Galuteolin

A mixture of natural products [0.01 mg galantamine, 0.01 mg jatrorrhizine, 0.03 mg galuteolin, 0.02 mg quercetin, 0.06 mg luteolin, and 0.03 mg isorhamnetin (Shanghai Yuanye biotechnology co.) in 1 mL storage buffer which contains 5% DMSO (Aladdin Chemistry), M-S₀] was added to the above freshly prepared NA-MB, and the mixture was gently mixed for 3 h at room temperature by a rotating mixer. The supernatant (M-S₁) was separated by magnetic separator. The beads were washed 4 times with storage buffer (1 mL), and each time mixed at room temperature for 8 min on a rotary mixer, the supernatant of each wash was M-S₂–M-S₅. In the extraction step, 50% methanol (MeOH) was first added to the beads, followed by extraction with methanol to obtain supernatants M-S₆–M-S₉. 500 μL of M-S₀–M-S₉ were filtered and analyzed by HPLC. HPLC separations were carried out using a Waters Symmetry C18 column (5 μm, 250 mm × 4.6 mm, Waters Corporation) with a 45 min linear gradient from 10% to 100% methanol containing 0.1% formic acid. UV detector was set at 280 nm.

NA from C. perfringens (Type V, 6 UN, lyophilized powder) and 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid sodium salt hydrate (MUNANA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The BeaverBeads™ Mag NH₂ amine-terminated magnetic beads (2 μm, 10 mg/mL) were the products of Beaverbio Co., Ltd. (Suzhou, China). Pyridine, Tris(hydroxymethyl) methyl aminomethane (Tris), 4-morpholineethanesulfonic acid (MES), 50% glutaraldehyde, ammonium acetate, and dimethylsulfoxide (DMSO) were all obtained from Aladdin Chemistry (Shanghai, China). Standard compounds, including galantamine, jatrorrhizine, galuteolin, quercetin, luteolin, and isorhamnetin were obtained from Shanghai Yuanye biotechnology Co., Ltd. (Shanghai, China). Acetonitrile and methanol (HPLC grade) were purchased from Merck (Germany). Deionized water purified by a Milli-Q water-purification system from Millipore (Bedford, MA, USA) was used for reagent and sample preparation and dilution. All buffer mentioned in this article were freshly prepared before experiments.