Studies On The Triterpenes From Ganoderma Lucidum And Their CYPs Inhibitory Activities

Objective:The triterpenoids were isolated and identified from the traditional Chinese medicine Ganoderma lucidum,and the CYP enzyme inhibitory activityof the triterpenoids compounds was evaluated,which will reveal the main active components of ganoderma lucidum inhibiting CYP enzyme,and provide reference for the safe and reasonable usage of Ganoderma lucidum.Methods:1)In this study,the Chinese medicinal Ganoderma lucidum was extracted with 95%ethanol under reflux for the further preparation of the dichloromethane extract,and its inhibition on CYP enzyme was evaluated by in vitro and in vivo methods.2)The extract of dichloromethane from Ganoderma lucidum was isolated and purified by silica gel column chromatography,medium pressure column chromatography and pre-HPLC chromatography.The structures of the isolated compounds were identified by the analysis of spectroscopic data,including ¹H NMR,¹³C NMR,2D NMR,MS and X-ray single crystal diffraction.Then,the triterpenoid library of Ganoderma lucidum was prepared by combining the existing compounds in our laboratory.3)The inhibitory effect of triterpenoids on CYPs was investigated by means of in vitro incubation.4)The metabolic inhibition effect of triterpenoids on related clinical drugs was evaluated by co-incubation in vitro.Results:In vitro evaluation of CYP enzymes inhibition showed that the dichloromethane extract of Ganoderma lucidum had significant inhibitory effects on CYP1A2,2B6,3A4 and 2C9,along with little difference in species.In vivo results showed that dichloromethane extract of Ganoderma lucidum inhibited the metabolism of phenacetin,bupropion,midazolam and diclofenac in Rats.In this study,thirty-seven compounds were isolated from Ganodia lucidum,and their chemical structures were identified by 1D NMR,2D NMR,HRESIMSR spectroscopic techniques,combined with CD and X-ray single crystal diffraction.Compounds 1-37 were all lanostane triterpenoids,Compounds 36 and 37 were oleanane triterpenoids,including 14 new compounds（1-7、18-21、23-25）and 23 known compounds（8-17、22、26-37）.The new triterpenoids were determined to be ganoderica A（1）,ganoderica B（2）,ganoderica C（3）,ganoderica D（4）,ganoderica E（5）,ganoderica F（6）,12β-acetoxy-3β,7β-dihydroxy-11,15,23-trioxo-5α-lanosta-8,20E-dien-26-oic acid（7）,ganoderione A（18）,ganoderione B（19）,ganoderione C（20）,ganoderione D（21）,ganodrol A（23）,ganodrol B（24）,ganodrol C（25）,The known triterpenoids were determined to be ganodrol D（8）,ganoderenic acid F（9）,ganoderic acid A（10）,ganoderenic acid A（11）,methyl ganoderenate D（12）,ganoderenic acid E（13）、12β-acetoxy-3,7,11,15,23-pentaoxo-lanost-8,20E-dien-26-oic acid.（14）、ganoderic acid C2（15）、ganoderenic acid C（16）、resinacein L（17）、lucidenic lactone（22）、5α-lanosta-7,9-（11）-dien-3β,24S,25R,26-tetraol（26）、ganolucidic acid D（27）、ganoderic acidγ（28）、ganolucidate F（29）、7-oxo-ganoderic acid Z（30）、lucidenic acid Q（31）、ganoderic acidε（32）、15α-acetoxyl ganorbiformin G（33）、ganodermic acid P1（34）、15-hydroxy-ganodericacid S（35）、3β-hydroxy-oleana-11,13（18）-dien-28-oic acid（36）、eupatoric acid（37）.Compounds 1-4,6 and 7 showed significant inhibitory effects on CYP1A2with IC₅₀ values of 10.3,16.03,13.96,11.6,8.61 and 12.8μM,respectively.Co mpound 25 inhibited CYP3A4,CYP2B6,CYP2C19,and CYP2C9 subtypes with IC₅₀ values of 4.78,25.28,15.40,and 12.57μM,respectively.Among them,co mpounds 6 and 25 both showed significant inhibitory effects on the metabolism of 16 related clinical drugs,such as riluzole and clozapine.Conclusion:We isolated and identified 37 triterpenoids from Ganoderma lucidum,including 14 novel compounds（1-7,18-21,23-25）and 23 known compounds（8-17,21,26-37）,and evaluated their inhibitory effects on CYP enzymes.The triterpenoids as the main bioactive compounds of Ganoderma lucidum displayed inhibitory effect on CYP enzymes,which provided important reference for the rational use of Ganoderma lucidum in clinical.