Extraction Of Diosgenin Based On Pressurized Biphase Acid Hydrolysis

Diosgenin is a very important steroid in pharmaceutical industry,which is a main precursor for synthesis of contraceptives,cortisone,sexual hormones,and anabolic agents.In addition,many pharmacology studies have suggested that the compound has various bioactivities including anti-cancer,anti-inflammation,improving immune system,anti-thrombosis,antiatheroscloresis,antiasthma,and anti-virus.In China,the tubers of Dioscorea zingiberensis C.H.Wright and Dioscorea nipponica Makino were main resources for diosgenin production because of high quantity.The diosgenin exits in plants as saponins,so glycosidic bond must be hydrolyzed while production.Conventional methods always lead to serious environmental pollution,low diosgenin yield and poor production efficiency in the utilization of those natural Dioscorea resources.The aim of this study was to establish a more ecological and efficient approach for producing diosgenin from the tubers of Dioscorea zingiberensis C.H.Wright and Dioscorea nipponica Makino,and improve efficiency of resource utilization.Work competed are as follows:Part I A HPLC-UV method for diosgenin quantification was establishedA HPLC-UV method was established,namely,chromatographic column:Waters XBridge Shield(150 mm × 4.6 mm,5 ?m),mobile phases: ACN : water(75 :25,v/v),flow rate: 1.0 mL/min,column temperature: 40.0°C,UV detection wavelength: 203 nm,injection volume: 20 ?L.Under the condition,diosgenin could be well separated from interference on baseline(R>1.5).Then linearity,precision,repeatability,stability and accuracy were investigated.Results indicated that linearity was good within the range of 0.002~1.024 mg /mL(r2 = 0.9999),and the method was precise(RSD = 1.02%),repeatable(RSD = 1.78%),and accurate because recoveries at three levels were 96.2%(RSD = 1.17%),100.4%(RSD = 2.47%)and 101.6%(RSD = 1.09%),respectively.And the prepared sample was stable within 24 hrs(RSD= 1.93%).Consequently,the established HPLC method was demonstrated to be stable,reliable and could be applied for diosgenin quantification.Part II Extraction of diosgenin from the tubers of Dioscorea zingiberensis by pressurized biphase acid hydrolysisReaction condition of pressurized biphase acid hydrolysis was optimized through orthogonal design,where four factors including temperature,H2SO4 concentration,reaction duration and sample to acidic solution ratio were investigated.Then the optimal condition was verified,and this novel method was compared to two traditional methods and pre-fermentation biphase acid hydrolysis.In addition,silica gel chromatographic column and re-crystallization was applied to purify diosgenin.As results,under the best conditions,namely,temperature was 140 oC,H2SO4concentration was 0.2 mol/L,reaction duration was 1.5 hrs,and sample to acidic solution ratio was 2.5 : 50(g/mL),diosgenin yield was achieved at 2.21%,that was63.7%,70.4,20.8% higher than two conventional methods and pre-fermentation biphase acid hydrolysis,respectively.And,H2SO4 concentration of novel method was only one tenth of traditional methods.In conclusion,the established novel method had higher diosgenin yield,lower H2SO4 consumption and shorter reaction duration.Part III Extraction of diosgenin from the tubers of Dioscorea nipponica by saponins extraction-pressurized biphase acid hydrolysisAt first,total saponins were extracted from Dioscorea nipponica in Soxhlet extractor using 70% EtOH.Then,the saponins were hydrolyzed in pressurized biphase acid hydrolysis system to produce diosgenin.Four factors including sample to acidic solution ratio,H2SO4 concentration,reaction temperature and hydrolysis duration were investigated through single factor experiment design,followed by further optimization through orthogonal design for the best reaction condition,namely,sample to acidic solution ratio was 8 : 50(g/mL),50 mL of H2SO4 aqueous solution at4 ?L/m L,reaction temperature was 150°C,and reaction duration was 2.5 hrs.In comparison,the yield of diosgenin by novel method was increased to 1.670%,66.02%higher than traditional method(1.006%),and H2SO4 consumption was only 1.497mL/g,97.00% lower than traditional method(49.760 mL/g).Consequently,the totalsaponins extraction-pressurized biphase acid hydrolysis was a more ecological and efficient method to extract diosgenin from the tubers of Dioscorea nipponica.And in general,pressurized biphase acid hydrolysis was demonstrated to be a better approach to extract diosgenin from Dioscorea medicinal plants.Part IV Extraction of diosgenin from the tubers of Dioscorea zingiberensis by comprehensive utilization-pressurized biphase acid hydrolysisIn the first stage,starches and cellulose were recycled after physical separation,and crude saponins were hydrolyzed in the pressurized biphase acid hydrolysis system.Afterwards,7 factors including sample to acidic solution ratio,H2SO4 concentration,volume of extraction solvent,reaction temperature,duration of hydrolysis,agitation speed and boiling point of petroleum ether,were sequentially optimized through single factor experimental design.Then,a comparison was made among traditional method,previously established method and this newly proposed method with diosgenin yield,H2SO4 consumption,COD and TOC as indexes.Consequently,under the optimal conditions,namely,a 1:100 ratio of sample to acidic solution(g/mL),50 mL of 2 ?L/mL H2SO4 aqueous solution,30 mL of petroleum ether(90~120°C),130°C,2.0 hrs and 100 r/min,the yield of diosgenin has been increased to 4.17%.By using this method,the yield was 22.29% higher than the conventional method,and H2SO4 consumption was reduced by 88.53%,meanwhile the COD and TOC were decreased by 50.15% and 64.39%.In conclusion,comprehensive utilizationpressurized biphase acid hydrolysis was a more ecological and efficient approach for producing diosgenin from tubers of Dioscorea zingiberensis,and it is a promising and potential strategy for application in industrial practice.