Study On Preparation Of Active Components From Eucommia Ulmoides Oliv.Leaves By Double Aqueous Phase-Macroporous Resin-Organic Solvent Extraction

Eucommia ulmoides Oliver,as a unique traditional and precious medicinal material in China,which has abundant bioactive ingredients and high medicinal value.However,there are some prominent problems,such as low extraction rate and low comprehensive utilization rate,which seriously restrict the development and utilization of E.ulmoides resources.In order to obtain a green and efficient extraction process,the green and efficient extraction technology of active ingredients from E.ulmoides leaves was studied in this paper,which was focusing on the objectives of high efficiency,green,energy saving,simple operation and industrialized production.The main results of this paper were as follows:1.Based on the active ingredients（8 monomeric compounds,total phenolics,total flavonoids and gutta percha）,biological activities（antioxidation,bacteriostatic）and HPLC fingerprint analysis,E.ulmoides of different varieties and parts were compared and analyzed.The results showed that the bark of Purple leaf E.ulmoides was better varieties.The contents of total phenolic and gutta-percha were higher.The antioxidant and antimicrobial activities were stronger.The leaves of Purple leaf E.ulmoides and Qinzhong No.1 were of better quality.The biological activities of Purple leaf E.ulmoides were stronger(the values of FRAP,ABTS,DPPH_IC50 and MTS were 6161.60±2.98μmol equiv.Trolox/g,7471.37±2.65μmol equiv.Trolox/g,0.95±0.73μg/m L and 2.54±0.13 mg/m L,respectively).The contents of total 6 active components of Qinzhong No.1 were higher（total content was302.37 mg/g）.For different parts,chlorogenic acid content in leaves was higher than bark.The quality of Purple leaf E.ulmoides and Qinzhong No.1 were the best by fingerprint and chemometric analysis.At the same time,chlorogenic acid,hyperoside,astragalin and geniposidic acid were identified as key compounds for quality evaluation of E.ulmoides leaves.Aucubin,geniposidic acid,geniposide and pinoresinol diglucoside were the key compounds to evaluate the quality of the bark of E.ulmoides.2.The extraction method and technology of E.ulmoides leaves active ingredients were studied.The optimum technological conditions for enzymatic extraction were:1.5%cellulase dosage with ratio of material to liquid 1:20.And extraction temperature was 55°C,p H was 2 with extraction time 1 h.The optimum process conditions of high-speed homogenization-ultrasonic extraction was 1:8 ratio of material to liquid with extraction temperature of 30°C,extraction power of 160 W,extraction speed of 10000 rpm,and extraction time of 15 min.Under the optimum conditions,the two methods were compared with ethanol reflux,ultrasound and shaker assisted ethanol extraction.Finally,the ethanol reflux method was determined to be the best method for extracting active ingredients from E.ulmoides leaves.Under this method,30.43 mg of chlorogenic acid can be extracted of 1 g E.ulmoides.The yield of crude extract was 39.60%,and the content of chlorogenic acid was76.84 mg/g（crude extract）.3.Enrichment of the above crude extracts by aqueous two-phase technology was carried out,and the optimum process was determined as follows:20%（w/w）Na H₂PO₄,30%（w/w）ethanol concentration and 10%（w/w）samples.The optimum system temperature was40°C and p H=4,the extraction time was 1 h.Under the optimum conditions,the yield of the concentrate was 3.71%of the raw material,the chlorogenic acid content in the concentrate was 689.98±2.67 mg/g,which was 8.98 times higher than that of the crude extract,and the extraction rate of chlorogenic acid was 84.17%of the raw material.The contents of other active ingredients were aucubin（79.42±0.65 mg/g）,geniposidic acid（82.35±0.24 mg/g）,rutin（23.52±0.96 mg/g）,hyperoside（8.64±0.39 mg/g）,and astragalin（5.32±0.61 mg/g）,respectively.The enrichment effects was good.4.Macroporous resin-organic solvent extraction technology was used to further refine the aqueous two-phase concentrate.Five kinds of resins were screened by static adsorption test,and NKA-II was the best resin.The optimum process was as follows:The sample concentration was 800 mg/m L and p H=3,the loading amount was 3 BV,and the flow rate was 3 BV/h.The flow rates of deionized water and 30%ethanol were 5 BV,50%ethanol and 80%ethanol were 10 BV with a flow rate of 3 BV/h.The optimum extraction process for ethyl acetate was p H 3（chlorogenic acid）and not regulated p H（flavonoids）for 3 times and extraction time was 5 min.Under the optimum conditions,the two-phase concentrate was refined by macroporous adsorption resin-ethyl acetate,and the purified product with chlorogenic acid content as high as 91.26%（the yield of the refined product was 1.30%of the raw material）,and the extraction rate of chlorogenic acid was 38.99%.At the same time,the refined iridoid with 30%content（the yield of refined product was 2.13%of raw material）,the extraction rate of iridoid terpene is 11.18%,the content of flavonoids is55.48%,and the extraction rate is 39.65%（the yield of refined product is 0.36%of raw material）.5.Based on the content of chlorogenic acid as the main line and taking into account the contents of various active ingredients as the evaluation index,a set of high-efficiency extraction and enrichment technology of active ingredients in E.ulmoides leaves was established.The problems of low extraction rate,low quality,low comprehensive utilization rate and single evaluation index of E.ulmoides active ingredients were solved.At the same time,three extracts with chlorogenic acid content as high as 91.26%,iridoid contents as 30%and flavonoid contents as 55.48%were obtained.The process is environmentally friendly,low cost,simple and suitable for industrial production.It is of great significance for extracting high purity chlorogenic acid from E.ulmoides leaves,and provides a new way for high value utilization of E.ulmoides leaves.