Separation,Identification And Quantification Of Triterpene Saponins In Seeds Of Camellia Sinensis

Saponins extracted from tea（Camellia sinensis）are important bioactive compounds,and they have attracted the attention of scientists all over the world.However,the separation and identification of saponin monomers are challenging because their chemical properties and molecular stuctures are very similar.In this study,UPLC-Q-TOF/MS/MS was applied to analyze the saponins extracted from tea（Camellia sinensis）seeds,and 51 different kinds of saponins were identified.6 saponin monomers were purified.In addition,an UPLC-PDA quantification method for tea seed saponins was developed.The main results are as follows:1.An UPLC-Q-TOF/MS analysis method was firstly established for tea seed saponins.The chromatographic separation of tea seed extracts was performed on a Waters UPLC HSS T₃（150 mm×2.1 mm,i.d.,1.8μm）.The mobile phase consisted of water 0.1%formic acid（A）and acetonitrile containing 0.1%formic acid（B）at a flow rate of 0.2 mL/min.A gradient elution was applied for chromatographic separation:0 min:35%B;4 min:37%B;32 min:37%B;58 min:45%B;60 min:35%B.The column temperature was set at 25℃,the injection volume was 5μL and the detection wavelength was 210 nm.51 different monomers of saponins were isolated and identified by UPLC-Q-TOF/MS/MS.Among the 51 monomers,6 monomers have not been reported in tea seeds.2.An efficient and stable preparation method with twice elution was established to purify 6 known saponin monomers using medium-low pressure preparative chromatography.At the first stage,the elution time is 26 min and the total yield is86.09%;at the second stage,the elution time is 60 min and the total yield is 92.0%.The purity of the 6 monomers are as follows:1,Assamsaponin G/Theasaponin E₁₂,92.2%;2,Theasaponin E₁,100%;3,Theasaponin B₅,96.16%;4,Assamsaponin A,98.88%; 5,Assamsaponin B/C or Theasaponin E₅/E₉,91.56%;6,Theasaponin E₂/E₄/E₇/E₈,92.43%。3.Theasaponin E₁ was used as a standard compound to establish the standard curve for quantification of tea seed saponins.The linear regression equation is y=8233.9x+43043（R²=0.9994）.The limits of detection and quantitation were0.03μg/m L and 0.09μg/m L,respectively.The linear range of the curve is between 0and 1000μg/m L.The saponin content in 70%methanol extract of tea seeds was determined to be 22.31%.