Studies On The Chromatography-mass Spectrometry Analysis And Isolation Of Glycosidic Aroma Precursors In Tea

Glycosidic aroma precursors are a kind of secondary metabolites in the leaves ofCamellia sinensis during growth,which have low content and no aroma.The aglyconeswill released from their respective glycosides by endogenous enzymatic hydrolysisunder the conditions of leaf plucking, pest damage, tissue damage or malady infectionetc.The released volatile aglycones demonstrate the specific odor,which are the basicpart of tea aroma.It is difficult to obtain standard sample of glycosidic aromaprecursors by the separation processing due to its low content.So the quantitativeanalysis can not be carried out,which restrict the depth of research in this field.In thispaper,a series methods adopted to identify and isolate the glycosidic aroma precursorsin tea leaves provided a valuable tool for the establishment of quantitative analysismethod and the preparative separation of glycosidic aroma precursors.They aresummarized as follows:1.The extraction and enrichment of glycosidic aroma precursorsThe components of green tea were successively extracted with petroleumether,dichloromethane,ethyl acctate,methanol by system solvent in order ofincreasing polarities by ultrasonic wave.The volatile aglycones by gaschromatography-mass spectrometry analysis released from the four different extractsunder the condition of enzymatic hydrolysis showed that the methanol extractenriched glycosidic aroma precursors. The fractions obtained from methanol extractseparated over a HPD-500macroporons resin by eluting gradiently with differentmethanol concentrations. The volatile aglycones were monitored by gaschromatograpy-mass spectrometry released from these fractions under the conditionof enzymatic hydrolysis.The results showed that glycosidic aroma precursors wereenriched in the fraction eluted by20%methanol and they can be accumulated bymacroporons(HPD-500) with excellent efficiency.Caffeine from20%methanolfraction was removed by extracting with chloroform and tea polyphenols wereprecipitated by utilizing lead subacetate.2.The identification of glycosidic tea aroma precursors①A qualitative method for the identification of eleven glycosidic tea aromaprecursors was developed by using time of flight massspectrometer(TOF/MS)providing the exact molecular mass and molecular formula and high performanceliquid chromatography-electrospray ionization multistage tandem mass spectrometry(HPLC-MSn).②We summarize the cracking law of glycosidic tea aroma precursors in the ESI(-) ion source mode.3.The isolation and purification of glycosidesChemical constituents were isolated by chromatograph of silica gel,ODS,sepadexLH-20,etc.The structures of isolated compositions were characterized byspectroscopicanalysis,six of which have been identified as three glycosides:kaempferol-3-glucoside(4),coniferin(5),adenosine(6);three nonglycosides: theobromine(1),3,5-dihydroxy-4-methoxy-benzoic acid(2),gallic acid(3).Compound5was isolated fromgreen tea for the first time.