Extraction and Purification of Quercitrin, Hyperoside, Rutin, and Afzelin from Zanthoxylum Bungeanum Maxim Leaves Using an Aqueous Two-Phase System.

In this study, an aqueous two-phase system (ATPS) based on ethanol/NaH2 PO4 was developed for the extraction and purification of quercitrin, hyperoside, rutin, and afzelin from Zanthoxylum bungeanum Maxim leaves. These 4 flavonoids were 1st extracted from dried Z. bungeanum leaves using a 60% ethanol solution and subsequently added to the ATPS for further purification. The partition behavior of the 4 flavonoids in ATPS was investigated. The optimal ATPS conditions were: 29% (w/w) NaH2 PO4 , 25% (w/w) ethanol concentration, 1% (w/w) added amount of leaf extracts, no pH adjustment, and repeated 1 h extractions at 25 °C. Under the optimal conditions for the 10 g ATPS, the absolute recovery of quercitrin, hyperoside, rutin, and afzelin reached 90.3%, 83.5%, 92.3%, and 89.1%, respectively. Compared to the 60% ethanol extracts, the content of quercitrin (44.8 mg/g), hyperoside (65.6 mg/g), rutin (56.4 mg/g), and afzelin (6.84 mg/g) in the extracts increased by 49.9%, 38.8%, 45.6%, and 36.8% respectively. The extracts after ATPS also exhibited stronger antioxidant activities, the 2,2-diphenyl-1-picrylhydrazyl IC50 value (10.5 μg/mL) decreased by 41.8%, and the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt value (966 μmol Trolox/g) and ferric reducing power value (619 μmol Trolox/g) increased by 29.8% and 53.7%, respectively. Furthermore, scale-up experiments indicated that a larger scale experiment was feasible for the purification of the 4 flavonoids.