Cell Membrane Chromatography Research Articles

A method for screening active components from Chinese herbs by cell membrane chromatography-offline-high performance liquid chromatography/mass spectrometry and an online statistical tool for data processing

Cell membrane chromatography (CMC) has been successfully applied to screen bioactive compounds from Chinese herbs for many years, and some offline and online two-dimensional (2D) CMC-high performance liquid chromatography (HPLC) hyphenated systems have been established to perform screening assays. However, the requirement of sample preparation steps for the second-dimensional analysis in offline systems and the need for an interface device and technical expertise in the online system limit their extensive use. In the present study, an offline 2D CMC-HPLC analysis combined with the XCMS (various forms of chromatography coupled to mass spectrometry) Online statistical tool for data processing was established. First, our previously reported online 2D screening system was used to analyze three Chinese herbs that were reported to have potential anti-inflammatory effects, and two binding components were identified. By contrast, the proposed offline 2D screening method with XCMS Online analysis was applied, and three more ingredients were discovered in addition to the two compounds revealed by the online system. Then, cross-validation of the three compounds was performed, and they were confirmed to be included in the online data as well, but were not identified there because of their low concentrations and lack of credible statistical approaches. Last, pharmacological experiments showed that these five ingredients could inhibit IL-6 release and IL-6 gene expression on LPS-induced RAW cells in a dose-dependent manner. Compared with previous 2D CMC screening systems, this newly developed offline 2D method needs no sample preparation steps for the second-dimensional analysis, and it is sensitive, efficient, and convenient. It will be applicable in identifying active components from Chinese herbs and practical in discovery of lead compounds derived from herbs.

Simultaneous identification of the anaphylactoid components from traditional Chinese medicine injections using rat basophilic leukemia 2H3 and laboratory of allergic disease 2 dual-mixed/cell membrane chromatography model.

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.

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography.

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)-expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293-EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (KD, 8.49±0.11×10-7 M)>erlotinib (KD, 1.07±0.02×10-6 M)>canertinib (KD, 1.41±0.07×10-6 M)>afatinib (KD, 1.80±0.12×10-6 M)>vandetanib (KD, 1.99±0.03×10-6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.

Improved cell membrane bioaffinity sample pretreatment technique with enhanced stability for screening of potential allergenic components from traditional Chinese medicine injections.

Aiming at improving reliability and tedious analysis time in conventional cell membrane chromatography, an improved bioaffinity sample pretreatment technique with enhanced stability was developed to fast screen and extract potential allergenic components from traditional Chinese medicine injections. In this study, rat basophilic leukemia-2H3 cell membrane coated silica particles (RBL-2H3/CMCSPs) were fabricated by irreversible adsorption between the cell membrane and silica and self-fusion of the cell membrane, which could simulate drug-receptor interactions in vitro. Also, benefiting from the use of paraformaldehyde, the average recoveries of the six batches of RBL/CMCSPs were 90.2% with a relative standard deviation of less than 7.8%, showing that the stability of the materials was remarkably improved compared to materials without fixation. After the successful characterization by spectroscopic and imaging instruments, the prepared RBL-2H3/CMCSPs exhibited desirable adsorption capacity and selectivity. The RBL-2H3/CMCSPs combined with high performance liquid chromatography coupled with time of flight mass spectrometry were then successfully applied to screen and identify two potential allergenic components from huangqi injection, the allergenic activities of which were further investigated by β-hexosaminidase release assay and histamine release assay in vitro. Overall, this work provides a good platform for the fabrication of bioaffinity sample pretreatment materials with high stability and a long lifespan, which can be a time-saving and energy-saving bioaffinity method to rapidly screen and preconcentrate target compounds from complex samples.

Front Cover: Osteoblast cell membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry for screening specific active components from traditional Chinese medicines

Front Cover: Osteoblast cell membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry for screening specific active components from traditional Chinese medicines

Osteoblast cell membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry for screening specific active components from traditional Chinese medicines.

A method using osteoblast membrane chromatography coupled with liquid chromatography and time-of-flight mass spectrometry was developed to recognize and identify the specific active components from traditional Chinese medicines. Primary rat osteoblasts were used for the preparation of the stationary phase in the cell chromatography method. Retention components from the cell chromatography were collected and analyzed by liquid chromatography with time-of-flight mass spectrometry. This method was applied in screening active components from extracts of four traditional Chinese medicines. In total, 24 potentially active components with different structures were retained by osteoblast cell chromatography. There were five phenolic glucosides and one triterpenoid saponin from Curculigo orchioides Gaertn, two organic acids and ten flavonoids from Epimedium sagittatum Maxim, one phthalide compound and one organic acid from Angelica sinensis Diels, and two flavonoids and two saponins from Anemarrhena asphodeloides Bunge. Among those, four components (icariin, curculigoside, ferulaic acid, and timosaponin BII) were used for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation, alkaline phosphatase activity, levels of bone gla protein and collagen type 1, and promoted mineralized nodule formation. The developed method was an effective screening method for finding active components from complex medicines that act on bone diseases.

Preparation and characterization of micro-cell membrane chromatographic column with N-hydroxysuccinimide group-modified silica-based porous layer open tubular capillary

Cell membrane chromatography (CMC) is an effective tool in screening active compounds from natural products and studying membrane protein interactions. Nevertheless, it always consumes a large amount of cells (e.g. 107-108) for column preparation. To overcome this, micro-CMC (mCMC), that employs a silica capillary as membrane carrier, was developed. However, both CMC and mCMC suffer from short column life span (e.g. 3days), mainly due to the falling-off of cellular membranes (CMs). This has greatly limited further application of CMC and mCMC, especially when the cells are hard to obtain. To solve this, N-hydroxysuccinimide (NHS)-modified silica-based porous layer open tubular capillary was first prepared for mCMC. The NHS groups can easily react with amino groups on CMs to form a stable covalent bond under a mild condition. So, CMs immobilized on the NHS-modified capillary are less likely to fall off. To verify this, SKBR3/mCMC (Her2 positive) and BALL1/mCMC (CD20 positive) columns were prepared. Two monoclonal antibody drugs, trastuzumab (anti-Her2) and rituximab (anti-CD20), were selected as analytes to characterize the columns. As a result, NHS-modified column for mCMC can afford higher chromatographic retention than non-modified column. Besides, the column life span was significantly improved to more than 16days for SKBR3/mCMC and 14days for BALL1/mCMC, while the compared column showed a sharp decline in retention factor in first 3days.

Ligand Fishing with Cellular Membrane-Coated Magnetic Beads: A New Method for the Screening of Potentially Active Compounds from Natural Products

Ligand fishing with target biomolecule-immobilized magnetic beads (MBs) has been established and developed for nearly 10 years. Advantages of this technique, such as the ease of operation, associated with a diversity of automated online approaches, make it a valuable tool for affinity studies. However, transmembrane proteins have not been used as the target biomolecules in the assay, since they are usually not available in a purified and bioactive form. In addition, few publications have reported the use of this method for screening active compounds derived from natural products. In this work, for the first time, cellular membrane-coated MBs, which to a large extent maintain the activity of the transmembrane proteins, were used for the fishing assay. We demonstrated application of red blood cell membrane-coated MBs for fishing potential active components from a natural product (Angelica dahurica). The potential active compounds, such as imperatorin, bergapten, and pabulenol, were detected. The result correlated well with cell membrane chromatography (CMC) coupled with HPLC. Comparisons of the developed MBs fishing assay with the CMC method showed the noteworthy advantages of the fishing technique regarding the consumption of cellular membranes, buffers as well as length of operation time.

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

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

Comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatography for screening anti-tumor components from Radix Sophorae flavescentis.

Radix Sophorae flavescentis is generally used for the treatment of different stages of prostate cancer in China. It has ideal effects when combined with surgical treatment and chemotherapy. However, its active components are still ambiguous. We devised a comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatography system for screening anti-prostate cancer components in Radix Sophorae flavescentis. Gefitinib and dexamethasone were chosen as positive and negative drugs respectively for validation and optimization the selectivity and suitability of the comprehensive two-dimensional chromatographic system. Five compounds, sophocarpine, matrine, oxymatrine, oxysophocarpine, and xanthohumol were found to have significant retention behaviors on the PC-3 cell membrane chromatography and were unambiguously identified by time-of-flight mass spectrometry. Cell proliferation and apoptosis assays confirmed that all five compounds had anti-prostate cancer effects. Matrine and xanthohumol had good inhibitory effects, with half maximal inhibitory concentration values of 0.893 and 0.137mg/mL, respectively. Our comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatographic system promotes the efficient recognition and rapid analysis of drug candidates, and it will be practical for the discovery of prostate cancer drugs from complex traditional Chinese medicines.

Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method

The equilibrium dissociation constant (KD) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the KD value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative KD values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The KD values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the KD values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity.

Screening allergic components of Yejuhua injection using LAD2 cell membrane chromatography model online with high performance liquid chromatography-ion trap-time of flight-mass spectrum system.

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.

Screening active compounds from Corydalis yanhusuo by combining high expression VEGF receptor HEK293 cell membrane chromatography with HPLC - ESI - IT - TOF - MSn method

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.

Quantitative analysis of allosteric effects for 5-hydroxytryptamine receptor by cell membrane chromatography

Quantitative analysis of allosteric effects for 5-hydroxytryptamine receptor by cell membrane chromatography

Screening active anti-breast cancer compounds from cortex magnolia officinalis by MCF-7 cell membrane chromatography coupled with UHPLC-ESI-MS/MS

MCF-7/CMC-HPLC-MS/MS for screening active anti-breast cancer compounds from Traditional Chinese Medicines.

Berberine inhibits the proliferation and migration of breast cancer ZR-75-30 cells by targeting Ephrin-B2

BackgroundBerberine, a plant-derived compound isolated from Coptis chinensis used in traditional Chinese medicine, has been shown to possess anti-cancer properties. However, no study has shown that berberine could target ephrin-B2, which plays a critical role in cell proliferation and migration. PurposeThe aim of this study is to investigate the effect of berberine on cancer cell growth and migration, through the regulation of ephrin-B2 and downstream signaling molecules. MethodsIn this study, a high ephrin-B2-expressing cell membrane chromatography method was developed to investigate 48 crude extracts from traditional Chinese medicine that could act on ephrin-B2. Cell proliferative and wound-healing assays were used to study the effect of berberine on cancer cell growth and migration. The mechanism of berberine was investigated using western blot. ResultsBerberine was isolated from C. chinensis extracts and showed activity on the HEK293/ephrin-B2 cell membrane chromatography column. Berberine showed a greater inhibitory effect in high-expressing ephrin-B2 cells (HEK293/ephrin-B2 cells) than in normal HEK293 cells, and decreased the expression of ephrin-B2 and its PDZ binding proteins, which indicates that ephrin-B2 is a target of berberine. Furthermore, berberine downregulates the phosphorylation of VEGFR2 and downstream signaling members (AKT and Erk1/2), which in turn downregulates the expression of MMP2 and MMP9. ConclusionThe above data confirm the inhibitory effects of berberine on ZR-75-30 cell proliferation and cell migration. Overall, our studies demonstrate that berberine inhibits cell growth and migration by targeting ephrin-B2.

Development of APTES-Decorated HepG2 Cancer Stem Cell Membrane Chromatography for Screening Active Components from Salvia miltiorrhiza.

Cell membrane chromatography (CMC) is an ideal method for screening potential active components acting on target cell membranes from a complex system, such as herbal medicines. But due to the decay and falling-off of membranes, the CMC column suffers from short life span and low reproducibility. This has greatly limited the application of this model, especially when the cell materials are hard to obtain. To solve this problem, a novel type of (3-aminopropyl)triethoxysilane (APTES)-decorated silica gel was employed. The silica gel was decorated with aldehydes with the help of APTES, which react with the amino groups on cell membranes to form a covalent bond. In this way, cell membranes were immobilized on the surface of silica gel, so it is not easy for membranes to fall off. According to our investigation, the column life of the APTES-decorated group was prolonged to more than 12 days, while the control group showed a sharp decline in column efficiency in the first 3 days. To verify this model, a novel APTES-decorated HepG2 cancer stem cell membrane chromatography (CSCMC) was established and applied in a comprehensive two-dimensional chromatographic system to screen potential active components in Salvia miltiorrhiza. As a result, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I were retained on this model and proved to be effective on HepG2 cancer stem cells by the following cell proliferation and apoptosis assay, with IC50 of 10.30 μM, 17.85 μM, and 2.53 μM, respectively. This improvement of CMC can significantly prolong its column life span and broaden the range of its application, which is very suitable for making invaluable or hard-to-obtain cell materials, such as stem cells, for specific drug screening.

Characterization of Interactions Between Taspine Derivate TPD7 and EGF Receptor by Cell Membrane Chromatography with Zonal Elution and Frontal Analysis

In this study, a high epidermal growth factor receptor (EGFR) expression cell membrane chromatography method was established to investigate the interactions between ligands and EGFR. The affinity of ligands for EGFR was evaluated by frontal analysis. Competition study using afatinib as the marker was used to evaluate the interactions that occurred at specific binding sites on EGFR. The results indicated that TPD7, HMQ1611 and afatinib may have direct competition at a single common binding site on EGFR. From the model of frontal analysis, the dissociation equilibrium constants (KD) were 6.05 × 10−7 M for afatinib, 6.91 × 10−7 M for TPD7, and 9.68 × 10−7 M for HMQ1611. In cells, HMQ1611 and TPD7 could both inhibit the growth of HEK293/EGFR cells and significantly decrease EGFR phosphorylation in HEK293/EGFR cells in a dose-dependent manner. The studies showed that TPD7 and HMQ1611 could bind EGFR as afatinib. TPD7 exhibited higher inhibitory effect than HMQ1611 while TPD7 and HMQ1611 had a similar effect in HEK293/EGFR cells, thus indicating that TPD7 might be the novel blocker for cancer with high-EGFR expression.

Characterization the affinity of α1A adrenoreceptor by cell membrane chromatography with frontal analysis and stoichiometric displacement model.

As a bionic chromatographic method, cell membrane chromatography (CMC) has been used widely in screening active components in traditional Chinese medicine. Nevertheless, few studies have characterized the affinity between drug and receptor by CMC model. In this study, the alpha 1 adrenoreceptor (α1A AR) high expression CMC method, combined with frontal analysis and stoichiometric displacement model respectively, was established for characterizing the affinity of seven alkaloids binding to the α1A AR. The results indicate that the seven alkaloids have similar interaction strengths with tamsulosin hydrochloride (α1A AR antagonist) between them and α1A AR. In addition, electrostatic force is the main intermolecular forces between tamsulosin hydrochloride and seven alkaloids and α1A AR. The study provides a versatile approach for the characterization the affinity between drug and receptor by CMC model.

Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

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).