模拟体内微环境筛选与肿瘤细胞作用的中药活性成分

利用三维(3D)细胞反应器模拟体内微环境,建立了一种与肿瘤细胞作用的活性分子的筛选和分析方法.利用药物与三维细胞反应器中活肿瘤细胞和固化肿瘤细胞分别作用后的HPLC生物指纹谱峰面积之间有无显著性差异,建立了与细胞结合的活性成分的筛选识别模型.已知抗肿瘤药物紫杉醇和白藜声醇的谱峰均具有显著性差异,而非抗肿瘤药物酮洛芬和青霉素G的谱峰均没有显著性差异,证明利用该模型筛选识别与细胞结合的活性成分是可行的.此外,应用该模型从中草药桃儿七提取物中筛选出了7种可作用于Lovo细胞的活性成分.此研究提供了一种模拟体内微环境下与肿瘤细胞作用的活性成分的筛选和分析方法,在药物发现环节,特别是中草药活性成分研究中具有潜在的应用价值.     

靶向亲和-液相色谱-质谱联用技术快速筛选黄藤总生物碱中乙酰胆碱酯酶抑制剂

采用靶向亲和-液相色谱-质谱联用技术(Target molecule affinity-LC-ESI-MSn)快速筛选黄藤总生物碱中能够抑制乙酰胆碱酯酶活性的成分,共筛选出12种具有潜在抑制乙酰胆碱酯酶的活性成分,并鉴定了6种成分,分别为黄藤素(Palmatine)、小檗碱(Berberine)、药根碱(Jatrorrhizine)、巴马汀红碱(Palmatrubine)、7,8-二氢-8-羟基小檗碱(7,8-Dihydro-8-hydroxyberberine)、Groenlandicine,结合体外酶学实验对这6种化合物进行了活性验证实验.结果表明,黄藤素抑制活性最强,其抑制作用强于阳性对照药盐酸多奈哌齐,说明黄藤素具有开发成抗阿尔茨海默症药物的潜力.本方法简单、快速、准确地从复杂的中药提取物中筛选出具有抑制乙酰胆碱酯酶活性的成分,适用于复杂体系中的高通量筛选.     

基于化学指纹图谱和抗血小板聚集效价的丹参质量评价

通过化学分析和生物活性评价考察丹参药材的品质差异,探讨丹参抗血小板聚集生物活性的主要贡献成分.采用高效液相色谱(HPLC)技术建立丹参药材HPLC指纹图谱,以抗血小板聚集相对效价作为指标,评价不同产地不同批次丹参药材的品质差异,构建基于化学表征及生物效价测定的评价模式.结果表明,不同批次丹参药材的HPLC指纹图谱相似度很高(相似度0.930~0.998),而其抗血小板聚集相对效价相差10倍,提示化学指纹图谱难以反映丹参的活性和质量差异.通过化学指纹图谱与抗血小板聚集生物效价进行谱效相关分析,筛选出与生物活性相关系数大于0.5的6个色谱峰:二氢丹参酮Ⅰ、隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA及2个未知化合物.对上述4种已知化合物单体进行活性验证发现,隐丹参酮的抗血小板聚集活性最强,而其它3种丹参酮类化合物几乎没有体外抗血小板聚集活性.进一步比较丹参中高含量成分丹酚酸B与低含量成分隐丹参酮的活性贡献,结果表明,两者的活性贡献基本相当,说明隐丹参酮是丹参中低含量高活性成分,对评价丹参质量具有重要贡献度.     

靶细胞提取和高效液相飞行时间质谱联用分析预测丹参中的活性成分

建立靶细胞提取和与高效液相飞行时间质谱联用(HPLC-ESI/TOFMS)分析相结合进行丹参活性成分研究的方法,对丹参中可能的活性成分进行了推测。将靶细胞和丹参样品一起孵育培养,根据活性成分可以与靶细胞膜有特异性的结合或者进入靶细胞内的原理,用HPLC-MS方法对培养后细胞破碎液中的成分进行分析。从加药Raw264.7细胞和Ecv304细胞破碎液中检测到丹参素、原儿茶醛、咖啡酸、丹酚酸D、紫草酸、迷迭香酸及丹酚酸B7种丹参中成分,在加药的HL7702细胞破碎液中检测出丹参素、原儿茶醛、咖啡酸及丹酚酸B4种丹参中的水溶性成分。结果显示:细胞破碎液中检测的成分为丹参在靶细胞中有吸收的成分,本方法可用于预测中药中的活性成分。     

3种色谱模式联用在中药活性成分初步筛选中的应用

提出了反相高效液相色谱(RP-HPLC)、固定化脂质体色谱(ILC)以及固定化载体蛋白色谱(ICPC)3种色谱模式联用筛选中药中活性成分的新思路，并用于传统中药川芎中的生物活性成分的初步筛选。从川芎的甲醇提取液中筛选出几种既有细胞膜的穿透能力又有与载体蛋白的结合能力的成分，并对其中两种主要的组分进行了初步的结构鉴定。     

直观推导式演进特征投影法测定中药丹参中3种丹参酮含量

利用化学计量学方法直观推导式演进特征投影法和高效液相色谱(二级管阵列检测器)产生的二维数据,解析丹参药材中重叠色谱峰,并对其中的活性成分-丹参酮ⅡA、隐丹参酮和丹参酮Ⅰ进行测定。该方法能够快速测定复杂体系中药内的活性成分,显著提高分析效率。     

中草药活性成分的高通量筛选技术研究进展

随着高通量筛选技术的不断发展,该技术已经成为发现新药物的重要途径之一。高通量筛选技术已大量应用于筛选药物活性成分的领域中,但是其中大部分为从化合物库中筛选活性成分,仅有十几篇文献应用于中药活性成分的筛选,而中国传统中草药却是探索和发展新药物的丰富来源。本文通过综述国内外2008年到2017年的相关文献,阐述了分子和细胞水平上的高通量筛选技术在中草药活性成分的筛选及其应用进展,为今后中草药新药研发提供参考。     

用高表达EGFR细胞膜色谱-HPLC/MS联用快速筛选独活中抗EGFR活性成分

报道了用高表达表皮生长因子受体细胞膜色谱与高效液相色谱/质谱在线联用方法(EGFR/CMC-online-HPLC/MS)快速筛选发现中药独活中的活性成分.实验中,采用高表达EGFR的细胞膜制备色谱固定相,建立EGFR/细胞膜色谱(EGFR/CMC)模型,利用柱切换和固相萃取技术,将EGFR/CMC模型与高效液相色谱/质谱(HPLC/MS)在线联用,构成一种新的可同时"识别-鉴定"目标成分的二维色谱系统,并应用于快速筛选独活中具有抗EGFR活性的目标成分.结果发现独活中的蛇床子素具有与对照药物达沙替尼类似的色谱保留特性,能够作用于EGFR;同时MTT及Elisa分析实验证实蛇床子素对HEK293EGFR细胞增殖及EGFR表达均有抑制作用.本文建立的EGFR/CMC-online-HPLC/MS二维色谱方法,可以选择性地从中药复杂体系中快速"识别-鉴定"目标组分,且筛选结果与特定生物效应显著相关.     

体外细胞模型和高效液相质谱联用分析预测黄芪中的活性成分

利用体外细胞模型模拟体内细胞对中药有效成分的特异性吸收,结合高效液相色谱/质谱分析筛选中药黄芪中的生物活性成分。将中药黄芪提取液与Caco-2细胞及红细胞分别混合培养,破碎与药材结合后的细胞,使之释放出结合的药材中的成分。运用高效液相色谱/飞行时间质谱(HPLC-ESI/TOFMS)分析中药黄芪提取液与活性细胞有结合的成分,并对其进行结构鉴定。结果显示:黄芪中有10个化合物与Caco-2细胞结合,14个化合物与红细胞结合。本方法可用于预测口服药物在体内的吸收以及与特定靶细胞的结合情况,特异性地筛选中药复杂体系中的药效物质基础。     

丹参酮ⅡA和丹参酮Ⅰ的电子轰击与电喷雾电离质谱分析

采用电喷雾电离质谱(ESI-MS)和电子轰击质谱(EI-MS)两种质谱技术分别对传统中药丹参的主要脂溶性活性成分丹参酮ⅡA和丹参酮Ⅰ的化学结构和裂解途径进行系统研究。采用EI-MS从丹参酮ⅡA获得m/z 294[M] 、279、261、233、207等特征质谱峰,从丹参酮Ⅰ获得m/z 276[M] 、248、233、219、205等特征质谱峰;采用ESI-MS从丹参酮ⅡA获得m/z 295[M H] 、280、278、262、249等特征质谱峰,从丹参酮Ⅰ获得m/z 277[M H] 、259、249、231、221、193等特征质谱峰,并用Mass Frontier 3.0软件辅助解析了其中的主要特征碎片离子以及可能的裂解途径;比较了丹参酮ⅡA和丹参酮Ⅰ的电喷雾电离质谱和电子轰击质谱裂解规律,本研究为研究丹参二萜醌类主要特征活性成分的生物转化与结构修饰提供了依据。     

Primary study on the application of Serum Pharmacology in Chinese traditional medicine

In the paper, two main methods, which are Serum Pharmacology and Traditional Pharmacology, were adopted to study Chinese traditional medicine, such as Ginkgo biloba extract (GBE), ginsenosides (GS) and compound GG (GBE + GS), pharmacology in vitro. The results showed that there were evident difference between the results of Serum Pharmacology and that of Traditional Pharmacology. There was no significant difference between the drug effect of crude GS on nitric oxide (NO) production in ECV304 and that of crude GBE, and the drug effect of GG was superior to that of GS and GBE, respectively. But, compared with GBE serum, the GS serum up-regulation of NO production in ECV304 increased significantly, and the GG serum up-regulation of the NO production in ECV304 was inferior to that of GS serum and GBE serum significantly. The results suggested that Serum Pharmacological study should be adopted in the pharmacological investigation on the Chinese traditional medicine and the drug screening of the Chinese traditional medicine.     

Characterization of a microfluidic in vitro model of the blood-brain barrier (μBBB)

The blood-brain barrier (BBB), a unique selective barrier for the central nervous system (CNS), hinders the passage of most compounds to the CNS, complicating drug development. Innovative in vitro models of the BBB can provide useful insights into its role in CNS disease progression and drug delivery. Static transwell models lack fluidic shear stress, while the conventional dynamic in vitro BBB lacks a thin dual cell layer interface. To address both limitations, we developed a microfluidic blood-brain barrier (μBBB) which closely mimics the in vivo BBB with a dynamic environment and a comparatively thin culture membrane (10 μm). To test validity of the fabricated BBB model, μBBBs were cultured with b.End3 endothelial cells, both with and without co-cultured C8-D1A astrocytes, and their key properties were tested with optical imaging, trans-endothelial electrical resistance (TEER), and permeability assays. The resultant imaging of ZO-1 revealed clearly expressed tight junctions in b.End3 cells, Live/Dead assays indicated high cell viability, and astrocytic morphology of C8-D1A cells were confirmed by ESEM and GFAP immunostains. By day 3 of endothelial culture, TEER levels typically exceeded 250 Ω cm(2) in μBBB co-cultures, and 25 Ω cm(2) for transwell co-cultures. Instantaneous transient drop in TEER in response to histamine exposure was observed in real-time, followed by recovery, implying stability of the fabricated μBBB model. Resultant permeability coefficients were comparable to previous BBB models, and were significantly increased at higher pH (>10). These results demonstrate that the developed μBBB system is a valid model for some studies of BBB function and drug delivery.     

Development of a blood-brain barrier model in a membrane-based microchip for characterization of drug permeability and cytotoxicity for drug screening

Since most of the central nervous system (CNS) drug candidates show poor permeability across the blood-brain barrier (BBB), development of a reliable platform for permeability assay will greatly accelerate drug discovery. Herein, we constructed a microfluidic BBB model to mimic drug delivery into the brain to induce cytotoxicity at target cells. To reconstitute the in vivo BBB properties, human cerebral microvessel endothelial cells (hCMEC/D3) were dynamically cultured in a membrane-based microchannel. Sunitinib, a model drug, was then delivered into the microchannel and forced to permeate through the BBB model. The permeated amount was directly quantified by an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS) after on-chip SPE (μSPE) pretreatment. Moreover, the permeated drug was incubated with glioma cells (U251) cultured inside agarose gel in the downstream to investigate drug-induced cytotoxicity. The resultant permeability of sunitinib was highly correlated with literature reported value, and it only required 30 min and 5 μL of sample solution for each permeation experiment. Moreover, after 48 h of treatment, the survival rate of U251 cells cultured in 3D scaffolds was nearly 6% higher than that in 2D, which was in accordance with the previously reported results. These results demonstrate that this platform provides a valid tool for drug permeability and cytotoxicity assays which have great value for the research and development of CNS drugs.     

Co-culture Based Blood-brain Barrier In Vitro Model,a Tissue Engineering Approach using Immortalized Cell Lines for Drug Transport Study

This study evaluated the feasibility of using commercially available immortalized cell lines in building an in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Astrocytes-derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel biomimetic basement membrane for the endothelial cells to form tight junctions. Trans-Endothelial Electrical Resistance (TEER) and solute mass transport studies quantitatively evaluated the tight junction formation. Immuno-fluorescence microscopy and Western blot analysis qualitatively verified the expression of occludin, one of the tight junction proteins on the samples. Experimental data from a total of 13 experiments conclusively showed that the novel BBB in vitro co-culture model with aECM (CO + aECM) is promising in terms of establishing tight junction formation represented by TEER values, transport profiles, and tight junction protein expression when compared with traditional co-culture (CO) model setup or the endothelial cells cultured alone (EC). In vitro colorimetric sulforhodamine B (SRB) assay also revealed that the “CO + aECM” samples resulted in less cell loss on the basal sides of the insert membranes than traditional co-culture models. Our novel approach using immortalized cell lines with the addition of aECM was proven to be a feasible and repeatable alternative to the traditional BBB in vitro modeling.     

On-line coupling of hollow fiber membranes with electrospray ionization mass spectrometry for continuous affinity selection, concentration and identification of small-molecule libraries.

Combinatorial chemistry has been widely employed in the pharmaceutical industry in the effort towards drug discovery. Rapid and sensitive screening of lead candidates among library compounds has thus imposed significant analytical challenges in recent years. This work involved the development of a continuous affinity capture and concentration system, providing cost-effective and structural analysis of drug candidates in a flow-through format. The system combines the strengths of a hollow fiber dialysis membrane of ease and speed of purification and concentration with the specificity of affinity interactions in solution. The complexes between the lead compounds and the affinity binding proteins are separated from other chemical components inside a dialysis hollow fiber as the result of their differences in size. The affinity complexes are further concentrated inside a second dialysis fiber. The concentrated drug candidates are liberated from the binding proteins in a microdialysis junction and can be directly identified using electrospray ionization mass spectrometry. Two model systems, including human serum albumin-warfarin-related compounds and anti-phenobarbital antibody-barbiturates, were employed for mechanistic studies of dialysis versus dissociation kinetics and competitive selection of drug candidates according to their binding strengths.     

Rapidly identify compounds from danshen by using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer and predict its mechanisms of intervening thrombotic diseases

Danshen, having good efficacies of dilating the coronary artery, improving microcirculation and preventing thrombosis, is widely used in clinic to treat various thrombotic diseases in China. This study aimed to identifying and analyzing the chemical constituents of Danshen from different planting areas based on UHPLC-LTQ-Orbitrap MS and comprehensively predicting the potential pharmacological targets and pathways of the main chemical constituents that involve in the treatment of thrombotic diseases by using network pharmacology. As a result, we identified 870 single peaks from total ion current and accurately identified 19 compounds from Danshen in this study. Further analysis showed that these compounds had three types of accumulation patterns and contents of most compounds had significant differences in samples from different planting areas. Network pharmacological analysis showed that Tanshinone IIA, Tanshinone IIB, Dehydromiltirone, Cryptotanshinone, Danshenxinkun A, Danshenxinkun B and Neocryptotanshinone might intervene thrombotic diseases by adjusting the targets mainly involved in pathways of endocrine system, signal transduction, signaling molecules and interaction, cell motility, environmental adaptation and nervous system. This study provides a scientific basis for exploring the mechanism of Danshen in treating thrombotic diseases and controlling the quality of Danshen-related preparations.     

一种基于液相色谱-质谱技术进行血清代谢组学研究的方法：从代谢指纹到潜在标志物

本文描述了一种基于液相色谱-质谱技术（LC-MS）的代谢组学发现疾病潜在标志物的方法.该方法利用LC-MS获得代谢指纹图谱,并通过多种统计分析方法对产生的海量数据进行分析,最终筛选出潜在标志物.数据分析过程包括：通过归一化、修正80%规则、数据集分割和数据缩放等方法对数据集进行预处理 通过正交校正的偏最小二乘（OPLS）模式识别方法对样品进行分型 根据模型的变量重要性因子（VIP值）、非参数检验结果和z值筛选潜在标志物.以宫颈癌血清样本为例,应用上述方法,15个变量被确认为潜在标志物,操作者接受曲线（ROC）下的面积为0.667～0.956.经过相关性分析和结构鉴定,发现这15个变量来自9个化合物.其中7个化合物被鉴定为色氨酸、硬脂酸、花生四烯酸、溶血磷脂酰胆碱（0：0/16：0,16：0/0：0,18：1/0：0和18：0/0：0）,说明在宫颈癌中花生四烯酸和溶血磷脂酰胆碱的代谢发生异常.     

HI-6人体血清白蛋白纳米粒的制备及其对梭曼中毒小鼠脑AChE重活化作用评价

迅速恢复中毒乙酰胆碱酯酶(acetylcholinesterase,AChE)活性是神经性毒剂中毒救治最关键的环节.血脑屏障的存在限制了具有重活化作用的抗毒药物以有效治疗剂量进入中枢.本研究以已知重活化作用最强的重活化剂酰胺磷定(HI-6)为目标药物,通过去溶剂化法制备了人体血清白蛋白纳米粒,通过静电作用将HI-6分子装载在纳米粒表面,合成装载HI-6的人体血清白蛋白纳米粒;在斑马鱼及神经性毒剂梭曼染毒小鼠上,对药物穿透血脑屏障能力及中枢中毒AChE重活化作用进行了评价.结果表明,装载HI-6人体血清白蛋白纳米粒在物理表征上符合纳米药物基本特征;相对于自由HI-6,HI-6人体血清白蛋白纳米粒能够透过血脑屏障,并将中枢中毒AChE活性提升2倍以上,表明人体血清白蛋白纳米粒成功携带HI-6分子进入中枢.本文建立了一种无毒、高效、小尺寸中枢靶向性纳米粒的制备方法,基于该方法制备的纳米重活化剂,在脑内可有效释放目标药物并迅速发挥解毒作用.