Study On Extraction, Separation&Purification And Its Effects Of Antioxidation&Hypolipidemic Of Chlorogenic Acid (CGA) In Leaves Of Eucommia

Objective:To investigate the extraction, separation and purification of CGA in leaves of Eucommia, and evaluate its antioxidant and lipid-lowering effects in vitro.Method:1. Paper chromatography-UV and HPLC was used to determine the content of CGA.2. The optimum extraction process were determined by using water, alcohol, ultrasonic and microwave extraction and compared their yield on CGA by single factor experiments and orthogonal design.3. The optimum separation conditions were designed by using lime precipitation, solvent extraction, precipitation of lead acetate and chitosan to separate CGA. Seven types of macroporous resin, including NKA-9, NKA-Ⅱ, D101, XA-8, XDA-8, HPD100and HPD826, were compared the purification efficiency, and the best one was screened. The extract of CGA was purified, frozen and dried.4. The antioxidant activity of CGA were evaluated by using DPPH free radical, superoxide anion free radical, hydroxyl radical and linoleic acid oxidation system induced by the alkyl free radicals and observing the rats’red blood cell hemolysis.5. The lipid-lowering effect and mechanism of CGA in Eucommia leaf were studied by influencing the formation of cholesterol micelles and inhibition of3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and pancreatic lipase in vitro.Result:1. Paper chromatographic conditions:Butanol-acetic acid-water, Xinhua Paper No.1, Exhibition15cm, UV light wavelength254nm, Detection wavelength327nm. HPLC conditions:Little (4.5×250mn:5u m), ODS column, acetonitrile-0.4%phosphate buffer (13:87),327nm, N≥2000, v=lml/min, column temperature30℃, injection volume10μl.2.Optimized conditions of ultrasonic extracting process:with60%ethanol of pH5at a ratio of1:12, and taken repeatedly at the frequency of25kHz for30min. The yield of chlorogenic acid was2.54%.3. Chitosan and NKA-9macroporous resin can be used in the separation and purification of crude extract of CGA. The content of extract of CGA was42.43%by HPLC.4. In0.01～0.6mg/ml, the scavenging effect of CGA extract on DPPH radical, superoxide anion radical, hydroxyl radical were better, but on linoleic acid system, induced by alkyl free radicals was poor, it had better inhibition on red blood cell hemolysis in rats. The maximal inhibition rates were79.53%,81.28%,65.15%,49.78%and77.46%, IC50were0.0140mg/ml,0.0383mg/ml,0.0611mg/ml,0.6487mg/ml and0.0214mg/ml respectively.5. In1×10-5～lmg/ml, the formation of cholesterol micelles, the HMG-CoA reductase and pancreatic lipase were restrained by the extract of CGA in a dose dependent manner in vitro. The maximal inhibition rates were96.66%,48.96%and95.20%. The IC50of CGA extract inhibiting on the formation of cholesterol micelles and pancreatic lipase were0.0648mg/ml and0.0026mg/ml respectively.Conclusion:1. Paper chromatography-UV combined with HPLC to determine the content of CGA in Eucommia leaf were simple, accurate, sensitive, and good reproducible.2. Ultrasonic extraction of CGA was high efficiency and suitable for large-scale industrial production.3. Using chitosan and NKA-9macroporous resin to separate and purify the extract of CGA was more effective, suitable for the production.4. The extract of CGA in Eucommia leaf has strong antioxidant activity and lipid-lowering effect, which can be used as a potential antioxidant activity lipidlowering drugs.