## 4 Discussion  
Excessive inflammation and oxidative stress is closely linked to the development of many human chronic illnesses. Up to date, little is known about okra's anti-inflammatory and antioxidative effects and modes of action. This study investigated the effects of four different okra ethanolic extracts (ROE, BOE, SOE, and SVOE) on regulation of LPS-induced inflammatory enzyme and cytokine expression and H\({}_{2}\)O\({}_{2}\)-induced ROS generation in RAW264.7 cells. We report that ROE, BOE, SOE, and SVOE strongly inhibit not only LPS-induced expression of iNOS, TNF-\(\alpha\), IL-1\(\beta\), and IL-6, but also H\({}_{2}\)O\({}_{2}\)-induced ROS generation in RAW264.7 cells. Figure 5: Effect of cooking method and okra extract on ROS production in H\({}_{2}\)O\({}_{2}\)-treated RAW264.7 cells. Cells were treated with ROE, BOE, SOE, and SVOE (50, 100, 200 \(\upmu\)g/mL) and 200 \(\upmu\)M H\({}_{2}\)O\({}_{2}\). The ROS production was analyzed by flow cytometry. Each bar represents the mean \(\pm\) SD. *, *** Significance of okra cooking method. Significant values are represented by \(\alpha\)-C (* \(p\) < 0.05, *** \(p\) < 0.001, compared to the 200 \(\upmu\)M H\({}_{2}\)O\({}_{2}\) group). ROS: reactive oxygen species, H\({}_{2}\)O\({}_{2}\): hydrogen peroxide, ROE: raw okra ethanol extract, BOE: blanched okra ethanol extract, SOE: steamed okra ethanol extract, and SVOE: sous-vide cooked okra ethanol extract. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Okra Extract** & **ROE** & **BOE** & **SOE** & **SVOE** & **F-Value (\(p\))** \\ \hline Control & \(100\pm 2.00\) & \(76.17\pm 5.64\) & \(100.00\pm 8.83\) & \(100\pm 4.56\) & \\
200 \(\upmu\)g/mL & \(76.17\pm 5.52\) & \(88.07\pm 5.20\) & \(81.3\pm 12.08\) & \(88.02\pm 8.40\) & \\ H\({}_{2}\)O\({}_{2}\) & \(531.38\pm 50.87\)\({}^{\text{abA}}\) & \(514.25\pm 60.19\)\({}^{\text{ac}}\) & \(605.85\pm 21.83\)\({}^{\text{bC}}\) & \(628.35\pm 32.93\)\({}^{\text{cC}}\) & \multirow{5}{*}{5.680 *} \\ H\({}_{2}\)O\({}_{2}\) + 50 & \(433.61\pm 8.98\)\({}^{\text{ab}}\) & \(423.36\pm 19.30\)\({}^{\text{ab}}\) & & & 80.059 *** \\  & & & & & (0.000) \\ H\({}_{2}\)O\({}_{2}\) + 100 & \(420.81\pm 9.65\)\({}^{\text{ab}}\) & \(440.67\pm 31.06\)\({}^{\text{ab}}\) & \(526.33\pm 22.52\)\({}^{\text{bB}}\) & \(535.43\pm 14.72\)\({}^{\text{bB}}\) & \multirow{2}{*}{32.174 ***} \\ H\({}_{2}\)O\({}_{2}\) + 200 & \(423.00\pm 20.84\)\({}^{\text{bB}}\) & \(333.35\pm 29.36\)\({}^{\text{aa}}\) & & & (0.000) \\ \hline F-value & \(44.23\)*** & \(11.39\)*** & \(35.43\)*** & \(280.56\)*** & \\ (\(p\)) & (0.000) & (0.003) & (0.000) & (0.000) & (0.000) \\ \hline \hline \end{tabular}
\begin{tabular}{c} Values represent mean \(\pm\) SD (m = 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 (m = 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. * \(p\) < 0.05 and *** \(p\) < 0.001, compared to the cells treated with H\({}_{2}\)O\({}_{2}\). ROS: reactive oxygen species, H\({}_{2}\)O\({}_{2}\): hydrogen peroxide, ROE: raw okra ethanol extract, BOE: blanched okra ethanol extract, SOE: steamed okra ethanol extract, and SVOE: sous-vide cooked okra ethanol extract.  
\end{table} 
Table 3: Effect of okra extract on reactive oxygen species (ROS) production in hydrogen peroxide (H\({}_{2}\)O\({}_{2}\))-induced RAW264.7 cells. It has been previously reported that ethanolic extracts of raw okra at \(\leq\)200 ug/mL concentrations inhibit LPS-induced expression of TNF-a and IL-1\(\beta\) in BV2 microglia cells with no cytotoxicity, addressing its potential utility against neuro-inflammatory illnesses [14]. Macrophages are key defensive cells in response to inflammation, producing NO and cytokines to serve as first-line protection against infection.