中国药物警戒 ›› 2020, Vol. 17 ›› Issue (2): 75-80.
DOI: 10.19803/j.1672-8629.2020.02.03

• 基础研究 • 上一篇    下一篇

制附子水提物的生物活性限值测定研究

刘蕾1, 张琼玲1, 王继永2, 丁世兰1, 游云1, 肖苏萍2*   

  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:2019-11-04 修回日期:2020-06-22 出版日期:2020-02-15 发布日期:2020-02-26
  • 通讯作者: *肖苏萍,女,副研究员,中药产品研发和质量控制。E-mail:xiaoladdy99@163.com
  • 作者简介:刘蕾,女,在读硕士,心血管药理学。
  • 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

Bioassay on Aquatic Extract of Processed Radix Aconiti Carmichaeli

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

  1. 1Institude of Chinese Materia Media, China Academy of Chinese Medical Science, Beijing 100700, China;
    2China National Traditional Chinese Medicine Co., Ltd, Beijing 100195, China
  • Received:2019-11-04 Revised:2020-06-22 Online:2020-02-15 Published:2020-02-26

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摘要： 目的 研究不同来源附子水提物对大鼠心肌细胞H9c2毒性的影响,探讨其毒性剂量限值及其检测限,拟从生物活性层面对附子进行质量控制。方法 由中国中药有限公司提供4个批次的附子水提物,批号分别为:HSPYN、HSPSXHZ、HSPSCJY和HSPSCJY1。首先对细胞试验条件进行优化,并从附子水提物抑制心肌细胞活力的量效关系、精密度、重复性等方面进行考察。以CCK-8法测定H9c2心肌细胞的生长曲线,确定优选的细胞密度及药物作用时间;确定附子水提物影响心肌细胞活力的量效曲线,确定药物作用的限值。对不同来源的4批附子药材水提物进行毒性评价,找出附子水提物剂量限值及抑制H9c2细胞活力的检测限。结果 在不同细胞种植密度条件下,H9c2细胞在0~48 h为增殖期,48~60 h到达平台期,60~72 h进入衰亡期;选用0.5×105/mL细胞密度种植于96孔板,药物作用12 h作为实验条件;批号为HSPYN的附子水提物浓度范围在15.6、31.3、62.5、125.0 μg·mL-1条件下,对 H9c2细胞毒性可呈现一定的量效关系,抑制率分别为24.5±3.4%、26.9±6.2%、27.3±8.8%和39.5±11.3%。阳性对照药选择新乌头碱(100μg·mL-1),抑制率范围在24.6%~32.4%。通过对4个批次附子水提物抑制心肌细胞活性的精密度及重复性进行考察,确定了药物浓度在15.6 μg·mL-1其抑制率稳定在21.4±1.9%范围内。结论 结合附子的临床功能主治及毒副作用,采用附子抑制心肌细胞活力的效应作为生物活性评价指标,在本研究确定的试验条件下,确定15.6 μg·mL-1可作为限值剂量。在该限值浓度下对大鼠心肌H9c2细胞活力抑制率为20%~24%,可判定为质量合格。

关键词: 附子, H9c2, 毒性, 质量控制

Abstract: Objective To set up the bioassay method for aquatic extract of Processed Radix Aconiti Carmichaeli as the supplementary quality control by its toxic effect on rat myocardial cells H9c2. Methods Aquatic extracts of Processed Radix Aconiti Carmichaeli were provided by China National Traditional Chinese Medicine Co., Ltd. with the batch numbers of HSPYN, HSPSXHZ, HSPSCJY, and HSPSCJY1 separately. The optimized experimental conditions were investigated from the points of cells seeding density, action duration, concentration-response relationship, precision and repeatability test. The growth curve of H9c2 cardiomyocytes was measured by the CCK-8 method to find the optimal cell density and drug action time; and the concentration-response relationship of Processed Radix Aconiti Carmichaeli on cardiomyocytes vitality was determined too. Toxicity evaluation of four batches of Processed Radix Aconiti Carmichaeli was performed and repeated for at least 5 times to determine the dose limit and toxic effect range. Results Batch of HSPYN was used to do the methodological investigation. The cell density was chosen as 5.0×104/ mL and 100μL was seeded in 96-well plates,and the drug action time of 12h showed more stable effects. With the concentrations at 15.6, 31.3, 62.5, and 125.0 μg•mL-1 of Processed Radix Aconiti Carmichaeli, the inhibition rates on H9c2 vitality were 24.5±3.4%, 26.9±6.2%, 27.3±8.8%, and 39.5±11.3% respectively. The main component of Processed Radix Aconiti Carmichaeli, neoaconitine (100 μg•mL-1) was selected as a positive control, and the inhibition rate ranged from 24.6% to 32.4%. With the precision and repeatability of bioassay experiments on all the four batches carried out, it was determined that the inhibition rate on cardiomyocytes vitality was stable within the range of 21.4±1.9% at the concentration of 15.6 μg•mL-1. Conclusion Associated with the clinical indications and toxicity of Processed Radix Aconiti Carmichaeli, its inhibited effect on cardiomyocytes viability might be used as an indicator for bioassay as the supplementary quality control. The testing sample with concentration of 15.6 μg•mL-1 with inhibition rate above 20.0% might be judged as qualified.

Key words: Processed Radix Aconiti Carmichaeli, H9c2, toxicity, quality control

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