川芎提取物的分离及其对大鼠心肌缺血/再灌注损伤的药理作用

Separation of Extracts from Chuanxiong Rhizoma and the Pharmacological Effects on Myocardium of Ischemia/Reperfusion Injury in Rats

关键词:  川芎提取物  川芎嗪  心肌缺血  再灌注损伤  液相色谱  高速逆流色谱  中药指纹图谱

Key words:  Chuanxiong Rhizoma extract  ligustrazine  myocardial ischemia  reperfusion injury  liquid chromatography  high-speed countercurrent chromatography  Chinese herbal fingerprint spectrum

摘要:

目的 评价采用大孔树脂方法分离的10种川芎组分对大鼠心肌缺血/再灌注损伤（myocardialischemia/reperfusion injury，I/R）后血栓形成、血小板功能和血管内皮因子的药理作用。方法 川芎饮片采用水提取后醇沉方法，分别采用大孔树脂分离和高速逆流色谱法分离得到10种不同的川芎组分。清洁级Wistar大鼠，♀♂各半，采用（按0.009 mL·g^-1体质量腹腔注射4%水合氯醛麻醉）结扎冠状动脉左前降支建立I/R模型。于结扎成功后（心电图显示）10 min时大鼠股静脉分别注射实验用药，川芎组分给药剂量为20 mg·kg^-1；阳性对照组给予川芎嗪（盐酸四甲基吡嗪二水化合物），给药剂量为20 mg·kg^-1；假手术组和模型组给予等量生理盐水，每组10只。于缺血40 min再灌注120 min时取血，测定体外血栓形成的长度、湿重和干重，全血分离后放射免疫测定血浆内皮素-1含量，硝酸盐还原法测定血浆一氧化氮（NO）含量，比浊法测定血小板最大聚集率。结果 与假手术组比较，模型组的血栓长度、湿重和干重均明显增加（P<0.05或P<0.01）；与模型组比较，川芎组分②、③、⑤号组对血栓长度、湿重和干重均有不同程度的降低（P<0.05或P<0.01）。血管内皮因子和血小板聚集的结果表明，与假手术组比较，模型组内皮素-1水平明显上升（P<0.01），NO水平有降低趋势；与模型组比较，川芎组分⑤、⑥、⑧和⑩号组对大鼠血浆内皮素-1水平具有显著降低作用（P<0.05或P<0.01），对大鼠NO水平具有显著升高作用（P<0.05或P<0.01）；川芎组分①至⑩号组均有显著降低大鼠血小板聚集率的作用（P<0.01）。结论 通过HPLC和高速逆流色谱仪建立的川芎全组分指纹图谱具有良好的对应性，分离的川芎提取物的药理作用具有差异性且作用靶点不同，具有开发新产品的药效特征和研究的示范性。

Abstract:

OBJECTIVE To evaluate the pharmacological effects of ten kinds of extracts from Chuanxiong Rhizoma, with macroporous resin method, on thrombosis, the platelet function and vascular endothelial factors in rat myocardium of ischemia/reperfusion injury(I/R). METHODS Ten different Chuanxiong Rhizoma component were extracted from Chinese herbal pieces Chuanxiong Rhizoma through water decoction alcohol sinking method and macroporous resin separation as well as high-speed countercurrent chromatography. Wistar rats of clean grade were randomly divided. I/R was established by ligation of coronary artery left anterior descending branch. Ten minutes after successful ligation, the different experimental drugs (20 mg·kg^-1 Chuanxiong Rhizoma component) were injected into rats from femoral vein; the rats in positive control group were injected with ligustrazine injection(including with 20 mg·kg^-1 tetramethylpyrazine hydrochloride dehydrate). The rats in sham group and the model group were injected with equivalent dosage of saline, ten rats in each group. The blood samples were extracted after 40 min myocardial ischemia and 120 min myocardial reperfusion, thrombus length, wet weight and dry weight of thrombosis in vitro were examined, plasma endothelin 1 was tested by radiation immunoassay method, plasma NO was tested by nitrate reduction method, the maximum platelet aggregation rate was tested by turbidimetric method. RESULTS Compared with sham control group, thrombus length, wet weight and dry weight of thrombosis in model group increased significantly (P<0.05 or P<0.01); Compared with model group, thrombus length, wet weight and dry weight of thrombosis in Chuanxiong Rhizoma component ② group and ③ group as well as ⑤ group had different degrees of reduction (P<0.05 or P<0.01). Compared with sham control group, endothelin 1 level in model group increased significantly(P<0.01), NO level in model group decreased. Compared with model group, endothelin 1 level in Chuanxiong Rhizoma component ⑤ group, ⑥ group,⑧ group and ⑩ group had significant reduction(P<0.05 or P<0.01), and NO level had significant increase(P<0.05 or P<0.01); and platelet aggregation rate in Chuanxiong Rhizoma component ① group and ⑩ group had also significant decrease(P<0.05 or P<0.01). CONCLUSION Chuanxiong Rhizoma fingerprint spectrum established with HPLC and high speed countercurrent chromatography has a good correspondence. Different pharmacological effects of Chuanxiong Rhizoma component has the differentiation and specific targeting, which has the pharmacodynamic characteristics of developing new products and demonstration study.