车前草和杜仲提取物对肝损伤的保护作用
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摘要
肝脏是人体内新陈代谢的中心站,是人体重要的解毒器官,是许多药物、外源性化学物质、氧化性毒物的重要靶点。肝损伤是多种严重肝脏疾病的发生、发展及最终走向肝功能衰竭的始动环节和共同途径。各种有害因素所致的肝损伤可表现为肝坏死、脂肪肝、胆汁郁积、肝纤维化、肝硬化及肝癌等。
本实验中我们通过小鼠体内试验研究车前草和杜仲提取物对四氯化碳(CCl4)和硫代乙酰胺(TAA)急性肝损伤的保护作用。通过体外测定中药提取物的抗氧化活性,以及血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)的活性,白蛋白(ALB)、总蛋白(TP)、胆固醇(CHO)、总胆红素(TB)、葡萄糖(GLU)的含量和肝组织中蛋白的表达变化探讨其保护机制。
实验结果表明：(1)用石油醚、乙酸乙酯、正丁醇对车前草乙醇提取物依次进行萃取后,水提取物含量最多；正丁醇提取物中黄酮类、多酚类化合物较高,乙酸乙酯萃取物中环烯醚萜类化合物含量较高。正丁醇提取物DPPH(1,1-二苯基-2-三硝基苯肼)、ABTS(2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐)自由基的清除作用最强,IC50分别为11.50、5.32μg/mL；乙酸乙酯提取物对超氧阴离子的清除作用最强,IC5o为149μg/mL；水提取物对猪油过氧化的抑制作用最强。乙醇总提物使CCl4肝损伤升高的ALT、AST、TP活性明显降低(P<0.01)。在TAA肝损伤实验中,乙醇总提物使升高的ALB、TB的活性明显降低(P<0.01)；乙酸乙酯和正丁醇提取物使升高的ALT、AST的活性明显降低(P<0.01)；水提取物使升高的TP的活性明显降低(P<0.01)。且乙酸乙酯和正丁醇提取物对CCl4和TAA肝损伤的肝脏组织蛋白的表达变化都有抑制作用。
(2)用石油醚、乙酸乙酯、正丁醇对杜仲乙醇提取物依次进行萃取后,水提取物含量最多；正丁醇萃取物中的烯醚萜类、黄酮类、多酚类化合物的含量较高。乙酸乙酯提取物对DPPH、O2-自由基的清除作用最强,IC5o分别为105.2、484μg/mL；正丁醇提取物对ABTS超氧阴离子的清除作用最强,IC50为43.27μg/mL；乙醇总提物对猪油过氧化的抑制作用最强。提取物对5%CCl4肝损伤没有明显的保护作用。在TAA肝损伤实验中,乙醇总提物使升高的ALB.TB的活性明显降低(P<0.01)；乙酸乙酯和正丁醇提取物使升高的ALT、AST、ALB的活性明显降低(P<0.01)；水提取物使升高的ALT、ALB的活性明显降低(P<0.01)。且经SDS-PAGE和2-DE表明,乙酸乙酯和正丁醇提取物对TAA肝损伤的肝脏组织蛋白的表达都有一定的修复作用。
结论：车前草和杜仲的乙酸乙酯和正丁醇提取物对TAA急性肝损伤有明显的保护作用,其作用机制可能与清除自由基,保护肝细胞膜,抑制肝细胞凋亡有关。
The liver is the central station of metabolism in vivo, the important detoxication organ, the important target of drugs, xenobiotics and oxidative toxicant. Liver injury is the starting link and common approach of serious liver disease occurrence, development and finally moving towards the liver function failure. The liver injury being caused by various harmful factors were manifested as hepatic necrosis, fatty liver, cholestasis, liver fibrosis, liver cirrhosis and liver cancer and so on.
The effects of compound isolated from Plantago asiatica L. and Eucommia ulmoides Oliv. by oral administration on carbon tetrachloride (CCl4) and thioacetamide (TAA) acute liver injury in mice were examined. Then, antioxidant activity of herbal extracts in vitro, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), the content of albumin (ALB), total protein (TP), cholesterol (CHO), total bilirubin (TB), glucose (GLU) and the chang of protein in liver were determinated to clarify the possible protection mechanisms.
Taken together, the results indicated:(1) Ethanol extract of Plantago asiatica L. was isolated with petroleum ether, ethyl acetate, n-butyl alcohol. The content of water extract, flavonoids and polyphenol compounds in n-butyl alcohol extract, iridoids compounds in ethyl acetate extract is higher. Scavenging effects on DPPH (1,1-Diphenyl-2-picrylhydrazyl radical2.2-Diphenyl-1-(2,4.6-trinitrophenyl)hydrazyl), ABTS (2.2'-Azinobis-3-ethylben-zthiazoline-6-sulphonate)) radical of n-butyl alcohol extract were the strongest, IC50were1.50,5.32μg/mL separately. Scavenging effects on superoxide anion of ethyl acetate extract was the strongest, IC50were149μg/mL. the inhibition effect on lard peroxidation of ethanol extract was the best. Ethanol extract had significantly suppressed the elevation of ALT. AST and TP activities which cased by CCl4(P<0.01). In TAA liver injury experiment, ethanol extract had significantly suppressed the elevation of ALB and TB activities (P<0.01), ethyl acetate and n-butyl alcohol extracts had significantly suppressed the elevation of ALT and AST activities (P<0.01). water extract had significantly suppressed the elevation of TP activities (P<0.01). The ethyl acetate and n-butyl alcohol extracts had inhibition effect on the chang of liver protein expression caused by CCl4and TAA.
(2) Ethanol extract of Eucommia ulmoides Oliv. was isolated with petroleum ether, ethyl acetate, n-butyl alcohol. The content of water extract, flavonoids, polyphenol and iridoids compounds in n-butyl alcohol extract is higher. Scavenging effects on DPPH, O2-radical of ethyl acetate extract were the strongest, IC50were105.2、484μg/mL separately. Scavenging effects onABTS radical of n-butyl alcohol extract was the strongest, IC50were43.27μg/mL. the inhibition effect on lard peroxidation of ethanol extract was the best. Extracts hadn't significantly protective effect on CCl4(5%) liver injury. In TAA liver injury experiment, ethanol extract had significantly suppressed the elevation of ALB and TB activities (P<0.01). ethyl acetate and n-butyl alcohol extracts had significantly suppressed the elevation of ALT、AST and ALB activities (P<0.01), water extract had significantly suppressed the elevation of ALT and ALB activities (P<0.01). The ethyl acetate and n-butyl alcohol extracts had inhibition effect on the chang of liver protein expression caused by TAA.
Conclusion:the ethyl acetate and n-butanol extract of Plantago asiatica L. and Eucommia ulmoides Oliv. had obviously protective effects on TAA acute liver damage, which might be related the effect of anti-oxidation, protection of liver cell membrane and inhibition of liver cell apoptosis.
本实验中我们通过小鼠体内试验研究车前草和杜仲提取物对四氯化碳(CCl4)和硫代乙酰胺(TAA)急性肝损伤的保护作用。通过体外测定中药提取物的抗氧化活性,以及血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)的活性,白蛋白(ALB)、总蛋白(TP)、胆固醇(CHO)、总胆红素(TB)、葡萄糖(GLU)的含量和肝组织中蛋白的表达变化探讨其保护机制。
实验结果表明：(1)用石油醚、乙酸乙酯、正丁醇对车前草乙醇提取物依次进行萃取后,水提取物含量最多；正丁醇提取物中黄酮类、多酚类化合物较高,乙酸乙酯萃取物中环烯醚萜类化合物含量较高。正丁醇提取物DPPH(1,1-二苯基-2-三硝基苯肼)、ABTS(2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐)自由基的清除作用最强,IC50分别为11.50、5.32μg/mL；乙酸乙酯提取物对超氧阴离子的清除作用最强,IC5o为149μg/mL；水提取物对猪油过氧化的抑制作用最强。乙醇总提物使CCl4肝损伤升高的ALT、AST、TP活性明显降低(P<0.01)。在TAA肝损伤实验中,乙醇总提物使升高的ALB、TB的活性明显降低(P<0.01)；乙酸乙酯和正丁醇提取物使升高的ALT、AST的活性明显降低(P<0.01)；水提取物使升高的TP的活性明显降低(P<0.01)。且乙酸乙酯和正丁醇提取物对CCl4和TAA肝损伤的肝脏组织蛋白的表达变化都有抑制作用。
(2)用石油醚、乙酸乙酯、正丁醇对杜仲乙醇提取物依次进行萃取后,水提取物含量最多；正丁醇萃取物中的烯醚萜类、黄酮类、多酚类化合物的含量较高。乙酸乙酯提取物对DPPH、O2-自由基的清除作用最强,IC5o分别为105.2、484μg/mL；正丁醇提取物对ABTS超氧阴离子的清除作用最强,IC50为43.27μg/mL；乙醇总提物对猪油过氧化的抑制作用最强。提取物对5%CCl4肝损伤没有明显的保护作用。在TAA肝损伤实验中,乙醇总提物使升高的ALB.TB的活性明显降低(P<0.01)；乙酸乙酯和正丁醇提取物使升高的ALT、AST、ALB的活性明显降低(P<0.01)；水提取物使升高的ALT、ALB的活性明显降低(P<0.01)。且经SDS-PAGE和2-DE表明,乙酸乙酯和正丁醇提取物对TAA肝损伤的肝脏组织蛋白的表达都有一定的修复作用。
结论：车前草和杜仲的乙酸乙酯和正丁醇提取物对TAA急性肝损伤有明显的保护作用,其作用机制可能与清除自由基,保护肝细胞膜,抑制肝细胞凋亡有关。
The liver is the central station of metabolism in vivo, the important detoxication organ, the important target of drugs, xenobiotics and oxidative toxicant. Liver injury is the starting link and common approach of serious liver disease occurrence, development and finally moving towards the liver function failure. The liver injury being caused by various harmful factors were manifested as hepatic necrosis, fatty liver, cholestasis, liver fibrosis, liver cirrhosis and liver cancer and so on.
The effects of compound isolated from Plantago asiatica L. and Eucommia ulmoides Oliv. by oral administration on carbon tetrachloride (CCl4) and thioacetamide (TAA) acute liver injury in mice were examined. Then, antioxidant activity of herbal extracts in vitro, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), the content of albumin (ALB), total protein (TP), cholesterol (CHO), total bilirubin (TB), glucose (GLU) and the chang of protein in liver were determinated to clarify the possible protection mechanisms.
Taken together, the results indicated:(1) Ethanol extract of Plantago asiatica L. was isolated with petroleum ether, ethyl acetate, n-butyl alcohol. The content of water extract, flavonoids and polyphenol compounds in n-butyl alcohol extract, iridoids compounds in ethyl acetate extract is higher. Scavenging effects on DPPH (1,1-Diphenyl-2-picrylhydrazyl radical2.2-Diphenyl-1-(2,4.6-trinitrophenyl)hydrazyl), ABTS (2.2'-Azinobis-3-ethylben-zthiazoline-6-sulphonate)) radical of n-butyl alcohol extract were the strongest, IC50were1.50,5.32μg/mL separately. Scavenging effects on superoxide anion of ethyl acetate extract was the strongest, IC50were149μg/mL. the inhibition effect on lard peroxidation of ethanol extract was the best. Ethanol extract had significantly suppressed the elevation of ALT. AST and TP activities which cased by CCl4(P<0.01). In TAA liver injury experiment, ethanol extract had significantly suppressed the elevation of ALB and TB activities (P<0.01), ethyl acetate and n-butyl alcohol extracts had significantly suppressed the elevation of ALT and AST activities (P<0.01). water extract had significantly suppressed the elevation of TP activities (P<0.01). The ethyl acetate and n-butyl alcohol extracts had inhibition effect on the chang of liver protein expression caused by CCl4and TAA.
(2) Ethanol extract of Eucommia ulmoides Oliv. was isolated with petroleum ether, ethyl acetate, n-butyl alcohol. The content of water extract, flavonoids, polyphenol and iridoids compounds in n-butyl alcohol extract is higher. Scavenging effects on DPPH, O2-radical of ethyl acetate extract were the strongest, IC50were105.2、484μg/mL separately. Scavenging effects onABTS radical of n-butyl alcohol extract was the strongest, IC50were43.27μg/mL. the inhibition effect on lard peroxidation of ethanol extract was the best. Extracts hadn't significantly protective effect on CCl4(5%) liver injury. In TAA liver injury experiment, ethanol extract had significantly suppressed the elevation of ALB and TB activities (P<0.01). ethyl acetate and n-butyl alcohol extracts had significantly suppressed the elevation of ALT、AST and ALB activities (P<0.01), water extract had significantly suppressed the elevation of ALT and ALB activities (P<0.01). The ethyl acetate and n-butyl alcohol extracts had inhibition effect on the chang of liver protein expression caused by TAA.
Conclusion:the ethyl acetate and n-butanol extract of Plantago asiatica L. and Eucommia ulmoides Oliv. had obviously protective effects on TAA acute liver damage, which might be related the effect of anti-oxidation, protection of liver cell membrane and inhibition of liver cell apoptosis.
引文
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