Establishment of cluster of differentiation 20 immobilized cell membrane chromatography for the screening of active antitumor components in traditional Chinese medicine.

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.

Screening of CPT1A-Targeting Lipid Metabolism Modulators Using Mitochondrial Membrane Chromatography.

Carnitine palmitoyltransferase 1A (CPT1A), which resides on the mitochondrial outer membrane, serves as the rate-limiting enzyme of fatty acid ß-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.

KIT-SNAP-tag/cell membrane chromatography model coupled with liquid chromatography-mass spectrometry for anti-GIST compound screening from Evodia rutaecarpa.

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.

Evaluation of Coenzyme Q10 (CoQ10) Deficiency and Therapy in Mouse Models of Cardiomyopathy.

ABSTRACT: Mitochondrial dysfunction plays a key role in the development of heart failure, but targeted therapeutic interventions remain elusive. Previous studies have shown coenzyme Q10 (CoQ10) insufficiency in patients with heart disease with undefined mechanism and modest effectiveness of CoQ10 supplement therapy. Using 2 transgenic mouse models of cardiomyopathy owing to cardiac overexpression of Mst1 (Mst1-TG) or ß 2 -adrenoceptor (ß 2 AR-TG), we studied changes in cardiac CoQ10 content and alterations in CoQ10 biosynthesis genes. We also studied in Mst1-TG mice effects of CoQ10, delivered by oral or injection regimens, on both cardiac CoQ10 content and cardiomyopathy phenotypes. High performance liquid chromatography and RNA sequencing revealed in both models significant reduction in cardiac content of CoQ10 and downregulation of most genes encoding CoQ10 biosynthesis enzymes. Mst1-TG mice with 70% reduction in cardiac CoQ10 were treated with CoQ10 either by oral gavage or i.p. injection for 4-8 weeks. Oral regimens failed in increasing cardiac CoQ10 content, whereas injection regimen effectively restored the cardiac CoQ10 level in a time-dependent manner. However, CoQ10 restoration in Mst1-TG mice did not correct mitochondrial dysfunction measured by energy metabolism, downregulated expression of marker proteins, and oxidative stress nor to preserve cardiac contractile function. In conclusion, mouse models of cardiomyopathy exhibited myocardial CoQ10 deficiency likely due to suppressed endogenous synthesis of CoQ10. In contrast to ineffectiveness of oral administration, CoQ10 administration by injection regimen in cardiomyopathy mice restored cardiac CoQ10 content, which, however, failed in achieving detectable efficacy at molecular and global functional levels.

Construction and application of covalently bonded CD147 cell membrane chromatography model based on polystyrene microspheres.

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.

Enhanced stability designs of cell membrane chromatography for screening drug leads.

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.

Magnetic nanoparticles covalently immobilizing epidermal growth factor receptor by SNAP-Tag protein as a platform for drug discovery.

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.

Targeting and Covalently Immobilizing the EGFR through SNAP-Tag Technology for Screening Drug Leads.

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 O6-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.

Screening and evaluation of anti-SARS-CoV-2 components from Ephedra sinica by ACE2/CMC-HPLC-IT-TOF-MS approach.

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 KD 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 ACE2h 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 ACE2h 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 ACE2h cells.

Pseudo-allergic compounds screened from Shengmai injection by using high-expression Mas-related G protein-coupled receptor X2 cell membrane chromatography online coupled with liquid chromatography and mass spectrometry.

Adverse drug reactions of traditional Chinese medicine injection mainly manifested as pseudo-allergic reactions. In the present study, ginsenoside Rd, Ro, and Rg3 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 Rg3 may be potential allergens that cause pseudo-allergic reactions. This study might be helpful for the safe use of Shengmai injection.

Construction of graphene quantum dots-decorated EGFR cell membrane chromatography for screening active components from Peucedanum praeruptorum Dunn.

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 (SiO2-NH2) to form a covalent bond, thereby preparing the GQD-decorated silica gel (SiO2-GQDs). The EGFR cell membrane was further immobilized on the SiO2-GQDs through the same covalent binding method to obtain the GQD-decorated cell membrane stationary phase (SiO2-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 (SiO2-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 SiO2-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.

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus.

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 (ACE2h 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 ACE2h cells. RESULTS: Results showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD = (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 ACE2h 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.

Cell membrane chromatography coupled online with LC-MS to screen anti-anaphylactoid components from Magnolia biondii Pamp. targeting on Mas-related G protein-coupled receptor X2.

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 ß hexosaminidase and histamine release assays on mast cells, and it was found that these two components could inhibit ß 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.

Liquid Chromatography Tandem Mass Spectrometry Based Label-Free Quantification Method for Assessment of Allergen-Induced Anaphylactoid Reactions.

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 ß-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 P14R 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 R2 > 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.

Multi targeted cell membrane chromatography: A comprehensive method for screening the anaphylactoid components from complex samples.

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 H1 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 Rb1, ginsenoside Rb2, 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.

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.

Screening the anti-allergic components in Saposhnikoviae Radix using high-expression Mas-related G protein-coupled receptor X2 cell membrane chromatography online coupled with liquid chromatography and mass spectrometry.

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-ß-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-ß-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.

Dual-mixed/CMC model for screening target components from traditional Chinese medicines simultaneously acting on EGFR & FGFR4 receptors.

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.

A high expression Mas-related G protein coupled receptor X2 cell membrane chromatography coupled with liquid chromatography and mass spectrometry method for screening potential anaphylactoid components in kudiezi injection.

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 ß-hexosaminidase and histamine from Laboratory of Allergic Disease 2 cells enabled evaluation of the anaphylactoid activities of these compounds in vitro. Both ß-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.

Screening of bioactive components from traditional Chinese medicines using cell membrane chromatography coupled with mass spectrometry.

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.