药学学报, 2019, 54(12): 2169-2177
引用本文:
张定堃, 赵志浩, 李春雨, 牛明, 谭鹏, 裴瑾, 杨明, 彭成, 张萍, 肖小河, 王伽伯. 基于生物毒效检测的附子不同炮制品质量评价研究[J]. 药学学报, 2019, 54(12): 2169-2177.
ZHANG Ding-kun, ZHAO Zhi-hao, LI Chun-yu, NIU Ming, TAN Peng, PEI Jin, YANG Ming, PENG Cheng, ZHANG Ping, XIAO Xiao-he, WANG Jia-bo. Quality evaluation of different processed products of Aconiti Lateralis Radix Praeparata combining bioassay and toxicity detection[J]. Acta Pharmaceutica Sinica, 2019, 54(12): 2169-2177.

基于生物毒效检测的附子不同炮制品质量评价研究
张定堃1,6, 赵志浩2, 李春雨3, 牛明2, 谭鹏4, 裴瑾1,6, 杨明5, 彭成1, 张萍2, 肖小河2, 王伽伯2
1. 成都中医药大学药学院, 西南特色中药资源国家重点实验室, 四川 成都 611137;
2. 解放军总医院第五医学中心, 全军中医药研究所, 北京 100039;
3. 中国医学科学院、北京协和医学院肿瘤医院, 北京 100021;
4. 四川省中医药科学院, 国家中医药管理局中药质量生物评价重点研究室, 四川 成都 610041;
5. 江西中医药大学, 江西 南昌 330004;
6. 四川好医生攀西药业有限责任公司, 四川 西昌 615000
摘要:
附子炮制品种类繁多,毒效差异不明确,亟需建立精准的质量评控方法,保障临床用药安全有效。采用引起大鼠室性早搏(premature ventricular contraction,PVC)的最小量测定法评价8种附子炮制品的生物毒性,结果发现生附片醇提液的最小中毒剂量(minimal toxic dose,MTD)为0.16 g·kg-1,毒性远强于其余附片;其次为黑顺片、白附片、刨附片、炮附子与蒸附片,醇提液的MTD为2.64~5.75 g·kg-1;炒附片与炮天雄未检测出心毒性,安全性好。建立急性心衰大鼠+dp/dtmax测定法评价8种附子炮制品的强心活性,并通过成分-活性相关分析筛选、验证强心质量标志物。结果显示,8种附片均具有明确的强心效果,其中,生附片强心效果最强,给药15 min后大鼠心功能恢复正常;黑顺片、白附片等5种附片具有中等强度强心活性;而炮附子与炮天雄强心活性最弱。采用LC-MS/MS法同时测定附子水煎液中13种生物碱的含量,结果发现乌头碱、新乌头碱与次乌头碱3种剧毒成分无法检测,去甲乌药碱仅在生附片中存在,去甲猪毛菜碱在生附片中的含量高出其余附片4~56倍。相关性分析表明,去甲猪毛菜碱与强心指标的相关性最好,相关性系数高达0.817,而3种单酯型生物碱与强心作用相关性不大。进一步直接对13种单体的强心活性验证评估,表明去甲乌药碱与去甲猪毛菜碱是强心活性物质,其余11种成分无强心活性。该研究建立了附子生物毒效精准评价的技术方法,揭示了8种常见炮制品毒效作用的强弱规律,发现了强心质量标志物,为附子炮制品的质量评控及临床合理选用提供了科学支撑。
关键词:    附子      心毒性      强心      质量标志物      生物评价     
Quality evaluation of different processed products of Aconiti Lateralis Radix Praeparata combining bioassay and toxicity detection
ZHANG Ding-kun1,6, ZHAO Zhi-hao2, LI Chun-yu3, NIU Ming2, TAN Peng4, PEI Jin1,6, YANG Ming5, PENG Cheng1, ZHANG Ping2, XIAO Xiao-he2, WANG Jia-bo2
1. State Key Laboratory of Characteristic Chinese Medicine Resources in Southwest China, College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China;
2. China Military Institute of Chinese Medicine, the Fifth Medical Centre of Chinese People's Liberation Army General Hospital, Beijing 100039, China;
3. Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021;
4. State Key Laboratory of Quality Evaluation of Traditional Chinese Medicine, Sichuan Academy of Traditional Chinese Medicine, Chengdu 610041, China;
5. Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China;
6. Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd., Xichang 615000, China
Abstract:
There are many kinds of processed products of Aconiti Lateralis Radix Praeparata (ALRP), but their differences in toxicity and efficacy have not been identified. The minimum premature ventricular contraction (PVC) method was used to evaluate the biological toxicity of eight processed products. The results showed that the minimal toxic dose (MTD) of an ethanol extract of Shengfupian (SFP) was 0.16 g·kg-1, which was much lower than that of Heishunpian (HSP), Baifupian (BFP), Baofupian (BAP), Paofuzi (PFZ) or Zhengfupian (ZFP), with MTDs ranging from 2.64 to 5.75 g·kg-1. No cardiotoxicity was detected with Chaofupian (CFP) and Paotianxiong (PTX). A novel +dp/dtmax assay for acute heart failure in rats was developed to evaluate the cardiac activity. It was found that all eight processed products had cardiac effects, with Shengfupian showing the strongest cardiotonic effect and the ability to restore damaged cardiac function to normal within 15 minutes of injection. Heishunpian, Baifupian and the three other products displayed moderate activity, while Paofuzi and Paotianxiong were the weakest. An LC-MS/MS method was utilized to determine the content of 13 alkaloids in water extracts. The results demonstrated that hypertoxic aconitine, mesaconitine, and hypaconitine could not be detected, higenamine was only present in Shengfupian, and salsolinol was about 4-56 times higher in Shengfupian than in other products. A correlation analysis showed that salsolinol had the best correlation with the cardiotonic index, with a correlation coefficient as high as 0.817, while the three monoester alkaloids failed to correlate with the cardiotonic effect. Higenamine and salsolinol were cardiotonic, while the 11 other components had no cardiotonic activity. This study establishes methods for precise evaluation of cardiotoxicity and cardiac activity, reveals the toxicity and efficacy of common processed products, and identifies the key quality markers for cardiac activity, providing scientific support for the quality evaluation and clinical application of processed products of aconite.
Key words:    Aconiti Lateralis Radix Praeparata    cardiotoxicity    cardiac activity    quality marker    biological evaluation   
收稿日期: 2019-08-20
DOI: 10.16438/j.0513-4870.2019-0658
基金项目: 国家重点研发计划—中医药现代化(2018YFC1707000);四川省中医药管理局科研创新专项(2018QN008);国家中药标准化项目(ZYBZH-Y-TJ-43);全军医学科技青年培育项目(16QNP151);国家自然科学基金项目(81630101,81573676);首都卫生发展科研专项(首发2018-2-5031).
通讯作者: 王伽伯,Tel:86-10-66933323,E-mail:wjb0128@126.com
Email: wjb0128@126.com
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