摘要
目的:筛选潜在的何首乌中致肝损伤的毒性成分,并探讨其初步损伤的机制。方法:采用4种人源肝细胞系(L-02、HepG2、HepaRG、hiHeps),建立CCK-8体外毒性高通量筛选方法,对何首乌的总醇提物及其分部位进行系统筛选,确定目标毒性分部位;基于建立的方法,筛选何首乌代表性的单体化合物16种成分,确定潜在毒性成分;进一步考察毒性分部位中毒性成分的含量,反推出目标毒性成分。最后通过高内涵的方法,对目标毒性成分进行在亚器官水平(活性氧、线粒体膜电位、内质网应激、中性脂代谢紊乱以及钙流稳态)进行初步的机制探索。结果:毒性部位的筛选,发现何首乌中最可能的毒性部位为Fr.4、Fr.5、Fr.6和Fr.7;毒性成分的筛选,发现何首乌中最可能的毒性成分为没食子酸、白藜芦醇、大黄素以及大黄酸。含量测定发现没食子酸在可能的毒性部位中含量最高,在何首乌的总醇提物中含量也是四个毒性成分中最高的一个,基本确定没食子酸为何首乌潜在致肝损伤的毒性成分。高内涵分析表明,活性氧、线粒体膜电位、中性脂以及钙流等指标变化不大,而没食子酸对内质网有一定程度的损伤,提示没食子酸是通过引起内质网应激达到影响肝细胞内稳态,进而引起损伤。结论:何首乌致肝损伤的物质基础为没食子酸,作用机制初步判断为引起内质网应激导致的细胞损伤。
Objective:our aim was to screen the toxic constituent from Polygonum multiflorum and explore the underlying mechanism.Method:we applied four human liver cell lines(L-02,HepG2,HepaRG,hiHeps) for screening hepatotoxic chemicals in vitro in a high throughput CCK-8 method.Systematic screening the total ethanol extract,eight fractions and sixteen chemical constituents from P.multiflorum was conduct and the target constituent was set based on determination of quantifications by UPLC.Finally,high content screening was used for preliminary mechanism exploration based on five indicators as ROS,MMP,ERS,neutral lipid metabolism disorders and calcium ion stabilization.Results:Fr.4,Fr.5,Fr.6 and Fr.7 were the most toxic parts of P.multiflorum on CCK-8 screening.And gallic acid,resveratrol,emodin and rhein were the main toxic constituents from P.multiflorum.Determination of contains of latent toxic parts,we found gallic acid was the highest proportion in the ethanol extract and other toxic parts,which reversely proved gallic acid was the target hepatotoxic constituent we've been searching for.High content screening analysis showed us that only ERS was happened notably followed by gallic acid treatment which was inferred that the hepatotoxic mechanism may be correlated with ERS and the main course of P.multiflorum induced liver injury.Conclusion:gallic acid was the main hepatotoxic constituent of P.multiflorum and the main hepatotoxic mechanism was correlated with ERS.
引文
1.Lin,L.;Ni,B.;Lin,H.;Zhang,M.;Li,X.;Yin,X.;Qu,C;Ni,J.,Traditional usages,botany,phytochemistry,pharmacology and toxicology of Polygonum multiflorum Thunb.:a review.Journal of ethnopharmacology 2015,159,158-83.
2.But,P.P.;Tomlinson,B.;Lee,K.L.,Hepatitis related to the Chinese medicine Shou-wu-pian manufactured from Polygonum multiflorum.Veterinary and human toxicology 1996,38(4),280-2.
3.Wu,X.;Chen,X.;Huang,Q.;Fang,D.;Li,G.;Zhang,G.,Toxicity of raw and processed roots of Polygonum multiflorum.Fitoterapia 2012,83(3),469-75.
4.Zhang,Y;Ding,T.;Diao,T;Deng,M.;Chen,S.,Effects of Polygonum multiflorum on the activity of cytochrome P450 isoforms in rats.Pharmazie 2015,70(1),47-54.
5.Yu,J.;Xie,J.;Mao,X.J.;Wang,M.J.;Li,N.;Wang,J.;Zhaori,G.T;Zhao,R.H.,Hepatoxicity of major constituents and extractions of Radix Polygoni Multiflori and Radix Polygoni Multiflori Praeparata.Journal of ethnopharmacology 2011,137(3),1291-9.
6.Zhu,Y.;Li,Y.G.;Wang,J.B.;Liu,S.H.;Wang,L.F.;Zhao,Y.L.;Bai,Y.F.;Wang,Z.X.;Li,J.Y.;Xiao,X.H.,Causes,Features,and Outcomes of Drug-Induced Liver Injury in 69 Children from China.Gut and liver2015,9(4),525-33.
7.Asnaashari,M.;Farhoosh,R.;Sharif,A.,Antioxidant activity of gallic acid and methyl gallate in triacylglycerols of Kilka fish oil and its oil-in-water emulsion.Food Chem 2014,159,439-44.
8.Verma,S.;Singh,A.;Mishra,A.,Gallic acid:molecular rival of cancer.Environmental toxicology and pharmacology 2013,35(3),473-85.
9.Daglia,M.;Di Lorenzo,A.;Nabavi,S.F.;Talas,Z.S.;Nabavi,S.M.,Polyphenols:well beyond the antioxidant capacity:gallic acid and related compounds as neuroprotective agents:you are what you eat!Curr Pharm Biotechnol 2014,15(4),362-72.
10.Hsieh,C.L.;Lin,C.H.;Wang,H.E.;Peng,C.C.;Peng,R.Y.,Gallic acid exhibits risks of inducing muscular hemorrhagic liposis and cerebral hemorrhage--its action mechanism and preventive strategy.Phytotherapy research:PTR 2015,29(2),267-80.