“新冠三方”的体外抗氧化作用比较

Comparing the Anti-oxidative Effects of "Three Decoctions for COVID-19"

关键词:  清肺排毒汤  化湿败毒方  宣肺败毒方  氧化应激  新型冠状病毒感染

Key words:  Qingfei Paidu decoction  Huashi Baidu decoction  Xuanfei Baidu decoction  oxidative stress  COVID-19

摘要:

目的 平行比较“新冠三方”(清肺排毒汤、化湿败毒方和宣肺败毒方，以下简称“三方”)的体外抗氧化作用。方法 在非细胞体系中，采用FRAP法考察三方的总抗氧化能力；通过DPPH法考察三方对DPPH自由基的清除作用；采用NBT还原法考察三方对超氧阴离子的清除作用；通过基于终产物丙二醛的荧光测定法考察三方对羟自由基的清除作用；用FeSO₄体外诱导大鼠肝匀浆MDA法考察三方对抗脂质过氧化的作用。依据以上5个抗氧化指标，平行比较三方提取物在非细胞体系的抗氧化能力。进一步地，在脂多糖活化的RAW264.7细胞模型上，分别使用L-012探针和MitoSOX线粒体超氧化物红色荧光探针检测胞质与线粒体活性氧(reactive oxygen species，ROS)的生成情况；通过光泽精探针检测胞质NADPH氧化酶活性，以平行比较三方提取物在细胞体系的抗氧化能力。结果 在非细胞体系中，三方均能剂量依赖地清除DPPH自由基、超氧阴离子和羟自由基，并且能有效对抗脂质过氧化的发生。在等提取物浓度下，三方对DPPH自由基与超氧阴离子的清除作用相当，总抗氧化能力相当，而化湿败毒方在清除羟自由基和对抗脂质过氧化方面表现更优。在细胞体系中，三方均能通过抑制NADPH氧化酶的活性来减少脂多糖刺激RAW264.7细胞质ROS的生成；不仅如此，三方对线粒体ROS的生成也有抑制作用，其中清肺排毒汤的作用最佳。结论 三方在非细胞体系和细胞体系均展现了多重体外抗氧化作用，且在不同指标上的作用强度各有优劣。考虑到抗氧化应激对COVID-19治疗的重要意义，上述结果提示三方的抗氧化能力是它们发挥治疗COVID-19作用的药效学基础之一。

Abstract:

OBJECTIVE To compare the anti-oxidative effects of "three decoctions for COVID-19" (Qingfei Paidu decoction, Huashi Baidu decoction, Xuanfei Baidu decoction) in parallel experimental models.METHODS In the cell-free system, the total antioxidant capacity was investigated by FRAP method. The scavenging effects of DPPH radicals and superoxide anions were evaluated by DPPH and NBT reduction method, respectively. The scavenging effect of hydroxyl radicals was determined by a fluorescence method based on the end-product MDA. The anti-lipid peroxidation activity was investigated using the FeSO₄-induced rat liver homogenate MDA method. Based on these five antioxidant indicators, the antioxidant capabilities of the extracts of three decoctions were parallelly compared in the cell-free system. Furthermore, in lipopolysaccharide-activated RAW264.7 cells, the productions of intracellular and mitochondrial reactive oxygen species(ROS) were detected using the L-012 probe and the MitoSOX mitochondrial superoxide red fluorescence probe, respectively; and intracellular NADPH oxidase activity was measured using the lucigenin probe. These three indicators were used to parallelly compare the antioxidant capabilities of the extracts of three decoctions. RESULTS In the cell-free system, three decoctions for COVID-19 could concentration-dependently scavenge DPPH radicals, superoxide anions and hydroxyl radicals, and potently inhibit the lipid peroxidation. At the equal extract concentration, their scavenging effects on DPPH radicals and superoxide anions and the total antioxidant capacity were comparable; while Huashi Baidu decoction exhibited the strongest ability to scavenge hydroxyl radicals and inhibit lipid peroxidation. In the cell system, three decoctions could reduce lipopolysaccharide-elevated intracellular ROS level by weakening NADPH oxidase activity; meanwhile, they could decrease mitochondrial ROS productions, among which Qingfei Paidu decoction possessed the most comprehensive effection. CONCLUSION Collectively, three decoctions for COVID-19 exert diverse antioxidant effects in both cell-free and cell systems, and each of them possesses the distinct advantages. Given that oxidative stress is pivotal during the pathological process of COVID-19, the results may suggest that the antioxidant ability of three decoctions is one of the pharmacodynamic basis for their clinical use.