Research On New Methods In Liquid Chromatography And Its Application On Analysis Of Bioactive Compounds In Traditional Chinese Medicines

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 (R² =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/H₂O mobile phase and C18-stationary phase (ODS) with ACN/H₂O 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.