Studies On The Triterpenoid Active Constituents Of Medicinal Fungi Ganoderma Lucidum Karst And Ganoderma Tsugae Murr.

In this paper,guided by the anti-gout active components of Ganoderma lucidum·karst and the anti-Alzheimer disease active components of Ganoderma tsugae Murr.,xanthine oxidase inhibitors and acetylcholinesterase inhibitors in Ganoderma lucidum·karst and Ganoderma tsugae Murr.were screened and separated.To clarify the inhibitory mechanism of anti-gout active components of Ganoderma lucidum·karst and anti-Alzheimer’s disease components of Ganoderma tsugae Murr..To provide theoretical and practical basis for resource development and related research of medicinal fungi Ganoderma lucidum·karst and Ganoderma tsugae Murr..The specific research work is as follows:The ultrasonic extraction process of triterpenoid active components from Ganoderma lucidum·karst was optimized by simulated annealing algorithm combined with response surface test design,and the ultrasonic extraction process of triterpenoid active components from Ganoderma tsugae Murr.was optimized based on genetic algorithm and response surface test design.Further analysis and identification of triterpenoid active components by chromatography-mass spectrometry.The results showed that the optimum extraction parameters of triterpenoids from Ganoderma lucidum·karst were as follows:the ratio of material to liquid 1:30 g/m L,extraction time1.0 h,ethanol concentration 65%,ultrasonic times 2 times,the extraction rate of triterpenes was（1.05±0.05）%.The optimum extraction parameters of triterpenoid active components of Ganoderma tsugae Murr.were as follows:material-liquid ratio 1:33g/m L,extraction time 1.0 h,ethanol concentration 88%,ultrasonic times 4 times,the extraction rate of triterpenes was（1.21±0.02）%.Using xanthine oxidase and acetylcholinesterase as targets,ultrafiltration mass spectrometry and molecular docking techniques were used to quickly screen xanthine oxidase inhibitors in Ganoderma lucidum·karst and acetylcholinesterase inhibitors in Ganoderma tsugae Murr..The results showed that four potential xanthine oxidase inhibitors were screened from Ganoderma lucidum·karst.They were Ganoderic acid C₂,Ganoderic acid B,Ganoderic acid A and Ganoderic acid D₂.The docking binding energies of xanthine oxidase to xanthine oxidase were-6.29 kcal/mol,-6.65 kcal/mol,-4.29 kcal/mol and-5.96 kcal/mol,respectively,which were consistent with the results of ultrafiltration.Molecular docking proved that the inhibitor had a good binding effect with xanthine oxidase.Three potential acetylcholinesterase inhibitors were screened from Ganoderma tsugae Murr.,namely Erythrone A,Ganoderic acid A and Erythritic acid A.The docking binding energies of them to acetylcholinesterase were-8.86kcal/mol,-7.5 kcal/mol and-9.21 kcal/mol,respectively,which were consistent with the results of ultrafiltration experiment.Based on the triterpenoid active components selected in the previous screening,the anti-gout active components of Ganoderma lucidum·karst and the anti-Alzheimer disease active components of Ganoderma tsugae Murr.were separated and purified by high speed countercurrent chromatography and semi-preparative liquid chromatography.The triterpenoid active components were analyzed and identified by chromatography-mass spectrometry.The results showed that four triterpenoids with purity of more than 90%were isolated from Ganoderma lucidum·karst,which were Ganoderic acid C₂,Ganoderic acid B,Ganoderic acid A and Ganoderic acid D₂,respectively.Three triterpenoid active components with purity of more than 90%were isolated from Ganoderma tsugae Murr..They are Erythrone A,Ganoderic acid A and Erythritic acid A.The in vitro activity of triterpenoids in Ganoderma lucidum·karst and Ganoderma tsugae Murr.were evaluated by enzymatic reaction kinetics.The results showed that Ganoderic acid C₂,Ganoderic acid B,Ganoderic acid A and Ganoderic acid D₂ from Ganoderma lucidum·karst showed reversible inhibition on xanthine oxidase.The inhibition rates were 59.43%,63.91%,55.69%and 56.71%,respectively.IC₅₀ was34.05,27.94,46.83 and 40.09μg/m L,respectively.Ganoderic acid A,Ganoderic acid C₂ and Ganoderic acid D₂ were competitive inhibitors.Ganoderic acid B is a non-competitive inhibitor.The triterpenoid active components of Ganoderma tsugae Murr.,Erythrone A,Ganoderic acid A and Erythritic acid A showed reversible inhibition on acetylcholinesterase,and their inhibition rates were 68.55%,66.54%and 69.12%,respectively.IC₅₀ was 15.49、22.79和10.44μg/m L,respectively.Ganoderic acid A is a competitive inhibitor,and Erythrone A and Erythritic acid A are non-competitive inhibitors.The network pharmacology was used to clarify the mechanism of anti-gout active components of Ganoderma lucidum·karst and anti-Alzheimer’s disease active components of Ganoderma tsugae Murr..The results showed that there were 184targets related to active components and 1780 targets related to gout in Ganoderma lucidum·karst.71 common targets were obtained by screening the intersection of the two targets.From the pathway enrichment analysis,it is known that the main metabolic pathways are involved in lipid and atherosclerosis,prostate cancer,cancer and other pathways.It is speculated that triterpenes in Ganoderma lucidum·karst treat gout through the above metabolic pathways.There are 189 targets related to active components in Ganoderma tsugae Murr.and 10236 targets related to Alzheimer’s disease.167 common targets were obtained by screening the intersection of the two targets.The pathway enrichment analysis showed that the main metabolic pathways were involved in TNF signal pathway,IL-17 signal pathway,lipid and atherosclerosis and other pathways.It is speculated that triterpenoids in Ganoderma tsugae Murr.treat Alzheimer’s disease through the above metabolic pathways.In this paper,the triterpenoid active components of Ganoderma lucidum·karst and Ganoderma tsugae Murr.were studied by mathematical model extraction process optimization,analysis and identification,activity screening,separation and purification,activity evaluation and action mechanism of medicinal fungi Ganoderma lucidum·karst and Ganoderma lucidum·karst.To provide theoretical and practical basis for the development of characteristic resource medicinal fungi Ganoderma lucidum·karst and Ganoderma tsugae Murr..