中药有效成分分析的新方法新技术研究
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摘要
本论文以寻找中药有效成分为出发点,以色谱、质谱技术为基础,尝试探索中药研究的新方法和新技术。建立了以提取手段、色谱质谱联用技术、基于细胞模型的活性成分筛选技术互相搭配的分离分析手段用于中药有效成分的提取、分离、筛选和鉴定；同时,针对中药复方方剂的配伍比例进行了优化探索,并采取先分解再组合的实验思路建立基于中药复方叠加效应的有效成分筛选方法,并搭建了两维色谱体系用于分离复方中药中的复杂成分,从而实现了在综合考量中药多组分、多靶点、多效应特点上的较为全面的活性物质分离鉴定；此外,以基质辅助激光解吸飞行质谱(MALDI-TOF-MS)为出发点,通过采用新基质成功实现了高通量的中药化合物筛选及长链脂肪酸富集；在此基础上,将MALDI-TOF-MS筛查扩展到了固定酶抑制剂的高通量筛查,从而扩宽了高通量MALDI-TOF-MS技术的筛查范围。相关研究工作及成果的摘要如下：
第一章文献综述部分充分描述了中药的丰富内涵,突出了中药有效成分在中药质量筛查中的重要性,以及探索中药药效机理对于药物筛选与研发的必要性；介绍了色谱技术作为一种现代高效分离工具在中药研究方面的发展与应用；阐述了针对中药有效成分研究的各种提取技术,围绕中药多组分、多靶点、多效应等特点展开的数学实验思考,以及对于中药有效成分筛查的体外生物模型研究方法；同时,详细描述了在化合物分离鉴定方面发挥重要作用的多维色谱、质谱技术,概述了当前应用于中药分离的多维色谱模式以及在中药研究中逐渐展露身手的质谱鉴定技术。从中药的提取预处理、到数学思维综合考量、到多维色谱高效分离、到生物模型科学筛分、到质谱技术高通量鉴定等多种化学生物技术相互结合的方式,对于中药有效成分的分析具有十分重要的意义。通过描述该研究的基本思路和进展状况,阐明了本课题选题及研究的学术意义和技术背景。
第二章将提取、分离、筛选、鉴定技术互相搭配,从而实现对中药有效成分的分离分析。以中药半枝莲为分析样本,探索不同提取方法对于其中有效成分-野黄芩苷的提取效率。通过纳升级的色谱＼质谱联用模式来定性鉴定和定量测量经过红外提取后的半枝莲中的成分野黄芩苷。同时,红外提取的各个过程各个实验参数都进行了优化,从而验证了该提取方法的可靠性及高效性。同时,通过与其他几种提取方法,例如常规的加热回流方法、超声提取方法、微波提取方法等进行比较可知,红外提取能够获得更高的提取效率。同时,中药半枝莲在纳升级色谱质谱联用模式下得到了有效分离。通过参照野黄芩苷标准品的色谱保留时间及质谱结构信息,即可以快速准确的确定半枝莲中野黄芩苷的相关信息,从而实现高效定性鉴定。此外,通过建立野黄芩苷标准曲线从而获得相应的定量信息,进而得到较为满意的方法有效性、线性以及检测限。结果显示,该方法在0.6至20ng mL-1范围内线性良好,相关系数(R2)为0.9973,检测限(LOD)为0.5ng mL-1(S/N=3)。
第三章将中药复方作为分析对象,以探索复方配伍合理性,考量复方药效叠加作用以及分析复方中药复杂组分为出发点,通过提取优化、生物检测细胞模型、两维色谱分离及高效准确质谱鉴定等技术,尝试从更为全面的角度来分析纷繁复杂的中药有效成分。首先,从探究复方方剂的配伍比例合理性出发,针对特定的中药复方方剂(该方剂由三种单味药),在结合细胞生物筛查效果的基础上,进行方剂配伍比例值的优化探索；其次,鉴于中药各个组分之间的药效叠加效应,从整体着眼,根据先分解再组合的思想重新进行细胞增殖生物筛查,进而从更为全面的角度对有效成分进行筛查；然后,以紫杉提取液为实验对象,继续按照先分解再整合的实验思路对其中的有效成分进行筛查,根据实验结果,紫杉醇被确定为其中的有效成分,从而验证了这一筛选方法的有效性；最后,将亲水色谱柱和反相色谱柱相结合,搭建两维色谱分离系统,经过验证,该系统具有更好的正交性和更高的峰容量,继而将此两维色谱分离体系与细胞模型、质谱鉴定相结合,从而实现高通量的药物筛选,对于药物研发及药物筛查具有重要意义。
第四章以基质辅助激光解吸飞行质谱(MALDI-TOF-MS)为出发点,将石墨烯和氧化石墨烯用作MALDI基质,来鉴定传统中药化合物。由于石墨烯和氧化石墨烯的表面积较大,从而避免了MALDI离子源与真空系统的污染。同时,由于石墨烯优异的电学、热学以及机械性能使得MALDI-TOF-MS可以在浓度为100nM时达到稳定的分析,且免除背景噪音干扰。实验采用当归和半枝莲做为检测样本,将当归中的阿魏酸(m/z194)、半枝莲中的野黄芩苷(m/z462)及汉黄芩素(m/z284)作为检测的目标化合物。实验表明,这两种MALDI基质对于中药中组分的不同浓度不具有歧视效果。此外,石墨烯和氧化石墨烯还可以作为样品的吸附剂展示其富集功效,从而使被测物的检测限获得较大幅度的降低。实验表明,这种MALDI-TOF-MS方法具有简单灵敏、快速经济、以及高通量等突出特点,可以为中药快速质量筛查提供新的技术手段。
此外,还将石墨烯和氧化石墨烯用于富集长链脂肪酸。将5种长链脂肪酸,即十二烷酸(C12)、十四烷酸(C14),十六烷酸(C16),十八烷酸(C18)和二十烷酸(C20),用作为代表性样本。由于石墨烯或氧化石墨烯较大的表面积和强烈相互作用,这5种长链脂肪酸被石墨烯和氧化石墨烯有效富集,实现了高通量的检测。同时,本方法还可以成功用于低浓度下检测实际生物样品中的5种长链脂肪酸样本,从而证明了这种MALDI-TOF-MS方法可以实现简单灵敏、快速经济并且高通量的检测长链脂肪酸。
第五章则在上述MALDI-TOF-MS检测技术的基础上,将这种高通量筛查技术用于酶抑制剂的筛选中,该筛选由固定酶磁性碳球和以氧化石墨烯为基质的MALDI-TOF-MS结合完成。首先,将乙酰胆碱酯酶(AChE)固定于3-glycidoxypropyltrimethoxysilane (GLYMO)修饰的磁性碳球上,该固定酶磁性碳球具有高效的酶活性和稳定性,并可以将酶从底物和产物中有效分离出来。固定化乙酰胆碱酯酶AChE的效率则通过固定酶磁性碳球和样品底物之间的反应加以验证,而后续的定量分析底物乙酰胆碱和产物胆碱则通过基于氧化石墨烯为基质的MALDI-TOF-MS方法完成,该MALDI-TOF-MS方法没有背景干扰。乙酰胆碱的检测限LOD是0.25fmol/μL,在0.5至250fmol/μL范围内有良好的线性值(R2=0.9998)。而胆碱则在0.05至15pmol/μL内有很好的线性(R2=0.9994)和低检测限LOD(0.15fmol/μL)。在标准曲线的浓度范围内有良好的准确度和精确度。共有8种化合物(4种已知乙酰胆碱酯酶AChE抑制剂和4种没有乙酰胆碱酯酶AChE抑制效果的对照化合物)用此方法进行验证,所得结果证明该高通量筛分方法可以大大推动日常酶抑制剂筛选的进程。
总体而言,本论文围绕中药有效成分研究为核心,以色谱、质谱技术为基础,着重发展新方法和新技术。首先,建立了包含提取、分离、筛选、鉴定的操作技术方法；其次,围绕中药复方这一复杂体系,着重优化复方配伍比例值,探索基于复方药效叠加作用基础上的有效成分筛选方法,建立两维色谱分离模式以实现更为全面的中药复方分离分析；同时,将MALDI质谱技术成功用于中药分子检测及长链脂肪酸富集,从而为高通量的中药质量筛查提供新的技术基础；最后,固定酶抑制剂的高通量MALDI检测的实现,大大拓宽了该MALDI质谱检测新技术的应用领域,具有极大潜力。本论文以色谱质谱等现代仪器手段为基础,结合生物模型,着重发展中药有效成分分离分析的新方法新技术。以高效灵敏、准确可靠的分离、筛选、鉴定中药中的有效成分为目标,发展了包含提取手段、分离鉴定、基于细胞模型的活性成分筛选、高通量中药化合物鉴定等新技术和新方法,为中药有效成分的分离分析提供了较为全面的研究方法。这些新方法和新技术的发展对于中药有效成分的分离分析十分重要,有利于中药的质量筛查及物质基础探索。这些针对中药有效成分分析的新方法和新技术的建立,有利于不断的在中药研究领域挖掘出更多的创新型解决方案。
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.
第一章文献综述部分充分描述了中药的丰富内涵,突出了中药有效成分在中药质量筛查中的重要性,以及探索中药药效机理对于药物筛选与研发的必要性；介绍了色谱技术作为一种现代高效分离工具在中药研究方面的发展与应用；阐述了针对中药有效成分研究的各种提取技术,围绕中药多组分、多靶点、多效应等特点展开的数学实验思考,以及对于中药有效成分筛查的体外生物模型研究方法；同时,详细描述了在化合物分离鉴定方面发挥重要作用的多维色谱、质谱技术,概述了当前应用于中药分离的多维色谱模式以及在中药研究中逐渐展露身手的质谱鉴定技术。从中药的提取预处理、到数学思维综合考量、到多维色谱高效分离、到生物模型科学筛分、到质谱技术高通量鉴定等多种化学生物技术相互结合的方式,对于中药有效成分的分析具有十分重要的意义。通过描述该研究的基本思路和进展状况,阐明了本课题选题及研究的学术意义和技术背景。
第二章将提取、分离、筛选、鉴定技术互相搭配,从而实现对中药有效成分的分离分析。以中药半枝莲为分析样本,探索不同提取方法对于其中有效成分-野黄芩苷的提取效率。通过纳升级的色谱＼质谱联用模式来定性鉴定和定量测量经过红外提取后的半枝莲中的成分野黄芩苷。同时,红外提取的各个过程各个实验参数都进行了优化,从而验证了该提取方法的可靠性及高效性。同时,通过与其他几种提取方法,例如常规的加热回流方法、超声提取方法、微波提取方法等进行比较可知,红外提取能够获得更高的提取效率。同时,中药半枝莲在纳升级色谱质谱联用模式下得到了有效分离。通过参照野黄芩苷标准品的色谱保留时间及质谱结构信息,即可以快速准确的确定半枝莲中野黄芩苷的相关信息,从而实现高效定性鉴定。此外,通过建立野黄芩苷标准曲线从而获得相应的定量信息,进而得到较为满意的方法有效性、线性以及检测限。结果显示,该方法在0.6至20ng mL-1范围内线性良好,相关系数(R2)为0.9973,检测限(LOD)为0.5ng mL-1(S/N=3)。
第三章将中药复方作为分析对象,以探索复方配伍合理性,考量复方药效叠加作用以及分析复方中药复杂组分为出发点,通过提取优化、生物检测细胞模型、两维色谱分离及高效准确质谱鉴定等技术,尝试从更为全面的角度来分析纷繁复杂的中药有效成分。首先,从探究复方方剂的配伍比例合理性出发,针对特定的中药复方方剂(该方剂由三种单味药),在结合细胞生物筛查效果的基础上,进行方剂配伍比例值的优化探索；其次,鉴于中药各个组分之间的药效叠加效应,从整体着眼,根据先分解再组合的思想重新进行细胞增殖生物筛查,进而从更为全面的角度对有效成分进行筛查；然后,以紫杉提取液为实验对象,继续按照先分解再整合的实验思路对其中的有效成分进行筛查,根据实验结果,紫杉醇被确定为其中的有效成分,从而验证了这一筛选方法的有效性；最后,将亲水色谱柱和反相色谱柱相结合,搭建两维色谱分离系统,经过验证,该系统具有更好的正交性和更高的峰容量,继而将此两维色谱分离体系与细胞模型、质谱鉴定相结合,从而实现高通量的药物筛选,对于药物研发及药物筛查具有重要意义。
第四章以基质辅助激光解吸飞行质谱(MALDI-TOF-MS)为出发点,将石墨烯和氧化石墨烯用作MALDI基质,来鉴定传统中药化合物。由于石墨烯和氧化石墨烯的表面积较大,从而避免了MALDI离子源与真空系统的污染。同时,由于石墨烯优异的电学、热学以及机械性能使得MALDI-TOF-MS可以在浓度为100nM时达到稳定的分析,且免除背景噪音干扰。实验采用当归和半枝莲做为检测样本,将当归中的阿魏酸(m/z194)、半枝莲中的野黄芩苷(m/z462)及汉黄芩素(m/z284)作为检测的目标化合物。实验表明,这两种MALDI基质对于中药中组分的不同浓度不具有歧视效果。此外,石墨烯和氧化石墨烯还可以作为样品的吸附剂展示其富集功效,从而使被测物的检测限获得较大幅度的降低。实验表明,这种MALDI-TOF-MS方法具有简单灵敏、快速经济、以及高通量等突出特点,可以为中药快速质量筛查提供新的技术手段。
此外,还将石墨烯和氧化石墨烯用于富集长链脂肪酸。将5种长链脂肪酸,即十二烷酸(C12)、十四烷酸(C14),十六烷酸(C16),十八烷酸(C18)和二十烷酸(C20),用作为代表性样本。由于石墨烯或氧化石墨烯较大的表面积和强烈相互作用,这5种长链脂肪酸被石墨烯和氧化石墨烯有效富集,实现了高通量的检测。同时,本方法还可以成功用于低浓度下检测实际生物样品中的5种长链脂肪酸样本,从而证明了这种MALDI-TOF-MS方法可以实现简单灵敏、快速经济并且高通量的检测长链脂肪酸。
第五章则在上述MALDI-TOF-MS检测技术的基础上,将这种高通量筛查技术用于酶抑制剂的筛选中,该筛选由固定酶磁性碳球和以氧化石墨烯为基质的MALDI-TOF-MS结合完成。首先,将乙酰胆碱酯酶(AChE)固定于3-glycidoxypropyltrimethoxysilane (GLYMO)修饰的磁性碳球上,该固定酶磁性碳球具有高效的酶活性和稳定性,并可以将酶从底物和产物中有效分离出来。固定化乙酰胆碱酯酶AChE的效率则通过固定酶磁性碳球和样品底物之间的反应加以验证,而后续的定量分析底物乙酰胆碱和产物胆碱则通过基于氧化石墨烯为基质的MALDI-TOF-MS方法完成,该MALDI-TOF-MS方法没有背景干扰。乙酰胆碱的检测限LOD是0.25fmol/μL,在0.5至250fmol/μL范围内有良好的线性值(R2=0.9998)。而胆碱则在0.05至15pmol/μL内有很好的线性(R2=0.9994)和低检测限LOD(0.15fmol/μL)。在标准曲线的浓度范围内有良好的准确度和精确度。共有8种化合物(4种已知乙酰胆碱酯酶AChE抑制剂和4种没有乙酰胆碱酯酶AChE抑制效果的对照化合物)用此方法进行验证,所得结果证明该高通量筛分方法可以大大推动日常酶抑制剂筛选的进程。
总体而言,本论文围绕中药有效成分研究为核心,以色谱、质谱技术为基础,着重发展新方法和新技术。首先,建立了包含提取、分离、筛选、鉴定的操作技术方法；其次,围绕中药复方这一复杂体系,着重优化复方配伍比例值,探索基于复方药效叠加作用基础上的有效成分筛选方法,建立两维色谱分离模式以实现更为全面的中药复方分离分析；同时,将MALDI质谱技术成功用于中药分子检测及长链脂肪酸富集,从而为高通量的中药质量筛查提供新的技术基础；最后,固定酶抑制剂的高通量MALDI检测的实现,大大拓宽了该MALDI质谱检测新技术的应用领域,具有极大潜力。本论文以色谱质谱等现代仪器手段为基础,结合生物模型,着重发展中药有效成分分离分析的新方法新技术。以高效灵敏、准确可靠的分离、筛选、鉴定中药中的有效成分为目标,发展了包含提取手段、分离鉴定、基于细胞模型的活性成分筛选、高通量中药化合物鉴定等新技术和新方法,为中药有效成分的分离分析提供了较为全面的研究方法。这些新方法和新技术的发展对于中药有效成分的分离分析十分重要,有利于中药的质量筛查及物质基础探索。这些针对中药有效成分分析的新方法和新技术的建立,有利于不断的在中药研究领域挖掘出更多的创新型解决方案。
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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