【作者】 陈军辉；

【导师】 王小如；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2008， 博士

【摘要】 将不同模式“液质”联用技术,包括:高效液相色谱-电喷雾飞行时间质谱(HPLC-ESI-TOF/MS),高效液相色谱-大气压化学电离质谱(HPLC-APCI-MS),高效毛细管电泳-电喷雾飞行时间质谱(HPCE-ESI-TOF/MS),超高效液相色谱-电喷雾串联质谱(UPLC-ESI-MS/MS)等,用于采用普通分析方法难以测定的复杂天然药物研究中,并成功建立快速分析、鉴别陆源及海洋药用生物中活性成分的方法学,以解决几种典型陆源和海洋天然药物已知、未知活性成分鉴别困难的难题,为我国陆源及海洋天然药物现代化研究进程的加速提供强大的技术推动力。本文研究内容与所得结果如下:第一章,对我国中药现代化、海洋药物现代化研究以及不同色谱-质谱联用技术相关的概念、理论和国内外研究现状进行了简要的介绍和评述。第二章,加速溶剂萃取技术(ASE)是近年来发展起来的一种全新的萃取技术,具有自动化程度高、萃取时间短、溶剂用量少、萃取效率高等显著特点。目前已逐渐应用于中药现代化研究领域,成为中药有效成分色谱分析样品前处理的强有力手段。本章首次将ASE用于中药材娑罗子和黄连有效成分的提取研究。首先,探讨ASE提取娑罗子中七叶皂苷的可行性,并比较该方法相比于回流和超声提取法的优越性。以娑罗子中四种七叶皂苷的提取率为指标,采用HPLC法测定,用单因素考察法对ASE从娑罗子中提取七叶皂苷的工艺条件进行优化。其次,以黄连中四种主要生物碱的提取率为评价指标,采用正交设计实验对ASE从黄连中提取生物碱的工艺条件进行优化,并与回流和超声提取法进行比较;对比实验结果表明ASE提取法四种主要生物碱提取率均高于传统方法。由此可知,ASE是娑罗子皂苷及黄连生物碱类化合物快速、高效提取的有效方法。第三章,采用HPLC-ESI-TOF/MS对中药材娑罗子和莲子心中的活性成分进行分析。首先,建立娑罗子中4种主要七叶皂苷定量测定的HPLC-UV方法,探讨4种七叶皂苷电喷雾质谱分析裂解规律,并对娑罗子中的其他七叶皂苷类化合物进行鉴别。在选定的色谱条件下,七叶皂苷类化合物得到较好分离,方法的精密度、重复性、稳定性均良好。通过ESI-TOF/MS分析获得娑罗子中各皂苷成分的精确分子量和分子式,采用质谱碰撞诱导解离技术获得各化合物碎片裂解信息,从而阐明了4种主要七叶皂苷的电喷雾质谱裂解规律,结合文献还对娑罗子中的14种皂苷类化合物进行了初步鉴定。其次,建立了ASE-HPLC-DAD-ESI-TOF/MS分析莲子心中生物碱类化合物的方法,在选定的最佳仪器条件下,鉴定了莲子心提取物中的6种生物碱。本章研究结果表明HPLC-ESI-TOF/MS是娑罗子皂苷类化合物、莲子心生物碱类化合物快速鉴别的有效工具。第四章,率先将HPCE-ESI-TOF/MS用于分析黄连中的8种生物碱类化合物。使用未涂层石英毛细管,优选出3种不同运行电解质溶液,并对联用技术的仪器工作条件进行了优化。在优化所得的最佳仪器条件下,使用3种运行缓冲溶液,进行黄连提取物的HPCE-DAD分析,黄连中的8种生物碱均能获得良好的分离结果;进行黄连提取物的HPCE-ESI-TOF/MS分析,能对黄连中的8种生物碱进行快速鉴别。此外,还建立了黄连中3种主要生物碱(小檗碱、巴马汀、药根碱)的HPCE-DAD测定方法,该方法的精密度、重复性、稳定性均良好。综上所述,说明HPCE-ESI-TOF/MS联用技术是快速分析鉴定黄连中生物碱类化合物的强有力工具,在中草药生物碱类化合物鉴定研究中具有很强的适用性。第五章,首次采用UPLC-ESI-MS/MS对黄连中生物碱类化合物进行分析。实验采用BEH C18色谱柱,以乙腈-水(含0.5%的乙酸和20 mmol/L的乙酸铵)为流动相,梯度洗脱,UV 350nm检测,能获得较好的分离结果,可作为黄连药材中生物碱类化合物含量测定的检测方法。采用ESI-MS/MS对黄连提取物中的生物碱类化合物进行鉴别,通过获得各生物碱的一级、二级质谱图,推测各生物碱的质谱裂解规律,结合相关文献,可对黄连中的8种生物碱进行了快速鉴别。另外,还建立了ESI-MS/MS测定黄连中3种生物碱的分析方法,结果表明,MS/MS检测器的灵敏度明显高于PDA检测器,更高于第四章发展的HPCE-DAD法,适于低浓度黄连生物碱的分析测定研究。此外,还对黄连生物碱UPLC指纹图谱进行了探索,UPLC指纹图谱和HPLC指纹图谱相比,大大缩短了指纹图谱的分析时间,有望解决HPLC指纹图谱分析时间过长的难题。第六章,采用HPLC-APCI-MS对烟叶、灵芝和川芎中的活性成分进行分析研究。首先,建立烟叶中茄尼醇定量测定的HPLC-UV分析方法,烟叶样品进行皂化及超声提取处理后,采用反相HPLC进行测定;结果表明,该方法操作简便、灵敏度高,重现性好,可作为烟叶中茄尼醇含量的检测方法。另外,对茄尼醇APCI-MS分析条件进行了系统优化,建立了HPLC-APCI-MS测定茄尼醇的方法,并与ESI-TOF/MS进行了比较;通过对茄尼醇的ESI-TOF/MS和APCI-MS的质谱分析特征比较可以发现,茄尼醇在ESI源分析中的信号强度远远小于在APCI源分析中的信号强度,说明APCI源更适于茄尼醇的定量分析。其次,采用HPLC-DAD和HPLC-APCI-MS对灵芝中含有的三萜类化合物进行了分析。用DAD检测器记录各个色谱峰的紫外吸收光谱,采用APCI-MS进行在线同步分析,记录总离子流色谱图(TIC)和各个色谱峰的质谱图,通过紫外光谱及质谱分析并与文献对照初步鉴定了灵芝中的32个三萜类成分。再次,建立川芎生药HPLC特征指纹图谱分析方法,并采用HPLC-DAD-APCI-MS对川芎提取物中活性成分进行快速鉴定。结果表明,本文所发展的色谱方法精密度、重复性、稳定性良好,采用APCI-MS共计鉴定出川芎甲醇提取物中10种有效成分。综上所述,HPLC-APCI-MS是天然产物中极性较小,电喷雾质谱不易分析的化合物测定的有力工具。第七章,采用体外DPPH(1,1-二苯基苦基苯肼)抗氧化模型对海马提取物的抗氧化性质进行评价,并建立小海马HPLC特征指纹图谱,用于小海马药材的鉴别及质量评价。首先,利用离线DPPH抗氧化评价体系,对海马不同提取物清除DPPH自由基的能力进行比较,结果表明海马水提物清除DPPH自由基的能力最强,在此基础上又探明了海马水提物清除DPPH自由基能力随时间和浓度的变化规律,为海马抗氧化活性提供了科学依据。其次,依据抗氧化活性实验结果,建立了海马水提物HPLC特征指纹图谱分析方法;在选定的最佳色谱条件下,海马水提物大部分化合物达到基线分离,方法的精密度、重复性、稳定性良好;对10批小海马样品进行分析,建立小海马药材HPLC指纹图谱,采用中药指纹图谱相似度计算软件,对小海马进行真伪辨别和质量评价,结果表明该方法简捷、有效,是小海马药材鉴别及质量控制的有效方法。第八章,将基于HPLC在线清除DPPH?自由基活性快速筛选自由基清除剂的方法与ESI-TOF/MS结合,发展一种复杂天然产物中抗氧化活性成分在线筛选、鉴别的技术体系,并用于中草药金银花、海洋药物海马提取物中抗氧化活性成分的快速筛选、鉴别。本章所发展的HPLC -ESI-TOF/MS-DPPH技术体系,适用范围广、简单,可靠,还可以用于评价抗氧化成分的抗氧化效果。利用该方法从金银花醇提物中筛选出4种具有明显抗氧化活性的化合物,结合文献和数据库可对其进行鉴定;从海洋药物小海马中筛选出一种具有明显抗氧化活性的化合物,为海马水提物抗氧化活性提供了更可靠的科学依据。通过以上研究,本文针对不同天然药物所含活性成分的结构特点,发展了多种色谱-质谱联用技术,解决了几种典中药(娑罗子、莲子心、黄连、灵芝、川芎等)及海洋药物小海马研究中存在的一些难题,为陆源中草药及海洋药物活性成分的快速分离、鉴别、筛选、测定提供了一系列新方法。此外,探索了ASE用于天然药物微量有效成分提取的可行性,证明了ASE在天然药物成分色谱分析样品前处理研究领域,具有很强的实用性。更多还原

【Abstract】 A host of modern hyphenated chromatography-mass spectrometric techniques have been developed and their performance evaluated for the identification and quantification of bioactive components of medicinal values in complex natural products. These techniques include high performance liquid chromatography- electrospray time of flight mass spectrometry (HPLC-ESI-TOF/MS), high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS), high performance capillary electrophoresis- electrospray time of flight mass spectrometry (HPCE-ESI-TOF/MS), ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS), etc. Centered surrounding these highly sensitive instrumental techniques, effective analytical methodology have been developed and optimized for the rapid determination of active components in selected traditional Chinese medicinal materials of both herbal and marine origins. The main studies and results in the paper are outlined as follows:In Chapter One, a brief review is given covering subjects concerning the basic concepts, theories and current advances, both in China and abroad, on traditional Chinese medicines and marine drugs, and different chromatography-mass spectrometry techniques.In Chapter Two, the recent advancement of accelerated solvent extraction (ASE), as a new extraction technique developed recently, will be described. The technique offers significant advantages over conventional extraction techniques because of its high analytical speed, low solvent consumption, ease of automation and high extraction efficiency. In recent years, ASE is gaining increasing popularity in the study of traditional Chinese medicines (TCMs). Results from these studies demonstrate that ASE is indeed a powerful tool in the preparation of herbal extracts for downstream chromatographic analysis. In the present chapter, the development of ASE extraction method for the analysis of active compounds in Semen Aesculi and Coptis chinensis Franch will be reported for the first time.Firstly, to develop ASE extraction method for escins in Semen Aesculi and compare the advantage of this method with other extraction methods. By making the single factor experiments, the optimum technological parameters for ASE extraction were obtained. Through the experiments, the most favorable conditions for ASE extraction of escins in Semen Aesculi were obtained. Secondly, the optimal parameters for ASE extraction of alkaloids in Coptis chinensis Franch were obtained by making the orthogonal experiments. Results showed that the optimal conditions are as following: 80% ethanol+ 0.5% hydrochloric acid as solvent, temperature at 130℃, 10 min duration and once extraction. It can be concluded that the ASE is a quickly and effectively extraction technique for escins in Semen Aesculi and alkaloid compounds in Coptis chinensis Franch.In Chapter three, the development of a convenient method based on HPLC and positive ion ESI-TOF/MS for the analysis and characterization of active compounds in extracts of Semen Aesculi and Semen Nelumbinis will be discussed. To begin with, a reliable HPLC method for quantification of four major saponins in Semen Aesculi was developed. The ESI-MS fragmentation patterns of four major saponins were explored, and other saponin compounds in Semen Aesculi were also identified by accurate molecule weight information and fragmentation behavior obtained by collision induced dissociation experiment combined with literature review. The results indicated that the developed quantification method is simple, accurate and reliable for the determination of four major saponins in Semen Aesculi. The [M+H]+ or [M+Na]+ ions of saponins in Semen Aesculi extract were observed by ESI-TOF/MS on-line detection, and used to obtain accurate molecular weight and molecular formula information of saponins. Fourteen saponins in Semen Aesculi extract could be primary identified. On the other hand, an ASE-HPLC-DAD-ESI-TOF/MS method was developed for the analysis of alkaloids in Semen Nelumbinis. By comparing the spectrogram and mass spectrum of the chromatogram peaks with the references value, six compounds in Semen Nelumbinis were identified. The HPLC-ESI-TOF/MS method developed in this chapter has the advantages of simple operation, rapid measurement and accurate determination, and it’s a powerful tool for identification of saponins in Semen Aesculi and alkaloids in Semen Nelumbinis.In Chapter 4, the development of a HPCE technique with dual diode array assay (DAD) and ESI-TOF-MS detection for the analysis of seven protoberberine alkaloids and one aporphinoid alkaloid in Coptis chinensis Franch will be described. Three background electrolytes (BGEs) were developed for HPCE-DAD and HPCE-MS analyses, and the HPCE-ESI-TOF-MS conditions were optimized. Eight main alkaloids in Coptis chinensis Franch could be separated with base-line resolution by HPCE-DAD with these three different BGEs, and identified by ESI-TOF-MS analysis. Moreover, three major alkaloids berberine, palmatine and jatrorrhizine could be quantified accurately by HPCE-DAD with the BGE system consisting of 50: 50 v/v water and acetonitrile containing 50 mM ammonium acetate at pH=6.8. The results indicated that the developed HPCE-DAD method is simple and reliable for the determination of three alkaloids in Coptis chinensis Franch. The HPCE-MS method, as a viable alternative to HPLC-MS, is found to be suitable for the rapid identification and quantification of basic compounds in herbal or natural product applications.In Chapter five, the development of a new UPLC-MS/MS method for the identification and quantification of major alkaloids in extracts of Coptis chinensis Franch will be described. The UPLC system consisted of a dual detection system of photodiode array detector (PDA) and positive ion ESI-MS/MS in sequential configuration. A tandem quadrupole spectrometer operating in either full scan mode or in MS/MS mode for multiple reaction monitoring (MRM) was used for the identification and quantitative analysis of eight major alkaloids in Coptis chinensis Franch extracts. The samples were also analyzed on a HPLC-ESI-TOF-MS system to confirm the identification results. Three of the eight major alkaloids, berberine, palmatine and jatrorrhizine were quantified by UPLC-PDA and UPLC-MS/MS. The results indicated that both UPLC-PDA and UPLC-MS/MS methods were simple, sensitive and reliable for the determination of alkaloids in Coptis chinensis Franch. Seven Coptis chinensis Franch samples from different locations were analysed using the established methods. UPLC fingerprints based on the distribution of the eight major alkaloids can serve as a rapid and reliable method for the authentication and quality evaluation of TCM herbs.In Chapter six, HPLC-APCI-MS has been used for the determination and identification of active compounds in tobacco leaves, Ganoderma Lucidum (Leyss. Ex Fr.)Karst and Ligusticum chuanxiong Hort. Firstly, an user-friendly HPLC analysis method with two detector including UV and APCI-MS was established for the determination of solanesol in tobacco leaves from different sources. The results indicated that the developed HPLC-UV method is simple, accurate and reliable for the determination of solanesol in tabacoo leaves with a good linearity. The contents of solanesol in tobacco leaves from different producing area were distinct. Moreover, the main instrumental parameters of APCI-MS for analysis of solanesol were optimized to provide the best possible sensitivity, and a comparison with ESI-TOF/MS was conducted. In the APCI+ mode, abundant stable [M–H2O+H]+ ion (m/z at 613.5) was also observed, but can not observe other ion and fragmentation. By comparing APCI-MS and ESI-TOF/MS for the analysis of solanesol in extract of tobacco leaf, it is could be find out that the APCI mode is more sensitive than ESI mode, so APCI mode is more suited for solanesol quantitative analysis. Secondly, HPCL-APCI has been applied to analyze the triterpenoids in extracts of Ganoderma Lucidum (Leyss. Ex Fr.)Karst. The detection wavelength is 253 nm and the UV spectra of peaks were obtained with a DAD detector. On-line APCI-MS in positive and negative mode was used to get the mass spectrum of the analytes. Thirty-two components of Ganoderma Lucidum (Leyss. Ex Fr.)Karst were primarily identified by comparison of the UV spectra and mass spectrum with literatures. Finally, a HPLC fingerprint method has been established for the quality control of Ligusticum chuanxiong Hort and its extract. HPLC-APCI-MS has been applied to identify the compound in methanol extracts of Ligusticum chuanxiong Hort. On-line APCI-MS in positive and negative mode was used to get the mass spectrum of the analytes. Ten components of Ligusticum chuanxiong Hort were primarily identified by comparison of the UV spectra and mass spectrum with literatures. In summary, the HPLC-APCI-MS method developed in this chapter is a powerful tool for identification of weak polar compounds in natural products, which is a hard work for ESI-MS.In Chapter seven, the antioxidant activity of the extracts of Hippocampus japonucus Kanp (Haima) was evaluated for the first time, and the HPLC fingerprint of Haima was established for the discrimination of true or false and the quality evaluation of Haima. Firstly, antioxidant properties of Haima extracts with different solvent were assayed in terms of antioxidant activity by scavenging activities on 1,1-diphenyl-2-picrylhdrazyl (DPPH). The results indicated that the antioxidant activity of water extract of Haima is higher than all the other extracts of Haima. The effect of time and concentration of Haima extract on the antioxidant activity was also studied and the results showed that Haima can provide a good source of antioxidant. Secondly, a chromatographic fingerprint method based on HPLC was developed. The results indicated that the developed HPLC method is simple, accurate and reliable for the development of Haima fingerprint. Ten Haima samples collected from different medicine hall were analyzed and the Haima HPLC fingerprint was established. The similarity of the HPLC chromatogram was performed for authentication and quality control of Haima. The HPLC fingerprinting techniques have high potential in authentication or source-tracing types of applications.In Chapter eight, the development of a method based on the HPLC online scavenging DPPH? radical activity and ESI-TOF/MS for the rapid screening and identification of radical scavengers from complex natural products such as TCM and marine drugs will be discussed. The HPLC-ESI-TOF/MS-DPPH method developed in this chapter is simple, reliable. The method was applied to Lonicera japonica Thunb and Haima extracts. The results showed that four compounds in Lonicera japonica Thunb have scavenging radical activity, and all the four compounds were identified as organic acid compounds by ESI-TOF/MS according to literatures and data base. While one compound in Haima extract was found having scavenging radical activity, which could provide a reliable and scientific evidence for the antioxidant activity of Haima.In the present dissertation, effective analytical methodology of different modes of hyphenated“liquid chromatography- mass spectrometry”techniques were developed according to the structure of active components in natural products. Some analytical problems of several typical herbal medicines (Semen Aesculi, Semen Nelumbinis, Coptis chinensis Franch, Ganoderma Lucidum (Leyss. Ex Fr.)Karst, Ligusticum chuanxiong Hort, etc) and Haima were solved, and a series of new methods for separation, identification, screening, determination of active components in selected TCM materials of both herbal and marine origins were provided. Moreover, results from our studies demonstrate that ASE is indeed a powerful tool in the preparation of herbal extracts for downstream chromatographic analysis.更多还原