【作者】 张玉峰；

【导师】 程翼宇；

【作者基本信息】 浙江大学 ， 药物分析学， 2008， 博士

【摘要】 中药是天然产物的宝库,里面蕴藏的秘密吸引着无数研究者前赴后继,而九十年代后期成熟的液相色谱质谱联用技术可能就是打开宝库大门的一把金钥匙!本论文以对治疗心血管疾病、抗肿瘤和抗炎效果较好的三味中药（灯盏细辛、苦参和金果榄）为对象,采用液质联用技术深入细致地分析了其中的天然活性化合物,示范了如何根据多级质谱碎片信息确定化合物的结构。围绕着如何提高数据分析的智能化、简便化,设计了多个实用的算法和工具,并初步建立了基于Web浏览器的天然产物质谱检索系统,有望成为新一代天然产物结构确证系统的原型。本文主要内容及学术贡献如下:（1）建立了分析灯盏细辛和雪莲果酚酸类成分的LC/DAD/MSⁿ方法;根据UV光谱和多级质谱,鉴定或推测了灯盏细辛中53个化合物,包括咖啡酰奎尼酸酯（CQAs）、CQA葡萄糖苷、丙二酸单酰CQA、乙酰CQA、咖啡酰-2,7-脱氢-3-去氧-2-辛酮糖酸衍生物（CDOAs）、咖啡酰-2,7-脱氢-2-辛酮糖酸衍生物（COAs）、黄酮、黄酮醇和二氢黄酮,大部分化合物为灯盏细辛中首次报道,其中19个为新化合物。首次研究了CDOAs和COAs类化合物的裂解途径,其MSⁿ中的离子^2,5O^-,^3,6O^-,^4,6O^-是它们的特征碎片。（2）通过从苦参中提取和合成的方法,制备得18个含异戊烯单元的二氢黄酮、二氢黄酮醇和查尔酮,详细研究了它们在正离子和负离子模式下的ESI-MSⁿ,再辅以高分辨质谱数据和模型化合物,建立了这一类化合物可能的裂解途径。研究发现,黄酮B环2’-OH在分子内与1位氧原子形成分子内氢键,使黄酮C环的RDA反应极易发生,在正离子模式生成^1,3A⁺,负离子模式生成^1,4A^-。此外,二氢黄酮在（+）ESI/MS²中的脱水也与2’-OH有关。用E2消除机制成功解释了极少见的负离子模式脱水和脱甲醇现象。二氢黄酮和对应的查尔酮异构体可通过比较（+）ESI/MS²中^1,3A⁺和S⁺离子的相对丰度来区分,而它们的负离子模式多级质谱则没有区别。（3）使用LC/DAD/ESI-MSⁿ技术,建立了鉴定和比较中药金果榄的两种基源植物青牛胆和金果榄中化学成分的方法,该方法可同时分析植物中的异喹啉生物碱、二萜内酯和植物甾体三大类成分。正离子模式下,根据这三类成分的各自不同的特征离子可快速判断提取物中化合物的类别:异喹啉生物碱为[M]⁺或[M+H]⁺,二萜内酯为[M+NH₄]⁺和/或[M+H-CO₂]⁺,植物甾体为[M+H-nH₂O]⁺（n=1.3）。紫外光谱也有助于化合物类别的判断。最终,通过与标准品对照确证了11个化合物,根据MSⁿ数据推断了另外13个化合物的结构,其中2个为新化合物。虽然青牛胆和金果榄两种植物中化合物的种类基本一致,但其含量差别很大,这可能会导致药理活性的不同。（4）编制了Finnigan原始raw文件的数据转化程序raw2txt,为后继开发数据处理相关的算法奠定基础。针对液质联用数据背景噪音的特点,设计了FMSA算法,比已报道的方法更能准确区分信号与噪音。为提高全扫描质谱中加合离子判断的速度和准确度,编写了AddCal软件,软件支持用户自定义可被加合的离子。MS2table可辅助研究者快速制作符合文章发表要求的多级质谱数据。（5）提出了一种全新的基于液质联用数据的中药质控方法,该方法同时包含了化合物的定量和定性信息,弥补了以往色谱指纹图谱定性方面的不足。（6）初步建立了天然产物多级质谱数据库,同时兼顾本实验室数据和文献已报道数据,既可作为实验数据的管理、查询系统,也可根据裂解途径快速判断未知化合物可能的结构。更多还原

【Abstract】 The Chinese herbal medicine is a treasury of natural products,and fascinating more and more researchers to explore the ancient secret in it.The development of LC/MS technology may be a key to this treasury after it’s mature in late 90s of last century.In this work,three herbs which have shown solid curative effects to cardiovascular diseases,cancers and inflammations were selected as our research subjects and analyzed by many LC/MS technologies.Our results will be a good example of how natural compounds are identified by ESI-MSⁿ.In order to improve the speed,reduce human labor,and make it more intelligent in handling LC/MS data,new algorithms and programs were coded and a database of multi-stage mass spectra also established even though the current version is very primary.The main contents and our contributions are summarized as following:（1） A LC/DAD/MSⁿ method was developed to analyze the phenolic compounds of Erigeron breviscapus and Smallanthus sonchifolius.A total of 53 compounds were identified or tentatively characterized based on their UV and mass spectra.These compounds included caffeoylquinic acids（CQAs）,CQA glucosides,malonyl-CQAs,acetyl-CQAs,caffeoyl-2,7-anhydro-3-deoxy-2-octulopyranosonic acids（CDOAs）,caffeoyl-2,7-anhydro-2-octulopyranosonic acids（COAs）, flavones,flavonols and flavonones.Most of them were reported for the first time from E. breviscapus and nineteen of them belonged to new compounds.The MSⁿ spectra of CQA glucosides were similar to CQAs and they were discriminated by their retention times.Fragment ions(^2’5O^-,^3,6O^- and ^4,6O^-) corresponding to ring cleavage were shown in MSⁿ spectra of CDOAs and COAs,characteristic of this class of compounds.（2） Eighteen isoprenylated flavonoids（8 flavanones,3 flavanols,and 7 chalcones） isolated from Kushen or synthesized were studied by positive and negative ion electrospray ionization multi-stage tandem mass spectrometry（ESI-MSⁿ）.Plausible fragmentation patterns were obtained by comparing their MSⁿ spectra with each other,which were further supported by high-resolution MS data and two model compounds.It was shown that the 2’-OH group would make the C-ring of flavonoids studied more labile through a six-membered mechanism,resulting in base peaks of ^1,3A+（positive mode） and ^1,4A-（negative mode）.In addition,the 2’-OH is also responsible for the neutral loss of water in（+）ESI/MS² of flavanones.The neutral loss of water（or methanol） in （-）ESI/MS² of flavanols was elucidated by a E2 elimination mechanism.Different relative abundances of ^1,3A⁺ and S⁺ in（+）ESFMS² spectra were used to discriminate flavanones with their open-ring products,chalcones,since the equilibrium for flavanone??chalcone isomerization in ESI ion source could not be obtained in positive mode.（3） The main components（isoquinoline alkaloids,diterpenoids and steroids） in crude extracts of Chinese herb Jin-Guo-Lan,prepared from the roots of Tinospora sagittata and Tinospora capillipes,were explored by liquid chromatography/electrospray ionization multistage mass spectrometry coupled with diode array detection（LC-DAD/ESI-MSⁿ）.In positive ion mode,the three types of compounds showed very different characteristic ions:strong[M]⁺ or[M+H]⁺ ions were observed for isoquinoline alkaloids;[M+NH₄]⁺ and/or[M-CO₂]⁺ for diterpenoids; [M+H-nH₂O]⁺（n=1-3） for steroids.These adduct ions and/or fragments were used to deduce the mass and categories of known and unknown components in crude extracts,and their structures were further confirmed by ESI-MSⁿ in positive ion mode.Moreover,UV absorption peaks obtained from diode array detection provided useful functional group information to aid the MSⁿ-based identification.As a result,11 compounds were unambiguously identified by comparing with standard compounds and 13 compounds were tentatively identified or deduced according to their MSⁿ data.Two of these compounds（13-hydroxycolumbamine and 13-hydroxyjatrorrhizine） were found to be new compounds and another one （13-hydroxypalmatine） was firstly detected as natural products.Although the roots of Tinospora sagittata and Tinospora capillipes have almost identical compounds,the content of the compounds in them is dramatically different,suggesting the necessity of further comparison the bioactivities of the two species.（4） A software "raw2txt" was programmed to extract full scan mass spectrum data from binary raw files of Finnigan.This is a basic tool for users to develop other algorithms about LC/MS data.The frequency-based mass selection algorithm（FMSA） can beat any other reported methods in discrimination of noise and signals.The programs "AddCal" and "MS2table" are helpful for users to determine the construction of adduct ions and format the MSⁿ data for publication.（5） A novel quality control method was developed based on the two-way data from LC/MS, in which both quantitative and qualitative information was included.（6） A multi-stage mass spectra database for natural products（MMSDNP） was constructed.It can be used as a laboratory MS information management system or assist users to characterize unknown compounds quickly.更多还原