大青叶提取物对HepG2细胞酒精性损伤的保护作用
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摘要
以大青(Clerodendrum cyrtophyllum Turcz)叶乙醇提取物和石油醚相、二氯甲烷相、乙酸乙酯相、正丁醇相、水相分级萃取物为研究对象,探讨其对肝细胞酒精性损伤的保护作用。采用酒精诱导人肝癌细胞(HepG2)氧化应激建立酒精性肝细胞损伤模型,通过噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测不同浓度大青叶醇提物和五相萃取物预处理对HepG2细胞酒精性损伤后存活率的影响,以2,7-二氢二氯荧光黄双乙酸钠(2,7-dichlorofuorescin diacetate,DCFH-DA)探针法测定细胞内活性氧(reactive oxygen species,ROS)水平,硫代巴比妥酸法测定细胞内丙二醛(malondialdehyde,MDA)含量,二硫代二硝基苯甲酸[5,5'-Dithiobis-(2-nitrobenzoic acid),DTNB]法检测还原型谷胱甘肽(glutathione,GSH)含量,水溶性四唑盐(water-soluble tetrazolium,WST)法检测超氧化物歧化酶(superoxide dismutase,SOD)活力。结果显示,大青叶醇提物和乙酸乙酯相、正丁醇相、水相萃取物可以明显提高HepG2细胞酒精性损伤后的存活率,通过抵御细胞内ROS生成,降低MDA浓度的方式对细胞进行保护,通过提高细胞内抗氧化物GSH含量和抗氧化酶SOD活力来恢复细胞抗氧化能力,石油醚相和二氯甲烷相无明显作用效果。综上,大青叶醇提物能够抵御HepG2细胞的酒精性损伤,具有护肝功能性食品应用研究价值;乙酸乙酯相、正丁醇相、水相是其有效部位,尤其是乙酸乙酯相,可以作为大青叶中天然抗氧化剂的主要研究对象。
The ethanol extracts and petroleum ether,dichloromethane,ethyl acetate,n-butanol and aqueous fractions of Clerodendrum cyrtophyllum Turcz leaves were studied to investigate the protective effects on alcohol-induced injury in hepatocyte. An alcoholic hepatocyte injury model was established by alcohol-induced hepatocellular carcinoma(HepG2)oxidative stress. The effect of pretreatment with different concentrations of ethanol extract and five fractions extracts on the survival rate of HepG2 cells after alcoholic inducing was detected by methyl thizaolyl tetrazolium(MTT)assay. The level of reactive oxygen species(ROS)in HepG2 cells was measured by 2,7-dihydrodichlorofluorescein diacetate(DCFH-DA)probe method. The thiobarbituric acid method was used to determine the content of malondialdehyde(MDA) in HepG2 cells. The dithiodinitrobenzoic acid(DTNB)method was used to detect the content of glutathione(GSH). Water-soluble tetrazolium(WST)method was used to detect the activity of superoxide dismutase(SOD). The results showed that ethanol extracts and ethyl acetate,n-butanol and aqueous fractions of C. cyrtophyllum Turcz leaves could significantly increase the survival rate of HepG2 cells after alcohol injury. The HepG2 cells were protected by reducing intracellular ROS production and MDA concentrations. The antioxidant capacity of HepG2 cells was restored by increasing the intracellular antioxidant GSH content and antioxidant enzyme SOD activity. The petroleum ether and dichloromethane fractions did not show a significant effect. In summary,the ethanol extract of C. cyrtophyllum Turcz leaves could resist the alcoholic damage in HepG2 cells,and had a research value in the application of hepatoprotective functional foods. Ethyl acetate,n-butanol and water fractions were the effective parts,especially the ethyl acetate fraction,which could be used as the main research object of natural antioxidants in C. cyrtophyllum Turcz leaves.
The ethanol extracts and petroleum ether,dichloromethane,ethyl acetate,n-butanol and aqueous fractions of Clerodendrum cyrtophyllum Turcz leaves were studied to investigate the protective effects on alcohol-induced injury in hepatocyte. An alcoholic hepatocyte injury model was established by alcohol-induced hepatocellular carcinoma(HepG2)oxidative stress. The effect of pretreatment with different concentrations of ethanol extract and five fractions extracts on the survival rate of HepG2 cells after alcoholic inducing was detected by methyl thizaolyl tetrazolium(MTT)assay. The level of reactive oxygen species(ROS)in HepG2 cells was measured by 2,7-dihydrodichlorofluorescein diacetate(DCFH-DA)probe method. The thiobarbituric acid method was used to determine the content of malondialdehyde(MDA) in HepG2 cells. The dithiodinitrobenzoic acid(DTNB)method was used to detect the content of glutathione(GSH). Water-soluble tetrazolium(WST)method was used to detect the activity of superoxide dismutase(SOD). The results showed that ethanol extracts and ethyl acetate,n-butanol and aqueous fractions of C. cyrtophyllum Turcz leaves could significantly increase the survival rate of HepG2 cells after alcohol injury. The HepG2 cells were protected by reducing intracellular ROS production and MDA concentrations. The antioxidant capacity of HepG2 cells was restored by increasing the intracellular antioxidant GSH content and antioxidant enzyme SOD activity. The petroleum ether and dichloromethane fractions did not show a significant effect. In summary,the ethanol extract of C. cyrtophyllum Turcz leaves could resist the alcoholic damage in HepG2 cells,and had a research value in the application of hepatoprotective functional foods. Ethyl acetate,n-butanol and water fractions were the effective parts,especially the ethyl acetate fraction,which could be used as the main research object of natural antioxidants in C. cyrtophyllum Turcz leaves.
引文
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[10]林东明,罗其葵,黄斌学,等.大青木水煎剂及山大青甙对狗血压的影响[J].右江民族医学院学报,1994(1):8-11
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[15]Wan L,Jiang J G.Protective effects of plant-derived flavonoids on hepatic injury[J].Journal of Functional Foods,2018,44(5):283-291
[16]曹佩琴,牛丽,黄建安,等.朝鲜蓟叶水提取物对酒精诱导人肝癌细胞株Hep G2损伤的影响[J].食品安全质量检测学报,2015(6):1973-1979
[17]Peng F C,Tang S H,Huang M C,et al.Oxidative status in patients with alcohol dependence:a clinical study in Taiwan[J].Journal of Toxicology&Environmental Health,2005,68(17/18):1497-1509
[18]杨建华,胡君萍,热娜·卡斯木,等.肉苁蓉属植物中六种苯乙醇苷类化合物抗氧化活性的构效关系研究[J].中药材,2009,32(7):1067-1069
[19]高原,王越,赵莹莹,等.白藜芦醇对H2O2诱导的Hep G2细胞氧化损伤保护作用研究[J].化学工程师,2017,31(6):16-18
[20]Hao L,Sun Q,Zhong W,et al.Mitochondria-targeted ubiquinone(Mito Q)enhances acetaldehyde clearance by reversing alcohol-induced posttranslational modification of aldehyde dehydrogenase 2:A molecular mechanism of protection against alcoholic liver disease[J].Redox Biology,2017,14:626-636
[21]Aytacoglu B N,Calikoglu M,Tamer L,et al.Alcohol-induced lung damage and increased oxidative stress[J].Respiration,2006,73(1):100-104
[22]Castro-Alférez M,Polo-López M I,Marugánc J,et al.Mechanistic model of the Escherichia coli inactivation by solar disinfection based on the photo-generation of internal ROS and the photo-inactivation of enzymes:CAT and SOD[J].Chemical Engineering Journal,2017,318(6):214-223
[23]Han D,Hanawa N,Saberi B,et al.Mechanisms of liver injury.III.Role of glutathione redox status in liver injury[J].American Journal of Physiology Gastrointestinal&Liver Physiology,2006,291(1):1-7
[2]张秋燕,张福平.粤东地区马鞭草科药用植物资源[J].食品与药品,2010,12(9):366-369
[3]Nhuong L C,Nguyen V L,Hoa D,et al.Chemical constituents and antioxidant activity of flavonoids from Clerodendron cyrtophyllum Turcz.[J].Tap Chi Duoc,2006,46(11):30-33
[4]Hoang V D,Ba T C,Hoa L,et al.Chemical study of clerodendron cyrtophyllum Turcz.Part I-chemical constituents of n-hexan extract of the roots[J].Journal of chemistry,2006,44(6):704-706
[5]李艳.大青根化学成分的研究[D].沈阳:沈阳药科大学,2008
[6]赵庆春.大青根化学成分的研究(Ⅱ)[D].沈阳:沈阳药科大学,2008
[7]吴明美,王利勤,华燕.大青属植物的化学成分及药理作用研究进展[J].化学工程与装备,2011(4):112-116
[8]赵良忠,王放银,段林东.大青叶抗菌物质提取及抗菌效果研究[J].食品科学,2004,25(11):138-140
[9]张惠勤,黄崇基,王育生.大青木药理作用的初步研究[J].华夏医学,1993(1):11-13
[10]林东明,罗其葵,黄斌学,等.大青木水煎剂及山大青甙对狗血压的影响[J].右江民族医学院学报,1994(1):8-11
[11]Zhou J,Zheng X,Yang Q,et al.Optimization of ultrasonic-assisted extraction and radical-scavenging capacity of phenols and flavonoids from Clerodendrum cyrtophyllum Turcz leaves[J].Plos One,2013,8(7):e68392
[12]Hagstr觟m H.Alcohol,smoking and the liver disease patient[J].Best Practice&Research Clinical Gastroenterology,2017,31(5):537-543
[13]Flora K,Hahn M,Rosen H,et al.Milk Thistle(Silybum marianum)for the Therapy of Liver Disease[J].American Journal of Gastroenterology,1998,93(2):139-143
[14]Asadi-Samani M,Kafash-Farkhad N,Azimi N,et al.Medicinal plants with hepatoprotective activity in Iranian folk medicine[J].Asian Pacific Journal of Tropical Biomedicine,2015,5(2):146-157
[15]Wan L,Jiang J G.Protective effects of plant-derived flavonoids on hepatic injury[J].Journal of Functional Foods,2018,44(5):283-291
[16]曹佩琴,牛丽,黄建安,等.朝鲜蓟叶水提取物对酒精诱导人肝癌细胞株Hep G2损伤的影响[J].食品安全质量检测学报,2015(6):1973-1979
[17]Peng F C,Tang S H,Huang M C,et al.Oxidative status in patients with alcohol dependence:a clinical study in Taiwan[J].Journal of Toxicology&Environmental Health,2005,68(17/18):1497-1509
[18]杨建华,胡君萍,热娜·卡斯木,等.肉苁蓉属植物中六种苯乙醇苷类化合物抗氧化活性的构效关系研究[J].中药材,2009,32(7):1067-1069
[19]高原,王越,赵莹莹,等.白藜芦醇对H2O2诱导的Hep G2细胞氧化损伤保护作用研究[J].化学工程师,2017,31(6):16-18
[20]Hao L,Sun Q,Zhong W,et al.Mitochondria-targeted ubiquinone(Mito Q)enhances acetaldehyde clearance by reversing alcohol-induced posttranslational modification of aldehyde dehydrogenase 2:A molecular mechanism of protection against alcoholic liver disease[J].Redox Biology,2017,14:626-636
[21]Aytacoglu B N,Calikoglu M,Tamer L,et al.Alcohol-induced lung damage and increased oxidative stress[J].Respiration,2006,73(1):100-104
[22]Castro-Alférez M,Polo-López M I,Marugánc J,et al.Mechanistic model of the Escherichia coli inactivation by solar disinfection based on the photo-generation of internal ROS and the photo-inactivation of enzymes:CAT and SOD[J].Chemical Engineering Journal,2017,318(6):214-223
[23]Han D,Hanawa N,Saberi B,et al.Mechanisms of liver injury.III.Role of glutathione redox status in liver injury[J].American Journal of Physiology Gastrointestinal&Liver Physiology,2006,291(1):1-7