| Objective To establish a new Diosgenin extraction method,which obtained Diosgenin by hydrolyzing the total saponins extracted from enzymes pretreated Dioscorea Zingiberensis C.H.Wright,in order to solve the traditional production process of Diosgenin yield is low and the serious environmental pollution problems.Methods 1.The research of total saponins and diosgenin content determination method Using dioscin as reference substance,established a ultraviolet-visible(UV-Vis)spectrophotometry method to determine the content of total saponins.Using dioscin as reference substance,established a high performance liquid chromatography(HPLC)method to determine the content of diosgenin.2.The research of total saponins extraction method Setting the yield of the total saponins as index,the 5 factors included temperature,p H,water consumption,dosage of enzyme and enzyme hydrolysis time in the process of enzyme pretreatment were inspected via single factor experiment,and the water consumption,dosage of enzyme and enzyme hydrolysis time were optimized via orthogonal experiment.In saponins extraction process the ethanol concentration,extracted time were inspected via single factor experiment,and the extracted time,ratio of solid to liquid and extraction degree were optimized via orthogonal experiment.3.The study of total saponins hydrolysis condition Setting the diosgenin yield as index evaluated the 3 factors ratio of solid to liquid,sulfuric acid concentration and acid hydrolysis time in the total saponins hydrolysis process via orthogonal experiment,to screen out the best hydrolysis condition.4.The study of Diosgenin extraction condition The diosgenin yield and purity were set as the indexes which were standardized as the comprehensive evaluation value.The results were obtained by comparing the diosgenin yield of the 3 kinds of extraction method soxhlet extraction,ultrasonic extractor,reflux extraction,and the comprehensive evaluation value of 5 kinds of extraction solvent methanol,ethyl acetate,ethanol,petroleum and cyclohexane.Screening the best extraction method and extraction solvent,and the 4 factors ratio of solid to liquid,ultrasonic frequency,extracted time and extraction degree of final extraction process were optimized via orthogonal experiment.5.The comparison of different methods for the diosgenin extraction The diosgenin yield and purity were set as the indexes which were standardized as the comprehensive evaluation value.The results were obtained by comparing the direct acid hydrolysis,ladder enzymatic,composite enzymatic,prefermentation,extraction separation and bio-enzyme pretreatment —alcohol extraction and acid hydrolysis methods with the comprehensive evaluation value,to evaluate the diosgenin extraction effect of bio-enzyme pretreatment —alcohol extraction and acid hydrolysis method.Results 1.The UV-Vis spectrophotometry determination method for the total saponins had been established,dioscin within the 0.01 ~0.05 mg·L-1 scope had good linear relationship with the absorbance(A),R2=0.9992.The HPLC determination method for the diosgenin had been established,the chromatography condition was as following: the mobile phase was methanol,the column temperature was 30?,the flow rate was 1m L·min-1,the detection wavelength was 203 nm.Diosgenin within 0.02~1.5 mg·m L-1 scope had good linear relationship with the peak area,R2=0.9995.2.The best conditions in the enzyme pretreatment are as follows: the temperature for the enzyme is 70?,p H is 5.5,water consumption is 4 L·kg-1,dosage of enzyme is 8 m L·kg-1,enzyme hydrolysis time is 24 hours;the best conditions in the total saponins extracted process are as follows: ethanol concentration is 60%,ratio of solid to liquid is 1:8,extracting time is 1.0 hour,extracting degree is twice.3.The best conditions of the total saponins hydrolysis was as follows: the ratio of solid to liquid was 1:4,concentration of sulfuric acid was 2.0 mol·L-1,hydrolysis time was 5hours.4.The diosgenin yield does not have significant difference of reflux extraction,soxhlet extraction and ultrasonic extraction,but the method of ultrasonic extraction efficiency is higher.The comprehensive evaluation value of the 5 extraction solvent: petroleum ether > ethyl acetate > cyclohexane > ethanol> methanol.Using the petroleum ether as extraction solvent,the best extraction condition of ultrasonic extraction method for diosgenin extraction was as follows: the ratio of solid to liquid was 1:50,extracting 3 times,each time extracted 20 minutes.5.The comprehensive evaluation value of the six methods were as follows: bio-enzyme pretreatment—alcohol extraction and acid hydrolysis > ladder enzymatic > composite enzymatic > extraction separation > prefermentation > direct acid hydrolysis.Conclusions 1.The UV-Vis spectrophotometry method for the total saponins determination and the HPLC method for the diosgenin determination had been established.According to the methodlogy study the content determination methods were stable and reliable,which could provide reference for the optimize of extraction conditions.2.The enzyme pretreatment method for the total saponins extracting from Dioscorea Zingiberensis had been established,compared with the traditional reflux method,the total saponin yield of enzyme pretreatment method was increased by 4.3 times,that could ensure the final yield of diosgenin at the optimal level.3.The optimum acid hydrolysis conditions for the total saponins had been established.Under this condition the total saponins could be hydrolysised to diosgenin stable.4.The best extracting method,extraction solvent and the corresponding extraction conditions for extraction of diosgenin had been determined.5.The bio-enzyme pretreatment —alcohol extraction and acid hydrolysis had the best effect in the 6 diosgenin extraction method,the diosgenin yield reached 3.71%,which was 2.18 times higher than the direct acid hydrolysis method,more suitable for the diosgenin extraction. |
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