| Dioscoreae Nipponicae Rhizoma is the tuber of Dioscorea nipponica Makino,which is rich in steroidal saponins,it is mainly used as raw materials to produce diosgenin in modern pharmaceutical industries.Diosgenin is an important precursor compound for the synthetic of steroids hormone drugs,such as sex hormone and adrenocortical hormone.In traditional industry,diosgenin was usually produced by direct acid hydrolysis.However,the resulted acid wastewater has caused serious environmental pollution and underutilized starch and cellulose in Dioscoreae Nipponicae Rhizoma has resulted in waste of resources.In this study,efficiently and environmental technology of producing diosgenin was established,and the comprehensive utilization of spent Dioscoreae Nipponicae Rhizoma residues was carried out,the main research contents are as follows:(1)A HPLC-UV method was established,chromatographic column:Agilent XDB-C18(150 mm L.×4.6 mm ID.,5μm),mobile phase:ACN:water(85:15,v/v),flow rate:1 mL/min,wavelength:203 nm,elution time:15 mins,column temperature:35℃and injection volume:20μL.The methodology was investigated followed this condition.Results showed that diosgenin and other interfering compounds could be separated at baseline(R>1.5);the linearity of diosgenin was good within the range of0.01211 to 1.550 mg/mL(r~2=0.9999);RSD of precision experiment is 1.87%,1.93%and 1.96%;the RSD of repeatability experiment is 1.63%;the sample solution was stability within 48 hrs,RSD=1.32%;and the average recovery rate of sample loading was 97.91%~100.01%with RSD less than 2%;the detection limit(S/N=3)was 0.58μg;the limit of quantification(S/N=10)is 1.93μg;Consequently,the constructed HPLC-UV method is stable and reliable for determination of diosgenin.(2)The technique of extracting diosgenin from Dioscoreae Nipponicae Rhizoma by pressurized biphasic acid hydrolysis was established.Then,Box-Behnken response surface experimental design was used to optimize the conditions of pressurized biphasic acid hydrolysis to obtain the best reaction conditions,temperature of 140℃,reaction time of 1.8 hrs and sulfuric acid concentration of 6μL/mL.The verification experiment results showed that the yield of diosgenin is 1.87%,while the relative error is 3.21%,as compared to the theoretically predicted value.The results indicated that the optimized hydrolysis conditions of pressurized biphasic acid are stable and reliable.Compared with the traditional direct acid hydrolysis method,the yield of diosgenin was increased by 85.1%and consumption of sulfuric acid was reduced by 92.5%.Meanwhile,the by-products of starch and cellulose were obtained,which significantly improved the extraction efficiency of diosgenin and benefited the rational utilization of Dioscoreae Nipponicae Rhizoma.(3)The technique of extracting diosgenin from Dioscoreae Nipponicae Rhizoma by pressure extraction combined with pressure biphasic acid hydrolysis was established.Single factor experiment investigation and orthogonal experiment optimization was performed to optimize extraction condition.Then optimum extraction condition was obtained with extraction temperature of 140℃,extraction volume of 350 mL,ethanol concentration of 60%and extracting for 80 mins.It was found that the yield of diosgenin by pressure extraction was 53.77%higher than that of the traditional method.Furthermore,by-products of starch and cellulose could be obtained.Moreover.Although the yield of diosgenin was slightly lower than that of pressurized biphasic acid hydrolysis method,the time consumption was greatly reduced and the extraction efficiency was significantly improved.In addition,pressure extraction combined with pressure biphasic acid hydrolysis was used to determine the content of diosgenin in Dioscoreae Nipponicae Rhizoma from 13 areas.It was found that the content of diosgenin from Zhangguangcailing and Xiaoxing’anling in Northeast China was higher,followed by the Qinling Mountains;and the content of diosgenin from Taishan and Luoyang is lower.Finally results of paired T test indicated that the yield of diosgenin prepared by pressure extraction combined with pressure biphasic acid hydrolysis increased significantly as compared to traditional methods(P<0.01).(4)The steroidal saponins in Dioscoreae Nipponicae Rhizoma were extracted,separated and purified by silica gel column chromatography and preparative high-performance liquid chromatography technology.Then,compound structures were identified by ESI-MS and NMR spectroscopy.Four kinds compounds,including protodioscin(I),protogracillin(II),dioscin(III)and pseudoprotodioscin(IV)were obtained.Subsequently,content of four saponins in Dioscoreae Nipponicae Rhizoma from 13 areas were determined,showing great difference between different areas.The content of protodioscin in Huadian of Jilin province was the highest;the content of protogracillin in Qinling of Shanxi province was the highest;the content of dioscin in Zhangguangcailing of Heilongjiang province was the highest and the content of pseudoprotodioscin in Xiaoxing’anling of Heilongjiang province was the highest.To evaluate whether the differences were statistically significant,one-way analysis of v Dioscoreae Nipponicae Rhizoma from 13 areas was performed based on the content of four steroidal saponins.The results showed that the content of four steroidal saponins in Dioscoreae Nipponicae Rhizoma from 13 areas was significant difference(P<0.001)at the significant level ofα=0.05.In addition,the results of correlation analysis showed that the content of four steroidal saponins had significant positive correlation at the significant level ofα=0.01(P<0.05).Finally,according to the content of four kinds steroidal saponins,the Dioscoreae Nipponicae Rhizoma from 13 areas was classified into three categories:Zhangguangcailing,Xiaoxinganling and Wuchang were the first category cause the content of four kinds of steroidal saponins was significantly higher than that of other areas and the quality was the best.;Huadian,Jilin,Yan’an,Chengde,Baishan,Qinling and Tonghua were the second category;Huludao,Taishan and Luoyang were the third category.(5)Biodegradable films were prepared by the comprehensively utilization of spent Dioscoreae Nipponicae Rhizoma residues(SDR)after extracting saponins,and the morphology and spectral properties of the SDR films were characterized to analyze its antibacterial properties.The results showed that the prepared SDR film was uniform and compact with a thickness of 0.0924 mm,which can effectively block the transmission of ultraviolet light with a wavelength of less than 375 nm;Moreover,the results suggested that the amount of SDR had different effects on moisture content,swelling degree and total soluble material.Examination of antimicrobial performance found that SDR films had a significant inhibitory effect on E.coli.To sum up,the SDR films prepared from spent Dioscoreae Nipponicae Rhizoma residues after extracting saponins have good applications prospect in the packing of food,feed and packaging film materials of photosensitive substances. |
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