Structural Elucidation And Lipid-lowering Activity Of The Alkali-extracted Polysaccharide From Cordyceps Militaris

Cordyceps milila,ris polysaccharide is one of the major bioactive components of the fruiting body of C.mililaris and has the effect of lowering lipids.However,the structural characteristics and the underlying mechanisms of action of these polysaccharides are not yet clear.In this study,one alkaline extracted polysaccharide,named as CM3-SII,was obtained from the fruiting bodies of C.militaris,and its structural characteristics were analyzed by methylation and NMR analysis.Of importance,the lipid-lowering effect and underlying mechanisms of CM3-SⅡ were investigated in humanized LDLR（+/-）;hamsters.In this study,the fruiting body powder of C.militaris was defatted with 95%ethanol twice,and then extracted with water for three times.Next,the residues were further extracted with 0.5 M NaOH to obtain the crude alkali-extracted polysaccharide.The crude polysaccharide was separated and purified by AKTA pure 150 fast protein lipid chromatography combined with a Q-SepharoseTM Fast Flow anion exchange column and then by a Sephacryl S-200 HR sizeexclusion column to obtain the polysaccharide designated as CM3-SⅡ.Chemical analysis showed that the sugar content was 93.3%,and the uronic acid was undetectable in CM3-SⅡ.The weight-average molecular weight of CM3-SⅡ was determined to be 25.2 kDa by high performance gel permeation chromatography（HPGPC）.High performance liquid chromatography（HPLC）showed that the monosaccharide composition of CM3-SⅡ was mannose,glucose and galactose（molar ratio 10.6:1.0:3.7）,and the content of mannose was up to 67.5%.Fourier transform infrared spectrum（FT-IR）analysis showed that CM3-SⅡ had characteristic absorption peaks of polysaccharide.After a complete methylation,the derivatives were subjected to GC-MS analysis.The methylation data suggested that polysaccharide CM3SⅡ was composed of[Manp(1→],[Glcp(1→],[Galf（1→],[Galp（1→],[→2）Galf（1→],[→4）Glcp（1→],[→4）Manp（1→],[→6）Manp（1→],[→4,6）Manp（1→],[→4,6）Glcp（1→],and[→2,6）Manp（1→]glycosidic linkages.1D and 2D-NMR analysis were performed using a JEOLJNM-ECP600MHz NMR spectrometer.The results showed that the backbone of CM3SⅡ was consists of[→4）-β-D-Manp（1→],[→6）-β-D-Manp（1→],and[→6）-α-D-Manp（1→]glycosyls and branching at the O-4 position of[→4,6）-（3-D-Manp（1→].The physiochemical results showed that the particle size distribution of CM3-SⅡ was 60.7,487.3 and 5416 nm,and the Zeta potential value was-15.6 mV.The thermal stability of CM3-SⅡ was analyzed by thermogravimetry and differential scanning calorimetry,and the results showed that CM3-SⅡhad a good heat resistant ability.The microstructures of CM3-SⅡ was analyzed by atomic force microscope and scanning electron microscope、and the results showed that CM3-SⅡ had a sheet-like structure.The expression of lipid metabolism-related proteins was detected by reverse transcription-polymerase chain reaction（RT-PCR）and Western blotting analysis to explore the lipid-lowering mechanism of CM3-SⅡ.Compared with the model group,the CM3-H group could significantly reduce the TG in plasma by 18.5%;the CM3-L and CM3-H groups decreased the TC level by about 10.6%and 23.2%,respectively.In plasma,CM3-SII upregulated the expression of Apolipoprotein A1（ApoA1）（P<0.05）,but had no effect on the expression of ApoB.In liver,CM3-SII significantly up-regulated the expression of liver X receptor（LXR）α mRNA（P<0.01）;up-regulated the protein expression levels of ATP-binding cassette transporter（ABC）G8 and peroxisome proliferators-activated receptor（PPAR）α（P<0.05）,down-regulated the expression Niemann-pick Cl-Like 1（NPC1L1）and sterolregulatory element binding protein（SREBP）1C protein levels,and the expression levels of SREBP2 and SREBP-1C mRNA（P<0.05）.However,the expression of low-density lipoprotein receptor（LDLR）,scavenger receptor class B type 1（SR-B1）,ABCG5,and cholesterol 7αhydroxylase（CYP7A1）was not affected.In summary,CM3-SII reduces the levels of TC and TG in the plasma of LDLR（+/-）hamsters.The lipid-lowering mechanisms are:1）CM3-SⅡ up-regulates the expression of ApoA1 in plasma and improves the LXRα/ABCG8 pathway in the liver,suggesting it may promote reverse cholesterol transport,thereby reducing TC level in circulation;2）CM3-SⅡreduces cholesterol synthesis and reabsorption by downregulating the expression of SREBP2 and NPC1L1,respectively;3）CM3-SⅡ inhibits the synthesis of TG and promotes fatty acid oxidation by regulating the expression of SREBP-1C and PPARα,respectively,leading to a reduction of TG level.This study demonstrated that the alkali-extracted polysaccharide CM3SⅡ of C.militaris has a good lipid-lowering effect in LDLR（+/-）hamsters fed a high-fat diet,suggesting CM3-SⅡ has a potential application in lipid-lowering as a food additive.