the bound form with protein or fibre in the butanol-HCL solution of SDS and 2-mercaptoethanol, and the bound PC content of grape seed and stem could be measured after coloring with better precision and accuracy. The PC content of free form, bound form with protein or fibre in the grape seed was 3.86%, 2.93% and 2.80% respectively. The bound PC exists in the form of hydrophobic bond, hydrogen bond and covalent bond. It can reduce the loss of polyphenol and free PC, and also inhibits its transformation to bounded PC with vacuum foil package of grape seed at lower temperature.The grape seed was vacuum dried at 40℃, and the PC was extracted from it with 70% ethanol. The best technical parameters were 80-150 mesh particle size of grape seeds, 55 min of stirring, 15℃as extracting temperature, and 0.75%(v/v) acid content. The adsorption yield of PC in HP-2MGL was 91.1%. However the desorption yield was 95.8%. The PC content of GSPP3 was 96.5% which was higher than that of GSPP1 and GSPP2. The result of LC-MS analysis of GSPP3 showed that there were 27 peaks of GSPP3 in the total ion current of mass chromatogram. This meant the compose of GSPP3 was very complex. The peaks 2 and 4 were all dimmers of PC according to their mass spectra. GSPP3 was further purified by SephadexLH-20, and two components GSPP3-SP1 and GSPP3-SP2 were got by elution with 70% acetone. LC-MS analysis showed that there were four peaks for GSPP3-SP1, and seven peaks for GSPP3-SP2. According to their mass chromatogram and fragment information, they contained the monomer and dimmer of PC.On analysis of infrared spectra of GSP,GSPP1,GSPP2 and GSPP3, it was found that the basic structural unit of grape seed PC was procyanidin. Alcoholyzing the grape seed PC and analysing it by LC-MS, it was proved to be cyanidin. Hence the main structural unit of grape seed anthocyanidin is procyanidin which has two hydroxyl groups with a B-ring.On determining the relative molecular weight of grape seed PC by laser light scattering with GPC, it was found that the average relative molecular weight for...