Extraction And Purification Of Triterpenes From Ganoderma Lucidum And Effect On Lipid Metabolic In High-Fat-Diet Induced Wistar Rats

Lipid metabolism disorder is closely linked with obesity,hyperlipidemia,hyperglycemia,hypertension and fatty liver.With the improvement of people’s living standard and material abundance,Animal food of rich in fat intake increased for the past years,leading to the high incidence of some diseases associated with lipid metabolism disorders increases year by year.such as diabetes,hyperlipidemia,atherosclerosis,coronary atherosclerosis Heart disease and hypertension and other diseases.The present hypolipidemic used to regulate lipid metabolism abnormalities is not ideal,because they could cause liver or kidney damage,gastrointestinal reactions,organism and other side effects of drug resistance.Therefore,The nutritional health food and biological medicine of natural biological resources of high-efficiency for improving the lipid metabolism,Having become a focus of global attention and research.Ganoderma triterpenes(GP)compounds is class of important bioactive ingredients isolated from ganoderma lucidum,Numerous studies have shown that that GP could enhanced immunity,anti-aging,liver protection,anti-cancer,hypolipidemic,Hypoglycemic,etc.At present,the research on the biological activity of GP has made some achievements at home and abroad,but the research on the regulation of lipid metabolism and the mechanism of action of GP are still few.Therefore,In this paper,the main chemical constituents of Ganoderma applanatumn were analyzed.Simultaneously,optimization of ultrasonic assisted extraction process condition of GP and purification methods with macroporous adsorption resins,and its main components were identified by UPLC-TOF-MS.The effects of GP on lipid metabolism in wistar rats induced by high-fat diet were studied at the same time.The effects of GP on gut microbiota composition by performing a high-throughput sequencing-based analysis of bacterial 16S rRNA(V3-V4 region)in caecal faeces and cecum and tnd the liver tissue transcriptase sequencing analysis based on the Illumina high-throughput sequencing platform.The possible mechanism of lipid metabolism was established,which provided the theoretical basis for the application of GP in functional food and drugs of the regulation of lipid metabolism.The main results are as follows:(1)The compositions of total sugar,total triterpenes,protein,crude fat,crude fiber,amino acid,fatty acid and trace elements of Ganoderma lucidum were determined,providing basic data for further study of Ganoderma lucidum.(2)The optimal technological parameters of GP ultrasonic extraction were determined by single factor experiments and Box Behnken experimental design:G.applanatum were extracted with 70%ethanol at the solid-liquid ratio of 1:20(g/mL)for 60 min at 55℃.Under these conditions,the average extraction rate of GP was 1.75%.To explore the adsorption and desorption characteristic to total riterpenoids from Ganoderma applanatum with 5 types of macroporous resins:AB-8,PHD700,S-8,NKA-9,D101,the results showed that D101 macroporous resin performed the best adsorption and desorption properties to GP.Through the dynamic adsorption analytical curve experiment,it was found that the adsorption and analysis of total triterpenes of D101 resin were rapid equilibrium,time-saving and efficient,and it had good static adsorption and desorption properties.It was suitable for industrial enrichment and purification of GP.The purification proces of GP was optimized.the optimum adsorption conditions was:the sample volume 10BV,sample flow rate 1.5 BV/h,the sample liquid pH6;The best elution conditions was:elution solvent 80%ethanol,elution speed 2 BV/h,elution volume 6 BV.The content of GP in the extract was increased from 19.31%to 70.69%,which indicated that D101 macroporous adsorption resin had a better effect of refining on the total triterpenoid compounds.80%ethanol analyte was identified by UPLC-TOF-MS,and its main components were Ganoderma acid,consisting of Ganoderic Acid A,Ganoderic acid B,Ganoderic acid G,Ganoderic acid H and Ganoderic acid F.(3)The effects of different doses of GP on lipid metabolism were studied on Wistar rats induced by high-fat diet.The results showed that:the weight of Wistar rats fed with 8 weeks of high-fat diet was significantly higher than the control group(p<0.05)The weight growth of different doses of GP group(GP50,GP100,GP150)were significantly slower than model group(p<0.01),which indicating that the GP could effectively control the body weight of HFD rats.The biochemical indexes,antioxidant enzymes and free fatty acid content of blood and liver in experimental rats were analyzed.The results showed that different doses of the test samples had a certain improvement in the treatment effect on lipid metabolism disorders,hyperlipidemia and nonalcoholic fatty liver disease.In particular,the animal serum(liver)levels of TG,TC,ALT,AST and FFA in high-dose test sample group(GP150)was significantly lower than those in the model group(HFD),and(HDL-c)was higher than that in HFD.Antioxidant enzymes in liver tissue of experimental ratswere analyzed,and compared with the model group(HFD),the levels of MDA of silymarin(Sym group),GP50 group,GP100 group and GP150 group were significantly decreased respectively.(p<0.05),Compared with HFD group,GSH-PX and SOD in GP 150 group were significantly increased(p<0.01).(4)The changes of the faecal and intestinal contents flora in each group were analyzed based on the high-throughput sequencing technique of of 16S rDNA V3-V4 variable region,the results showed that Firmicutes-to-Bacteroidetes ratio of HFD was significantly higher than NFD,Firmicutes-to-Bacteroidetes ratio with gavage GP were lower compared with HFD.high-fat diet can lead to significant increased in the abundance of Coprococcus,Globicatella,Enterococcus and Desulfovibrio.On the other hand,decreased in the abundance of Bifidobacterium,Lactobacillus,Anaeroplasma and so on.The results showed that different doses of GP had a certain effect on the balance of intestinal microecology in high fat diet rats.microorganisms related with lipid metabolism was Found out by the correlation analysis of ntestinal flora structure with blood biochemical and liver biochemical indicators,(5)The liver tissue of each experimental group was subjected to transcriptome sequencing analysis based on Illumina high-throughput sequencing platform,and the differentially expressed genes were analyzed by GO enrichment and KEGG enrichment.Compared with the model group,it was found that the differentially expressed genes were mainly enriched in PPAR signaling pathway,Biosynthesis of amino acids,Non-alcoholic fatty liver disease,Type Ⅱ diabetes mellitus and AMPK signaling pathway.Compared with the model group,the differentially expressed genes closely related to lipid metabolism mainly include Gk2,MEF,Scd1,IRS1IRM,SREBP-1c,PKL,Jun,IRS1IRM,Tkfc,PKL,Gadd45,enoyl-CaA hydratase,Hadhsc,Cyp2c70,Acaa2,Cyp4all,RT1-A,Ins1,HRSL3,Phlpb,Fadsd6,Muscpho,Xbp1,CYP7A1,PPARa,FAS and pe-CoA.The expression of genes related to lipid metabolism in the liver of the experimental rats was analyzed by RT-qPCR.The possible mechanism of the regulation with lipid metabolism in Ganoderma lucidum was elucidated.