S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Ph.D. / Sc.D._농생명공학부)
Metabolomic approach for the discrimination of species, age and processing of ginseng by UPLC-QTOF MS and GC-MS
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- "metabolomics"; "UPLC-QTOF MS"; "GC-MS"; "ginseng species"; "in-situ steaming process"; "age of ginseng"
- 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 김정한.
- An 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.