Studies On Extraction Technics, Separation And Purification Of Chlorogenic Acid From Arctium Lappa Linn Leaves And Its Activities To Pathogenic Fungus

Chlorogenic acid (CHA) is a compound of phenylpanoids produced in the process of aerobic respiration and acts as phytoalexins of some plants. CHA possesses extensive and remarkable pharmacological activities, such as anti-bacterium, anti-oxidation, anti-inflammation, and scavenging effect on free radicals. In the present, CHA is also the quality control index of many Chinese traditional medicines and teas. CHA is mainly extracted from Eucommia ulmoides Oliv, honeysuckle, sunflower seeds, coffee and cocoa trees. The CHA content of honeysuckle in Guizhou province is stable and about 5.3-5.7%, while it is 1.5-3.3% of sunflower seeds and 2.1-3.5% of sunflower seed kernels. But all these materials are expensive. Plenty of CHA was explored in A. lappa leaves, which may be a fresh source of CHA, by our task team for the first time. In view of its extensive application in food, medicine and daily-use chemical industry, we investigated the extraction and purification technics of CHA from A. lappa leaves, and studied its effects on several fungus. List the results as follows:(1) CHA was extracted by water and ethanol from A. lappa leaves. The yield rate of CHA is 0.32% and 0.58%, respectively. When treated at 80℃ for 15min before extraction, the yield rate would rise evidently to 0.58% and 1.22%, respectively.Based on treatment at 80℃, the optimal condition of water extraction from A. lappa leaves by single-factor experiment is: pH4.0, solid/liquid ratio 1: 16, whisking or shaking extraction at 60rpm, extraction temperature 40℃, and extraction times 1; as for honeysuckle, it is: pH4.0, extraction times 2, and not crush. Through single factor experiment and orthogonal test to obtain the ideal condition of ethanol extraction from A. lappa leaves: treatment temperature 80℃, treatment time 20min, ethanol concentration 70%, extraction temperature 40℃, extraction time 3.5h, solid/liquid ratio 1: 20.(2) By macroporous adsorption resin AB-8 column chromatography and acetacetic ester extraction to purify CHA from both A. lappa leaves and honeysuckle, the CHA content of the crude extracts by water extraction is 31.14% and 47.65%, respectively, while it is 84.69% and 94.37% by ethanol extraction.CHA white acerate crystal was obtained by ethanol extraction, macroporous adsorption resin AB-8 and Sepharese LH-20 column chromatography, acetacetic ester extraction and recrystallization from A. lappa leaves, and was identified to be single-constituent by RP-HPLC, TLC and melting point determination.(3) CHA was able to inhibit the mycelium diffusion of Coletotrichum lagenarium, Phytophthora capsici and Fusarium oxysporum, and also restrain the growth of C. lagenarium mycelium in solid medium. In liquid medium, CHA promoted the mycelium growth of C. lagenarium, and decrease the utilization of reducing sugar; CHA also protected cell membrane. If CHA solution was irradiated with ultraviolet radiation for 15min before employed, the protective effect on cell membrane would be enhanced prominently. Maybe the protective effect of CHA was not performed by itself, but the intermediate produced from it, and UV irradiation helped to bring intermediate.