| Aim Polysaccharide, a biological response modifier, arouses interests in antitumor and antisenium aspect. Previous researches have proved that polysaccharide from Angelica sinenesis has radioprotective, hematopoietic and antitumor effects . In the present study, the polysaccharide was isolated and purified from Angelica sinenesis (Oliv.) Diels grown in Minxian County, Gansu Province. The immunomodulating activities of Angelica polysaccharide and its mechanism were investigated.Methods (1) The fresh roots of Angelica sinensis were washed, smashed, and refluxed with ethanol to remove the essential oils. Water extraction with alcohol precipitation was used to prepare for Angelica. The white powder of Angelica polysaccharides (AP-0) was obtained by dialyzing of the crude polysaccharide, protein depletion and freeze-drying. To purify the polysaccharide, gel filtration chromatography was carried out by using a Saphacry S-400 column eluted with 0.1mol/L sodium chloride. (2) Phenol-sulfuric acid method, m-hydroxyldiphenyl method and Serva blue-G method were used to determine the content of carbohydrate, uronic acid and protein in Angelica polysaccharides respectively. The average molecular weight(MW) was determined by using HPLC method. After hydrolysis with trifluoroacetic acid, paper chromatography was carried out to determine the monosaccharide composition of Angelica polysaccharides. The ER. and UV spectrum were also performed. (3) The effects of AP-0 on normal andimmunosuppressing mouse model were investigated, which were induced either by subcutaneous injection of cyclophosphamide or by intragastric hydrocortisone. After intraperitoneal administration of AP-0 for 7 days, mouse thymus and spleen were weighed, and phagocytosis of mononuclear macrophage (MO) were tested with carbon clearance assay. The production of specific IgG and IgM against sheep red blood cell(SRBC) in serum was estimated by spectrophotometric method. (4) The effects of AP-3, the major fraction of AP-0, on proliferation of murine splenocyte, macrophage and mixed lymphocyte reaction were tested by MTT assay. T and B lymphocytes were separated with immune magnetic beads, then the proliferation stimulated by AP-3 was determined. (5) Total splenocyte was cultured in the presence of AP-3 and the change of CD4+ lymphocyte percentage was detected by flow cytometric method, the production of IL-2 and IFN-r in supernatant were determined by ELISA, and mRNAs expression of these two cytokines was detected by quantitative RT-PCR.Results (1) Three fractions with good solubility and appearence were obtained, which were designated as AP-1, AP-2 and AP-3. Among the three fractions, AP-3 with average molecular weight of 50 kDa was the major fraction. (2)The analytic results were as follows, AP-0: content of carbohydrate was 67.9%, uronic acid was 26.7%, protein was 0.8%; AP-1: content of carbohydrate was 82.3%, uronic acid was 18.1%, protein was 0.5%, MW was (33-51)+104; AP-2: content of carbohydrate 73.7%, uronic acid was 38.1%, protein was 0.3%, MW was (13-22)+104; AP-3 content of carbohydrate was 68.3%, uronic acid was 37.5%, protein was 0.7%, MW was (2-7)+104. There was no obvious difference in monosaccharide composition among the three fractions, and each sample was composed of Glu, Ara andGluA. IR spectrum showed a typical absorptive manner of polysaccharide, and had absorption at 770cm-1 and 840cm-1, which suggested that the structure of Angelica polysaccharide be a-D-pyranoglucose. (3) At the range of 10-100mg/kg, AP-0 increased phagocytic function of MO and spleen index significantly in both normal and immunosuppressive mice but showed no obvious influnence on thymus index. The production of specific IgG and IgM in serum was remarkably decreased. (4) The results in vitro studies showed that AP-3 directly stimulated proliferation of murine splenocyte, macrophage, mixed lymphocyte reaction and T lymphocyte, but inhibited B lymphocyte proliferation at the range of 30-300g/ml. The percentage of CD4+ subfractioned lymphocytes in total...
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