Extraction, Purification, Structure And Bioactivity Of Polysaccharides From The Peduncles Of Hovenia Dulcis

Hovenia dulcis, belongs to a small genus of Rhamnaceae, is a tall deciduous tree that widely distribute in China. The peduncles of Hovenia dulcis have been used as a traditional Chinese herbal medicine for more than a millennium. The main effect including clear away heat, diuretic, slak thirst, and detoxify alcoholic intoxication. Modern pharmaceutical researches revealed that the peduncles of Hovenia dulcis could reduce alcohol toxicity, protect liver injury, resist fatigue and have anti-lipidperoxidation activities.Recent studies about the peduncles of Hovenia dulcis were mainly concentrated on its’ crude extracts and some of low molecular weight chemical constituents, there is no report about the water-soluble polysaccharide from peduncles of Hovenia dulcis (HDPS). In this thesis, HDPS was studied, including optimization of extraction parameters, isolation and purification, chemical characterization, preliminary analysis of structures, antioxidant activity, hepatoprotective activity against acute alcohol-induced liver injury, immunomodulatory activity and inhibition activity of tumor proliferation. Main results are listed as follows:1. Optimization of extraction parameters of HDPSExtraction temperature, extraction time and liquid-raw ratio were selected in single-factor experiments. Based on the single-factor experiments, response surface methodology (RSM) experiments were applied for the optimization of extraction parameters of HDPS. Then the data was analyzed by Design-Expert Software, and the optimum extraction parameters were obtained as follows:extraction temperature96℃, extraction time200min, liquid-raw ratio21mL/g and extraction times3. Under optimum extraction conditions, the real extraction yield of HDPS was10.47%.HDPS obtained with the optimum extraction conditions, was treated by freeze-thaw action and S-8macroporous resin to remove starches and decolor, respectively. After two step treatment, HDPS with a little color and high soluble, the yield of crude HDPS was methods. The results showed that crude HDPS could scavenge O2· free radicals, inhibit lipid-peroxidation and chelate Fe2+in vitro. Based on its’ antioxidant activities in vitro, hepatoprotective activity against acute alcohol-induced liver injury were evaluated by acute alcohol administration mice model. The results showed that crude HDPS pretreatment for20days could remarkably prevent alcohol-induced elevation of ALT and AST, enhance the activity of antioxidant enzymes (SOD and GSH-Px) and decrease the generation of lipid-peroxidation production (MDA). These results suggested that crude HDPS possessed a hepatoprotective effect against acute alcohol-induced liver injury via enhancing activity of antioxidant enzymes, scavenging reactive oxygen species, inhibiting lipid-peroxidation and chelating metal irons against the oxidative stress.4. Immunomodulatory activity of HDPSFirstly, Immunomodulatory activities in vitro of HDPS-1, HDPS-2and HDPS-3were evaluated by using the cell model experiments. Then according to the screening results of in vitro experiments, the purified fraction possessing best in vitro immunomodulatory activity was further studied for its’ immunomodulatory activity in vivo.Immunomodulatory activities in vitro results demonstrated that all the three fractions had potent immunostimulatory activity, resulting in stimulating proliferation of splenocytes, enhancing phagocytosis, nitric oxide production and acid phosphatase activity of peritoneal macrophages. Furthermore, the three polysaccharide fractions had significant differences in stimulatory effect at every evaluation item, and their effects were in the order of HDPS-1> HDPS-2>HDPS-3(from high to low).Immunomodulating activities in vivo results showed that different dose HDPS-1could resist mice spleen weight decrease induced by Cyclophosphamide and enhance activity of LZM in spleen, and a certain dose HDPS-1could significantly enhance the content of IL-6and IFN-y in serum of immune suppress mice. In addition, HDPS-1plays an immunomodulatory activity in vivo with a high dose-dependent manner, that is, the low or medium dose could promote the immune function, and in high dose has an immunosuppressive activity to a certain degree. These results suggested that HDPS-1possessing a double immunomodulatory activity via regulating the generation of cytokine related with cellular and humoral immunity.5. Proliferation inhibitory activities of HDPS on tumor cellsProliferation inhibitory activities of HDPS for three tumor cell lines (A549, HepG2and BGC-823) were evaluated by MTT assay. For the most sensitive cell line to HDPS, the effects of HDPS on its’morphology and apoptosis were further studied.Results showed that HDPS-1, HDPS-2and HDPS-3have no significant inhibitory effect on the proliferation of tumor cell line A549and Hep G2, but have significant inhibitory effect for BGC-823. Three purified fractions have proliferation inhibition on BGC-823at significant time and dose dependent manner. Under drug administration time72h and dose320μg/mL, the inhibition rates of HDPS-1, HDPS-2and HDPS-3on BGC-823proliferation were93.94%,84.38%and78.33%,respectively. Among them HDPS-1showed the strongest inhibitory effect on the proliferation of BGC-823. At drug administration time24,48and72h,50%concentration of inhibition of HDPS-1was207.05,44.13and5.02μg/mL.Study on the cell morphology showed that HDPS-lcould significantly inhibit proliferation of BGC-823,the cell density decreased with the increase of sample dose, cellular atrophy and even apoptosis. Hoechst33258stain and flow cytometry detection confirmed that HDPS-1-induced cell apoptosis plays a main effect on its’ proliferation inhibitory effect to BGC-823.