In vitro screening and mechanism study of Ganoderma lucidum compounds with protective activity against neuronal oxidative stress injury

【Objective】Screening for compounds in Ganoderma lucidum that had neuroprotective activity and exploring their corresponding mechanisms of action，to provide scientific basis for the research and development of functional foods，health products and drugs with the effects of preventing and treating Alzheimer disease（AD）.【Method】This study established a cellular model based on β-amyloid protein（Aβ）induced oxidative damage，and screened the antioxidant activity of G. lucidum alcohol extracts preliminarily assessed by the 1,1-diphenyl-2-trinitrophenylhydrazine（DPPH） free radical scavenging method，iron ion reduction/antioxidant capacity test，and 2,2-azinobis（3-ethylbenzothiazoline -6- sulfonic acid）ammonium salt（ABTS）free radical scavenging method，and evaluated their neuronal protective activity. Furthermore，the 4 triterpenoids compounds related to antioxidants in G. lucidum alcohol extracts were used to verify their activity and to explore their related mechanisms through surface plasma resonance technology.【Result】The results of in vitro physicochemical primary screening and cellular level screening experiments on DPPH and ABTS free radical scavenging，iron ion reduction/antioxidant capacity indicated that 6 G. lucidum alcohol extracts（108，161，106-2，171， 173 and 109）had strong antioxidant protective activity for neurons，among which samples 108，109 and 106-2 showed the best protective effects against oxidative stress damage in rat adrenal pheochromocytoma（PC12）cells. The G. lucidum alcohol extracts could exert neuroprotective effects by alleviating cell cycle disruption，inhibiting early cell apoptosis，enhancing superoxide dismutase（SOD）activity and glutathione（GSH）content，and suppressing the increase of malondialdehyde（MDA）content. The average contents of 4 triterpenoids（ganoderal A，ganoderic acid B，ganoderic acid C2，and ganoderic acid LM2）in G. lucidum alcohol extracts numbered 108，106-2 and 109 were relatively high. Activity validation demonstrated that these 4 compounds all exhibited obvious reparative effects against cellular oxidative stress damage. Among them，ganoderal A and Aβ_1-42 showed strong affinity，with a KD value of 15.62 μmol/L，and could inhibit the binding of Aβ_1-42 to RAGE through competitive binding with Aβ_1-42.【Conclusion】The antioxidant neuronal activity of G. lucidum ethanol extracts is achieved through the synergistic effects of 4 compounds：ganoderic acid C2， ganoderic acid B，ganoderic acid LM2 and ganoderal A. Ganoderal A can alleviate or inhibit the cytotoxic effects of Aβ_1-42 by competitively binding with RAGE. G. lucidum has the potential for developing into a functional product for the prevention and treatment of AD.