Study on Difference of Tea Cultivars Planted in Xinyang by Non-targeted Metabolomics with Chemometrics Based on Ultrahigh Performance Liquid Chromatography£­High Resolution Mass Spectrometry

KeyWord:tea cultivar  ultrahigh performance liquid chromatography£­high resolution mass spectrometry  non-targeted metabolomics  chemometrics  automatic data analysis strategy

Abstract:

      A non-targeted metabolomics with automatic chemometric data analysis platform based on ultrahigh performance liquid chromatography£­high resolution mass spectrometry£¨UHPLC£­HRMS£© was developed for investigation of the differences of chemical components in five tea cultivars£®In this study£¬a total number of forty£­five fresh tea samples £¨one bud and one leaf£© from five cultivars£¬ie£®Wuniuzao£¬Shuchazao£¬Baihaozao£¬Pingyangtezao and Shaancha 1 were collected directly from the same tea garden in Xinyang£¬Henan province of China£¬which is famous for the main producing area of Xinyang Maojian green tea£®The samples were ultrasonically extracted with methanol£­water £¨1¡Ã9£¬by volume£© for 30 min£¬and centrifuged twice£®The supernatant was separated on a Waters Acquity UPLC HSS T3 £¨2.1 mm ¡Á 100 mm£¬1.8 ¦Ìm£© column by gradient elution£¬with acetonitrile£­water£¨both with 0.1% formic acid£© as mobile phase£®The analysis was performed using a UHPLC£­Q Exactive£­HRMS system with an injection volume of 1 ¦ÌL£®The mass spectra was acquired using an ESI source in the FULL MS/DD£­MS2 £¨TOP4£© under positive ionization mode over the range of mass to charge£¨m/z£© 120£­1 000£®The automatic non-targeted data analysis strategy was introduced to screen the metabolites that expressed significantly among different cultivars£®The chromatographic and mass spectrographic features of the metabolites could be extracted and registered automatically£®Meanwhile£¬metabolite identification was performed based on high-resolution mass values and mass spectra of screened peaks£®The raw UHPLC£­HRMS files were analyzed in the automatic data analysis platform and a set of thirty£­three metabolites were identified£¬including one alkaloid£¬five amino acids£¬three catechins£¬four phenolic acids£¬six nucleotides£¬and fourteen flavonoids£¬which primarily enable the varietal discrimination£®Finally£¬hierarchical clustering analysis £¨HCA£©£¬partial least squares£­discriminant analysis £¨PLS£­DA£© and heatmap analysis were used for discrimination of the samples from various tea cultivars£®The results showed that each cultivar has its own characteristics£¬in which Wuniuzao£¬Shuchazao and Baihaozao show a certain similarity to each other£¬but obviously different with the other two cultivars£®Furthermore£¬the metabolites of five tea cultivars are significantly different under the same growth environment and agronomic conditions£®Wuniuzao has higher levels of £¨£­£©-epigallocatechin and other eight flavonoids including isoquercetin£¬quercetin-3-O-xyloside and naringenin£¬etc£®Shuchazao has higher levels of catechins and nucleotides£®Baihaozao has higher levels of phenolic acids£®Shaancha 1 has higher levels of theobromine£¬phenolic acids and flavonoids£®The contents of theanine and arginine in Pingyangtezao were significantly higher than the other four varieties£®The results obtained could offer a theoretical guidance for the quality evaluation£¬breeding and promotion of tea cultivars£®