Search

Mas-related G protein-coupled receptor X2 (MrgprX2) plays a crucial role in anaphylactoid reactions and allergic diseases. Some antagonists with reasonable potency and selectivity have been reported. Cell membrane chromatography (CMC) is effective for discovering ligands. Protein-tag-based CMC models (e.g., SNAP tags and HALO tags) have enhanced performance but also increased nonspecific adsorption of small molecules. The Avi tag, a short peptide sequence, binds biotin specifically via BirA catalysis. Our study showed that 2-iminobiotin (IB) can be a BirA substrate, enabling the development of a new cell membrane stationary phase (CMSP) based on the chemical properties (modifying carboxyl silica gel and specifically labeling the Avi tag) of IB. First, we constructed the MrgprX2-Avi-tag HEK293T cell line. Next, we synthesized IB-modified silica gel (SiO 2 -IB) stepwise. Finally, we immobilized Avi-tagged MrgprX2 cell membranes on SiO 2 -IB under BirA catalysis. We characterized the developed CMSP and used it to establish a MrgprX2-Avi-tag/CMC-HPLC/MS two-dimensional screening platform, successfully screening vitexicarpin from Viticis Fructus extract via a 2D/CMC platform. In vitro and in vivo experiments confirmed that vitexicarpin targets the MrgprX2 receptor, demonstrating antiallergic effects. Our IB-Avi tag-based CMC approach effectively decreased nonspecific adsorption of the screening materials. The Avi-tag-based 2D/CMC platform is suitable for screening potential drug candidates.

Mitochondrial Membrane Chromatography (MMC) is a bioaffinity chromatography technique developed to study the interaction between target proteins embedded in the mitochondrial membrane and their ligand compounds. However, the MMC stationary phases (MMSP) prepared by chemical immobilization are prone to nonspecific binding in candidate agent screening inevitably. To address these challenges, Twin Strep-Tag/Strep Tactin was employed to establish a specific affinity system in the present study. We prepared a carnitine palmitoyltransferase 1A (CPT1A) MMSP by specifically linking Strep-tactin-modified silica gel with the Twin Strep-Tag on the CPT1A-oriented mitochondrial membrane. This Twin Strep-Tag/Strep Tactin modified CPT1A/MMC method exhibited remarkably better retention behavior, longer stationary phase lifespan, and higher screening specificity compared with previous MMC systems with glutaraldehyde immobilization. We adopted the CPT1A-specific MMC system in screening CPT1A ligands from traditional Chinese medicines, and successfully identified novel candidate ligands: ononin, isoliquiritigenin, and aloe-emodin, from Glycyrrhiza uralensis Fisch and Senna tora (L.) Roxb extracts. Biological assessments illustrated that the compounds screened promote CPT1A enzyme activity without affecting CPT1A protein expression, as well as effectively reduce the lipid droplets and triglyceride levels in the high fat induction HepG2 cells. The results suggest that we have developed an MMC system, which is promising for studying the bioaffinity of mitochondrial membrane proteins to candidate compounds. This system provides a platform for a key step in mitochondrial medicine discovery, especially for bioactive molecule screening from complex herbal extracts.

Non-small cell lung cancer (NSCLC) brain metastases present a significant treatment challenge due to limited drug delivery efficiency and severe adverse reactions. In this study, we address these challenges by designing a "on/off" switchable crosslinked paclitaxel (PTX) nanocarrier, BPM-PD, with novel ultra-pH-sensitive linkages (pH 6.8 to 6.5). BPM-PD demonstrates a distinct "on/off" switchable release of the anti-cancer drug paclitaxel (PTX) in response to the acidic extratumoral microenvironment. The "off" state of BPM-PD@PTX effectively prevents premature drug release in the blood circulation, blood-brain barrier (BBB)/blood-tumor barrier (BTB), and normal brain tissue, surpassing the clinical PTX-nanoformulation (nab-PTX). Meanwhile, the "on" state facilitates precise delivery to NSCLC brain metastases cells. Compared to nab-PTX, BPM-PD@PTX demonstrates improved therapeutic efficacy with a reduced tumor area (only 14.6%) and extended survival duration, while mitigating adverse reactions (over 83.7%) in aspartate aminotransferase (AST) and alanine aminotransferase (ALT), offering a promising approach for the treatment of NSCLC brain metastases. The precise molecular switch also helped to increase the PTX maximum tolerated dose from 25 mg/kg to 45 mg/kg This research contributes to the field of cancer therapeutics and has significant implications for improving the clinical outcomes of NSCLC patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024 Wiley‐VCH GmbH.)

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignant tumors occurring in B or T lymphocytes, and no small molecule-positive drugs to treat NHL have been marketed. Cluster of differentiation 20 (CD20) is an important molecule regulating signaling for the life and differentiation of B lymphocytes and possesses the characteristics of a drug target for treating NHL. 2-Methoxyestradiol induces apoptosis in lymphoma Raji cells and CD20 protein is highly expressed by Raji lymphoma cells. Therefore, in this study, a CD20-SNAP-tag/CMC model was developed to validate the interaction of 2-methoxyestradiol with CD20. 2-Methoxyestradiol was used as a small molecule control compound, and the system was validated for good applicability. The cell membrane chromatography model was combined with high-performance liquid chromatography ion trap time-of-flight mass spectroscopy (HPLC-IT-TOF-MS) in a two-dimensional system to successfully identify, analyze, and characterize the potential active compounds of Schisandra chinensis (Turcz.) Baill. extract and Lysionotus pauciflorus Maxim. extract, including Schisandrin A, Schizandrol A, Schizandrol B, Schisantherin B, and Nevadensin, which can act on CD20 receptors. The five potential active compounds were analyzed by non-linear chromatography. The thermodynamic and kinetic parameters of their interaction with CD20 were also analyzed, and the mode of interaction was simulated by molecular docking. Their inhibitory effects on lymphoma cell growth were assessed using a Cell Counting Kit-8 (CCK-8). Nevadensin and Schizandrin A were able to induce apoptosis in Raji cells within a certain concentration range. In conclusion, the present experiments provide some bases for improving NHL treatment and developing small molecule lead compounds targeting CD20 with low toxicity and high specificity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Carnitine palmitoyltransferase 1A (CPT1A), which resides on the mitochondrial outer membrane, serves as the rate-limiting enzyme of fatty acid β-oxidation. Identifying the compounds targeting CPT1A warrants a promising candidate for modulating lipid metabolism. In this study, we developed a CPT1A-overexpressed mitochondrial membrane chromatography (MMC) to screen the compounds with affinity for CPT1A. Cells overexpressing CPT1A were cultured, and subsequently, their mitochondrial membrane was isolated and immobilized on amino-silica gel cross-linked by glutaraldehyde. After packing the mitochondrial membrane column, retention components of MMC were performed with LC/MS, whose analytic peaks provided structural information on compounds that might interact with mitochondrial membrane proteins. With the newly developed MMC-LC/MS approach, several Chinese traditional medicine extracts, such as Scutellariae Radix and Polygoni Cuspidati Rhizoma et Radix (PCRR), were analyzed. Five noteworthy compounds, baicalin, baicalein, wogonoside, wogonin, and resveratrol, were identified as enhancers of CPT1A enzyme activity, with resveratrol being a new agonist for CPT1A. The study suggests that MMC serves as a reliable screening system for efficiently identifying modulators targeting CPT1A from complex extracts.

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 K D 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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

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., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fusion with host cell membrane is the main mechanism of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we propose that a new strategy to screen small-molecule antagonists blocking SARS-CoV-2 membrane fusion. Using cell membrane chromatography (CMC), we found that harringtonine (HT) simultaneously targeted SARS-CoV-2 S protein and host cell surface TMPRSS2 expressed by the host cell, and subsequently confirmed that HT can inhibit membrane fusion. HT effectively blocked SARS-CoV-2 original strain entry with the IC 50 of 0.217 μM, while the IC 50 in delta variant decreased to 0.101 μM, the IC 50 in Omicron BA.1 variant was 0.042 μM. Due to high transmissibility and immune escape, Omicron subvariant BA.5 has become the dominant strain of the SARS-CoV-2 virus and led to escalating COVID-19 cases, however, against BA.5, HT showed a surprising effectiveness. The IC 50 in Omicron BA.5 was even lower than 0.0019 μM. The above results revealed the effect of HT on Omicron is very significant. In summary, we characterize HT as a small-molecule antagonist by direct targeting on the Spike protein and TMPRSS2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Patients have different responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and these may be life-threatening for critically ill patients. Screening components that act on host cell receptors, especially multi-receptor components, is challenging. The in-line combination of dual-targeted cell membrane chromatography and a liquid chromatography-mass spectroscopy (LC-MS) system for analyzing angiotensin-converting enzyme 2 (ACE2) and cluster of differentiation 147 (CD147) receptors based on SNAP-tag technology provides a comprehensive solution for screening multiple components in complex samples acting on the two receptors. The selectivity and applicability of the system were validated with encouraging results. Under the optimized conditions, this method was used to screen for antiviral components in Citrus aurantium extracts. The results showed that 25 µmol /L of the active ingredient could inhibit virus entry into cells. Hesperidin, neohesperidin, nobiletin, and tangeretin were identified as antiviral components. In vitro pseudovirus assays and macromolecular cell membrane chromatography further verified the interaction of these four components with host-virus receptors, showing good effects on some or all of the pseudoviruses and host receptors. In conclusion, the in-line dual-targeted cell membrane chromatography LC-MS system developed in this study can be used for the comprehensive screening of antiviral components in complex samples. It also provides new insight into small-molecule drug-receptor and macromolecular-protein-receptor interactions., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this paper, a new nanoscale metal Ti particle-reinforced Mg-3Al-1Zn matrix composite was successfully designed and prepared, which is mainly characterized by the fact that in addition to the "light" advantages of magnesium matrix composite, it also realizes bidirectional improvement of strength and ductility of the composite, and can be used as an alternative material for military light vehicle armor and individual armor. The SEM test shows that the nano-Ti particles are uniformly distributed at the grain boundary under the extruded state, which nails the grain boundary, inhibits the grain growth, and significantly refines the grain. XRD tests show that the addition of nano-Ti particles increases the crystallinity of the composite, which is consistent with the SEM test results. In addition, the EBSD test shows that the weakening of the texture of Ti/Mg-3Al-1Zn matrix composites and the increase in the starting probability of slip system are the main reasons for the improvement in ductility. Mechanical tests show that the yield strength, tensile strength, and elongation of the 0.5 wt% Ti/Mg-3Al-1Zn matrix composites exceed the peak values of ASTM B107/B107M-13 by 38.6%, 26.7%, and 20%, respectively.

In this study, a novel cell membrane chromatography (CMC) model was developed to investigate cluster of differentiation 147 (CD147) targeted anti-tumor drug leads for specific screening and ligand-receptor interaction analysis by SNAP-tagged CD147 fusion protein conjugation and polystyrene microspheres (PS) modification. Traditional Chinese medicines (TCMs) are widely used in the treatment of cancer. CD147 plays important roles in tumor progression and acts as an attractive target for therapeutic intervention; therapeutic drugs for CD147-related cancers are limited to date. Thus, a screening method for active components in TCMs is crucial for the further research and development of CD147 antagonists. However, improvement is still needed to perform specific and accurate drug lead screening using the CMC-based method. Recently, our group developed a covalently immobilized receptor-SNAP-tag/CMC model using silica gel as carrier. Besides the carboxyl group on multi-step modified silica particles, the amino group of benzyl-guanine (BG, substrate of SNAP-tag) also possesses reactivity towards the carboxyl group on available carboxyl-modified PS. Herein, we used PS as carrier and an extended SNAP-tag with CD147 receptor to construct the PS-BG-CD147/CMC model for active compound investigation coupled with HPLC/MS and applied this coupled PS-BG-CD147/CMC-HPLC/MS two-dimensional system to drug lead screening from Nelumbinis Plumula extract (NPE) sample. In addition, to comprehensively verify the pharmacological effects of screened ingredients, a cell proliferation inhibition assay was performed, and the interaction between the ingredients and CD147 was studied by the frontal analysis method. This study developed a high-throughput PS-based CMC screening platform, which could be widely applied and utilized in chromatographic separation and drug lead discovery., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

In the field of metal matrix composites, it is a great challenge to improve the strength and elongation of magnesium matrix composites simultaneously. In this work, xTC4/AZ31 (x = 0.5, 1, 1.5 wt.%) composites were fabricated by spark plasma sintering (SPS) followed by hot extrusion. Scanning electron microscopy (SEM) showed that nano-TC4 (Ti-6Al-4V) was well dispersed in the AZ31 matrix. We studied the microstructure evolution and tensile properties of the composites, and analyzed the strengthening mechanism of nano-TC4 on magnesium matrix composites. The results showed that magnesium matrix composites with 1 wt.%TC4 had good comprehensive properties; compared with the AZ31 matrix, the yield strength (YS) was increased by 20.4%, from 162 MPa to 195 MPa; the ultimate tensile strength (UTS) was increased by 11.7%, from 274 MPa to 306 MPa, and the failure strain (FS) was increased by 21.1%, from 7.6% to 9.2%. The improvement in strength was mainly due to grain refinement and good interfacial bonding between nano-TC4 and the Mg matrix. The increase in elongation was the result of grain refinement and a weakened texture.

With the rapid development of therapeutic monoclonal antibody drugs, it is increasingly difficult to meet clinical needs using traditional antibody purification techniques. In this study, epidermal growth factor receptor (EGFR)-SNAP-tag was expressed in HEK293 cells. Then we captured the EGFR-SNAP-tag from the cell membrane suspension onto a O 6 -benzylguanine-modified silica gel to prepare a new EGFR stationary phase as a bioactive material, which could specifically recognize its antibody in bio-samples. The EGFR stationary phase was systematically characterized via scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and fourier transform infrared spectroscopy. Then we used EGFR stationary phase to establish a new EGFR cell membrane chromatography (CMC) model. The EGFR/CMC-online-ion exchange chromatography (IEC)/high performance liquid chromatography (HPLC) was established for the efficient purification and specific identification of cetuximab, nituzumab, and panizumab from cell culture medium and human serum. The results show that the EGFR stationary phase prepared by one-step immobilized technique can maintain biological activity and stability like EGFR in cell membrane. The EGFR/CMC-online-IEC/HPLC method has a high specificity, accuracy and sensitivity. Therefore, it will present a valuable method for the purification, identification, and analysis of monoclonal antibody drugs., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pseudo-allergic reactions (PARs) are IgE-independent hypersensitivity reactions. Mas-related G protein-coupled receptor-X2 (MrgX2) was proved the key receptor of PAR. The anti-pseudo-allergic compound discovery based on MrgX2 was of great value. Cell membrane chromatography (CMC) based on MrgX2 provides a convenient and effective tool in anti-pseudo-allergic compound screening and discovery, and further improvements of this method are still needed. In this work, SNAP-tag was introduced at C-terminal of Mas-related G protein-coupled receptor (MrgX2-SNAP-tag), and an MrgX2-SNAP-tag/CMC model was then conducted using CMC technique. Comparative experiments showed that the new model not only satisfied the good selectivity and specificity of screening but also exhibited more stable and longer life span than traditional MrgX2/CMC model. By coupling with HPLC-MS, two compounds were screened out from Arnebiae Radix and identified as shikonin and acetylshikonin. Nonlinear chromatography was performed to study the interactions between two screened compounds and MrgX2, and binding constant (K A ) of shikonin and acetylshikonin with MrgX2 were 2075.67 ± 0.34 M -1 and 32201.36 ± 0.35 M -1 , respectively. Furthermore, β-hexosaminidase and histamine release assay in vitro demonstrated that shikonin (1-5 μM) and acetylshikonin (2.5-10 μM) could both antagonize C48/80-induced allergic reaction. In conclusion, the MrgX2-SNAP-tag/CMC could be a reliable model for screening pseudo-allergy-related components from complex systems., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography., (© 2022 Wiley-VCH GmbH.)

Magnetic nanoparticles (NPs) cloaked with cell membranes expressing high levels of the epidermal growth factor receptor (EGFR) have been used to screen for EGFR-targeting active compounds in traditional Chinese medicine (TCM) formulations. However, previous strategies involved physical immobilization of the biomaterials on the surface of the nanocarrier, resulting in highly unstable platforms since the biological materials could dislodge easily. Chemical bonding of biomaterials to the nanoparticles surface can improve the stability of the biomimetic platforms. In this study, membrane fragments from cells expressing SNAP-Tag-EGFR (ST-EGFR) were immobilized on the surface of magnetic NPs. The ST-EGFR magnetic cell membrane nanoparticles (ST-EGFR/MCMNs) showed greater stability, and higher binding capacity, selectivity adsorption of gefitinib after 7 days compared to the un-immobilized magnetic cell membrane nanoparticles (EGFR/MCMNs). The ST-EGFR/MCMNs were used to screen for the EGFR-targeting active compounds of Zanthoxyli Radix (ZR), and identified toddalolactone and nitidine chloride. The latter significantly inhibited the proliferation of EGFR-overexpressing cancer cells, and was more effective compared to gefitinib. This innovative technology can be used to rapidly screen for active compounds from complex extracts, and aid in drug discovery., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate)., (© 2021 Wiley-VCH GmbH.)

Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH 2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Membrane protein immobilization is particularly significant in in vitro drug screening and determining drug-receptor interactions. However, there are still some problems in the immobilization of membrane proteins with controllable direction and high conformational stability, activity, and specificity. Cell membrane chromatography (CMC) retains the complete biological structure of membrane proteins. However, conventional CMC has the limitation of poor stability, which results in its limited life span and low reproducibility. To overcome this limitation, we propose a method for the specific covalent immobilization of membrane proteins in cell membranes. We used the SNAP-tag as an immobilization tag fused to the epidermal growth factor receptor (EGFR), and Cys145 located at the active site of the SNAP-tag reacted with the benzyl group of O 6 -benzylguanine (BG). The SNAP-tagged EGFR was expressed in HEK293 cells. We captured the SNAP-tagged EGFR from the cell membrane suspension onto a BG-derivative-modified silica gel. Our immobilization strategy improved the life span and specificity of CMC and minimized loss of activity and nonspecific attachment of proteins. Next, a SNAP-tagged EGFR/CMC online HPLC-IT-TOF-MS system was established to screen EGFR antagonists from Epimedii folium . Icariin, magnoflorine, epimedin B, and epimedin C were retained in this model, and pharmacological assays revealed that magnoflorine could inhibit cancer cell growth by targeting the EGFR. This EGFR immobilization method may open up possibilities for the immobilization of other membrane proteins and has the potential to serve as a useful platform for screening receptor-binding leads from natural medicinal herbs.

Traditional Chinese medicines played an important role in the treatment of COVID-19 in 2020. Ephedra sinica, one of the major constituent herbs of multi-component herbal formula, has been widely used to treat COVID-19 in China. However, its active components are still unclear. The objectives of this study are to screen and evaluate active components from the traditional Chinese medicine Ephedra sinica for the treatment of COVID-19. In our study, we established an ACE2/CMC bioaffinity chromatography model, and then developed an ACE2/CMC-HPLC-IT-TOF-MS system for the active compounds screening and identification from Ephedra sinica extract. We performed molecular docking and surface plasmon resonance (SPR) assays to assess the binding characteristics (binding mode and K D value). We used CCK-8 staining to assess the toxicity of screened compounds, and also used SARS-CoV-2 pseudovirus to observe the viropexis effect of screened compounds in ACE2 h cells. In this current work, one fraction was fished out, separated and identified as ephedrine (EP), pseudoephedrine (PEP), and methylephedrine (MEP). Binding assays showed that the three compounds could bind with ACE2 in a special way to some amino acid residues, similar to the way SARS-CoV-2 bound with ACE2. Additionally, the three compounds, especially EP, can inhibit the entrance of SARS-CoV-2 spike pseudovirus into ACE2 h cells because they can reduce the entrance ratio of pseudovirus in the pseudovirus model. Overall, the ACE2/CMC-HPLC-IT-TOF-MS system was established and verified to be suitable for ACE2-targeted bioactive compound screening. EP, PEP, and MEP with ACE2-binding features were screened out from Ephedra sinica, and acted as blockers inhibiting SARS-CoV-2 spike pseudovirus entering ACE2 h cells.

Adverse drug reactions of traditional Chinese medicine injection mainly manifested as pseudo-allergic reactions. In the present study, ginsenoside Rd, Ro, and Rg 3 were identified as pseudo-allergic components in Shengmai injection by a high-expression Mas-related G protein-coupled receptor X2 cell membrane chromatography coupled online with high-performance liquid chromatography and mass spectrometry. Their pseudo-allergic activities were evaluated by in vitro and in vivo assay. The three compounds were further found to induce pseudo-allergic reaction through Mas-related G protein-coupled receptor X2. Therefore, we concluded that ginsenoside Rd, Ro and Rg 3 may be potential allergens that cause pseudo-allergic reactions. This study might be helpful for the safe use of Shengmai injection., (© 2021 Wiley-VCH GmbH.)

A novel stability-enhanced graphene quantum dot (GQD)-decorated epidermal growth factor receptor (EGFR) cell membrane chromatography was constructed to study the potential application of GQDs in bioaffinity chromatography, and to screen active components acting on EGFR from traditional Chinese medicine (TCM). The carboxyl groups on the surface of GQDs reacted with the amino groups of the amino-silica gel (SiO 2 -NH 2 ) to form a covalent bond, thereby preparing the GQD-decorated silica gel (SiO 2 -GQDs). The EGFR cell membrane was further immobilized on the SiO 2 -GQDs through the same covalent binding method to obtain the GQD-decorated cell membrane stationary phase (SiO 2 -GQDs-CMSP). In this way, the cell membrane was firmly immobilized on the decorated silica carrier. The life span and stability of the GQD-decorated cell membrane chromatographic (SiO 2 -GQDs-CMC) column were both enhanced, and the optimal immobilization conditions of the EGFR cell membrane were also determined. This model was then verified by establishing a SiO 2 -GQDs-CMC online liquid chromatography-ion trap-time-of-flight (LC-IT-TOF) system to screen possible active components in Peucedanum praeruptorum Dunn. As a result, praeruptorin B (Pra-B) was screened out, and its inhibitory effect against EGFR cell growth was evaluated by the cell counting kit-8 (CCK-8) assay. Molecular docking assay was also conducted to further estimate the interaction between Pra-B and EGFR. Overall, this research indicated that GQDs may be a promising nanomaterial to be used in prolonging the life span of the CMC column, and Pra-B could be a potential EGFR inhibitor so as to treat cancer.

The recent emergence of the novel pathogenic coronavirus disease 2019 (COVID-19) is responsible for a worldwide pandemic. In sight of this, there has been growing interest in the use of chloroquine (CQ) and hydroxychloroquine (HCQ) as potential treatments. In this study, we use angiotensin converting enzyme 2 (ACE2) over-expressed cell membrane chromatography (CMC) to study the interaction of CQ and HCQ with ACE2 receptor. Both CQ and HCQ were retained on the ACE2/CMC column. Then we analyzed the binding character of CQ and HCQ to ACE2 by CMC frontal analysis, ionic force investigation and competitive binding experiment. Results showed that CQ and HCQ KD values obtained from the CMC frontal analysis method were 8.22(±0.61) × 10 -7 M and 11.70(±2.44) × 10 -7 M. Compare to CQ, HCQ has the weaker affinity with ACE2. The action force of CQ, HCQ and ACE2 is mainly ionic force. CQ and HCQ have different degrees of competitive binding relationship with ACE2. Our study revealed the interaction of CQ and HCQ with ACE2 receptor, which provides new insights for the use of CQ and HCQ in the treatment of COVID-19. Moreover, this biomimetic drug screening method is expected to open the door for rapid targeting and separating bioactive ingredients active towards ACE2 receptor., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating . The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial., Purpose: The objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection., Methods: In our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2 h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2 h cells., Results: Results showed that HCQ is slightly more toxic to ACE2 h cells than CQ. Both CQ and HCQ could bind to ACE2 with K D  = (7.31 ± 0.62)e -7  M and (4.82 ± 0.87)e -7  M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2 h cells., Conclusions: CQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Mas-related G protein-coupled receptor X2 was a mast cell-specific receptor mediating anaphylactoid reactions by activating mast cells degranulation, and it was also identified as a target for modulating mast cell-mediated anaphylactoid and inflammatory diseases. The anti-anaphylactoid drugs used clinically disturb the partial effect of partial mediators released by mast cells. The small molecule of Mas-related G protein-coupled receptor X2 specific antagonists may provide therapeutic action for the anaphylactoid and inflammatory diseases in the early stage. In this study, the Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography was coupled online with liquid chromatography and mass spectrometry and successfully used to screen anti-anaphylactoid components from Magnolia biondii Pamp. Fargesin and pinoresinol dimethyl ether were identified as potential anti-anaphylactoid components. Bioactivity of these two components were investigated by β hexosaminidase and histamine release assays on mast cells, and it was found that these two components could inhibit β hexosaminidase and histamine release in a concentration-dependent manner. This Mas-related G protein-coupled receptor X2 high expression cell membrane chromatography coupled online with liquid chromatography and mass spectrometry system could be applied for screening potential anti-anaphylactoid components from natural medicinal herbs. This study also provided a powerful system for drug discovery in natural medicinal herbs., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mast cells are essential in mediating inflammatory processes. When activated, mast cells can rapidly release characteristic granules and various mediators into the interstitium. Tryptase (TPS) and β-hexosaminidase (HEXB) are typical protease mediators stored in granules and released upon activation. They have been recognized as important biomarkers of anaphylaxis, and the released level is associated with the severity of allergic reactions. In this study, a sensitive, accurate, and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneously quantifying the two biomarkers was developed and validated in LAD2 cell culture supernatant, and P 14 R was used as internal standard. Good linearity was observed in the range of 50-2500 ng/mL for TPS and 10-2000 ng/mL for HEXB both with R 2 > 0.99. The matrix effect and recovery were both within acceptable limits. We quantified TPS and HEXB released from Laboratory of Allergic Disease 2 (LAD2) mast cells treated with several potential allergens, and the results demonstrate that the method can be used to investigate TPS and HEXB levels in LAD2 mast cell model during allergy research. We anticipate our approach to be a robust and sensitive assessment method for more biomarkers with similar kinetics characteristics and to be a major tool of allergic drug assessment or antiallergic drug development in research.

Anaphylactoid reactions induced by drugs are serious and can be life-threatening, and screening the anaphylactoid ingredients especially in complex samples is challenging. Here, a multi targeted cell membrane chromatography method, based on the Mas-related G protein coupled receptor X2, Fc epsilon RI and H 1 receptors, online coupled with LC-MS system provides a comprehensive solution for screening the anaphylactoid components from complex samples. The validation including selectivity and suitability of this system has been evaluated, and it shows promising results. With optimized conditions, this method has been utilized to screen the anaphylactoid ingredients from Shenmai Injection. Ginsenoside Rb 1 , ginsenoside Rb 2 , ginsenoside Rc, ginsenoside Rd and 20(S)-ginsenoside Rg3 were identified as anaphylactoid components. The anaphylactoid effects of these five components were further verified by the in vitro sensitization assay, showing promising effects on some or all sensitization cells. In conclusion, the multi targeted cell membrane chromatography online coupled with LC-MS system developed throughout this study could be used to fully screen anaphylactoid components in complex samples. Moreover, it also provides new insights for drug quality control., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Intrathecal morphine infusion is often applied to treat chronic pain related to cancer and other conditions. However, persistent pain can be caused by nerve compression because of granuloma formation. In this study, a mouse model of morphine-induced granuloma formation by intrathecal catheterization morphine infusion into the atlanto-occipital membrane of the foramen magnum was established in wild-type mice, MrgprB2 mutant (MrgprB2 -/- ) mice, and in mast cell-deficient W-sash c-kit mutant (Kit W-sh/W-sh ) mice. Heat-related pain after surgery was performed to investigate the antipain effect of morphine. H&E staining and immunofluorescence staining of the spinal cord were applied to analyze the mechanism of granuloma formation. Morphine-induced mast cell degranulation was assessed by measuring the Ca 2+ influx and mediator release. Anaphylactoid reactions were measured after s.c. morphine infusion to the paws. Chemokine release by mast cells was determined by Human XL Cytokine Array. Experiments with wild-type, MrgprB2 mutant, and mast cell-deficient W-sash c-kit mutant mice demonstrated that morphine activated mast cells and inflammatory cell aggregation through MrgprB2 in intrathecal infusion sites. The chemokine production of human mast cells demonstrated that granuloma formation is correlated with chemokines release. In addition, morphine activated mouse primary mast cells and de novo chemokine synthesis via the MRGPRX2 in human LAD2 cells. We concluded that granuloma formation during intrathecal morphine infusion was associated with MrgprB2/X2. Reducing MRGPRX2 potentially blocks morphine-induced side effects, including granuloma formation., (Copyright © 2019 by The American Association of Immunologists, Inc.)

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., (© 2019 John Wiley & Sons, Ltd.)

Saposhnikoviae Radix, the dried root of Saposhnikoviae divaricata, is commonly used in the traditional Chinese anti-allergic preparations, like Bofutsusho-san and Yupingfeng granules. A high-expression Mas-related G protein-coupled receptor X2 cell membrane chromatography coupled online with high-performance liquid chromatography combined with an ion trap time-of-flight multistage mass spectrometry system was established and used for screening and identifying the anti-allergic components in Saposhnikoviae Radix. The system was validated for excellent specificity and suitability using the appropriate standards. Two retained fractions were obtained on the cell membrane chromatography column, and three main components were identified as prim-O-glucosylcimifugin, cimifugin, and 4'-O-β-d-glucosyl-5-O-methylvisamminol. Next, the molecular docking study was conducted, which confirmed that these three components could effectively bind to MRGPRX2 through hydrogen bonds with its amino acid residues. Finally, histamine release assay was performed to investigate the bioactivities of prim-O-glucosylcimifugin, cimifugin, and 4'-O-β-d-glucosyl-5-O-methylvisamminol. Results showed that these three components could exert anti-allergic effects by inhibiting the histamine release in a dose-dependent manner (from 10 to 100 µM). In conclusion, the high-expression Mas-related G protein-coupled receptor X2 cell membrane chromatography is an effective tool for discovering the anti-allergic components in Saposhnikoviae Radix., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pseudo-allergic reactions occurred in patients administered drugs for the first time, seriously threaten man's survival. Due to the frequent reports of pseudo-allergic reactions to Danshen injection, in our previous study, isosalvianolic acid C in Danshen injection was found to trigger off mast cell degranulation. However, the direct involvement and the mechanisms underlying pseudo-allergic reactions have not been elucidated. In this study, the pseudo-allergic reactions induced by isosalvianolic acid C were confirmed by an ear swelling assay, a hindpaw swelling and extravasation assay in vivo and mast cell degranulation assays in vitro. We also evaluated whether the pseudo-allergic effect is related to MRGPRX2, Isosalvianolic acid C induced Ca 2+ mobilization was verified as MRGPRX2-related by Ca 2+ imaging using mouse peritoneal mast cells (both wild-type and MrgprB2 knockout mice), MRGPRX2-expressing HEK293 and MrgprB2-expressing HEK293 cells. MRGPRX2-related pseudo-allergic reactions induced by Isosalvianolic acid C were further confirmed by MrgprB2 knockout mice and MRGPRX2 knockdown mast cells both exhibited reduced isosalvianolic acid C-induced pseudo-allergic effects. Furthermore, both the frontal analysis and molecular docking assays showed that isosalvianolic acid C has a considerable affinity with MRGPRX2. Based on the above experiments, the western blot analyses were conducted, the results indicated that isosalvianolic acid C induced Ca 2+ mobilization and degranulation via the activation of PLC-γ and IP3R, and releasing chemokines via the activation of PLC-γ, PKC and P38. This study should alarm many clinicians that medicines containing isosalvianolic acid C might induce pseudo-allergic reactions, and it may provide guidance on safe dosage of these medicines in the process of production and use., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Radix Salviae Miltiorrhiae (also known as DanShen (DS) in China), a popular herbal drug in traditional Chinese medicine (TCM) for promoting blood circulation and treating blood stasis, has been reported to possess potential anti-tumor effects. The aim of the study was to develop an effective and practical method for screening and identifying bioactive compounds from Radix Salviae Miltiorrhiae. In this work, the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptors 4 (FGFR4) dual-mixed/cell membrane chromatography (CMC) coupled with high performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrum (HPLC-ESI-IT-TOF-MSn) was established and successfully used to identify the active components from Radix Salviae Miltiorrhiae. Salvianolic acid C (SAC), tanshinone I (Tan-I), tanshinone IIA (Tan-IIA), and cryptotanshinone (C-Tan) were identified as bioactive components with EGFR and FGFR4 activities. MTT and kinase assay were performed to investigate inhibitory effects of these compounds against EGFR and FGFR4 cells growth in vitro. Both cell viability and kinase activity showed that cryptotanshinone acting on EGFR receptor and tanshinone IIA acting on FGFR4 receptor. In conclusion, the EGFR & FGFR4 dual-mixed/CMC can simultaneously screen the bioactive components from TCMs that act on both EGFR and FGFR4 receptors, which significantly improve the efficiency of specific bioactive components identification from a complex system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Kudiezi injection is a traditional Chinese medicine injection used widely in China to alleviate blood stasis and to stimulate blood circulation. Its use has been associated with a high rate of adverse effects including anaphylactoid reactions. In the present study, a two-dimensional system comprised of high expression Mas-related G protein-coupled receptor X2 cell membrane chromatography coupled online with HPLC-ESI-IT-TOF-MS was established, and used to screen and identify anaphylactoid components in kudiezi injection. Luteolin-7-O-glucuronide, apigenin-7-O-glucronide, luteoloside and luteolin were identified as the potential anaphylactoid components. The release of β-hexosaminidase and histamine from Laboratory of Allergic Disease 2 cells enabled evaluation of the anaphylactoid activities of these compounds in vitro. Both β-hexosaminidase and histamine release were enhanced markedly with increasing concentrations of the anaphylactoid components. The molecular docking assay showed excellent interactions between the potential anaphylactoid constituents and MRGPRX2. In general, the two-dimensional system developed in this study is effective in screening for the anaphylactoid components in Kudiezi injection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 John Wiley & Sons, Ltd.)

Traditional Chinese medicine (TCM) has been used for prevention and treatment of various diseases for many decades. TCM injection is a new dosage form, with incidence of anaphylactoid reactions increasing every year. In this study, the rat basophilic leukemia 2H3 (RBL-2H3) and laboratory of allergic disease 2 (LAD2) dual-mixed/CMC was established and was coupled with an HPLC-ESI-IT-TOF-MS system to identify the potential allergenic components in Haqing injection. Cinobufagin, piperine, osthole, praeruptorin A, and schizandrin A were screened from Haqing injection via this coupled system. Competitive binding assay showed piperine, praeruptorin A, and schizandrin A acting on MrgprX2 and cinobufagin and osthole act on the IgE receptor. The release of mediators of anaphylaxis results showed cinobufagin and osthole can cause anaphylactoid reactions by triggering the release of β-hexosaminidase and histamine via IgE-R. Praeruptorin A and schizandrin A could promote the release of β-hexosaminidase and histamine via MrgprX2 receptor. In summary, the dual-mixed/CMC model can significantly improve the efficiency of target component identification from a complex sample. When combined with competitive binding assay and validation of biological activities, this model enables accurate determination of the dual-target components, offering improved methods for quality control of TCM injections., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mast cells are unique immune cells that function as sentinels in host defence reactions, including immediate hypersensitivity responses and allergic responses. The mast cell-specific receptor named MAS-related G protein-coupled receptor X2 (MRGPRX2) triggers mast-cell degranulation, a key process in anaphylactoid reactions. It is widely observed that antimicrobials can induce pseudo-allergic reactions (i.e. IgE-independent mechanism) with symptoms ranging from skin inflammation to life-threatening systemic anaphylaxis. However, their direct involvement and the mechanisms underlying anaphylactoid reactions caused by antimicrobials have not been demonstrated. Structurally different antimicrobials were screened by Ca 2+ imaging using MRGPRX2 overexpressing HEK293 cells. MRGPRX2 related anaphylactoid reactions induced by these components were investigated by body temperature drop and mast cell degranulation assays. We showed that MRGPRX2 is involved in allergic-like reactions to three types of antimicrobials in a dose-dependent manner. However, mast cells lacking the receptor show reduced degranulation. Furthermore, mice without MAS-related G protein-coupled receptor B2 (the orthologous gene of MRGPRX2) exhibited reduced substance-induced inflammation. Interestingly, β-lactam and antiviral nucleoside analogues did not induce anaphylactic reactions, which were also observed in vitro. These results should alarm many clinicians that such drugs might induce anaphylactoid reactions and provide guidance on safe dosage of these drugs., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Anaphylactoid reactions are common clinical acute adverse drug reactions that can exacerbate a patient's condition and produce effects that may become life-threatening. Therefore, it is important to establish a novel method to evaluate drugs for anaphylactoid reactions. In this study, we developed a sensitive and rapid method to detect histamine release from LAD2 cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and constructed a relative release index based on various release curve parameters, including allergen release time and sudden change rate, to evaluate the potential and strength of allergen-induced anaphylactoid reactions. This LAD2 release model was used to evaluate anaphylactoid reactions induced by ciprofloxacin, norfloxacin, lomefloxacin, moxifloxacin, and baicalin. The results positively correlated with those obtained with an Evans blue ear test and negatively correlated with the Ca 2+ influx EC 50 . In summary, the current study established a novel in vitro method to analyze the properties of histamine release from LAD2 cells and characterize the sensitization and strength of sensitization of drugs or components that may induce anaphylactoid reactions.

Yejuhua (YJH) injection is a traditional Chinese medicine (TCM) injection and has a widely application in clinical practice. However, adverse drug reactions (ADRs) caused by YJH injection, majorly manifested as allergic reactions, have been reported. Hence, Effective and practical method for allergen screening and identification is needed. In this work, a LAD2/CMC model coupled online with HPLC-IT-TOF-MS system was developed to screen, analyze, and identify the allergenic components of YJH injection. A fraction was retained on the LAD2/CMC column, and identified as linarin (LN). Histamine release assay was performed by the multiple reaction monitoring (MRM) method of UPLC-ESI-MS/MS. Results showed that YJH injection and LN were in accord with their allergic effects by increasing histamine release. In conclusion, the LAD2/CMC-HPLC-IT-TOF-MS system developed in this study could be used to screen allergenic components in complex systems., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Corydalis Thizoma,or Yuanhu in China, is a common herbal drug used for thousands of years as analgesic in Chinese medicine that has been reported to have potential anti-angiogenic effects. In this study, a VEGFR/cell membrane chromatography (VEGFR/CMC) coupled with HPLC- ESI-IT-TOF-MSn system was developed and successfully applied for identifying active components from YuanHu extract acting on VEGFR. We identified tetrahydropalmatine and corydaline as bioactive components with VEGFR activity, thus confirming their inhibitory activity on VEGFR engineered HEK293 cell growth by MTT assay. The activity of tetrahydropalmatine and corydaline was compared with the positive control sorafenib in a range of concentration from 6.25 to 50.0μM, showing a dose-dependent inhibitory trend. These results indicate that the VEGFR/CMC coupled with HPLC- ESI-IT-TOF-MSn system can purify and identify specific bioactive components from complex systems, thus representing a promising tool for screening molecules active towards VEGFR from natural herbs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 John Wiley & Sons, Ltd.)

Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016)., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

MaiLuoNing injection is a traditional Chinese medicine that used clinically since the 1950s in China. However, anaphylactic reactions, through the potentiation of mast cell degranulation, have been reported. In the present study, a rat basophilic leukemia-2H3 cell membrane chromatography coupled with high-performance liquid chromatography and electrospray ionization-ion trap-time of flight-mass spectrometry method was established for screening, analyzing, and identifying the potential anaphylactic components of MaiLuoNing injection. Harpagoside, a potential degranulator of rat basophilic leukemia-2H3 cells, was retained in rat basophilic leukemia-2H3 cell membrane chromatography. We aimed to evaluate the retained components to determine which of those were capable of inducing degranulation of basophilic leukemia cells. A β-hexosaminidase assay revealed that harpagoside can induce rat basophilic leukemia-2H3 cell degranulation in a dose-dependent manner. BLBA/c mice also exhibit passive cutaneous anaphylaxis in response to harpagoside. These results indicate that rat basophilic leukemia-2H3 cell membrane chromatography coupled with high-performance liquid chromatography and electrospray ionization ion trap time-of-flight mass spectrometry is effective in screening for the anaphylactic components of MaiLuoNing injection., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Context: Leonurus artemisia (Lour.) S.Y.Hu (Lamiaceae) (YiMuCao in Chinese) is a traditional Chinese medicine. Leonurus artemisia has been shown to have many pharmacological effects such as increasing uterine contraction amplitude, and tension, but the active components are still unknown., Objective: The objective of this study is to determine active components of L. Artemisia that are responsible for the biological activity using HPLC and cell membrane-based system., Materials and Methods: The whole L. artemisia ethanol extract and its eight fractions were screened using Sprague-Dawley rat uterus cell membrane chromatography (CMC) combined with the HPLC/MS system. Oxytocin was used to investigate the activity of CMC column. The effect of active components screened from L. artemisia was studied by tension measurement of isolated rat uterine strips in vitro at a dose of 10(-7)-10(-4 )mol/L with oxytocin as a control., Results: The acetone extract showed obvious activity when compared with the eight extracts of L. artemisia. From the acetone extract, in the negative ionization mode, the active compound was identified as genkwanin, with a molecular weight of 283. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions at a dose from 10(-7) to 10(-4 )mol/L. The EC50 value was 4.86 ± 4.21 μmol/L for genkwanin and 4.30 ± 3.65 μmol/L for oxytocin on the contractile amplitude of uterine strips isolated from rats., Discussion and Conclusion: Genkwanin was identified as the active compound in L. artemisia by this method. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions. Genkwanin may be used to uterine inertia and may have an effect on postpartum hemorrhage.

Carthamus tinctorius, used in traditional Chinese medicine, has many pharmacological effects, such as anticoagulant effects, antioxidant effects, antiaging effects, regulation of gene expression, and antitumor effects. However, there is no report on the antiallergic effects of the components in C. tinctorius. In the present study, we investigated the antiallergic components of C. tinctorius and its mechanism of action. A rat basophilic leukemia 2H3/cell membrane chromatography coupled online with high-performance liquid chromatography and tandem mass spectrometry method was developed to screen antiallergic components from C. tinctorius. The screening results showed that Hydroxysafflor yellow A, from C. tinctorius, was the targeted component that retained on the rat basophilic leukemia 2H3/cell membrane chromatography column. We measured the amount of β-hexosaminidase and histamine released in mast cells and the key markers of degranulation. The release assays showed that Hydroxysafflor yellow A could attenuate the immunoglobulin E induced release of allergic cytokines without affecting cell viability from 1.0 to 50.0 μM. In conclusion, the established rat basophilic leukemia 2H3 cell membrane chromatography coupled with online high-performance liquid chromatography and tandem mass spectrometry method successfully screened and identified Hydroxysafflor yellow A from C. tinctorius as a potential antiallergic component. Pharmacological analysis elucidated that Hydroxysafflor yellow A is an effective natural component for inhibiting immunoglobulin E-antigen-mediated degranulation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Analyses of four endocrine disrupting chemicals (EDCs) in water and milk samples were undertaken by using magnetic molecularly imprinted polymers (MMIPs). These were prepared via the surface molecular imprinting technique using super paramagnetic core-shell nanoparticle as support. Diethylstilbestrol (DES), which is a typical EDC, was employed as the template molecule. The obtained MMIPs were characterized using transmission electron microscope, Fourier transform infrared, X-ray diffraction, and vibrating sample magnetometer. Accordingly, the adsorption capacity and selectivity of prepared MMIPs were investigated. The binding isotherms were obtained for DES and fitted by the Freundlich isotherm model. A corresponding analytical method to determine four EDCs was developed. The recoveries of the spiked samples in pond water and pure milk range from 67.8 to 93.2% and from 65.3 to 92.5%, respectively. Coupled with high-performance liquid chromatography analysis, the prepared MMIPs were successfully applied to the analysis of EDCs in water and milk samples.

Allergy is an abnormal immune response to an allergen. Type I hypersensitivity is an immunoglobulin (Ig) E-mediated allergic disorder. Fructus piperis is derived from the ripe fruit of the pepper, which is widely used as a spice in human diets and is also administered as a medicine in many countries. Piperine has been shown to have anti-oxidant, anti-depressant, anti-tumor, and anti-inflammatory activities. However, the effect of piperine on IgE-mediated allergic responses has not been reported. Here, the rat basophilic leukemia cells by membrane chromatography (RBL-2H3/CMC) coupled to high performance liquid chromatography/mass spectrometry (HPLC/MS) to discover and identify piperine can bind to RBL-2H3 cell membranes. Piperine inhibited the expression of cytokines, and the release of both β-hexosaminidase and histamine, which could be stimulated by antigen in RBL-2H3 mast cells. We found that the levels of intracellular Ca(2+) also decreased. Furthermore, RT-PCR showed that the mRNA expression levels of IL-4, IL-13, and TNF-α were significantly suppressed by piperine. The inhibitory effect of piperine on IgE-mediated degranulation and cytokine production by RBL-2H3 cells may be caused by the inhibition of IgE-mediated signaling pathways, including the phosphorylation of Lyn, p38, Erk, and Ras. In summary, piperine can inhibit antigen-induced allergic reactions that control degranulation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

It has been reported that the histamine H1 receptor (H(1)R) gene is up-regulated in patients with allergic rhinitis and H(1)R expression level strongly correlates with the severity of allergy symptoms. Drugs for therapy should avoid allergy symptoms, especially for patients with over-expressed H(1)R. Therefore, screening of the components which could induce H(1)R activation is urgently needed for drug safety evaluation. Homoharringtonine injection is a preparation for acute nonlymphocytic leukemia, which is approved by China Food and Drug Administration (CFDA) and US Food and Drug Administration. However, severely adverse reactions often occur with intravenous injection of the preparation. In present study, an H(1)R/CMC model was applied for capturing membrane retained components which could induce H(1)R activation. Retention components were enriched and analyzed by H(1)R/CMC-HPLC/MS. Homoharringtonine was recognized, separated and identified in homoharringtonine injection. Ca(2+) flux assay and p-IP3R expression founded that homoharringtonine retained by the H1 R/CMC model increased phosphorylation of IP3R and promoted cytosolic free Ca(2+) elevation in a dose-dependent manner which further verified the activity of homoharringtonine in activating the H1 R. In conclusion, homoharringtonine was screened and identified as a potential allergic factor. This provides an indication that a patient with over-expressed H1 R should be aware of possible allergic reaction when applying homoharringtonine injection., (Copyright © 2014 John Wiley & Sons, Ltd.)