Efficient Detection And Separation Of Puerarin And The Research Of Its Stability And Solubilization

Pueraria is the dry root of leguminous plants Pueraria lobata, which is a medicinal and edible plants approved by the Ministry of Health. Pueraria are widely distributed in our country, its variety is rich and varied. Pueraria has a good value in medical and health care,but due to the limitations of production technology has limit its better development and utilization. Based on the experimental study, we take a in-depth system research for the production and application of puerarin, a major functional isoflavones components in pueraria; And provide theoretical basis for the comprehensive exploitation and utilization of pueraria resources. First by spectrophotometry and high performance liquid chromatography(HPLC) method to detecting the content of pueraria isoflavones, using standard compounds as a reference standard, then establishing a rapid accurate testing method for pueraria isoflavones content; After, to optimize the extraction process of pueraria isoflavones, to obtain the best extraction process; The following, to separate and purificate pueraria isoflavones by resin adsorption and acid hydrolysis process, we can get high purity products of puerarin and daidzein;Finally, we carry a systemic research on the stability and solubility of puerarin, provide theoretical basis for its production and processing(1) Besides isoflavones, Puerariae radix also contains many other constituents that have absorbance in UV range. Thus, the total isoflavones content in Puerariae radix detected by direct spectrophotometer method is incorrect. In the present study, we found that there is a linear relation between the results determined by HPLC and direct spectrophotometer method. The linear equation was obtained by the analysis of 16 batches of Puerariae radix sample. With the correction of HPLC, the total isoflavones in Puerariae radix can be simply determined spectrophotometer method. The method was successfully applied for the analysis of 10 batches of Puerariae radix sample.(2) To optimize the extraction of pueraria isoflavones, ethanol reflux extraction,microwave assisted extraction and ultrasonic assisted extraction were compared, concluded that microwave assisted extraction is the best. The optimum technological parameters were microwave power 400 w, irradiation time 80 s, the extraction temperature 80℃, the ethanol concentration was 75%. Using reversed-phase high performance liquid chromatography(RP-HPLC) method to detect three kinds of isoflavones content in radix puerariae samples, the results as follows: puerarin 30.49 mg/g, daidzin 3.40mg/g, daidzein 0.80mg/g.(3) Puerarin is the major isoflavone of Puerariae Lobatae Radix. A method for efficient separation and purification of puerarin was developed through resins adsorption and acid hydrolysis. The absorption properties of six resins(D101, S-8, H103, X-5,HPD600, AB-8) were compared through the adsorption kinetics and equilibrium adsorption isotherms. Results showed that H103 resin had the best adsorption rate and capacity. The mass transfer zone motion model was further used for analyzing the fixed bed adsorption of H103 resin. Its length of mass transfer zone with 2 mg/mL of puerarin in water and 10% ethanol at flow rate of 10 mL/min were 41.6 and 47.5 cm, while the equilibrium adsorption capacity was 90.44 and 60.34 mg/g, respectviely. By using 75%ethanol, puerarin could be well desorbed from the resin with recovery of 97.4%.Subsequently, H103 resin was successfully used for puerarin purification from Puerariae Lobatae Radix. The content of total isoflavones and puerarin in the resin absorption product were 69.25% and 41.78%, respectively, which were about three times increased compared to the crude extract. Then, the product was hydrolyzed by 2.5 M HCl. Puerarin with purity of 90% and a byproduct daidzein with purity of 78% were obtained.(4) Puerarin is the dominant isoflavone of leguminous plants Pueraria lobata(wild).As a kind of estrogen, it can improve blood circulation, strengthen the organic immunity.There are few literatures about the stability study of puerarin, this paper studied the influence of pH, temperature, metal ion, H2O2, Na2SO3 and extraction solvents on the stability of puerarin. Results showed that temperature, Cu2 + and Na2SO3 had major impact on the stability of puerarin; With the increase of storage temperature and duration, puerarin content decreased significantly; Cu2+ affect the UV absorption spectra of puerarin; Na2SO3 made the maximum UV absorption wavelength of puerarin shifted, and with the increase of Na2SO3 concentration, the absorbance values increased.(5) Puerarin is the major isoflavone of Puerariae Lobatae Radix, which has a good medicinal value. The development and utilization of puerarin had been limited due to its smaller solubility in aqueous solution. In this paper, through the study of the ultraviolet absorption spectrum and phase solubility, compared the characteristics of the inclusion complex of puerarin with α-CD, β-CD, γ-CD. The β-CD and puerarin can form a relatively stable molecular microcapsules by the stoichiometric ratio of 1:1. Through coprecipitation to preparation the inclusion complex of puerarin with β- CD, and to optimize the package process parametersn including ethanol concentration, solid-liquid ratio, time, and temperature, the best process parameters as follows: 25% ethanol, solid-liquid ratio 1:5,package for 6 h, temperature at 20 ℃.The solubility of microcapsules was increased by more than 7 times, and 3 times in vitro dissolution rate increase. Through the analysis of infrared spectra, the inclusion complex has all the spectral characteristics of puerarin and cyclodextrin, which proved the formation of microcapsules of puerarin and β- CD.