## 4 Discussion
LPSs found in the cell membranes of macrophages have been used to induce inflammation in related experiments [2; 3]. Nitric oxide (NO), as an indicator of inflammatory response, is produced mainly by inducible nitric oxide synthase (iNOS) and is associated with the virulence of inflammatory diseases [2; 4; 23]. In this study, we have demonstrated that ethanolic extracts of raw okra (ROE) at \(\leq\)200 ug/mL largely suppresses not only LPS-induced expression of iNOS, TNF-\(\alpha\), IL-1\(\beta\), and IL-6, but also LPS-induced NO production in RAW264.7 cells with no cytotoxicity, supporting ROE's anti-inflammatory effect. As previously mentioned, the nutrients and functional compounds in foods changed according to the cooking method used, such as moist, or dry heat cooking [19; 20; 24], which may thus influence their biological activity. Of importance, we herein have shown that other ethanolic extracts of okra (BOE, SOE, and SVOE) prepared from blanching, steaming, and sous-vide cooking methods, respectively, are also able to greatly inhibit LPS-induced iNOS expression and NO production in RAW264.7 cells. These results point out that BOE, SOE, and SVOE also have strong anti-inflammatory activities. Given that SVOE has higher anti-inflammatory (and antioxidant) activities than those of ROE herein, it is suggested that the sous-vide cooking method is useful in not only preserving the nutrients and functional compounds in okra but also improving okra's biological (anti-inflammatory) activity. There is a wealth of information illustrating presence of a variety of active components in okra (extracts), including polysaccharide [8; 9; 13]. Because both ROE and SVOE have strong anti-inflammatory (and antioxidant) activities herein, it will be interesting to identify and compare, in future, functional components in ROE and SVOE and elucidate how they trigger anti-inflammatory (antioxidant) activities. COX-2 and iNOS are absent in normal cells, but can be induced by LPS or inflammatory cytokines, resulting in inflammatory tissue damage, gene mutation, and nerve damage [25; 26]. The present decrease in COX-2 and iNOS expression by okra extracts from all cooking methods is indicative of okra's anti-inflammatory effects. LPS induced high iNOS mRNA expression in RAW264.7 cells, pointing out that the iNOS proteins induced by LPS in these cells were due to its transcriptional upregulation. In addition, a concentration-dependent inhibitory effect of okra extracts on the production of iNOS and inflammatory cytokines (TNF-\(\alpha\), IL-6, and IL-1\(\beta\)) was shown across all four cooking methods. TNF-\(\alpha\) is a cytokine produced by white blood cells, including lymphocytes and macrophages. It is generated upon stimulation of macrophages, by substances such as LPS, but is not produced in a normal state [25; 26]. TNF-\(\alpha\) plays a critical role in natural immune responses and is associated with the onset and progress of acute and chronic inflammatory diseases [27; 28]. IL-6 has a crucial role in the early immune response and overexpression could cause various diseases such as infectious diseases, malignant tumors, or autoimmune diseases; hence, IL-6 is always increased in various inflammatory diseases. IL-6 is also important in the functional regulation of T cells and B cells [29; 30]. IL-1\(\beta\), on the other hand, promotes T cell activation, B cell maturation, and natural killer cell activity [31]. The infiltration of inflammatory cells such as lymphocytes and neutrophils is therefore increased in the inflammatory area [32] and, when facilitated with IL-1\(\beta\) overexpression, results in autoimmune abnormality, pain or fever, suggestive of IL-1\(\beta\)'s crucial role in inflammation. In this study, the okra extracts prepared using all four different cooking methods were shown to have effectively inhibited the production of iNOS, TNF-\(\alpha\), IL-6 and IL-1\(\beta\), the main inflammatory cytokines, when their levels were measured after treating RAW264.7 cells simultaneously with LPS and different okra extracts.