Inhibition of nitric oxide (NO) production (Griess assay)

Following MTT assay and confirmation of the non-toxicity of the tested concentrations, RAW 264.7 cells were seeded into 96-well opaque plates at a density of 2.5 × 10⁵ cells/well; after 24 h of incubation, the culture medium was replaced with 90 μl of medium containing different concentrations of extracts (for final concentrations of 5, 10, 25, 50 μg/ml), diclofenac or prednisolone (for final concentrations of 5, 10, 25, 50 μM). Following 1 h of incubation, 10 μl of 10 μg/ml LPS was added to the wells containing the concentrations of the extracts or positive control compounds (prednisolone and diclofenac), as well as to the vehicle-treated wells (negative controls without LPS or extract/compound were also included). The final concentration of LPS to which cells were exposed was 1 μg/ml. The cells were cultured for a further 24 h, after which nitric oxide (NO) levels were assessed by nitrite quantification as previously described [17] using Promega’s Griess Reagent System. Briefly, experimental samples (50 μl supernatant from the wells) as well as nitrite concentration standards prepared from the supplied nitrite stock solution were incubated in the dark, at room temperature, with 50 μl of suphanilamide (1% in 5% H₃PO₄). After 10 min of incubation, 50 μl of napthylethylenediamine (0.1% in distilled H₂O) was added and a further 10 min incubation was carried out in the dark at room temperature. Absorbance was read at 550 nm on a microplate reader (CLARIOstar Microplate Reader, BMG Labtech, UK). Each experiment was repeated three times, with each treatment assessed in triplicate in each experiment. The nitrite concentration was determined by comparison to a nitrite standard reference curve.

The ability of the extracts to inhibit NO production following LPS stimulation potentiated by Interferon gamma (IFNγ) was also investigated. RAW 264.7 cells were seeded into 96-well opaque plates at a density of 2.5 × 10⁵ cells/well; after 24 h of incubation, the culture medium was replaced with 80 μl medium containing different concentrations of extracts (final concentrations of 5, 10, 25, 50 μg/ml), diclofenac or prednisolone (final concentrations of 5, 10, 25, 50 μM). Diclofenac and prednisolone are anti-inflammatory agents and so were used as positive controls. Following 1 h of incubation, 10 μl of 1 μg/ml LPS was added to the wells, followed by 10 μl of 50 ng/ml IFNγ. The final concentrations of LPS and IFNγ were 0.1 μg/ml and 5 ng/ml, respectively, and negative controls were included. Cells were cultured for a further 24 h, after which nitric oxide (NO) levels were assessed by nitrite quantification as described earlier.