A novel multi-hyphenated approach to screen and character the xanthine oxidase inhibitors from saffron floral bio-residues.

Recently, the incidence of hyperuricemia increased with patient rejuvenation, searching for new xanthine oxidase (XOD) inhibitors from natural products becomes important. In our previous work, a flavonoid extract of saffron floral bio-residues (SFB) was found to alleviate hyperuricemia via inhibiting XOD. In this study, an integrated approach combining two-dimensional liquid chromatography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) was developed to online screen and character the potential XOD inhibitors from SFB. The two-dimensional liquid chromatography consisted of affinity chromatography and reverse phase chromatography (2D-AR), in which an XOD column, an inactive XOD column, and a control column were used in the first dimensional liquid chromatography to avoid phenomena of "false positive" and "missing screen of compounds with weak affinity to XOD" that often occur in the screening process, and a C column was used in the second dimensional liquid chromatography to separate the mixed XOD binders. Four flavonoid glycosides, i.e., quercetin-3-O-sophoroside (QS), kaempferol-3-O-sophoroside (KS), kaempferol-3-O-rutinoside (KR), and kaempferol-3-O-glucoside (KG), were thus successfully screened and identified from SFB extract by the 2D-AR method. The affinity of QS, KS, KR, KG, kaempferol (aglycone of KS, KR and KG), and quercetin (aglycone of QS) binding to XOD was investigated using SPR method, with K ranged from 4.8 μM to 47.6 μM. The inhibitor constant (K) of KS, KR, KG, quercetin and kaempferol were 4.92 mM, 1.11 mM, 0.294 mM, 4.93 μM and 3.27 μM, respectively, determined using ITC method. Finally, the anti-XOD activities of KS, the most abundant flavonoid in SFB extract, and kaempferol in hyperuricemia mice were verified, which suggested that the multi-hyphenated approach established herein can be applied for screen and character the XOD inhibitors in natural products.