基于细胞和斑马鱼胚胎试验的5种新疆香阿魏毒性研究

Toxicity study on 5 species of Ferula sinkiangensis K.M.Shen based on cells and zebrafish embryos experiments

目的探讨5种新疆香阿魏(准噶尔阿魏、多伞阿魏、山地阿魏、全裂叶阿魏、荒地阿魏)在体外试验中的毒性及其毒性部位。方法用95%乙醇回流法制备5种香阿魏乙醇提取物。将不同浓度的5种香阿魏乙醇提取物分别加入人胚肾上皮细胞(HKC)、人正常肝细胞(L02细胞)和斑马鱼胚胎培养液中,同时设空白组和正常对照组,计算37.0℃培养24 h的HKC和L02细胞增殖抑制率和培养24、48和72 h斑马鱼胚胎存活率。从5种香阿魏中选择毒性最强的1种,制备其不同极性部位(石油醚、二氯甲烷、乙酸乙酯、水饱和正丁醇和水)提取物进行上述细胞毒性和斑马鱼胚胎毒性试验,以明确其不同部位的毒性。结果细胞毒性试验显示,5种香阿魏乙醇提取物对HKC和L02细胞增殖的半数抑制率(IC50)均以准噶尔阿魏为最低(70.92µg/ml和42.33µg/ml),以多伞阿魏为最高(107.80µg/ml和97.92µg/ml)。斑马鱼胚胎毒性试验显示,1.0µg/ml给药组培养48 h时准噶尔阿魏组即出现胚胎死亡,存活率为70%(14/20),其他4组胚胎存活率均为100%(20/20);10.0µg/ml给药组胚胎培养24 h时多伞阿魏组胚胎存活率为50%(10/20),其他4组胚胎存活率均为0(0/20)。采用准噶尔阿魏的石油醚、二氯甲烷、乙酸乙酯、水饱和正丁醇和水部位提取物进行细胞毒性试验,HKC和L02细胞增殖的IC_(50)均以石油醚部位组为最低(77.35µg/ml和67.73µg/ml),以水部位组为最高(731.52µg/ml和364.09µg/ml);斑马鱼胚胎毒性试验中,石油醚部位组的斑马鱼胚胎存活率最低,给药浓度为3.0µg/ml在72 h时下降到50%,10.0µg/ml在24 h时为50%,48 h和72 h以及在30.0µg/ml给药浓度的所有观察时间胚胎存活率均为0。结论5种新疆香阿魏中准噶尔阿魏的毒性最大,多伞阿魏毒性最小。准噶尔阿魏毒性成分可能多聚集在石油醚部位。

Objective:To explore the toxicities and their toxic sites of 5 species of Ferula sinkiangensis K.M.Shen,including Ferula songarica(F.songarica),Ferula feruloides(F.feruloides),Ferula akitschkensis(F.akitschkensis),Ferula dissecta(F.dissecta),and Ferula syreitschikowii(F.syreitschikowii)in vitro experiments.Methods:Five ethanol extracts of Ferula sinkiangensis K.M.Shen were prepared using 95%ethanol reflux method.The 5 ethanol extracts were added to human embryonic kidney epithelial cells(HKC),human normal liver cells(L02 cells),and zebrafish embryo culture medium at 5 different concentrations,respectively.The relevant blank group and normal control group were set up meanwhile.The inhibition rates of HKC and L02 proliferation at 24 hours of culture under 37.0℃and survival rates of zebrafish embryos at 24,48,and 72 hours of culture under 37.0℃were calculated.The most toxic specie of the 5 Fragrant Ferula L.was selected and the extracts from different polar parts(petroleum ether,dichloromethane,ethyl acetate,water saturated n-butanol,and water parts)were prepared for the above mentioned cytotoxicity and zebrafish embryotoxicity tests to clarify the toxicities.Results:Cytotoxicity tests showed that among 5 ethanol extracts of Fragrant Ferula L.,the half inhibitory concentration(IC_(50))of the F.songarica(70.92μg/ml and 42.33μg/ml)to HKC and L02 cells were lowest,and the IC50 of F.feruloides(107.80μg/ml and 97.92μg/ml)were highest.The zebrafish embryotoxicity test showed that at concentration of 1.0μg/ml,embryonic death occurred when cultured 48 hours in the F.songarica group,with a survival rate of 70%(14/20),while it had a survival rate of 100%(20/20)in the other 4 groups;at concentration of 10.0μg/ml,the embryo survival rate in the F.feruloides group was 50%(10/20)when cultured 24 hours,while it had a survival rate of 0(0/20)in the other 4 groups.Cytotoxicity tests were conducted using extracts from the petroleum ether,dichloromethane,ethyl acetate,water saturated n-butanol,and water parts of F.songarica,the IC50 of the petroleum ether parts(77.35μg/ml and 67.73μg/ml)to HKC and L02 cell proliferation were the lowest and of the water parts(731.52μg/ml and 364.09μg/ml)were the highest.In the zebrafish embryotoxicity test,the survival rate of zebrafish embryos in the petroleum ether group was the lowest.The survival rate was 50%at concentration of 3.0μg/ml for 72 hours′culture and at concentration of 10.0μg/ml for 24 hours′culture.The survival rate was 0 at concentration of 10.0μg/ml for 48 and 72 hours′culture,as well as at 30.0μg/ml at all observation times.Conclusions:Among the 5 Ferula sinkiangensis K.M.Shen,the toxicity of F.songarica is relatively high,while that of F.feruloides is relatively low.The toxic parts of F.songarica may mostly accumulate in petroleum ether parts.