Study On Separation And Purification Of Polysaccharides From Salvia Miltiorrhiza And Their Pharmbiological Activities

BACKGROUNDPolysaccharide, one class of natural polymers in Chinese herbs, is the basic material to maintain normal operation of the lives, and has better regulation of the immune system function and anti-aging effects. So, there is a tremendous potential development in the herbal plant polysaccharides. The research of polysaccharides has aroused great interest during recent years. Polysaccharide is not only used in the treatment of cancer but also of immunodeficiency disorders. Polysaccharides as immunomodulation agents had being investigated for potential uses in neoplasm and AIDS et al. With the rapid evolution and adoption of molecular biology and new analytical techniques, the methods of isolating and analyzing, polysaccharides develop greatly too. So far establishing product-quality-standard is the key point of polysaccharides research, especially for the characteristic index and the action mechanism of Polysaccharides.Salvia miltiorrhiza Bunge belongs to the salvia plant, which is one of the Chinese traditional herbs usefully, its dry rootstalk could be used to be medicine, it could cure many diseases such as coronary heart disease, apoplexy and microcirculation disturbance and so on. Salvia earliest record found in "Shen Nong's Herbal Classic", was listed as the top grade Grass. Several studies have indicated that Salvia is a famous medicine which promoting blood circulation by removing blood stasis effectively. And it was widely used to treat coronary heart disease, angina, myocardial infarction and cerebral vascular disease in clinical practice. Tanshinones is its main lipo-soluble effective components which have the antibacterial activity; salvianolic acid is its main water-soluble active components which has the pharmacological activity such as antioxidation. By far the study for the effective components in Salvia miltiorrhiza Bunge is be only limited to these small molecule substances, tanshinones and salvianolic acid, while the study for its active polysaccharides and glycoproteins hasn't been reported.OBJECTIVEPolysaccharide, one of the effective parts in Salvia, has begun to be studied in recent years. The extraction of polysaccharides from Salvia were studied and optimized by our early research group. It has been able to extract high content and strong activity Salvia polysaccharides now. The future research was designed to evaluate the hepatoprotective effects of S. miltiorrhiza Polysaccharides (SMPS) in immunological liver injury. The results suggest that SMPS play a protective role against immunological liver injury. Based on these studies, it is proposed as an immune enhancer to develop, further study of its refined purification technology and to complete some pre-clinical studies. To make the further use of the Salvia miltiorrhiza Bunge resources, we took Salvia mmiorrhiza Bunge as material and investigated its extraction process, separation and purification using modern separation and analysis techniques, the immune activity of the glycoprotein according to the modern way of evaluating function firstly.METHODS AND RESULTSThe primary methods and research results were as follows:1. The root was extracted by water purified preliminarily by alcohol precipitation, and then the crude polysaccharide can be got. Using phenol-sulfuric acid method to determine the polysaccharide content, the results indicated that there was a good linearity of glucose in the range of 0～80μg-mL-1. The regression equation was Y=105.26 X-1.2998 (R2=0.9971); Using G250 method to determine the protein content, the results indicated that there was a good linearity of glucose in the range of 0～0.1 mg·mL-1. The regression equation was Y=167.62X-1.4735 (R2 =0.9991). Methodological experiments showed that the method had high precision and good repeatability and can be used as the detection means of polysaccharide and protein in S.miltiorrhiza. The total polysaccharide of crude extracts is 40.35%, and protein content amount to 8.96%.2. Two methods for removal of protein were discussed, in order to find the right technology conditions. The method of using Sevag has a low percentage of removed protein, which is 48.65%, but a high percentage of decomposed polysaccharides, which is 21.38%.The method of using CC13COOH, has a higher percentage of removed protein, which is 52.30%, but a lower percentage of decomposed polysaccharides, which is 12.43%. The optimum conditions of the Sevag were obtained by the orthogonal experiment design. The results showed that trichloroacetic acid (TCA) exhibited higher removal percentage of protein and lower loss of polysaccharide.3. Four different types Macro porous adsorption resin and one ion-exchange resin were comparative investigated in the purification and isolation of Salvia polysaccharides. Results The total polysaccharide of crude extracts is 40.35%,and protein content amount to 8.96%. Compared with the traditional methods, AB-8, DB-301 type of resin used in purification of polysaccharide can simply the working process and obtain better effect. Then the obtained crude polysaccharide though AB-8 resin purified by ion-exchange resin DEAE-52. Three portions of powder were obtained through lyophilization and named as SMP1, SMP2, and SMP3.4. The effect of polysaccharides extracted from Salvia miltiorrhiza Bge. was explored. Sixty KM mice were randomly divided into the normal control group, Cyclophosphamide(CY) group, lentinus edodes mycelia polysaccharide tablets group, low-dose of SMPS+ CY group, middle-dose of SMPS+ CY group and high-dose of SMPS+ CY group (n=10). The immune inhibited mice model were made with the method of inject CY (50 mg·kg-1) in the mice s abdominal for 4 days. The normal control was treated with NS, and the other groups were treated with Polysacc-haridile lentinus edodes(10 mg·kg-1, ig), SMPS (100,200 and 300 mg·kg-1, ig) respectively for 10 days. The effects of SMPS on Liver index, thymus index, spleen index and carbon clearance index in both normal mice and immunosuppression mice induced by CY were examined.A. Effects of polysaccharide on the weight of the immune organsIn comparison with the control group, the spleen and thymus index was decreased significantly in model CY group (P<0.05). It was shown that CY could atrophy the immune organs and the immune suppression model was established successfully. Compared with model group, the spleen index in 3 three doses groups were increased significantly (P<0.05), but all treatment groups have no significant differences compared to control group (P>0.05). That tells us each dose of SPMS could against the splenic atrophy induced by CY and repair the damage to normal. The thymus index in both medium dose and high dose of SPMS groups (200 and 300 mg·kg-1, ig) were increased significantly compared to the CY group (P<0.05), but have no significant differences in comparison with the control group (P>0.05), which indicated that the dose of SPMS could against the thymus atrophy induced by CY and make it normal.B. Effects of polysaccharide on phagoeyte function of miceIn comparison with the control group, the carbon clearance index K and phagocytic index a in model CY group were significantly reduced. The results showed us CY could effectively decrease the mouse peritoneal macrophage phagocytosis. Compared with model group, index K and a in positive drug group were significantly enhanced (P<0.05), but have no significant differences compared to control group (P>0.05). Those tell us positive drug could make the mice phagocytosis function back to normal. The index K and a in low (100 mg·kg-1) and medium dose (200 mg·kg-1) of SPMS groups were promoted greatly compared to the CY group (P<0.05), which indicate that the low and medium dose of SPMS could recover the mice phagocytosis function destroyed by CY.5. IL-2, IL-10 and IFN-γin blood serum of Cyelophosphamide-immunosuppressant mice were observed by the techniques of ELISA. According to the different treatment, we set the 80 NIH mice into eight groups for test:the normal control group, CY model group, CY +Lentinan Positive group, CY+low-dose of SMPc treatment group (100 mg·kg-1), CY+ high-dose of SMPc treatment group (300 mg·kg-1),CY+low-dose of SMPS treatment group(100 mg·kg-1), CY +middle-dose of SMPS treatment group(200 mg·kg-1) and CY +high-dose of SMPS treatment group(300 mg·kg-1) (n=10). The normal control group was given with saline once a day for 10 consecutive days. Other groups were given with CY(75 mg·kg-1)once a day for 4 consecutive days to established immunosuppression model, other process just same as the above experiments. After the last treatment, the mice were killed for serum, in which the content of cytokines(IL-2,IL-10,IFN-γ) were measured by ELISA method. As it can be seen from the experimental results:A. Effect of polysaccharide on IL-2 in mice serumIn comparison with the control group, the IL-2 expression in model CY group was significantly reduced (P<0.05).The result told us CY could significantly lower IL-2 in mice serum. The two SMPc treatment groups(SMPc of 100,200 mg·kg-1) were all no significantly different compare to CY group, and all content of IL-2 were lower than control. Data showed SMPc treatment groups had few obvious regulation of IL-2.But for SMPS, compared with the CY group,IL-2 in all three doses of SMPS treatment groups (100,200,300mg·kg-1) were significantly increased (P<0.05),while the low and medium dose groups had no significantly different (P<0.05), but the high dose group were significantly enhanced compare to the control. It was verified from the experiment that SMPS treatment groups could effectively promote the expression of IL-2.B. Effect of polysaccharide on IL-10 in mice serumFor the content of IL-10 in mice serum, there is no significantly different in all 8 groups (P>0.05). It suggested that the polysaccharide could not regulate the conent of IL-10 in mice serum.C. Effect of polysaccharide on IFN-γin mice serumCompared with the control group, IFN-γin model CY group were significantly decreased (P<0.05).It told us that CY could significantly lower IFN-γin mice serum and the Immunosuppression model were established, successfully. For two SMPc treatment groups (SMPc of 100,300mg·kg-1), IFN-γwere all no significantly different compare to CY group, which indicate that we cannot believe SMPc enhance the expression of IFN-γin serum. But for SMPS, compared with the CY group, IFN-γin all three doses of SMPS treatment groups (100,200,300mg·kg-1)were significantly increased (P<0.05),while compare to the control group, the three were all no significantly different(P>0.05). As it can be seen from the results:all dose of SMPS could regulate the expression of IFN-y to return back to normal effectively.CONCLUSION1. Research in the refining and purification we comparative investigated some different types resin in the purification and isolation of Salvia polysaccharides. It was finally found purification of Salvia polysaccharides can be effectively conducted by adopting AB-8 or DB-301 type of resin and DEAE-52 ion-exchange resin.2. Research in immune activity The immune modulation activities of the Salvia polysaccharides were investigated via establishing the mice immunosuppression model, the results were as follows:(1) SMPS increased spleen index, thymus index and carbon clearance index K and phagocytic index a significantly. It was verified from the experiment that SMPS could not only restore the damaged immune organs but also increased macrophage phagocytic function in immunosuppressed mice induced by CY. (2) The IL-2 and IFN-y expression in immunosuppressed mice were significantly improved by Salvia polysaccharides. It found that SMPS could upgrade the immune cytokines expression.Note:The statistical analyses in this thesis were processed with SPSS software. One-way ANOVA followed by Least Significant Difference test. (Or Dunnett's T3 test, if equal variances not assumed) KEYWORDS:alvia miltiorrhiz; Polysaccharides; Purification; Immunomdulatory; Carbon clearance;...