Studies On The Isolation, Purification And Bioactivity Of Glycoprotein From Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge belongs to the salvia plant, which is one of theChinese traditional herbs usefully, its dry rootstalk could be used to be medicine, itcould cure many diseases such as coronary heart disease, apoplexy and microcirculationdisturbance and so on. Tanshinones is its main lipo-soluble effective components whichhas the antibacterial activity, salvianolic acid is its main water-soluble activecomponents which has the pharmacological activity such as antioxidation. By far thestudy for the effective components in Salvia miltiorrhiza Bunge is be only limited tothese small molecule substances, tanshinones andsalvianolic acid, while the study for itsactive polysaccharides and glycoproteins hasn't been reported. To make the further useof the Salvia miltiorrhiza Bunge resource, I took Salvia miltiorrhiza Bunge as materialand investigated its extraction process, separation, purification, composition and strctureusing modern separation and analysis techniques, the antioxidative and immune activityof the glycoprotein according to the modern way of evaluating function firstly, the mainresearch results were as follows:1. The optimum conditions with the hot-water extraction method obtained by theorthogonal experiment design were as follows: the ratio of sample to water was 1:20,the temperature was 70℃, the time was 4 hours, and the NaCl concentration was0.1mol/L. Under the conditions, the extraction ratio of the glycoprotein were 4.68%.2. The optimum conditions with the ultrasonical-assisted extraction methodobtained by the orthogonal experiment design were as follows: the ultrasonic power of250W, the ultrasonic time of 20 min, the NaCl concentration of 0.05mol/L, the ratio ofsample to water 1:20. Under the conditions, the extraction ratio of the glycoprotein was6.89%.3. The optimum conditions of the refination were as follows: the removal ofprotein with the Sevage method for 7 times, the precipitation with 80% alcohol.underthe best technological conditions, the extraction rate of the glycoprotein was 5.03%.4. The physical and chemical determination showed this glycoprotein extracts wasa kind of acidic compound containing no starch, no reduction sugar and no polyphenolrather than protein with the 17 determined amino acids and 8 monosacharides andexisted a great deal of O-glycosidic linkage, the content of polysaccharide and protein was 51.24% and 37.34% respectively, the spectrograms was obtained by UV andinfra-red spectra analysis.5. Four fractions named respectively CSGP-1-a, CSGP-1-b, CSGP-3'and CSGP-4'were isolated and purified from the glycoprotein extracts by the DE-52 ion-exchangeand Sephadex G-200 gel filtration chromatogram, the purity were identified by agarosegel electrophoresis and Sepharose CL-6B gel filtration chromatogram and the resultsshowed four fractions were all homogeneous fractions.6. Gas chromatography analysis, UV and infra-red spectra analysis, physical andchemical characters showed that CSGP-1-a is a kind of sulfate polysaccharidecomposed of the glucose withα,β-glycoside linkage containing 95.4% polysaccharide,its molecular weight is 87.6kD and the specific optical rotation is +52.3°; CSGP-1-b,CSGP-3', CSGP-4' are all the acidic glycoconjugates containing no starch, no reductionsugar and no polyphenol rather than protein, the total sugar content is 89.5%, 79.3%,92.5% respectively and their contents of protein was 2.5%, 19.6%, 7.9%; theirmolecular weight determined by SDS-PAGE was 65.4kD, 55.8kD, 42.3kD respectively,the specific optical rotation was +89.6°, +132.5°, +99.5. All of the three fractions weremainly composed of glucose and galactose, existed O-glycosidic linkage and containedpyran ring; CSGP-3' and CSGP-4' were all sulfate glycoproteins.7. Amino acids analysis showed CSGP-1-b, CSGP-3' and CSGP-4' contained the17 determined amino acids except CSGP-1-a, among which the contents of Asp, Glu,Pro, Gly, Phe, Ser and Tyr were higher.8. The glycoproteins, CSGP, from Salvia miltiorrhiza Bunge showed strongerscavenging efficiency on hydroxyl free radical(·OH) from Fenton reaction andsuperoxide anion radical(O₂^-·) generated by NADH/PMS, the scavenging efficiency wasrelative to the glycoprotein contents and equal to Vc at the same concentration, theglycoproteins showed weaker scavenging efficiency on 1, 1-diphenyl-2-picrylh-yrazylfree radical(DPPH.), the scavenging efficiency wasn't relative to the glycoproteincontents.9. The fractions of CSGP-A, CSGP-B and CSGP-C obtained by the alcoholfractionated precipitation showed different scavenging efficiency on·OH and O₂^-·According to the scavenging concentration of 50%(IC₅₀), the sequences of scavengingefficiency for·OH and O₂^-·were: CSGP-A＞CSGP＞CSGP-B＞CSGP-C, the IC₅₀ for·OH and O₂^-·was 0.0970mg·mL^-1 and 0.0716mg·mL^-1 respectively, this result showedCSGP-A has stonger scavenging radical efficiency and was a stonger antioxidative fraction.10. The immune modulation activities of the glycoproteins, CSGP, from Salviamiltiorrhiza Bunge were investigated via establishing the mice model inhibitidbyCyclophosphamide(Cy), the results showed the glycoprotein extracts could effectivelystimulate the growth of immune organs, improve phagocytosis of peritonealmacrophages, accelerate distinctly the level of serum hemolys, enhance the PFC andincrease markedly the splenic lymphocyte proliferation in mice inhibitid by Cy, so theglycoprotein extracts possessed the immune modulation activities.The research indicated that the glycoproteins in Salvia miltiorrhiza Bunge not onlywere greatly high in content but also had-preferable bioactivity, so it is a plant activecomponent with a markedly developed prospect.