基于大鼠室性早搏心脏毒性的附子质量生物评价方法研究

Establishment of biological assess for quality control of Fuzi based on determination of premature ventricular contractions in rats

附子是确有疗效的毒剧类中药,构建关联临床毒副反应的质量评价方法对提高其炮制减毒质控水平和保障临床安全性具有重要意义。该文针对附子可导致室性早搏这一早期心脏中毒反应,构建基于大鼠室性早搏(premature ventricular contractions,PVC)最小中毒量(minimal toxic dose,MTD)测定的附子炮制品质量生物评价方法,通过影响因素考察优化方法学(动物性别、体重、对照品稳定性与供试品稳定性),检测结果的重现性符合药品质量生物评价要求。应用该方法检测生附子及其不同炮制品黑顺片、白附片、蒸附片、刨附片、炮天雄致大鼠室性早搏MTD,结果表明,生附子炮制后MTD均显著升高(P<0.05),其中黑顺片、蒸附片、刨附片与白附片的MTD分别为生附子的15.76,22.36,19.65,20.97倍;而炮天雄几乎无毒,用该方法检测不出MTD。该方法可以比较好的反映附子及其炮制品的整体心脏毒性。进一步结合附子炮制品的化学成分分析,发现炮制品中双酯型生物碱乌头碱、新乌头碱、次乌头碱,以及单酯型生物碱苯甲酰乌头原碱与PVC呈显著相关。此外,可能还存在其他与致心律失常相关的未测成分,值得深入研究。上述研究为附子临床早期心脏毒性评价与质量控制提供了整体、综合的方法,可作为化学评价方法的有益补充,为保障附子临床应用的安全性提供了新的方法。

Aconiti Lateralis Radix（ Fuzi） is a toxic traditional Chinese medicine with definite efficacy. In order to improve the quality control of its different prepared products and ensure the security in clinic,it is significant to establish a method of quality evaluation related to clinic adverse effects. Aiming at the important biological marker of early cardiac toxicity reaction,there was no method to detect it. In this manuscript,a novel approach for measuring the minimal toxic dose（ MTD） of premature ventricular contractions（ PVC） poisoning of rats was established. Then,the determination methodology and conditions were optimized to meet the needs of the quality and biological assessment,including animal sex,weight,stability of standards and test solutions. Using this method,the MTD value of different Fuzi products were determined,such as Heishunpian,Baifupian,Zhengfupian,Baofupian,and Paotianxiong. The results showed that the MTD of Fuzi was significantly decreased after detoxification processed（ P〈0. 05） and the MTD of Heishunpian,Zhengfupian,Baofupian and Baifupian was as much as 15. 76,22. 36,19. 65 and 20. 97 times to that of unprocessed Shengfuzi. In addition,Paotianxiong could not induce PVC in rats,which indicated that Paotianxiong was nontoxic and safe. This method could appropriately reflects the cardiotoxity of Fuzi and its prepared samples. Together with the chemical composition analysis,the contents of diester alkaloids were explored including aconitine,mesaconitine and hypaconitine as well as monoester alkaloids in Fuzi and its prepared products were significantly associated with PVC. Furthermore,there may be some components undetermined facilitating arrhythmia to be worth exploring. This research provides an overall and comprehensive approach to diagnose early clinical cardiotoxity and control the quality of Fuzi,which could not only be a complementary solution for the chemical evaluation,but a new method to ensure its efficacy and security of clinical application.