HPLC同时测定红芪中4种黄酮类成分的含量

目的:建立HPLC法测定红芪中毛蕊异黄酮葡萄糖苷、金雀异黄酮、芒柄花素、美迪紫檀素4种主要黄酮类成分的方法。方法:采用HPLC法,样品经甲醇回流,采用Waters公司Sun Fire C18色谱柱(250 mm×4.6 mm,5μm),乙腈-0.2%磷酸水为流动相进行梯度洗脱,流速为1.0 ml·min-1,检测波长为240 nm,柱温为35℃,进样量为20μl。结果:毛蕊异黄酮葡萄糖苷在0.035~1.042μg(r=0.999 6),金雀异黄酮在0.027~0.821μg(r=0.999 7),芒柄花素在0.031~0.941μg(r=0.999 9),美迪紫檀素在0.025~0.745μg(r=0.999 2)范围内均成良好的线性关系。毛蕊异黄酮葡萄糖苷、金雀异黄酮、芒柄花素和美迪紫檀素的加样回收率分别为100.32%(RSD=1.87%),99.3%(RSD=1.76%),100.5%(RSD=1.48%),99.2%(RSD=1.45%)(n=6)。结论:该方法分析时间短、稳定性和准确度良好,对红芪药材的质量控制具有一定的参考价值。     

芒柄花素抑制RANKL诱导破骨细胞分化的实验研究

目的:建立RANKL诱导的破骨细胞体外研究模型,阐述活血化瘀中药鸡血藤有效组分芒柄花素(formononetin,FO)调控小鼠骨髓单核-巨噬细胞(BMMs)向破骨细胞分化和功能的影响,探讨其抑制破骨细胞分化的分子机制。方法:取20只4~6周龄清洁级C57/BL6小鼠,雌雄各10只,体重(20±2)g,无菌条件下分离出股骨和胫骨内BMMs,用α-MEM培养基进行体外培养和增殖。BMMs在加入M-CSF和不同浓度的芒柄花素(5~50?滋M)分别培养4 d后进行细胞增殖与毒性的CCK8检测。将生长状态良好的BMMs依次加入M-CSF和RANKL诱导破骨细胞分化,对照组无特殊处理,DMSO对照组加入DMSO溶剂,各观察组分别加入不同浓度芒柄花素(1~20?滋M),分别进行培养6 d后进行抗酒石酸酸性磷酸酶(TRAP)染色,对破骨细胞的进行计数和统计分析。分别在破骨细胞培养的1、2 d收取总蛋白和磷酸化蛋白,Western blot检测破骨细胞分化中关键转录因子NFATc1和c-Fos的表达以及磷酸化蛋白ERK表达;在培养的4 d提取RNA,Real-Time PCR检测破骨细胞相关基因CTSK、TRAP、MMP9和Car2的活性。结果:CCK8检测结果提示芒柄花素能够剂量依赖性地抑制BMMs的活性,在≤20?滋M的安全浓度范围内对BMMs细胞生长无明显毒性效应(P=0.278>0.05)。TRAP染色结果发现芒柄花素在(1~20?滋M)浓度范围内能够剂量依赖性的抑制破骨细胞的生成,尤其是10?滋M能够显著抑制破骨细胞的生成(P=0.000<0.05)。Western blot检测表明芒柄花素(10?滋M)能显著抑制破骨细胞分化关键蛋白NFATc1和c-Fos的表达,而对磷酸化蛋白ERK的表达未见明显的作用。在破骨细胞功能上,Real-Time PCR检测芒柄花素(10?滋M)能著抑制破骨细胞功能相关基因CTSK(P=0.000<0.05)、TRAP(P=0.000<0.05)、MMP9(P=0.000<0.05)和Car2(P=0.000<0.05)的表达。结论:鸡血藤有效组分芒柄花素能够抑制原代骨髓单核-巨噬细胞向破骨细胞增殖和分化,并下调破骨细胞骨吸收功能相关蛋白和基因的表达,可能是其防治股骨头坏死中骨破坏及塌陷的机制之一。     

测定红车轴草中刺芒柄花素含量的新方法

目的:建立了一种用同步荧光测定红车轴草中刺芒柄花素含量的新方法。方法:研究了检测条件对刺芒柄花素荧光强度的影响,以及实验的准确性、重现性和回收率。结果:该方法的线性回归方程为y=1440x-0.263,r=0.999,检出限为1.50×10-9mol/L。本方法测得红车轴草中刺芒柄花素的含量与HPLC法的结果相对偏差为1.65%。结论:说明本方法结果准确,可以满足科研和生产的要求。     

PAMPA permeability, plasma protein binding, blood partition, pharmacokinetics and metabolism of formononetin, a methoxylated isoflavone

Formononetin (FMN) is a methoxylated isoflavone which is the major constituent in red clover and in commercially available extracts of this plant. In this study, we investigated the parallel artificial membrane permeability assay (PAMPA) permeability, protein binding, blood uptake characteristics, pharmacokinetics and metabolism of FMN. The permeability study samples were analyzed by HPLC-PDA method; whereas the pharmacokinetic study, protein binding and whole blood partitioning samples were analyzed by LC-MS/MS method. The PAMPA permeability of FMN was found to be high at pH 4.0 and 7.0. Plasma protein binding of FMN was found to be 93.61 ± 0.44% and 96.14 ± 0.15% at the tested concentration of 50 and 150 ng/mL, respectively. FMN reached equilibrium fast between red blood cells (RBCs) and plasma, and the partition coefficients between RBCs and plasma (K_RBC/PL) were independent of the initial rat blood concentrations of FMN. The bioavailability of unchanged/free FMN was found to be poor, i.e. approximately 3%. FMN was found to have a high clearance (5.13 L/h/kg) and a large apparent volume of distribution (14.16 L/kg). Circulating conjugates (glucuronides/sulfates) of FMN and daidzein (DZN) were quantified using enzymatic hydrolysis of plasma samples. The levels of isoflavone glucuronides/sulfates were found to be much greater than that of the corresponding aglycones.     

芒柄花黄素对超氧阴离子及羟自由基清除作用的研究

目的:探讨芒柄花黄素体外对氧自由基的抑制和清除作用。方法:采用分光光度法检测芒柄花黄素对超氧阴离子自由基(O2-)和羟自由基(.OH)的清除及抑制作用。结果:芒柄花黄素终浓度为10、20、40μg.ml-1时,在体外对O2-的清除率分别为42.19%,56.64%及65.43%,对O2-生成的抑制率分别为31.06%,44.72%及63.35%;而对.OH的清除率分别为34.98%,41.06%及61.60%。结论:芒柄花黄素对O2-具有明显的抑制及清除作用,其对.OH也有明显的清除作用。     

HPLC法同时测定黄芪中4种黄酮类成分的含量

目的:建立以高效液相色谱法同时测定11个产地黄芪中毛蕊异黄酮葡萄糖苷、芒柄花苷、毛蕊异黄酮和芒柄花素4种黄酮类成分含量的方法,从有效成分含量探讨药材道地性内在因素。方法:色谱柱为Zorbax Eclipse XDB-C1(8250mm×4.6mm,5μm),流动相为乙腈-0.2%甲酸水溶液(梯度洗脱),检测波长为280nm。结果:毛蕊异黄酮葡萄糖苷、芒柄花苷、毛蕊异黄酮和芒柄花素的检测浓度分别在0.0051～0.510、0.0050～0.300、0.0049～0.294、0.0046～0.276mg·mL-1范围内与各自峰面积积分值呈良好的线性关系(r均为0.9999)。内蒙古、山西等4个产地的黄芪中毛蕊异黄酮苷和芒柄花苷的含量较高;河北安国、赤城和甘肃定西产黄芪中毛蕊异黄酮和芒柄花素含量较高。结论:本方法简便、快速、准确,试验结果与黄芪本草考证、道地沿革的文献基本相符。     

Formononetin attenuates H2O2-induced cell death through decreasing ROS level by PI3K/Akt-Nrf2-activated antioxidant gene expression and suppressing MAPK-regulated apoptosis in neuronal SH-SY5Y cells

Formononetin is an isoflavone, found in herbs like Trifolium pratense, which executes a variety of physiological activities including anti-neurodegenerative effect. However, the molecular mechanism of formononetin-mediated neuroprotection remains unclear. In this study, we investigated the protective effect of formononetin on hydrogen peroxide (H₂O₂)-induced death of human neuroblastoma SH-SY5Y cells and its underlying molecular mechanism. Formononetin suppressed H₂O₂-induced cytotoxicity. H₂O₂-induced increase in the intracellular reactive oxygen species (ROS) levels was decreased by formononetin, together with the enhanced expression of the antioxidant genes. H₂O₂-induced elevation of the Bax/Bcl-2 ratio and cleaved caspase-3 and caspase-7 levels were lowered by formononetin treatment. Moreover, formononetin repressed H₂O₂-induced phosphorylation of mitogen-activated protein kinases (MAPKs). Nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA decreased antioxidant gene expression and elevated the H₂O₂-induced ROS level in the formononetin-treated cells. Furthermore, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling is involved in the activation of the nuclear translocation of Nrf2. These results indicate that the neuroprotective effect of formononetin against H₂O₂-induced cell death is due to a decrease in the ROS level with the enhanced expression of the antioxidant genes through activation of the PI3K/Akt-Nrf2 signaling. In addition, formononetin suppressed apoptosis through inhibition of phosphorylation of MAPKs in SH-SY5Y cells. Thus, formononetin is a potential therapeutic agent for the treatment of neurodegenerative diseases.     

芒柄花黄素应用于抗癌药物开发的研究进展

 芒柄花黄素（Formononetin, FN）及其衍生物已被证明具有多种生物学功能，包括抗氧化、抗病毒和保护作用等，近年来它的抗癌活性得到国内外学者越来越多的关注。本文总结了芒柄花黄素在抗癌药物开发方面的研究进展，并探讨了当前存在的一些问题。     

HPLC测定新疆红芪中芒柄花素的含量

目的建立新疆红芪中芒柄花素(7-羟基-4′-甲氧基异黄酮)的含量测定方法。方法采用HPLC测定。结果平均回收率为99.48%,RSD=0.83%。结论所建方法快速、灵敏、准确,样品处理简单。