中药竹叶椒镇痛抗炎物质基础及作用机制研究

Zhuyejiao tablet(from the root of Zanthoxylum armatum DC.), a Traditional Chinese Medicine, possesses an obvious and high efficacy in the treatment of abdominal pain, stomach ache and acute appendicitis pain in clinic. But its substance basis of analgesic activities remain unclear, which seriously restricted the further development of its medicinal value. Our group previous studies indicated that the periphery anti-inflammatory effect might be major responsible for analgesic activity of Zanthoxylum armatum DC. We also identified 27 compounds from the active fractions. In this study, Z. armatum in Longnan area of Gansu Province is uesd as raw materials, we systematically and deeply study substance basis of analgesic and anti-inflammatory effets. These major chemical constituents in active fractions are quickly detected by using hyphenated chromatographic techniques, and rapidly isolated and purificated by using semi-preparative high preformance liquid chromatography, high-speed countercurrent chromatography and conventional phytochemical separation technique. With effects of main compounds on inflammatory pain as a breakthrough point. The anti-inflammatory activities of main compounds obtained from active fractions are evaluated by inflammatory model in vitro using high-throughput and multi-target methoed, and then the effects of these compounds with high activities on the expression of iNOS and COX-2, on activation of NF-κB and MAPK signaling pathways，and on the effect of TRPV1(a key target of peripheral inflammatory pian) are also investigated to elucidate the possible mechanisms. This study provide a scientific basis for development and utilization of Z. armatum, and contribute to development of the modernization of Traditional Chinese Medicine of Gansu Province.

中药竹叶椒片在临床上对胃脘痛、腹痛及急性阑尾炎痛有显著疗效，但是由于其镇痛物质基础不明确，严重影响了药用价值的进一步开发。本课题组前期实验证实竹叶椒镇痛作用主要涉及外周抗炎镇痛，并从中鉴定了27种单体化合物。 本项目拟对甘肃陇南产竹叶椒镇痛抗炎物质基础进行系统研究。采用色谱联用技术快速检测活性组分群中主要化学成分，使用半制备高效液相色谱、高速逆流色谱以及常规分离手段快速分离纯化主要成分。以其对炎性疼痛的作用为切入点，通过体外细胞试验高通量、多靶点评价主要成分抗炎活性。考察具有显著活性的化合物对COX-2、iNOS蛋白表达、对NF-κB和MAPK两条通路的激活作用，并探讨这些化合物对外周炎性疼痛关键靶点TRPV1离子通道的影响，进而阐述其可能的镇痛抗炎作用机制。本项目为竹叶椒的开发利用提供科学依据，为甘肃省中药现代化快速发展出做贡献。

竹叶椒在临床上对胃脘痛、腹痛及急性阑尾炎痛有显著疗效。课题组前期实验证实竹叶椒镇痛作用主要涉及外周抗炎镇痛。本项目期望通过研究进一步阐述竹叶椒镇痛抗炎的物质基础及其可能机制。一方面，对竹叶椒活性部位化学成分进行分离、鉴定。通过柱层析以及各种现代分离纯化技术手段 (半制备液相色谱，逆流色谱) 分离、鉴定了包括生物碱、木脂素、苯酚苷等61种化合物，其中3个新化合物，16个为芸香科中首次分离，3个从花椒属首次分离，16个首次从该植物中分离。另一方面进行了单体化合物的抗炎作用及其机制研究。针对木脂素和生物碱为主的八种量大的单体成分开展了对LPS诱导RAW264.7细胞的炎性介质释放影响的实验。结果表明，这些化合物对NO，IL-6，TNF-α，PGE2的一种或者多种的生成有一定的抑制作用，其中都对PGE2的产生有显著的抑制作用。Western blot法检测结果显示部分单体成分降低LPS 致RAW264.7 巨噬细胞炎症反应作用机制与调控MAPK和NF-κB信号通路有关；同时竹叶椒提取物镇痛抗炎作用也与调控TRPV1离子通道相关。本研究为竹叶椒药材质量标准制定提供实验依据，也为中药竹叶椒片的临床合理、有效使用以及竹叶椒新药开发提供依据。