2.10. Determination of Nitric Oxide (NO) and Proinflammatory Cytokines (IL-6 and TNF-α) Production

The anti-inflammatory activities of O. indicum were evaluated by measuring the level of NO and proinflammatory cytokines (IL-6 and TNF-α) production in LPS plus IFN-γ-activated RAW264.7 cells. The cells were seeded at a density of 6 × 10⁵ cells/well in a 6-well plate and then incubated overnight. After incubation, the culture medium was removed, and the cells were pretreated with different concentrations of O. indicum (50, 100, and 200 μg mL⁻¹) or the anti-inflammatory agent, DEX (1 μM), for 3 h. Then, the cells were activated with 1 μg mL⁻¹ LPS plus 10 ng mL⁻¹ IFN-γ and incubated for 24 h. The supernatant was collected for further analysis of NO using Griess reagent and determined the level of TNF-α and IL-6 with the ELISA kits.

The level of NO in the culture media was detected as nitrite, a major stable product of NO, using Griess reagent as described by Sittisart et al. [24]. Briefly, 100 μL of cell culture medium was mixed with an equal volume of Griess reagent in a 96-well plate and incubated at room temperature for 10 min in the dark. The intensity of the pink color of the samples was measured at 540 nm using a microplate reader (Bio-Rad Laboratories, Inc.). The amount of nitrite in the samples was determined using the linear sodium nitrite calibration curves at a concentration range of 2.5–100 μM.

Proinflammatory cytokine levels (IL-6 and TNF-α) were quantified by Mouse IL-6 or Mouse TNF-α DuoSet® ELISA Kits (R&D systems Inc., Minneapolis, USA) according to the manufacturer's instructions. Optical density was measured at 450 nm with a microplate reader (Benchmark Plus, Bio-Rad, Japan). By preparing the standards of known cytokine concentrations, the number of cytokines in the samples was quantified from a standard curve.