细胞膜色谱技术应用于中药活性成分筛选的研究进展

目的了解细胞膜色谱技术在中药活性成分筛选应用中的研究进展,为该技术的进一步开发和应用提供参考。方法通过查阅国内外相关文献,进行总结、分析,对细胞膜色谱技术在中药活性成分筛选中的应用进行综述。结果细胞膜色谱技术已成功应用于中药药效物质基础研究,对于中药活性成分的筛选涉及到多个方面,包括抗炎镇痛、抗心血管疾病、抗糖尿病和抗肿瘤等活性成分,并在不断发展和完善。结论细胞膜色谱技术还存在一些问题,随着对该技术研究的不断深入,其在中药药效物质基础研究等方面具有广阔的前景。     

高效液相色谱法在合成多肽分离与纯化中的应用

近年来，多肽药物化学形成并迅速发展成为药物化学的重要分支。而随着分离纯化技术的进展，又大大加快了新活性多肽的发现和合成速度。在多肽合成中．许多杂质显示与产物类似的性质，随着肽链的增长，分离的难度也增大，因此纯化的方法和工艺显得异常重要。本文综述了凝胶过滤色谱、离子交换色谱和反相色谱等高效液相色谱技术在合成多肽分离与纯化中的设计和应用。     

细胞膜色谱法及其在中药活性成分研究中的应用

细胞膜色谱法作为一种新兴的色谱技术,将药物效应成分的分离和活性筛选结合在一起,因此特别适用于中药及天然药物的物质基础研究。对细胞膜色谱技术在中药活性成分研究方面的应用做一简要综述,为该技术的进一步研究和应用提供参考。     

用SMMC-7721和Hep-G2全二维细胞膜-整体柱色谱联用技术筛选中药鸦胆子的抗癌活性物质

目的筛选中药鸦胆子中的抗癌活性成分。方法同时使用全二维色谱技术和细胞膜色谱技术联用质谱技术。结果经初步分离筛选,得到了腺苷和鸦胆子苦素B两种潜在的抗癌活性成分。结论该方法对中药中活性成分的筛选,具有快速、高效和在线的特色和优点。     

用CD40高表达细胞膜色谱模型筛选抗动脉粥样硬化中药的活性成分

目的：细胞膜色谱（Cell Membrane Chromatograph。CMC）技术是一种研究药物与靶体（包括受体）相互作用的亲和色谱分析技术。它是将活性细胞膜固定在特定载体表面,制备成细胞膜固定相（CMSP）,用液相色谱的方法研究药物或化合物与固定相上细胞膜及膜受体的相互作用。所以特别适用于中药药物有效部位及活性成分的筛选。但是CMC法使用的自然生物膜中存在多种活性蛋白,化合物可能与多种受体或通道蛋白结合,所以缺乏特异性、靶向性。而CD40-CD40L是As疾病相关基因,阻断CD40-CD40L炎症信号通路为抗As药物作用靶点,因此建立CD40高表达的CMC模型,其方法更具有应用价值。我们建立了CD40高表达细胞膜色谱模型,对经典抗As中药丹参进行特异性、靶向性筛选,以期发现其活性抗动脉粥样硬化成分。从而为中药筛选提供了方法和途径,为中药现代化提供了新思路。方法：构建CD40高表达的内皮细胞（ECV-304）,制备细胞膜固定相,应用CD40高表达的内皮细胞膜色谱模型筛选丹参中抗动脉粥样硬化的有效成分。     

HEK293α1A、HEK293α1B细胞膜色谱系统的构建及其与配体的细胞膜色谱研究

目的：作为一种新型生物亲和色谱技术，细胞膜色谱（Cell membrane chromatography，CMC）始创于1996年。细胞膜固定相（The cell membrane stationary phase，CMSP）由多孔硅胶包被生物活性细胞膜组成。本研究了细胞膜色谱系统中受体和配体之间的相互作用，尤其是评价了细胞膜色谱法用于亚型受体研究的精确性和可行性。提供受体高表达HEK293α1A、HEK293α1B。细胞膜色谱系统和技术路线，为肾上腺素受体亚型高选择性配体的研究提供新的方法。     

细胞膜色谱与HPLC-ESI-IT-TOF-MS二维在线联用筛选苍耳子中抗过敏组分

目的建立细胞膜色谱在线联用高效液相色谱质谱方法筛选苍耳子提取物中的抗过敏组分。方法采用MrgprX2-HEK293细胞膜色谱模型,二维在线联用HPLC-ESI-IT-TOF-MS系统对苍耳子中抗过敏组分进行筛选鉴定,并利用β-氨基己糖苷酶释放及组胺释放实验验证所筛出成分的抗过敏活性。结果利用所建立的方法从苍耳子筛选出的潜在抗过敏组分为噻嗪双酮苷,β-氨基己糖苷酶及组胺释放实验发现噻嗪双酮苷在一定浓度范围对由C48/80引起的LAD2细胞的β-氨基己糖苷酶及组胺释放起到抑制作用,并呈剂量依赖性,验证了噻嗪双酮苷的抗过敏活性。结论成功构建了Mrgpr X2受体的细胞膜色谱在线联用HPLC-ESI-IT-TOF-MS系统,利用此系统筛选出的抗过敏组分噻嗪双酮苷具有一定的抗过敏活性,所建立的细胞膜色谱与HPLC-ESI-IT-TOF-MS二维在线联用技术为抗过敏药物的开发提供了新的思路。     

膜活性多肽(MAPs)的抗菌活性及其作用机制的研究进展

膜活性多肽(MAPs)是一类具有较好抗菌活性的抗菌肽。作为先天宿主防御分子,广泛的分布于细菌、植物、无脊椎动物、脊椎动物中。膜活性多肽具有抗菌肽的结构特征,肽链通常较短,带正电荷,具有两亲性的α-螺旋或β-折叠结构,通过破坏膜的通透性杀死细菌、真菌和部分肿瘤细胞。膜活性多肽在细胞膜或细胞内部存在特定的分子靶点,并因其独特的作用机制而成为一种新型的肽类抗生素。本文主要对膜活性多肽的抗菌活性及其作用机制的研究现状和发展情况做一综述。     

利用高分辨质谱结合生物信息学分析预测注射用心肌肽中的生物活性肽

目的:基于高分辨率质谱多肽鉴定技术，分析注射用心肌肽中多肽类物质组成，并采用生物信息技术预测多肽的生物活性。方法:注射用心肌肽净化除盐后，以Orbitrap Fusion Lumos三合一高分辨质谱系统对混合多肽进行鉴定，利用BIOPEP-UWM数据库工具预测肽兰克评分≥0.80的肽段的潜在生物活性。结果:注射用心肌肽经分析鉴定出799条肽段，分别来源于70种蛋白质，主要为肌动蛋白、血红蛋白亚基α、ADP/ATP移位酶3等。肽兰克评分≥0.80的10条肽段中具有丰富的血管紧张素转化酶抑制活性(ACEi)、二肽基肽酶-4抑制活性(DPP4i)及抗氧化活性(AO)氨基酸片段。结论:超高效液相色谱-高分辨质谱联合技术及生物信息技术为多组分生化药分析鉴定及活性预测提供便捷手段。注射用心肌肽的心肌保护等作用的物质基础可能源于其中的多肽影响血管紧张素转化酶、抗氧化及二肽基肽酶-4等多种生物活性。     

蛇毒膜毒素抗肿瘤机制的研究进展

蛇毒是从毒蛇的毒腺中分泌出来的一种毒液,主要成份为蛋白质和多肽。其中的膜活性多肽（membrane active polypeptide）是一种碱性蛋白,其毒性通过破坏细胞膜而实现，又被称为膜毒素（membrane toxin，MT）。因膜毒素具有多种生物学活性，是一类对心脏具有毒性又有抗癌活性的组份，又被称作心脏毒素（cardiotoxin，CTX），[第一段]     

Cardiotoxicity of Kenyan green mamba (Dendroaspis angusticeps) venom and its fractionated components in primary cultures of rat myocardial cells

The cardiotoxic actions of Kenyan green mamba (Dendroaspis angusticeps) venom have been investigated using primary myocardial cell cultures isolated from neonatal rat hearts. The cardiotoxic actions of the whole venom and its fractionated components were evaluated on the basis of leakage of lactate dehydrogenase (LDH), changes in morphology, cell membrane lysis, decreases in viability and inhibition of spontaneous beating activity. The whole venom caused time- and concentration-dependent arrest of myocardial contraction, leakage of LDH, extensive disruption of cell monolayer, and decreases in viability. The venom was separated into 6 (DaI to DaVI) fractions by gel permeation chromatography on Sephadex G-50. Spontaneous beating activity was abolished by DaI to DaVI at high concentrations, while at lower doses they induced progressive depression of beating frequency after a 3-h treatment period. DaI to DaIV caused significant leakage of LDH, morphological damage, and decreases in viability after a 6-h incubation period. The most cardiotoxic fraction (DaIV), which also contains about 54% of the total protein of the whole venom, was fractionated into 18 polypeptides (Da1 to Da18) by ion exchange chromatography on Bio-Rex 70. On the basis of their ability to abolish myocardial contractility, release LDH, alter cellular structure, lyse cell membranes and reduce viability, the 18 fractions have been divided into 4 arbitrary subgroups of cytotoxins: cardiotoxins, Da1 to Da3; cardiotoxin-like polypeptides, Da4 to Da12, Da14; less active membrane lytic polypeptides, Da13, Da15 to Da17; and membrane lytic polypeptide, Da18. Marked synergistic cell membrane lysis occurred in myocardial cell cultures treated simultaneously with 2 cardiotoxin-like polypeptides, Da7 and Da11. It is suggested that the additive and synergistic cardiotoxic effects of high molecular weight cytotoxic proteins (DaI to DaIII), very low molecular weight cholinomimetic substances (DaV to DaVI) and the 4 subgroups of cardiotoxins may directly contribute to the pronounced cardiovascular problems observed in victims of green mamba bites.     

地锦草有效部位抗真菌作用及其机制研究

目的初步研究维吾尔药材地锦草有效部位体外抗真菌作用及其机制。方法采用透射电镜观察地锦草有效部位对红色毛癣菌超微结构的影响;高效液相色谱法研究地锦草有效部位对真菌细胞膜麦角甾醇生物合成的影响。结果透射电镜下可见,真菌细胞壁不完整,厚薄不均且局部有缺损,细胞膜轮廓不清,局部有破损,胞内细胞器损伤严重,多呈空泡化,细胞内成分聚集成电子密度较高的团块;高效液相色谱法检测,红色毛癣菌中细胞膜中麦角甾醇含量与其生长对照菌相比,含量降低,差异有显著性,并且有明显的剂量依赖性。结论地锦草有效部位作用于红色毛癣菌后,可使其形态和超微结构发生明显的改变,通过影响真菌细胞膜麦角甾醇的生物合成发挥抗真菌作用。     

SH-SY5Y细胞膜固相色谱法的建立及其对远志皂苷碱水解产物效应物质的研究

目的建立SH-SY5Y细胞膜固相色谱法,并运用该方法分析远志皂苷碱水解产物中的活性成分。方法应用SH-SY5Y细胞膜作为固定相选择性地结合远志皂苷碱水解产物中的活性成分,排除未与细胞膜靶点结合的非活性成分后,使细胞碎裂,被结合的活性成分释放出来,样品经SPE固相萃取处理后用HPLC对这些成分进行分析。结果远志皂苷碱水解产物中的活性成分初步确定为4个,分别为细叶远志皂苷、黄花倒水连皂苷A、3,4,5-三甲氧基肉桂酸和对甲氧基肉桂酸。结论细胞膜固相色谱法基本可以反映化合物与细胞膜生物靶点的相互作用,该体系中的保留成分和其药理作用有显著的相关性。     

Tests of some peptides isolated from residue after insulin isolation from the pancreas. II. Establishment of heterogeneity level

Heterogeneity of the active substance of Lipotropin-Polfa preparation was investigated by the method of gel column chromatography. The isolated polypeptide hormone has been found to possess the average molecular weight nine times smaller than that reported in the literature. This polypeptide was separated into six fractions by chromatography on Sephadex G-10. These fractions were found to differ in respect of the qualitative and quantitative amino acid composition. With the purpose of comparison, an analogous study of Lipormone-Labor. Choay (France) was performed. Similarity of this preparation to fraction I of Lipotropin-Polfa has been shown.     

Study of mangiferin-receptor affinity by cell membrane chromatography using rat pancreas

The aim of this study was to ascertain affinity binding of antidiabetic drug (mangiferin) with cell membrane protein receptor by frontal affinity chromatography. The cell-membrane stationary phase (CMSP) was prepared by immobilizing rat islet pancreas cell membrane on the surface of silica. The obtained CMSP had enzymatic activity and stability. The dissociation constant K _d and the amount of active binding sites (B _t) for mangiferin on cell membrane were determined using frontal chromatography. The calculated K _d values was 400.2?nM and the average B _t value was 22.68?±?7.30?μmol bed^?1. These results suggested that mangiferin possessed strong affinity with membrane protein and abundant binding sites on cell membrane.     

A high expression EGFR/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Rhizoma Polygoni Cuspidati

The epidermal growth factor receptors (EGFRs) in some tumor cells are significant targets for drug discovery. In this work, we have developed an EGFR cell membrane chromatography and online high performance liquid chromatography/mass spectrometry system for screening active component from Rhizoma Polygoni Cuspidati. As a result, resveratrol from Rhizoma Polygoni Cuspidati was found to be the active component acting on EGFR like gefitinib. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. The EGFR/CMC-online-HPLC/MS system demonstrated fast and effective characteristics for screening leading compounds from traditional Chinese medicine.     

Cytotoxicity of Corylifoliae fructus. I. Isolation of the effective compound and the cytotoxicity

The ethanol extract of Psoraleae Fructus (Psoralea corylifolia L.) was found to have cytotoxic activity against L929-cells in cell culture. The active compound was isolated by column chromatography on silica gel and identified as bakuchiol by means of spectral evidence. The cytotoxic activity of bakuchiol in cell culture was observed in short time and found to be unreversible. The mechanism of the cytotoxic activity was considered to be due to an injury of cell membrane from electron microscopic observation and hemolytic activity.     

Some biochemical characteristics and cell membrane actions of a toxic phospholipase A2 isolated from the venom of the pit viper Agkistrodon halys (Pallas)

A toxic component (AgTx) from the venom of Agkistrodon halys (Pallas) was isolated using DEAE-cellulose DE11 and CM-Sephadex C50 column chromatography and finally purified to homogeneity by FPLC on a MonoQ column. The toxin is a neutral (pI 6.9) single chain polypeptide with a mol. wt of 14,000 and an amino acid composition (123 residues) roughly similar to that of notexin. AgTx was found to have phospholipase A2 activity which was dependent on calcium and stimulated by sodium deoxycholate. The toxin caused efflux of 2-deoxy-(1-3H)-glucose-6-phosphate (a cell membrane integrity probe) as well as of [3H]acetylcholine from rat brain synaptosomes. No cell membrane damage was induced by AgTx on cultured N1E 115 neuroblastoma cells and chick myotube cultures. The LD50 ws 150 micrograms/kg (i.p.) in mice. The main symptom observed was respiratory paralysis. The results obtained show that AgTx can be classified as a toxic phospholipase A2 with a presynaptic site of action.     

Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum.

Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P(app) values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and alpha-CHC (alpha-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium. One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor alpha-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.     

A tetravalent RGD ligand for integrin-mediated cell adhesion

Monovalent RGD (arginine-glycine-aspartic acid) peptides or polymers furnished with RGD in random distributions are employed as cell-scaffolds and gene delivery vehicles. However, integrin binding to RGD is dependent on the spatial distribution (clustering) of the ligand and intrinsic integrin affinity via conformational changes (avidity). Here we have designed and expressed a polypeptide consisting of a tetrameric coiled coil and spacer facilitating polyvalent (clustered) display of integrin ligands; the RGD motif was used as proof of principle. Size-exclusion chromatography and circular dichroism showed that the polypeptide self assembled as a tetramer in solution with a defined secondary structure. Cell adhesion to surfaces coated with the polypeptide was up to 3-fold greater than that for (monovalent) RGDS peptide at equivalent concentrations. Moreover, the polypeptide in solution at concentrations >or= 1 microM inhibited cell adhesion to fibronectin-coated surfaces, while RGDS peptide in solution at concentrations up to 500 muM did not. These cell data demonstrate that the polypeptide bound integrin receptors in a polyvalent manner. The polypeptide will therefore be of use in the engineering of tissue-culture scaffolds with increased cell adhesion activity, or to targeted gene delivery vehicles, and could incorporate protein ligands in place of the RGD motif.