温阳扶正膏中双酯型生物碱的限量测定及急性毒性实验研究

Limit determination of diester alkaloids in Wenyang Fuzheng ointment and experimental study on acute toxicity

关键词:  附子  双酯型生物碱  限量检查  急性毒性实验

Key words:  aconite  diester alkaloids  limit test  acute toxicity experiments

摘要:

[目的] 为初步探索含附子的外用制剂在临床使用中的安全性，现以我院含附子的外用制剂温阳扶正膏为研究对象建立双酯型生物碱的限量测定方法，同时开展单次给药急性毒性实验及皮肤刺激实验和皮肤致敏性实验。[方法] 采用Agilent ZORBAX Extend C₁₈色谱柱（4.6×250 mm，5 μm），以乙腈-四氢呋喃（25∶15）为流动相A，以0.1 mol/L醋酸铵溶液为流动相B，梯度洗脱；流速为1 mL/min，柱温为25℃，进样体积为10 μL，检测波长为235 nm。以SD大鼠为实验对象，进行单次给药急性毒性实验，观察并记录SD大鼠的体质量、行为活动、皮肤反应并对各组大鼠的皮肤进行病理学检查。以豚鼠为实验对象，进行皮肤刺激实验和皮肤致敏性实验，观察完整皮肤和破损皮肤动物接触受试物后所产生的局部刺激反应和致敏作用。[结果] 乌头碱、新乌头原碱、次乌头原碱3种对照品检测限浓度分别为5.73、5.92和5.56 μg/mL。3批温阳扶正膏样品及阴性样品均未检测出乌头碱、新乌头原碱、次乌头原碱。给药组SD大鼠未见中毒症状和毒性反应，病理学检查均未见异常明显的炎症病变，与正常对照组比较无明显差异。温阳扶正膏对完整皮肤和破损皮肤豚鼠单次及多次用药均无明显皮肤刺激反应。温阳扶正膏对豚鼠皮肤外用接触后无致敏作用。[结论] 本研究从双酯型生物碱限量检查和单次给药急性毒性实验及皮肤刺激实验和皮肤致敏性实验方面初步验证了含附子的外用制剂的安全性，为后续这类含有附子的外用制剂的临床应用和研发奠定了实验基础。

Abstract:

[Objective] In order to preliminarily explore the safety of external preparations containing aconite in clinical use，we established a limit test method for diester alkaloids in our hospital’s external preparation containing aconite，Wenyang Fuzheng Ointment. At the same time，we conducted single dose acute toxicity experiments，skin irritation experiments，and skin sensitization experiments. [Methods] An Agilent ZORBAX Extend C₁₈ column（4.6× 250 mm，5 μm）was used，with acetonitrile-tetrahydrofuran（25∶15）as mobile phase A and 0.1 mol/L ammonium acetate solution as mobile phase B，for gradient elution；The flow rate was 1 mL/min，the column temperature was 25 ℃，the injection volume was 10 μL，and the detection wavelength was 235 nm. Using SD rats as experimental subjects，single dose acute toxicity experiments were conducted to observe and record the body weight，behavioral activity，and skin reactions of SD rats，and pathological examination was performed on the skin of each group of rats. Using guinea pigs as experimental subjects，skin irritation experiments and skin sensitization experiments were conducted to observe the local irritation reactions and sensitization effects of intact and damaged skin animals in contact with the test substance. [Results] The detection limits of three reference standards，namely aconitine，new aconitine，and secondary aconitine，were 5.73，5.92 and 5.56 μg/mL，respectively. No aconitine，new aconitine，or secondary aconitine was detected in the three batches of Wenyang Fuzheng Ointment samples and negative samples. The SD rats in the treatment group showed no toxic symptoms or toxic reactions，and pathological examination showed no obvious inflammatory lesions. There was no significant difference compared to the normal control group. The Wenyang Fuzheng Ointment showed no significant skin irritation reactions to guinea pigs with intact or damaged skin after single or multiple doses of medication administration. The Wenyang Fuzheng Ointment had no sensitizing effect on guinea pig skin after external contact. [Conclusion] This study preliminarily verified the safety of external preparations containing aconite from the aspects of limit test of diester alkaloids，single dose acute toxicity experiments，skin irritation experiments，and skin sensitization experiments，laying an experimental foundation for the clinical application and research and development of such external preparations containing aconite in the future.