The Isolation And Identification On The Enzymatic Hydrolysate Of PGG And Research On Its Bioactivity

1,2,3,4,6-penta-O-galloyl-beta-D-glucose(PGG)is a natural compound contained in Chinese herbal medicine plants,fruits and vegetables.PGG has become a hot research focus with many attractive pharmacologic and physiologic activities such as anti-tumor activity,anti-diabetic activity,antivirus and antibacterial activities,anti-allergy activity and anti-inflammation activity.In this dissertation,PGG was hydrolyzed by tannase to obtain the degradation product of PGG.The degradation products were isolated from PGG hydrolyzate by high performance preparative liquid chromatography and has its identified through MS and NMR analysis.The present work provided many significant findings for reasonable development and utilization of polyphenols.It also estabilisded the theoretical foundation used to investigate on antoxidant,anti-tumor and anti-inflammatory mechanism.The ma in results were concluded as follows:1.The activity of tannase was 200 U/g tested by spectrophotometer,and its purity was above 95% tested by HPLC.Taking the maximum enzyme activity as reference,the optimal pH value of the tannase was determined in the range of 4.5 ~6.0,and the optimum temperature was between 45 ~50 ?.The effect of tannase degradation of PGG was similar to human intestinal flora when the additive amount of enzyme was between 0.10% ~ 0.20% and the enzymolysis time was between 40 ~ 60 min.2.The degradation products were isolated from PGG hydrolyzate using high performance preparative liquid chromatography.Five products were obtained including P1,P2,P3,P4 and P5.The purity of P1 was about 76.5%,the P2 was about 79.2%,the P3 was about 71.5%,the P4 was about 92.3% and the P5 was about 98.0% by HP LC.3.The chemical structure of PGG degradation products was analyzed by HPLC-MS.The results indicate that the molecular weight of PGG degradation products is 787,635,483,169 successively,and there are many isomers.The possible structures of the compounds were determined wirh the comparison to the literature data.The molecular weights of P1 and P2 were both 787,the isomers of TGG(tetragalloyl glucose),P3 was TriGG(trigalloyl glucose),P4 was DGG(digalloyl glucose),P5 conforms to the molecular weight of gallic acid,after comparing with the standard,P5 is determined to be GA(gallic acid).4.Comparison among antioxidant activity of PGG and its degradation products.The antioxidant capacity was evaluated by eliminating DPPH,ABTS radical and O RAC.It shows that all of the degradation products of PGG have strong antioxidant ability,and the antioxidant ability of degradation products was even stronger than PGG itself.5.The study on antitumor activity of PGG and its degradation products.HCT-116 cells were selected as anti-tumor activity.Cell viability was determined by MTT assay.After comparison,we cam conclude that the anti-tumor activity of PGG was the strongest,and GA was stronger than others comparing with other degradation products.PGG and its degradation products have strong anti-tumor activity.6.Finally,anti-inflammatory effect of purified extracts with degradation products was investigated by using mouse macrophage cell line RAW264.7 pretreated by LPS.Neutral red method shows that PGG has less effect on phagocytosis than its degradation products.PGG and its degradation products both inhibited the secretion of IL-1?,TNF-? and NO secreted by RAW264.7 macrophages significantly.There was a dose effect on the inhibitory effect and the concentration of the sample.PGG inhibit the secretion of IL-6 significantly,but the degradation of IL-6 products is relatively weak.In summary,PGG and degradation products have a strong anti-inflammatory activity,their anti-inflammatory effect is better than dexamethasone.