瑞香素的抗疟作用及其高效液相色谱分析方法  

Antimalarial effect and high performance liquid chromatography analyzation of daphnetin

作　　者：王琴美[1] 刘云光[1] 陶奕[1] 王鸣杰[1] 倪奕昌[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,200025

出　　处：《中国药物与临床》2004年第1期20-24,共5页Chinese Remedies & Clinics

基　　金：国家自然科学基金资助项目(39970669)

摘　　要：目的研究中药瑞香素在体外和体内的杀疟原虫裂殖体作用、抗红细胞外期疟原虫作用及其高效液相色谱(HPLC)分析方法.方法在恶性疟原虫FCC1株常规体外培养中测试瑞香素杀裂殖体活性,并按'4 d抑制试验'在感染伯氏疟原虫ANKA株的小鼠中测定不同剂量瑞香素的体内抗疟活性.于癌症研究所(ICR)小鼠腹腔注射约氏疟原虫子孢子后0.5 h灌胃给药,连续4 d.不同剂量的瑞香素及瑞香素与伯氨喹(PQ)配伍用药的抗疟作用分别以第8天ICR小鼠阴性率及第12天或第13天ICR小鼠每千个红细胞被原虫感染数评价.再以高效液相色谱及质谱的方法分析鉴定瑞香素及其两个结构类似物:香豆素,7-羟基香豆素.结果体外试验中瑞香素在1～10μmol/L剂量范围内有明显杀裂殖体作用,而体内试验中按第5天减虫率与感染鼠在30 d内的平均生存天数评价,50或100 mg·kg-1·d-1×4 d瑞香素灌胃以及10、50或100 mg·kg-1·d-1×4 d瑞香素腹腔注射给药在伯氏疟原虫ANKA株感染鼠中的抗疟作用与10 mg·kg-1·d-1×4 d氯喹(CQ)灌胃的疗效相似.瑞香素的剂量范围为每天10～100 mg/kg,连服4 d,第8天原虫阴性小鼠数及第12天红细胞被感染程度与对照组相比差异均无显著性;瑞香素每天50 m/kg和每天PQ 5 mg/kg配伍组的第8天小鼠阴性率与PQ每天10 mg/kg组相当.运用特定的高效液相色谱方法,瑞香素与其结构类似物可达完全的基线分离,分析灵敏度为10～20 ng;经质谱分析确定各化合物的分子量正确.结论瑞香素在体外和体内均有一定的杀裂殖体活性.瑞香素单独用药,无明显抗红细胞外期疟原虫作用,但瑞香素每天50 mg/kg与PQ每天5 mg/kg配伍用药的抗疟效果与PQ每天10 mg/kg相当.高效液相色谱方法可实现对中药瑞香素的准确、精密的定量分析.Objective To investigate the in vitro and in vivo schizonticidal activity of daphnetin,to investigate the activity of daphnetin on the exo-erythrocytic stage of rodent malaria and to develop a high performance liquid chro-matography(HPLC)method for analyzing daphnetin.Methods Schizonticidal activity of daphnetin was tested with an in vitro assay based on the routine in vitro cultivation of Plasmodium falciparum FCC 1 strain.The in vivo antimalarial effects of daphnetin at various dosages were assessed in mice infected with Plasmodium berghei ANKA according to'4-day suppress assay'.Groups of ten male ICR mice were infected by intraperitoneal(ip)injection with sporozoites of Plasmodi-um yoelii.Mice were given daphnetin0.5h postinfection for four consecutive days intragastrically(ig).The effects of daph-netin at various dosages and those of the combination of daphnetin with primaquine(PQ)were assessed by the number of mice with negative Giemsa-stained slides from tail blood on the seventh day after infection and by the average number of red blood cells(RBC)infected in1000RBC observed on the eleventh or twelfth day after infection;daphnetin and its two structure-related compounds:coumarin and umbelliferone were analyzed and identified with HPLC and mass spec-trum(MS)methods.Results In vitro,daphnetin exhibited potent schizontocidal activity comparable to chloroquine(CQ)at the dose range of1~10μmol/L.In vivo,50or100mg·kg -1 ·d -1 ×4d daphnetin ig and10,50or100mg·kg -1 ·d -1 ×4d daphnetin ip showed antimalarial efficacy comparable to CQ10mg·kg -1 ·d -1 ×4d ig in mice infected with P.berghei AN-KA,evaluated by both reduction rate of parasitemia on D 4 and average survival days in30days.Daphnetin exhibited no detectable antimalarial effect on the exo-erythrocytic stage of Plasmodium yoelii,but when daphnetin50mg·kg -1 ·d -1 combined with5mg·kg -1 ·d -1 PQ,the antimalarial efficacy was camparable to PQ10mg·kg -1 ·d -1 igin

关 键 词：瑞香素士 抗疟作用 高效液相色谱 疟原虫 伯氏 约氏 高压液相 HPLC 

分 类 号：R285[医药卫生—中药学]