摘要
目的优化小鼠大脑乙酰胆碱酯酶(AChE)提取、测定方法,评价异喹啉类生物碱的AChE抑制活性。方法采用蔗糖密度梯度离心及Ellman法评价不同提取方法对小鼠大脑AChE亚型表达及提取影响,优化测定方法,并在此基础上对异喹啉类生物碱的AChE抑制活性及构效关系进行考察。结果匀浆+超声、匀浆提取法对大脑AChE亚型表达一致,但前者AChE提取效果较优。随着反应温度、显色时间增加,药物AChE抑制活性增强,但显色时间超过30 min时,药物AChE抑制活性趋于平缓。随着大脑裂解液稀释倍数增加,药物AChE抑制活性未明显变化,且稀释10~20倍时,空白组AChE吸光度值为0.6~1.5。异喹啉类生物碱具有较好AChE抑制活性,而叔胺型生物碱AChE抑制活性降低。结论匀浆联合超声对小鼠大脑AChE有较好提取效果;反应温度37℃,显色时间30 min,大脑裂解液稀释10~20倍时,药物AChE抑制活性灵敏度、准确性好。异喹啉类生物碱的AChE抑制活性与其季胺型结构密切相关。
Objective To optimize the extraction and determination of AChE in mouse brain and study the inhibitory activity of isoquinoline alkaloids on AChE.Methods The sucrose density gradient centrifugation and Ellman method were selected to determine AChE isoform expression and optimize the extraction and determination of AChE in the mouse brain.On this basis,the AChE inhibitory activity and structure-activity relationship of isoquinoline alkaloids were investigated.Results The isoform expressions of AChE in brain tissue by homogenization and ultrasonic extraction were same,while the homogenization combined with ultrasonic extraction had a better effect on AChE extraction.The AChE inhibitory activity of drugs enhanced with the increase of reaction temperature and color development time.However,the drug’s AChE inhibition tends to stabilize when the color development time exceeds 30 min.The drug’s AChE inhibition did not change significantly with increased brain lysate dilution at the same drug concentration.Besides,the AChE absorbance value of the control group was 0.6-1.5.The isoquinoline alkaloids showed better AChE inhibitory activity,while the AChE inhibitory activity of tertiary amine alkaloids reduced.Conclusion The homogenization combined with ultrasonic extraction had a better effect on mouse brain AChE than homogenization extraction solely.The AChE inhibitory activity sensitivity of drugs was better when keeping the measurement condition as following:reaction temperature at 37℃,color development time for 30 min,brain lysate dilution for 10-20 times.In addition,the quaternary amine structure of isoquinoline alkaloids closely related to the AChE inhibitory activity.
作者
孔祥鹏
陈志从
夏英杰
任海琴
董婷霞
詹华强
KONG Xiangpeng;CHEN Zhicong;XIA Yingjie;REN Haiqin;DONG Tingxia;ZHAN Huaqiang(Shenzhen Key Laboratory of Edible&Medicinal Bioresources,HKUST Shenzhen Research Institutey,Shenzhen 518057,Guangdong,China;Institute of Pharmaceutical&Food Engineering,Chinese Medicine Master Studio of WANG Shimin,Shanxi University of Chinese Medicine,Jinzhong 030619,Shanxi,China;Division of Life Science and Center for Chinese Medicine,Hongkong University of Science and Technology,Hongkong 999077,China)
出处
《辽宁中医药大学学报》
CAS
2021年第7期35-39,共5页
Journal of Liaoning University of Traditional Chinese Medicine
基金
中国博士后科学基金项目(2019M653087)
广东省深圳市科技计划项目(JCYJ20170413173747440,20180306174903174,ZDSYS201707281432317)
山西中医药大学科技创新能力培育计划项目(2019PY-110,2020PY-YC-05)
关键词
乙酰胆碱酯酶
提取测定
方法优化
异喹啉类生物碱
构效关系
AChE
extraction and determination
method optimization
isoquinoline alkaloids
structure-activity relationship
