| Anthocyanin is the water-soluble plant pigment,and widely found in a variety of organs of plants.Modern pharmacological experiments showed that anthocyanin has a variety of biological activities.It has broad prospects on clinical application.In the present study,cyanidin-3-O-galactoside was separated from the peel of hawthorn fruit(Crataegus pinnatifida Bge.var.major N.E.Br.)(PHF).In addition,the anthocyanin extract of PHF was prepared and its phytochemical composition and biological activity was studied.The following is the main content of this study:1.To separate cyanidin-3-O-galactoside from PHF by conventional column chromatography combined with high-speed counter-current chromatography(HSCCC).A sample of PHF powder was extracted with 80% methyl alcohol containing 0.1%(v/v)hydrochloric acid.The extract solution was concentrated at 40°C,and then chromatographed on a Diaion HP-20 macroporous resin column,eluted subsequently with water,30%,and 70% ethanol all containing 0.1%(v/v)hydrochloric acid.The fraction eluted by 30% ethanol containing 0.1%(v/v)hydrochloric acid was collected and concentrated.Subsequently,the concentrate was chromatographed on a polyamide adsorption resin column,eluted with 20% ethanol containing 0.1%(v/v)hydrochloric acid.This fraction was collected,concentrated and lyophilized.The concentrate was further separated by HSCCC using a solvent system containing methyl tert-butyl ether,1-butanol,acetonitrile,water and trifluoroacetic acid(1: 3: 1: 5: 0.01;v/v),yielding 56 mg of cyanidin-3-O-galactoside with purity of 87.0%.The structure of the compound was identified by proton nuclear magnetic resonance(1H-NMR).2.To prepare the anthocyanin extract of PHFTo get a high extraction efficiency of anthocyanin,different extraction method(immersion,ultrasonic,reflux,and infrared-assisted reflux extraction),extraction solvents(50%,60%,70%,80% EtOH)and pH value(1,2,3,4),solid-liquid ratios(1:10,1:15,1:20,1:25),extraction time(5,10,30,60,90 min)and temperature(20,30,40,50°C)were studied using the contents of cyanidin-3-O-galactoside and cyanidin-3-O-arabinoside as the index.Accordingly,a sample of 1.0 g of PHF powder was extracted with 60% ethanol(trifluoroacetic acid,pH 2).And ultrasonic extraction for 10 min at 30°C was found to be suitable for the extraction of the target compounds.The purification of the anthocyanin extract was performed by Diaion HP-20 adsorption chromatography.The different sample concentration(200,100,50,25,12.5 mg/mL)and pH value(1,2,3,4,5),desorption solvents(30%,40%,50%,60%,70% EtOH)and pH value(1,2,3,4,5),absorption and elution flow rate(1,2,3 BV/h)were studied using the contents of cyanidin-3-O-galactoside and cyanidin-3-Oarabinoside as the index.The optimum conditions of purifying of the anthocyanin extract were as follows: the concentration of sample concentrate was 200 mg/m L(trifluoroacetic acid,pH 2)and absorptive flow rate was 1 BV/h.The desorption solvent was 40% EtOH(trifluoroacetic acid,p H 2),the elution flow rate was 3 BV/h,and the target compounds mainly in the first two elution BV.3.Qualitative and quantitative analysis of the anthocyanin extract of PHFPhytochemical composition of the anthocyanin extract was analyzed qualitatively and quantitatively using HPLC and liquid chromatography tandem mass spectrometry(LC-MS)method.Eight kinds of components,i.e.cyanidin-3-O-galactoside,cyanidin-3-O-arabinoside,procyanidin B2,hyperoside,isoquercitrin,procyanidin C1,(-)-epicatechin and chlorogenic acid were identified,and the contents of cyanidin-3-O-galactoside,cyanidin-3-O-arabinoside,hyperoside,isoquercitrin,(-)-epicatechin and chlorogenic acid were determined to be 140.7,2.5,1.7,0.2,51.1 and 10.5 mg/g,respectively.It was found that the contents of the six compounds were higher than those in PHF(0.9,0.1,0.4,0.2,0.6 and 0.8 mg/g,respectively),which showed that macroporous resin was useful for enrichment of the flavonoids from PHF.4.Biological activity of the anthocyanin extract of PHFThe antioxidant capacity and acetylcholinesterase(AChE)inhibitory activity of the anthocyanin extract were evaluated in this study.Radical scavenging capacity of the anthocyanin extract was estimated using 2,2-diphenyl-1-picryhydrazyl(DPPH)assay and oxygen radical absorbance capacity(ORAC)assay.The ORAC value of the anthocyanin extract was 12.14 ± 0.96 μM Trolox equivalents(TE)/mg and the DPPH IC50 value of the anthocyanin extract was 5.79 μg/m L.The AChE inhibitory activity of the anthocyanin extract was evaluated by Ellman method and the AChE inhibitory IC50 value of the anthocyanin extract was 6.99 μg/mL.The results indicated that the anthocyanin extract exhibited strong antioxidant and AChE inhibitory activity.Above all,the cyanidin-3-O-galactoside was separated from PHF by conventional column chromatography combined with HSCCC for the first time.Meanwhile,this is the first time to get the anthocyanin extract from PHF and to evaluate its biological activity.The results of this study laid the foundation for further research on hawthorn and its exploitation and utilization. |
