A simple and efficient protocol for large‐scale preparation of three flavonoids from the flower of Daphne genkwa by combination of macroporous resin and counter‐current chromatography

Abstract

In this paper, macroporous resin column chromatography and counter-current chromatography (CCC) were applied for large-scale preparative separation of three flavonoids from the flower of Daphne genkwa, a famous Chinese medicinal herb. Nine kinds of resins were investigated by adsorption and desorption tests and D101 macroporous resin was selected for the first cleaning-up, in which 40% aqueous ethanol was used to remove the undesired constituents and 90% aqueous ethanol was used to elute the targets. The crude extract after the first step was directly subjected to the preparative CCC purification using the solvent system composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v). The compounds apigemin (823 mg), 3-hydroxyl-genkwanin (842 mg) and genkwanin (998 mg) with the purities of 98.79, 97.71 and 93.53%, respectively, determined by HPLC were produced from 3-g crude extract only in one CCC run. Their chemical structures were identified by MS, UV and the standards.

STUDIES ON A SIMPLE AND EFFICIENT METHOD FOR LARGE-SCALE PREPARATION OF GENKWANIN FROM DAPHNE GENKWA SIEB. ET ZUCC. USING NORMAL-PHASE FLASH CHROMATOGRAPHY

“…[18-21] However, there are some problems in these methods, such as tedious, time consuming, and multiple step developments and high capital requirement. Since genkwanin is a low polarity flavonoid and has poor solubility in the solvent system of water-methanol, HPLC has a problem of long retention and low sample recovery.…”

“…It was used for the purification of polymethoxyflavones from citrus in 50 minutes, yielding up to a gram level with high purities. [22] NPFC has the advantages of simple sample pretreatment, large handling capacity and easy operation. Compared with silica gel column chromatography, it has a better separation effect by using smaller particle size of silica gel as stationary phase and saves labor while the solvent is transported by pump.…”

PREPARATIVE ISOLATION AND PURIFICATION OF FOUR FLAVONOIDS FROM DAPHNE GENKWA SIEB. ET ZUCC. BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

“…Some papers have been reported for detection of the chemical from D. genkwa by HPLC [25–27] , in which only three compounds (apigenin, 3′-hydroxygenkwanin, genkwanin) have been determined. Various combinations of mobile phase (methanol–water, methanol–aqueous acetic acid with different concentrations of acetic acid), flow rate, and detection wavelength were tested in both the isocratic- and gradient-elutionmodes.…”

“…The method also permits introduction of crude samples directly into the column without extensive preparation, and has been successfully applied for isolation and purification of a large number of natural products [16-24] . In the past, three flavonoids have been purified from D. genkwa by combined use of macroporuous resin column chromatography and HSCCC [25] . In this paper, four flavonoids were purified by HSCCC by a sinle run, thus eliminating the sample loss by irreversible adsorption onto the solid support.…”

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high‐speed counter‐current chromatography using stepwise elution

“…As an advanced separation technique, it offers various advantages including high sample loading capacity, high sample recovery, and high purity of fractions. So, it is especially suitable for the separation and purification of active components from natural products such as alkaloids [7, 8], coumarins [9, 10], flavonoids [11], anthraquinones [12], polyphenols [13], and so on. The two-phase solvent systems, composed of CHCl 3 –MeOH–dilute HCl (or phosphate buffer), had been widely used for the HSCCC separation of alkaloids [7, 14].…”