Characterization Of Chemical Composition And Polysaccharide Structure From Natural Cordyceps Sinensis And Cultured Mycelium

Cordyceps sinensis(Berk.)Sacc.(C.sinensis)is a famous traditional Chinese medicine and has a long history being used as food/medicine.It is becoming increasingly popular in public and scientific research due to its nutritive and medicinal value.Researches indicated that C.sinensis contained many bioactive constituents,including polysaccharide,amino acid,protein,nucleoside,mannitol,etc.However,in recent years the yield of wild C.sinensis is declining due to its strict growing habitat.In order to meet the increasing demand,modern researchers tried to isolate several mycelial strains from wild C.sinensis,and then combined with fermentation technology so as to get cultured mycelia.What interested researchers are whether the cultured C.sinensis had similar chemical composition and bioactivity with the natural ones or whether the cultured mycelium could be regarded as a substitute of natural counterpart? Therefore,in this study,three types of natural C.sinensis collected from different regions and one cultured mycelium were compared and contrasted to characterize the amino acid composition,minerals,carbohydrates and nucleosides,further to evaluate the antioxidant properties.Afterwards,polysaccharides from natural or cultured C.sinensis were isolated and purified,respectively.Their physicochemical properties and structural features were further analyzed,laying theoretical fundation on the research of structure-bioactivity relationship of Cordyceps polysaccharide.The main results were summarized as follows:1.Cultured mycelium had a higher proportion of essential amino acid.Glutamic acid was the most abundant amino acid in both natural and cultured C.sinensis.The most abundant fatty acid in natural samples was oleic aicd(>50%),while it was linoleic acid in cultured mycelium.The contents of minerals,except for Cr,Mo,Sn,V and Cd,were significantly different between natural and cultured samples.The content of polysaccharide(10.43%)was significantly higher in cultured mycelium but it had a lower mannitol content(4.75%).The amount of adenine and adenosine in cultured sample were 0.50 mg/g and 2.61 mg/g,respectively,considerably higher than the natural ones.There was no significant difference in content of guanosine among four samples.Cordycepin was not detected in both natural and cultured C.sinensis.Additionally,water extracts of cultured mycelium had stronger scavenging abilities against DPPH and hydroxyl radicals,as well as a higher reducing power.2.The effects of drying and storage process on the contents of the bioactive ingredients in natural C.sinensis were also investigated.No significant effect in terms of amino acid composition and fatty acid composition was observed.The contents of crude fat and trehalsoe were increased significantly after freezing dry or storage.The content of mannitol was increased obviously only after storing for a long time.With regard to nucleosides,except for adenosine of which content was increase significantly,the levels of all the other four nucleosides(guanosine,uridine,hypoxanthine and adenine)was decreased obviously after freezing drying or storage.Cordycepin was not detected in all these samples as well.3.The water-extracted polysaccharides from three natural C.sinensis and one cultured mycelium were compared in terms of structural characteristics and in vitro antioxidant potentials.Results revealed that the chemical structure of the major polysaccharide fraction from three natural C.sinensis was similar,but it was significantly different with that from cultured mycelium.Three major differences were observed: a)Polysaccharides from natural C.sinensis were predominately high molecular weight fraction(>60%)of around 950 kDa.On the contrary,the average molecular weight of cultured polysaccharide was around 28 kDa.b).Polysaccharide from natural C.sinensis was mainly composed of glucose,with small amount of galactose and mannose,and no urnoic acid was detected.The cultured polysaccharide,on the other hand,was mainly composed of galactose,glucose and mannose,with small amount of ararbinose and galactournic acid.c)Polysaccharide from natural C.sinensis was probably had a backbone of 1,4-linked Glcp and braning at C-6,while the structure of cultured polysaccharide was highly complex,and 1,4-glucose and 1,4-galactose was supposed to exist in the main chain.For in vitro antioxidant assays,all the four polysaccharides showed similar scavenging capacity against DPPH and hydroxyl radicals,and have certain ferric reducing ability,which was possibly related to a combination of multi-factors such as the phenolic compounds and amino acids that conjugated in polysaccharides.4.A water-soluble polysaccharide(NCSP-50)was obtained from natural C.sinensis by hot water extraction and ethanol fractionation precipitation,with a molecular weight of 9.76 × 105 Da.The structure was analyzed by monosaccharide composition,methylation analysis,1D/2D NMR spectroscopy,enzymatic hydrolysis coupled with MALDI-TOF mass spectrometry.Results showed that NCSP-50 was an ?-(1 ? 4)-linked glucan,and branched at C-6.The repeating unit of this polysaccharide was proposed as follows:Unlike starch,another kind of ?-(1?4)-glucan,NCSP-50 showed potent immunostimulatory activity on promoting macrophage proliferation,enhancing NO production,as well as cytokines(IL-1? and TNF-?)secretion.5.A low-molecular weight polysaccharide(ANCSP-S50)with a molecular weight of 7207 Da was isolated and purified from the alkali extraction of natural C.sinensis.The fine structure of ANCSP-S50 was elucidated based on partial acid hydrolysis,monosaccharide composition,methylation analysis and 1D/2D nuclear magnetic resonance(NMR)spectroscopy.Results showed that ANCSP-S50 was a novel ?-(1?6)-linked galactomannan,mainly consisted of a mannan skeleton and galactofuranosyl chains.The structure of galactofuranosyl part was formed by alternating ?-(1?5)-Galf and ?-(1?6)-Galf or a single ?-(1?6)-Galf,attaching to the O-2 and O-4 of the mannose chain,and terminated at ?-T-Galf.The mannan core was revealed by analyzing the partial acid hydrolysate of the galactomannan and the structure was composed of ?-(1?6)-linked mannopyranosyl backbone,with substitution at O-2 by short chains of 2-substituted mannopyranoses or galactofunanosyl branches.The propsed structure was showed as follows:6.The high-molecular weight polysaccharide(ANCSP-50)from natural C.sinensis by alkali extraction was characterizated by methylation analysis and 1D/2D NMR spectroscopy.Results showed that ANCSP-50 was a ?-(1?3)-glucan,with branching point at C-6.The molecular weight was 4.77 × 105 Da and the instrinic viscosity [?] was 2.8 dL/g.The proposed structure was as follows:Then Dynamic light scattering(DLS)and static light scattering(SLS)were conducted to analyze the solution properties of ANCSP-50 in different solvents(0.5 M NaOH and DMSO).Results showed that ANCSP-50 in DMSO had the tendency to aggregates,while 0.5 M Na OH could eliminate the aggregates effectively.SLS analysis revealed that the parameters of ANCSP-50 in 0.5 M Na OH was Mw 424 kDa,Rg 17.3 nm and A2 was 5.0 × 10-3 cm3 mol/g2,but in DMSO the respective parameter was 2200 k Da,56.7 nm and 1.79 ×10-5 cm3 mol/g2.DLS results revealed that Rh was 27.9 nm in 0.5 M Na OH solution,revealing that ANCSP-50 had a highly branched chain conformation in that solvent.7.A novel polysaccharide fraction(CSP2-F0.05)was isolated and purified from the mycelium of cultured C.sinensis by Sephadex G-200 gel permeation chromatography and DEAE-52 cellulose chromatography.The structure of the polysaccharide was investigated by monosaccharide composition,partial acid hydrolysis,methylation analysis and 1D/2D NMR spectroscopy.The results showed that CSP2-F0.05 was mainly composed of mannose,glucose and galactose,and had a highly complex structure features.Several structural fragments were found in the polysaccharide,which was proposed as follows:In conclusion,this study fully compared the chemical composition,bioactive component and in vitro antioxidant activity between natural and cultured C.sinensis.Besides,the chemical structure of different polysaccharide fractions was characterized.Results showed that the chemical consistutents between natural and cultured C.sinensis was obviously different,especially the contents of polysaccharides,mannitol,adenine,adenosine,etc.The polysaccharides from natural C.sinensis were ?-(1?4)-glucan,?-(1?3)-glucan and ?-(1?6)-linked galactomannan.However,the structure of polysaccharides from cultured C.sinensis was very different,which was heteopolysaccharides mainly consisted of galactose,glucose and mannose.