Research On Chlorogenic Acid And Flavonoids From Eucommia Ulmoides Leaves: Extraction, Isolation, Purification And Structural Identification

Eucommia ulmoides Oliv. is kind of healthy effective medicinal plant. In recent years, some people had found that the bark and leaf of Eucommia ulmoides contain similar main components and have same pharmacological effect. On this study, the raw materials were dry leaves of Eucommia ulmoides, with different methods of extraction, separation, purification to obtain the components. In order to understand the functional compositions of Eucommia ulmoides leaves. Then identified the structures by thin-layer chromatography (TLC), ultra violet spectrum (UV), electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Ultimately, selected the best extraction and isolation methods, additionally found the active compounds'molecular structures from Eucommia ulmoides leaves. The research lays foundation for development and utilization of Eucommia ulmoides leaves. The major results as follows:1. Crude extract from Eucommia ulmoides leavesThe crude extract from Eucommia ulmoides leaves was evaluated by means of reflux extraction, ultrasonic extraction, microwave extraction, respectively. Results showed that the ultrasound-assisted reflux extraction is best. Its optimal extracting conditions are derived from orthogonal experimental design, as follows: solid-liquid ratio of 1:10, temperature 80℃, the concentration of ethanol 30%, pH value of solvent 5, firstly ultrasonic extraction 30min, and then reflux extraction 30min, repeat this process again.According to the optimum conditions to conduct the expanded experiment by 5 kg Eucommia leaves. The crude extract of flour yield was 24%, which contained chlorogenic acid 22.29%, total flavonids 54.67%.2. Iisolation and purification of crude extractAt first, screened NKA-Ⅱfrom 9 types of macroporous resins, while the chlorogenic acid adsorption capacity of 17.17mg/g, adsorption rate of 90.92%, desorption rate of 75.63%, and the total flavonoids adsorption capacity of 49.16mg/g, adsorption rate of 90.44%, desorption rate of 70.02%. Then the static adsorption-desorption experiments indicated that NKA-Ⅱresin belonged to a rapidly balanced adsorption resin in 4h, the best adsorption solution pH of 4, 70% ethanol as desorption liquid for chlorogenic acid and flavonoids. Lastly, by dynamic adsorption-elution experiments showed that the sample concentration of chlorogenic acid 0.9012mg/mL, flavonoids 3.4406mg/mL; the sample liquid flow rate of 1.0mL/min, the sample solution volume not more than 10 times of the column volume.With the optimization parameters to carry out extension dynamic adsorption-elution experiments on NKA-Ⅱcolumn. 600g crude extract was taken gradient elution by pure water (18L), 30% ethanol (9L), 50% ethanol (15L), 70% ethanol (12L), 90% ethanol (3L). Three sections were collected. FA(64.93g) was the section one which contained large amounts of chlorogenic acids. FB(36.06g) was the section two which contained both chlorogenic acid and flavonoids. FC(30.27g) was section three which mainly contained flavonoids, so chose FA and FC for further purification.On polyamide column 25g FA was taken gradient elution by pure water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol. Section FA-1, FA-2, FA-3 were detected, so collected the 21~70 eluated fractions and concentrated, then obtained compound FA-2 by Sephadex LH-20 column purification.On polyamide column 25g FC was taken gradient elution by pure water, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 90% ethanol. Section FC-1, FC-2, FC-3, FC-4, FC-5 were detected, so collected the 27~73, 105～150, 295～315 eluated fractions and concentrated, then obtained compounds FC-2, FC-3, FC-4 by Sephadex LH-20 column purification.3. Identification the structures of componentsThrough thin-layer chromatography analysis, the four compounds' spots were clear and focused under this chromatography agent system. FA-2 Rf value was similar to chlorogenic acid as 0.265. FC-2,FC-3,FC-4 Rf values were 0.632, 0.211, 0.737 respetivily,which were belonged to flavonoids.UV spectrum showed that the UV absorption characteristic of FA-2 was consistent with chlorogenic acid standard, maximum absorption wavelength at 324nm. FC-2,FC-3,FC-4 had the flavonoids'typical UV absorption peak , with the bandⅠUVmax were 349nm, 345nm, 367nm, with the bandⅡUVmax 256nm, 254nm, 254nm, respectively. So inferred FC-2 and FC-3 were flavonoid glycosides, FC-4 could be quercetin.On this basis, these compounds were identified by means of electrospray ionization mass spectrometry (ESI-MS) and nuclear magne- tic resonance (NMR). Comprehensive speculated that the study on the functional constituents of Eucommia ulmoides leaves led to the purifica- tion of four compounds, as chlorogenic acid (FA-2), quercetin-3-O-β-glu- coside(FC-2), quercetin-3-O-acetyl-glucoside (FC-3) and quercetin (FC-4).