琥珀酸半醛脱氢酶抑制剂筛选方法的建立及其在天麻成分筛选中的应用

目的建立琥珀酸半醛脱氢酶(SSADH)抑制剂类抗癫痫神经系统药物筛选模型,并运用此模型对中药天麻有效成分及其类似物进行体外活性筛选和构效关系分析。方法制备琥珀酸半醛脱氢酶(SSADH)酶系液,并以紫外分光光度法评价SSADH活力与吸光度相关性;优化酶催化反应温度、反应时间、底物NAD+浓度、底物琥珀酸半醛(SSA)浓度、缓冲液p H、底物SSADH浓度等因素对SSADH活性的影响,建立SSADH酶系活性筛选方法;采用文献已有报道的对羟基苯甲醛(HBA)为阳性对照验证此方法可靠性,同时,对HBA结构类似物测定结果及作用机制进行分析。结果成功构建SSADH酶抑制剂活性筛选方法,HBA测定结果与前期文献报道抗癫痫作用一致。确定SSADH的紫外光谱(UV)活性检测体系及其检测缓冲液配置组成,其反应条件为37℃,温孵30 min,酶活力相关吸光度在340 nm处测量。HBA和香草醛(Va)对GABA-T的构效关系一致,—OH及苯环上的—CHO为SSADH酶抑制作用必须药效团。结论该模型可应用于SSADH酶抑制剂的高通量筛选,也为研究中药中SSADH酶抑制剂类活性成分的筛选及其作用机制研究提供参考。

Screening succinate semialdehyde dehydrogenase inhibitors:an estab-lished model and its application in Tall Gastrodia Tuber

Objective To establish a model of screening succinate semialdehyde dehydrogenase (SSADH)inhibitors with anti-epilepsy effects,then using this model to find out the effective components in Tall Gastrodia Tuber (Latin:Rhizoma Gastrodiae,pinyin:Tianma)and some analogues and to study their structure-activity relationship.Methods First,SSADH enzyme system fluid was prepared,and the correlation of activity and optical density of SSADH was evaluated using UV spectrophotometry.Then,af-ter the screening method of SSADH activity was established by optimization of reaction temperature,reac-tion time,concentration of NAD +,SSA and SSADH,and pH of buffer on the SSADH activity,p-hydroxybenzaldehyde (HBA),a positive drug recorded in literature,was applied to verify the model. And the structural analogues of HBA were measured and the mechanism was analyzed.Results The screening model was established successfully,proved by HBA test.The UV detective system and its buff-ers of SSADH activity were determined with reaction temperature at 37 ℃ for 30 min,and detect wave at 340 nm.The structure-activity of HBA on GABA-T was as the same as that of vanillic aldehyde,and—OH and —CHO of benzene ring were the essential groups for inhibition SSADH.Conclusion The model established in this paper can be used for high throughput screening SSADH inhibitors and may guide the study of analogues of SSADH inhibitors and their mechanism.