Prostate Cell Membrane Chromatography�CLC/MS Method for Screening ��1A-Adrenoceptor Antagonists from Lotus Plumule

The ??_1A-adrenoceptor (??_1A-AR) plays an important role in drug discovery and development. An online analytical method coupling prostate cell membrane chromatography (CMC) with high-performance liquid chromatography/mass spectrometry (LC/MS) was established to screen ??_1A-AR antagonists in traditional Chinese medicines (TCMs). The prostate cell membrane stationary phase (CMSP) was prepared by immobilizing the prostate cell membrane onto the surface of the silica carrier. The surface and chromatographic characteristics of the prostate CMSP were studied using tamsulosin as a model molecule. The retained fractions from the prostate CMC were analyzed by transferring them to an LC/MS system through a 10-port switch valve. The active component, which could act on the prostate cell membrane and receptor on it (such as ??_1A-AR), was determined using a displacement experiment. The results indicated that liensinine, isoliensine, and neferine from Lotus Plumule exhibited similar retention characteristics to the control drug tamsulosin when utilizing the prostate CMC model. This new prostate CMC?CLC/MS method is applicable for screening ??_1A-AR antagonists from TCMs such as Lotus Plumule and could be employed as a drug discovery tool for natural medicinal herbs.     

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.     

Establishment of a High Expression of α1A Adrenergic Receptor Cell Membrane Chromatography-RPLC Method for Screening Target Components from Radix Caulophylli

We describe here an analytical method of HEK293 α_1A AR cell membrane chromatography (HEK293 α_1A AR/CMC) combined with reverse phase liquid chromatography (RPLC) for recognition, separation and identification of target components from Traditional Chinese Medicines (TCMs) Radix Caulophylli. The HEK 293 α_1A cells with high expressing α_1A adrenergic receptors were used to prepare the stationary phase in the CMC model. Retention fractions on the α_1A AR–CMC model were collected using an automated fraction collection and injection module (FC/I). And each fraction was analyzed by RPLC under optimized conditions. 5-Methylurapidil (5-MU) and tamsulosin hydrochloride were used as standard compounds to investigate the suitability and reliability of the HEK 293 α_1A AR–CMC–RPLC method prior to screening target component from Radix Caulophylli total alkaloid. The results indicated that caulophine was the target component acting on the α_1A AR. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds.     

EGFR/cell membrane chromatography-online-high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Radix Angelicae Pubescentis

The intracellular kinase domains of the epidermal growth factor receptor (EGFR) in some tumor cells are significant targets for drug discovery. We have developed a new EGFR cell membrane chromatography (EGFR/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening anti-EGFR antagonists from medicinal herbs such as Radix Angelicae Pubescentis. In this study, the HEK293 EGFR cells with high expression of EGFR were used to prepare cell membrane stationary phase (CMSP) in the EGFR/CMC model. The retention fractions on the EGFR/CMC model were directly analyzed by combining a 10 port columns switcher with a HPLC/MS system online. As a result, osthole from Radix Angelicae Pubescentis was found to be the active component acting on EGFR like dasatinib as the control drug. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. This new EGFR/CMConline-HPLC/MS method can be applied for screening anti-EGFR antagonists from TCMs, for instance, Radix Angelicae Pubescentis. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

Screening potential antagonists of epidermal growth factor receptor from Marsdenia tenacissima via cell membrane chromatography model assisted by HPLC–ESI–IT–TOF–MS

Marsdenia tenacissima, or Tongguanteng in Chinese, is a traditional Chinese herb and has a broad application in clinical practice for its pharmacological effects of treating asthma, pneumonia, tonsillitis, pharyngitis tumors, etc. However, few studies have reported the screening of the active components of this medicine for tumor therapy. In this work, a two‐dimensional analytical system was developed to screen antagonists of epidermal growth factor receptor (EGFR) from M. tenacissima. A fraction was retained on the EGFR cell membrane chromatography (CMC) column, separated and identified as tenacissoside G (TG), tenacissoside H (TH) and tenacissoside I (TI) by two‐dimensional HPLC–IT–TOF–MS. Molecular docking and 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) assay were carried out to assess the activity of TS (including TG, TH and TI). Molecular docking results showed that the binding mode of TS on EGFR is similar to that of gefitinib. The MTT assay demonstrated that gefitinib and TS (especially TI) could inhibit the growth of EGFR highly expressed cell lines in a dose‐dependent manner in the range of 5–50 μmol/L. In conclusion, the two‐dimensional EGFR/CMC–HPLC–IT–TOF–MS system could be a useful approach in drug discovery from traditional Chinese medicines for searching for potential antitumor candidates.     

An online coupled peritoneal macrophage/cell membrane chromatography and high‐performance liquid chromatography/mass spectrometry method to screen for anti‐inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei

Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)–online‐high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti‐inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor‐α in lipopolysaccharide‐stimulated mice and peritoneal macrophages in a dose‐dependent manner. The results demonstrated that the PM/CMC‐online‐HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Screening active anti‐breast cancer compounds from Cortex Magnolia officinalis by 2D LC–MS

Most of the anti‐breast cancer drugs are often limited owing to drug resistance and serious adverse reactions. Therefore, development of more targeted and low toxic drugs from traditional Chinese medicines for breast cancer are needed. At the same time, establishment of fast and effective drug screening methods are urgently required. We describe here a 2D LC method of MDA‐MB‐231 cell membrane chromatography combined with HPLC/MS for recognition, separation, and identification of target components from traditional Chinese medicine Cortex Magnolia officinalis. The MDA‐MB‐231 cells membrane was used to prepare the chromatographic stationary phase in the first dimension. The active compounds had a retention characteristic on the cell membrane chromatography model (10 × 2.0 mm, 5 μm). The retention fractions were enriched using an online C₁₈ column (10 × 1.0 mm, 5 μm) and were analyzed by the second dimension RP chromatography. Finally, the activity of the retention fractions was tested through in vitro experiments. Results showed that the retention fractions were honokiol and magnolol and the inhibition rate on MDA‐MB‐231 cell growth were 23 and 64 μM, respectively. These results support the conclusion that this coupled analytical technique could be an efficient method in drug discovery.     

Simultaneous screening of four epidermal growth factor receptor antagonists from Curcuma longa via cell membrane chromatography online coupled with HPLC–MS

The epidermal growth factor receptors (EGFRs) are significant targets for screening active compounds. In this work, an analytical method was established for rapid screening, separation, and identification of EGFRs antagonists from Curcuma longa. Human embryonic kidney 293 cells with a steadily high expression of EGFRs were used to prepare the cell membrane stationary phase in a cell membrane chromatography model for screening active compounds. Separation and identification of the retention chromatographic peaks was achieved by HPLC–MS. The active sites, docking extents and inhibitory effects of the active compounds were also demonstrated. The screening result found that ar‐turmerone, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from Curcuma longa could be active components in a similar manner to gefitinib. Biological trials showed that all of four compounds can inhibit EGFRs protein secretion and cell growth in a dose‐dependent manner, and downregulate the phosphorylation of EGFRs. This analytical method demonstrated fast and effective characteristics for screening, separation and identification of the active compounds from a complex system and should be useful for drug discovery with natural medicinal herbs.     

Activity ranking of synthetic analogs targeting vascular endothelial growth factor receptor 2 by an integrated cell membrane chromatography system

Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well‐developed biological chromatographic technique. In this study, we have developed combined SMMC‐7721/CMC and HepG2/CMC with high‐performance liquid chromatography and time‐of‐flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R² = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery.     

用高表达EGFR细胞膜色谱-HPLC/MS联用快速筛选独活中抗EGFR活性成分

报道了用高表达表皮生长因子受体细胞膜色谱与高效液相色谱/质谱在线联用方法(EGFR/CMC-online-HPLC/MS)快速筛选发现中药独活中的活性成分.实验中,采用高表达EGFR的细胞膜制备色谱固定相,建立EGFR/细胞膜色谱(EGFR/CMC)模型,利用柱切换和固相萃取技术,将EGFR/CMC模型与高效液相色谱/质谱(HPLC/MS)在线联用,构成一种新的可同时"识别-鉴定"目标成分的二维色谱系统,并应用于快速筛选独活中具有抗EGFR活性的目标成分.结果发现独活中的蛇床子素具有与对照药物达沙替尼类似的色谱保留特性,能够作用于EGFR;同时MTT及Elisa分析实验证实蛇床子素对HEK293EGFR细胞增殖及EGFR表达均有抑制作用.本文建立的EGFR/CMC-online-HPLC/MS二维色谱方法,可以选择性地从中药复杂体系中快速"识别-鉴定"目标组分,且筛选结果与特定生物效应显著相关.     

Research on drug–receptor interactions and prediction of drug activity via oriented immobilized receptor capillary electrophoresis

Oriented covalent immobilized β₂‐adrenergic receptor (β₂‐AR) CE (OIRCE) was developed to determine the interactions between a set of natural extracts of Radix Paeoniae Rubra (NERPR) and β₂‐AR, and to predict the activity of NERPR. The inner capillary surface is chemically bonded with stable β₂‐AR coating via microwave‐assisted technical synthesis. The modified capillaries were characterized via infrared spectroscopy and fluorescence microscopy. Furthermore, the bonding amounts of β₂‐AR were first obtained via fluorescence spectroscopy method. In determining the amount of bonded β₂‐AR, the regression equation A  =  576 707C + 35.449 and the correlation coefficient 0.9995 were obtained. This result revealed an excellent linear relationship in the range of 2 × 10^?4 mg/mL to 1 × 10^?3 mg/mL. The normalized capacity factor (K_RCE) was obtained using OIRCE in evaluating drug–receptor interactions. Related theories and equations were used to calculate K_RCE values from apparent migration times of a solute and EOF. The order of K_RCE and the binding constant (K_b) values between drugs and β₂‐AR was well consistent. The results confirmed that the OIRCE and K_RCE values can be effectually used to investigate drug‐receptor interactions, and OIRCE has the potential to predict drug activity and to select leading compounds from natural chemicals.     

Recognition and identification of active components from Radix Bupleuri using human neuroblastoma SH‐SY5Y cells

The aim of the study was to screen active components of Radix Bupleuri (a traditional Chinese herb) and discover novel anti‐schizophrenic candidate drugs using human neuroblastoma SH‐SY5Y cells. SH‐SY5Y cells were used for preparation of the stationary phase in the cell membrane chromatography model. Retention components by the SH‐SY5Y/CMC model were collected and then analyzed by GC/MS under the optimized conditions in offline conditions. After investigating the suitability and reliability of the SH‐SY5Y/CMC method using amisulpride and haloperidol as standard compounds, this method was applied to screening active components from the extracts of Radix Bupleuri. Retention components of SH‐SY5Y/CMC model were saikosaponin A, saikosaponin B1, saikosaponin B2, saikosaponin C and saikosaponin D, which were identified by the GC/MS method. In vitro pharmacological trials‐MTT, saikosaponin B1, saikosaponin B2 and saikosaponin C could protect SY5Y cells. The protective effects of saikosaponin B1 and saikosaponin C were concentration dependent. Saikosaponin A and saikosaponin D inhibited cell viability at concentrations >30 µg/mL (p < 0.05). Via SH‐SY5Y/CMC method and SH‐SY5Y MTT trial, we rapidly detected target components from Radix Bupleuri, accurately identified them and determined their different effects on SH‐SY5Y cells. Saikosaponin B1, saikosaponin B2 and saikosaponin C may be anti‐schizophrenic candidate drugs. Copyright © 2015 John Wiley & Sons, Ltd.     

成骨细胞膜色谱/超高效液相色谱-飞行时间质谱法快速筛选六味地黄汤抗骨质疏松活性成分

采用成骨细胞建立了细胞膜色谱/超高效液相色谱-飞行时间质谱（CMC/UPLC-TOF/MS）的分析方法。该法可快速筛选中药方剂六味地黄汤中潜在的抗骨质疏松活性成分，通过细胞试验和斑马鱼骨质疏松模型试验验证了筛选结果梓醇的体内外药效作用。以六味地黄汤水提物（90 g/L）为样品，通过CMC/UPLC-TOF/MS分析，快速鉴别细胞膜色谱柱保留成分群，高选择性获取六味地黄汤中16种潜在活性成分。该文以前沿色谱法分析梓醇、丹皮酚、齐墩果酸与细胞膜色谱固定相的亲和强度，选择亲和强度和含量较高的梓醇进行体内外药效验证，发现梓醇在对小鼠成骨细胞有显著促生长作用，能提高骨质疏松斑马鱼头部骨矿化面积。CMC/UPLC-TOF/MS筛选方法能在复杂中药方剂中快速得到抗骨质疏松活性成分，具有操作简便、快速、高效灵敏的优势。     

The human mast cell line‐1 cell membrane chromatography coupled with HPLC‐ESI‐MS/MS method for screening potentical anaphylactic components from chuanxinlian injection

Chuanxinlian injection is a traditional Chinese medicine injection widely used in China to treat sore throat, cough and dysentery, although a high occurrence of severe adverse reactions has been reported in clinical practice in recent years. In the present study, a human mast cell line‐1 cell membrane chromatography coupled with HPLC‐ESI‐MS/MS method was established to screen and identify potentical anaphylactic components in chuanxinlian injection, and the dehydroandrographolide was identified as a potential anaphylactic component. In vitro anaphylactic assay showed that intracellular Ca²⁺ concentration clearly increased under dehydroandrographolide (100 μm ) treatment. β ‐Hexosaminidase and histamine release in human mast cell line‐1 cells were both markedly enhanced with increased concentrations of dehydroandrographolide, confirming the anaphylactic activity of dehydroandrographolide. The application for chuanxinlian injection in this study suggested that the developed human mast cell line‐1 cell membrane chromatography coupled with HPLC‐ESI‐MS/MS system may be effective and rapid for screening the potentical anaphylactic components from complex samples.     

Using open‐tubular capillary electrochromatography with part‐coating column for binding constants determination of β2‐adrenergic receptor with seven drugs

An open‐tubular capillary electrochromatography method has been developed for the determination of binding constants between β₂‐adrenergic receptor (β₂‐AR) and seven drugs. β₂‐AR was oriented immobilized onto one part of inner surface of capillary via microwave‐assisted technical synthesis. According to the linear relationship between coating length and the apparent mobility of analyte, the binding constant (K_b) can be obtained by related theories and equations. The order of K_b values between drugs such as adrenaline hydrochloride, norepinephrine bitartrate, and propranolol hydrochloride with β₂‐AR is well consistent with that reported in the literature. By the method, K_b values between four extracts of Radix Paeoniae Rubra and β₂‐AR were also successfully obtained. Subsequently, computer models were applied to interpret the CEC experiments. And the results proved to be in good agreement with the method. The work, herein, demonstrates the potential of the method in drug‐receptor affinity interactions evaluation and screening of lead compounds from natural sources.     

Bioactivity‐based ultra‐performance liquid chromatography‐coupled quadrupole time‐of‐flight mass spectrometry for NF‐κB inhibitors identification in Chinese Medicinal Preparation Bufei Granule

Traditional Chinese medicine (TCM) preparations have become effective treatments for many diseases. However, their active ingredients are still uncertain and difficult to identify. In this study, we propose a strategy that integrates ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) and bioactive (NF‐κB inhibitor) luciferase reporter assay systems for the rapid determination of various anti‐inflammatory compounds of TCM preparations. In this way, Bufei Granule (BFG), a TCM preparation used for the clinical therapy of asthma, was analyzed by the two ways of component identification and activity detection. Potential anti‐inflammatory constituents were screened by NF‐κB activity assay systems and simultaneously identified according to the mass spectrometry data. Three structural types of NF‐κB inhibitors (caffeic acid derivatives, flavonoids and Pentacyclic triterpenes) were characterized. Further cytokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q‐TOF‐MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data‐based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in TCM preparations. Copyright © 2016 John Wiley & Sons, Ltd.     

A vascular smooth muscle/cell membrane chromatography–offline-gas chromatography/mass spectrometry method for recognition,separation and identification of active components from traditional Chinese medicines

We describe an analytical method of vascular smooth muscle cell membrane chromatography (VSM/CMC) combined with gas chromatography/mass spectrometry (GC/MS) for recognition, separation and identification of active components from traditional Chinese medicines (TCMs). VSM cells by means of primary culture with rat thoracic aortas were used for preparation of the stationary phase in the CMC model. Retention components by the VSM–CMC model were collected and then analyzed by GC/MS under the optimized conditions in offline conditions. After investigating the suitability and reliability of the VSM/CMC–offline-GC/MS method using nifedipine and nitrendipine as standard compounds, this method was applied in screening active components from the extracts of TCMs such as Radix Angelicae Dahuricae (RAD), Rhizomza Seu Radix Notopterygii (RSRN), Radix Glehniae (RG) and Fructus Cnidii (FC). Retention components from the extracts in the VSM–CMC model were imperatorin and osthole identified by the GC/MS method. In vitro pharmacological trials indicated that imperatorin and osthole could concentration dependently relax the rat thoracic artery pre-contracted by KCl (P < 0.05). The maximum relaxation effects (R_max) were 63 ± 5% and 40 ± 6% for imperatorin and osthole, respectively. The VSM/CMC–offline-GC/MS method is an effective screening system that can rapidly detect and enrich target components from a complex sample and then accurately identify them.     

Screening anti‐allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti‐allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC‐ESI‐MS/MS method was developed to screen, analyze and identify the anti‐allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC‐ESI‐MS/MS. The coupled system was then used to screen anti‐allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose‐dependent manner from 1 to 100 μm . The LAD2 cell membrane chromatography online with UHPLC‐ESI‐MS/MS method is an effective technique for screening the anti‐allergic components of Huangqi.     

Establishment of A431 cell membrane chromatography-RPLC method for screening target components from Radix Caulophylli

We describe here an analytical method of A431 cell membrane chromatography (A431/CMC) (CMC, cell membrane chromatography) combined with RPLC for recognition, separation, and identification of target components from traditional Chinese medicines (TCMs) Radix Caulophylli. The A431 cells with high expressed epidermal growth factor receptor (EGFR) were used to prepare the stationary phase in the CMC model. Retention fractions on the A431-CMC model were collected using an automated fraction collection and injection module (FC/I). Each fraction was analyzed by RPLC under the optimized conditions. Gefitinib and erlotinib were used as standard compounds to investigate the suitability and reliability of the A431 cell membrane chromatography-RPLC method prior to screening target component from Radix Caulophylli total alkaloids. The results indicated that caulophine and taspine were the target component acting on the epidermal growth factor receptor. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds.     

Screening epidermal growth factor receptor antagonists from Radix et Rhizoma Asari by two‐dimensional liquid chromatography

Radix et Rhizoma Asari is a traditional Chinese medicine, and has many pharmacological effects, such as calming, analgesia, anti‐inflammation, antiarrhythmic, antihypertensive, antivirus, etc. But few studies have screened the active compounds from extracts of Radix et Rhizoma Asari for tumor therapy. In this study, a two‐dimensional liquid chromatography system was built to screen active compounds acting on epidermal growth factor receptor (EGFR) from Radix et Rhizoma Asari. The screening result showed that asarinin from Radix et Rhizoma Asari was the targeted component that could act on EGFR specificity. The competitive binding assay and molecular docking assay results showed asarinin binding with EGFR in similar manner as with gefitinib, which was used as a positive control drug. Then the antitumor effect of asarinin was studied through cell growth assay in vitro. The results showed that gefitinib and asarinin could inhibit highly expressed EGFR cell growth in a dose‐dependent manner in the range of dose from 0.10 to 102.4 μM. This two‐dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates.