Plant metabolomics for classification of two Isodon medicinal plants and red ginseng

Abstract

Traditional Chinese medicine (TCM) is the traditional medicine used in China mainly based on plant materials. The quality control of TCM is a topic of current concerns. The difficulty mainly originates from the chemical compositions of plants that are highly diverse due to the difference in species, habitat, climate etc. and in some cases, multi-species are used in the same herb. Plant metabolomics is the holistic study of metabolite profiles in plants in response to genetic or environmental changes. This thesis aimed to apply metabolomics to provide evidence to support the development of quality control methods for TCM. Two applications were selected to demonstrate that metabolomics could be used to devise effect quality control. The two applications were 1) species differentiation of Isodon Herba and the establishment of quality control for the two species of Isodon Herba, and 2) discrimination of the geographical location of Radix ginseng rubra (red ginseng) using plant metabolomics. In Chinese Pharmacopeia, there are two botanical sources of Isodon Herba; Isodon lophanoides (Buch.-Ham. ex D. Don) H. Hara and I. serra (Maxim.) Kudo. These two species are commonly known as sweet Xihuangcao and bitter Xihuangcao. Samples from each botanical source were collected and authenticated. Untargeted metabolomics profiling using UPLC-Orbitrap-MS was used to compare the metabolite composition of the two species. With reference to online databases, in-house library and standards, a number of diterpenoids, such as nodosin, gerardianin A and horminone, other compounds e.g. rosmarinic acid were found to contribute the differences in the chemical compositions of the two species. Based on the metabolomics study, nodosin and rosmarinic acid were proposed to serve as chemical markers for the quality control of Isodon Herba. An HPLC-DAD quantitative analysis method was developed to separate and quantify nodosin and rosmarinic acid in samples. The HPLC-method could be used for the quality control of Isodon Herba. The second study was the discrimination of the geographical origin of red ginseng. As red ginseng from South Korea has higher market price, a method that could differentiate the difference between the red ginseng from South Korea and China would be essential to prevent the adulteration of red ginseng from different production site on the market. Currently the study of ginseng mainly focuses on the ginsenoside content. Although ginsenoside content varies during the steaming process of red ginseng, there is no specific ginsenoside that is only present in Korean ginseng that people can use to identify Korean ginseng. Plant metabolomics with multiple analytical techniques of GM-MS and UPLC-Orbitrap-MS is employed to obtain information on the primary and secondary metabolites that could be used to identify Korean ginseng. Samples of red ginseng from China and South Korea were first extracted using liquid-liquid extraction method (LLE) to analyze their primary metabolites. The aqueous layer from LLE was analyzed subsequently using GC-TOF-MS after derivatization. The organic layer from LLE was analyzed using UPLC-Orbitrap-MS for lipids and non-polar molecules. A separate set of the methanol extracts of all the samples was prepared for secondary metabolites analysis. Multivariate analysis was carried out to identify the key metabolites for differentiating between the red ginsengs from these two locations. The result showed that ginsenosides G-Rg1, G-Re, G-Rb1, G-Rf, gypenoside XVII and chikusetsusaponin IVa, AFG, arginine, pyroglutamic acid, β-D-glucopyranose, and fatty acids PC(16:1/18:2), PI(16:0/18:1), PI(16:0/18:2), PA (16:0/18:2) and AS 1-5 (glycosyl-n-acylsphingosines) could be tentatively identified as the metabolites to differentiate the red ginseng from these two origins. It is concluded that plant metabolomics is a powerful tool in revealing metabolic changes from the complex chemical profiles in plants, and it could be a tool to solve the challenges on the quality control of TCM.