Separation Of Active Components From Ganoderma Atrum,Ganoderma Lucidum,and Ganoderma Amboinense By Active Guided Coupling Chromatography And Analysis Of Their Interaction With AChE

Alzheimer’s disease（AD）is a central nervous system degenerative disorder characterized by progressive cognitive impairment and behavioral disturbances.According to the World Alzheimer Report published by the Alzheimer’s Disease International（ADI）,there are currently at least 50 million dementia patients worldwide,and this number is projected to reach 152 million by 2050,with approximately 60%to 70%of them suffering from Alzheimer’s disease.The increasing trend of AD has become a significant health challenge,imposing enormous economic burdens on families and societies.Although the etiology of AD remains unclear,various hypotheses have been proposed around different causative factors.Among them,the cholinergic hypothesis has provided the earliest pharmacological basis for the drug development of AD.Ganoderma,a medicinal fungus belonging to the family Polyporaceae,has a long history of medicinal use in China.Ganoderma contains a variety of active ingredients,such as ganoderic acids and ergosterol,which have multiple pharmacological effects,including alleviating neuroinflammation,exerting antidepressant effects,reducing hippocampal neuron apoptosis,and alleviating brain physiological decline.It may achieve targeted therapy for Alzheimer’s disease by regulating key proteins and important pathways.However,due to the diverse species of Ganoderma and the complexity of its chemical components,especially in terms of the mechanism of action in neurodegenerative diseases,research is still unclear.Therefore,the screening,isolation,and study of the mechanism of action of active chemical components of Ganoderma,particularly their interaction with AChE,are of great significance.This paper mainly focuses on the screening,isolation,and mechanism of action of active chemical components with acetylcholinesterase inhibitory activity in Ganoderma atrum,Ganoderma lucidum,and Ganoderma amboinense.For the first time,a combination of affinity ultrafiltration mass spectrometry,molecular docking,molecular dynamics simulations,enzyme kinetics,and in vivo animal models were employed for the active screening methods.The obtained active chemical components were screened and isolated using online"seperation and detection"coupling technology.Furthermore,their multi-target and multi-level interaction mechanisms were explored at the molecular level.This study provides new research ideas for seeking acetylcholinesterase inhibitors in Ganoderma and thus developing potential drugs for the treatment of AD.The specific research work of this paper is as follows:Firstly,Ganoderma atrum was selected as the research subject,and active chemical components from Ganoderma atrum were isolated using activity-guided complex chromatography techniques,followed by studying their interactions with AChE.Experimental results revealed that four active components with strong affinity to AChE were screened from the Ganoderma atrum[BMIM]Br extract using affinity ultrafiltration mass spectrometry.Their structures were identified via high-resolution mass spectrometry as ergosterol,ergosterol-7,22-diene-3-one,ergosterol-7-en-3β-ol,and ergosterol-4,6,8（14）,22-tetraene-3-one.These four active components were isolated as monomers using a combination of continuous counter-current chromatography（ethyl acetate/methanol/water,4.0:1.0:7.0,v/v/v）and semi-preparative high-performance liquid chromatography（4%methanol,0-30 min;4-100%methanol,30-40 min）.Their purities were determined to be 92.23%,90.19%,90.52%,and 96.72%,respectively.Their IC₅₀values for acetylcholinesterase inhibition were found to be 79.89,67.91,29.79,and 37.61μmol/L,indicating that these four active components possess significant AChE inhibitory effects.This suggests the potential of Ganoderma atrum extract in preventing and treating neurodegenerative diseases.The research methodology adopted in this chapter,ranging from efficient extraction of raw materials,affinity ultrafiltration screening of active components,structural confirmation,to isolation and purification of active components,as well as evaluation of their AChE inhibitory activities,has rendered the systematic study of active components in Ganoderma atrum more scientifically efficient.Secondly,focusing on Ganoderma lucidum,a"computer-aided virtual screening combined with enzyme inhibition experiment-based efficient screening"strategy was developed,coupled with complex chromatography techniques to isolate active components.Experimental results demonstrated the successful screening of six active components from Ganoderma lucidum through enzyme inhibition experiments,molecular docking.These components were identified via high-resolution mass spectrometry as ganoderic acid I,C₂,G,B,A,and ganoderenic acid A.Subsequently,these six active components were isolated as monomers using complex chromatography techniques,with purities exceeding 90%.Further investigation into the mechanisms of action of these six active components on acetylcholinesterase revealed their significant inhibitory activity against AChE at different concentrations.Additionally,the AChE protein’s active sites and target molecules were found to interact strongly via hydrogen bonds,hydrophobic interactions,and salt bridges.The binding energies of ganoderic acid I,C₂,G,B,A,and ganoderenic acid A with AChE were determined to be-9.5,-8.4,-9.5,-8.5,-9.4 and-7.7 kcal/mol,respectively,confirming the consistency between experimental and virtual screening results.These findings illustrate that the strategy combining computer-aided virtual screening with enzyme inhibition experiments enables the analysis of active chemical components and their interactions with AChE in Ganoderma lucidum.This study provides a solid foundation for the further research and development of medicinal resources from Ganoderma species and offers valuable insights into the isolation and purification of bioactive substances using complex chromatography techniques.Finally,building upon the preliminary experimental research,this study continued to focus on Ganoderma amboinense and established an efficient screening strategy combining in vitro and in vivo approaches,along with the application of complex chromatography techniques to isolate active components.The study delved into the mechanism of triterpenoid chemical components from Ganoderma amboinense in inhibiting AChE and providing neuroprotection.An Alzheimer’s disease model in mice induced by D-galactose/Al Cl₃was established,and the efficacy of the treatment on AD model mice was evaluated using behavioral analysis,H&E pathological examination,immunohistochemistry,ELISA,and other in vivo experimental techniques.Combined with in vitro enzyme inhibition experiments and high-resolution mass spectrometry,twelve active chemical components were screened out,namely ganoderic acid I,C₂,G,B,K,A,ganoderenic acid A,deacetyl ganoderic acid F,lucidenic acid F,ganoderic acid D,ganoderic acid F,and ganoderic acid J.These twelve active components were then isolated as monomers using complex chromatography techniques,with purities exceeding 90%.Subsequent research into the multi-level AChE inhibition mechanism of these twelve active components revealed their significant AChE inhibitory activity.Compared to the positive control group,the high-dose Ganoderma amboinense group significantly reduced AChE activity and increased Ch AT levels,stabilizing and repairing cholinergic deficits.Additionally,it was found that the triterpenoid chemical components from Ganoderma amboinense exerted neuroprotective effects by protecting neuronal cells,reducing Aβprotein deposition,inhibiting glial cell stress expression,and mitigating hippocampal damage caused by neurotoxic substances.The results demonstrate that the combined application of in vitro and in vivo experiments can efficiently screen for active chemical components interacting with AChE in Ganoderma amboinense,providing a more precise basis for active screening and targeted isolation.In conclusion,this thesis established a rapid and high-throughput screening system combining in vitro and in vivo approaches,achieving targeted isolation and structural identification of bioactive substances.Moreover,a comprehensive evaluation system was constructed from different levels,including target proteins,computer simulations,and animal models,to evaluate the mechanism of action of active chemical components.This provides new research ideas,new methods and newscientific basis for the application of Ganoderma species in the treatment of Alzheimer’s disease.