3.3. NO and β-Hexosaminidase Release Assay

RAW264.7 cells (5 × 104 cells/well, 24-well plates) were seeded and incubated for 24 h. The cells were stimulated with LPS (1 μg/mL) and treated with various concentrations of saponarin (20, 40, 60, and 80 μM) for 24 h. After incubation, the supernatant was mixed with Griess reagent for 10 min, and the optical absorbance was measured at 530 nm using a microplate reader. The amount of NO produced was calculated using a sodium nitrite (NaNO₂) standard curve.

RBL-2H3 cells (2 × 105 cells/well, 24-well plates) were seeded and treated with 0.5 μg/mL DNP-IgE for 24 h. After incubation, the cells were washed with Tyrode buffer (2.5 mM calcium chloride [CaCl₂], 119 mM sodium chloride [NaCl], 1.19 mM magnesium sulfate [MgSO₄], 5 mM potassium chloride [KCl], 5 mM glucose, 10 mM HEPES, and 1 mg/mL BSA, pH 7.3) and incubated with various concentrations of saponarin (5, 10, 20, and 40 μM) for 20 min, then with 100 ng/mL DNP-BSA for 1 h. The supernatants (50 μL) were incubated with a substrate buffer (3.3 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide, pH 4.5) at 37 °C for 1 h. The reaction was terminated using a stop solution (0.1 M sodium bicarbonate [NaHCO₃] and sodium carbonate [Na₂CO₃], pH 10.2), and the optical absorbance was measured at 407 nm using a microplate reader.