Research On The Pretreatment Of Secondary Metabolites In Plants And The Imaging Of Lipid Metabolites In Neuroembryos

With the development of the research in metabolites,it is increasingly recognized that metabolite research is of great significance to life and health.For example,secondary metabolites in plant are important source for drug development,and many diseases are closely related to abnormalities in lipid metabolites.Therefore,metabolite analysis has resulted in a great interest in recent years.In this thesis,sample preparation of secondary metabolites in plant and mass spectrometry imaging of lipid metabolites in Neurula were studied.The pretreatment of sample has been playing a dominant role in the analysis of secondary metabolites,especially the extraction.Conventional extraction methods are slow,inefficient,and complicated in operation,and the temperature changes during the extraction process may cause inactivation or decomposition of unstable metabolites.Although the methods of pretreatment evolve all the time,there is still a need to explore a novel method to overcome the shortcomings of traditional method.A novel and green solvent assisted mechanochemical extraction(MCE)was developed for extraction of secondary metabolites in plant.MCE,a room temperature mechanical treatment,with small ceramic balls inside extracted tube,used in this system can provide a unique motion to disrupt biomolecular lipid layers and cell walls using a multidirectional,simultaneous mechanical in-liquid smashing with specialized beads on the sample to achieve full release of bioactive compounds into the surrounding solvents for extraction.Compared to other extraction methods,it saves time during the sample preparation stage,provides higher extraction yields,decreases the consumption of solvents and gentle.Meanwhile,some cellulose or lignin of the cell wall may dissolve in Ionic liquid(IL)and Deep eutectic solvent(DES).In this study,IL-MCE and DES-MCE were developed as a novel method for sample pretreatment.It is a room-temperature extraction method that can prevent structural alteration.Tanshinones from Salvia Miltiorrhiza Bunge(SMB)and bioactive compounds including alkaloids,flavonoids,and catechins from tea were selected to evaluate the feasibility of this method.The results showed that the extractions can be completed within dozens of seconds.Moreover,MCE-based method is more ecofriendly,faster,gentler,and more efficient than conventional methods.For many compounds,we could simply use the method without optimization.On the other hand,various compounds can be extracted from various interferences because of high extraction efficiency.Lipid metabolites are up-regulated during the early stage of development of the nervous system.The Neurula stage is characterized by the start of development of the nervous system.Lipid metabolites play an important role in the neural embryo stage.So far,the distribution of lipid metabolites in Neurula has not been studied.In this study,Xenopus laevis was selected as model to perform the lipid metabolites distribution in Neurula.We present the first MALDI mass-spectrometry images of metabolites within the developing embryo.These images were generated at 133 m/z values.The images were subjected to clustering algorithm,which revealed a remarkably consistent co-localization of the metabolites between slices and stages of development.Three clusters of metabolites were identified.One small cluster is localized near the outer membrane of the embryo and appears to be contaminants or the compounds related to the outer membrane.A large cluster of metabolites was found in cavities destined to form the neural tube and gut,and contains a number of ceramide species,which tend to be associated with cellular signaling,including differentiation,proliferation,and programmed cell death.Another large cluster of metabolites is found in tissue and is dominated by phosphatidylcholines,which are common components of cell membranes.Surprisingly,few metabolites appear to be homogeneously distributed across the slices;all of these metabolites are localized either within tissue or in cavities.The study of spatial and temporal distribution and identification of lipids in neurula stage embryos can produce a deeper understanding of their function in developmental process.