A New Tetraglycosylated Flavonoid from Leaves of Platycyamus regnellii Benth. Isolated by High-Speed Countercurrent Chromatography

A new tetraglycosylated flavonoid derivative of kaempferol was isolated from the butanol extract of leaves of Platycyamus regnellii (Fabaceae) using high-speed countercurrent chromatography (HSCCC). The butanol extract (960.5 mg) was initially fractionated with the solvent system EtOAC-BuOH-H2O 2:8:10 (v/v/v) (elution in normal phase mode). The fractions containing the major tetraglycosylated flavonoid were further purified with the same solvent system by modifying the ratios to 3:7:10 (v/v/v) and alternating the elution to reversed phase mode. The structure of the isolated flavonoid (48.1 mg, 99.7% purity by ultra-high performance liquid chromatography with diode array detector (UHPLC-DAD)) was characterized by spectrometric and spectroscopic methods and identified as kaempferol 3-O-β-D-glucopiranosyl-(1→2)-α-L-rhamnopiranoside-(1→6)-α-Lrhamnopiranoside-7-O-α-L-rhamnopiranoside. Other minor flavonoids were also detected and tentatively identified using ultra-high performance liquid chromatography with diode array detector coupled to mass spectrometry using the electrospray ionization interface (UHPLC-DAD-ESI-MS/ MS). By using the latter technique, a second tetraglycosylated flavonol with an O-methyl substituent (either rhamnetin, 3-O-methyl-quercetin or isorhamnetin) was detected, together with a triglycosylated kaempferol and diglycosylated flavonoids (either rhamnetin, 3-O-methyl-quercetin or isorhamnetin).