A novel strategy by combining “magnified” matrix solid phase dispersion extraction with high-speed countercurrent chromatography for the rapid and efficient isolation of flavonoids isomers with anti-inflammatory effect from Lindera aggregata (Sims) Kosterm Leaves

A rapid and efficient isolation strategy by combining matrix solid phase dispersion extraction (MSPD) with high-speed countercurrent chromatography (HSCCC) had been proposed in this study for the separation of four flavonoids with anti-inflammatory effect in high edible value plant food, leaves of Lindera aggregata (Sims) Kosterm (L.aggregata-L). MSPD was firstly developed as an efficient extraction methodology to obtain a “clean” extract for the purpose of further isolation of target compounds. Then, two runs of HSCCC isolation were consecutively performed on MSPD extract to purify the target compounds, in which an environmental-friendly biphasic solvent system composed of ethyl acetate-n-butanol-0.05 mol/L of phosphate buffer (pH 3.16) containing 0.05 mol/L of hydroxypropyl-β-cyclodextrin (HP-β-CD) was established for the successful separation of the flavonoids isomers, for the first time. As a result, 21.3 mg of quercetin-3-O-rhamnopyranoside (purity of 96%), 18.7 mg of kaempferol-7-O-α-l-rhamnopyranoside (purity of 89%), 5.5 mg of quercetin-3-O-β-D-glucoside (purity of 90.3%) and 6.4 mg of quercetin-5-O-β-D-glucoside (purity of 86.4%) were obtained from 5 g plant materials. Further cellular assays (RAW264.7 mouse macrophage cells) revealed that the isomers demonstrated good anti-inflammatory activities, of which quercetin-5-O-β-D-glucoside exhibited better anti-inflammatory effect (330% higher than quercetin-3-O-β-D-glucoside at the concentration of 25 μM) but lower cytotoxicity (50% lower than quercetin-3-O-β-D-glucoside at the concentration of 400 μM). In summary, this research indicated a promising isolation strategy of natural compounds which would provide a solid surport for the subsequent further study and development of L. aggregata-L.