目的 采用电化学模拟技术制备天麻素体内I相氧化代谢产物，并确认其为潜在起效成分。方法 采用电化学模拟技术(μPrepcell电池及Synthesis^TM制备型电池)，优化电压、电解液、反应物浓度和体积流量等，制备天麻素体内I相氧化代谢产物。构建缺糖缺氧/复氧(oxygen and glucose deprivation/reperfusion，OGD/R)诱导的PC12细胞模型，考察天麻素及其5个代谢产物的神经保护作用。结果 通过优化参数，确定薄层电化学电池(μPrepcell)模拟天麻素代谢为对羟基苯甲醛-O-β-D-吡喃葡萄糖苷和对羟基苯甲酸-O-β-D-吡喃葡萄糖苷的反应条件为：掺硼金刚石(borondopeddiamond，BDD)为工作电极，最佳底物浓度为100 μmol/L，最佳电解液浓度为20 mmol/L甲酸铵水溶液-甲醇(1∶9)，最佳体积流量为50 μL/min，最佳电压为1.6 V。再利用电化学合成电池(SynthesisCell)进行大量制备，玻碳(reticulated glassy carbon，RGC)电极为工作电极，从240 mg天麻素中制备得到77 mg对羟基苯甲醛-O-β-D-吡喃葡萄糖苷。神经保护活性评价表明天麻素及其5个代谢产物对OGD/R诱导的PC12细胞均具有较好的保护作用(P＜0.05、0.01)，且浓度为50 μmol/L时，与天麻素相比，代谢产物对羟基苯甲醛-O-β-D-吡喃葡萄糖苷和对羟基苯甲酸-O-β-D-吡喃葡萄糖苷神经保护作用相对显著(P＜0.05)，存活率分别提高了8.63%、9.42%，细胞凋亡率分别降低了29.27%、45.30%。Western blotting结果表明对羟基苯甲醛-O-β-D-吡喃葡萄糖苷和对羟基苯甲酸-O-β-D-吡喃葡萄糖苷均可显著增加OGD/R诱导的PC12细胞B淋巴细胞瘤-2(B-cell lymphoma-2，Bcl-2)蛋白表达水平(P＜0.01)，降低Bcl-2相关X蛋白(Bcl-2 associated X protein，Bax)和半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3，Caspase-3)蛋白表达水平(P＜0.01)。结论 天麻素及其代谢产物均具有神经保护作用，首次应用电化学模拟新技术制备天麻素体内I相氧化代谢产物并初步阐明了其发挥神经保护作用的机制，为阐明天麻素的起效物质奠定基础。

Objective To prepare the phase I oxidative metabolites of gastrodin in vivo by electrochemical technology, and then confirm the potential active components. Methods Electrochemical technologies (μPrepcell and Synthesis^TM cell) were used to optimize the voltage, electrolyte, reactant concentration and flow rate, etc., and prepare phase I oxidation metabolites of gastrodin in vivo. Oxygen and glucose deprivation/reperfusion (OGD/R) induced PC12 cells model was constructed to investigate the neuroprotective effects of gastrodin and its five metabolites. Results By optimizing the parameters, μPrepcell of electrochemical was determined to simulate the metabolism of gastrodin to p-hydroxybenzaldehyde-O-β-D-glucopyranoside and p-hydroxybenzoic acid-O-β-D-glucopyranoside. The optimized reaction conditions: Borondopeddiamond (BDD) working electrode was used as the working electrode; Optimal substrate concentration was 100 μmol/L; Optimal concentration of electrolyte was 20 mmol/L ammonium formate water-methanol (1∶9), optimal flow rate was 50 μL/min, and optimal voltage was 1.6 V. Using electrochemical SynthesisCell, reticulated glassy carbon (RGC) electrode for bulk preparation, 77 mg p-hydroxybenzaldehyde-O-β-D-glucopyranoside was prepared from 240 mg gastrodin. The neuroprotective activity evaluation showed that gastrodin and its five metabolites had a good protective effect on OGD/R induced PC12 cells (P＜0.05, 0.01), and when the concentration was 50 μmol/L, p-hydroxybenzaldehyde-O-β-D-glucopyranoside and p-hydroxybenzoic acid-O-β-D-glucopyranoside had a relatively significant neuroprotective effect (P＜0.05), survival rate increased by 8.63% and 9.42%, and apoptosis rate decreased by 29.27% and 45.30%, respectively. The results of Western blotting showed that p-hydroxybenzaldehyde-O-β-D-glucopyranoside and p-hydroxybenzoic acid-O-β-D-glucopyranoside could obviously increase the protein expression level of B-cell lymphoma-2 (Bcl-2) (P＜0.01), and reduce the protein expression levels of Bcl-2 associated X protein (Bax) and cystein-asparate protease-3 (Caspase-3) in OGD/R induced PC12 cells (P＜0.01). Conclusion For the first time, electrochemistry was used to simulate the in-vitro phase I oxidative metabolism of gastrodin and the mechanism of its neuroprotective effect was preliminarily elucidated. It was preliminarily found that gastrodin had neuroprotective effects, which laid a foundation for clarifying the active substances of gastrodin.

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国家自然科学基金资助项目(82173957)；国家自然科学基金资助项目(81774155)；国家自然科学基金资助项目(81803703)；中央高校基本科研基金资助项目(2020-JYB-XJSJJ-022)