A new A431/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening epidermal growth factor receptor antagonists from Radix sophorae flavescentis

The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A₄₃₁ cells) are an important target for drug discovery. We have developed a new A₄₃₁/cell membrane chromatography (A₄₃₁/CMC)-online–high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A₄₃₁ cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A₄₃₁/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A₄₃₁ cell growth in vitro. This new A₄₃₁/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

Simultaneous Separation and Determination of Benzoic Acid Compounds in the Plant Medicine by High Performance Capillary Electrophoresis

A simple and inexpensive high performance capillary electrophoresis (HPCE) was applied to separate five benzoic acid compounds simultaneously. The investigation was carried out by micellar electrokinetic capillary chromatography (MECC). To avoid a time‐consuming and tedious procedure, orthogonal experimental design OA₉ (3⁴) for separation experiments was applied to find the optimal conditions in terms of the resolution and analytical time. The best conditions for separation were obtained using a 20 mM borax and 30 mM sodium dodecyl sulfate (SDS) buffer (pH 9.8) containing 2 mM β‐CD and 4% methanol (v/v). Online UV detection was performed at 250 nm. A voltage of 16 kV was applied and the temperature was controlled at 25 °C. Injection was performed for 5 s. The method was validated for the quantification of benzoic acid, salicylic acid and ortho‐aminobenzoic acid in Radix Isatidis, a traditional plant medicine with removal of endotoxin. The separation and determination were satisfactory and quick.     

高效液相色谱—质谱法鉴定葛根有效成分及测定小儿腹泻宁泡腾片中葛根素的

应用反相高效液相色谱－电喷雾－质谱－质谱（Ｒ－ＨＰＬＣ－ＥＳＩ－ＭＳ－ＭＳ）方法对中药葛根提取物的化学成分进行分析，分离并鉴定出葛根中的葛根素、大豆甙元、大豆甙、葛甙Ｃ等４种有效成分，应用ＨＰＬＣ紫外和质谱检测方法测定了一种改剂型四类新药“小儿腹泻宁泡腾片”中葛根素的含量分别为２．２７ｍｇ．ｇ＾－１和２．２６ｍｇ．＾－１，结果表明ＨＰＬＣ－ＭＳ可以作为葛根及其相关制剂的指纹临别和质量控制的一种方法。     

东毕血吸虫幼虫期在宿主体内发育的组织化学和扫描电镜观察

在内蒙古呼伦贝尔草原检获土耳其斯坦东毕吸虫幼虫期发育程度各不相同的阳性萝卜螺，通过组织学和组织化学方法对其幼虫期进行了观察． 参考周述龙（１９９１）对日本血吸虫尾蚴幼虫期进行的分期，把土耳其斯坦东毕吸虫的尾蚴发育过程也分为 ５ 期，尾蚴发育的胚细胞期、胚球期、尾蚴雏体期、尾蚴成熟前期和尾蚴成熟期． 子胞蚴育腔内胚细胞分裂，形成胚球，胚球中出现体细胞和胚细胞的分化，胚细胞结合疏松，胞浆中含酸性粘液和大量汞溴酚蓝反应阳性的碱性蛋白成分． 尾蚴成熟前后在三种腺体的呈色反应方面和日本血吸虫相似，其后钻腺腺体部分 Ｐ Ａ Ｓ反应呈阳性的含糖类物质在雏体期后期开始分泌，逐渐由腺管通向头器末端． 两种尾蚴在尾蚴的体实质部分，对 Ｐ Ａ Ｓ和汞溴酚蓝反应的呈色结果有着较大的差异． 另外，通过扫描电镜对土耳其斯坦东毕吸虫的尾蚴进行了比较观察，它们在头器的结构、体棘的形态以及感觉器的分布上存在着一定的差异．两种血吸虫的相似性表明在系统演化上有着密切的联系．     

The comparative pharmacokinetics of four bioactive ingredients after administration of Ramulus Cinnamomi–Radix Glycyrrhizae herb pair extract,Ramulus Cinnamomi extract and Radix Glycyrrhizae extract

Ramulus Cinnamomi (RC)–Radix Glycyrrhizae (RG) is a classic herb pair, which is commonly used as a fixed form to treat cardiovascular disease in the clinic. Our work aimed to compare the pharmacokinetic difference of cinnamic acid, liquiritin, isoliquiritigenin and glycyrrhetinic acid in rats after oral administration of the RC–RG herb pair extracts [Guizhigancao Decoction (GGD) and Lingguizhugan Decoction (LGZGD)] and the single RC or RG extract. A HPLC‐MS method was developed and validated to study comparative pharmacokinetics. The pharmacokinetic parameters (C_max, AUC, MRT) of four compounds between the RC–RG herb pair group and the single herb (RC or RG) group showed significant differences (p < 0.05). Compared with the single herb (RC or RG) group, higher peak concentration, slower elimination and larger exposure could be observed after giving the RC–RG herb‐pair extracts. The pharmacokinetic differences might indicate the relativity of remedy in the RC–RG herb pair and provide scientific information for rational administration of the drug in the clinic. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of anti‐Coxsackie virus B3 constituents of Radix Astragali by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

To profile the anti‐Coxsackie virus B3 constituents of Radix Astragali, an HPLC‐DAD‐MSⁿ analytical method, combined with an in vivo test, has been developed to identify the constituents of the active part, which has been demonstrated to have potency to inhibit the proliferation of virus in cardiac muscle, alleviate infraction in heart and elevate the survival rate of the animal. By comparing their retention time and MS data with those obtained from the authentic compounds and the published data, a total of 19 compounds, including 11 isoflavonoids and eight saponins, were identified, among which one pterocarpane glucoside was reported for the first time. The present study provides an approach to rapidly screening bioactive constituents in traditional Chinese medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

Fragmentation study of iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae by rapid resolution liquid chromatography with diode‐array detection and electrospray ionization time‐of‐flight mass spectrometry

Rapid resolution liquid chromatography (RRLC) coupled with diode array detection (DAD) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF MS) method was applied to the mass spectral study of a series of naturally occurring iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae, which provides higher speed and increased sensitivity without loss of resolution. With dynamic adjustment as the key role of the fragmentor voltage and confirmed with authentic standards, valuable structural information regarding the nature of both the glycoside skeletons was thus obtained. Most compositions were found to possess organic acid moiety such as cinnamoyl, caffeoyl and ferulyol. Besides extensive fragmentation of the carbohydrate moiety, losses of the hydroxyl and glucose residue units showed in the spectra, permitting the exploration of the skeleton and the identity of substituents in the molecule. Ten major iridoid glycosides and 10 phenylpropanoid glycosides were identified or tentatively characterized based on their retention times, UV and TOF MS data. The major fragmentation pathways of PGs in Radix Scrophulariae obtained through the MS data was schemed systematically for the first time, which provides a reference for other PGs derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and application of mesoporous molecular sieve for miniaturized matrix solid‐phase dispersion extraction of bioactive flavonoids from toothpaste,plant, and saliva

This article describes the use of the mesoporous molecular sieve KIT‐6 as a sorbent in miniaturized matrix solid‐phase dispersion (MSPD) in combination with ultra‐performance LC for the determination of bioactive flavonoids in toothpaste, Scutellariae Radix, and saliva. In this study, for the first time, KIT‐6 was used as a sorbent material for this mode of extraction. Compared with common silica‐based sorbents (C18 and activated silica gel), the proposed KIT‐6 dispersant with a three‐dimensional cubic Ia3d structure and highly ordered arrays of mesoporous channels exhibits excellent adsorption capability of the tested compounds. In addition, several experimental variables, such as the mass ratio of sample to dispersant, grinding time, and elution solvent, were optimized to maximize the extraction efficiency. The proposed analytical method is simple, fast, and entails low consumption of samples, dispersants and elution solvents, thereby meeting “green chemistry” requirements. Under the optimized conditions, the recoveries of three bioactive flavonoids obtained by analyzing the spiked samples were from 89.22 to 101.17%. Also, the LODs and LOQs for determining the analytes were in the range of 0.02–0.04 μg/mL and 0.07–0.13 μg/mL, respectively. Finally, the miniaturized matrix solid‐phase dispersion method was successfully applied to the analysis of target solutes in real samples, and satisfactory results were obtained.     

不同产地黄芪的红外光谱鉴别研究

采用红外光谱、二阶导数红外光谱和二维相关红外光谱,对四种不同产地的黄芪原药材进行了鉴别研究。结果表明:不同产地黄芪的红外光谱和二阶导数红外谱具有一定的相似度,与淀粉的红外谱图比对,4个不同产地的黄芪均含有淀粉,其中陕西绥德产黄芪的淀粉含量比其它3个产地黄芪的都要高。山西浑源和山西天镇产黄芪谱图的1 510、1 425cm-1木质素特征峰比内蒙古固阳和陕西绥德产黄芪的更为明显,说明前二者产黄芪中木质素含量高于后二者产黄芪。在二维相关红外谱图上,根据4个产地黄芪的相对峰强度的差异,可进行产地的鉴别。研究结果表明对于不同产地黄芪的鉴别,红外三级鉴定法是一种快速有效的新方法。     

An improved cell membrane chromatography method for the simultaneous screening of two epidermal growth factor receptor antagonists from radix scutellariae

A high‐expression epidermal growth factor receptor cell membrane chromatography using the silica gel with the average particle size of 3 μm as the stationary phase carrier coupled with high‐performance liquid chromatography and mass spectrometry was established for the online screening of epidermal growth factor receptor antagonists from Radix Scutellariae (Huang Qin in Chinese), a traditional Chinese medicine. In this study, the growth factor receptor cell membrane chromatography model using the smaller particle size carrier showed a higher efficiency for simultaneous screening baicalein, another one of the potential epidermal growth factor receptor antagonists from Radix Scutellariae extract besides wogonin, which was found in our previous work. The molecular docking result showed the occupancy site and binding mode of baicalein and wogonin with epidermal growth factor receptor tyrosine kinase were similar to gefitinib. The result of the assay for the in vitro inhibitory activity showed that baicalein and wogonin inhibited the growth of the high‐expression epidermal growth factor receptor cell in a dose‐dependent manner and even achieved a better inhibition effect than gifitinib in the low‐dosage range.