The Study On Extraction And Purification And Biological Activity Of Total Tritepenoidic Acid From Fomes Officinalis Ames

Objective: To establish determination method of content in total tritepenoidic acid from Fomes officinalis ames,to optimize the extraction technology and purification process conditions,to research acute and toxicity of triterpenoid acid of purification product from Fomes officinalis ames,study the cough expectorant effect of total tritepenoidic acid from Fomes officinalis ames,for Fomes officinalis ames provide theoretical basis for the further research and clinical application. Methods: 1. To establish a content determination method Ursolic acid as reference substance,determine the detection wavelength,use the 5% vanillin-ice acetic acid solution, perchlorate solution chromogenic system,screening conditions of chromogenic system,using ultraviolet pectrophotometry to measure absorbance of samples,and methodology. 2. Extraction process To optimize the extraction of total tritepenoidic acid from Fomes officinalis ames with the help of central composite design-response surface methodology. Screening of extraction times of single factor experiment,With extraction time, the quantity of solvent and the concentration of solvent as the independent variable,the extract yield of total tritepenoidic acid from Fomes officinalis ames as dependent variable.multiple linear regression and binomial of each level of independent variables were fitted.Optimum technology was selected by central composite design and response surface methodology,and prediction was carried out through comparing the observed and predicted values. 3. Purification process With adsorption and desorption rate of total tritepenoidic acid from Fomes officinalis ames as index,to inspect ability of adsorption and desorption of 10 different types of macroporous adsorption resin for total tritepenoidic acid from Fomes officinalis ames,determining which one macroporous adsorption resin is the best,and optimizing its purification conditions. 4. The purified structure identificationUsing the HPLC-MS to analyze purification product of total tritepenoidic acid from Fomes officinalis ames,according to related literature compare chromatogram and mass spectrogram of each composition,infer the types and content discretion of triterpenoid acid in preliminary. 5. The studies of acute and toxicity of the total triterpenoid acid of purification product from Fomes officinalis ames According to the preliminary experimental results, to determine the LD50, observe the mice look after the treatment,the change of behavior, record secretions and deaths. 6. The pharmacodynamics research of the total tritepenoidic acid from Fomes officinalis ames 6.1 By the method of ammonia led to cough experiment and phenol red excretion in mice,using cough rate and expectoranting index as evaluation index,and watching the cough expectorant effect of total tritepenoidic acid from Fomes officinalis ames. 6.2 Using lipopolysaccharide(LPS)to inject into trachea of mice, observe the signs,gross specimen are made in pathological lungs, and observed under light, confirm the mice with chronic bronchitis(CB) model is successful, watched the total tritepenoidic acid purification from Fomes officinalis ames on the effects of the lung tissue pathology morphology in mice on chronic bronchitis.Results: 1. The results of the content determination show that,Ursolic acid in the range of0.0019-0.0114 mg/mL insider sex is good,r=0.9995,the average recovery rate of sample was 101.08%,RSD was 1.13%. 2. The optimum extraction conditions consisted of extraction two times,145 min for each time with thirteen fold of 91% ethanol. 3. DM301 macroporous adsorption resin is the best resin, the best purification process conditions sample liquid is pH value of 5,the sample liquid concentration is 12.18 mg/mL,the sample flow rate of 0.6 mL/min, the best sample amount is 2 Bv, static adsorption time is 1 hour,by 5.5 BV, pH = 8 and 85% ethanol elution to elute with 0.75 mL/min flow velocity. 4. A preliminary estimate of the triterpenoid acid species includes dehydrosulpurenic acid,2-(1-carboxyhexadecylamino)- 2- aminosuccinic acid, dehydroeburicoic acid,fomefficinic acid F,fomefficinic acid G,fomefficinic acid E, etc. 5. The total tritepenoidic acid purification from Fomes officinalis ames has certain toxicity,the LD50 is 7.3440g/Kg.6. The total tritepenoidic acid from Fomes officinalis ames has cough effect and expectoranting action,and therapeutic effect on chronic bronchitis. Conclusion: The extraction and purification processof total tritepenoidic acid from Fomes officinalis ames is stable, simple, feasible, and has a certain cough and expectorant effect,and have a certain therapeutic effect for chronic bronchitis.