Study On Isolation, Purification, Identification Of Structure Of The Isoflavones From Puerariae Flos

Puerariae flos is the dried bud of Pueraria lobata or pueraria thomsonii.It is a traditional Chinese herbal medicine,and can be used in therapy to counteract problems associated with alcohol drinking.Isoflavones such us kakkalide and tectoridin as main components in the Puerariae flos,has the most potent inhibitory activities against ethanol, hepatic injueries and resistance to menopause in the traditional medicine clinic.Therefore, it is interesting to find an effective method to prepare isoflavones from Pueraia lobata flowers.The technologies of the extraction and purification of the total isoflavones from Puerariae flos were studied in this thesis.The chemical and spectral methodologies(UV, HPLC-MS,¹H NMR,¹³C NMR) were used to research the structure of isoflavones. Meanwhile primary standards for contents of total isoflavonoids and tectoridin of Pueraia lobata flowers had been established.1.Optimization for extraction process of isoflavones in Puerariae flos。Extraction of isoflavones from Puerariae flos using n-butanol/water two-phase solvent system as solvents was studied.The optimal conditions for isoflavones extraction were determined by response surface methodology.Box-Behnken design was applied to evaluate the effects of four independent variables（extraction time,temperature,n-butanol/water composition and solvent-solid ratio） on the isoflavones yield of Puerariae flos. Correlation analysis of the mathematical-regression model indicated that a quadratic polynomial model could be employed to optimise the n-butanol/water two-phase solvent system extraction of isoflavones.The optimal conditions to obtain the highest isoflavones yield of Puerariae flos were a time of 1.30 h,a temperature of 70℃,n-butanol/water composition of 1:1（v/v） and solvent-solid ratio of 55:1.Under these optimal conditions, the extraction yield of total isoflavones is up to 96.24%and the purity of isoflavones sample is about 73.89%.2.Study on purification identification of tectoridin in Puerariae flos.Here we propose a simple and rapid extraction method involving an n-butanol/water two-phase solvent system and rime-afesh time,suitable for separating tectoridin （4’,5,7-trihydroxy-6-methoxyisoflavone-7-O-β-D-glucopyranoside） in Pueraia lobata flowers.One compound was extracted from the n-butanol site of the Puerariae flos, which exhibited colorless needles.On the base of chemical and spectral methodologies (UV,HPLC-MS,¹H NMR,¹³C NMR) and by checking with the literature,the compounds were identified as tectoridin(C₂₂H₂₂O₁₁).Its purity exceeds 99%（HPLC） and yield is up to 0.62%.The method has been applied to separate tectoridin without column purification.The method is simple and convenient,yield is high.3.Preliminary contents standard study on total isoflavonoids and tectoridin in Puerariae flos.In this paper,Pueraia lobata flowers were qualitative investigation by the chemical way and TLC chromatographic,and quantitative investigation by UV method and HPLC method.In qualitative investigation,total isoflavonoids of Pueraia lobata flowers was indicated by chemical way,and tectoridin identified by TLC chromatographic method.In quantitative investigation,the UV method was used to determine the content of total isoflavonoids in Pueraia lobata flowers.Moreover,the HPLC method was used to determine the content of tectoridin in Pueraia lobata flowers.All the methods of determination were accurate and feasible.This research has provided the scientific basis for laying the foundation of the raw material for medicine and the preparation standardization.