Study On HPLC Characteristic Chromatogram And Separation,Purification,Immunomodulatory Activity Of Polysaccharides From Mongolian Medicine Rubus Sachalinensis

Objective: The HPLC characterastic chromatogram and content determination methods of Rubus sachalinensis from different producing areas were established for quality evaluation.To extract,separate and purify homogeneous polysaccharides from Rubus sachalinensis and their monosaccharides were analyzed.To study the effect of polysaccharide on immunomodulatory activity of spleen lymphocytes in mice.This paper will supplement the previous quality standard research and form a relatively complete basic research on the medicinal materials of Rubus sachalinensis stirp of Mongolian medicine,so as to clarify the internal relationship between the efficacy of rubus promoting epidemic heat maturation and the enhancement of immune function activity in modern biomedicine.Method: 5um)with gradient elution of acetonitrile(A)~ 0.1% phosphoric acid(B)(0~18min,10% A~13% A;18~33min,11% A~20% A;33~48min,20% A~30% A;48~78min,30% A ~85% A;78~90min,85% A~10% A)at a flow rate of 1.0 mL·min-1.The detection wavelength was set at 254 nm.The column temperature was 35?and injection volume of 15?L.The characterastic chromatogram of Rubus sachalinensis from 17 different producing areas were analyzed by HPLC characterastic chromatogram coupled with similarity evaluation of Chinese medicine chromatographic fingerprinting(2012 version)software.The cluster analysis were performed using SPSS software.The contents of caffeic acid(324nm),epicatechin(280nm)and ellagic acid(254nm)were determined.2)The crude polysaccharides of Rubus sachalinensis were extracted by hot water.Protein removal rate and polysaccharide loss rate were used to evaluate and compare the effectiveness of Sevage method,trichloroacetic acid(TCA)method,papain method and papain-Sevage method in deproteinizing Rubus sachalinensis polysaccharides.The Cellulose DE-52,Sephadex G-100 columns were used to separate and purify homogeneous polysaccharides.The relative molecular mass was analyzed by high-performance gel permeation chromatography and the monosaccharide composition and structure were preliminarily identified by GC,IR and NMR.3)The effects on proliferation function of mice spleen lymphocyte proliferation were determined by CCK-8,and the effects on the release capacity of IL-2,IFN-? and TNF-? were determined by the ELISA kit.Result: 1)The characterastic chromatogram of 12 batches of samples identified a total of 11 peaks as common peaks.The similarity software was used to analyze the characterastic chromatogram of 17 batches of samples.The similarity of some sample s from 17 batches of samples varied largely and the similarity was 0.286~0.972.The similarity of >0.8 batches of medicinal materials was established and clustering analysis was performed.Cluster analysis classified 12 batches of Rubus sachalinensis into 2 categories.The linear ranges of caffeic acid,epicatechin and ellagic acidwere 0.54~2.700?g(r = 0.9995),1.122~5.610?g(r = 0.9996)and 0.045~0.225?g(r = 0.9998).The recoveries of the samples were 99.41%~100.25%,95.69%~99.09%,97.97%~100.90%,respectively.The average recoveries were 99.56%,RSD 0.47%;96.83%,RSD 1.54%;99.66%,RSD 1.73%.2)The crude polysaccharides were obtained by water extraction and alcohol precipitation with a yield of 2.2%.Papain-Sevage method was found to be the best method for the deproteinization of RSP,and the optimal operating conditions were 86.62 min hydrolysis with papain at an enzyme-to-substrate of 0.40mg/mL and 62.46? followed by two treatment with Sevage.Two homogeneous polysaccharides,RSP1-1 and RSP1-2,were separated and purified,with molecular weights of 13227 Da and 9343 Da determined by HPGPC.They mainly contained arabinose,mannose,glucose and galactose,with the mole ratios 9.5:7.0:10.3:18.6 and 5.7:11.1:10.3:14.2,respectively.The structure of RSP1-1and RSP1-2 was analyzed by IR and NMR,and RSP1-1 might mainly contain ?-1,3-Ara,?-1,4Gal,?-1,6-Glc,?-1,3-Man,and RSP1-2 might mainly contain?-1,4Gal,?-1,6-Glc,?-1,3-Man.3)At 5 ? 200?g/mL,RSP1-1,RSP1-2 and RSP2 stimulated the proliferation of spleen lymphocytes(P<0.05)and promote lymphocytes to secrete IFN-? and TNF-?(P<0.05).At 5?g/mL,RSP1-1,RSP1-2 and RSP2 promote lymphocytes to secrete IL-2(P<0.05).Conclusion: This paper is complementary to the previous quality standard research,and forms a relatively complete basic research on the medicinal materials of Mongolian medicine raspberry wood.The established methods of HPLC characteristic spectrum detection and quantitative determination analysis of raspberry wood can effectively evaluate the quality of this medicinal material.RSP1-1,RSP1-2 and RSP2 are natural homogeneous polysaccharides that are obtained from this plants for the first time.The two homogeneous polysaccharides could promote the proliferation of splenic lymphocyte in different degrees and promote lymphocytes to secrete IL-2,IFN-? and TNF-? and all have immunomodulating activity.