苍耳子水萃取物对大鼠肝脏毒性作用的实验研究

Experiment study on hepatotoxicity induced by the water extract of cocklebur fruit in rats

目的:观察不同浓度苍耳子水萃取物对大鼠肝脏功能与形态的影响,为临床安全使用苍耳子提供实验依据。方法:取苍耳子碎粉22 kg,用65%乙醇提取,用石油醚、乙酸乙酯、正丁醇萃取,回收有机溶剂后得到水萃取物743.6 g。水提取物用含3%吐温80生理盐水配制成28.00 mg/mL和1.12 mg/mL混悬液。144只SPF级雄性SD大鼠,体重(200±10)g,用随机数字表法分为苍耳子水萃取物高剂量组、低剂量组和对照组,每组48只。高、低剂量组分别给予28.00 mg/mL和1.12 mg/mL苍耳子水萃取物混悬液2.5 mL每日2次灌胃,对照组以3%吐温80生理盐水2.5 mL灌胃;均连续28 d。分别于给药开始后7、142、12、8 d和停药后71、4 d观察大鼠被毛、摄食量和活动情况,测量大鼠体重,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)水平,取肝脏计算肝脏指数(肝重/体重×100%),进行肝脏病理分级和评分。结果:苍耳子水萃取物高剂量组大鼠给药28 d内先后出现竖毛、脱毛、疲倦少动、摄食量减少、对外界刺激反应缓慢等症状;低剂量组仅少数大鼠出现少动喜卧、精神萎靡等症状。停药14 d后高剂量组大鼠活动量和摄食量均增加,少动喜卧症状减轻,被毛光泽度恢复。给药212、8 d和停药7 d时,高剂量组大鼠体重([10.5±4.2)(、10.2±3.1)(、12.1±4.5)g]明显低于低剂量组([15.3±2.1)(、16.7±4.2)(、17.6±3.2)g]和对照组([18.6±3.4)(、20.5±5.2)(、19.6±2.5)g],血清ALT([42.8±3.2)(、49.3±5.9)(、43.2±3.2)U/L]和AST([108.8±11.7)(、119.8±16.3)(、110.5±17.6)U/L]水平均明显高于低剂量组([33.7±4.60)(、34.8±5.4)(、33.5±4.9)U/L(,94.7±12.6)(、95.4±10.7)(、96.8±12.8)U/L]和对照组([31.2±4.3)(、32.5±6.3)、(31.7±5.7)U/L(,92.3±16.2)(、92.9±20.3)(、93.7±16.3)U/L;]血清AKP([197.2±25.7)(、210.4±41.8)(、189.3±17.6)U/L]水平明显高于低剂量组([174.3±22.6)(、175.3±27.4)(、176.3±22.8)U/L]和对照组[(171.3±25.6)、(172.5±28.7)(、172.8±26.3)U/L;]给药21、28 d和停药7 d时高剂量组肝脏指数(4.2±0.45、.0±0.7、4.9±0.3)明显高于低剂量组(3.4±0.63、.6±0.53、.9±0.6)和对照组(3.0±0.4、3.2±0.3、3.4±0.5);给药14、21、28d和停药7d时高剂量组大鼠肝脏病理评分中位数分别为(1.0、1.5、3.0、1.5),明显高于低剂量组(0.2、0.5、0.9、0.5)和对照组(0.1、0.1、0.2、0.1)。以上各指标高剂量组与低剂量组、对照组间差异均有统计学意义(P<0.05,P<0.01);而低剂量组与对照组间差异均无统计学意义(均P>0.05)。结论:苍耳子水萃取物致大鼠肝损害与其浓度和作用时间有关;大剂量和长时间用药可加重肝损害。

Objective： To observe the effects of different concentrations of water extracts of cocklebur fruit on liver function and morphology in rats,in order to provide experimental evidence for safe use of cocklebur fruit in clinical practice.Methods： Twenty-two kilogram of ground cocklebur fruit was extracted with 65% alcohol.The alcohol extractions was extracted with petroleum ether,ethylacetate and n-butanol in turn.The organic solvents were retrieved.Seven hundred and forty-three point six gram of water extracts of cocklebur fruit was obtained.The water extracts was mixed with normal saline solution containing 3% Tween-80,and two different concentrations of suspensions（28.00 mg/mL and 1.12 mg/mL） was prepared.One hundred and forty-four SPF SD male rats,weighing（200±10）g,were divided into the high-dose group,the low-dose group and the control group by random-digit table method,each group comprised 48 rats.The 2.5mL of 28.00 g/m L suspension and 2.5mL of 1.12 g/mL suspension were perfused into the rats ＇stomach in the high-dose and the low-dose groups twice daily,respectively.The rats in the control group was perfused with same volume of normal saline solution containing 3% Tween-80 twice daily.The duration of gavage feedin g was 28 days.The appearance,intake of food,and activities of rats were observed.The body weight of the rats were weighed,the serum levels of ALT,AST and AKP were measured 7,14,21,and 28 days after drug initiation and 7,14 days after drug withdrawal,respectively.Subsequently,the rats were sacrificed,the l iver index was calculated（liver weight/body weight×100%） and the liver histopat hology was scored and graded.Results： During a 28-day period of medication,the rats in the high-dose group developed erect hair,epilation, lassitude,anorexia,and were unresponsive to stimuli.Only a few rats in the low-dose group developed lassitude and listlessness.Fourteen days after drug withdrawal,the rats＇activities and intake of food were increased,degree of las situde improved and the gloss of coat normalized in the high-dose group.Twenty-one and 28 days after drug initiation and 7 days after drug withdrawal,the body weight of rats in the high-dose group[（10.5±4.2）,（10.2±3.1）,（12.1±4.5） g ] were markedly lower than those in the low-dose group[（15.3±2.10）,（16.7 ±4.2）,（17.6±3.2） g] and the control group[（18.6±3.4）,（20.5±5.2）,（19.6±2.5）g],the serum levels of ALT[（42.8±3.2）,（49.3±5.9）,（43.2±3.2）U/L],AST[（108.8±11.7）,（119.8±16.3）,110.5±17.6）U/L] were markedly higher than those in the low-dose group [（33.7±4.6）,（34.8±5.4）,（33.5±4.9）U/L,（94.7±12.6）,（95.4±10.7）,（96.8±12.8）U/L]and the control group[（31.2±4. 3）,（32.5±6.3）,（31.7±5.7）U/L,（92.3±16.2）,（92.9±20.3）,（93.7±16.3）U/L], the serum levels of AKP [（197.2±25.7）,（210.4±41.8）,（189.3＋17.6）U/L] were markedly higher than those in the low-dose group [（174.3±22.6）,（175.3±27.4）,（176.3±22.8）U/L]and the control group [（171.3±25.6）,（172.5±28.7）,（172.8 ±26.3）U/L].Twenty-one and 28 days after drug initiation and 7 days after with drawal,the liver indexes in the high-dose group（4.2±0.4,5.0±0.7,4.9±0.3）were markedly higher than those in the low-dose group and the control group[（3.4±0.6,3.6±0.6,3.9±0.6）,（3.0±0.4,3.2±0.3,3.4±0.5）].Fourteen,21 a nd 28 days after drug initiation and 7 days after drug withdrawal,the median score of liver pathology in the high-dose group（1.0,1.5,3.0,1.5） were marked ly higher than those in the low-dose group（0.2,0.5,0.8,0.5） and the control group（0.1,0.1,0.2,0.1）.The differences of each index mentioned above betwee n the high-dose group and the low-dose group as well as the control group were statistically significant（P0.05,P0.01）,but the differences betw een the low-dose group and the control group were no statistically significant（ P0.05）.Conclusion： The rat＇s liver damage due to the water extract of cocklebur fruit is related to its dosage and duration of drug use;high-dose and prolonged use may increase liver damage.