Enzymatic Hydrolysis Of Theasaponins And The Structural Analysis Of New Theasaponins

Theasaponins have good surface activity and bioactivity, and there is a close relationbetween the structure and the activity. Studies on theasaponin indicate that structure oftheasaponins influence their activities. In order to obtain saponins with high activities, theenzyme hydrolysis of theasaponin were studied in this paper. Enzyme produced frommicrobial strain of Aspergillus Niger sp.48s was used to hydrolyze theasaponin. The conditionof enzyme hydrolysis was studied through orthogonal design with four factors and four levels.The theasaponins from seeds of camellia oleifera were purified by AB-8resin. Theasaponinswere determined by Thin Layer Chromatography （TLC） and High Pressure LiquidChromatography （HPLC）. The chemical structures of new theasaponins were determined byLC-Q-TOF-MS.The purification of theasaponins was finished by AB-8resin. The effect of concentrationsof eluting reagent and NaOH were studied. The optimal process of purification has beenobtained, which is80%eluting alcohol concentration and0.1%eluting NaOH concentration.The optimal pH, temperature, time and theasaponin concentration was6.0,45℃,10h and6.0mg/ml respectively in enzyme hydrolysis. The saponins were analyzed by TLC and HPLC,and two new theasaponins were obtained.The new saponins were analyzed by LC-Q-TOF-MS/MS to determinate the chemicalstructure. Molecular weight1120、1262、1232、1190and1202were determinated in the theasaponins. They are Desacyl-camellisaponin B, Theasaponin A5, Theasaponin A2, TheasaponinA1and Camellisaponin B1respectively. The molecular weight of two new saponins was967and981respectively. They were products of Theasaponin A2or Theasaponin A5hydrolyzedby enzyme from sp.48s. The molecular formula was C₄₉O₂₂H₇₃and C₄₉O₁₉H₇₅respectively.The structure of981was3-O-[β-D-glucopyranosiduronic acid-（1-2）-β-D-glucopyranosiduronic acid]21-O-angeloyl-28-O-acetyltheasapogenol A. It was produced from Theasaponin A2,which was hydrolyzed to lose β-D-xylopyranosyl, α-L-arabinopyranosyl andβ-D-galactopyranosyl and link β-D-glucopyranosiduronic acid. Or it was produced fromTheasaponin A5, which was hydrolyzed to lose β-D-galactopyranosyl, β-D-glucopyranosy and α-L-arabinopyranosyl and link β-D-glucopyranosiduronic acid. The structure of967was3-O-[β-D-glucopyranosiduronic acid-（1-2）-β-D-galactopyranosyl]21-O-angeloyl-28-O-acetyltheasapogenol A. It was produced from Theasaponin A2, which hydrolyzed to loseβ-D-xylopyranosyl and α-L–arabinopyranosyl, or Theasaponin A5was hydrolyzed to loseβ-D-glucopyranosy and α-L-arabinopyranosyl.