Study On The Efficient Extraction And Separation Of Five Active Compounds From Eucommia Ulmoides Leaves

Eucommia ulmoides leaves(EUL)is the mature leaves of Eucommia(Eucommia ulmoides Oliv.).It is included in the 2005 edition of "Chinese Pharmacopoeia".It has mild and pungent taste,warm nature,and goes to liver and kidney meridian It has the effects of invigorating the liver and kidney,strengthening the muscles and bones,invigorating the vital energy,and strengthening the aspiration.It is mainly used for the treatment of weakness of the waist and knees,insufficiency of the liver and kidney,and weak muscles and bones.The main components of EUL are iridoids flavonoids and lignans.Modern pharmacological studies have shown that EUL has the effects of lowering blood pressure,regulating blood glucose,lowering lipids,anti-inflammatory,anti-viral,anti-fatigue,and enhancing immunity.EUL is rich in resources and has high economic value.Therefore,it is of great significance to carry out comprehensive development and study of EUL,a plant resource.In this paper,EUL as the research object,through the efficient extraction of EUL and the separation and purification of active ingredients,the establishment of a scalable production method provides a material basis for the in-depth development of Eucommia resources.The results of were as follows:1.A high HPLC method was developed for the simultaneous determination of geniposide,chlorogenic acid,quercetin 3-O-sanguinoside,rutin and isoquercitrin in EUL.The determination was performed on Diamonsil C18(5 μm,250 mm × 4.6 mm I.D.)column with mobile phase consisted of 0.2%formic acid(A)-acetonitrile(B).Gradient elution conditions:0-1 min,5-7%B;1-8 min,7-7%B;8-10 min,7-10%B;10-18min,10-10%B;18-45 min,10-30%B;Sample size:5μL,column temperature:30 oC,flow rate:1 mL/min;Detection wavelength:238 nm,254 nm,330 nm.The results showed that the five target compounds showed good linear correlation in the detection range,good repeatability,precision and stability,and accurate recoveries.2.The optimization of extraction parameters of target compounds in EUL with a novel ternal-deep eutectic solvent combined with microwave assisted extraction was determined.The optimum extraction conditions were as follows:microwave extraction power 400 W,extraction temperature 54℃,extraction time 20 min,liquid-solid ratio 19 mL/g.The composition of the new deep eutectic solvent is:choline chloride:1,4-butanediol:L-ascorbic acid=1/1/0.2(molar ratio),water content of 20%.Under these optimal conditions,the content of the five target components was about 1.23 times that of the traditional organic solvent extraction method,and the extraction process is green,efficient and energy saving.3.The optimum process parameters were determined for the enrichment of five target components in EUL with macroporous resin:AB-8 macroporous resin;The concentration of impurity removal eluent was 40%ethanol.The eluent concentration was 60%ethanol.The loading volume was 50 mL;The total volume of the eluent was 6 BV.Under these conditions,the deep eutectic solvent can be recovered and reused,and the contents of the five target compounds in EUL are about 4.7 times higher than that in the untreated crude extract.In addition,the adsorption rate and desorption rate were high,which realized the goal of enrichment of target compounds in EUL.4.A preparative chromatographic method for the separation and purification of five target components from EUL was established.Two separation methods were used.In the first step,Hedera ODS-2(250 mm ×20.0 mm,10 μm)column was selected for separation.Methanol-water as the elution solvent;The elution procedure was 0-80 min,7-100%methanol;Elution flow rate:8 mL/min;The detection wavelength was 210 nm and 254 nm.In the second step,Thermo Fisher(250 mm ×21.2 mm,5μm)was selected to prepare the column.Methanol-water=45/55(V/V)as the elution solvent;Equivalent elution was used;The elution flow rate was 5 mL/min.The detection wavelength was 210 nm and 254 nm.Under these optimized conditions,a total of five compounds were obtained,namely,geniposidic acid,chlorogenic acid,quercetin 3-O-sangudiglycoside,rutin and isoquercitrin.The recoveries of the five compounds were more than 85%,and the purities of the five compounds were more than 94%.This paper successfully developed a new type of natural ternary deep eutectic solvent,and the first joint microwave-assisted technology to extract the main active ingredient in EUL,macroporous adsorption resin technology was adopted to realize the recycling of solvent and enrichment of active compounds,and combined with the preparative chromatography separation technology for five kinds of target composition has carried on the selective separation and purification.This study provides a theoretical support for the rational application of Eucommia ulmoides resources,and also provides a technical reference for the efficient extraction and purification of other medicinal plant components.