Studies On Bioactive Polysaccharides Isolated From Gastrodia Elata Blume And Morchella Esculenta (L.) Pers. Grown In Yunnan

Two polysaccharides were isolated and purified from the fruiting body of Gastrodia elata Blume and wild Morchella esculenta(L.)Pers,edible-medicinal fungi grown in Yunnan.Then,anticaner ability,blood lipids reducing activity and congitive activity of two polysaccharides were investigated by high-throughput screening and animal experiments in vitro or in vivo.The following results were obtained.(1) The optimal parameters for extraction of two crude polysaccharides were achieved.Highest recovery(9.76%db) of crude polysaccharide from Gastrodia elata Blume could be obtained with liquid-material ratio 10:1,50℃,3 h and precipitating alcohol concentration 80%.When the pruiting body of Morchella esculenta(L.)Pers. extracted with liquid-material ratio 50:1,85℃for 2 h,and precipitated with 80%,the highest recovery of crude polysaccharide(4.96%,db) was gained.(2) Two HPLC proved purified polysaccharides,PGEB-3-H from Gastrodia elata Blume and PMEP-1 from Morchella esculenta(L.)Pers.,were obtained from two crude polysaccharides successicvely purified by Sevag deprotein,dialysis,DEAE-52 column and Sephadex G-100 column.The recovery of two purified polysaccharides were 0.824%and 1.58%respectively.The molecular weight of PGEB-3-H and PMEP-1 were determined with column chromography as 28800 and 43600 respectively. Physico-chemical characteristics analysis indicated that two polysaccharides do not contain monosaccharose,uronic acid,protein and polyphenols.Therefore,it can be concluded that they are non-starch neutral pure polysaccharides. (3) Using chemical methods(methylation reaction,periodate oxidation,Smith degradation) and instrument analysis(IR,GC-MS,¹H-NMR and ¹³C-NMR),the structural characterics of PGEB-3-H and PMEP-1 were investigated.It was found that PGEB-3-H was mainly composed by glucose,and it had aα-1,4-linked glucan main chain occasionally branched withα-1,6 glycosidic linkage PMEP-1 was identified as a high-ramified heteropolysaccharide,whose main chain was a mixing linkage ofα-1,4-glucose,α-1,6-galactose,α-1,2-mannose andα-1,5-arabose,and its side chain attached atα-1,2,6-mannose,α-1,4,6-glucose andα-1,2,6-galactose.(4) The possible bologival activities of PGEB-3-H and PMEP-1 were tested with blood lipids matablism and obesity related PPARs and cancer related A549 models by high-throughput screening(HTS).The result showed PGEB-3-H and PMEP-1 had slight activation effects on PPARδmodel but they are not significantly differed from control ones.No effects were obsereved of testing polysaccharides on PPARαmodel. Then,it can be concluded on the basis of this experiment that the tested two polysaccharides exerted a slight bioacticity,which were evry low as compared to positive controls,2-Bro and WY14643.The high-throughput screening proved that neither PGEB-3-H nor PMEP-1 has obvious inhibitory effect on tumour cell A549 in vitro.(5) The effet of PGEB-3-H and PMEP-1 on reducing serum blood lipids of rats was studied,and the results indicated that the high-fat diet useded in this experiment could successfully induced formation of hyperlipemia(increase in TC,TG and LDL-C, decrease in HDL-C) in rats,and the model of rat was properly bulited.Low,medium and high dose of PGEB-3-H and PMEP-1 could significantly(P＜0.05) reduce TG in serum of rats.The higher the dose,the stronger function in reducing blood lipids was founded.It was very interesting that serum TG level was maintained at normal level when rats fed with high-fat diet as well as medium or high dose of tested polysaccharides.However,for TC in serum,marked reduction,but still higher than normal level,was only observed with low dose of PGEB-3-H when compared to high-fat diet control.Low or medium dose of PMEP-1 could significantly(P＜0.05) reduce LDL-C when compared to high-fat diet control.However,medium dose of PGEB-3-H could significantly(P＜0.05) increase total cholesterin HDL-C to normal level when compared to high-fat diet control.Although,no significant difference was observed between PMEP-1 groups and high-fat diet control in HDL-C,the level of HDL-C in PMEP-1 groups incereased to a higher level which caoul not be significantly differed from normal control.So PGEB-3-H and PMEP-1 could significantly reduce the blood lipids(or inhibit the formation of hyperlipemia) of rats,and they have stronger effecs on TG than TC.(6)The congitive activity of PGEB-3-H was conducted in this paper.Morris water maze showed that scopolamine treated mice need loner time to corss maze than noral and positive control.PGEB-3-H had a positive effect scopolamine treated mice to corss maze and high dose of PGEB-3-H could significantly shorten the crossing time.At the same time,it was founded that high dose of PGEB-3-H could significantly could significantly increase(P＜0.05) acetylcholine(Ach) content in mice cerebra,which even higher than that of positive control(20 g·kg^-1·d^-1 Gastrodia elata Blume powder).So,it can be concluded that PGEB-3-H could enhance congitive ability and its mechanism may be regarded as increasing Ach content.The creative aspects of this paper were summarized into five points as following. (1) In order to make basis for 3^rd generation healthy foods,two purified polysaccharides were firstly and successfully obtained from edible-medicinal fungi Gastrodia elata Blume and wild Morchella esculenta(L.)Pers.,and the physico-chemical characteristics were also systematically studied.(2) The primary structure of PGEB-3-H and PMEP-1 was researched firstly by chemical and instrument methods such as gas chromagraphy, IR,GS-MS and NMR,the structural character of PMEP-1 and the repeating unit of PGEB-3-H were firstly identified.On this basis,3D structure of PGEB-3-H was therotical modelled with MM2 programme.(3) High-throughput screening(HTS) technique was subjected for possible boiactivity of PGEB-3-H and PMEP-1.(4) A polysaccharide,PGEB-3-H,was firstly proved as a new substance member for congitive function of Gastrodia elata Blume besides gastrodine,which incoporated new infromation into the studies in congitive activity of Gastrodia elata Blume.(5) PGEB-3-H and PMEP-1 was approved as hepful drugs to act against the formation of hyperlipidemia,especially TG in serum,when a high fat diet was consumed.In this paper,the research methods are normative and reliable,and the conclusion can be effectively used for production of the 3^rd generation healthy foods.