Extraction, Purification And Preparation Of Effective Parts Of Rebirth

Purpose: To improve the active effections of Chinese herb Fanhuncao and rational use Fanhuncao,we extracted and purified the active ingredients from Fanhuncao,Senecio cannabifolius Less,and prepared and evaluated the Fanhuncao ophthalmic thermosensitive in situ gel delivery system.Material and method:(1).Central composite design-response surface methodology was used to evaluate the extraction of the effective constituents from Senecio cannabifolius Less.T HPLC was to used to mesure and analyze protocatechuate,4-hydroxyphenylacetic acid,chlorogenic acid and caffeic acid in Senecio cannabifolius Less.Agilent Zorbax 5 TC-C18 column(250×4.6 mm,5 ?m)was used with acetonitrile-4% methanoic acid as the mobile phase in gradient elution(0~10 min,94%?94%B;10~50 min,96%?86%B).The flow rate was 1 m L/min.The column temperature was maintained at 25?,at a detection wavelength of 280 nm.Four phenolic acids were separaed on baseline.Central composite design and response surface method was used to evaluate the effects of influence factors such as solvent fold ethanol concentration and extract time on extraction rate of protocatechuate,4-hydroxyphenylacetic acid,chlorogenic acid and caffeic acid.The date was imitated using multi-linear equation and second-older polynomial equation.Response surface methodologh was used to optimize the process of extraction and predict the response in the optimal region.(2).Seven types of macroporous adsorption resin were selected with static adsorption usingadsorption capacity and resolution rate of protocatechuate,4-hydroxyphenylacetic acid,chlorogenic acid and caffeic acid as index.The isolation and purification condition was optimized with dynamic adsorption using the elution volume of protocatechuate,4-hydroxyphenylacetic acid,chlorogenic acid and caffeic acid as index.The maximum sample volume was investigated,and the samples were collected with distilled water,and draw leakage curve;On the investigation of the water removal and impurity removal,the sample was completely adsorbed,and then washed with distilled water,draw wash off curve.The concentration of eluent and the amount of eluent were investigated,respectively,the optimum ethanol concentration was determined by the amount of 4 kinds of components in the eluent,and the amount of the 4 kinds of components in the eluent was calculated.(3).To screen an optimal formulation of ophthalmic thermosensitive in situ gel of Senecio cannabifolius Less and evaluate its release behavior in vitro.The mass fraction of poloxamer P407 and P188 were used as independent variables,and the gelation temperatures before and after simulated tear fluid dilution were selected as response variables in the research.Response surface methodologh was used to optimize the prescription and predict the response in the optimal region and investigated the properties of the preparation in vitro.Results: 1.Multiple correlation coefficients from second-order polynomial equation were better than those from multi-linear equation.The optimum conditions of extraction process were as followed the Senecio cannabifolius Less herbs were crushed to medium powder;the extraction temperature was set at 80?,and the extraction time was 1.7 h;64% ethanol concentration,solvent ratio was 21,3 times of extraction.We got 549.33 ?g·g-1 of protocatechuic acid,995.49 ?g·g-1 of p-hydroxyphenylacetic acid,1965.73 ?g·g-1 of chlorogenic acid,11.38 ?g·g-1 of caffeic acid.The rate of extractum was 17.71%.2.The adsorption and desorption effect of AB-8 macroporous resin was better than that of the active part.The optimum concentration of the sample solution was 25 mg·m L-1,and the sample volume was about 4 BV.After the sample was adsorbed,the optimized impurity removal rate was about 3 BV,and the elution volume was about 4 BV with a solution of 60% ethanol as eluent.3.Multiple linear regression and second-order polynomial equation were used to establish mathematical model between independent variable and dependent variable.The second-order polynomial equation fitting degree was high,and had a good predictability.According to the provisions of the scope of the gelation temperature,through the optimization from the central composite design of Senecio cannabifolius Less thermosensitive ophthalmic in situ gel prescription.As the optimal mass fraction of P407 was 24%,and the mass fraction of P188 was 4.5%.The preparation is stable in vitro and had a good sustained-release effect.Conclusion: 1.Central composite design-response surface methodology is convenient and highly predictive for the optimization of the extraction process of Senecio cannabifolius Less.2.The effect of macroporous adsorption resin on the purification of the extract was obvious,and the content of phenolic acid was obviously higher than that of the other extractions.3.The ophthalmic thermosensitive in situ gel of Senecio cannabifolius Less is stable in vitro and had a good sustained-release effect.has a certain sustained-release effect.This study will lay a foundation to assess the safety and efficacy for the future study of this gel product.