基于模拟炮制研究黄草乌碱甲不同炮制方法的转化产物及其毒效变化

Studies on the transformation products of vilmorrianine A under different processing methods based on simulated processing and their toxicities and analgesic activities

关键词:  黄草乌碱甲  炮制  油炸  pyrovilmorrianine A  镇痛  Prodynorphin

Key words:  vilmorrianine A  processing  frying  pyrovilmorrianine A  analgesic  prodynorphin

摘要:

摘要: 目的 研究黄草乌碱甲油炸（油浴模拟）炮制产物结构，并比较其与水煮炮制产物的毒性和镇痛活性差异。方法 采用HPLC法筛选出黄草乌碱甲主要炮制产物转化的温度和时间参数，通过制备薄层和核磁共振、质谱技术，分离鉴定出黄草乌碱甲油炸的主要转化产物；利用大鼠心肌细胞和神经细胞比较原型成分和转化产物的毒性强弱，利用大鼠小胶质细胞比较不同炮制产物的镇痛活性。结果 通过比较不同炮制温度、时间样品的HPLC色谱图，筛选出黄草乌碱甲油炸炮制主要产物在180℃-20min时含量达到最高；分离、鉴定出黄草乌碱甲的油炸产物pyrovilmorrianine A；体外细胞毒性实验结果表明，黄草乌碱甲水煮炮制产物南乌碱乙和Ezochasmaine的心肌和神经毒性依次降低，而油炸产物pyrovilmorrianine A毒性与南乌碱乙相当。对强啡肽A基因表达影响实验结果显示，所有药物均能增加HAPI细胞中Prodynorphin mRNA表达量，其中黄草乌碱甲和南乌碱乙组Prodynorphin mRNA表达量与正常组相比有显著性差异（P＜0.001），且优于pyrovilmorrianine A（P＜0.05）。 结论 黄草乌碱甲油炸过程中较为稳定的转化产物是pyrovilmorrianine A，能够达到炮制减毒的目的，且保留了一定的镇痛活性。 关键词：黄草乌碱甲；炮制；油炸；pyrovilmorrianine A；镇痛；Prodynorphin

Abstract:

ABSTRACT: OBJECTIVE To study the structure of transformation product of vilmorrianine A by fried processing (oil-bath simulation) and to compare its difference in toxicity and analgesic activity with that of the product under boiled processing. METHODS The temperature and time parameters for the frying transformation of vilmorrianine A were screened by HPLC analysis, and the main transformed product was obtained through prepared thin layer chromatography (TLC) and identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques. The toxicities between the prototypical component and its boiled and fried transformation products were compared through cytotoxicity assay on H9c2 and PC12 cells in vitro, and their analgesic activities was determined by the expression of prodynorphin mRNA on HAPI microglia after drug administration. RESULTS The main fried processing product of vilmorrianine A, which was isolated and named pyrovilmorrianine A, could reach the highest content at 180℃-20 min based on HPLC analysis of the samples under different processing temperatures and times. The cardiotoxicity and neurotoxicity of pyrovilmorrianine A and austroconitine B were virtually the same, while Ezochasmaine exhibited no toxicity by cytotoxicity assay in vitro. The results of the analgesic experiment indicated that all the drugs could increase the expression of prodynorphin mRNA in HAPI cells, in which vilmorrianine A and austroconitine B had significant differences compared with normal group(P<0.001), while pyrovilmorrianine A showed remarkable difference (P<0.05). CONCLUSION Pyrovilmorrianine A was a stable transformation product of vilmorrianine A under fried processing, which method could achieve the purpose of toxicity reduction and also retain the analgesic activity partly. KEY?WORDS: vilmorrianine A; processing; frying; pyrovilmorrianine A; analgesic; prodynorphin