Purification And Isolation Of Flavonoids In Water Dropwort And Their Bioactivity

As a kind of common vegetable in China water dropwort (Oenanlhe javanica), has been widely used in traditional Chinese medicine for the treatment of hepatic diseases. Previous studies have shown that it has liver-protective, anti-arrhythmic, anti-anaphylactic, antidiabetic effects. Water dropwort contains various kinds of amino acids, volatile oil and flavonoids. Investigation on functional components of Water dropwort is significant in the research of its development and biological activity such as antioxidant, anti-cancer properties.1. To optimize the process for flavonoids extraction from the leave of water dropwort using water bath heating, a series of single-factor experiments were made to investigate the effects of ethanol concentration, extraction duration, temperature and liquid-to-solid ratio. Based on the results, a response surface design of four factors and three levels was generated by the Design-Expert software and experimental data obtained were subjected to quadratic regression analysis to create a mathematical model. The optimal extraction conditions were found to be:extracting for ethanol concentration of70%and liquid-to-solid ratio of25:1for2.5h at73.8℃.Validation experiments indicated that the flavonoid yield of leave was31.48mg/g DW (approximated the prediction of32.0mg/g DW) and that of stem was6.21mg/g DW under the same condition.2. Six types of macro-porous adsorption resin were selected to compare their performance in absorbing and desorbing flavonoids of water dropwort. Experimental results showed that AB-8resin was optimal choice for flavonoids from water dropwort with static adsorption rate of71.37%and desorption rate of76.57%. The dynamic adsorption separating condition including loaded rate, ethanol concentration and amount of wash solvent, desorption velocity was studied. The optimized condition was found to be loading rate of1mL/min, desorption rate of1.5mL/min,4BV80%ethanol as eluent. Under this condition the purity of the water dropwort extracts with42.60mg flavone content was improved from7.1%to58.63%and the enrichment amount of AB-8macroporous adsorption resin was7.6multiple. High performance liquid chromatography (HPLC) analysis results indicated that the purity of water dropwort flavonoids was improved from6.44%to55.65%with the enrichment coeffient of8.65. In addition, rutin was highly concentrated with the enrichment coeffient of10.3. Total flavonoids components were initially obtained by ethanol-water extraction of water dropwort followed by using AB-8resin based column chromatography and High Speed Counter-Current Chromatography (HSCCC). In the solvent system composed of ethyl acetate-ethanol-water (5:1:5, V:V:V),29.6mg rutin with the purity of97.6%and5.2mg kaempferol-3-O-rutinoside with the purity of97.1%were obtained from the100mg water dropwort extracts.7.1mg quercetin with the purity of93.4%was obtained using the solvent system composed of n-hexane-ethyl acetate-ethanol-water (5:7:5:5, V:V:V:V). The structure of these compounds was confirmed by HPLC and MS technique in comparision with flavonoid standards.4. Antioxidant activity of three flavonoids monomers and total flavonoids were evaluated by DPPH method, ABTS method, FRAP method and ORAC method. The trolox equivalent antioxidant capability values of these samples are:rutin4.46±0.35g TE/g, kaempferol-3-O-rutinoside1.25±0.35g TE/g, quercetin4.961±0.39g TE/g, total flavonoids powder1.521±0.32g TE/g.