Isolation, Purification And Structural Identification Of Flavonoids From Celery Leaves

Celery(Apium graveolens L.),is a plant of Apiaceae(Umbelliferae),which has a long history and wide planting area.Celery flavonoids have a variety of biological activity,including antioxidant,anti-inflammatory,analgesic,hypolipidemic,blood pressure and so on.Content of celery leaves flavonoids is the most compared to other parts of celery.However,there are few studies on the separation and purification of the main flavonoid glycosides and their aglycones in celery leaves,so the variation of flavonoid glycosides and the structure of flavonoid glycosides could not be fully explained in celery leaves.So,the extraction process of flavonol glycosides from celery leaves was optimized.Then,the hydrolysis conditions of flavonoid glycosides from celery leaves were optimized.What’s more,the two components were separately by preparative high performance liquid chromatography.Finally,the structures of the isolated compounds were identified.The results were as follows:(1)The adsorption and desorption characteristics of polyamide resin were studied including static and dynamic of polyamide resin to obtained the optimal extraction conditions of extraction of flavonoids from celery leaves.The optimal conditions were as follows:the concentration,the maximum sample volume,and pH were set as 0.3125 mg/mL,8 BV and 5.0,respectively.The sample flow rate and elution flow rate,the washing volume,the volume fraction of ethanol were 2 BV/h,4 BV and 40%,respectively.The ethanol elution volume was 6 BV.Under this condition,the contents of flavone glycoside were increased from 6.15%to 78.5%before purification and the recoveries of total flavonoids were 82.3%.(2)The optimal separation conditionds of flavonol glycosides from celery leaves and its hydrolyzate were preliminarily optimized by high performance liquid chromatography(HPLC),and the optimal separation conditions of flavonoid glycosides from celery leaves and its hydrolyzate were further optimized by preparative high performance liquid chromatography(Prep-HPLC).The optimum conditions of flavonoid glycosides were as follows:Chromatographic column was SinoChrom ODS-BP(10 μm,30 mm×250 mm).The mobile phase A was 100%methanol and the mobile phase B was 0.1%formic acid and the ratio of mobile phase was 50:50.The UV detection wavelength was 267 and 333 nm.Injection volume and elution flow rate were 100 mL and 20 min/min.Under this condition,seven celery flavonoid monomers were isolated.The optimum conditions of the hydrolysis product of flavonoid glycosides from celery leaves were as follows:Chromatographic column was SinoChrom ODS-BP(10 μm,30 mm×250 mm).The mobile phase A was 100%methanol and the mobile phase B was 0.1%formic acid and the ratio of mobile phase was 60:40.The UV detection wavelength was 267 and 333 nm.Injection volume and elution flow rate were 100 mL and 20 min/min.Under this condition,five celery flavonoid monomers were isolated.(3)Eleven compounds about celery flavonoids were identified by UV,HPLC,ultra-high resolution time-of-flight mass spectrometry(UHPLC-Q-TOF-MS/MS)and nuclear magnetic resonance(NMR),including chlorogenic acid,luteolin 7-O-β-D-apiofuranosyl(1 →2)-β-D-glucopyranoside,luteolin 7-O-β-D-glucopyranoside,apiin,chrysoeriol 7-O-β-D-apiofuranosyl(1→2)-β-D-glucopyranoside,luteolin 7-O-[β-D-apiofuranosyl(1 →2)-(6"-O-malonyl)]-β-D-glucopyranoside,apigenin 7-O-[β-D-apiofuranosyl(1→2)-(6"-O-malonyl)]-β-D-glucopyranoside,apigenin 7-O-β-D-glucopyranoside,luteolin,apigenin and chrysoeriol.The purity of the monomer compounds was higher than 97%.