Isolation,Purification,and Activity Evaluation Of Active Compounds From The Leaves Of Malus Domestica Borkh

Natural antioxidants,which have the advantages of high efficiency,low toxicity,and good biocompatibility,are gradually replacing synthetic antioxidants used in cosmetic,food,and pharmaceutical industries.Polyphenols are one kind of natural antioxidants that are widely distributed in plant resources.They have anti-oxidation,anti-aging,anti-tumor,and other biological activities,leading to the potential research value in the prevention and treatment of various chronic human diseases,such as diabetes,cardiovascular diseases,cancers,and neurodegeneration.Apple leaves,which are abundant and inexpensive agriculture wastes,are characterized by a high content of polyphenols.The utilization of apple leaves can also increase the additional economic values of the apple industry.Nowdays,most researches have been focused on the contents and compositions of polyphenols in apple leaves.Other respects still need to be further studied,such as the separation and purification techniques of polyphenols,biological activities of monomer and combined polyphenols,and practical application of polyphenols.Therefore,the extraction,isolation,purification,and activity evaluation of polyphenols from apple leaves were done in this thesis.Most of the research conclusions and results are as follows:1.The petroleum ether,ethyl acetate,and 75%ethanol extracts of apple leaves were obtained by stepwise reflux extraction method with respective organic solvent.Then,their total phenolic and flavonoid contents were evaluated by UV-vis spectrophotometer,followed by the in vitro antioxidant activities,including scavenging 2,2-diphenyl-1-picrylhydrazyl(DPPH)and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid(ABTS)anion free radical capacities and ferrice reducing antioxidant power.Among the three extracts,the 75%ethanol extract contained the highest phenolic and flavonoid contents and had the highest antioxidant activities.The contents of total phenolic and flavonoid were positively correlated with antioxidant activities.Then the DPPH free radical scavenging assay was combined with high performance liquid chromatography(HPLC)to rapidly screen the antioxidant polyphenols from the 75%ethanol extract and the screened antioxidant components were identified by HPLC combined with ion trap time of flight mass spectrometry.The main five antioxidant polyphenols in the 75%ethanol extract were identified as phloretin-2'-O-glucoside,quercetin-3-O-glucoside,quercetin-3-O-xyloside,quercetin-3-O-arabinoside,and quercetin-3-O-rhamnoside.2.The chromatographic technique combining high speed countercurrent chromatography(HSCCC)with preparative HPLC(prep-HPLC)was developed to isolate the five main polyphenols.Optimization of HSCCC conditions was realized by response surface methodology considering two response indexes including retention of stationary phase and analysis time,which were merged using the Derringer's desirability function.The optimal HSCCC conditions were flow rate of 2.10 mL/min,revolution speed of 725 rpm,and temperature of 25?.Under the optimal HSCCC conditions,from 200 mg 75%ethanol extract,2.13 mg quercetin-3-O-glucoside,2.01 mg quercetin-3-O-rhamnoside,and 1.89 mg quercetin-3-O-xyloside with the purities over 90%and 20.31 mg mixtures of phloretin-2'-O-glucoside and quercetin-3-O-arabinoside were obtained.The mixtures were further isolated using prep-HPLC and the other three compounds were further purified to make sure all purities over 99%.With the qualitative and quantitative analysis,homogeneity test,stability test,and purity test of eight laboratories,the purities of phloridin-2'-O-glucoside and quercetin-3-O-xyloside were final defined as(99.63�0.09)%and(98.89�0.06)%,respectively.These two compounds are used to apply for national standard samples.Establish a HPLC method for the determination of phloridin-2'-O-glucoside and quercetin-3-O-xyloside in apple pomace and other products.The method is stable and reproducible and used to apply for national standard method.3.The in vitro antioxidant activities of the five polyphenolic monomers,including phloretin-2'-O-glucoside,quercetin-3-O-glucoside,quercetin-3-O-xyloside,quercetin-3-O-arabinoside,and quercetin-3-O-rhamnoside,were evaluated using the DPPH free radical scavenging assay.The antioxidative activities of the four quercetin glycosides were strong,especially quercetin-3-O-glucoside(IC50 2.92 mg/L),while phloretin-2'-O-glucoside,the high content and characteristic component of apple,has relatively weak antioxidant activity(IC50 119.19 mg/L).The antioxidant activities of quercetin-3-O-glucoside combined with phloretin-2'-O-glucoside,2,6-di-tert-butyl-4-methylphenol(BHT),and tert-butyl benzenediol(TBHQ)at a ratio of 1:25,1:5,1:1,5:1,and 25:1 were determined.The synergistic antioxidant activities were found in the combinations of quercetin-3-O-glucoside with phloretin-2'-O-glucoside and BHT at all ratios,and the strongest at a ratio of 1:1.The mechanism was that quercetin-3-O-glucoside could regenerate phloretin-2'-O-glucoside and BHT in the antioxidant activity test.A hydrogen peroxide-induced oxidative stress in rat hippocampal neurons was estabilished as in vitro cell model,for investigating the inhibitory effects of the five polyphenolic monomers and their combinations on oxidative stress.Quercetin-3-O-glucoside,quercetin-3-O-xyloside,quercetin-3-O-arabinoside,and quercetin-3-O-rhamnoside all significantly inhibited oxidative stress at the concentrations of 0.5 mg/L and 1.0 mg/L,while the combined antioxidant activity of quercetin-3-O-glucoside with phloretin-2'-O-glucoside or BHT was just additive.4.An automatic on-line DPPH-HPLC method for high-throughput screening of antioxidants from natural products was established and validated with nine standards including organic acids(4-hydroxyphenylacetic acid,p-coumaric acid,ferulic acid,and benzoic acid),alkaloids(coptisine and berberine),and flavonoids(quercetin-3-O-rhamnoside,astragalin,and quercetin).The established method has been successfully applied to the extracts of Saccharum officinarum rinds,Coptis chinensis,and apple leaves,consecutively.This established method is cheap and automatic,and could not only be used as an efficient tool for high-throughput antioxidant screening from various complex natural products,but also preliminarily quantify the antioxidant capacity of each component.