Isolation, Activity And Application Of Poylsaccharides From Camellia Oleifera Seed Cake And Fruit Hull

In order to reveal the relationship between structure and activity of the polysaccharides from Camellia oleifera seed cake and Camellia oleifera fruit hull (the polysaccharides from seed cake are called CCP and that from fruit hull are called CFP), and make full use of Camellia oleifera residues, then the chemical constituents of Camellia oleifera seed cake and Camellia oleifera fruit hull were studied respectively, and then the isolation and extraction process, the physicochemical properties and structure, the scavenging free radicals and hypoglycemic activity of CCP&CFP were investigated systemly,and effervescence tablets for diabetic patients were prepared with CCP as active ingredient.The results obtained have some certain theory and practical significance on revealing the structure-activity relationship of CCP and CFP, expanding the utilization of Camellia oleifera by-products and the Camellia oleifera industry chain. The main contents and conclusions of this paper are described as follows:(1) The main chemical constituents of Camellia oleifera seed cake and fruit hull.The contents of total sugar in Camellia oleifera seed cake and fruit hull were19.65%,23.34%respectively. The results showed that he active polysaccharide owing to its feasible to extract higher contents of total sugar in Camellia oleifera seed cake and fruit hull. The two flavonoid glycosides in Camellia oleifera seed cake and fruit hull were identified to be kaempferol-3-O-[2-O-B-D-galactopyranosy-]-6-O-a-L-rhamnopyranosyl]-β-D-glucopyranoside kaempferol-3-O-[2-O-B-D-xylopyranosy-]-6-O-a-L-rhamnopyranosyl]-β-D-and glucopyranoside, respectively, and the flavonoid glycosides content in Camellia oleifera seed cake was much higher than that in fruit hull with the content ratio of2.00:0.18.(2) The extraction process of CCPThe measuring conditions of total sugar and reduced sugar by PSA (phenylhydrate sulfuric acid method) and DNS (3,5-dinitrosalicylic acid method) were studied.The response surface methodology was used to optimize the extraction conditions of CCP with enzyme-assisted extraction. The results showed that the polysaccharide yield of26.62%under optimum condition extraction temperature50℃, extraction time3.0h, amount of enzyme0.35% (3) Purification of CCP&CFPThe yield of crude CCP&CFP were1.8%and1.1%respectively by water extraction-alcohol precipitation.80.13%free protein was removed, loss of polysaccharide was13.10%and the decolorization rate was94.40%, when polyamide method was used to get rid of free protein in the polysaccharide liquid, which is better than sevage method and trichloroacetic acid method.Crude CCP&CFP were purified by DEAE-52column and sephadexG-100column chromatography respectively. Five components (CCPA-1-5&CFPA-1-5) from DEAE-52and one component (CCPB&CFPB) from sephadexG-100were obtained.(4) Physicochemical properties and structure of CCP&CFPPhysicochemical properties and structure of four components with higher yield (CCPA-3,CCPB,CFPA-3,CFPB) were studied. The results showed that relative molecular weight of polysaccharide were10248,4736,118262and179790, containing aminogalactose, galactose, glucuronic acid, xylose and arabinose.There were backbone of (1,3)-linked glycosidic bond, protein and uronic acid in the structure of polysaccharides. Polysaccharides were observed fold and uneven structures on the surface by SEM. Thermal analysis indicated that polysaccharide had higher aggregation and would decompose range200～500℃.(5) Scavenging free radical activity of CCP&CFP in vitroScavenging free radical activity of CCP&CFP in vitro with different doses was studied and compared with tea polysaccharide (TP). There were certain scavenging free radical activity in vitro with CCP&CFP and the activity increased with higher concentration. The IC50of CCP&CFP and TP were1017.31μg/mL,25.31μg/mL,36.74μg/mL on DPPH free radical and185.50μg/mL,11.23μg/mL,10.18μg/mL on ABTS free radical.(6) Hypoglycemic activity of CCP&CFP in VitroTo investigate the hypoglycemic activity of CCP&CFP and their purified components, liver cancer HepG2cells consumption efficiency andα-Glucosidase(AGC) inhibitory capacity were used in vitro. Results of experiment of AGC showed that the inhibitory capacity increased with higher concentration. The IC50of TP,CCP,CCPA-3,CCPB, CFP,CFPA-3,CFPB were4.37μg/Ml,629μg/mL,54.41μg/mL, 57.10μg/mL,23.15μg/mL,10.92μg/mL and11.86μg/mL respectively.Glucose uptake ability of HepG2cells by CCP&CFP was confirmed in different concentrations. Samples with0.125mg/mL had75.08～85.06%consumption efficiency of metformin hydrochloride, as same as that of TP. Cell survival rate was closed to95.00%on the concentration. By structure-activity analysis, relative molecular weight, glycosidic bond,protein and uronic acid on the structure of polysaccharides were probably the main reason of hypoglycemic effect.(7) Preparation of CCP effervescent tabletThe prescription of CCP effervescent tablets was optimized by orthogonal experiment with the disintegration time and hardness. The optimal prescription contained10%CCP,40.5%mannitol,12.5%citric acid,12.5%tartaric acid,20.0%sodium bicarbonate,4.0%polyethyleneglycol6000,0.25%aspartame and0.25%cyclamate.