## 3 Results and Discussion
### COX-2 and iNOS in Protein Level  
The effect of okra ethanol extracts, prepared using different cooking methods, on COX-2 and iNOS protein expression in LPS-treated RAW264.7 cells was examined Western blot analysis, and the result is shown in Figure 3. In comparison to the controls without LPS treatment, the ROE, BOE, SOE, and SVOE groups showed a decrease in _COX-2_ and iNOS expression at 200 ug/mL, while a marked increase was observed in the LPS-treated groups. Strikingly, ROE inhibited LPS-induced iNOS protein expression in a concentration-dependent manner in RAW264.7 cells. Notably, ROE at 200 g/mL could further inhibit endogenous COX-2 and iNOS expression in RAW264.7 cells. BOE also suppressed LPS-induced COX-2 and iNOS protein expression in a dose-dependent manner in RAW264.7 cells. However, BOE at 200 g/mL had no effect on endogenous COX-2 and iNOS expression in RAW264.7 cells. In addition, treatment with SOE and SVOE, respectively, led to a concentration-dependent blockage of LPS-induced COX-2 and iNOS protein expression  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Okra Extract** & & & & & & \\ (\#g/mL) & **ROE** & **BOE** & **SOE** & **SVOE** & **F-Value** \\ \hline Control & 100.00 \(\pm\) 2.16 & 100.00 \(\pm\) 20.55 & 100.00 \(\pm\) 1.40 & 100.0490 \(\pm\) 8 & \\
200 & 74.86 \(\pm\) 8.80 & 43.78 \(\pm\) 1.02 & 47.85 \(\pm\) 3.04 & 54.26 \(\pm\) 0.10 & \\ LPS + 1 & 264.67 \(\pm\) 9.57 \({}^{\text{bC}}\) & 221.36 \(\pm\) 36.7 \({}^{\text{aC}}\) & 254.06 \(\pm\) 0.52 \({}^{\text{abD}}\) & 267.52 \(\pm\) 12.82 \({}^{\text{bD}}\) & 3.47 \\ LPS + 50 & 206.7 \(\pm\) 35.3 \({}^{\text{bB}}\) & 179.0 \(\pm\) 17.88 \({}^{\text{abB}}\) & 172.55 \(\pm\) 8.18 \({}^{\text{ab}}\) & 200.43 \(\pm\) 6.83 \({}^{\text{aC}}\) & 3.611 \\ LPS + 100 & 191.49 \(\pm\) 8.37 \({}^{\text{bB}}\) & 182.85 \(\pm\) 1.53 \({}^{\text{bB}}\) & 194.63 \(\pm\) 3.81 \({}^{\text{bC}}\) & 132.71 \(\pm\) 15.26 \({}^{\text{aB}}\) & (0.000) \\ LPS + 200 & 141.34 \(\pm\) 8.80 \({}^{\text{cA}}\) & 92.43 \(\pm\) 1.02 \({}^{\text{aA}}\) & 112.51 \(\pm\) 3.04 \({}^{\text{bA}}\) & 97.56 \(\pm\) 3.23 \({}^{\text{aA}}\) & (0.000) \\ \hline F-value & 35.88 *** & 21.24 *** & 451.217 *** & 149.592 *** & \\ (\(p\)) & (0.000) & (0.000) & (0.000) & (0.000) & \\ \hline \hline \end{tabular} Values represent mean \(\pm\) SD (n = 3). ROE: raw okra ethanol extract, BOE: blanched okra ethanol extract, SOE: steamed okra ethanol extract, and SVOE: sous-vide cooked okra ethanol extract. Values are mean (n = 3) \(\pm\) SD. Means in the common letter (a-c: the level of significance for each cooking method; A-D: the level of significance for each concentration) with the different superscripts are significantly different. *** \(p<0.01\) and *** \(p<0.001\), compared to the control.  
\end{table}
Table 2: Effect of okra extract on nitric oxide (NO) production in LPS-induced RAW264.7 cells.  
Figure 2: Cooking methods and okra extract on nitric oxide (NO) production in LPS-induced RAW264.7 cells. Cells are treated with ROE, BOE, SOE, and SVOE (25, 50, 100, 200 ug/mL), then with or without LPS (1 ug/mL) for 24 h. The cultured supernatant of the treated cells were used to measure NO level. Each bar represents the mean \(\pm\) SD. *** Significance of okra cooking method. Significant values are represented by a-c (*** \(p<0.001\)) compared to the group treated LPS alone. NO: nitric oxide, LPS: lipopolysaccharide, ROE: raw okra ethanol extract, BOE: blanched okra ethanol extract, SOE: steamed okra ethanol extract, and SVOE: sous-vide cooked okra ethanol extract.  
in RAW264.7 cells. Of interest, while SOE at 200 g/mL could inhibit endogenous COX-2 and iNOS expression in RAW264.7 cells, SVOE at 200 g/mL had no effect on it. Among the extracts tested, SVOE at 200 g/mL showed the highest suppressive effect on LPS-induced COX-2 and iNOS protein expression in RAW264.7 cells. The decrease in COX-2 and iNOS expression by okra extracts from all cooking methods is indicative of okra's anti-inflammatory effect.