中国药物警戒 ›› 2020, Vol. 17 ›› Issue (10): 659-664.
DOI: 10.19803/j.1672-8629.2020.10.03

• 基础与临床研究 • 上一篇    下一篇

基于RAW264.7巨噬细胞吞噬功能的甘草水提物生物限值测定研究

张琼玲1, 肖苏萍2, 刘蕾1, 张权2, 丁世兰1, 闫惠捷1, 王继永2, 游云1,*   

  1. 1中国中医科学院中药研究所,北京 100700;
    2中国中药有限公司,北京 100195
  • 收稿日期:2020-10-14 修回日期:2020-10-14 出版日期:2020-10-15 发布日期:2020-10-13
  • 通讯作者: *游云,女,博士,研究员·博导,中药药理学。E-mail：youyunrice@126.com
  • 作者简介:张琼玲,女,在读硕士,中药药理学。
  • 基金资助:
    北京市科技计划项目（Z171100001717028) ：十病十药研发-中药品质评价关键技术体系与平台建设

Biological Evaluation for Quality Control of Water Soluble Extracts of Radix Glycyrrhizae Based on Macrophage Phagocytosis Model

ZHANG Qiongling1, XIAO Suping2, LIU Lei1, ZHANG Quan2, DING Shilan1, YAN Huijie1, WANG Jiyong2, YOU Yun1,*   

  1. 1Institute of Chinese Materia Media, China Academy of Chinese Medical Sciences, Beijing 100700, China;
    2China National Traditional Chinese Medicine Co., Ltd, Beijing 100195, China
  • Received:2020-10-14 Revised:2020-10-14 Online:2020-10-15 Published:2020-10-13

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摘要： 目的 建立基于小鼠巨噬细胞系RAW264.7吞噬功能的甘草水提物生物限值测定方法,从生物活性层面对甘草进行质量控制。方法 由中国中药有限公司提供4个批次的甘草水提物冻干粉,批号分别为：GCGSQ、GCGSC、GCNMG、GCXJ,GCGSC作为验证使用。首先对细胞实验条件进行优化,并从甘草水提物对巨噬细胞吞噬功能影响的量效关系、精密度、重复性等方面进行考察。采用CCK-8法考察细胞密度、LPS浓度、3个批次甘草水提物对巨噬细胞增殖活性的影响,确定合适的给药浓度范围;采用中性红吞噬实验考察3个批次药物对巨噬细胞吞噬功能的影响,确定药物作用限值及检测限。结果 实验结果提示巨噬细胞优选接种密度为3×105个/mL、药物作用时间为24 h、LPS浓度为0.25μg/mL,当甘草水提物质量浓度为125~500μg/mL时对巨噬细胞增殖及吞噬功能的影响呈剂量依赖性,当浓度为125μg/mL时促增殖作用和促吞噬功能最强;通过对3个批次甘草水提物对巨噬细胞吞噬功能作用的精密度及重复性进行考察,确定当甘草水提物浓度为125μg/mL为限值剂量,其吞噬指数范围为115%~129%。结论 本实验结合甘草免疫调节的药理活性,建立了基于RAW264.7巨噬细胞吞噬功能的甘草水提物生物限值评价方法,在本研究确定的试验条件下,确定125μg/mL为限值剂量,在该限值浓度下巨噬细胞的吞噬指数在115%~129%范围内,可判断为质量合格。

关键词: 甘草水提物, RAW264.7, 吞噬, 生物评价, 巨噬细胞, 限值测定

Abstract: Objective To establish a method for evaluating the biological activity of water soluble extracts of Radix Glycyrrhizae based on a macrophage phagocytosis model. Methods Aquatic extracts of Radix Glycyrrhizae were provided by China National Traditional Chinese Medicine Co., Ltd. with the batch numbers of GCGSQ, GCGSC, GCNMG and GCXJ, and GSGSC is used as a verification batch. The optimized experimental conditions were investigated. The seeding density of cells, LPS concentration, action duration, drug concentration-response curves were determined by CCK-8 assay. The phagocytosis indexes of RAW 264.7 macrophages were determined by neutral red uptake assay and repeated at least 5 times to set the dose limit. Results The optimum cell density was chosen to be 3.0×105/mL and 100μL was seeded in 96-well plates, the drug action time was 24h and LPS concentration was 0.25μg/mL, which obtained more stable effects. The aquatic extracts of Radix Glycyrrhizae stimulated proliferation and phagocytic activity in RAW264.7 in a concentration-dependent manner when the concentrations ranged from 125 to 500μg/mL. At the concentration of 125μg/mL, better activity with good precision (RSD=0.9%) and repeatability (RSD=2.15%) was observed in the extracts of bathes of GCGSQ, GCGSC, GCXJ. Under this experimental condition, the concentration of 125 μg/mL was selected as the dose limit, and the phagocytosis indexes of macrophages that ranged from 115% to 129% were set as the bioassay quality control supplement. Conclusion Combined with clinical indications, the effects of Radix Glycyrrhizae on phagocytic activity in RAW264.7 macrophages might be used as an indicator for bioassay as the supplementary quality control.

Key words: water soluble extracts of Radix Glycyrrhizae, RAW264.7, phagocytosis, biological evaluation, macrophage, quality control

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