Investigation On The Chlorogenic Acid Of Eucommia Ulmoides Leaves And Its Biological Activities

Duzhong（Eucommia ulmoides Oliver）is a plant of Eucommia ulmoides,which has a wide range of pharmacological effects including anti-hypertension,hypoglycemic,anti-hyperlipidemia,anti-oxidation,anti-osteoporosis,anti-tumor,immunomodulative and neuroprotective activities.The content of chlorogenic acid in eucommia ulmoides leaves can reach 1%～5.5%.Compared with common raw materials such as honeysuckle and coffee beans,eucommia ulmoides leaves are cheap,with an annual harvest of more than 4 million tons.Therefore,it is of great economic significance to extract and purify chlorogenic acid from eucommia ulmoides leaves.In this paper,the mass（W）volume（V）ratio（mg/m L）between water extract and water was determined to be 100:1 through the screening of extraction conditions,and the ethyl acetate extraction was performed by adjusting p H=2,and then the water extraction was performed by adjusting p H=8,so as to increase the content of chlorogenic acid from 8.8%to more than 60%.Then it is further purified by macroporous resin.HP-20 macroporous resin was selected by screening 6 macroporous resins.The chlorogenic acid with a purity greater than 95%was obtained by elution with 2 columns of deionized water,then with 4 columns of 20%ethanol solution,and finally with 2 columns of ethanol solution.Through the screening of the HSCCC solvent system,the final selection of ethyl acetate-n-butanol-water（4:1:5）（containing 0.05%TFA）as the purification of chlorogenic acid solvent system.Chlorogenic acid with a purity greater than 99%was obtained by HSCCC purification.At the cellular level,the anti-neuritis and neuroprotective activities of chlorogenic acid were studied,and the results showed that chlorogenic acid had certain anti-neuritis and neuroprotective activities.Chlorogenic acid can inhibit LPS activation of BV-2 cells to produce NO,and its IC₅₀value is 12.7μM,indicating that chlorogenic acid has anti-inflammatory activity.Using H₂O₂to stimulate PC12 cells to generate oxidative stress,it was found that the cell vitality of PC12 cells under oxidative stress under the action of 10μM of chlorogenic acid was 87%,slightly higher than that of positive control captopril（79%）,indicating that chlorogenic acid had neuroprotective activity.To sum up,chlorogenic acid was separated and purified from eucommia ulmoides leaves by means of extraction,macroporous resin separation and HSCCC,and the purity of the product could reach more than 99%.In addition,the activity evaluation showed that chlorogenic acid had anti-neuritis and neuroprotective effects.This research has important guiding significance to the development and application of eucommia ulmoides resources and products.