药食两用物质效用成分对人孕烷X受体的激活效应及细胞增殖抑制筛选

Activating Effect of Effective Components in Medicinal and Edible Substances on Human Pregnane X Receptor and Cytotoxicity Screening

目的:探讨药食两用物质甘草(甘草苷、甘草酸)、鱼腥草(槲皮素、鱼腥草素)、夏枯草(迷迭香酸)、决明子(橙黄决明素)、茯苓(茯苓酸)、百合(百合皂苷、秋水仙碱)、枸杞子(枸杞多糖)7种药食两用物质中10种效用成分对人孕烷X受体(PXR)的激活效应并筛选其潜在的毒性成分。方法:利用细胞增殖与活性检测(CCK-8)法考察了甘草苷、甘草酸、槲皮素、鱼腥草素、迷迭香酸、橙黄决明素、茯苓酸、秋水仙碱(10、20、50μmol·L^(-1))、百合皂苷、枸杞多糖(10、20、50 mg·L^(-1))10种成分对正常的人肝细胞(L02)增殖抑制的影响;通过生化分析仪检测药物处理后对L02细胞乳酸脱氢酶(LDH)释放的影响;Hoechst 33342染色考察了10种效用成分对L02细胞凋亡的影响;利用分泌型荧光素酶报告基因系统,将PXR表达载体和含细胞色素P4503A4(CYP3A4)转录调控区的报告基因载体共转染L02细胞,10μmol·L^(-1)利福平(RIF)为阳性对照,用甘草苷、甘草酸、槲皮素、鱼腥草素、迷迭香酸、橙黄决明素、茯苓酸、秋水仙碱(5、10、20μmol·L^(-1))和百合皂苷、枸杞多糖(5、10、20 mg·L^(-1))10种成分分别处理24 h,进行荧光素酶活性检测。结果:与空白组比较,秋水仙碱、百合皂苷、槲皮素会导致细胞活力呈浓度依赖性降低(P<0.05,P<0.01),槲皮素、迷迭香酸、甘草酸、秋水仙碱、橙黄决明素、茯苓酸可以增加L02细胞中LDH的释放率(P<0.05,P<0.01),迷迭香酸、甘草苷、枸杞多糖处理后部分细胞的高亮浓染细胞核的比例逐渐上升(P<0.05,P<0.01);pcDNA3.1-PXR与pGLuc-CYP3A4共转染L02细胞时,与空白组比较,橙黄决明素、茯苓酸对PXR有激活效应(P<0.05),甘草苷、甘草酸对PXR有抑制效应(P<0.05)。结论:在长期服用药食两用物质时,应注意其对药物代谢酶的影响及可能产生的相互作用,提高药食两用物质的安全性。

Objective:To explore the activating effects of ten important effective components in seven medicinal and edible substances on human pregnane X receptor(PXR),including Glycyrrhizae Radix et Rhizoma(liquiritin and glycyrrhizic acid),Houttuyniae Herba(quercetin and houttuyfonate),Prunellae Spica(rosmarinic acid),Cassiae Semen(aurantio-obtusin),Poria(pachymic acid),Lilii Bulbus(Lilium brownii saponin and colchicine),and Lycii Fructus(Lycium barbarum polysaccharide)and screen potentially toxic components.Method:Cell counting kit-8(CCK-8)assay was used to investigate the cytotoxic effect of liquiritin,glycyrrhizic acid,quercetin,houttuyfonate,rosmarinic acid,pachymic acid,aurantio-obtusin,and colchicine(10,20,and 50μmol·L^(-1)),and L.brownii saponin and L.barbarum polysaccharide(10,20,and 50 mg·L^(-1))on normal human hepatocyte cell line(L02).The release of lactate dehydrogenase(LDH)in L02 cells after drug treatments was detected by the biochemical analyzer.The apoptosis induced by ten effective components was explored by Hoechst 33342 staining.The secreted luciferase reporter system was used to cotransfect the PXR expression vector and reporter gene vector containing cytochrome P4503A4(CYP3A4)transcriptional regulatory region into L02 cells,with 10μmol·L^(-1)rifampicin(RIF)as a positive control.After treated with liquiritin,glycyrrhizic acid,quercetin,houttuyfonate,rosmarinic acid,aurantio-obtusin,pachymic acid,and colchicine(5,10,and 20μmol·L^(-1))and L.brownii saponin and L.barbarum polysaccharide(5,10,and 20 mg·L^(-1))for 24 h,the cells were tested for secreted luciferase activity.Result:Compared with the control group,colchicine,L.brownii saponin,and quercetin decreased the cell viability(P<0.05,P<0.01).Compared with the control group,quercetin,rosmarinic acid,glycyrrhizic acid,colchicine,aurantio-obtusin,and pachymic acid increased the release rate of LDH in L02 cells(P<0.05,P<0.01).The proportion of hyperchromatic nuclei increased gradually after rosmarinic acid,liquiritin,and L.barbarum polysaccharide treatments as compared with the control group(P<0.05,P<0.01).In terms of co-transfection of pcDNA3.1-PXR and pGLuc-CYP3A4 into L02 cells,compared with the control group,aurantio-obtusin and pachymic acid showed activating effects on PXR(P<0.05),whereas liquiritin and glycyrrhizic acid showed inhibitory effects(P<0.05).Conclusion:The findings suggest that when medicinal and edible substances are taken for a long time,attention should be paid to their influence on drug-metabolizing enzymes and possible interactions,so as to improve their safety.