| Fig leaf has been used for thousands of years in traditional medicine,and has been found with excellent clinical efficacy in clinical practice for a long time,being an important Chinese medicinal material.But the current development and utilization of it is not enough.Many of them are as the by-products of fig planting,resulting in a great waste of resources.Therefore,the study of the development and utilization of fig leaves can promote the added value of the fig planting industry and increase the economic benefits.Psoralen has many important clinical uses.Fig leaves contain abundant psoralen and psoralic acid-glucoside as a precursor of psoralen.The production of psoralen from fig leaves can enrich the source of raw materials and reduce production costs.However,the study of the extraction and separation of psoralen in the leaves of fig is still very backward and lacking of efficient methods.In order to make better use of fig leaves and develop a highly efficient method of obtaining psoralen from the leaves of fig,the main work of this paper is three aspects:1.A high performance liquid chromatography(HPLC)method has been developed for quantitative analysis of psoralen and psoralic acid-glucoside in fig leaves:Mobile phase:0.1%formate water(A)-acetonitrile(B);Chromatographic column:Luna C18(5μm,250 mm × 4.6 mm i.d.);Elution conditions:0-10 min,13-16%(B);10-11 min,16-17%(B);11-25 min,17%(B);25-55 min,and B(25-55%);Injection volume:5 uL;flow rate:1mL/min;Detection wavelength:254 nm;Column temperature:30 ℃.In the detection range of psoralen and psoralen glycosides,the method has good linear relationship(R2 = 0.99),the limits of detection were 0.22 and 0.17 g/mL the limits of quantitation were 0.68 and 0.52 g/ml,precision,reproducibility and stability were less than 5%.The yield of addition recovery was 97.88%-99,01%,and R.S.D.was less than 4%.These results showed that the method was sensitive,accurate and stable,which could be used for quantitative determination of psoralen and psoralic acid-glucoside in fig leaves.2.For the first time,we developed and optimized a new ultrasound assisted ionic liquid based pH dependent aqueous two-phase system,which could achieve simultaneously extraction and enrichment of psoralen and transformation of psoralic acid-glucoside into psoralen from fig leaves by on step.The process parameters were optimized and the optimal parameters were determined.Composition of ionic liquid acid solution:1-butyl-3-methyl imidazolium bromide([Bmim]Br),citric acid,water.The optimal parameters in the process of extraction and transformation:pH:2.21,ionic liquid concentration:2.01 M,liquid-solid ratio:15.35 ml/g.The optimal parameters in the purification process:1-butyl-3-methyl imidazolium bromide([Bmim]Br)content:37.0 4%wt,Citric acid content:30.21%wt,pH:7.0.Under the above optimum parameters,the extraction yield of psoralen was 31.22 mg/g(n=3),and the recovery yield was 96.32%(n=3).These results indicated that the method developed in this paper can achieve the simultaneous extraction and purification of psoralen and transformation of psoralic acid-glucoside into psoralen from fig leaves by adjusting the pH of the system.In this method,three independent operation units,which were the extraction,transformation and purification of natural products in traditional natural products production were integrated into one process by adjusting pH.It has the advantages of high extraction yield,low cost and simple operation.3.A method of high speed counter current chromatography was established for the preparation of psoralen and bergapten from fig leaves:Bergapten was one of the main compounds in Fig leaves extracts.In this part,the extraction and enrichment solution mentioned above was extracted by the mixture of petroleum ether and ethyl acetate,and the samples of psoralen and bergamot were obtained.The most suitable solvent system for separating psoralen and bergapten was screened out and was composed of n-hexane:ethyl:acetate methanol:water(7:3:5:5,v:v:v).When the speed of was 800 rmp,the flow rate was 2 mL/min and the amount of sample loaded were 200 mg,40.93 mg psoralen and 4.09 mg bergapten were prepared at one run.The results of high performance liquid chromatography(HPLC)showed that the purity of psoralen was 97.9%,the recovery rate was 99.1%,and the yield of psoralen in the leaves of fig per gram was 3.01%.The purity of bergapten was 96.76%,the recovery rate was 98.8%,and the yield of bergapten in the leaves of fig per gram was 0.644%.The results showed that compared with the conventional separation method,high speed countercurrent chromatography had the advantages of simple operation,saving separation time,high purity of the prepared compounds,and difficult decomposition of the prepared compounds in preparation process.In summary,by the combination of extraction and enrichment technology of ultrasonic assisted ionic liquid based pH dependent aqueous two-phase system with high-speed countercurrent chromatography,the purity of obtained psoralen was 97.9%and the yield in the leaves of fig per gram was 3.01%,the purity of obtained bergapten was 96.76%and the yield in the leaves of fig per gram was 0.644%.This research is beneficial to the development and utilization of fig leaves,and also provides a new idea for the application of ionic liquids in the extraction and separation of natural products. |
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