Speration,Purification,Structural Elucidation And Their Biological Activity Of Polysaccharides From Paris Polyphylla Var.Yunnanensis

Paris polyphylla var.yunnanensis(Franch.)Hand.-Mazz.,belonging to the genus Paris of the family Liliaceae,is a perennial herb.It is fairly difficult to plant P.polyphylla on large areas because its seeds have a long dormancy period.Due to the long-term harvest and the increasing demands,the wild P.polyphylla,becomes extreme shortage at present.The roots of P.polyphylla are mainly used for extracting saponins.while the residues after extraction and aerial parts of P.polyphylla are discarded directly,which cause the serious waste of P.polyphylla resource.In the present study,to provide theoretical foundations and technical supports for a new approach to utilization of P.polyphylla resource,the study was conducted on the polysaccharide of P.polyphylla.Firstly,the P.polyphylla was divided into aerial part and underground part,and the extraction conditions were optimized for the two parts,respectively.Secondly,the two polysaccharides were purified,and the polysaccharide structure of aerial part was elucidated.At last,the biological activities of two polysaccharides were evaluated respectively.The main results of this study were as follows:1.Based on single factor analysis,the extraction conditions were optimized by central composite design(CCD)for aerial part polysaccharides(PPLPs)and underground part polysaccharides(PPRPs),respectively.The optimal extraction condition of PPLPs was extraction temperature 95 ℃,ratio of water to material 29:1 mL/g,and extraction time 4.5 h,and the average yield efficiency of PPLPs was 13.16%under the optimized extraction condition.The optimal extraction condition of PPRPs was extraction temperature 52 ℃,extraction time 20 min,ultrasonic power 210 W,and ratio of water to material 49:1 mL/g,and the average yield efficiency of PPRPs was 6.68%under the optimized condition.2.PPLPs and PPRPs were dried by hot air drying,vacuum drying at room temperature and vacuum freeze-drying,respectively,and then their scavenging abilities against DPPH,·OH and O2·-were evaluated in vitro respectively.The results indicated that the antioxidant activities of PPLPs-F(PPLPs dried by vacuum freeze-drying)and PPRPs-F(PPRPs dried by vacuum freeze-drying)were the strongest than those of PPLPs and PPRP dried by other methods.The highest DPPH,·OH and O2·-scavenging efficiencies of PPLPs-F were 79.89%,72.75%and 72.49%,respectively,and the highest DPPH,-OH and O2·-scavenging efficiencies of PPRPs-F were 83.89%,74.05%and 76.61%,respectively.At the same concentration,the antioxidant activities have no significant difference between PPLPs-F and PPRPs-F.Therefore,vacuum freeze-drying was the optimal method for drying of PPLPs and PPRPs.The proteins of PPLPs and PPRPs were removed by Sevag method,and the relationship between times of deproteinization,deproteinization efficiency,and loss efficiency of PPLPs and PPRPs were investigated respectively.The optimal times of’ deproteinization for PPLPs was 5.and the deproteinization and loss efficiencies of PPLPs were 74.47%and 31.49%,respectively.The optimal times of deproteinization of PPRPs was 3,and the deproteinization and loss efficiencies of PPRPs were 72.70%and 20.88%,respectively.PPLPs and PPRPs were purified through DEAE-52 cellulose and Sephadex G100 columns successively.PPLPs-D(purified PPLPs with DEAE-52 cellulose)was eluted with 0.3 mol/L NaCl,and PPRPs-D(purified PPRPs with DEAE-52 cellulose)was eluted with 0.5 mol/L NaCl.which indicated that the polarity of PPRPs-D was stronger than that of PPLPs-D.PPLP and PPRP were collected after PPLPs-D and PPRPs-D had been purified by Sephadex G100 column,respectively.The elution volume of PPLP was greater than that of PPRP.which indicated that the molecular weight of PPRP was larger than that of PPLP.4.The molecular weight and purity of PPLP were determined by HPGPC.The elution curve was a symmetric peak,which indicated that PPLP was a homogeneous polysaccharide.The molecular weight of PPLP was 2.95 x 104 Da.The monosaccharide composition of PPLP was determined by GC,and the results indicated that PPLP consisted of L-Ara and D-Gal at a molar ratio of 0.42:0.58.Based on analysis of methylation,partial hydrolysis and NMR data,the backbone of PPLP consisted of(1,6)-β-D-Galp,and the side chains,mainly comprised of a-L-Araf residues,were linked to backbone at O-3.5.The antioxidant activities of PPLP and PPRP were evaluated in vitro.The highest DPPH,·OH and O2·-scavenging efficiencies of PPLP were 83.01%,74.54%and 82.50%,respectively,and their corresponding IC50 values were 0.26 mg/mL,0.55 mg/mL and 0.32 mg/mL.The highest DPPH,·OH and O2·-scavenging efficiencies of PPRP were 78.94%、67.75%and 77.92%,respectively,and their corresponding IC50 values were 0.34 mg/mL,0.58 mg/mL and 0.42 mg/mL.The results indicated that PPLP and PPRP possessed strong antioxidant abilities in vitro.6.In order to evaluate the antioxidant activities of PPLP and PPRP in vivo,the D-gal induced aging mice model was constructed by injection with D-Gal.The high dose groups of PPLP and PPRP(400 mg/Kg/day)significantly increased the bodyweight(p<0.05),markedly improved the thymus and spleen indices(p<0.01),dramatically enhanced the activities of T-SOD,GSH-Px,CAT and T-AOC(p<0.01),and significantly decreased the MDA content in serum,brain,liver and heart(p<0.01),which indicated that PPLP and PPRP possessed strong antioxidant ability in vivo.7.The hypolipidemic activities of PPLP and PPRP were evaluated through high-fat diet induced hyperlipidemic mice model.Compared with model group,the high dose groups of PPLP and PPRP(400 mg/Kg/day)significantly reduced the bodyweight(p<0.05),markedly decreased AI and the levels of TC,TG and LDL-c(p<0.01),and dramatically increased the level of HDL-c(p<0.05),which suggested that PPLP and PPRP possessed the strong hypolipidemic abilities.The hypolipidemic abilities are no significant difference between high dose PPLP and PPRP.