Preparative Isolation Of Polysaccharides From Auricularia Polytricha And Their Bioacitiveties

Polysaccharides in animals and plants and microorganisms have various bioactivities, such as anti-tumor, reducing blood sugar and blood fat, adjusting immune function and antioxidationt. The present studies aim to extraction, separation and purification of polysaccharides from Auricularia polytricha by high speed countercurrent chromatography and column chromatography. Finally,5 purified polysaccharides were obtained, and their biological activities of tumor inhibition, immune regulation, and antioxidant activity were investigated. The main results are summarized as follows:1. Two aqueous two-phase HSCCC solvent systems were established for the separation of polysaccharides from Auricularia polytricha, i.e. PEG 1000:Dextran:K2HPO4:KH2PO4:H2O=8:3:20:30:120 (w/w) and PEG1000:K2HPO4:KH2PO4:H2O=0.5:1.25:1.25:7.0 (w/w). The PEG1000:K2HPO4:KH2PO4:H2O=0.5:1.25:1.25:7.0 (w/w) was the best system by orthogonal test.2. The HSCCC separation conditions were determined by experimental methods. The optimum operation condition is:column rotation speed,700rpm; flow rate of mobile phase,2.5mL/min; sample loading size,2.0g of the crude polysaccharides (AAFRA). The separation yielded three polysaccharides:AAPS1 (192 mg), AAPS2 (173 mg) and AAPS3 (98 mg).3. Two polysaccharides AAFRB (120.5 mg) and AAFRC (92.7 mg) were obtained by DEAE-cellulose and Sephacryl S-500 HR column chromatography.4. The structures of the five polysaccharides AAPS1, AAPS2, AAPS3, AAFRB and AAFRC were elucidated by using chromatographic (HPGPC, PMP-HPLC, UPLC) spectral (1D NMR,2D NMR, GC-MS and FTIR) and chemical methods. All the five polysaccharides are composed of glucose. The molecular weights of AAPS1, AAPS2, AAPS3, AAFRB and AAFRC are 1.62×105,2.59×105,4.83×105,9.30×105 and 1.20×106Da, respectively. The specific rotations [α]D in 20℃are+89.3°,+24.7°,+112.6°.+49.6°and +61.2°, orderly for AAPS1, AAPS2, AAPS3, AAFRB and AAFRC. The structures are as follows:AAPS1:composed of a main chian (1→6)-a-D-glucopyranosyl, and (1→3,6)-β-D-glucopyranosyl and a side chain β-D-glucopyranosyloccurred O-3 of→4)-α-D-glucopyranosyl-(1→. AAPS1:AAPS2:composed of a backbone (1→3)-linked-β-D-glucopyr-anosyl and (1→3,6)-linked-β-D-glucopyranosyl with a ratio of 2:1, and the single terminal (1→)-β-D-glucopyranosyl links to O-6 position of (1→3,6)-linked-p-D-glucopyranosyl along the main chain. AAPS2:AAPS3:composed of residues→4-β-D-Glc-1→3-β-D-Glc-1→,→3-β-D-Glc-1→4-β-D-Glc-1→6-p-D-Glc-1→fragments andβ-D-1,4-Glc,β-D-1,3-Glc and p-D-1, and 6-Glc constitute the main chain. AAPS3: AAFRB:composed of five residues:→4)-α-D-Glcp-(1→,→3)-α-D-Glcp-(1→,→3,6)-α-D-Glcp-(1→,α-D-Glcp-(1→,->3)-β-D-Glcp-(1→with a ratio of 6:1:1:2:1, and the O-6 of→3)-α-D-Glcp-(1→residueslinks to the branched chainα-D-Glcp-(1→; AAFRB:AAFRC:composed of five residues→>4)-α-D-Glcp-(1→,→3)-α-D-Glcp-(1→,α-D-Glcp-(1→,→4)-β-D-Glcp-(1→and→3)-β-D-Glcp-(1→fragments, and the ratio is 10:3:4:1:3. The O-6 of→3)-α-D-Glcp-(1→links to the branched chainα-D-Glcp-(1→. AAFRC:The above five polysaccharides have not been reported in any literatures.5. The anti-tumor activity of the polysaccharides on the tumor cell membrane characteristics and immunological activity were studied by using S180 sarcoma mice. Among the five polysaccharides AAPS2 and AAFRB could significantly improve the liver index. Also AAFRB could significantly increase the thymus index. The five polysaccharides could promote the degradation of membrane phospholipids and showed better changes in tumor cell membrane structure characteristics. On the base of membrane lipid fluidity (LFU), AAPS2, AAFRB, AAFRC could significantly alter membrane fluidity (p<0.01) while AAPS3, AAFRB could significantly promote lymphocyte proliferation. The low dose group of AAPS1 showed a significantly increase of serum half of the hemolytic HC50, which indicated that the polysaccharide can promote serum antibody production with a correlation of dose-activity. The results of scavenging radicals DPPH-,02-·,·OH, NO2-·and reducing power showed that the five polysaccharide had different antioxidant activity. AAPS2 has a stronger activities of scavenging DPPH·radical, 02-·while AAFRC possessed a stronger activity of scavenging·OH radicals capacity and AAFRB has a stronger effect of removal N02 and reducing power. The structures of polysaccharides have directly effects on their mechanism of activities. The mechanism of anti-tumor activity can be divided into two kinds. The first is mainly a direct cytotoxicity against tumor cells; the second is indirect immunological regulation. The analysis of structure-activity relationship showed that polysaccharides withβconfiguration and the glycosidic bonds containing 1→3,1→6 have higher activity than those of a configuration, and the the molecular weight of the polysaccharides related to the activity. The polysaccharides with molecular weight range 200-300kDa showed the higher activity.