Isolation, Purification, Structural Characterization And Antitumor Activity Of Polysaccharides From Ganoderma Capense

This article systematically studied on structural characterization and antitumor activity of polysaccharides from mycelia powder of Ganoderma capense. The content was summarized as follows:1. Extraction, isolation and purification of crude polysaccharides: The four crude polysaccharides were obtained from mycelia powder of Ganoderma capense by water extraction or base extraction, ethanol precipitation. The yield of four crude polysaccharides were 13.4%, 1.87%, 1.2%, 1.1%, after deproteinization, dialysis and lyophilization. The six polysaccharides（GCP50-2, GCP50-3, GCP70-1, GCP70-3, GCP90-2 and GCP90-3） were obtained after successively purifing by DEAE Sepharose CL-6B and Sephadex G-75, respectively. The high performance gel permeation chromatography（HPGPC） and optical rotation identification proved that the six polysaccharides were homogenous. The Optical rotation [α]_D²⁵ of the homogenous polysaccharides were +113°, +129°, +131°, +168°, +146°, +142°, separately. The average Mw were 3167, 8123, 10685, 32436, 6381, 5808 Da, separately.2. Structural characterization of six polysaccharides: The homogenous polysaccharides were analysized by HPLC. The results showed GCP50-2, GCP50-3 and GCP70-1 were all glucan, with the main backbone chain of（1→4）-α-D-Glc residue interspersed with（1→4,6）-α-D-Glc residue and the side chain attached to the O-6 position with（1→）-α-D-Glc. While GCP70-3 was heteropolysaccharide concluding mannose, glucose, galactose, xylose and arabinose, comprising a backbone of 2,3,4)-β-D-Xyl-（1→, →3,6）-β-D-Man-（1→, →3）-β-L-Ara-（1→ and →6）-β-D-Gal-(1→ with branches consisting of β-D-Xyl-(1→ and β-D-Glc-（1→. GCP90-2 and GCP90-3 mainly composed of glucose, galactose, xylose and arabinose, and also a small amount of mannose and rhamnose. The structure of GCP90-2 comprises a backbone of →4,6）-β-D-Glc-（1→, →3）-β-D-Gal-（1→, →3,4）-β-D-Xyl-（1→ and →3,6）-β-D-Man-（1→ with branches consisting of →3）-β-L-Ara-(1→, β-D-Xyl-(1→, β-L-Ara-(1→ and β-L-Rha-（1→. The structure of GCP90-3 comprises a backbone of →6）-α-D-Glc-（1→, →3）-α-L-Ara-（1→, →3）-α-D-Gal-（1→, →3,4）-α-D-Xyl-（1→ and →3,6）-α-D-Man-(1→ with branches consisting of α-L-Ara-(1→ and α-L-Rha-(1→. On the basis of hydrolysis, methylation and GC-MS analysis, IR spectra and NMR spectra, the primary structure analysis of six pure polysaccharides were established. Meanwhlie, the advanced structures of the six pure polysaccharides were primarily studied using circular dichroism spectroscopy（CD）, scanning electron microscopy（SEM）, Congo-red assay and atomic force microscope（AFM）. Untill now, they have not been reported.3. Determination of in vitro bioactivities: Proliferation inhibitory activities of polysaccharides from G. capense for six tumor cell lines（MGC-803, SGC-7901, Hela, CNE-1, Hep G-2 and A549） were evaluated by MTT assay. Results showed that crude polysaccharide PB and pure polysaccharide GCP70-3 could inhibit proliferation of four cell lines（MGC-803, SGC-7901, Hela and CNE-1） in dose-dependent manner. Furthermore, we also evaluated the proliferation inhibitory activities with time and dose dependent manner of the pure polysaccharides（GCPB-1b, GCPB-1b, GCPB-2, GCPB-3） from base extraction and GCP70-3 for gastric tumor cell（MGC-803）. Results indicated GCPB-1a, GCPB-1b, GCPB-2 and GCP70-3 showed the proliferation inhibitory activities with time and dose dependent manner.