基于化学成分差异的不同干燥方式熊胆粉镇痛作用研究

目的采用超高效液相-三重四极杆-线性离子阱质谱(UPLC-QTRAP-MS)方法鉴定常压及减压干燥熊胆粉的化学成分,明确其差异成分,并以网络药理学理念为指导,探索熊胆粉镇痛作用机制。方法基于UPLC-MS/MS检测平台、数据库以及多元统计分析相结合的手段研究熊胆粉的化学成分差异;借助PharmMapper、Uniprot、GeneCards数据库及文献,获取熊胆粉成分和病毒性肝炎、胆囊炎及冠心病的作用靶点,并采用动物试验对其药效进行验证;对疾病靶标-药物靶标集做拓扑分析获得核心靶标,并进行基因本体(GO)功能和基因与信号通路分析(KEGG)通路富集,通过Cytoscape3.8.2软件构建成分-靶标-通路网络图。结果常压干燥、减压干燥熊胆粉共有成分721个,差异成分8个(L-焦谷氨酸、鸟苷5′-单磷酸水合物、5,6-二氢胸腺嘧啶、7-甲基黄嘌呤、左旋甲状腺素、甲基间酪氨酸、大黄素8-葡萄糖苷、益母草碱),搜索得到差异成分潜在靶点共1605个;检索得到病毒性肝炎靶点7444个、胆囊炎靶点636个、冠心病靶点7563个;熊胆粉对病毒性肝炎、胆囊炎及冠心病的共同靶点有282个,GO功能富集分析得到GO条目759个(P0.05),其中生物过程(BP)条目717个,细胞组成(CC)条目21个,分子功能(MF)条目21个;熊胆粉中的鸟苷5′-单磷酸水合物、甲基间酪氨酸、左旋甲状腺素及大黄素8-葡萄糖苷等成分可能通过二氢硫辛酸脱氢酶(dihydrolipoyl dehydrogenase,DLD)、过氧化氢酶(atalase,CATC)及一氧化氮合酶2(nitric oxide synthase 2,NOS2)等靶点,调节代谢通路和癌症通路等信号通路发挥其镇痛作用。药效试验结果表明常压、减压干燥熊胆粉均对热板及醋酸所致小鼠疼痛有抑制作用,且减压组镇痛作用强于常压组,有显著性差异(P0.05)。结论干燥方式使得熊胆粉化学成分变异,从而导致其镇痛作用差异,初步探索了熊胆粉镇痛作用的机制,建议大生产中采用减压干燥制备熊胆粉,为熊胆粉合理应用提供了一定依据。

Analgesic effects of bear bile powder by different drying methods based on differences in chemical components

Objective To identify the chemical components of normal pressure and reduced pressure dried bear bile powder by UPLC-QTRAP-MS method, clarify the differential components, so as to explore the analgesic mechanism of bear bile powder with the concept of network pharmacology as a guide. Methods Based on the combination of UPLC-MS/MS detection platform, database and multivariate statistical analysis, the composition differences of bear bile powder were studied. With the help of PharmMapper, Uniprot, GeneCards databases and literatures, the composition of bear bile powder and targets related to viral hepatitis, cholecystitis and coronary heart disease were obtained, and its efficacy was verified by animal experiments; the core target of the "disease target-drug target set" was obtained by topological analysis, and the GO function and KEGG pathway enrichment were carried out, Cytoscape3.8.2 software was use to construct a "component-target-pathway" network diagram. Results A total of 721 common components in bear bile powder by atmospheric pressure drying and vacuum drying methods. There were eight differential composition including L-pyroglutamic acid, guanosine 5''-monophosphate hydrate, 5,6-dihydrothymine, 7-methyl xanthine, levothyroxine, methyl-tyrosine, emodin 8-glucoside, leonurine. A total of 1605 differential composition potential targets, 7444 viral hepatitis targets, 636 cholecystitis targets, 7563 coronary heart disease targets were retrieved. There were 282 common targets of bear bile powder against viral hepatitis, cholecystitis and coronary heart disease, and 759 GO items were obtained by GO function enrichment analysis (P<0.05), including 717 BP items, 21 CC items and 21 MF items. Guanosine 5''-monophosphate hydrate, methyl-tyrosine, levothyroxine and rhein 8-glucoside in bear bile powder may exerted its analgesic role by regulating signaling pathways such as metabolic and cancer pathways through dihydrolipoyl dehydrogenase (DLD), catalase (atalase, CATC), nitric oxide synthase 2 (NOS2). The efficacy test results showed that both atmospheric pressure and decompression drying bear bile powder had inhibitory effect on the pain induced by hot plate and acetic acid in mice, and there was significant difference between atmospheric pressure and decompression group (P<0.05), and the analgesic effect of decompression group was stronger than atmospheric pressure group. Conclusion This study shows that the drying method changes the chemical composition of bear bile powder, which leads to differences in its analgesic effects. The mechanism of the analgesic effect of bear bile powder is initially explored, and it is recommended to use reduced pressure drying to prepare bear bile powder in large-scale production, which provides a certain basis for reasonable application of bear bile powder.