Structure And Conformation Characterization Of Polysaccharide From Ganoderma Lucidum And The Structure-Activity Relationship On Immunoregulation

The medicinal mushroom Ganoderma lucidum(G.lucidum)has been widely used in the development of functional foods and pharmaceuticals due to its bioactivities.Recent studies demonstrate that polysaccharides from G.lucidum play an important role in immunomodulating and anti-tumor activities.However,the complexity of polysaccharide structures from Ganoderma and the differences on the material sources and purification methods lead to the diversity of polysaccharides.The molecular structures of polysaccharides from Ganoderma were not well elucidated and the structure-function relationship studies were not investigated deeply enough.In this study,a bioactive purified polysaccharide was obtained from the fruit bodies of G.lucidum based on the screen of activity.The structure and conformation characteristics were systematically investigated.The structure-function relationship on immunoregulating activity was elucidated with the evaluation of in vivo and in vitro activity.Based on these results,high efficiency preparation technology and determination method were developed to provide support for further development,application and quality control of G.lucidum polysaccharide products.Molecular weight(M_w)distributions and monosaccharide compositions of polysaccharides from the fruiting bodies of different Ganoderma lucidum strains and Ganoderma sinense were investigated and compared.Results showed that there were big differences on the distributions of high M_w polysaccharide fractions from two official species of Ganoderma.Analysis on the polysaccharides from two groups of G.lucidum strains with different genetic similarity indicated that no obvious relationship between molecular weight distributions and the genetic similarity of G.lucidum occurred.All crude polysaccharides were mainly composed of glucosamine,galactose,glucose,mannose and glucuronic acid.Glucose possessed the highest molar percentage among all monosaccharides.A high molecular weight bioactive polysaccharide coded as GLP20 was purified and obtained by precipitating a hot-water extract from Ganoderma lucidum fruiting bodies with20%(V/V)ethanol.HPSEC-MALLS-RI analysis showed that the M_w and polydispersity of GLP20 were 2.42×10~6 g/mol and 1.32,respectively.The total carbohydrate content of GLP20 was determined to be 95.9%using the phenol sulfuric acid method.Monosaccharide composition,methylation and NMR analysis showed that GLP20 was a?-(1?3)-linked D-glucan with a(1?6)-?-D-glucopyranosyl side-branching unit on every third residue.Static and dynamic light scattering and HPSEC-MALLS-RI analysis were applied to characterize the conformation of GLP20 solutions.Results indicated that GLP20 existed as triple-helical chains in PBS aqueous solution.A conformation transition occurred in the alkaline solution with NaOH concentration larger than 0.15 M.The transition from ordered structure to single chain happened when GLP20 was heated above 135?in water solution and was irreversible as demonstrated by differential scanning calorimetry.Rheological properties of GLP20 in water at different concentrations were studied.Shear-thinning behavior occurred at lower concentrations.The results from dynamic experiments revealed that GLP20 in pure water displayed liquid-like behavior at low concentrations(?0.5%)and could form weak gels with the increase of concentration.The dynamic strain sweep measurements proved that the gelation of GLP20 in water was induced by the extremely entangled and stiff triple helices forming continuous network.The heating-cooling process proved that the sol-gel transition of GLP20 in water was thermally reversible.These cgaracteristics were related to its triple-helix conformation in solutions.In order to interpretate the structure-activity relationship on immunoregulating activity of?-D-glucan from Ganoderma lucidum,?-D-glucan fractions with similar repeat units and different molecular weights range from 3.5×10~4 to 2.42×10~6 g/mol were obtained using ultra sonic degradation,and sulfated derivatives with different M_w and degrees of substitution were also obtained by chemical modification.The effects of M_w and sulfated modification on the immune-enhancing and anti-inflammatory activity were investigated.The glucan fractions with M_w higher than 1.80×10~6 g/mol exhibited better activity on immunoenhancement and lower M_w fraction with M_w of 3.5×10~4 g/mol exhibited better activity on anti-inflammatory effects.Sulfated modification could change the conformation characteristics and improve the anti-inflammatory activity.Derivatives with higher degree of substitution showed better activity.Among sulfated derivatives with similar degree of substitution,derivatives with a lager molecular weight displayed higher inhibitory activity.The results indicated that the in vitro anti-inflammatory activity was related to the degree of substitution and the molecular weight of sulfated derivatives.Based on the structure and conformation analysis of GLP20,an HPSEC-MALLS-RI method was developed to provide support for the screen of Ganoderma lucidum fruit body materials and the quality control.A Ganoderma lucidum cultivar riched in?-D-glucan with content of 8.2 mg/g was screened out for processing.Comparison of enzymatic assay kit and the aniline blue fluorometric method on the determination of?-1,3-glucan indicated the aniline blue fluorometric method demonstrated good selectivity for?-1,3-glucan determination.For improving the extraction efficiency of?-D-glucan from Ganoderma lucidum,different solvents were used for the extraction of?-D-glucan.Results showed that KOH alkaline solution were efficient in sequential extraction for extracting?-1,3-glucan from the fruit bodies of G.lucidum,indicating that?-1,3-glucan mainly existed in the cell wall of the fruit bodies.Superfine grinding combined with the hot water extraction followed with ethanol precipitation could improve the yield and purity of?-D-glucan,which was suitable for the high efficiency preparation of?-D-glucan from the fruit bodies of Ganoderma lucidum.