Study On The Degree Of Polymerization And Bioactivities Of Grape Seed Procyanidins

Grape, which has a long history in china, is widely cultivated with an increasing production in recent years. As the by-product of grape juice and wine making, grape seeds are a rich source of procyanidins (a kind of natural antioxidants), but due to the hard hull, they are usually discarded as unwanted or used as crude feed, which is a waste of resources. For better use of the grape seeds, it is of great importance to analysis the procyanidins in grape seeds and study its biological activities and extraction.Grape seed procyanidins are oligomers and polymers of (+)-catechin, (-)-epicatechin, and (-)-epicatechin-O-gallate linked by C4→C8 or C4→C6 B-type bonds. They have attracted great attention in the fields of nutriology and pharmacology because of their potent radical-scavenging activity, broad biological and pharmacological effects and excellent security. The pharmacological properties of procyanidins largely depend on their structure and especially on their degree of polymerization (DP). But due to the diversity of procyanidin structures, the studies on the analysis methods as well as the relationship between the structure and activity are not thorough, which limits the utilization of procyanidins. In this work, feasible qualitative and quantitative methods were established, and the procyanidins were fractionated according to their degree of polymerization. And then the effects of DP on the antioxidant and anti-tumor activity in vitro were investigated systematically. Furthermore, studies on the extraction and isolation of procyanidins were also carried out for commercial preparation. The main results of this dissertation were described as following:The qualitative analysis of oligomeric procyanidins in crude grape seed extracts can be performed by reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (RP-HPLC-ESI-MS). Pseudomolecular ion and fragment information of ESI-MS made it possible to analyze the composition of oligmeric procyanidins directly without standards. By RP-HPLC-ESI-MS, three monomers, seven dimers, seven trimers and one tetramer were tentatively identified in crude grape seed extract, but due to the large number of isomers and their similar ESI-MS characters, the isomers are not identified precisely except monomers. In fact, only oligomers with DP below 4 can be well detected by RP-HPLC-MS, the higher polymers, with more isomers and more diverse polarity, can neither be separated nor be analyzed by RP-HPLC.Acid-catalyzed vanillin assay was improved for quantitation of procyanidins in grape seed. By the use of H2SO4 as acid medium, proper reaction conditions were obtained as following: after 0.5mL aqueous solution of grape seed extract was mixed with 2.5mL 3% vanillin in methanol and 2.5mL 30% H2SO4 in methanol, the reaction was carried out at 30℃ for 15 minutes and the absorbance at 500nm was measured. Evaluation of the method proved that it has good repeatability and high recovery, and is also more sensitive and stable compared with previous methods. Procyanidins of different grape seeds can be quantified and compared by this method. The assay was then used to determine the procyanidin content of several kinds of grape seeds from different district and different fermentation process. Results showed that the procyanidin content of grape seed was affected by the variety, clime and wine-making methods. Procyanidins should be extracted from unfermented grape seeds. Besides grape seeds, this assay can also be used to quantify the procyanidin of other plant in which vitamin C and dihydrochalcone are absent or scarce. Because of the complexity of procyanidin structure, the proyanidins were separated according to their degree of polymerization (DP) for further study of the relationship between structure and activity. By use of thiolysis for determining the mean degree of polymerization (mDP) and normal-phase HPLC, gel permeation chromatography (GPC) and mass spectrometry (MS) for analysis of molecule profile of procyan...