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大鼠胆汁中黄芩苷代谢物的分离、纯化及其对肝癌细胞 HepG2

增殖的影响研究 Δ

5
2，4
2，3
2
2，3
朱亚南 1，2＊ ，张 硕 ，张 敏 ，孟小夏 ，杨七妹 ，汤 磊 ，张荣平 ，高秀丽 1，2，3 # （1.省部共建药用植物功效与
2，3
利用国家重点实验室，贵阳 550025；2.贵州医科大学药学院，贵阳 550025；3.贵州省高校微生物与生化药学
工程中心，贵阳 550004；4.贵州省化学合成药物研发利用工程技术研究中心，贵阳 550004；5.昆明医科大学
药学院，昆明 650500）
中图分类号 R284.1；R284.5 文献标志码 A 文章编号 1001-0408（2020）07-0800-06
DOI 10.6039/j.issn.1001-0408.2020.07.07

摘 要 目的：从大鼠胆汁中分离纯化黄芩苷的代谢物，并研究其对肝癌细胞HepG2增殖的影响。方法：取6只SD大鼠，麻醉后
进行胆管插管，待大鼠清醒后，灌胃给予黄芩苷（168 mg/kg），收集灌胃给药后24 h的胆汁样品1，经处理后，采用高效液相色谱法
进行初步分析；另取5只SD大鼠，同上述方法麻醉、插管后，灌胃给予黄芩苷（400 mg/kg），并收集灌胃给药后48 h的胆汁样品2，
经处理后，采用半制备型高效液相色谱法对代谢物进行分离纯化；采用紫外光谱、红外光谱、质谱、核磁共振技术并结合其理化性
质对分离得到的代谢物进行鉴定；采用MTT法及高内涵细胞成像分析法研究代谢物对HepG2细胞增殖的影响。结果：黄芩苷通
过胆汁主要代谢成代谢物1和代谢物2，经分离纯化后分别鉴定为千层纸素A-7-O-β-D-葡萄糖醛酸苷和黄芩素6-O-β-D-葡萄糖醛
酸苷。MTT法结果显示，代谢物2对HepG2细胞具有显著的增殖抑制作用，其半数抑制浓度（IC50 ）为90 µg/mL；高内涵细胞成像
分析法结果显示，在一定浓度下，代谢物2可能通过改变HepG2细胞线粒体分布及膜通透性从而抑制细胞增殖（代谢物1的药理
活性已有报道，本研究略去）。结论：成功分离并鉴定出2个黄芩苷的胆汁代谢物，其中代谢物2黄芩素6-O-β-D-葡萄糖醛酸苷对
HepG2细胞具有显著的增殖抑制作用。
关键词 黄芩苷；代谢物；千层纸素A-7-O-β-D-葡萄糖醛酸苷；黄芩素6-O-β-D-葡萄糖醛酸苷；分离纯化；高内涵细胞成像分析；肝
癌细胞HepG2

Study on the Isolation and Purification of Baicalin Metabolites from Rat Bile and Its Effect on the
Proliferation of Liver Cancer HepG2 Cells
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ZHU Yanan ，ZHANG Shuo ，ZHANG Min ，MENG Xiaoxia ，YANG Qimei ，TANG Lei ，ZHANG
1，2
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2，3
2
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Rongping ，GAO Xiuli 1， 2， 3 （1.State Key Laboratory of Functions and Applications of Medicinal Plants
Co-constructed by Province and Ministry，Guiyang 550025，China；2.School of Pharmacy，Guizhou Medical
University，Guiyang 550025，China；3.Guizhou Provincial College of Microbiology and Biochemical Pharmacy
Engineering Center，Guiyang 550004，China；4.Guizhou Provincial Engineering Technology Research Center for
Chemical Drug R&D，Guiyang 550004，China；5.School of Pharmacy，Kunming Medical University，Kunming
650500，China）
ABSTRACT OBJECTIVE：To isolate and purify baicalin metabolites from rat bile，and to study its effect on the proliferation of
liver cancer HepG2 cells. METHODS：Totally of 6 SD rats were collected，anesthetized and then intubated with bile ducts. They
were given baicalin（168 mg/kg）intragastrically after the rats were awake as well as the bile sample 1 was collected during 24 h
after intragastric administration. After processed，bile sample 1 was preliminarily analyzed by HPLC. Another 5 SD rats were
anesthetized and intubated in the same way as above，and then given baicalin intragastrically（400 mg/kg）. The bile sample 2 was
collected during 48 h after intragastric administration. After processed， the bile sample 2 was isolated and purified by
semi-preparative HPLC. The isolated metabolites were identified by using UV，IR，MS and NMR，as well as based on physical
and chemical properties. MTT method combined with high content cell imaging analysis system was used to study the effect of the
metabolites on the proliferation of HepG2 cells. RESULTS：
Δ 基金项目：国家自然科学基金资助项目（No.81160413）；贵州省
Baicalin was mainly metabolized into metabolite 1 and
科技厅项目（No.黔科合成转字〔2015〕5213 号）；贵州省普通高等学校
metabolite 2 through bile. After isolation and purification，
工程研究中心课题项目（No.黔教合KY字〔2015〕338）
they were identified as oroxylin A-7-O-β-D-glucuronide and
＊硕士研究生。研究方向：新药研发及体内药物分析。E-mail：
2726719792@qq.com baicalein 6-O-β-D-glucuronide. The results of MTT assay
# 通信作者：教授，硕士生导师。研究方向：新药研发及体内药物 showed that the metabolite 2 had a significant inhibitory effect
分析。E-mail：gaoxl@gmc.edu.cn on the proliferation of HepG2 cells， and its IC50 was 90

·800 · China Pharmacy 2020 Vol. 31 No. 7 中国药房 2020年第31卷第7期