Epimedium Grandiflorum And Korea And Epimedium Glycoside Of Extraction, Separation And Purification Technology Was Studied

As a traditional Chinese herbal medicine. Epimedium has many pharmacological functions such as norishing liver and kidney, strengthening bones and muscles, reinforcing yang, benefiting essence and anti-rheumatism, etc. Its main active ingredient icariin has been extensively studied. With the in-depth studies on chemical constituents of epimedium. epimedin A. B and C have been recognized as the marker componds of epimedium besides icariin. Since epimedin have been paid more attention, research about their extraction process and medical functions is of great importance to the effective protection and sustainable development of epimedium resources.Since the chemical structures and characters of epimedin A, B and C are similar, there are few extraction and separation technologies of epimedin monomers, besides, the existing processes are usually complex, high-cost and loboratory limited. A simpler and more effective process, which can be used for the industrialized production of epimedin. will be the foundation for functional study of epimedin and effective use of epimedium.In this study, we chose E. brevicornu and E. pubescens as materials, which are widely distributed, of rich wild resources and stable quality. The methods included solvent extraction, macroporous resin column chromatography, organic solvent extraction and RP-silica gel chromatography. Single factor experiment, orthogonal test and response surface methodology (RSM) were used to optimize the extraction, seperation and purification of epimedin A,B, C and icariin. The results were as follows:(1) Comparing with water extraction, ethanol extraction had a higher extraction rate of flavonoids from epimedium. The main influencing factors of ethanol extraction were investigated. With RSM, the optimum conditions were:in the first extraction, ratio of liquid/material25mL/g, ethanol concentration44%. extraction tempatature80℃extraction time56min;in the second extraction, ratio of liquid/material20mL/g, ethanol concentration44%, extraction tempatature80℃. extraction time40min. For E. pubescens and E. hrevicornu, the extraction rate of total flavonoids was89.56%and87.91%, the yield was0.09g/g herbs and0.08g/g herbs, and the purity of total flavonoids was28.39%and25.51%. respectively.(2) Through static adsorption and desorption experiments of eight kinds of macroporous adsorption resins and three kinds of polyamide. D-101-â…  macroporous resin was selected as an excellent adsorbent with high adsorption and desorption capacities for epimedium flavonoids. The impact factors of dynamic column chromatography were investigated. For Φ1.0×25cm macroporous adsorption resin column, the optimum parameters were:in adsorption process, sample concentration2.5mg/mL. feeding rate1.0mL/min, feeding weight0.39g; in desorption process,2.5BV10%ethanol was used to remove inpurities. eluant of4BV70%ethanol was collected, elution rate1.5mL/min. For E. pubescens and E. brevicornu, the purity of flavonoids achieved53.01%and60.55%, with recoveries of68.68%and48.24%, respectively.(3) In organic solvent extraction, solvent types, ratio of lipid/aqueous phases and extraction time were investigated. The results showed that icariin mainly existed in ethyl acetate extract, and epimedin mainly existed in n-butanol extract after ethyl acetate extraction. The optimum extraction process was:first extracted by ethyl acetate with ratio of lipid/aqueous phases2:1, extraction time10min for5times, then extracted by n-butanol with ratio of lipid/aqueous phases1:2, extraction time2min for3times. In ethyl acetate extract of E. pubescem and E. brevicornu, the purity of icariin was8.09%and21.36%. with recoveries of77.05%and66.75%, respectively; in n-butanol extract after ethyl acetate extraction, the purity of epimedin was24.38%and40.65%, with recoveries of84.80%and77.06%, respectively.(4) The n-butanol extraction product was further separated and purified by RP-silica gel chromatography. For Φ1.2×40cm column, the optimum process was:feeding weight10mg, gradient elution program50%â†'60%â†'100%methanol. elution rate0.5mL/min. The result showed that different species of epimedium have different compositions, which induce varied effects of separation and purification. After RP-silica gel chromatography, for E. pubescens, the purity of epimedin A. B and C was23.04%.33.57%and54.92%. with recoveries of20.49%.17.19%and15.52%. respectively;for E. brevicornu, the purity of epimedin A. B and C was12.89%.64.50%and34.74%. with recoveries of37.68%.64.50%and8.06%. respectively, indicating that E. pubescens could be suitable for extraction and separation of epimedin C. while E. brevicornu could be the most appropriate material to produce epimedin B.(5) The ethyl acetate extraction product of E. brevicornu. which has a higher relative content of icariin. was further purified by RP-silica gel chromatography. In final product, the purity of icariin was77.54%. with recovery of14.54%. Above all. the process supposed by this study, which can extract epimedin A. B. C and icariin simultaneously, is simple, convenient, low-cost and reliable to operate. With this method, epimedin and icariin with high purity are aquired, epimedin A. B and C were also separated to a certain extent. It will laid a foundation for the further purification and functional study of epimedin monomers.