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Metabolomic approach for the discrimination of species, age and processing of ginseng by UPLC-QTOF MS and GC-MS

DC Field Value Language
dc.contributor.advisor김정한-
dc.contributor.author박희원-
dc.date.accessioned2017-07-13T08:20:26Z-
dc.date.available2017-07-13T08:20:26Z-
dc.date.issued2014-02-
dc.identifier.other000000018230-
dc.identifier.urihttps://hdl.handle.net/10371/119460-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 김정한.-
dc.description.abstractAn ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS) based metabolomic approach was developed in different species, steaming process and cultivation period of ginseng, respectively. And simultaneous determination method of 30 ginsenosides was established for metabolomic analysis data confirmation and getting basic information of ginseng samples using ultra performance liquid chromatography (UPLC). For the separation of 30 ginsenosides, optimization of eluent condition of UPLC and method validation was performed. The detection limits were 0.4 ~ 1.7 mg/L and calibration curves of peak area for the 30 ginsenosides were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 89-118%.
Metabolomic approach was performed in order of decreasing phenotype difference of ginseng because it was hard to discriminate groups having similar phenotypes. At first, the validity of metabolomic approach method was verified by comparison to targeted analysis result of different ginseng species.
As a result, all known biomarkers, ginsenoside Rf and pseudoginsenoside F11 were identified by the proposed metabolomic method and additional potential biomarker, 20-gluco-ginsenoside Rf was extracted from the huge amounts of global analysis data.
Metabolomic analysis of in-situ steaming process samples, fresh ginseng, steamed ginseng and red ginseng, was performed with established method. Principal component analysis (PCA) result showed clear separation between three types of ginseng, it means that there were substantial differences in chemical compositions according to steaming process. Four potential markers, (ginsesnoside 20(S)-Rg2, 20(R)-Rh1, Rh4 and arginyl-fructose-glucose), were identified. Targeted analysis of arginyl-fructose-glucose represent arginyl-fructose-glucose (AFG) generated in steaming process. There was no AFG in fresh ginseng and the average contents of AFG in steamed ginseng and red ginseng was 26.15 ± 6.99 and 65.90 ± 13.10 mg/g, respectively.
Metabolomic analysis of fresh ginseng roots by GC-MS clearly indicates that there are substantial differences in non-polar metabolites compositions according to cultivation period (4-6 years). PCA of non-polar metabolites showed clear separation between 4 and 6 years old fresh ginseng. Major constituents in non-polar fraction were polyacetylenes (30%), free fatty acids (38%), and monoacyl glycerols (17%). Short chain organic acids, terpens, and plant steroid comprised approximately 3% of total metabolites. Correlation analysis between metabolites suggested that some fatty acids (C18:2) and monoacylglycerol are important precursors of ginseng polyacetylenes. In addition, strong positive correlation between dehydrocrepenynic acid and others polyacetylenes precursors indicates that polyacetylene biosynthesis in ginseng root may follow the previously reported pathways in other Araliaceae plants.
Finally, UPLC-QTOF MS based metabolomic approach was performed to differentiate of processed ginseng (red ginseng) with different cultivation period (4 and 6 year). Multivariate analysis, including PCA and orthogonal partial least squared discriminant analysis (OPLS-DA) of metabolites showed clear separation between 4 and 6 years old ginseng roots. Ginsenoside malonyl Rb1 was confirmed that important metabolites of this differentiation. In targeted analysis, the contents of ginsenoside malonyl Rb1 were high in all of the 6 years cultivation samples than in 4 years samples. The average contents of malonyl ginsenoside were 1.84 and 2.58 mg/g at 4 year and 6 year group respectively.
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dc.description.tableofcontentsABSTRACT i
CONTENTS iv
LIST OF FIGURES viii
LIST OF TABLES xi
INTRODUCTION 1

I. Simultaneous determination of 30 ginsenosides in Panax ginseng preparations using UPLC 1

1. Purpose of the present study 10

2. Materials and methods 12
2.1 Materials 12
2.1.1 Chemicals and Reagents 12
2.2 Methods 13
2.2.1 Sample preparation 13
2.2.2 Chromatographic condition 13
2.2.3 Validation of developed method 14

3.Result and discussion 16
3.1 Optimization of extraction condition 16
3.2 Optimization of UPLC-PDA conditions 16
3.3 Validation of the developed method 21
3.4 Analysis of 30 ginsenosides in Panax ginseng preparations 27

II. Metabolomic approach for the discrimination of processed ginseng species (Panax ginseng and Panax quiquefolius) 31

1. Purpose of the present study 31

2. Materials and methods 33
2.1 Materials 33
2.1.1 Ginseng samples 33
2.1.2 Chemicals and Reagents 33
2.2 Methods 34
2.2.1 Sample preparation 34
2.2.2 Liquid chromatography 34
2.2.3 Mass spectrometry 35
2.2.4 Multivariate analysis 36

3. Results and discussion 37
3.1 Targeted analysis 37
3.2 Non-targeted analysis 41

III. Metabolomic approach for in-situ monitoring of chemical changes in Panax ginseng during steaming process 49
1. Purpose of the present study 49
2. Materials and methods 51
2.1 Materials 51
2.1.1 Ginseng samples 51
2.1.2 Chemicals and Reagents 51
2.2 Methods 52
2.2.1 Malonyl ginsenosides analysis 52
2.2.2 Amino-sugar analysis 53
2.2.3 Liquid chromatography and QTOF MS conditions 54
2.2.4 Multivariate analysis 55

3. Results and discussion 57
3.1 Non-targeted analysis 57
3.2 Targeted analysis 66

IV. Comparative metabolomics for Panax ginseng of different cultivation period 71

1. Purpose of the present study 71

2. Materials and methods 72
2.1 Materials 72
2.1.1 Ginseng samples 72
2.1.2 Chemicals and Reagents 72
2.2 Methods 73
2.2.1 Sample preaparation for GC-MS 73
2.2.2 Sample preparation for identifying dehydrocrepenate 74
2.2.3 GC-MS condition for analyizing non-polar metabolites 74
2.2.4 GC-MS condition for identifying dehydrocrepenate 75
2.2.5 Sample preparation for UPLC-QTOF MS 75
2.2.6 UPLC-QTOF MS data processing and statistical analyses 76
2.2.7 UPLC-QTOF MS condition for analyzing metabolites 77
2.2.8 Malonyl ginsenosides analysis 78

3. Results and Discussion 80
3.1 GC-MS analyses of non-polar metabolites of fresh ginseng 80
3.2 Statistical analyses of non-polar metabolite profiles 89
3.3 Biosynthesis of panaxynol and panaxydol 90
3.4 Nontargeted UPLC-QTOF MS analysis of processed ginseng sample with different cultivation period 96
3.5 Targeted analysis 100

CONCLUSIONS 103
REFERENCES 107
ABSTRACT IN KOREAN 125
CURRICULUM VITAE 127
PUBLICATIONS 128
ACKNOLEDGEMENTS 131
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dc.formatapplication/pdf-
dc.format.extent21650267 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectmetabolomics-
dc.subjectUPLC-QTOF MS-
dc.subjectGC-MS-
dc.subjectginseng species-
dc.subjectin-situ steaming process-
dc.subjectage of ginseng-
dc.subject.ddc630-
dc.titleMetabolomic approach for the discrimination of species, age and processing of ginseng by UPLC-QTOF MS and GC-MS-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesxi, 131-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2014-02-
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