Microwave Synthesis Cordyceps Megalosaccharide And Its Inhibition On High-fat Diet Induced Obesity

Cordyceps Megalosaccharide(CM) has good physiological function. Microwave has been widely used in organic synthesis due to its rapid, high efficient, lower energy consumption, and environment friendly features. Therefore, we used heteropolyacid as catalyst under the condition of microwave irradiation to rapidly and high efficiently synthesize CM. Then we studied the structure of megalosaccharides synthesized by microwave irradiation, and inferred the formation of glycosidic bond in the chemical reaction. Furthermore, we studied the effect of CM on gut microbiota and its inhibition of obesity on long-term high-fat diet fed mice. The main results of this study are as follow: 1. Preparation of Cordyceps Megalosaccahrides and its structure analysisPolydextrose(PDX) was synthesized using glucose and sorbitol(molar ratio 8.9:1) as substrate, and water as initiator, heteropolyacid as catalyst, under the condition of microwave irradiation. The optimum conditions were reaction temperature 120 oC, microwave irradiation time 2.5 min, the amount of initiator and catalyst were 12% and 1.0%(v/w), respectively. The actual maximum yield was 92.31%. The average DP was 13. The results of methylation analysis and NMR spectra assignments indicated that the structure of PDX had a branched backbone composed of T-b-D-Glcp, →2)-b-D-Glcp, →6)-a-D-Glcp, →4)-a-Glcp, and T-a-D-Glcp, the backbone were branched at the O-2, O-4, O-6 position.Poly-Galactose(PGal) was synthesized using galactose as substrate, sodium chloride solution as initiator, and heteropolyacid as catalyst, under the condition of microwave irradiation. The optimum conditions were reaction temperature 130 oC, microwave irradiation time 4.5 min, initiator concentration 0.25 mol/L, and catalyst 1.1%(v/w). The actual maximum yield was 97.22%. The average DP was 17. The structure of PGal had a branched backbone composed of →6)-b-D-Galp,→4)-b-D-Galp,→4)-a-D-Galp,→2,4)-a-D-Galp, and T-a-D-Galp,the backbone of PGal were branched at the O-2, O-4, O-6 position.Poly-Mannose(PMan) was synthesized under the condition of reaction temperature 115 oC, microwave irradiation time 4.5 min, and catalyst concentration 2.5 mol/L. The actual maximum yield was 89.42%. The average DP was 15. The structure of PMan had a branched backbone composed of →3)-b-D-Manp, →6)-a-D-Glcp, and →3,6)-a-Glcp,and PMan backbone were branched at the O-3, O-6 position. Using mannose as substract we synthesized PMan with the DP of 15, 27 and 36, respectively.Cordyceps Megalosaccharide(CM) was synthesized using glucose, galactose, and mannose as substrate, heteropolyacid as catalyst, under the condition of microwave irradiation. The optimum conditions were reaction temperature 120 oC, microwave irradiation time 3.5 min, the amount of catalyst 1.2%(v/w). The actual maximum yield was 87.79%. The average DP was 14. The structure of CM had a branched backbone composed of →4)-b-D-Galp,→2,6)-b-D-Galp,→,2)-a-D-Galp,T-b-D-Glcp,T-a-D-Glcp,→6)-a-D-Glcp,→3)-b-D-Manp, and T-a-D-Manp,the main chain were branched at the O-2, O-6 position. 2. Effect of Cordyceps Megalosaccharide on the gut microbiotaMegalosaccharides reduced the p H of fermentation liquid, and increase lactate acid and the amount of SCFA. The Terminal-restriction fragment length polymorphism(T-RFLP) analysis of gut microbiota in SHIME showed that megalosaccharides reduced the polymorphism of microbiota and promote advantage bacteria proliferation. RT-PCR studied showed that CM is better in proliferation of Bifidobacteria spp and Lactobacteria spp, and inhabitation of Enterobacteria spp compared with PDX, PGal, and PMan. Megalosaccharides fermentated by gut microbiota produced different amount of SCFA, CM produced much more SCFA than PGal, PMan and PDX.Fluorescence in situ hybridization(FISH) combined with flow cytometry(FCM) analysis the gut microbiota showed that megalosaccharides can significant increase the relative abundance of Bif164、Lab158、Bac303、Mib724, the effect of megalosaccharides on Bif164 was CM>PMan>PDX>PGal, and Lab158 was PDX>CM>PMan>PGal. Megalosaccharides significantly increased the ratio of Firmcuties to Bacterodies in HFD fed mice. 3. Effect of Cordyceps Megalosaccharide on HFD induced obese miceIn this study, megalosaccharides significantly reduced body weight(BW), Lee obesity index, fat to BW, and liver to BW of mice fed with HFD. It significantly reduced blood LPS, TG, and LDL-C level coampared with HFD fed mice. PDX, PMan, and CM significantly reduced blood TC level of HFD fed mice. Megalosaccharides can modulate the redox state of mice fed HDF, decrease ROS and MDA level, and improve total antioxidant capacity(TAOC) and antioxidant enzyme secretion.The effect of megalosaccharides on gut functional study showed that megalosaccharides decreased ileum and colon ROS and MDA level, improved the T-AOC, and increased CAT and GSH/GSSG activity. CM has higher ability than PDX, PGal and PMan in decrease ROS and MDA level, and improves T-AOC. Additionaly, megalosaccharides significantly up regulated the Nrf2 gene expression in HFD fed mice.Studied on the inflammatory cytokines and its relative gene expression showed that megalosaccharides significantly decrease TNF-a level and increased IL-10 level in HFD fed mice. PMan and CM significantly decreased the colon IL-6 and TNF-a level. Megalosaccharides significantly down regulate colon IL-6 and TNF-a relative gene expression, and significantly up regulate IL-10 gene expression. Althought megalosaccharides down regulated IL-12 gene expression, only CM significantly down regulted IL-12 gene expression compared with HFD fed mice. Furthermore, megalosaccharides significantly improved the thickness of muscular and mucous layers in HFD fed mice.Gut tight junction(TJ) protein relative gene expression analysis showed that megalosaccharide significantly up regulated the gene expression of Claudin-1, JAM, and Zo-1. But only CM up regulated Occludin gene expression in HFD fed mice.Live fatty acid metabolism study showd that megalosaccharides significantly decreased hepar cell fatty infiltration, up regulated liver lipolysis gene Cyp7 al expression, and down regulated LPl and SCD-1 gene expression. In addition, megalosaccharides up reglulated antioxidant gene Nrf2 expression in liver compared with HFD fed mice.In summary, PDX 、 PGal 、 PMan and CM were synthesized under the condition of microwave-assisted and heteropolyacid catalyst. The megalosaccharides were anti-digested in stomach and small intestinal, and reached to the colon, then fermented by gut microbiota to produce lactate and SCFA, which reduced the colon p H. The four megalosaccharides proliferated Bifidobacteria spp and Lactobacteria spp, and inhibitd Enterobacteria spp. Furthermore, the metabolites of megalosaccharides could regulate the body redox states, decrease gut inflammatory, improve gut barrier function, and reduce blood LPS level and heper cell fatty infiltration. Accordingly, megalosacchardes could alleviate obesity which was induced by long-term HFD.