| As one of the greatest achievements, Traditional Chinese medicines (TCMs) play a vital role in history. With the development of modern scientific technology, TCMs have aroused many attentions from all over the world. To understand TCMs, the priority thing is to analysis their bioactive compounds, because it is the bioactive compounds that function a lot in terms of pharmacological effect and metabolic process. And also, bioactive compounds are key targets in TCMs’quality control. As two powerful analytical techniques, liquid chromatography (LC) and mass spectrometry (MS) are very important to the separation and identification of TCMs’ compounds. Bid the aid of LC and MS, the quality control of TCMs would work out well in the aspect of qualification and quantification. However, the intrinsic characteristics of TCMs, especially the Compound TCMs make the analysis of TCMs not that much easy. For example, TCMs tend to have numerous compounds which differ with each other not only in structure but also in amount. Besides, all those uncountable compounds would react with each other, so as to exhibit some co-effects, like synergistic effect. Hence, to get an all-around understanding of TCMs, more comprehensive method or techniques are imperative.In this study, both LC and MS were optimized so as to gain a better perform in terms of bioactive compounds analysis. First, to have a rapid and effective analysis of bioactive compounds from TCMs, a workflow made up of the extraction, nano-LC/MS as well as cell bioassay model in vitro was established; second, on considering the complicated factors in compound TCMs, two mathematical models was designed here, which are the triangle model to investigate the proportion value of Compound TCMs and the additional model to integrate and mimic the synergistic effect, followed by the yew extraction was also utilized here to verify the additional model, and then, the pipeline of multidimensional LC coupled with cell bioassay model as well as electro-spray MS was established here to gain a high throughput screening of the bioactive compounds in Compound TCMs; third, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was introduced here to identify the small compounds from TCMs successfully, which means a lot for the high throughput TCMs screening; last but not least, the brand new MALDI-TOF MS method was also applied to the screening of enzyme inhibitor and long chain fatty acid, which shows great potential in terms of the high throughput screening of small compounds. This dissertation can be divided into the following five parts.In chapter one, a brief review was introduced as to the importance of TCMs analysis. By describing the mostly utilized chemical and analytical techniques, the different LC separation and extraction methodology unfold their respect characteristics gradually. Also, the progresses of mathematical calculation and biological screening models as well as MS technology were also described. Thus, from extraction to separation, from mathematical calculation to bioassay tests, and finally, to MS identification, the overall process for TCMs study presented here. Based on all this awareness, the importance and meaning of this dissertation were illustrated here.In chapter two, the workflow of infrared-assisted extraction (IAE) coupled with nano-LC/MS and cell bioassay test was established to analyze TCMs. Scutellaria barbata D. Don was used as the model TCM in the study, in which scutellarin is the main active component. The extraction of scutellarin in Scutellaria barbata D. Don was carried out in a home-made IAE system, and the IAE parameters were studied. It is found that, as a brand new sample preparation method for Scutellaria barbata D. Don, IAE gives the best result when compared with conventional methods including reflux extraction (RE), ultrasonic extraction (USE) and microwave-assisted extraction (MAE). The high efficiency of IAE can be due to that IR can not only be a heating resource, but also vibrate the inner molecules whether they are polar or non-polar ones. Here, scutellarin was analyzed by nano-LC/MS. The results showed that the combined IAE and nano-LC-MS method exhibits a good linearity (R2=0.9973,0.6-20ng mL-1) and a low limit of detection (0.5ng mL-1, S/N=3). This combinatory method demonstrates itself to be simple, rapid, effective and safe. Besides, fractions were also collected after LC separation and undergone cell bioassay test to screen out the bio-effective fraction, followed by MS identification. And scutellarin demonstrated to be the bioactive components in its original herb, which fully demonstrated the high efficient of the established workflow.In chapter three, apart from the combination of traditional biological and chemical techniques, two innovative mathematical designations were introduced for the first time to compound TCM research, a compound TCM model which consist three kinds of herbs was utilized as the model. Firstly, triangle mathematical mode and cell bioassay model method were tied up to obtain the optimized prescription proportion of compound TCM model; secondly, LC/cell bioassay model/MS pipeline was employed, according to the promoted addition principle, to screen and identify the bioactive compounds from compound TCM model. Compared with other methodologies, this mathematical appended method outstanding itself by simultaneously possessed the advantaged of mathematically simplification, biologically validation as well as high throughput. A reliable result was obtained here after taking the synergistic effect into consideration; thirdly, the yew extraction was introduced here as the check point to verify the addition principle; finally, an offline two dimensional LC system was established here, which demonstrated excellent orthogonality by the satisfying separation of polar and medium-polarity compounds that cannot be resolved by uni-dimensional chromatography, also, the notable cell bioassay model was again utilized to screen the bioactive compounds from compound TCM, so as to provide powerful scientific evidence for the medical effect, followed by the capable MS identification. And reliable result was obtained here, which opened a more executive and valid door for future compound TCM research.In chapter four, graphene or graphene oxide was utilized, for the first time, to identify small molecular components from TCM herbs, by acting as matrix of MALDI-TOF-MS. Due to the large surface area of graphene or graphene oxide, the analytes were trapped tightly to the matrix, which avoids the contamination of the ion source and vacuum system. Besides, their excellent electronic, thermal, and mechanical properties make them desired matrices for MALDI-TOF-MS. Stable analysis was achieved with no background inference even at the concentration of100nM. Moreover, the limit of detection could be greatly lowered by utilizing graphene or graphene oxide as a pre-enrichment adsorbent. In summary, the promoted MALDI-TOF-MS methodology was demonstrated to be simple, sensitive, fast, cost effective and most importantly--high throughput.In chapter five, the above mentioned MALDI-TOF-MS method was utilized to two other targets.The first target is enzyme inhibitors. The experiment was carried out by combining enzyme immobilized magnetic carbonaceous microspheres and MALDI-TOF-MS with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection for ACh was0.25fmol/μL, and excellent linearity (R2=0.9998) was maintained over the range of0.5and250fmol/μL. Choline was quantified over the range of0.05and15pmol/μL, also with excellent linearity (R2=0.9994) and low limit of detection (0.15fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.Another target was long chain fatty acid. And graphene or graphene oxide was utilized to enrich and ionize long chain fatty acids. All together five long chain fatty acids were selected as models here, which are n-dodecanoic acid (C12), n-tetradecanoic acid (C14), n-hexadecanoic acid (C16), n-octadecanoic acid (C18), and n-eicosanoic acid (C20). Due to the large surface area and strong interaction force of G or GO, all the five long chain fatty models were effectively enriched by graphene or graphene oxide. On the other hand, the excellent electronic, thermal, and mechanical properties enable graphene and graphene oxide to be prefect energy receptacles for laser radiation, which make the ionization steps more effectively. Eventually, the promoted graphene and graphene oxide methodology can sensitively detect the five long chain fatty acid models from real biological samples even at low concentrations. Meanwhile, by adopting our promoted methodology, the detection of long chain fatty acids by MALDI-TOF-MS was demonstrated to be simple, sensitive, fast, cost effective and high throughput, which is meaningful as to practical usage.To sum up, this dissertation aims at the analysis of bioactive compounds from TCMs. By utilizing LC and MS, brand new methods and technology were promoted. A pipeline consists of extraction, separation, bio-screening and identification was firstly established; and then, two mathematical models and multi-dimensional LC separation as introduced to obtain a comprehensive understanding of Compound TCM; besides, MALDI-TOF-MS was successfully utilized not only in TCM screening, but also in enzyme inhibitors as well as long chain fatty acids screening, this methodology means a lot for the high throughput screening of small compounds. All those new methods and techniques were designed for bioactive compounds analysis in TCMs, so as to inspire the emergence of more innovative solutions to TCMs research. |
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