基于色谱联用技术的沉香标志性差异成分分析研究
发布时间：2018-08-19 15:10
【摘要】：目的:沉香主要来源于瑞香科沉香属(Aquzlaria)和拟沉香属(Gyrinops)含有树脂的木材,主要分布于我国广东,海南,广西等地及印尼、越南、柬埔寨、马来西亚等东南亚国家。沉香分为进口沉香和国产沉香两种,国产沉香基原植物为沉香属Aquilari a sinensis,而进 口沉香基源植物多为沉香属 Aquilaria malaccensi、Aquilaria crassna。沉香品质好坏跟种源、结香方式等密切相关,现代分子鉴定技术虽然能够鉴别出沉香基原,但对同一来源不同结香方式的样品的区分尚未有有效方法。特定的种源和特定的结香方式会产生特定的化学成分,因此通过分析沉香内在化学成分的差异可能是沉香种源鉴别、质量控制、品质分类的一条很好的途径。目前尚未不清楚不同类型沉香间有存在哪些差异,也尚未建立筛选识别这些差异成分的方法,因此本文拟结合沉香内在成分的特点,利用现代先进分析仪器和统计学方法,采用GCMS和LCMS结合多元统统计和单维分析方法对沉香成分进行全面系统分析,辅以主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)等统计方法,找出不同沉香组间的差异成分,同时对不同类型的沉香成分进行分类比较和聚类分析,为沉香快速鉴别、质量控制和品质分类提供参考和依据。方法:(1)按2015版中国药典一部沉香鉴别项下相关要求测定样品醇浸出物的含量,采用HPLC法(色谱柱Altima C18(150 mm×4.6 mm,5 μm),流动相为乙腈(A)-0.1%甲酸水(B),梯度洗脱,流速0.7 mL·min-1,检测波长252 nm,柱温30℃)测定样品中的沉香四醇含量,结合醇浸出物和沉香四醇含量结果初步分析探讨不同类型沉香间的差异。(2)采用GCMS联用仪(HP-5弹性石英毛细管柱(0.25 mm×30 m,0.25 μm),载气为高纯氦气,流速为1.0 mL.in-1,不分流,进样量为5 μl,进样口温度250℃,传输线温度280℃,起始温度90℃,保持3 min,然后以10 ℃.min-1升至180℃,然后以3 ℃·mir-1升至280℃,保持10 min,然后以5℃.min-1升至300℃,保持8 min。EI电离70 eV,离子源温度为230℃,四级杆温度150℃,扫描方式为全扫描,扫描范围m/z为50-550)分析测定样品中的化学成分,采用标准数据库NIST14和保留指数对沉香挥发油进行定性鉴别,将经过预处理的数据导入Simca-P软件进行数据分析,分析不同类型沉香间的差异,找出其差异标志物,同时对沉香特征性成分进行归纳分类比较,分析内在质量差异的原因。(3)采用 LC-QTOFMS 联用仪(InetrSustainSwift C18 色谱柱(2.1×150 mm,1.9μn);流速:0.3ml/min;柱温:40℃;流动相:乙腈(A):0.1%甲酸水溶液(B)梯度洗脱(0-3min,10%A;3-8min,10%-30%A;8-25min,30%-50%A;25-32min,50%-100%A,32-35min,100%A);进样量:10μL;ESI离子源,正离子模式,一级质谱(m/z 100～2000,DP100,CE 10,采集时间:0.2s),二级质谱(m/z 50～2000,DP100,CE 45,采集时间:0.01s)分析测定样品中的化学成分,对采集数据进行预处理后导入Simca-P软件进行数据处理分析,分析不同类型沉香间的差异,找出其差异标志物,同时对沉香特征性成分进行归纳分类比较,分析内在质量差异的原因。结果:(1)52份沉香样品中沉香四醇含量为0.10-6.60%,醇浸出物含量为6.07-57.06%,沉香醇浸出物与结香方式、种源无相关性,沉香四醇含量高低与种源、结香方式有一定的相关性(A.sinensis人工和天然沉香的沉香四醇平均含量分别为0.67%、0.15%,两者比较有显著差异,天然沉香中种源为A.malaccensis与A.crasna中沉香四醇平均含量分别为2.57%、0.70%,两者有差异,且与A.sinensis(0.15%)有显著差异)。(2)基于GCMS联用技术经过多元统计和单维统计分析筛选出不同种源和结香方式沉香的差异标志物共19个,分别为5个2-2-苯乙基色酮类成分、5个倍半萜类和其他类。通过OPLS-DA分析,A.sinensi 人工结香和天然沉香样品有13个差异标志成分为色酮类、倍半萜类、烷烃类,其中2-(2-苯乙基)色酮、6,7-二甲氧基-2-(2-苯乙基)色酮、5,8-Dihydroxy-4a-methyl-4,4a,4b,5,6,7,8,8a,9,10-decahydro-2(3H)-phenanthreno ne(isomer 1)、三十一烷(isomer 3)4个成分在两组间具有显著差异;A.crassna人工结香和天然结香样品中的差异成分为2个倍半萜类成分;同时两种不同种源人工结香沉香(A.sinensis和A.crassna人工结香沉香)的差异标志物有2个,其中5,8-Dihydroxy-4a-methyl-4,4a,4b,5,6,7,8,8a,9,10-decahydro-2(3H)-phenanthrenone(isomer1)在两组间具有显著差异;通过对三个种源天然沉香对比分析,A.sinensi 沉香与A.c rassna沉香和A.malaccensis沉香的均有7个差异标志物,它们中具有显著差异的标志性成分均为三十一烷(isomer 3),而其他组间并未发现具有显著差异的成分。对52个沉香样品中烷烃类、倍半萜和2-(2-苯乙基)色酮类成分峰面积进行归纳分类并进行统计分析,结果表明天然沉香中倍半萜含量较高多为A.crassna、A.mal accensis沉香,同时2-(2-苯乙基)色酮含量较高的也同样为A.crassna A.malaccen sis沉香,而烷烃类较高的则主要A.sinensis天然沉香;对各组间不同类型成分的总峰面积比较分析表明在A.sinensi 沉香中人工和天然结香样品中在烷烃类成分无显著差异,而倍半萜和2-(2-苯乙基)色酮均存在差异,且人工结香沉香均高于天然沉香;A.crassn 沉香中人工和天然沉香间则显示各类成分并无显著差异;从两组人工沉香(A.sinensis和A.crassna)样品比较分析,各类成分并无显著差异;三种天然沉香对比分析,其中A.sinensis天然沉香与A.malaccensis天然沉香在烷烃类和倍半萜类均存在差异,而A.sinensis天然沉香与A.c crassna天然沉香仅在烷烃类成分存在差异,其他组别和成分间并无显著差异。聚类分类显示人工沉香多能聚为一类,多数天然沉香也能够聚为一类。(3)基于LCMS联用仪对不同组别沉香样品进行多元统计学和单维统计分析,找出123个差异标志物,通过一级和二级质谱结合已有文献,鉴别出62个化合物,其中23个为潜在新化合物。对同一种源人工沉香和天然沉香的比较分析,在A.sinensis沉香中发现45个差异物,15个具有显著差异,而其中6个成分(2-(2-苯乙基)色酮、7-羟基-2-(2-苯乙基)色酮、6-甲氧基-2-(2-苯乙基)色酮(isomer 2)、6,8-二轻基-2-(2-苯乙基)色酮(isomer 1)、6-甲氧基-2-[2-(4'-甲氧基苯基)乙基]色酮、dehydroxy AH21)是A.sinensis中人工沉香和天然沉香间的主要标志性差异物,而A.crassna中人工沉香和天然沉香间的差异物有29个,具有显著差异有7个,其主要标志性差异物为6,8-二羟基-2-(2-苯乙基)色酮(isomer1),AH21(isomer1)、methoxy AH21(isomer1)、2,3-二羟基-5-苯乙基-2,3-二氢-1ah-oxireno[2,3-f]chromen-7(7bh)-one(isomer 2)、dehydroxy AH21(isomer 2)5个成分。对同一结香方式不同种源沉香的比较分析,A sinensis与A.crassna的人工沉香间的差异物有36个,其中具有显著差异的3个,AH12(isomer 3)为该两组人工沉香中的主要标志性差异物。在天然沉香中,A.sinensis与A.malaccensis沉香的差异物有35个,具有显著差异13个,其主要标志性差异物为6-甲氧基-2-[2-(4'-羟基-3'-甲氧基苯基)乙基]色酮(isomer 2)、6,8-二羟基-2-[2-(3'-羟基-4'-甲氧基苯基)乙基]色酮(i somer 2)、沉香四醇、6-羟基-7-甲氧基-2-[2-(4'-羟基-3'-甲氧基苯基)乙基]色酮(isomer 2)、2,3-二羟基-5-苯乙基-2,3-二氢-1ah-oxireno[2,3-f]chromen-7(7bh)-one(isomer 1)等5个成分;A.crassna与A.malaccensis沉香中的差异物有20个,具有显著差异2个,其主要标志性差异物则为6-甲氧基-2-[2-(4'-羟基-3'-甲氧基苯基)乙基]色酮(isomer 2)、6,8-二羟基-2-[2-(3'-羟基-4'-甲氧基苯基)乙基]色酮(isomer 1)),其中其中6-甲氧基-2-[2-(4'-羟基-3'-甲氧基苯基)乙基]色酮(isomer 2)是A.malaccensis与A.sinensis和A.c rassna种天然沉香共同的主要标志性差异物;而A.sinensis与A.crassna沉香的差异物有6个,并未发现有显著差异成分。同时结果显示具有显著共性特征苯环上羟基和甲氧基取代的2-(2-苯乙基)色酮类成分可能为有效辨识人工和天然沉香的标志性成分,而5,6,7,8-四氢-2-(2-苯乙基)色酮类和双2-(2-苯乙基)色酮类成分则是区分不同种源沉香的关键物质。对不同组别沉香的四种类型的2-(2-苯乙基)色酮类成分的峰面积进行比较研究,结果表明各组之间差异有所不同,5,6,7,8-四氢-2-(2-苯乙基)色酮类成分、双2-(苯乙基)色酮类、三2-(苯乙基)色酮类成分在各组间多具有显著差异;同时基于2-(2-苯乙基)色酮类成分对所有样品进行聚类分类显示人工和天然沉香多各自聚为一类。结论:本文基于GCMS和LCMS技术,结合多维和单维统计建立了筛选沉香差异成分的方法。通过GCMS结合NIST 14质谱库鉴定出19个差异成分,其中具有显著差异的有4个成分;本文首次基于LC-ESI-QTOF高分辨质谱结合文献从沉香中鉴定出62个差异成分,其中23个为潜在的新双2-(2-苯乙基)色酮类化合物,通过多维和单维统计分析的方法,筛选出具有显著差异的有27个;这些标志性成分可以作为沉香的品种鉴定、质量控制及化学分类学指标成分。人工沉香和天然沉香的差异可能与苯环上有轻基和甲氧基取代的2-(2-苯乙基)色酮类成分密切相关;5,6,7,8-四氢-2-(2-苯乙基)色酮类和双2-(2-苯乙基)色酮类成分则是区分不同种源沉香的关键物质。对不同类型成分进行归类分析,烷烃类、倍半萜类和2-(2-苯乙基)色酮类成分的高低在一定程度上能够反映沉香内在质量差异,烷烃类成分越低,倍半萜类和2-(2-苯乙基)色酮类含量越高,其沉香树脂含量越多,其质量可能越好,反之,其质量则越差;通过沉香内在成分聚类研究,人工和天然沉香多各自聚为一类,不同组别的多能聚为一类,该方法能很好地将不同质量的沉香进行聚类和区分。本文能够为沉香的品种鉴别、质量控制、品质分类研究新的思路和方法。
[Abstract]:OBJECTIVE: The resin-containing wood of the genera Aquzlaria and Gyrinops mainly distributes in Guangdong, Hainan, Guangxi, Indonesia, Vietnam, Cambodia, Malaysia and other Southeast Asian countries. A. sinensis, and the imported source plants are mostly Aquilaria malaccensi, Aquilaria crassna. The quality of L. sinensis is closely related to provenance, aroma-forming methods and so on. Although modern molecular identification technology can identify L. sinensis primordia, but there is no effective method to distinguish the samples from the same source with different aroma-forming methods. It is not clear what differences exist among different types of aloes, and no screening method has been established to identify these components. Methods: According to the characteristics of the internal components of Chinese aloes, this paper makes use of modern advanced analytical instruments and statistical methods, uses GCMS and LCMS combined with multivariate statistics and single-dimensional analysis method to analyze the components of Chinese aloes comprehensively, supplemented by principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and other statistical methods to find out if not. Methods: (1) The content of alcohol extract was determined by HPLC (column Al) according to the relevant requirements of the Chinese Pharmacopoeia (2015 edition). TIMA C18 (150 mm x 4.6 mm, 5 micron), mobile phase acetonitrile (A) - 0.1% formic acid water (B), gradient elution, flow rate 0.7 mL min 1, detection wavelength 252 nm, column temperature 30 C) was used to determine the content of agaric tetraol in samples. The difference between different types of agaric incense was preliminarily analyzed by combining the results of alcohol extract and agaric tetraol content. Quartz capillary column (0.25 m m *30 m, 0.25 um), carrier gas is high-purity helium, flow rate is 1.0 mL.in-1, no diversion, injection volume is 5 microl, inlet temperature 250, transmission line temperature 280, starting temperature 90, holding for 3 minutes, then 10.M IN-1 to 180, then 3.Mir-1 to 280, holding 10 m in, then 5.M IN-1 to 300. EI was ionized at 70 eV for 8 min. The temperature of ion source was 230 C, the temperature of four-stage rod was 150 C. The scanning mode was full scanning, and the scanning range m/z was 50-550. The volatile oil was identified qualitatively by standard database NIST14 and retention index. According to the analysis, the differences among different types of aloes were analyzed to find out the markers of the differences, and the characteristic components of aloes were classified and compared to analyze the reasons for the internal quality differences. (3) LC-QTOFMS column (InetrSustain Swift C18 column) (2.1 *150 mm, 1.9 mun); flow rate: 0.3 ml/min; column temperature: 40; mobile phase: acetonitrile (A): 0.1% Formic acid aqueous solution (B) gradient elution (0-3 min, 10% A; 3-8 min, 10% - 30% A; 8-25 min, 30% - 50% A; 25-32 min, 50% - 100% A, 32-35 min, 100% A); sample size: 10 muL; ESI ion source, positive ion mode, first-order mass spectrometry (m/z 100-2000, DP100, CE 10, acquisition time: 0.2 s), secondary mass spectrometry (m/z 50-2000, DP100, CE 45, acquisition time: 0.01s) determination of samples. The chemical components were analyzed by Simca-P software after the data were pretreated. The differences between different types of aloes were analyzed to find out the markers of the differences. Meanwhile, the characteristic components of aloes were classified and compared, and the reasons for the internal quality differences were analyzed. 60% and 6.07-57.06% of the total alcohol extract, respectively. There was no correlation between the extract and the way of aroma formation, provenance, provenance, and the way of aroma formation. (2) Based on GCMS, 19 markers of different provenances and flavoring patterns were screened out by multivariate and single-dimensional statistical analysis, which were 5 2-2-phenylethyl chromones and 5 2-phenylethyl chromones, respectively. Sesquiterpenes and other compounds. According to OPLS-DA analysis, there are 13 different markers in A. sinensis artificial and natural aloes, including chromones, sesquiterpenes and alkanes, including 2-(2-phenylethyl) chromones, 6,7-dimethoxy-2-(2-phenylethyl) chromones, 5,8-Dihydroxy-4a-methyl-4,4a, 4b, 5,6,7,8,8a, 9,10-hydrodecane (3H) -phenanthreno ne (phenanthrene) There were significant differences between the two groups in the four components of Omer 1 and isomer 3, two sesquiterpenes in artificial and natural aroma-forming samples of A. crassna, and two different markers in artificial aroma-forming aroma of two different provenances (A. sinensis and A. crassna, artificial aroma-forming aroma), of which 5,8-Dihydroxy-4a-methyl-4, 4a, 4b, 5, 6, 7, 8, 8a, 9, 10-decahydro-2 (3H) - phenanthrenone (isomer1) had significant differences between the two groups; through the comparative analysis of natural aloes from three provenances, there were seven different markers between A. sinensis and A. C. rassna aloes and A. malaccensis aloes, and the significant differences in the marker components were isomer 3. The peak areas of alkanes, sesquiterpenoids and 2-(2-phenylethyl) chromones in 52 samples were classified and statistically analyzed. The results showed that the content of sesquiterpenoids in natural aloes was mainly A.crassna, A.mal accensis and 2-(2-phenylethyl) chromone. The higher was A. crassna A. malaccen sis, while the higher alkanes was A. sinensis. The total peak area of different types of components in A. sinensis showed no significant difference in alkanes between artificial and natural samples, while sesquiterpenes and 2 - (2-phenylethyl) chromone were both present. There was no significant difference between artificial and natural aromas of A. crassn. There was no significant difference between artificial and natural aromas of A. sinensis and A. crassna. There was no significant difference between the two groups of artificial aromas of A. sinensis and A. crassna. There were differences in alkanes and sesquiterpenes in SIS natural aloes, but only differences in alkanes were found between A. sinensis natural aloes and A. C. crassna natural aloes. There was no significant difference between other groups and components. 123 differential markers were identified by multivariate and single-dimensional statistical analysis. 62 compounds were identified by first-and second-order mass spectrometry combined with the existing literature, 23 of which were potential new compounds. 45 differences were found in the same provenance of artificial and natural aloes. Among them, 6 components (2-(2-phenylethyl) chromone, 7-hydroxy-2-(2-phenylethyl) chromone, 6-methoxy-2-(2-phenylethyl) chromone (isomer 2), 6,8-di-light-2-(2-phenylethyl) chromone (isomer 1), 6-methoxy-2-[2-(4'-methoxyphenylethyl) ethyl] chromone, dehydroxy AH21) are artificial precipitation and natural precipitation chromone in A.sinensis. Among them, 29 were the main markers, and 7 were significant differences. The main markers were 6,8-dihydroxy-2-(2-phenylethyl) chromone (isomer1), AH21 (isomer1), methoxy AH21 (isomer1), 2,3-dihydroxy-5-phenylethyl-2,3-dihydro-1ah-oxireno [2,3-f]chromen. 5 components of - 7 (7bh) - one (isomer 2), dehydroxy AH21 (isomer 2). Comparing and analyzing the different provenances of the same way of aroma formation, there were 36 differences between A. sinensis and A. crassna, of which 3 were significantly different. AH12 (isomer 3) was the main marker of the two groups of artificial aroma. There were 35 differences between NSIS and A. malaccensis, 13 of which were significant. The main markers were 6-methoxy-2-[2-(4'-hydroxy-3'-methoxyphenyl) ethyl] tryptone (i Somer 2), 6,8-dihydroxy-2-[2-(3'-hydroxy-4'-methoxyphenyl) ethyl] tryptone (somer 2), aloe tetraol, 6-hydroxy-7-methoxy-2-[2'-hydroxy-3'-hydroxy-3'-hydroxy-3'-hydroxy-ethyl] tryptone (i Somer 2). 5 constituents were isomer 2, 2,3-dihydroxy-5-phenylethyl-2,3-dihydro-1ah-oxireno [2,3-f] chromen-7 (7bh) -one (isomer 1). There were 20 differences between A. crassna and A. malaccensis, and the main marker difference was 6-methoxy-2-[2-(4'-hydroxy-3'-methoxyphenyl) ethyl] one (isomer 1). Isomer 2, 6,8-dihydroxy-2-[2-(3'-hydroxy-4'-methoxyphenyl) ethyl] chromone (isomer 1), in which 6-methoxy-2-[2-(4'-hydroxy-3'-methoxyphenyl) ethyl] chromone (isomer 2) is the main marker difference between A. The results showed that the hydroxyl and methoxy substituted 2-(2-phenylethyl) chromones on the phenyl ring may be the effective markers for the identification of artificial and natural aloes, while 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and bis-2-(2-phenylethyl) chromones are formed. The peak areas of 4 types of 2-(2-phenylethyl) chromones in different groups were compared. The results showed that the differences among the four groups were different, including 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones, bis-2-(phenylethyl) chromones, and tri-2-(phenylethyl) chromones. At the same time, all samples were clustered and classified based on 2-(2-phenylethyl) chromones. Conclusion: Based on GCMS and LCMS techniques, a method for screening the different components of Chinese traditional medicinal herbs was established by combining GCMS with NIST 14 mass spectrometry library. Nineteen different components were identified, of which four were significantly different. Based on LC-ESI-QTOF high-resolution mass spectrometry and literature, 62 different components were identified for the first time, 23 of which were potential new bis-2-(2-phenylethyl) chromones, and significant differences were screened out by multidimensional and single-dimensional statistical analysis. The differences between artificial and natural aloes may be closely related to 2-(2-phenylethyl) chromones with light and methoxy substitutes in the benzene ring; 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and bis-2-(2-phenylethyl) chromones. The contents of alkanes, sesquiterpenoids and 2-(2-phenylethyl) chromones can reflect the internal quality differences of Chinese aroma to some extent. The lower the alkanes, the higher the contents of sesquiterpenoids and 2-(2-phenylethyl) chromones, the higher the content of Chinese aroma. The more the resin content is, the better the quality may be, on the contrary, the worse the quality will be. Through Clustering Study on the intrinsic components of Chinese aloes, artificial and natural Chinese aloes are mostly clustered into one group, and the various groups are clustered into one group. This method can well classify and distinguish different quality of Chinese aloes. New ideas and methods for quality classification.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R284.1
本文编号：2192032
[Abstract]:OBJECTIVE: The resin-containing wood of the genera Aquzlaria and Gyrinops mainly distributes in Guangdong, Hainan, Guangxi, Indonesia, Vietnam, Cambodia, Malaysia and other Southeast Asian countries. A. sinensis, and the imported source plants are mostly Aquilaria malaccensi, Aquilaria crassna. The quality of L. sinensis is closely related to provenance, aroma-forming methods and so on. Although modern molecular identification technology can identify L. sinensis primordia, but there is no effective method to distinguish the samples from the same source with different aroma-forming methods. It is not clear what differences exist among different types of aloes, and no screening method has been established to identify these components. Methods: According to the characteristics of the internal components of Chinese aloes, this paper makes use of modern advanced analytical instruments and statistical methods, uses GCMS and LCMS combined with multivariate statistics and single-dimensional analysis method to analyze the components of Chinese aloes comprehensively, supplemented by principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and other statistical methods to find out if not. Methods: (1) The content of alcohol extract was determined by HPLC (column Al) according to the relevant requirements of the Chinese Pharmacopoeia (2015 edition). TIMA C18 (150 mm x 4.6 mm, 5 micron), mobile phase acetonitrile (A) - 0.1% formic acid water (B), gradient elution, flow rate 0.7 mL min 1, detection wavelength 252 nm, column temperature 30 C) was used to determine the content of agaric tetraol in samples. The difference between different types of agaric incense was preliminarily analyzed by combining the results of alcohol extract and agaric tetraol content. Quartz capillary column (0.25 m m *30 m, 0.25 um), carrier gas is high-purity helium, flow rate is 1.0 mL.in-1, no diversion, injection volume is 5 microl, inlet temperature 250, transmission line temperature 280, starting temperature 90, holding for 3 minutes, then 10.M IN-1 to 180, then 3.Mir-1 to 280, holding 10 m in, then 5.M IN-1 to 300. EI was ionized at 70 eV for 8 min. The temperature of ion source was 230 C, the temperature of four-stage rod was 150 C. The scanning mode was full scanning, and the scanning range m/z was 50-550. The volatile oil was identified qualitatively by standard database NIST14 and retention index. According to the analysis, the differences among different types of aloes were analyzed to find out the markers of the differences, and the characteristic components of aloes were classified and compared to analyze the reasons for the internal quality differences. (3) LC-QTOFMS column (InetrSustain Swift C18 column) (2.1 *150 mm, 1.9 mun); flow rate: 0.3 ml/min; column temperature: 40; mobile phase: acetonitrile (A): 0.1% Formic acid aqueous solution (B) gradient elution (0-3 min, 10% A; 3-8 min, 10% - 30% A; 8-25 min, 30% - 50% A; 25-32 min, 50% - 100% A, 32-35 min, 100% A); sample size: 10 muL; ESI ion source, positive ion mode, first-order mass spectrometry (m/z 100-2000, DP100, CE 10, acquisition time: 0.2 s), secondary mass spectrometry (m/z 50-2000, DP100, CE 45, acquisition time: 0.01s) determination of samples. The chemical components were analyzed by Simca-P software after the data were pretreated. The differences between different types of aloes were analyzed to find out the markers of the differences. Meanwhile, the characteristic components of aloes were classified and compared, and the reasons for the internal quality differences were analyzed. 60% and 6.07-57.06% of the total alcohol extract, respectively. There was no correlation between the extract and the way of aroma formation, provenance, provenance, and the way of aroma formation. (2) Based on GCMS, 19 markers of different provenances and flavoring patterns were screened out by multivariate and single-dimensional statistical analysis, which were 5 2-2-phenylethyl chromones and 5 2-phenylethyl chromones, respectively. Sesquiterpenes and other compounds. According to OPLS-DA analysis, there are 13 different markers in A. sinensis artificial and natural aloes, including chromones, sesquiterpenes and alkanes, including 2-(2-phenylethyl) chromones, 6,7-dimethoxy-2-(2-phenylethyl) chromones, 5,8-Dihydroxy-4a-methyl-4,4a, 4b, 5,6,7,8,8a, 9,10-hydrodecane (3H) -phenanthreno ne (phenanthrene) There were significant differences between the two groups in the four components of Omer 1 and isomer 3, two sesquiterpenes in artificial and natural aroma-forming samples of A. crassna, and two different markers in artificial aroma-forming aroma of two different provenances (A. sinensis and A. crassna, artificial aroma-forming aroma), of which 5,8-Dihydroxy-4a-methyl-4, 4a, 4b, 5, 6, 7, 8, 8a, 9, 10-decahydro-2 (3H) - phenanthrenone (isomer1) had significant differences between the two groups; through the comparative analysis of natural aloes from three provenances, there were seven different markers between A. sinensis and A. C. rassna aloes and A. malaccensis aloes, and the significant differences in the marker components were isomer 3. The peak areas of alkanes, sesquiterpenoids and 2-(2-phenylethyl) chromones in 52 samples were classified and statistically analyzed. The results showed that the content of sesquiterpenoids in natural aloes was mainly A.crassna, A.mal accensis and 2-(2-phenylethyl) chromone. The higher was A. crassna A. malaccen sis, while the higher alkanes was A. sinensis. The total peak area of different types of components in A. sinensis showed no significant difference in alkanes between artificial and natural samples, while sesquiterpenes and 2 - (2-phenylethyl) chromone were both present. There was no significant difference between artificial and natural aromas of A. crassn. There was no significant difference between artificial and natural aromas of A. sinensis and A. crassna. There was no significant difference between the two groups of artificial aromas of A. sinensis and A. crassna. There were differences in alkanes and sesquiterpenes in SIS natural aloes, but only differences in alkanes were found between A. sinensis natural aloes and A. C. crassna natural aloes. There was no significant difference between other groups and components. 123 differential markers were identified by multivariate and single-dimensional statistical analysis. 62 compounds were identified by first-and second-order mass spectrometry combined with the existing literature, 23 of which were potential new compounds. 45 differences were found in the same provenance of artificial and natural aloes. Among them, 6 components (2-(2-phenylethyl) chromone, 7-hydroxy-2-(2-phenylethyl) chromone, 6-methoxy-2-(2-phenylethyl) chromone (isomer 2), 6,8-di-light-2-(2-phenylethyl) chromone (isomer 1), 6-methoxy-2-[2-(4'-methoxyphenylethyl) ethyl] chromone, dehydroxy AH21) are artificial precipitation and natural precipitation chromone in A.sinensis. Among them, 29 were the main markers, and 7 were significant differences. The main markers were 6,8-dihydroxy-2-(2-phenylethyl) chromone (isomer1), AH21 (isomer1), methoxy AH21 (isomer1), 2,3-dihydroxy-5-phenylethyl-2,3-dihydro-1ah-oxireno [2,3-f]chromen. 5 components of - 7 (7bh) - one (isomer 2), dehydroxy AH21 (isomer 2). Comparing and analyzing the different provenances of the same way of aroma formation, there were 36 differences between A. sinensis and A. crassna, of which 3 were significantly different. AH12 (isomer 3) was the main marker of the two groups of artificial aroma. There were 35 differences between NSIS and A. malaccensis, 13 of which were significant. The main markers were 6-methoxy-2-[2-(4'-hydroxy-3'-methoxyphenyl) ethyl] tryptone (i Somer 2), 6,8-dihydroxy-2-[2-(3'-hydroxy-4'-methoxyphenyl) ethyl] tryptone (somer 2), aloe tetraol, 6-hydroxy-7-methoxy-2-[2'-hydroxy-3'-hydroxy-3'-hydroxy-3'-hydroxy-ethyl] tryptone (i Somer 2). 5 constituents were isomer 2, 2,3-dihydroxy-5-phenylethyl-2,3-dihydro-1ah-oxireno [2,3-f] chromen-7 (7bh) -one (isomer 1). There were 20 differences between A. crassna and A. malaccensis, and the main marker difference was 6-methoxy-2-[2-(4'-hydroxy-3'-methoxyphenyl) ethyl] one (isomer 1). Isomer 2, 6,8-dihydroxy-2-[2-(3'-hydroxy-4'-methoxyphenyl) ethyl] chromone (isomer 1), in which 6-methoxy-2-[2-(4'-hydroxy-3'-methoxyphenyl) ethyl] chromone (isomer 2) is the main marker difference between A. The results showed that the hydroxyl and methoxy substituted 2-(2-phenylethyl) chromones on the phenyl ring may be the effective markers for the identification of artificial and natural aloes, while 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and bis-2-(2-phenylethyl) chromones are formed. The peak areas of 4 types of 2-(2-phenylethyl) chromones in different groups were compared. The results showed that the differences among the four groups were different, including 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones, bis-2-(phenylethyl) chromones, and tri-2-(phenylethyl) chromones. At the same time, all samples were clustered and classified based on 2-(2-phenylethyl) chromones. Conclusion: Based on GCMS and LCMS techniques, a method for screening the different components of Chinese traditional medicinal herbs was established by combining GCMS with NIST 14 mass spectrometry library. Nineteen different components were identified, of which four were significantly different. Based on LC-ESI-QTOF high-resolution mass spectrometry and literature, 62 different components were identified for the first time, 23 of which were potential new bis-2-(2-phenylethyl) chromones, and significant differences were screened out by multidimensional and single-dimensional statistical analysis. The differences between artificial and natural aloes may be closely related to 2-(2-phenylethyl) chromones with light and methoxy substitutes in the benzene ring; 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and bis-2-(2-phenylethyl) chromones. The contents of alkanes, sesquiterpenoids and 2-(2-phenylethyl) chromones can reflect the internal quality differences of Chinese aroma to some extent. The lower the alkanes, the higher the contents of sesquiterpenoids and 2-(2-phenylethyl) chromones, the higher the content of Chinese aroma. The more the resin content is, the better the quality may be, on the contrary, the worse the quality will be. Through Clustering Study on the intrinsic components of Chinese aloes, artificial and natural Chinese aloes are mostly clustered into one group, and the various groups are clustered into one group. This method can well classify and distinguish different quality of Chinese aloes. New ideas and methods for quality classification.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R284.1
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