摘要
[目的]筛选灵芝中具有保护神经元活性的化合物,并探究其相应的作用机理,为研发具有预防和治疗阿尔茨海默病(AD)功效的灵芝功能性食品、保健品和药品提供科学依据.[方法]基于β-淀粉样蛋白(Aβ)诱导的氧化损伤建立细胞模型,对1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除法、铁离子还原/抗氧化能力检测法和2,2-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)自由基清除法初步评估得到的抗氧化活性较强的灵芝醇提物进行筛选,并对其神经元保护活性进行评价.进一步利用灵芝醇提物中与抗氧化有关的4种三萜类化合物,验证其活性并通过表面等离子共振技术探究其相关作用机理.[结果]DPPH、ABTS自由基清除及铁离子还原/抗氧化能力等体外理化初筛与细胞水平筛选试验结果表明,6种灵芝醇提物(108、161、106-2、171、173和109)抗氧化保护神经元活性较强,其中108、109和106-2样品对大鼠肾上腺髓质嗜铬瘤细胞(PC12细胞)氧化应激损伤的保护效果最好;灵芝醇提物可通过缓解细胞周期紊乱、抑制细胞早期凋亡、提高超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)含量,抑制丙二醛(MDA)含量上升来发挥神经元保护作用.灵芝醇提物108、106-2和109中4种三萜类化合物(灵芝醛A、灵芝酸B、灵芝酸C2和灵芝酸LM2)的平均含量占比相对较高.活性验证表明4种化合物对细胞氧化应激损伤均有明显修复效果,其中灵芝醛A与Aβ1～42具有较强的亲和力,其KD值为15.62 μmol/L,可通过与Aβ1～42竞争性结合来抑制Aβ1～42与RAGE结合.[结论]灵芝醇提物的抗氧化神经元活性通过灵芝酸C2、灵芝酸B、灵芝酸LM2和灵芝醛A 4种化合物的协同效应来实现.灵芝醛A能通过与RAGE竞争性结合来减轻或抑制Aβ1～42细胞毒性作用.灵芝具有开发防治AD功能性产品的潜力.
Abstract
[Objective]Screening for compounds in Ganoderma lucidum that had neuroprotective activity and explo-ring 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 antioxi-dant 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.lu-cidum alcohol extracts could exert neuroprotective effects by alleviating cell cycle disruption,inhibiting early cell apopto-sis,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.Ac-tivity validation demonstrated that these 4 compounds all exhibited obvious reparative effects against cellular oxidative stress damage.Among them,ganoderal A and Aβ1-42 showed strong affinity,with a KD value of 15.62 μmol/L,and could inhibit the binding of Aβ1-42 to RAGE through competitive binding with Aβ1-42.[Conclusion]The antioxidant neuronal ac-tivity 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 Aβ1-42 by competitively binding with RAGE.G.lucidum has the potential for developing into a functional product for the preven-tion and treatment of AD.
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