对神经元氧化应激损伤具有保护活性的灵芝化合物体外筛选及作用机理  

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

作　　者：马迎春 张劲松[2] 贾薇[2] 于子行 杨妍 刘艳芳[2] 张赫男[2] 唐传红[2] 张亚平[1] 汪雯翰[2] MA Ying-chun;ZHANG Jing-song;JIA Wei;YU Zi-hang;YANG Yan;LIU Yan-fang;ZHANG He-nan;TANG Chuan-hong;ZHANG Ya-ping;WANG Wen-han(College of Life Sciences,Shihezi University,Shihezi,Xinjiang 832003,China;Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences/Key Laboratory of Edible Fungal Resources and Utilization(South),Ministry of Agriculture and Rural Affairs/National Engineering Research Center of Edible Fungi/Key Laboratory of Agricultural Genetics and Breeding of Shanghai,Shanghai 201403,China;College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]石河子大学生命科学学院,新疆石河子832003 [2]上海市农业科学院食用菌研究所/农业农村部南方食用菌资源利用重点实验室/国家食用菌工程技术研究中心/上海市农业遗传育种重点实验室,上海201403 [3]上海海洋大学食品学院,上海201306

出　　处：《南方农业学报》2024年第7期1935-1948,共14页Journal of Southern Agriculture

基　　金：上海市现代农业产业技术体系项目(沪农科产字〔2023〕第9号);上海市农业科学院卓越团队建设计划项目(沪农科卓〔2022〕003)。

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Aβ_(1~42)具有较强的亲和力,其KD值为15.62μmol/L,可通过与Aβ_(1~42)竞争性结合来抑制Aβ_(1~42)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Aβ_(1~42)细胞毒性作用。灵芝具有开发防治AD功能性产品的潜力。【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 onβ-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

关 键 词：灵芝醇提物 淀粉样β蛋白肽 灵芝三萜 自由基 阿尔茨海默病 

分 类 号：S567.310.99[农业科学—中草药栽培]