Ionic-liquid-based ultrasound-assisted extraction combined with countercurrent chromatography and semipreparative LC for the preparation of monoamine oxidase B inhibitors from Pueraria thomsonii

A simple and efficient method was developed for the rapid screening and identification of ligands for monoamine oxidase B. A new ionic-liquid-based ultrasound-assisted extraction method for medicinal herbs was also developed and validated. In addition, the hyphenated technique of countercurrent chromatography and semipreparative-LC was developed and applied to the isolation of the chemical constituents for Pueraria thomsonii Benth. Three potent monoamine oxidase B inhibitors, namely, daidzein-4 ',7-diglucoside (42.2 mg), puerarin 6 ''-O-xyloside (88.3 mg), and 3 '-hydroxypuerarin (48.5 mg) with purities of 98.2, 96.3, and 97.1%, respectively, were obtained from 500 g of P. thomsonii raw material using semi-HPLC, whereas 3 '-methoxypuerarin (76.2 mg), daidzein-8-C-apiosyl (1 -> 6) glucoside (84.2 mg), and tectorigenin (75.1 mg) with purities of 98.5, 96.4, and 96.8%, respectively, were obtained from 500 g raw material via countercurrent chromatography using a two-phase solvent system comprising n-hexane-ethyl acetate-methanol-water at a volume ratio of 1.85:1.00:0.86:3.69 (v/v/v/v). Then, the anti-Alzheimer activity of the phytochemicals was assessed using a PC12 cell model. Treatment with tectorigenin, daidzein-4 ',7-diglucoside, puerarin 6 ''-O-xyloside, 3 '-hydroxypuerarin, 3 '-methoxypuerarin, and daidzein-8-C-apiosyl (1 -> 6) glucoside (100 mu g/mL), resulted in cell viabilities of 69.00, 65.81, 59.69, 57.90, 55.61, and 54.59%, respectively (p < 0.001). The protocol was proved to be very accurate and efficient.