苍耳子醇提取液的乙酸乙酯、正丁醇、水萃取物对大鼠肝脏的毒性作用

Toxic effects of ethyl acetate,n-butanol,and water extracts from alcohol extractions of cocklebur fruit on liver in rats

目的:研究苍耳子65%乙醇提取液的乙酸乙酯、正丁醇和水萃取物对大鼠肝脏的毒性作用,为探讨苍耳子的毒性成分提供实验依据。方法:苍耳子碎粉22kg,用8倍量乙醇浸泡6h回流加热提取2h,共提取2次,合并提取液,减压回收乙醇至无醇味,浓缩,浓缩液用石油醚萃取,回收石油醚;依次用乙酸乙酯、正丁醇萃取,回收有机溶剂,水层减压干燥。取苍耳子干燥乙酸乙酯萃取物4.8g,正丁醇萃取物24g和水萃取物56g,各加含3%吐温-80生理盐水2000mL,分别配成浓度为0.0024、0.0120和0.0280g/mL的混悬液。将SPF级雄性大鼠40只随机分成4组,每组10只。3个给药组大鼠分别用乙酸乙酯、正丁醇和水萃取物的混悬液2.5mL灌胃,2次/d(剂量分别为每天0.06g/kg、0.3g/kg、0.7g/kg),空白对照组给予等体积的吐温80生理盐水灌胃,2次/d,均连续灌胃28d。观察给药后大鼠外观、饮食和活动情况;并于给药前和给药开始后7、14、21、28d称体重。第29天检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(AKP)、总胆红素(TBil)和直接胆红素(DBil)的水平;之后,处死大鼠,计算肝脏指数,并进行肝脏组织学形态观察。结果:水萃取物组大鼠给药后7d皮毛无光泽,活动及摄食量减少。正丁醇和水萃取物组大鼠给药后14、21d出现皮毛枯黄,精神萎靡,28d出现竖毛,倦卧少动。乙酸乙酯萃取物组大鼠未见明显改变。正丁醇萃取物组大鼠给药开始后21、28d体重分别为(240.6±24.1)和(255.1±21.3)g,水萃取物组大鼠给药开始后14、21、28d体重分别为(214.4±20.5)、(230.7±21.2)和(239.1±18.5)g,均明显低于同时间点空白对照组大鼠体重[分别为(251.7±27.2)、(280.7±38.2)和(306.2±36.5)g,(P<0.05,P<0.01)]。正丁醇萃取物组AST为(112.6±24.3)U/L,明显高于空白对照组[(79.9±20.4)U/L,P<0.01];水萃取物组ALT、AST和AKP分别为(51.1±3.9)、(112.9±16.6)和(198.4±41.8)U/L,明显高于空白对照组[(44.3±6.2)、(79.9±20.4)和(152.2±39.9)U/L,(P<0.01,P<0.05)];正丁醇萃取物组和水萃取物组肝脏指数分别为4.71±0.89和5.80±0.64,明显高于空白对照组3.14±0.33(P<0.01)。各给药组大鼠的TBil和DBil值有所增加,但与空白对照组比较差异无统计学意义(P>0.05)。光镜观察可见正丁醇萃取物和水萃取物组大鼠肝细胞间隙增大、细胞核溶解、炎细胞浸润等病理改变。结论:苍耳子乙醇提取液的正丁醇萃取物及水萃取物对大鼠具有明显肝毒性作用。

Objective：To study the toxic effects of ethyl acetate,n-butanol,and water extracts from 65% alcohol extractions of cocklebur fruit on liver in rats in order to provide a basis for a further investigation of the toxic constituents of cocklebur fruit（Fructus Xanthii）.Methods：Twenty-two kilogram of ground cocklebur fruit was macerated in 8-fold amounts of 65% alcohol for 6 hours,and then was extracted by heating under a reflux condenser for 2 hours.The total number of extracting was two times.The alcohol extractions were combined,and the alcohol were retrieved with vaccum distillation till no smell of alcohol.The extractions were concentrated further.The concentrated solution was extracted with petroleum ether,followed by retrieval of petroleum ether.The extractions were extracted with ethyl acetate and n-butanol in turn.The solvents were retrieved and then the water layer was evaporated to dryness with vaccum evaporator.Four point eight gram of ethyl acetate extracts,24 g of n-butanol extracts,and 56 g of water extracts were mixed with 2000 mL of normal saline solution containing 3% Tween-80,respectively.The concentration of the suspensions were 0.0024,0.012,and 0.028 g/ml,respectively.Forty SPF male rats were randomly divided into 4 groups and each group comprised 10 rats.The three drug-exposed groups were gavaged with 2.5 mL of ethyl acetate,n-butanol,and water extract suspensions twice daily,respectively（the dosage was 0.06 g/kg,0.3 g/kg,0.7 g/kg daily,respectively）.The empty control group was gavaged with same volume of normal saline solution containing Tween-80 twice daily.The duration of gavage was 28 days.The appearance,diet,and activities of rats were observed.The body weight of the rats were weighted before drug administration and 14,21,and 28 days after drug initiation.On day 29,serum levels of ALT,AST,AKP,TBil,and DBil were measured.Subsequently,the rats were sacrificed and the liver index was calculated and changes in histomorphology were observed.Results：The rats in the water extract group developed lusterless fur and decrease in diet and activities 7 days after drug initiation.The rats in the n-butanol extract group developed lusterless fur and listlessness 14 and 21 days after drug initiation as well as erect hair and lassitude 28 days after drug initiation.The rats in the ethyl acetate extract group had no marked changes.The body weight of rats in the n-butanol extract group were（240.6±24.1） and（255.1±21.3）g 21 and 28 days after drug initiation,respectively.The body weight of rats in the water extract group were respectively（214.2±20.5）,（230.7±21.2）,and（239.1±18.5）g 14,21,and 28 days after drug initiation,and their body weight were all lower than that of the rats in the empty control group at the same time points [（251.7±27.2）,（280.7±38.2）,and（306.2±36.5）g,（P0.05,P0.01）].The AST levels in the n-butanol extract group was（112.6±24.3）U/L,which was marked higher than that [（79.9±20.4）U/L] in the empty control group（P0.01）.The levels of ALT,AST,and AKP in the water extract group were respectively（51.1±3.9）,（112.9±16.6）,and（198.4±41.8）U/L,which were marked higher than those [（44.3±6.2）,（79.9±20.4） and（152.2±39.9）U/L] in the empty control group（P0.05,P0.01）.The liver indexes in the ethyl acetate extract and water extract groups were respectively 4.71±0.89,and 5.80±0.64,which were marked higher than that（3.14±0.33） in the empty control group（P0.01）.The levels of TBil and DBil in all the drug-exposed groups increased,but there were no statistically significant differences（P0.05）.In the n-butanol extract and water extract groups,the pathological changes such as enlarged hepatic cell space,karyolysis,and inflammatory cell infiltration were observed under the light microscope.Conclusion：The n-butanol extracts and water extracts from alcohol extractions of cocklebur fruit have marked hepatotoxicity to rats.