ABSTRACT
Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell acti-vators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside Ⅳ,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory compo-nents of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.
ABSTRACT
The chemical components (groups) contained in traditional Chinese medicine(TCM) and its compound preparations are the material basis for its curative effect, because of the integrity of the action of TCM and the complexity of its compositions and mechanism. The separation and analysis of chemical constituents in TCM and its compound prescriptions by various methods is always a key problem to be solved in the research of disease prevention and treatment of TCM. The binding of drug molecules to receptors at the cellular level was explored to provide a new idea for the screening of active components of TCM or its compound preparations. Traditional methods have some drawbacks, such as cumbersome operation, time-consuming, waste of solvents and irreversible adsorption of samples. The basic information of pharmacological parameters cannot be given in the separation process, and the active ingredients cannot be efficiently and accurately located. At present, cell membrane chromatography (CMC), one of the methods, is used to study the interaction between drug molecules and receptors, and can combine the existing chromatography and mass spectrometry technology, cell biology and receptor pharmacology, and correctly reflect the interaction between active parts, active components and cell membrane and membrane receptors, so it has unique advantages in screening effective parts, separation of active components and high-throughput screening from complex TCM system. The principles and characteristics of CMC, the cell membrane model in the field of active ingredient selection of TCM and its research status in combination with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were reviewed, and its development prospects and future research methods were discussed, which provides theoretical basis and practical guidance for the research and utilization of CMC in the field of TCM.
ABSTRACT
OBJECTIVE: To establish a platelet cell membrane chromatographic model and investigate the retention behaviors of anti-platelet aggregation drugs on chromatographic column at different temperatures, and simulate the interactions between drugs and platelets under normal and febrile pathological conditions. METHODS: The platelet cell membrane chromatographic stationary phase was constructed by physical adsorption method. The column was packed with wet method. The protein content was determined by BCA protein concentration assay kit. The biological activity was determined by Na+, K+-ATPase assay kit. The chromatographic model was used to investigate the specificity of the column and the retention characteristics of drugs in the temperature range of 35.0-42.0℃. RESULTS: The activity of platelet ATPase was 0.214, and the concentration of platelet membrane protein was 0.340 9 mg•mL-1 before bonding and 0.080 5 mg•mL-1 after bonding. The retention characteristics of clopidogrel, dipyridamole and cilostazole on platelet cell membrane chromatographic column and blank silica gel column were quite different. The retention time of the three drugs on platelet cell membrane chromatographic column was the maximum at 36.0℃, and then decreased with the increase of temperature. CONCLUSION: A platelet cell membrane chromatographic model is successfully constructed, and the retention characteristics of antiplatelet aggregates at different temperatures are studied for the first time. The chromatographic retention behaviors of antiplatelet aggregates at normal and febrile body temperatures are simulated.
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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 po-sitive 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 mm). 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-lacto-globulin 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 con-clusion was consistent with other studies.
ABSTRACT
Objective:To screen anti-EGFR components from Aconitum soongaricum Stapf. and investigate their inhibitory effect on HEK293 cells and EGFR/HEK293 high expression cell lines. Methods:A two-dimensional online system (EGFR/CMC-HPLC-MS) was built to screen anti-EGFR active compounds from Aconitum soongaricum Stapf.;molecular docking assay was performed to determine the binding affinity of the components to EGFR;MTT was used to confirm the inhibition effect of the screened compounds on HEK293 cells and EGFR/HEK293 high expression cell lines.Results:The screening results showed that aconitine,12-epi-napellin and songorine were the active components acting on EGFR like gefitinib; molecular docking assay showed the targeting components acting on EGFR in the similar manner with gefitinib used as a positive control drug; MTT assay confirmed the screened components had inhibitory effects on HEK293 cells and EGFR/HEK293 high expression cell lines in a dose-dependent manner within the dose range of 0.4- 50.0 μmol·L-1. Conclusion:The screening results are apparently consistent with the pharmacological effect. The online EGFR/CMC-HPLC-MS method has a good application prospect in the rapid screening of the active compounds in the complex system of traditional Chinese medicines,and fully demonstrates such advantages of cell membrane chromatography as simple and rapid with high efficiency.
ABSTRACT
Cell membrane chromatography (CMC) was first proposed by Professor He in 1996. As one of bio-affinity chromatography technique, CMC was a simple and convenient technology in the study of interactions of active components in traditional Chinese medicines (TCMs) with membrane receptors in vitro, and screen active components from complicated TCMs. Recently, the CMC technology was developed rapidly, and widely applied in the discovery of lead compounds from nature product. This review article is focused on the application of cell membrane chromatography in the identification of active components in traditional Chinese medicine, together with the recent development of CMC methodology. Combining with our previous works in the analysis of the composition of complex substances, biochromatography and TLC bioautography for quality evaluation of TCM, we proposed a new holistic quality evaluation strategy of TCM related with bioactivity, which could be summarized as the integration of screen (screening of quality control marker by CMC), macroscopic characterization (characterizing the chemical material basis by multi-dimension and multi data fingerprinting) and microscopic description (multi-component quantification). The proposed strategy would provide a new idea for the holistic quality evaluation of TCM in the composition and concentration of bioactive components as quality evaluation indicators.
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Objective To screen potential active anti-cancer components of Brucea javanica.Methods This research has em-ployed comprehensive two dimensional chromatographic technology and cell membrane chromatographic technology simultaneously with mass spectrometry as detector .Results Adenosine and Bruceine B were found to be potentially anti-cancer active .Conclusion This study has combined the advantages of online , high speed and high throughput for the screen of potential active components of traditional Chinese medicine .
ABSTRACT
It is very difficult to comprehensively achieve the quality control of traditional Chinese medicine (TCM), because some problems such as indicator simplification and lack of correlation between efficacy and indicator are ex-isted in the common quality control of TCM. Combined with the practical experience of our research work, this paper outlined the characteristics and applications of cell membrane chromatography (CMC) and discussed that CMC method can be used for quality control of TCM.
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With the continuous development of modern chemistry, biology and pharmacy, researches on TCM modernization has witnessed a wide range of application of related modern technology, especially in researches of TCM monomer efficacy and pharmacokinetics. This can provide basis to internationalization of traditional Chinese medicine. The current research focus is how to obtain targeted TCM monomer. This article expounded the screening methods for TCM monomer at home and abroad, the principles of screening, the limitation and meanings.
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Objective To screen the active component from Fructus Citri Sarcodactylis which have aortic vascular relaxant effect. Methods The active components from Fructus Citri Sarcodactylis were screened by combining vascular smooth muscle/cell membrane chromatography (VSM/CMC) and liquid chromatography/mass spectrometry (LC/MS) with pharmacological assay in vitro. Results Bergapten was the active component of Fructus Citri Sarcodactylis that functions in vascular. The pharmacological experiment showed that bergapten had vasodilating effect. The capacity factor of this compound on the VSM/CMC model was found to be significantly correlated with its pharmacological effect. Conclusion Association between compound and membrane protein (receptor) can be reflected by using the VSM/CMC model. VSM/CMC-offline-LC/MS technology can be used to quickly screening new active components from natural medicine.
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An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.
ABSTRACT
An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli.On the basis of the analysis of the first dimension,retention components of the mine sample were collected into 30 fractions (one fraction per minute).Then offline analysis of the second dimension was carried out.34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine,and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method.As a result,binding affinities decreased along with the process of deacylation,debenzoylation and demethylation,which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism.Moreover,some minor components in rat urine (Songorine,14-benzoylneoline,Deoxyaconitine,etc.) exerted relatively strong affinity on cell membranes are worth exploring.The results delivered by the system suggest that the CMC can be applied to in vivo study.
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Objective To discover the effective component of Rhizoma Tupistrae chinensis with inhibiting angiogenesis. Methods Several parts of Rhizoma Tupistrae chinensis were obtained by using different extraction solvents. They were screened by the model of ECV304 cell membrane chromatography with anti-VEGF antibody as a control drug. The inhibition of effective component (ZGQ-C) on ECV304 proliferation in vitro was examined by MTT assay. Results The inhibition rate (40.51%,51.11%,63.54%,74.32%,81.26%) was correlated with the ZGQ-C concentration. The ECV304 cell had significantly changed in morphology. Conclusion The ZGQ-C is an effective component for inhibiting angiogenesis. The screening results on the model have a good correlation with that of MTT assay.
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Aim To investigate the bio-affinities of ligustilide and butylidenephthalide to rat aortic smooth muscle cells and the inhibitory effects of them on bFGF-stimulated proliferation of rat vascular smooth muscle cell (VSMC). Methods VSMCs were cultured from rat aorta pectoralis and identified by an immunohistochemical method. The bio-affinities between solute (ligustilide or butylidenephthalide) and cell membrane were measured by rat aortic cell membrane chromatography (CMC). The inhibitory effects of ligustilide and butylidenephthalide on bFGF-stimulated VSMC proliferation were evaluated by MTT colorimetric method. Results Both ligustilide and butylidenephthalide had selective affinities to rat aortic smooth muscle cell as the same as verapamil, one of the calcium ion antagonists. They could potently separately (P ＜ 0.05 ), but had no effects on the normal VSMC growth. Conclusion Both ligustilide and butylidenephthalide can inhibit the abnormal proliferation of VSMC induced by bFGF.