Research On Extraction,Purification And Activities Against Chondrocyte Of Polysaccharides From Bauhinia Championii Benth

ObjectiveThe objective of this project was to study the optimum extraction conditions and purification technology of Bauhinia championii Benth polysaccharides (BCBP) and analyse the monosaccharide composition of BCBP. On this foundation, we evaluated the effects of BCBP on the proliferation of chondrocyte. This experiment was to provide theoretical basis for the clinical application of the BCBP.Methods1. The water extraction and alcohol precipitation method was adopted to polysaccharide, phenol-sulfuric method was used to detect the polysaccharide content. The main influence factors were determined by single factor test. Response surface methodology was applied to optimize the main extraction conditions of BCBP; Coomassie brilliant blue method was used to detect the protein content, take the percentage of the removed protein and the loss rate of polysaccharides as the investigation index, study the purification technology of BCBP; BCBP was formed monosaccharidedes by TFA hydrdyzation. The hydrolysate reacted with1-phenyl-3-methyl-5-pyrazolone (PMP) to produce the PMP derivatives that were analyzed by HPLC.2. Cartilage was isolated from the knee joint of the4-week-old SD rats, and used to establish cultured primary chondrocytes. Immunohistochemical staining was used to identify chondrocytes, and MTT assay was used to evaluate cell viability. Flow cytometry was used for cell cycle analysis. Moreover, the mRNA and protein expression levels of CyclinDl, CDK4and CDK6in chondrocyte were detected by RT-PCR and Western blot analysis, respectively.Results1. The results showed that optimum extraction conditions were as follows:the extraction temperature was100℃and extraction time was4h, the liquid-solid ratio was23and the extraction times was2; The percentage of removed protein with methods Sevage, Enzyme-Sevage emethod, TCA-Sevage and TCA-butyl alcohol were46.21%,71.43%,81.12%and88.11%respectively. The loss rate of polysaccharides were33.73%,18.51%,43.40%,28.15%, respectively. The results of HPLC analysis showed that BCBP consisted of glucose, rhamnose, galactose.2. Immunohistochemical staining was used to identify chondrocytes, the results showed that the cytoplasm of cells was stained brownish yellow. BCBP promote chondrocytes proliferation in effective concentration of100μg/ml and effective time of48h.3. The results of flow cytometry demonstrated that BCBP treatment reduced cell population in G0/G1phase, whereas increased that in S phase. Furthermore, BCBP treatment enhanced the expression of CyclinDl, CDK4and CDK6than untreated control chondrocytes (P<0.05).Conclusios1. Response surface methodology is accurate and easy, thus suitable for the analysis of the optimum extraction conditions of BCBP. The combined methods of papain and Sevage are the best for removing protein. The results of HPLC analysis showed that BCBP consisted of glucose, rhamnose, galactose.2. The present study demonstrates that BCBP promotes chondrocyte proliferation by accelerating the Gl/S transition, and upregulating the expression of CyclinDl, CDK4and CDK6.