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Mas-related G protein-coupled receptor X2 (MrgprX2) is acknowledged as a mast cell-specific receptor, playing a crucial role in orchestrating anaphylactoid responses through mast cell degranulation. It holds promise as a target for regulating allergic and inflammatory diseases mediated by mast cells. Polygonum cuspidatum (PC) has shown notable anti-anaphylactoid effects, while its pharmacologically active components remain unclear. In this study, we successfully utilized MrgprX2 high-expressing cell membrane chromatography (CMC), in conjunction with liquid chromatography-mass spectrometry (LC-MS), to identify active anti-anaphylactoid components in PC. Our study pinpointed polydatin, resveratrol, and emodin-8-O-β-d-glucoside as potential anti-anaphylactoid compounds in PC. Their anti-anaphylactoid activities were evaluated through β-aminohexosidase and histamine release assays, demonstrating a concentration-dependent inhibition for both β-aminohexosidase and histamine release. This approach, integrating MrgprX2 high-expression CMC with LC-MS, proves effective in screening potential anti-anaphylactoid ingredients in natural herbal medicines. The findings from this study illuminated the anti-anaphylactoid properties of specific components in PC and provided an efficient method for the drug development of natural products., (© 2024 Wiley‐VCH GmbH.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

An affinity two-dimensional chromatography method was developed for the recognition, separation, and identification of allergic components from tubeimu saponin extracts, a preparation often injected to treat various conditions as indicated by traditional Chinese medicine. Rat basophilic leukemia-2H3 cell membranes were used as the stationary phase of a membrane affinity chromatography column to capture components with affinity for mast cells that could be involved in a degranulation reaction. The retained components were enriched and analyzed by membrane affinity chromatography with liquid chromatography and mass spectrometry via a port switch valve. Suitability and reliability of the method was investigated using appropriate standards, and then, the method was applied to identify components retained from tubeimu saponin extracts. Tubeimoside A was identified in this way as a potential allergen, and degranulation assays confirmed that tubeimoside A induces RBL-2H3 cell degranulation in a dose-dependent manner. An increase in Ca(2+) influx indicated that degranulation induced by tubeimoside A is likely Ca(2+) dependent. Coupled with the degranulation assay, RBL-2H3 cell-based affinity chromatography coupled with liquid chromatography and mass spectrometry is an effective method for screening and identifying allergic components from tubeimu saponin extracts., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Allergy is an abnormal reaction of the body to an allergen. Histamine is responsible for many of the acute symptoms of allergic diseases. Many of the allergic and inflammatory actions of histamine are mediated by the histamine H1 receptor. In the present study, we established a two-dimensional histamine H1 receptor/cell membrane chromatography with online high-performance liquid chromatography and mass spectrometry method for screening potential histamine-activating components in a traditional Chinese medicine injection. The specification of the method was validated by screening, separating, and identifying a mixed standard solution of diphenhydramine hydrochloride, gefitinib, tamsulosin, and nitrendipine. The Yujin injection, an example of traditional Chinese medicine injection, was screened and potential allergic components acting on the histamine H1 receptor were identified. A Ca(2+) flux assay showed that houttuyfonate and Yujin injection induced calcium release in a dose-dependent manner. This suggests that houttuyfonate is an activator of the histamine H1 receptor. The mechanism of houttuyfonate activation involves phosphorylation of the inositol-1,4,5-trisphosphate receptor. In conclusion, this two-dimensional method can rapidly detect and enrich target components isolated from the Yujin injection. This indicates that individuals with an overexpression of the histamine H1 receptor should be aware of possible allergic reactions when receiving the Yujin injection., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Radix et Rhizoma Asari is a traditional Chinese medicine, and has many pharmacological effects, such as calming, analgesia, anti-inflammation, antiarrhythmic, antihypertensive, antivirus, etc. But few studies have screened the active compounds from extracts of Radix et Rhizoma Asari for tumor therapy. In this study, a two-dimensional liquid chromatography system was built to screen active compounds acting on epidermal growth factor receptor (EGFR) from Radix et Rhizoma Asari. The screening result showed that asarinin from Radix et Rhizoma Asari was the targeted component that could act on EGFR specificity. The competitive binding assay and molecular docking assay results showed asarinin binding with EGFR in similar manner as with gefitinib, which was used as a positive control drug. Then the antitumor effect of asarinin was studied through cell growth assay in vitro. The results showed that gefitinib and asarinin could inhibit highly expressed EGFR cell growth in a dose-dependent manner in the range of dose from 0.10 to 102.4 μM. This two-dimensional liquid chromatography system will be a useful method in drug discovery from natural medicinal herbs for searching potential antitumor candidates., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)