Study Of Digging The Active Factors Of Ganoderma Lucidum Against Alzheimer’s Disease And Spectrum-effect Relationship

Alzheimer’s disease（AD）has become an important disease affecting the health of the Chinese people as the proportion of the aging population is increasing.Currently,some drugs have been developed to treat AD,but these drugs have shown certain side effects.According to the characteristics of AD,we should not only strengthen the development of therapeutic drugs,but also develop functional products that can prevent and delay the development of the disease.At present,there are several effective anti-AD drugs originated from medicinal fungi,which have few side effects and are suitable for long-term use.Ganoderma lucidum is one of the most widely studied medicinal fungi.Some studies have found that Ganoderma lucidum is a valuable resource for further developing AD drugs.Ganoderma lucidum triterpenoids are considered as one of the main active components with the function of preventing AD.Because the triterpenoids in Ganoderma lucidum are very complex,it is difficult to quickly find the active triterpenoids from the complex mixture of Ganoderma lucidum by the traditional separation and purification method combined with activity tracking method.This study intended to find triterpenoids that has ACh E inhibitory activity and antioxidant activity in Ganoderma lucidum by introducing the Genetic Algorithms combined with Partial Least Squares Regression Analysis,the spectrum-effect relationship of ACh E inhibitory activity and antioxidant activity of Ganoderma lucidum triterpenoids was established,and a method for rapid locking of active components in complex mixtures was established.Finally,ACh E was used to capture the active triterpenoids from Ganoderma lucidum,and the interaction and binding sites between the triterpenoids and ACh E were determined by molecular docking,so as to verify the accuracy of the equation of spectrum-effect relationship established.The results are as follows:（1）Optimization of screening model for ACh E inhibitory activityIn this experiment,a screening model for ACh E inhibitory activity based on Amplex red regent fluorescence was established,in which concentration of ACh E was determined to be 25 m U/m L,reaction time was 40min,concentration of substrate was100μM,concentration of sample solvent was 2.5%DMSO,concentration of Amplex Red regent was 400μM,buffer was tris-HCl solution of PH=8.0.The IC₅₀value of the positive drug physostigmine was estimated to be 409 nmol/L by the fixed screening model,and the technical parameters under the corresponding conditions were calculated.The results of technical parameter evaluation and positive drug verification based on the model showed that the screening model is of high accuracy.（2）Study on the spectrum-effect relationship of ACh E inhibitory activity of Ganoderma lucidum fruiting body triterpenoids based on Genetic Algorithms combined with Partial least squares regression analysisIn this study,Genetic Algorithms was firstly used to screen the characteristic peaks of fingerprint of Ganoderma lucidum triterpenoid,and Partial least squares regression analysis was used to analyze the correlation between ACh E inhibitory activity of Ganoderma lucidum alcohol extract and its selected characteristic peaks of triterpenoid.Among the 25 triterpenoid characteristic peaks selected from fingerprint of Ganoderma lucidum triterpenoid,the peaks S21,S23,S24,S25 and S29 were significantly positively correlated with ACh E inhibitory activity.Peaks S2,S3,S6 and S28 were negatively correlated with ACh E inhibitory activity.Triterpenoids corresponding to peaks were identified by comparing with triterpenoid standards,ACh E inhibitory activity of identified triterpenoid compounds were determined respectively,it was found that the ACh E inhibitory activity of Ganodermanontriol（peak S21）,Ganoderic acid DM（peak 23）,Ganederol F（peak S24）,Ganodermanondiol（peak S25）and Ganederol B（peak S29）was significantly higher than that of other compounds,which was in line with the result predicted by the spectrum-effect relationship equation.（3）Ultrafiltration affinity combined with liquid chromatography and molecular docking technology to screen triterpenoid with ACh E inhibitory activity in Ganoderma lucidumFour potential ACh E inhibitors were identified from Ganoderma lucidum fruiting body alcohol extract by Ultrafiltration affinity combined with liquid chromatography,and identified as Ganodermanontriol,Ganederol F,Ganodermanondiol and Ganederol B.This study estimated ACh E inhibitory activity of these four compounds,the binding sites and docking scores of the four ligands and ACh E were analyzed by the molecular docking technology.The results verified the accuracy of established spectrum-effect relationship equation.（4）Study on the spectrum-effect relationship of antioxidant activity of Ganoderma lucidum fruiting body triterpenoids based on Genetic Algorithms combined with Partial least squares regression analysisIn this study,the spectrum-effect equation of antioxidant activity of Ganoderma lucidum triterpenoids was established based on the Genetic Algorithms-Partial least squares regression analysis method.Among the 26 characteristic peaks selected from the fingerprint of Ganoderma lucidum triterpenoid,the peaks S1,S5,S9,S10,S13and S32 were significantly positively correlated with activity.S17,S22,S25 and S31were negatively correlated with activity.The scavenging DPPH free radical activity of the identified triterpenoids were determined respectively,and it was found that the scavenging activity of the Ganoderic acid C2（S5）,Ganoderic acid B（S9）,Ganoderic acid LM2（S10）and Ganoderal A（S32）with significantly positive correlation were significantly higher than those of other compounds,and the scavenging DPPH free radical activity of the Ganodermanondiol（S25）with significantly negative correlation were close to 0%.In addition,H₂O₂ damage PC12 cell model was used to further verify the antioxidant activity of Ganoderic acid C2,Ganoderic acid B,Ganoderic acid LM2,Ganodermanondiol in cell level,found that Ganoderic acid C2,Ganoderic acid B,ganoderic acid LM2,Ganoderal A has obvious protective activitiy against oxidative damage of nerve cells.Ganodermanondiol has no activity to protect nerve cells from oxidative damage.The results show that the spectrum-effect equation of antioxidant activity of Ganoderma lucidum triterpenoids has high accuracy.