High-speed Countercurrent Chromatography Purification Hawthorn And Almond Peel The Main Active Ingredient

In this paper, high-speed counter-current chromatography (HSCCC) was developed for the separation and purification of polyphenols from hawthorn and almond skin. Polyphenols of hawthorn had important physiological activities, mainly in the prevention and treatment of cardiovascular diseases; polyphenols of almond skin had the prevention of cardiovascular disease, regulation of the immune, antioxidant, antibacterial and antiviral, and many other effects. Chemical constituents of hawthorn and almond skin had been reported, but the separation and purification of polyphenols from hawthorn and almond skin by HSCCC had not been reported yet. This paper was in-depth research about the extraction, separation and purification of hawthorn and almond skin, meanwhile structure characterization of compounds were by means of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic. There are five parts in this paper:1. ReviewFirstly, from five aspects, high-speed counter-current chromatography was introduced, which included principles, characteristics, instrument types, how to select two-phase solvent system and the current research status; then, did a summary of the active ingredient and application of hawthorn and almond skin; Finally did an overview of the extraction, separation, function and application of polyphenols.2. Extraction, separation and LC-MS analysis of polyphenol of hawthornThe extract obtained under reflux, the optimized extraction conditions were:70% ethanol, a solid-liquid ratio of 1:15, extracting temperature of 80℃and extracting time of 2 h. Under the optimized conditions, a quality of crude extract 62.4 g was obtained from hawthorn(100 g). The crude extract of hawthorn was separated by high performance liquid chromatography, four main phenolic compounds obtained, which were identified as chlorogenic acid, procyanidin B2, epicatechin, and procyanidin C1 by LC-MS.3. Separation and purification of polyphenol from hawthorn by HSCCCBy LC-MS analysis of four phenolic components from hawthorn, we did a further research about extract of hawthorn by HSCCC; according to the K values of four target components, we choosed solvent systems composed of n-butanol-ethyl acetate-water at a volume ratio of 1:1:2 (v/v/v) for our purification in HSCCC. Optimizing HSCCC conditions with RSM was first time in this paper. The optimal condition obtained using response surface methodology (RSM) as follows:revolution speed, 850 rpm; flow-rate,1.5 mL/min; and separation temperature,25℃. The upper phase was used as the stationary phase while the lower phase was used as the mobile phase.9.7 mg of a product from the crude sample (500 mg)was obtained with purity over 94.8%. The product was identified with UV, LC-MS and H1NMR as chlorogenic acid.4. Extraction, separation and LC-MS analysis of polyphenol of almond skinwe use microwave assisted extraction of polyphenolic compounds from almond skin, by optimizing the extraction conditions, the results were as follow:70% ethanol, solid to liquid ratio of 1:30, microwave extraction power of 400 W and microwave irradiation time of 10 min. Under the optimized conditions, the content of polyphenol of almond skin was up to 8.5 mg/g. The crude extract of almond skin was separated by high performance liquid chromatography, three main phenolic compounds obtained, which were identified as chlorogenic acid dimer, procyanidin dimers and procyanidin trimers were identified by LC-MS.5. Separation and purification of polyphenol from almond skin by HSCCCBy LC-MS analysis of three phenolic components from almond skin, we did a further research about extract of almond skin by HSCCC; according to actual separation condition, we choosed solvent systems composed of n-butanol-ethyl acetate-1% acetic acid solution at a volume ratio of 1:1:2 (v/v/v) for our purification in HSCCC. The optimal condition were as follows:revolution speed,1000 rpm; flow-rate,1.5 mL/min; and separation temperature,25℃. The upper phase was used as the stationary phase while the lower phase was used as the mobile phase.300 mg extract could be introduced into the HSCCC system and 20.6 mg of a product was obtained with purity over 97% in one-step. The product was identified with LC-MS as chlorogenic acid dimer.