Objective:To investigate the osteoblastogenic activity of the ethyl acetate(EtOAc)extract of Smilax glabra Roxb roots and its major active compound astilbin.Methods:Astilbin was isolated from EtOAc extract using silic...Objective:To investigate the osteoblastogenic activity of the ethyl acetate(EtOAc)extract of Smilax glabra Roxb roots and its major active compound astilbin.Methods:Astilbin was isolated from EtOAc extract using silica gel chromatography combined with fraction crystallization.Chemical structure of astilbin was determined by analysis of the spectroscopic data in comparison with the literature.MTT method was used to detect the toxicity.Alkaline phosphatase(ALP)activity was determined by the spectrophotometric method at 405 nm using p-nitrophenyl phosphate as a substrate.Calcium deposition was stained with alizarin red-S,distained with cetylpyridium chloride,and quantified at 562 nm.In silico model for astilbin-ALP interaction was analyzed using AutoDock 4.2.6.The changes in expression of osteoblast differentiation related genes were determined using quantitative real-time PCR.Results:Both the EtOAc extract and astilbin had no toxicity toward osteoblast MC3T3-E1 cells at 5.0,10,25,and 50μg/mL.At 25μg/mL,they enhanced ALP activity and mineralization of osteoblasts up to 30%and 55%for the EtOAc extract and 22%and 41%for astilbin,respectively.Molecular docking analysis of astilbin-ALP interaction revealed Arg167,Asp320,His324,and His437 were key residues participating in hydrophobic interaction;meanwhile,His434 and Thr436 residues were involved in hydrogen bond formation in the active site of human tissue-nonspecific ALP.Moreover,the expression level of genes opn,col1,osx,and runx2 were up-regulated in astilbin treated samples with the fold changes as 2.2;3.7;4.1;2.3,respectively at 10μg/mL(P<0.05).Conclusions:The EtOAc extract and its major compound astilbin exhibit osteoblastogenic activity by up-regulating important markers for bone cell differentiation.It could be a new and promising osteogenic agent with dual actions for therapeutic applications.展开更多
目的:采用反相高效液相色谱法,测定不同来源土茯苓药材中落新妇苷和白藜芦醇的含量。方法:采用 YMC-PackODS-A(5 μm,4.6 mm×250 mm)色谱柱,乙腈(A)-0.05%磷酸水溶液(B)梯度洗脱(0～10 min 时25%A,10～15 min 时25%→35%A,15～20 m...目的:采用反相高效液相色谱法,测定不同来源土茯苓药材中落新妇苷和白藜芦醇的含量。方法:采用 YMC-PackODS-A(5 μm,4.6 mm×250 mm)色谱柱,乙腈(A)-0.05%磷酸水溶液(B)梯度洗脱(0～10 min 时25%A,10～15 min 时25%→35%A,15～20 min 时35%A);流速0.8 mL·min^(-1);检测波长306 nm。结果:落新妇苷、白藜芦醇浓度分别在0.018～0.288 mg·mL^(-1)和0.0025～0.025 mg·mL^(-1)范围内,与峰面积呈良好线性关系。落新妇苷平均回收率为99.6%～102.1%,RSD<1.9%;白藜芦醇平均回收率为99.2%～101.8%,RSD<2.2%。结论:该方法简便、准确,重复性好,可作为土茯苓药材的质量控制方法。展开更多
基金supported by the the Vietnam Academy of Science and Technology under grant NCCC 08.10/20-20the Institute of Biotechnology under grant CS20-01。
文摘Objective:To investigate the osteoblastogenic activity of the ethyl acetate(EtOAc)extract of Smilax glabra Roxb roots and its major active compound astilbin.Methods:Astilbin was isolated from EtOAc extract using silica gel chromatography combined with fraction crystallization.Chemical structure of astilbin was determined by analysis of the spectroscopic data in comparison with the literature.MTT method was used to detect the toxicity.Alkaline phosphatase(ALP)activity was determined by the spectrophotometric method at 405 nm using p-nitrophenyl phosphate as a substrate.Calcium deposition was stained with alizarin red-S,distained with cetylpyridium chloride,and quantified at 562 nm.In silico model for astilbin-ALP interaction was analyzed using AutoDock 4.2.6.The changes in expression of osteoblast differentiation related genes were determined using quantitative real-time PCR.Results:Both the EtOAc extract and astilbin had no toxicity toward osteoblast MC3T3-E1 cells at 5.0,10,25,and 50μg/mL.At 25μg/mL,they enhanced ALP activity and mineralization of osteoblasts up to 30%and 55%for the EtOAc extract and 22%and 41%for astilbin,respectively.Molecular docking analysis of astilbin-ALP interaction revealed Arg167,Asp320,His324,and His437 were key residues participating in hydrophobic interaction;meanwhile,His434 and Thr436 residues were involved in hydrogen bond formation in the active site of human tissue-nonspecific ALP.Moreover,the expression level of genes opn,col1,osx,and runx2 were up-regulated in astilbin treated samples with the fold changes as 2.2;3.7;4.1;2.3,respectively at 10μg/mL(P<0.05).Conclusions:The EtOAc extract and its major compound astilbin exhibit osteoblastogenic activity by up-regulating important markers for bone cell differentiation.It could be a new and promising osteogenic agent with dual actions for therapeutic applications.
文摘目的:采用反相高效液相色谱法,测定不同来源土茯苓药材中落新妇苷和白藜芦醇的含量。方法:采用 YMC-PackODS-A(5 μm,4.6 mm×250 mm)色谱柱,乙腈(A)-0.05%磷酸水溶液(B)梯度洗脱(0～10 min 时25%A,10～15 min 时25%→35%A,15～20 min 时35%A);流速0.8 mL·min^(-1);检测波长306 nm。结果:落新妇苷、白藜芦醇浓度分别在0.018～0.288 mg·mL^(-1)和0.0025～0.025 mg·mL^(-1)范围内,与峰面积呈良好线性关系。落新妇苷平均回收率为99.6%～102.1%,RSD<1.9%;白藜芦醇平均回收率为99.2%～101.8%,RSD<2.2%。结论:该方法简便、准确,重复性好,可作为土茯苓药材的质量控制方法。
