Research On Phenolic Compounds In Fresh Tobacco Leaves

Twenty-five phenolic compounds in tobacco were analyzed using high performance liquid chromatography-ultraviolet/mass spectrometry (HPLC-UV/MS), including absolute quantification with internal standard curve method of11main phenols by HPLC-UV and relative quantification with internal calibration of other14phenols by HPLC-MS. The11main phenols were separated on a Symmetry C18(250mmx4.6mm,5μm) column using methanol/water/acetic acid (5:44:1, v/v/v) and water/methanol/acetic acid (5:44:1, v/v/v) as mobile phase by gradient elution, and identified at UV340nm. The other14phenols were separated on an Agilent SB-AQ (100mmx2.1mm,1.8μm) column and detected by Electrospray Ionization-Quadrupole-Time of Flight Mass (ESI-Q-TOF). Gradient elution was employed with a mobile phase consisting of acetonitrile and water (containg0.1%acetic acid). Eleven phenol standards showed linearity with correlation coefficients (r2) of0.9993-0.9999over the mass concentration range from0.90to99.00μg/mL (the range of chlorogenic acid and rutin was from0.95to380.00μg/mL). The recoveries of the11phenol standards were91.0%-112.4%with the RSD of0.33%-8.11%at the levels of22.5-24.8,45.0-49.5and67.5-74.3μg/mL. The RSD of the reproducibility of the method was1.48%-13.40%. In addition, the intra-day and inter-day precision were also detected with the RSD of0.35%-15.54%. This method has been applied in batch sample analysis of tobacco leaves.Data of phenols in94samples of full aroma, fresh aroma and medium aroma mature fresh tobacco leaves was analysised by SIMCA-P11.5and SPSS16.0. Partial least squares discriminant analysis (PLS-DA) showed a clear separation of the three flavors, and full aroma was closer to medium aroma, fresh aroma samples into a separate class. Hierarchical clustering (HCA) got the same results. Non-parametric test (p<0.05) and PLS-DA (VIP>1) were combined to get12phenolic metabolites which is different among different flavors. Besides that, correlation analysis between phenolic compounds and climatic conditions of growing period showed that Quercetin-O-rhamnoglucoside, Quercetin-O-rhamnoglucoside II, Kaempferol-3-O-rutinoside, Kaempferol-3-O-rutinoside Ⅱ and Scopolin were significantly associated with climatic conditions (Pearson correlation coefficient r>0.6).Phenolic compounds in197samples of fresh tobacco leaves of different growth stages were researched. These samples were from three kinds of tobacco, Hongda, K326and Zhongyan100, and three different origins, Guizhou, Yunnan and Henan. Genetic factors, environmental factors and the interaction between Genetic and environmental factors had different impacts on accumulation of phenolic compounds. The PLS-DA scores plot and the variation curve which described how content of phenols changed with growth period showed that the accumulation rule of phenolic compounds of different varieties of tobacco leaves from the same origin was discriminate, but the discrimination was more apparent among the same variety samples from different origns, which illustrated the influence of environmental factors on the accumulation of phenolic compounds was more significant than genetic factors.A quality discrimination method based on phenolic compounds was established to distinguish fresh tobacco samples for metabolomics studies. The ratio of Rutin and Kaempferol-3-O-rutinoside content was set as a criterion for distinguishing qualified samples, metamorphic unqualified samples and partially metamorphic unqualified samples. The samples with a ratio of Rutin and Kaempferol-3-O-rutinoside content above6.4were qualified, which could be used for metabolomics analysis. The samples with the ratio below2.0were metamorphic unqualified, and the samples with the ratio between2.0and6.4were partially metamorphic unqualified. All350samples were distinguished using this method, metamorphic samples accounting for8.28%, partially metamorphic samples accounting for6.86%, and qualified samples accounting for84.28%.