Study On Extraction, Isolation, Structure Analysis And Antitumor Activities Of Polysaccharides Extracted From Parmelia Tinctorum Despr.

Objective:To extracte,isolate and purificate polysaccharides from Parmelia tinctorum Despr.in Mianning county,a mountain area of Sichuan Province.To find the optimum condition of extraction,and study on the structure of polysaccharides and antitumor activity of polysaccharide component WPT-B.Methods:Classical hot water extracting and ethanol precipitation method was used to isolate polysaccharides from Parmelia tinctorumDespr.The content of total polysaccharides was determined by the phenol-sulfuric acid method.Four factors were choosed by Orthogonal test to analyze the influence of extraction on the content of polysaccharide,including temperature,extracting time,the number of extraction, and the volume of water.Orthogonal test was designed for optimizing the extraction process of water-soluable polysaccharides from Parmelia tinctorum Despr.By comparing several deproteinization and decolorization methods,we optimized the process of purifing the crude polysaccharides from Parmelia tinctorum Despr. Further separation and purification were carried on by DEAE-Dextran gel A-25 column chromatography and Sephadex G-200 column chromatography,two polysaccharides named as WPT-A and WPT-B were obtained and identified.Their molecular weights were measured.Their chemical compositions were analyzed by using Ultra-violet spectroscopy and Infrared absorption spectroscopy.After hydrolyzation and acetylization reaction,the GC analysis were performed.The acute toxicity and antitumor activity of WPT-B were studied.Results:According to the results of orthogonal experiment,the optimum extraction conditions were determined as follows:polysaccharides are extracted three times at 100℃with decuple times of water for 3 hours.Under this condition,the average content of Polysaccharide was 17.040%.The crude polysaccharide was fractionated by DEAE-Dextran gel A-25 and Sephadex G-200 column chromatography.The two pure-polysaccharides named as WPT-A and WPT-B were obtained from Parmelia tinctorum Despr.WPT-A was composed of glucose,xylose and mannose,which the ratio was 3:2.5:6:1;and WPT-B was composed of glucose,xylose and mannose, which the ratio was 5:5:3.The results of HPLC analysis showed that the average molecular weight of WPT-A was 51414,and WPT-B was 62830.The results of the acute toxicity experiment on WPT-B indicated that no mouse died by the one time maximum dose method.In the antitumor experiment,the average inhibiting ratios of WPT-B is 58.04%,and the antitumor effect of 12 mg/kg dose group is the best.Compared with the model group,each group has significant difference.The inhibiting-tumor ratio in WPT-B and DDP combined group was better than that in DDP group.12mg/kg WPT-B+4mg/kg DDP and 24mg/kg WPT-B+4mg/kg DDP had more significant effects.Both drugs showed well synergistic action.Conclusion:We have got the optium conditions to extract and purify by using orthogonal test,the resign decolorization law and TCA method,The yield has been enhanced.It is so easy to operate and industrialize.The two polysaccharides extracted with water from Parmelia tinctorum Despr.are both homopolysaccharides.WTF-B has no obvious toxicity and may inhibit tumor growth of H₂₂on the mice.WTF-B combined with DDP shows well synergistic action.