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Mas‐related G protein‐coupled receptor X2 (MrgprX2) is acknowledged as a mast cell‐specific receptor, playing a crucial role in orchestrating anaphylactoid responses through mast cell degranulation. It holds promise as a target for regulating allergic and inflammatory diseases mediated by mast cells. Polygonum cuspidatum (PC) has shown notable anti‐anaphylactoid effects, while its pharmacologically active components remain unclear. In this study, we successfully utilized MrgprX2 high‐expressing cell membrane chromatography (CMC), in conjunction with liquid chromatography‐mass spectrometry (LC‐MS), to identify active anti‐anaphylactoid components in PC. Our study pinpointed polydatin, resveratrol, and emodin‐8‐O‐β‐d‐glucoside as potential anti‐anaphylactoid compounds in PC. Their anti‐anaphylactoid activities were evaluated through β‐aminohexosidase and histamine release assays, demonstrating a concentration‐dependent inhibition for both β‐aminohexosidase and histamine release. This approach, integrating MrgprX2 high‐expression CMC with LC‐MS, proves effective in screening potential anti‐anaphylactoid ingredients in natural herbal medicines. The findings from this study illuminated the anti‐anaphylactoid properties of specific components in PC and provided an efficient method for the drug development of natural products. [ABSTRACT FROM AUTHOR]

Gastrointestinal mesenchymal tumors, as the most common mesenchymal tumors in the gastrointestinal tract, are adjuvantly treated with multi-targeted tyrosine kinase inhibitors, such as imatinib and sunitinib, but there are problems of drug resistance and complex methods of monitoring therapeutic agents. The pathogenesis of this disease is related to mutations in tyrosine kinase (KIT) or platelet-derived growth factor receptor α, an important target for drug therapy. In recent years, the screening of relevant tyrosine kinase inhibitors from traditional Chinese medicine has become a hotspot in antitumor drug research. In the current study, the KIT-SNAP-tag cell membrane chromatography (KIT-SNAP-tag/CMC) column was prepared with satisfying specificity, selectivity, and reproducibility by chemically bonding high KIT expression cell membranes to the silica gel surface using the SNAP-tag technology. The KIT-SNAP-tag/CMC-HPLC-MS two-dimensional coupling system was investigated using the positive drug imatinib, and the results showed that the system was a reliable model for screening potential antitumor compounds from complex systems. This system screened and identified three potential active compounds of evodiamine (EVO), rutaecarpin (RUT), and dehydroevodiamine (DEVO), which possibly target the KIT receptor, from the alcoholic extract of the traditional Chinese medicine Evodia rutaecarpa. Then, the KD values of the interaction of EVO, RUT, and DEVO with KIT receptors measured using nonlinear chromatography were 7.75 (±4.93) × 10−6, 1.42 (±0.71) × 10−6, and 2.34 (±1.86) × 10−6 mol/L, respectively. In addition, the methyl thiazolyl tetrazolium assay validated the active effects of EVO and RUT in inhibiting the proliferation of high KIT-expressing cells in the ranges of 0.1–10 µmol/L and 0.1–50 µmol/L, respectively. In conclusion, the KIT-SNAP-tag/CMC could be a reliable model for screening antitumor components from complex systems. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Cell membrane chromatography (CMC), as a highly selective type of affinity chromatography, has been demonstrated as an effective method to screen bioactive components acting on specific receptor from a complicated biological system. Objective: To review the recent research progress and the technical applications of these analytical methods using CMC combined with gas chromatography‐mass spectrometry, (GC/MS) and liquid chromatography‐mass spectrometry (LC/MS). Methodology: In this review, we briefly introduce the CMC offline GC/MS, CMC online GC/MS, CMC offline LC/MS, and CMC online LC/MS system. And the practical application of these technologies is also enumerated. Then the future of these technologies and research methods were discussed. Results: Many bioactive components interacting with specific receptors have been screened and identified in traditional Chinese medicines. Conclusion: CMC technique has been combined with GC/MS and HPLC/MS and these combined systems have been successfully used to screen bioactive components acting on specific receptors from a complicated biological system. [ABSTRACT FROM AUTHOR]

Peucedanum praeruptorum Dunn (BaiHuaQianHu in Chinese) is a traditional Chinese medicine that has a long history of use in China. In this study, HEK 293 α1A adrenergic cell membrane chromatography was coupled with UHPLC-ESI-MS/MS and successfully used to identify active components from Peucedanum praeruptorum Dunn. Paeruptorin A, paeruptorin B, and paeruptorin C were identified with α1A adrenergic receptor activity. Pharmacological assays showed that tamsulosin hydrochloride, paeruptorin A, paeruptorin B, and paeruptorin C in concentrations of 1 × 10 −8 to 1 × 10 −4 mol/mL could relax prostate strips pre-contracted with adrenalin in a concentration dependent manner. Therefore, the HEK293 α1A cell membrane chromatography coupled UHPLC–ESI–MS/MS system may be a potentially useful drug discovery method for screening for medicinal herbal components with α1A adrenergic receptor inhibitory activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] CMC was first used to investigate the security of TCM injection. [•] The sensitization of the screening result was verified. [•] Baicalin was identified as an allergenic component. [ABSTRACT FROM AUTHOR]

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography–mass spectrometry (LC–MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC–MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors. [ABSTRACT FROM PUBLISHER]

Cell membrane chromatography (CMC) is an effective method for studying receptors with multiple transmembrane structure such as MAS-related G protein-coupled receptor X2 (MrgX2). CMC relies on the maintenance of the complete biological structure of a membrane receptor; however, it needs to be further improved to obtain a more convenient and stable CMC model. In the present study, the haloalkane dehalogenase protein tag (HALO-tag) technology was used to construct a new MrgX2/CMC model. The fusion receptors of MrgX2 with HALO-tag at the C terminus were expressed in HEK293 cells. The silica gel was modified with a substrate of HALO-tag (chloroalkanes) via one-step acylation for the rapid capture of fusion receptors. The new CMC model (MrgX2-HALO-tag/CMC model) was not only quicker to prepare but also more stable and had a longer lifespan than a previous MrgX2-SNAP-tag/CMC model. In combination with the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system, the MrgX2-HALO-tag/CMC model was used to screen and identify bioactive components in traditional Chinese medicine. Using this combination, sanggenon C and morusin were identified from Mori Cortex as anti-pseudo-allergic components. The MrgX2-HALO-tag/CMC model alone was also applied to analyze ligand–receptor interaction. The affinity order of four ligands to MrgX2 was as follows: desipramine < imipramine < amitriptyline < clomipramine. This was consistent with the results obtained using the MrgX2-SNAP-tag/CMC model. The MrgX2-HALO-tag/CMC model provides ideas and application prospects for the immobilization of cell membrane that contains receptors with more transmembrane structures. [Display omitted] • MrgX2 was immobilized in an easy and convenient way through HALO-tag technology. • The MrgX2-HALO-tag/CMC model improves performances of MrgX2-SNAP-tag/CMC model. • Sanggenon C and morusin were screened out as anti-pseudo-allergic compounds. • The affinity of four ligands with MrgX2 were successfully determined. [ABSTRACT FROM AUTHOR]

Highlights • Accurate quantification of β-hexosaminidase by LC–MS/MS method. • An attractive alternative method to traditional enzyme-based "substrate method". • An approach used to detecting β-hexosaminidase released from LAD2 cells. • A method for the preliminary assessment of anaphylactic reactions. • A reference technique for scientific research and clinical testing. Abstract β-Hexosaminidase is one of the enzymes that is secreted from mast cells via antigen-induced degranulation and has frequently been used as an indicator of anaphylactic reactions. The main method for determining β-hexosaminidase is indirect, "substrate-based" and shows limitations. Hence, development of an accurate detecting method is particularly important and urgently needed. In this study we developed a liquid chromatography tandem mass spectrometry (LC–MS/MS) method for measuring β-hexosaminidase. Laboratory of allergic diseases 2 (LAD2) cells were stimulated with compound 48/80 (C48/80), the supernatant was collected and subjected to in-solution protein digestion; the obtained peptides were desalted, concentrated, separated and analyzed with an LC-tandem MS instrument. A peptide with the sequence "LAPGTIVEVWK" was selected for quantitative analysis, and four other peptides for qualitative research. The time-effect relationship curve was studied, and the results of the LC–MS/MS method were found to be almost consistent with those obtained via the conventional method. The method was then employed to measure β-hexosaminidase released from LAD2 cells stimulated with potential allergens, and the results showed that it can be applied to determine the potential allergenicity of drugs. This new method showed good specificity, high sensitivity and a wide application range. It could be used to evaluate allergic reactions, providing a guide for medication safety during clinical testing. [ABSTRACT FROM AUTHOR]