芍药苷-6-O'-苯磺酸酯在大鼠体内代谢及其活性代谢物研究

Total glucosides of paeony(TGP) ,extracted from the roots of Paeonia lactiflora Pall, which possess widely pharmacological activities, has anti-inflammatory and immunomodulatory activity. TGP has obviously therapeutic action,fewer side effects and slow effects in treating rheumatoid arthritis in clinic .Paeoniflorin, as the the main active ingredients in TGP, is assimilated poorly after oral administration, which causes the slowly effects of TGP. The poor lipophilicity of Pae was one of the main reasons for its poor absorption. Our previous studies have found that Paeoniflorin-6-0'-benzenesulfonate(CP-25), derived from structure modification of Pae,displayed the superiority in increasing absorption, improving anti-inflammatory and immunomodulatory actions ,as well as more rapid anti-inflammatory effects compared with those of TGP and Pae. Furthmore,studies have suggest that the anti-inflammatory and immunomodulatory activity of CP-25 was probably associated with it's active metabolites in vivo. However, the active metabolite of Pae and it's tissue distribution features are poorly understood. Based on previous works, the present study is mainly to investigate the metabolic characteristics of CP-25,the metabolite's distribution and its anti-inflammatory & immunomodulatory activity in normal and collagen induced arthritis rats by using selective inhibitors of hepatic microsomal enzyme,solid Phase Extraction and applying flow cytometry & LC-MS, all of which provide the experiment evidences to guide the further researches on CP-25 anti-inflammatory & immunomodulatory actions and its mechanisms.

芍药药理作用广泛，其根部提取的白芍总苷（TGP）具有抗炎免疫调节活性。TGP在临床治疗类风湿关节炎疗效肯定，不良反应小，但起效较慢。芍药苷（Pae）是TGP主要活性成分，其口服吸收较差致TGP起效慢。Pae吸收差主要原因之一是其亲脂性较弱。课题组前期研究发现，Pae结构修饰物Pae-6-O'-苯磺酸酯（代号CP-25）口服吸收较好，其抗炎免疫作用的强度和起效时间明显好于TGP和Pae，研究还表明，CP-25的抗炎免疫调节作用可能主要来源于其体内的代谢产物。但CP-25代谢物活性及组织分布特点尚不清楚。本项目在以往工作基础上，采用液质联用、流式细胞术等，利用肝药酶选择性抑制剂和固相萃取柱等工具，研究CP-25在正常大鼠和胶原性关节炎大鼠体内代谢特点，探寻其体内代谢物和组织分布特点以及抗炎免疫调节活性，为指导CP-25的抗炎免疫调节作用及其机制的深入研究提供实验依据。

白芍总苷（TGP）在临床治疗类风湿关节炎疗效肯定，不良反应小，但起效较慢。芍药苷（Pae）是TGP主要活性成分，其口服吸收较差致TGP起效慢。研究发现Pae结构修饰物Pae-6-O'-苯磺酸酯（CP-25）口服吸收较好，其抗炎免疫调节作用明显强于TGP和Pae。当前研究主要考察CP-25在大鼠体内主要代谢途径、活性代谢物，同时明确CP-25在大鼠体内的吸收、分布、排泄等特点。结果表明CP-25灌胃给药后，在大鼠体内可检测到芍药苷、芍药苷单甲基化产物、芍药苷双甲基化产、芍药苷脱醛基化产物、芍药苷元等产物，代谢物活性大小依次为芍药苷>芍药苷元>单甲基化Pae>双甲基化Pae, 其中活性代谢物含量最大为芍药苷；CP-25给药能显著降低大鼠血清炎性细胞因子含量，降低Th1、Th2、Th17比例，提高Treg比例; CP-25、 CP-25代谢物和Pae代谢物体外给药能显著降低AA大鼠CD3+T细胞上清液IL-2，IL-4和IL-17的含量水平，提高IL-10含量水平，降低Th1、Th2、Th17比例，提高Treg的比例；CP-25在小肠全肠段均有吸收, 且吸收速率参数显著高于Pae；CP-25在大鼠、人以及犬血浆中均具有较高的血浆蛋白结合率，在浓度为40μg•mL -1 时，CP-25在大鼠、人、犬血浆中结合率依次为(92.23±0.23)%、(95.28±0.41)%、(93.62±0.17)%；CP-25口服给药后在大鼠主要脏器，如心、脾、肝、肺、肾和胃中均有较高的药物浓度，且相应浓度显著高于Pae；0-96h内，CP-25在雄性大鼠粪便累积排泄量为1923.20 μg，占总给药量 18.40%，雌性大鼠粪便累积排泄量为 1442.34 μg，占总给药量 14.04%。CP-25 在大鼠体内以粪便排泄为主，尿液、粪便和胆汁中的排泄情况具有明显的雌性差异；食物摄取会增加CP-25在大鼠体内的吸收，减慢其清除率。疾病的状态会降低CP-25在大鼠体内的吸收；CP-25在单核细胞和滑膜细胞吸收程度远多于Pae。本课题首次明确CP-25比Pae具有优异的体内过程，从动力学角度揭示CP-25抗炎免疫调节活性强于Pae，CP-25是具有自主知识产权的新型活性单体，有潜在医用价值。当前研究首次探讨CP-25体内过程和药动学特点，对深入研究其药理作用以及作用机制具有重要理论意义.