目的采用高内涵技术筛选补骨脂潜在肝毒性成分及其可能的作用机制。方法采用CCK-8法测定补骨脂中异补骨脂二氢黄酮、补骨脂二氢黄酮甲醚、补骨脂二氢黄酮、异补骨脂查尔酮、4’-O-甲基补骨脂查尔酮、补骨脂宁、补骨脂素、异补骨脂素、...目的采用高内涵技术筛选补骨脂潜在肝毒性成分及其可能的作用机制。方法采用CCK-8法测定补骨脂中异补骨脂二氢黄酮、补骨脂二氢黄酮甲醚、补骨脂二氢黄酮、异补骨脂查尔酮、4’-O-甲基补骨脂查尔酮、补骨脂宁、补骨脂素、异补骨脂素、补骨脂定、补骨脂酚10种成分的IC_(50)值。在相同浓度药物处理HepG 2细胞24 h后,进行高内涵检测,通过阳性细胞的平均荧光强度评价各组细胞活性氧、还原型谷胱甘肽、线粒体膜电位及线粒体超氧化物水平4个指标的变化情况,初步筛选补骨脂中主要肝毒性成分。结果CCK-8细胞活力实验结果显示,异补骨脂二氢黄酮、补骨脂二氢黄酮甲醚、补骨脂二氢黄酮、异补骨脂查尔酮、4’-O-甲基补骨脂查尔酮、补骨脂宁、异补骨脂素、补骨脂定、补骨脂酚对Hep G 2细胞的IC_(50)值分别为52.69、42.72、83.63、42.29、57.43、110.80、1420.00、23.58和25.34μmol·L^(-1)。高内涵结果显示,在20μmol·L^(-1)浓度下,补骨脂二氢黄酮甲醚、补骨脂二氢黄酮、异补骨脂查尔酮、补骨脂定和补骨脂酚可显著提高细胞内活性氧水平;异补骨脂查尔酮、4’-O-甲基补骨脂查尔酮、补骨脂宁、异补骨脂素、补骨脂定和补骨脂酚均可导致细胞中还原型谷胱甘肽水平保护性升高;异补骨脂查尔酮、补骨脂定和补骨脂酚均显著降低线粒体膜电位,造成线粒体超氧化物累积。结论异补骨脂查尔酮、补骨脂定和补骨脂酚是补骨脂中造成线粒体功能损伤的主要成分,具有潜在的肝脏毒性,其具体作用机制有待进一步研究。展开更多
Objective: To test the role of psoralidin in human liver cancer HepG2 cells in vitro. Methods: Cell viability was assessed by methylthiazolyldiphenyl-tetrazolum bromide assay and apoptotic cells were labeled by annexi...Objective: To test the role of psoralidin in human liver cancer HepG2 cells in vitro. Methods: Cell viability was assessed by methylthiazolyldiphenyl-tetrazolum bromide assay and apoptotic cells were labeled by annexin V then sorted by flow cytometry. Protein expressions of caspase-3, caspase-8, caspase-9, Bax, Bid, Bcl-2, Bcl-xL and p53 were examined by western blot while activity of caspase-3,-8 and -9 were also determined. Results: Psoralidin reduces cell viability greatly in a time dependent manner(64%, 40%, 21%, 12% at 2, 6, 24 and 48 h treatment with 64 μmol/L psoralidin respectively) and up-regulates activities of caspase-3,-8 and-9 in a concentration dependent manner(between 4 to 64 μmol/L). Psoralidin also increases the expression of pro-apoptosis genes Bax, Bid and p53 while decreases the expression of pro-survival genes Bcl-2 and Bcl-xL, both in a concentration dependent manner between 4 and 64 μmol/L(P<0.05 at 16 and 64 μmol/L). Caspase-3 inhibitor(Ac-DEVD-CHO at concentrations between 10 to 20 μmol/L), p53 inhibitor(pifithrin-α at 5 μmol/L) and cyclosporin A can attenuate the apoptotic effect of psoralidin. Conclusion: The cytotoxic role of psoralidin might work through both intrinsic and extrinsic apoptotic pathway.展开更多
目的为提高补骨脂定的水溶性和稳定性,用体外酶法糖基化反应对其进行结构修饰。方法通过UDP-糖基转移酶对补骨脂定进行糖基化修饰,合成一种新的葡萄糖苷化合物(1)。使用高分辨电喷雾电离质谱(HR-ESI-MS)和核磁共振(NMR)分析,鉴定化合物...目的为提高补骨脂定的水溶性和稳定性,用体外酶法糖基化反应对其进行结构修饰。方法通过UDP-糖基转移酶对补骨脂定进行糖基化修饰,合成一种新的葡萄糖苷化合物(1)。使用高分辨电喷雾电离质谱(HR-ESI-MS)和核磁共振(NMR)分析,鉴定化合物1的结构;利用高效液相色谱峰面积计算出样品溶液的浓度;MTT法检测化合物对3种肿瘤细胞(SMMC7721、MCF-7、SW480)增殖的影响。结果根据波谱分析,鉴定制备出的新型葡萄糖苷化合物为psoralidin-6',7-di-O-β-Dglucopyranoside(1)。水溶性检测结果表明,化合物1的水溶性是底物(补骨脂定)水溶性的32.6倍。此外,化合物1在p H 8.8和高温条件下较补骨脂定更加稳定。在抗肿瘤细胞增殖实验中,只有补骨脂定对3种肿瘤细胞都显示出较强的抑制能力。结论体外酶法糖基化是进行结构修饰、改善水溶性和稳定性的强有力方法。展开更多
文摘目的采用高内涵技术筛选补骨脂潜在肝毒性成分及其可能的作用机制。方法采用CCK-8法测定补骨脂中异补骨脂二氢黄酮、补骨脂二氢黄酮甲醚、补骨脂二氢黄酮、异补骨脂查尔酮、4’-O-甲基补骨脂查尔酮、补骨脂宁、补骨脂素、异补骨脂素、补骨脂定、补骨脂酚10种成分的IC_(50)值。在相同浓度药物处理HepG 2细胞24 h后,进行高内涵检测,通过阳性细胞的平均荧光强度评价各组细胞活性氧、还原型谷胱甘肽、线粒体膜电位及线粒体超氧化物水平4个指标的变化情况,初步筛选补骨脂中主要肝毒性成分。结果CCK-8细胞活力实验结果显示,异补骨脂二氢黄酮、补骨脂二氢黄酮甲醚、补骨脂二氢黄酮、异补骨脂查尔酮、4’-O-甲基补骨脂查尔酮、补骨脂宁、异补骨脂素、补骨脂定、补骨脂酚对Hep G 2细胞的IC_(50)值分别为52.69、42.72、83.63、42.29、57.43、110.80、1420.00、23.58和25.34μmol·L^(-1)。高内涵结果显示,在20μmol·L^(-1)浓度下,补骨脂二氢黄酮甲醚、补骨脂二氢黄酮、异补骨脂查尔酮、补骨脂定和补骨脂酚可显著提高细胞内活性氧水平;异补骨脂查尔酮、4’-O-甲基补骨脂查尔酮、补骨脂宁、异补骨脂素、补骨脂定和补骨脂酚均可导致细胞中还原型谷胱甘肽水平保护性升高;异补骨脂查尔酮、补骨脂定和补骨脂酚均显著降低线粒体膜电位,造成线粒体超氧化物累积。结论异补骨脂查尔酮、补骨脂定和补骨脂酚是补骨脂中造成线粒体功能损伤的主要成分,具有潜在的肝脏毒性,其具体作用机制有待进一步研究。
基金Supported by the National Natural Science Foundation of China(No.81202991)Tianjin City Application Basis,Cutting-edge Technology Research Program(Tianjin Municipal Science and Technology Commission,No.13JCYBJC38500)
文摘Objective: To test the role of psoralidin in human liver cancer HepG2 cells in vitro. Methods: Cell viability was assessed by methylthiazolyldiphenyl-tetrazolum bromide assay and apoptotic cells were labeled by annexin V then sorted by flow cytometry. Protein expressions of caspase-3, caspase-8, caspase-9, Bax, Bid, Bcl-2, Bcl-xL and p53 were examined by western blot while activity of caspase-3,-8 and -9 were also determined. Results: Psoralidin reduces cell viability greatly in a time dependent manner(64%, 40%, 21%, 12% at 2, 6, 24 and 48 h treatment with 64 μmol/L psoralidin respectively) and up-regulates activities of caspase-3,-8 and-9 in a concentration dependent manner(between 4 to 64 μmol/L). Psoralidin also increases the expression of pro-apoptosis genes Bax, Bid and p53 while decreases the expression of pro-survival genes Bcl-2 and Bcl-xL, both in a concentration dependent manner between 4 and 64 μmol/L(P<0.05 at 16 and 64 μmol/L). Caspase-3 inhibitor(Ac-DEVD-CHO at concentrations between 10 to 20 μmol/L), p53 inhibitor(pifithrin-α at 5 μmol/L) and cyclosporin A can attenuate the apoptotic effect of psoralidin. Conclusion: The cytotoxic role of psoralidin might work through both intrinsic and extrinsic apoptotic pathway.
基金Supported by National Natural Science Foundation of China(81302671)Grant for Innovative Science Research for Graduates of Bengbu Medical College(Byycx1557)~~
文摘目的为提高补骨脂定的水溶性和稳定性,用体外酶法糖基化反应对其进行结构修饰。方法通过UDP-糖基转移酶对补骨脂定进行糖基化修饰,合成一种新的葡萄糖苷化合物(1)。使用高分辨电喷雾电离质谱(HR-ESI-MS)和核磁共振(NMR)分析,鉴定化合物1的结构;利用高效液相色谱峰面积计算出样品溶液的浓度;MTT法检测化合物对3种肿瘤细胞(SMMC7721、MCF-7、SW480)增殖的影响。结果根据波谱分析,鉴定制备出的新型葡萄糖苷化合物为psoralidin-6',7-di-O-β-Dglucopyranoside(1)。水溶性检测结果表明,化合物1的水溶性是底物(补骨脂定)水溶性的32.6倍。此外,化合物1在p H 8.8和高温条件下较补骨脂定更加稳定。在抗肿瘤细胞增殖实验中,只有补骨脂定对3种肿瘤细胞都显示出较强的抑制能力。结论体外酶法糖基化是进行结构修饰、改善水溶性和稳定性的强有力方法。
