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Sicen Wang - One of the best experts on this subject based on the ideXlab platform.
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application of a stepwise frontal analysis method in cell Membrane Chromatography
Journal of Chromatography B, 2020Co-Authors: Yue Sui, Sicen WangAbstract:Abstract Bio-affinity Chromatography is used in the study of drug-receptor interactions. A stepwise frontal analysis (SFA) method was developed based on frontal analysis (FA). A high expression alpha 1A adrenergic receptor (α1A AR) cell Membrane Chromatography (CMC) method was then developed and combined with SFA to investigate the affinity of three model α1A AR-binding drugs towards α1A AR. Equilibrium dissociation constant (Kd) values for drug-receptor interactions were determined by FA and SFA; results showed that these methods were highly consistent. The results demonstrate that the CMC/SFA method is a time-saving and less wasteful method than traditional method for the evaluation of drug-receptor binding characteristics, and could be used to study the interactions between drugs and Membrane receptors.
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stepwise frontal analysis coupled with cell Membrane Chromatography for affinity screening and characterization analysis of bioactive constituent from the mature fruits of schisandra chinensis
Journal of Chromatography B, 2020Co-Authors: Fen Wei, Sicen WangAbstract:Abstract Cell Membrane Chromatography (CMC) is effective and widely used in drug screening, especially for the analysis of complex matrixes. However, it is time-consuming and costly given that cells or animals are employed for activity confirmation, which leads to a large amount of waste being produced if the result is negative. Stepwise frontal analysis is employed to saturate the affinity stationary phase, by using a series of low- to high-concentration solutions which resultantly form a staircase pattern. In doing so, the waste of samples, caused by the balancing process, can be avoided. In this study, stepwise frontal analysis coupled with a CMC system was performed for screening and characterizing the affinity of an active compound from wuweizi. Schizandrin A was screened and identified by α1A AR /CMC coupled with UHPLC-MS/MS. By comparing the values obtained with those related to the equilibrium dissociation constant (Kd) calculated by zonal elution, the accuracy of the stepwise frontal analysis was verified. Subsequently, the type of affinity force between Schizandrin A and α1A AR was studied by thermodynamic parameters. Moreover, schizandrin A showed an antagonistic effect on phenylephrine-induced contractions, which relax prostate muscle strips in a non-competitive antagonism manner. It has already suggested that the active compound, schizandrin A, could be used as a lead compound for the treatment of benign prostate hyperplasia (BPH) and should be further studied. Thus, the findings of this study are significant given that they could result in an online screening and affinity analysis method being utilized for the discovery of medicinal compounds as well as clarify the interaction characteristics between a drug and a receptor.
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screening of bioactive components from traditional chinese medicines using cell Membrane Chromatography coupled with mass spectrometry
Phytochemical Analysis, 2018Co-Authors: Shengli Han, Fen Wei, Sicen WangAbstract: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.
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screening active anti breast cancer compounds from cortex magnolia officinalis by mcf 7 cell Membrane Chromatography coupled with uhplc esi ms ms
Analytical Methods, 2017Co-Authors: Ping Zhang, Xiaofang Hou, Muhammad Saqib, Sicen WangAbstract:Breast cancer is one of the most frequent malignant tumors in females. Extracts from Cortex Magnolia Officinalis (CMO) have shown the ability to inhibit the growth of breast cancer cells. In the present study, we developed MCF-7 cell Membrane Chromatography (CMC) combined with a UHPLC-ESI-MS/MS method to screen and identify the active components from the extract of CMO. Sorafenib and dexamethasone acetate were used as positive and negative control drugs, respectively, to investigate the suitability of this system. Under optimum experimental conditions, two components showed strong retention characteristics on the MCF-7 cell Membrane chromatographic column. These compounds were identified as magnolol and honokiol by UHPLC-ESI-MS/MS. Cytotoxicity assay and Hoechst 33258 staining were done to investigate the anti-proliferative activity of the two compounds. The MTT results showed that MCF-7 cell viability decreased gradually in a dose-dependent manner during 48 h. The IC50 of magnolol and honokiol were 85.4 μM and 58.1 μM, respectively. After Hoechst 33258 staining, the cells were observed with bright blue nuclei due to karyopyknosis and chromatin condensation. The screening results showed that both magnolol and honokiol were promising active components of CMO extract having an inhibitory effect on MCF-7 cell growth. This two-dimensional method CMC-UHPLC-ESI-MS/MS could have potential utility for screening target components from traditional Chinese medicines.
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screening active compounds from corydalis yanhusuo by combining high expression vegf receptor hek293 cell Membrane Chromatography with hplc esi it tof msn method
Journal of Pharmaceutical and Biomedical Analysis, 2017Co-Authors: Fen Wei, Shengli Han, Jing Huang, Sicen WangAbstract: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.
Raja Ghosh - One of the best experts on this subject based on the ideXlab platform.
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preparative separation of monoclonal antibody aggregates by cation exchange laterally fed Membrane Chromatography
Journal of Chromatography B, 2017Co-Authors: Pedram Madadkar, Rahul Sadavarte, Michael Butler, Yves Durocher, Raja GhoshAbstract:Cation exchange (CEX) Chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the current study we address this challenge through the use of cation-exchange laterally-fed Membrane Chromatography (LFMC). Three different LFMC devices, each containing a bed of strong cation-exchange (S) Membranes were used for preparative-scale removal of mAb aggregates. Trastuzumab (IgG1) biosimilar derived from human embryonic kidney 293 (293) cells was used as the primary model mAb in our study. The other mAbs investigated were Chinese hamster ovary (CHO) cell line derived Alemtuzumab (Campath-1H) and a heavy chain chimeric mAb EG2-hFc. In each of these case-studies, aggregates were well-resolved from the respective monomer. The separated and collected monomer and aggregate fractions were analyzed using techniques such as hydrophobic interaction Membrane Chromatography (HIMC), native polyacrylamide gel electrophoresis (or PAGE), and size-exclusion high-performance liquid Chromatography (SE-HPLC). The high efficiency of separation obtained in each case was due to a combination of the small Membrane pore size (3-5μm), and the use of LFMC technology, which has been shown to be suitable for high-resolution, multi-component protein separations. Also, the LFMC based separation processes reported in this study were more than an order of magnitude faster than equivalent resin-based, cation exchange Chromatography.
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on the workings of laterally fed Membrane Chromatography
Journal of Membrane Science, 2016Co-Authors: Raja Ghosh, Pedram MadadkarAbstract:Abstract Membrane Chromatography has significant potential for use in biopharmaceutical purification processes. However, currently available Membrane Chromatography devices give poor resolution and therefore their use is restricted to polishing applications, typically carried out in the flow-through mode. Laterally-fed Membrane Chromatography (or LFMC) was developed specifically for carrying high-resolution multi-component protein separations. Recent studies have shown that resolution obtained with LFMC is comparable to that obtained with equivalent resin-based packed bed Chromatography. Therefore, LFMC combines high-resolution with high-productivity, a highly desirable attribute in any purification technique. In this paper we explain the workings of LFMC based on theoretical and experimental analysis of hydraulic flow-path and residence time distribution. Factors likely to affect efficiency of LFMC are discussed using a simple electrical circuit analogy.
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high resolution protein separation using a laterally fed Membrane Chromatography device
Journal of Membrane Science, 2016Co-Authors: Pedram Madadkar, Raja GhoshAbstract:Radial-flow Membrane Chromatography devices which are used for flow-through separation are generally unsuitable for bind-and-elute Chromatography, particularly where multiple components need to be separated. We discuss a laterally-fed Membrane Chromatography device, suitable for high-resolution, multi-component protein separation in the bind-and-elute mode. In the current study, a stack of cation exchange Membranes was housed within the Membrane device and its performance was compared with an equivalent commercial radial-flow device having the same Membrane bed volume and bed height. Tracer experiments were carried out using sodium chloride solution to compare their residence time distributions. The laterally-fed device showed superior flow distribution characteristics, which could be attributed to a lower variability in solute-flow path-lengths, and a smaller dead volume. Single protein bind-and-elute experiments carried out using lysozyme showed that the peaks obtained with the laterally-fed device were significantly sharper and more symmetrical. Excellent separation of three model proteins ovalbumin, conalbumin and lysozyme demonstrated that the laterally-fed Membrane Chromatography device was indeed suitable for carrying out high-resolution multi-component protein purification. These proteins could be fractionated in about 10 Membrane bed volumes using the laterally-fed device as opposed to 25 bed volumes with the radial-flow device. The design of the laterally-fed device is simple and its flat shape gives it significantly lower footprint and offers additional advantages such as stackability and ease of multiplexing.
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a laterally fed Membrane Chromatography module
Journal of Membrane Science, 2015Co-Authors: Pedram Madadkar, Raja GhoshAbstract:Module design is of critical importance in Membrane Chromatography as the efficiency of separation is highly dependent on fluid flow distribution and collection within the Membrane device. We discuss a novel, laterally-fed module, designed specifically for flat-sheet Membrane Chromatography. The performance of the novel module was compared with that of a conventional, centrally-fed, circular Membrane module. Experiments were carried out with both devices using anion-exchange Membrane sheets having the same surface area and thickness, and thereby the same bed volume. Tracer experiments using either a dye or a protein (lysozyme) under non-binding condition clearly indicated superior flow distribution and collection within the novel module. This could be attributed to greater uniformity in solute flow path length. The protein binding capacities of Membrane sheets of identical surface area and bed volume housed in the novel and conventional modules were compared in the breakthrough and pulse modes, using bovine serum albumin (or BSA) as the model adsorbed protein. The breakthrough experiments showed that at the same experimental conditions, the 1% breakthrough binding capacity of the Membrane housed in the novel module was 5.12 times higher than that housed in the conventional module. Moreover, flow-through and elution peaks obtained with the novel Membrane module were significantly sharper and more symmetrical, with lower peak width.
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purification of chimeric heavy chain monoclonal antibody eg2 hfc using hydrophobic interaction Membrane Chromatography an alternative to protein a affinity Chromatography
Biotechnology and Bioengineering, 2014Co-Authors: Rahul Sadavarte, Natalie Okun, Maureen Spearman, Michael Butler, Raja GhoshAbstract:Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A Chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity Chromatography does not remove aggregates already present in the feed. Hydrophobic interaction Membrane Chromatography (or HIMC) has already been studied as an alternative to protein-A Chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc. Biotechnol. Bioeng. 2014;111: 1139–1149. © 2014 Wiley Periodicals, Inc.
Yifeng Chai - One of the best experts on this subject based on the ideXlab platform.
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a stop flow comprehensive two dimensional hk 2 and hk 2 ciki cell Membrane Chromatography comparative analysis system for screening the active ingredients from pyrrosia calvata bak ching against crystal induced kidney injury
Journal of Pharmaceutical and Biomedical Analysis, 2021Co-Authors: Pengchao Pan, Yifeng Chai, Zhenyu Zhu, Jin Cheng, Wei Chen, Jiebin Hou, Tingting Zhao, Zhanying Hong, Zhiyong Guo, Xiaofei ChenAbstract:Crystal-induced kidney injury (CIKI) is the fundamental pathological change during nephrolithiasis, although the molecular mechanism is still unclear. Pyrrosia calvata (Bak.) Ching has been used in folk medicine to treat urolithiasis for years. To clarify the pharmacodynamic substances and the mechanism of its antiurolithiasis effects, in this study, a novel, stop-flow, comprehensive, two-dimensional (2D) HK-2 and HK-2/CIKI cell Membrane Chromatography (CMC) comparative analysis system was developed to screen for the potential active ingredients from Pyrrosia calvata (Bak.) Ching against CIKI. The comprehensive 2D CMC comparative analysis system showed satisfactory selectivity, and eight ingredients were screened and identified by this system. Among them, mangiferin exhibited higher affinity for the HK-2/CIKI CMC column than the HK-2 CMC column and was selected for further efficacy verification. Cell proliferation assays showed that mangiferin could protect HK-2 cell viability after stimulation with sodium oxalate (NaOX). Additionally, in a rodent model of CIKI, mangiferin decreased the deposition of calcium oxalate (CaOX) crystals in mouse kidneys, alleviated the pathological damage to kidney tissue, and inhibited the upregulation of OPN, MCP1, and CD44 expression caused by CaOX crystals. The established comprehensive 2D CMC comparative analysis system can be applied to screen active ingredients with disease specificity from traditional Chinese medicine (TCM) and is suitable for other cell models.
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development of 3 mercaptopropyltrimethoxysilane mpts modified bone marrow mononuclear cell Membrane Chromatography for screening anti osteoporosis components from scutellariae radix
Acta Pharmaceutica Sinica B, 2020Co-Authors: Xiao Chen, Yao Wang, Yue Liu, Leyi Zheng, Rong Wang, Shaozhan Wang, Yifeng Chai, Yongfang Yuan, Xiaofei ChenAbstract:Abstract Osteoporosis is a bone metabolic disease caused by the imbalance between osteoblasts and osteoclasts due to excess osteoclastogenesis, manifesting in the decrease of bone density and bone strength. Scutellariae Radix shows good anti-osteoporosis activity, but the effective component is still unclear. Cell Membrane Chromatography (CMC) is a biological affinity Chromatography with Membrane immobilized on a silica carrier as the stationary phase. It can realize a dynamical simulation of interactions between drugs and receptors on cell Membrane, which is suitable for screening active compounds from complex systems. In this study, the components of Scutellariae Radix with potential anti-osteoporosis activity through inhibiting the differentiation from bone marrow mononuclear cells (BMMCs) to osteoclast were screened by a BMMC/CMC analytical system. Firstly, a new 3-mercaptopropyltrimethoxysilane (MPTS)-modified BMMC/CMC stationary phase was developed to realize covalent binding with cell Membrane fractions. By investigating the retention time (tR) of the positive drug, the life span of the MPTS-modified CMC columns was significantly improved from 3 to 12 days. Secondly, 6 components of Scutellariae Radix were screened to show affinity to Membrane receptors on BMMCs by a two-dimensional BMMC/CMC–TOFMS analytical system. Among them, tectochrysin demonstrated the best anti-osteoporosis effect in vitro, which has never been reported. We found that tectochrysin could inhibit the differentiation of BMMCs into osteoclasts induced by receptor activator of nuclear factor-κΒ ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in a concentration-dependent manner in vitro. In vivo, it significantly reduced the loss of bone trabeculae in ovariectomized mice, and decreased the level of C-terminal cross-linking telopeptides of type 1 collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRAP-5b), interleukin 6 (IL-6) in serum. In conclusion, tectochrysin serves as a potential candidate in the treatment of osteoporosis. The proposed two-dimensional MPTS-modified BMMC/CMC-TOFMS analytical system shows the advantages of long-life span and fast recognition ability, which is very suitable for infrequent cell lines.
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comparative two dimensional hepg2 and l02 cell Membrane Chromatography c18 time of flight mass spectrometry for screening selective anti hepatoma components from scutellariae radix
Journal of Pharmaceutical and Biomedical Analysis, 2019Co-Authors: Xiaofei Chen, Leyi Zheng, Rong Wang, Shaozhan Wang, Yifeng Chai, Xiaoyu Wang, Bin Zhang, Zhenyu Zhu, Yongfang YuanAbstract:Abstract Screening active components from Chinese traditional medicine is an effective approach to discover new drugs or active structures. Cell Membrane Chromatography (CMC), developed rapidly because of its high sensitivity and effectiveness, has achieved a wide application in screening active components on pathological cells or tissues. However, it is hard to clarify the selectivity between pathological and normal tissues through simply using pathological cells. In this study, a novel comparative two-dimensional (2D) cell Membrane Chromatography system was established. Briefly, hepatic carcinoma HepG2 CMC columns and normal hepatic L02 CMC columns were simultaneously loaded to screen potential selective antitumor components from Scutellariae Radix by comparing the retention behaviors on two kinds of cells. Totally 13 components in Scutellariae Radix retained on both HepG2/ CMC and L02/ CMC columns. Among them, three components, oroxylin A, wogonin and chrysin, were screened out to perform stronger affinity on HepG2 columns, and in further cell proliferation assay, IC50 of these three compounds of HepG2 cells were 9.66 μM, 66.77 μM and 36.26 μM respectively, while of L02 cells, IC50 of chrysin was 59.10 μM and over 200 μM of the other two components. On the whole, the toxity of these three compounds to hepatoma cells was stronger than to normal cells. It can be supposed that oroxylin A, wogonin, and chrysin own the potential to be developed as selective anti-hepatoma active components, which expects further research to validate.
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comprehensive two dimensional hepg2 cell Membrane Chromatography monolithic column time of flight mass spectrometry system for screening anti tumor components from herbal medicines
Journal of Chromatography A, 2012Co-Authors: Xiaofei Chen, Diya Lv, Junping Zhang, Yifeng ChaiAbstract:Abstract Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using specific cell Membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and Membrane receptors. In this study, a novel comprehensive two-dimensional (2D) Chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell Membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p
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comprehensive two dimensional hepg2 cell Membrane Chromatography monolithic column time of flight mass spectrometry system for screening anti tumor components from herbal medicines
Journal of Chromatography A, 2012Co-Authors: Xiaofei Chen, Zhenyu Zhu, Yan Cao, Junping Zhang, Yifeng ChaiAbstract:Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using specific cell Membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and Membrane receptors. In this study, a novel comprehensive two-dimensional (2D) Chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell Membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p<0.05). The result demonstrated that the proposed comprehensive 2D HepG2/CMC/monolithic column/TOFMS system has the advantages of strong recognition and rapid analysis abilities for the total screening procedure, which will be selectable and practical in drug discovery from complex HM samples and can also be applied to other bioChromatography models.
Xiaofei Chen - One of the best experts on this subject based on the ideXlab platform.
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a stop flow comprehensive two dimensional hk 2 and hk 2 ciki cell Membrane Chromatography comparative analysis system for screening the active ingredients from pyrrosia calvata bak ching against crystal induced kidney injury
Journal of Pharmaceutical and Biomedical Analysis, 2021Co-Authors: Pengchao Pan, Yifeng Chai, Zhenyu Zhu, Jin Cheng, Wei Chen, Jiebin Hou, Tingting Zhao, Zhanying Hong, Zhiyong Guo, Xiaofei ChenAbstract:Crystal-induced kidney injury (CIKI) is the fundamental pathological change during nephrolithiasis, although the molecular mechanism is still unclear. Pyrrosia calvata (Bak.) Ching has been used in folk medicine to treat urolithiasis for years. To clarify the pharmacodynamic substances and the mechanism of its antiurolithiasis effects, in this study, a novel, stop-flow, comprehensive, two-dimensional (2D) HK-2 and HK-2/CIKI cell Membrane Chromatography (CMC) comparative analysis system was developed to screen for the potential active ingredients from Pyrrosia calvata (Bak.) Ching against CIKI. The comprehensive 2D CMC comparative analysis system showed satisfactory selectivity, and eight ingredients were screened and identified by this system. Among them, mangiferin exhibited higher affinity for the HK-2/CIKI CMC column than the HK-2 CMC column and was selected for further efficacy verification. Cell proliferation assays showed that mangiferin could protect HK-2 cell viability after stimulation with sodium oxalate (NaOX). Additionally, in a rodent model of CIKI, mangiferin decreased the deposition of calcium oxalate (CaOX) crystals in mouse kidneys, alleviated the pathological damage to kidney tissue, and inhibited the upregulation of OPN, MCP1, and CD44 expression caused by CaOX crystals. The established comprehensive 2D CMC comparative analysis system can be applied to screen active ingredients with disease specificity from traditional Chinese medicine (TCM) and is suitable for other cell models.
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comparative two dimensional gpc3 overexpressing sk hep1 cell Membrane Chromatography c18 time of flight mass spectrometry for screening selective gpc3 inhibitor components from scutellariae radix
Journal of Chromatography B, 2021Co-Authors: Chun Chen, Rong Wang, Shaozhan Wang, Xiaofei Chen, Zhenyu Zhu, Xinyi Chai, Shuya Jiang, Yongfang YuanAbstract:Abstract Screening active components targeting Membrane proteins is important for drug discovery from traditional Chinese medicine. Cell Membrane Chromatography (CMC) has achieved a wide application in screening active components on pathological cells due to its high sensitivity and effectiveness. However, it is hard to clarify the specific target protein through simply using pathological and normal cells. In this study, a novel comparative two-dimensional (2D) cell Membrane Chromatography system was established. Based on the construction of hepatocellular carcinoma cell line SK-Hep1-GPC3 with high expression of protein Glypican-3 (GPC3), SK-Hep1-GPC3/CMC column was loaded to screen selective antitumor components from Scutellariae Radix according to the retention behaviors on column. Viscidulin I was retained on SK-Hep1-GPC3/CMC column, and showed 4.33 μM affinity to GPC3 according to surface plasmon resonance (SPR). The IC50 of viscidulin I on SK-Hep1-GPC3 cells was 18.01 μM in cell proliferation assay. Thus, this method can be applied to screen complex herbal medicines for ligands bound to specific target protein receptor related to hepatic carcinoma.
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development of 3 mercaptopropyltrimethoxysilane mpts modified bone marrow mononuclear cell Membrane Chromatography for screening anti osteoporosis components from scutellariae radix
Acta Pharmaceutica Sinica B, 2020Co-Authors: Xiao Chen, Yao Wang, Yue Liu, Leyi Zheng, Rong Wang, Shaozhan Wang, Yifeng Chai, Yongfang Yuan, Xiaofei ChenAbstract:Abstract Osteoporosis is a bone metabolic disease caused by the imbalance between osteoblasts and osteoclasts due to excess osteoclastogenesis, manifesting in the decrease of bone density and bone strength. Scutellariae Radix shows good anti-osteoporosis activity, but the effective component is still unclear. Cell Membrane Chromatography (CMC) is a biological affinity Chromatography with Membrane immobilized on a silica carrier as the stationary phase. It can realize a dynamical simulation of interactions between drugs and receptors on cell Membrane, which is suitable for screening active compounds from complex systems. In this study, the components of Scutellariae Radix with potential anti-osteoporosis activity through inhibiting the differentiation from bone marrow mononuclear cells (BMMCs) to osteoclast were screened by a BMMC/CMC analytical system. Firstly, a new 3-mercaptopropyltrimethoxysilane (MPTS)-modified BMMC/CMC stationary phase was developed to realize covalent binding with cell Membrane fractions. By investigating the retention time (tR) of the positive drug, the life span of the MPTS-modified CMC columns was significantly improved from 3 to 12 days. Secondly, 6 components of Scutellariae Radix were screened to show affinity to Membrane receptors on BMMCs by a two-dimensional BMMC/CMC–TOFMS analytical system. Among them, tectochrysin demonstrated the best anti-osteoporosis effect in vitro, which has never been reported. We found that tectochrysin could inhibit the differentiation of BMMCs into osteoclasts induced by receptor activator of nuclear factor-κΒ ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in a concentration-dependent manner in vitro. In vivo, it significantly reduced the loss of bone trabeculae in ovariectomized mice, and decreased the level of C-terminal cross-linking telopeptides of type 1 collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRAP-5b), interleukin 6 (IL-6) in serum. In conclusion, tectochrysin serves as a potential candidate in the treatment of osteoporosis. The proposed two-dimensional MPTS-modified BMMC/CMC-TOFMS analytical system shows the advantages of long-life span and fast recognition ability, which is very suitable for infrequent cell lines.
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comparative two dimensional hepg2 and l02 cell Membrane Chromatography c18 time of flight mass spectrometry for screening selective anti hepatoma components from scutellariae radix
Journal of Pharmaceutical and Biomedical Analysis, 2019Co-Authors: Xiaofei Chen, Leyi Zheng, Rong Wang, Shaozhan Wang, Yifeng Chai, Xiaoyu Wang, Bin Zhang, Zhenyu Zhu, Yongfang YuanAbstract:Abstract Screening active components from Chinese traditional medicine is an effective approach to discover new drugs or active structures. Cell Membrane Chromatography (CMC), developed rapidly because of its high sensitivity and effectiveness, has achieved a wide application in screening active components on pathological cells or tissues. However, it is hard to clarify the selectivity between pathological and normal tissues through simply using pathological cells. In this study, a novel comparative two-dimensional (2D) cell Membrane Chromatography system was established. Briefly, hepatic carcinoma HepG2 CMC columns and normal hepatic L02 CMC columns were simultaneously loaded to screen potential selective antitumor components from Scutellariae Radix by comparing the retention behaviors on two kinds of cells. Totally 13 components in Scutellariae Radix retained on both HepG2/ CMC and L02/ CMC columns. Among them, three components, oroxylin A, wogonin and chrysin, were screened out to perform stronger affinity on HepG2 columns, and in further cell proliferation assay, IC50 of these three compounds of HepG2 cells were 9.66 μM, 66.77 μM and 36.26 μM respectively, while of L02 cells, IC50 of chrysin was 59.10 μM and over 200 μM of the other two components. On the whole, the toxity of these three compounds to hepatoma cells was stronger than to normal cells. It can be supposed that oroxylin A, wogonin, and chrysin own the potential to be developed as selective anti-hepatoma active components, which expects further research to validate.
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comprehensive two dimensional hepg2 cell Membrane Chromatography monolithic column time of flight mass spectrometry system for screening anti tumor components from herbal medicines
Journal of Chromatography A, 2012Co-Authors: Xiaofei Chen, Diya Lv, Junping Zhang, Yifeng ChaiAbstract:Abstract Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using specific cell Membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and Membrane receptors. In this study, a novel comprehensive two-dimensional (2D) Chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell Membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p
Xiaofang Hou - One of the best experts on this subject based on the ideXlab platform.
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study on screening potential allergenic proteins from infant milk powders based on human mast cell Membrane Chromatography and histamine release assays
Journal of Pharmaceutical Analysis, 2019Co-Authors: Ping Zhang, Wei Sun, Yingdi Shi, Xiaofang HouAbstract:Abstract Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell Membrane chromatographic (CMC) method based on human mast cells (HMC-1) for screening potential allergens in infant formula milk powders (IFMP). HMC-1 cell Membranes were extracted and mixed with silica to prepare cell Membrane Chromatography columns (10 mm × 2 mm i.d., 5 µm). Under the conditions of 0.2 mL/min flow rate and 214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic (RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.
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Study on screening potential allergenic proteins from infant milk powders based on human mast cell Membrane Chromatography and histamine release assays
Elsevier, 2019Co-Authors: Ping Zhang, Wei Sun, Yingdi Shi, Xiaofang HouAbstract:Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell Membrane chromatographic (CMC) method based on human mast cells (HMC-1) for screening potential allergens in infant formula milk powders (IFMP). HMC-1 cell Membranes were extracted and mixed with silica to prepare cell Membrane Chromatography columns (10 mm × 2 mm i.d., 5 µm). Under the conditions of 0.2 mL/min flow rate and 214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic (RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies. Keywords: Allergenic proteins, Cell Membrane Chromatography, Milk powder
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screening active anti breast cancer compounds from cortex magnolia officinalis by mcf 7 cell Membrane Chromatography coupled with uhplc esi ms ms
Analytical Methods, 2017Co-Authors: Ping Zhang, Xiaofang Hou, Muhammad Saqib, Sicen WangAbstract:Breast cancer is one of the most frequent malignant tumors in females. Extracts from Cortex Magnolia Officinalis (CMO) have shown the ability to inhibit the growth of breast cancer cells. In the present study, we developed MCF-7 cell Membrane Chromatography (CMC) combined with a UHPLC-ESI-MS/MS method to screen and identify the active components from the extract of CMO. Sorafenib and dexamethasone acetate were used as positive and negative control drugs, respectively, to investigate the suitability of this system. Under optimum experimental conditions, two components showed strong retention characteristics on the MCF-7 cell Membrane chromatographic column. These compounds were identified as magnolol and honokiol by UHPLC-ESI-MS/MS. Cytotoxicity assay and Hoechst 33258 staining were done to investigate the anti-proliferative activity of the two compounds. The MTT results showed that MCF-7 cell viability decreased gradually in a dose-dependent manner during 48 h. The IC50 of magnolol and honokiol were 85.4 μM and 58.1 μM, respectively. After Hoechst 33258 staining, the cells were observed with bright blue nuclei due to karyopyknosis and chromatin condensation. The screening results showed that both magnolol and honokiol were promising active components of CMO extract having an inhibitory effect on MCF-7 cell growth. This two-dimensional method CMC-UHPLC-ESI-MS/MS could have potential utility for screening target components from traditional Chinese medicines.
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recent advances in cell Membrane Chromatography for traditional chinese medicines analysis
Journal of Pharmaceutical and Biomedical Analysis, 2014Co-Authors: Xiaofang Hou, Sicen Wang, Tao Zhang, Jie Zhang, Yanmin ZhangAbstract:Traditional Chinese medicines (TCMs) have been used for preventative care for thousands of years. The active components are the basis for the pharmacodynamics of TCMs, and they can be an important source of lead compounds. As a bioaffinity Chromatography technique, cell Membrane Chromatography (CMC) has been developed for almost 20 years since 1996. It has been proven to be a useful method for studying drug-receptor interactions and screening active components from medicinal herbs. In our review in 2007 (Drug Discov. Ther., 1 (2007) 104-107), the preparation, identification, evaluation, and preliminary applications of CMC stationary phases were presented. In this article, we briefly review some of the latest progress and applications about CMC including instrument development, research on drug-receptor interactions, screening active components from TCMs, and quality control of TCMs.
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screening active anti breast cancer compounds from cortex magnolia officinalis by 2d lc ms
IEEE Journal of Solid-state Circuits, 2013Co-Authors: Xiaofang Hou, Sicen Wang, Xilong Yuan, Bing Zhang, Qinhua ChenAbstract:Abstract Most of the anti-breast cancer drugs are often limited owing to drug resistance and serious adverse reactions. Therefore, development of more targeted and low toxic drugs from traditional Chinese medicines for breast cancer are needed. At the same time, establishment of fast and effective drug screening methods are urgently required. We describe here a 2D LC method of MDA-MB-231 cell Membrane Chromatography combined with HPLC/MS for recognition, separation, and identification of target components from traditional Chinese medicine Cortex Magnolia officinalis. The MDA-MB-231 cells Membrane was used to prepare the chromatographic stationary phase in the first dimension. The active compounds had a retention characteristic on the cell Membrane Chromatography model (10 × 2.0 mm, 5 μm). The retention fractions were enriched using an online C(18) column (10 × 1.0 mm, 5 μm) and were analyzed by the second dimension RP Chromatography. Finally, the activity of the retention fractions was tested through in vitro experiments. Results showed that the retention fractions were honokiol and magnolol and the inhibition rate on MDA-MB-231 cell growth were 23 and 64 μM, respectively. These results support the conclusion that this coupled analytical technique could be an efficient method in drug discovery.