Isolation, Structural Investigation And Biological Activity Of Several Polysaccharides

China has rich resources of TCM and microbe, from which the polysaccharides possessing high activity and curative effect can be isolated and purified. We may use them as materials to carry out the research on chemical structure and biological activity. This work is significant in two aspects. Firstly, it contributes to improve the glycochemistry and glycobiology and the glyco-bank can be enriched. Secondly, it helps to develop polysaccharide drugs in China.Cudrania tricuspidata (Carr.) Bur. is a deciduous shrub or tree distributed over China, Korea, and Japan. Its roots are applied in clinic for the treatment of digestive apparatus tumor, especially gastric carcinoma, and are also used to heal gonorrhea, rheumatism, jaundice, boils, scabies, bruising, and dysmenorrhea. As a widely used traditional Chinese medicine, it can stimulate the immune function, and show antioxidative, anti-inflammatory, and other effects in pharmacological studies. The chemical constituents of C. tricuspidata include xanthones, flavones, isoflavones, benzophenones, polysaccharides, etc. Previous reports on polysaccharides of C. tricuspidata are weak in the preparation of homogeneous fractions, so it is impossible to learn their types, contents, and their relationship with pharmacological effects. This dissertation is focused on the isolation, purification, chemical characterization, and immunological activity of the polysaccharide fractions of C. tricuspidata.The roots of C. tricuspidata were extracted with hot water, giving one crude preparation, from which 4 homogeneous polysaccharide fractions (CPS-OA, CPS-1A, CPS-2B, CPS-3A) were isolated using anion-exchange chromatography and gel permeation chromatography. Their chemical structures and physicochemical properties were investigated using HPLC, glycosyl analysis, methylation analysis, elemental analysis, GC, GC-MS, partial hydrolysis, enzymatic hydrolysis, and spectroscopic methods, such as IR, UV, ¹³C NMR, ¹H NMR, HMQC and HMBC. The structure formula or characteristics were inferred as follow:1) CPS-OA contains glucose residue solely, with an average repeating unit of decasaccharide, having a backbone consisting of 1, 4-linked α-D-glucopyranosyl residues, to which the side chain consisting of terminal and 1, 4-linked α-D-glucopyranosyl residues is attached at position 0-6 of the branching residues.2) CPS-1A has a backbone mainly consisting of α-1, 4-D-glucopyranosyl residues, and the terminals are galactose, glucose, and arabinose residues. Minor branches are attached to galactosyl and glucosyl residues, and CPS-1A also contains trace xylose residue.3) The major composition part of CPS-2B is a kind of pectic polysaccharide called type RG-I. The side chain, composed of complex type II AG, is attached to the backbone consisting of rhamnose at position 0-4. AG itself also has branch structure, which has a backbone consisting of 1, 3-linked β-D-galactosyl residues, and at position 0-6 of partial backbone there were further branches composed of terminal, 1, 5-linked, 1, 3, 5-linked arabinosyl residue and 1,6-linked galactosyl residues.4) CPS-3A is composed of 1, 6-linked β-D-galactosyl residues, 1, 4-linked β-D-galactosyl residues, 1, 4-linked β-D-galacturonic acid residues, 1, 3, 6-linked β-D-galactosyl residues, and 1, 2, 4-linked β-L-rhamnosyl residues. There are branch points at position 0-2 or 0-4 of 1, 2, 4-linked β-L-rhamnosyl residues, and at position 0-3 of 1, 3, 6-linked β-D-galactosyl residues, furthermore, the terminal is 1-linked galactosyl residues. The side chain consists of 1-linked arabinosyl residues, 1, 5-linked arabinosyl residues, and 1, 3, 5-linked arabinosyl residues. The side chain is attached to the backbone at position 0-3 of 1, 6-linked β-D-galactosyl residues.It is the first time that the presence of the four polysaccharides in C. tricuspidata is reported.Arctium lappa L. is a member of the Compositae family, which is a kind of biennial giant plant and possesses excellent value for medical care and special nutrition. The roots of A. lappa L. contain rich proteins, amino acids, vitamins, miner- als, and inulin, which is special for Compositae plants. The roots were extracted with hot water, giving one crude preparation, from which one homogeneous oligosacch-aride fraction (ALO) was isolated using gel permeation chromatography. Its chemical structure and physicochemical property were investigated using HPLC, glycosyl analysis, methylation analysis, GC, GC-MS, and spectroscopic methods, such as IR, UV, ¹³C NMR, ¹H NMR, HMQC and HMBC. ALO is composed of D-fructose and D-glucose in the molar ratio of about 13:1, which is confirmed by the composition of 12 fructose residues linked by β (2→1) glycosidic bond and 1 glucose residue linked by a (1→2) glycosidic bond at the end of linear straight sugar chain. The structure formula was inferred as follow:The extracellular polysaccharide produced by Trichoderma pseudokoningii was isolated and purified using anion-exchange chromatography and gel permeation chromatography. Its chemical structure and physicochemical property were elementarily investigated. The homogeneous polysaccharide named TPP-0 was composed of rhamnose and glucuronic acid in the molar ratio of 1.6:1.0. There were nine anomeric carbon signals in the ¹³C NMR spectrum, which suggested that TPP-0 has many linkage types. Its detailed structure is still in research.The effects of CPS-OA, CPS-1 A, CPS-2B, and CPS-3A on proliferation of lymphocytes and lymphocytes induced by ConA or LPS were studied with MTS-PMS assay. They were also studied on phagocytosis of peritoneal macrophages (MO) of mice in vitro with colorimetric method of neutral red. At the same time, the effects of CPS-OA, CPS-1 A, CPS-2B, and CPS-3A were compared with that of lentinan widely used in clinic.The results showed that CPS-1 A, CPS-2B, and CPS-3A directly stimulated lymphocytes proliferation at concentrations of 6.25 ?100 礸/mL. Compared with the control group, the mean differences of these effects were very significant. Effects of CPS-1A and CPS-2B at 50 μg/mL and 100 μg/mL were better than that of lentinan at the best concentration of 25 μg/mL and the mean differences were very significant. Effects of CPS-3A at 25 μg/mL, 50 μg/mL, and 100 μg/mL were better than that of lentinan and the mean differences were very significant.CPS-0A, CPS-1A, CPS-2B, and CPS-3A stimulated lymphocytes proliferation induced by ConA at concentrations of 6.25 ～100 μg/mL. Compared with the control group, the mean differences of these effects were very significant. The effect of CPS-1A at 50 μg/mL was better than that of lentinan and the mean difference was very significant. The effects of CPS-2B at 25 μg/mL and 50 μg/mL were better than that of lentinan. The mean difference was significant at 25 μg/mL and very significant at 50 μg/mL. Effect of CPS-3A at 12.5 μg/mL was better than that of lentinan, the mean difference was significant and the concentration was less than that of lentinan.In lymphocytes proliferation induced by LPS, effects of CPS-1 A at concentrations of 50 μg/mL and 100 μg/mL were better than those of the control group and lentinan. Compared with the control group, the mean differences were very significant. Compared with lentinan, the mean difference was significant at 50 μg/mL and very significant at 100 μg/mL. CPS-2B stimulated lymphocytes proliferation at concentrations of 6.25～100 μg/mL. Compared with the control group, the mean differences were very significant. Effects of CPS-2B at 6.25 μg/mL, 50 μg/mL and 100 μg/mL were better than that of lentinan, the mean differences were significant and the concentration of 6.25 μg/mL was less than that of lentinan. CPS-3 strengthened lymphocytes proliferation at concentrations of 6.25～100 μg/mL. Compared with the control group, the mean difference was significant at 12.5μg/mL and very significant at 6.25μg/mL, 25μg/mL, 50μg/mL and 100μg/mL. Effect of CPS-3 A at 25 μg/mL was better than that of lentinan and the mean difference was significant.In phagocytosis of peritoneal MO experiments, CPS-1 A promoted phagocytosis of MO on neutral red. Compared with the control group, the mean difference was significant at 100 μg/mL and very significant at 50 μg/mL. The effect of CPS-1 A at 50 μg/mL was equal with that of lentinan at the best concentration of 50 μg/mL. CPS-2B promoted phagocytosis of MO on neutral red. Compared with the control group, the mean difference was significant at 100μg/mL. The effect of CPS-2B at 100 μg/mL and that of lentinan were insignificant. Compared with the control group, CPS-3 A had no effect on phagocytosis of MO.In the above immunological assays, CPS-OA showed no activity. CPS-1 A, CPS-2B, and CPS-3A might be better immunomodulators than lentinan.The polysaccharide fractions showing high activity from C. tricuspidata are those highly branched or possessing uronic acid at nonreducing terminals. CPS-OA with few branches and without uronic acid showed no activity. The results suggested that complex branches and uronic acid have significant effect on immune activity for polysaccharides.Three commonly used methods in elimination of proteins, i.e. Sevag method, TCA method, and papain-Sevag method, were compared in this dissertation. The result showed that papain-Sevag method had the best effect on the elimination of proteins in preparing the polysaccharide from C. tricuspidata.