摘要
随着中药现代化进程的不断前进,中药药效的物质基础、药理学、药物代谢过程及相关的生物学、药物的质量控制等等方面的研究越来越成为中药现代化的关键问题。运用现代分析仪器,发展高效的分离分析手段和方法,为解决此类问题提供了一个契机。色谱作为一种现代分离分析手段,在中药的开发和研究方面发挥了非常重要的作用,尤其是在中药有效成分的定性定量分析及质量控制上。但是中药成分复杂,结构多样性、含量差别等等干扰分析的因素诸多。单纯采用单一色谱系统,不能提供中药全面的信息。利用多种色谱模式的串联,以及其与其他化学、生物技术的有效结合,可以从不同角度探讨中药的化学组成,以及相关的药效关系,更好地解决中药复杂体系的分离与分析,并为高通量筛选中药中有效组分提供物质依据。
本论文主要发展和研究了以色谱分离技术为基础的多种分离分析模式,包括建立纳升液质联用系统用于中药中标准物质的质量分析;优化多维色谱系统用于中药分离;以两维色谱为基础结合体外酶模型和细胞模型查找相关的有效成分;利用新型材料固定靶酶,通过色谱/质谱等技术检测固定酶的活性并运用于相关抑制剂的筛选。具体的研究工作和成果的摘要如下:
第一章作为前言简单回顾了中药现代化的发展,阐述了中药药效研究的基本思路和策略;综述了中药研究中的各类化学分析方法;重点描述了色谱在中药研究中的重要作用和近几年来的发展,以色谱分析前的预处理提取方法为始,举例说明了新型色谱技术在中药定性定量剖析、复杂体系分离分析以及高通量药物筛选中的研究和应用。同时也概述了其他新技术新方法与色谱分析技术结合在中药组分分析中的新进展和应用。通过现状描述和前景评估,阐明了本课题选题和研究的学术背景和技术意义。
第二章主要研究和考察了色谱分析技术在中药质量控制中的应用,建立了纳升级液相色谱质谱联用系统,利用色谱的高效分离能力和质谱的灵敏性,用于中药复杂体系的定性定量分析。此前,关于液质联用系统用于中药有效成分定性定量分析的报道较少,尤其是低浓度中药的痕量物质分析。以金银花为例,我们首次使用纳升色谱/质谱联用(nano-LC/MS)鉴定和定量中药中的标准物质。比较色谱保留时间和质谱结构解析的信息,快速专一地确认金银花中标准物质绿原酸的定性信息;通过对绿原酸标准曲线的制作,建立绿原酸外标法的定量标准。作为分离前预处理方法的优化,考察了不同提取方法,包括微波辅助萃取的新方法,常规提取方法,如回流萃取,超声萃取等对金银花中绿原酸的萃取效率。比较显示微波辅助萃取能够在较短时间内,即4分钟之内,提供较好的提取效率。同时,液质联用系统用于定性定量分析的有效性和可靠性也经过考察方法的线性、检测限、准确性和回收率得到确认。结果显示方法的线性范围在0.8-20 ng/mL,相关指数R~2在0.991,检测限为0.5 ng/mL,标准偏差2.54%,回收率84.8%。nano-LC/MS和微波辅助萃取的结合,综合了两者快速分析、减少样品损耗、节约时间、降低试剂成本等优点,可用于中药标准物质的定量和定性评估,尤其对于那些含量较少的痕量物质更能体现其优势。
第三章建立和优化了离线的两维色谱系统用于中药组分的分离,提高了色谱分离的峰容量和分辨力,解决了两维间兼容性问题。首先将中药千层塔在第一维色谱进行初步分离,这里选用分辨率相对较低的CN柱和甲醇/水流动相作为第一维。按照时间搜集各组流分后,再将这些流分通过第二维色谱进行进一步的细分。第二维色谱采用ODS和乙腈/水的反相色谱模式。为了考察这种色谱模式的有效性和兼容性,将不同提取时间和不同产地的千层塔作为目标物进行分离。不同的谱图表现证实了这种离线两维系统的高分辨能力。相对一维色谱,两维色谱系统能可靠地提供中药更多的信息,为药物的鉴定和后期的药效药理研究提供必要的分离基础。
第四章重点开发了以酶为药物靶点的有效组分筛选系统,利用酶的特异性结合和色谱、质谱的高效分离鉴定能力快速有效地确认中药组分中的有效成分,实现了有效成分确定、分离的嫁接。将糖尿病治疗中的关键酶α-葡萄糖苷酶,通过乙醛基团与氨基基团的作用,借助戊二醛的螯合基团固定在氨基磁性纳米微球上。固定化的酶用于底物的水解反应,通过高效液相色谱考察产物产量的变化评估酶的催化活性。与传统的生物检测方法比较,这种预制备的固定化酶在活性的高低和系统稳定性上都有更好的表现,并且能够简单地重复利用10次左右。在确认固定酶的系统稳定性前提下,将固定酶和色谱检测结合的方式用于考察已知临床药物和几味中药对该酶的抑制作用。通过色谱检测产物增加和减少,可以简单直观地评价固定酶的活性和抑制剂的抑制作用的强度。该方法通用于各类蛋白酶活性监测,并且具有产物易分离,成本较低,试剂消耗更有效率的优点。
此外,还选用了治疗老年痴呆症的关键活性酶,乙酰胆碱酯酶作为对象,考察磁性微米硅球固定酶的性能。乙酰胆碱酯酶固定于3-(2,3-环氧丙氧)丙基三甲氧基硅烷(GLYMO)修饰的磁性微米硅球上,与标准底物乙酰胆碱一起反应后,利用磁性将底物与产物快速分离。电喷雾质谱作为快速定性和半定量方法用于测定产物量的变化,并以此作为酶活性的评价标准。比较同一组固定酶的5次重复酶促反应,没有明显的活性变动,固定酶的稳定性得以确认。磁球固定的酶也用于酶抑制剂的筛查,已知乙酰胆碱酯酶抑制剂石杉碱甲和石杉碱乙,与固定酶一起参与酶促水解反应,酶活性的钝化和产物离子峰的改变证明了固定酶和质谱检测在筛选酶抑制剂中的可行性和可靠性。
另外,新型介孔材料FDU-12也用于制作固定酶的微酶反应器。介孔的三维立体有序排列促进了分子的扩散和运输,从而形成了酶浓度增高和底物富集效应。酶反应器的有效应用为发展有效、生物兼容、多功能的纳米反应器用于高通量多靶点药物的筛选和研究提供了基础。
第五章则拓展了多维色谱与生物技术的联用,运用两维色谱的高分辨分离和正交性,简化中药初筛步骤,降低筛选成本。结合酶模型、细胞模型的高特异选择性,不仅可以发现中药中的有效成分,而且还能从抑制酶活性和诱导细胞凋亡两条路径解释中药作用的机理。同时借助多孔板、自动流分收集器、酶标仪等现代自动化、产量化技术,建立中药活性筛选的高通量模型。在酶分子水平上,我们选用了糖尿病代谢中的关键酶α-葡萄糖苷酶模型和离线两维色谱结合,作为两维色谱和酶模型联用的方法研究。首先建立和优化离线两维色谱分离黄芪中的各类组分,用于体外酶解实验,初步确定有效区域。然后将有效区域通过色谱进一步分离,细分后的流分再通过体外酶试验进一步确认更准确的有效物质。最后将已经确认的几个有效物质进行质谱的结构解析,将这些结果与已知标准物进行对比,其中主要为已知的有效成分黄酮和异黄酮类。这是首次将两维色谱与体外生物实验结合筛查中药中的有效成分,充分利用了两维色谱的高分辨率和生物试验的灵敏性。
将这种系统扩展到体外细胞模型上,利用两维色谱分离黄连中的各个流分,然后将其与肝癌细胞一起培养,通过观察细胞的凋亡情况,找到促进肝癌细胞凋亡的有效物质。从细胞水平上更有力地证明了两维色谱分离系统在药物开发和研究中的重要作用。
简言之,本论文的主要工作是考察了色谱/质谱技术在质量控制中的应用,建立了两维色谱分离系统,并首次将其与体外靶酶和细胞模型结合,筛查中药中的有效组分。同时也合成了一些新型材料,固定糖尿病和老年痴呆的关键酶,通过色谱和质谱的定性定量监测,评估酶活性的改变,筛选不同中药中潜在的有效成分。这些方法和系统的建立,有助于提供中药有效成分研究中的新技术新思路。
With the development and modernization of traditional Chinese medicines (TCMs), research on substantial foundation, pharmacology, metabolism process and quality control of bioactive compounds is becoming a key resolution. It offers an opportunity by developing and establishing high effective separation and analysis methods based on the modern analytical instruments and techniques. As a modern measurement technique, liquid chromatography (LC) plays an important role in exploration and development of TCMs, especially in qualification and quantification assessment of their components, which helps quality control of them. But due to the complication of these components, there are various factors interfering the determination and identification, such as diversity in structure, difference in amount. Simple and sole LC may not provide complete and throughout information of TCM. Hyphenating LC with other chemical methods and bioassays may be helpful in probing TCM and understanding their bioactivity through different aspects, then leads to better solution to separation and analysis for TCMs together with substantial foundation of bioactive compounds.
In this study, we focus on research of various separation and analysis modes based on LC, including establishment of nano-LC/MS for quality control and multidimensional LC for bioactive compounds determination. The latter is fundamental for screening bioactive compounds in TCM through target enzyme and cell models in vitro. Meanwhile, a novel immobilized enzyme strategy is created by magnetic microspheres for monitoring enzyme activity and screening inhibitors followed by LC and electrospray mass spectrometry. This dissertation is divided into following five parts.
In chapter one, a simple review of the development of modernization in TCMs is made in order to describe the basic idea and strategy of the research on bioactive compounds in TCMs. A variety of chemical and analytical methods are summarized in details. The description is focused on recent development of LC, comprising extraction methods, examples from new LC and its application in analyzing complex system and rapid screening bioactive compounds. Furthermore, the combination of LC with other techniques and its application on analysis of TCMs is also represented. Under this background, the intention and meaning of this dissertation are explained.
In chapter 2, we study the system of nano-LC/MS and applied this system onto the quality and quantity assessment of chlorogenic acid (CA) in Honeysuckle. The nano-LC/MS takes the advantages of high resolution of LC and high sensitivity of MS. So far, there are limited reports of nano-LC/MS and its application on quantitative evaluation on bioactive compounds in complex TCM system. The chlorogenic acid (CA) in Honeysuckle is determined and identified through nano-liquid chromatography-electrospray mass spectrometry (nano-LC-ESI/MS) by comparison of the intention time and structural information of the authentic standard. Meanwhile, CA is measured with the assistance of the calibration curve of its authentic standard. The sample preparation methods before nano-LC/MS are investigated. As a new sample preparation method for Honeysuckle, the MAE procedure is optimized, validated and compared with conventional methods including reflux extraction (RE) and ultrasonic extraction (USE). It is found that MAE gives the best result due to the highest extraction efficiency within shortest extraction time (only 4 min). The method linearity, detection limit, precision and recovery are studied. The results show that the combined MAE and nano-LC-ESI/MS method has a linearity (R~2 =0.991, 0.8-20 ng/mL), a low limit of detection (0.5 ng/mL), good precision (R.S.D = 2.54%) and a recovery (84.8%). The MAE method followed by nano-LC-ESI/MS determination, which takes comprehensive effect such as rapid analysis, reduced sample, time and solvent consumption, is a simple, rapid, and reliable method for the quality and quantify assessment of CA in Honeysuckle.
In chapter three, a two-dimensional liquid chromatographic method for the study of the total alkaloids in traditional Chinese herb Huperzia serrata is developed. This system improves the peak capacity and resolution of LC separation techniques and meanwhile dissolves the compatible problem. Firstly the extracts are separated on a CN column by using the mixture of methanol and water as the mobile phase. Then the fractions of the effluent are further separated on an ODS column by using the mixture of acetonitrile and water as the mobile phase. This method is used to separate the extracts obtained with different heating time. Also the differences between the herbs in two different areas of China are investigated. It iss found that this method is reliable and can provide more information about the compounds in the herb than the one-dimensional methods. Thus it may be used for future identification and pharmacological studies of the alkaloids in H. serrata.
In chapter four, a novel strategy based on enzyme immobilization for monitor enzyme activity and screening enzyme inhibitors through LC and MS is demonstrated. This is the first combination of bioactive compounds determination, separation and identification. Firstly,α-glycosidase is selected as the target. Through the reaction of the aldehyde groups with amine groups,α-glycosidase is simply and stably immobilized onto magnetic nanospheres by the cross-linking agent glutaraldehyde. In order to profiling the activity of the immobilizedα-glycosidase, the natural substrate is hydrolyzed by it and the yield of product is determined by HPLC. Compared with traditional bioassay approach, the prepared immobilizedα-glycosidase displays a high activity and stability which allows it to be easily reused for 10 times. Enzyme inhibition assays by known inhibitor glucobay and three candidate traditional Chinese medicines (TCMs) are then investigated using a similar methodology. This assay is able to readily detect the change of the immobilized enzyme activity based on measuring a decrease of product formation using HPLC. The approach is general and offers many attractive advantages including easy product isolation, inexpensive cost, and high efficiency in terms of reagent consumption.
Similarly, a technique for screening inhibitors by electrospray mass spectrometry (ESI-MS) with immobilized enzyme on magnetic microspheres has been established. First, the model enzyme acetylcholinesterase (AChE) is immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic silica microspheres. AChE activity is monitored by biochemical assay that is based on mixing of AChE immobilized microspheres and model substrate acetylcholine, separating and detecting the product through ESI-MS. Stability of the enzyme-immobilized microspheres is investigated. No apparent loss of enzyme activity is observed after fivefold reuse of AChE-immobilized microspheres. The enzyme immobilized bioassay is used to effectively identify AChE inhibitors among two standard samples, huperzine A and huperzine B, and their source herbal Huperzia serrata, all of which are spiked into the substrate. The inhibition is determined by measuring a decrease of product formation using ESI-MS.
Additionally, a nanoreactor has been developed to rapidly monitor enzyme activity and screen enzyme inhibitor candidates from complex mixture. Acetylcholinesterase is firstly immobilized onto the 3-glycidoxypropyltrimethoxysilane -modified mesoporous silica FDU-12. The optimization of immobilized enzyme assay is also made in terms of temperature and time. Then, a library standard compound, including previous known enzyme inhibitors is tested to demonstrate the capability of mesoporous silica nanoreactors in screening enzyme inhibitors. Moreover, the enzyme-immobilized nano device is applied to detect the potential inhibitors from the extract of various traditional Chinese medicines. This is an excellent example of using mesoporous silica nanospheres to develop nano-enzymatic reactor for enzymatic studies.
In chapter five, a novel system, two-dimensional liquid chromatography (2D-LC) assisted by bioassay is developed and demonstrated for separating enzyme and cancer cell inhibitory components from traditional Chinese herb. The coupling of biological techniques and multi-LC methods leads to the high throughout screening in TCMs. First,α-glucosidase and Radix astragali are exemplified for 2DLC and enzyme model. Reverse phase chromatographic separation modes, cyano-stationary phase (CN) with MeOH/H_2O mobile phase and C18-stationary phase (ODS) with ACN/H_2O mobile phase, are combined as off-line 2DLC separation to achieve orthogonal resolution due to their selectivity difference. To mark general active regions in 2D chromatogram, 30 fractions from above two separation modes are submitted to post-column enzymatic hydrolysis. Inhibitory ability of each fraction is determined by measuring a decrease of hydrolyzate formation using UV spectrophotometer. Further to separate active components in these active regions, refined chromatographic fractions are separated and again detected by post-column hydrolysis. Several tentativeα-glucosidase inhibitors are identified, subjected to MS/MS analysis and elucidated as flavonoids and isoflavones. This application establishes as a new method for separating bioactive components seems quite effective and simple to apply in R. astragali as well as other TCMs. The similar method is also applied onto screening bioactive compounds through liver cancer cell model. Coptis is firstly separated by same 2DLC system and then fractions are collected and cultivated with live cancer cell 7703. The cell model may support the reliability of the system which is composed of 2DLC and biotechnology.
In summary, the main contributes of this dissertation is that we construct LC/MS for quality control of TCMs and 2DLC for separation and further pharmaceutical study. We firstly hyphenate the 2DLC with enzyme and cell model for detecting bioactive compounds by high throughput screening method. Besides, we also synthesize several functional magnetic materials and successfully utilized them for rapid monitoring enzyme activity and finding enzyme inhibitors. We aim at exploring and finding out new techniques for bioactive compounds research in TCM fields, so that more breakthroughs can be obtained in the TCM modernization.
引文
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