# Evaluation of In Vitro Antihypertensive and Anti-Inflammatory Properties of Dairy By-Products
## 3 Results and Discussion
### Effect of D-P3 on Nitric Oxide Production in LPS-Stimulated RAW 264.7 Cells  
To evaluate the effect of digested whey in preventing induced NO formation, LPS-stimulated RAW 264.7 macrophage cells were used due to the potential interaction of bioactive components with inducible NO, since the iNOS molecule is expressed in these cells [53]. NO production was evaluated for the resultant fraction after digestion, referred to as SW-D-P3 and YAW-D-P3, and the results are shown in Figures 3 and 4, respectively. Figures 3 and 4 illustrate that both aforesaid dairy by-products fractions reduced the NO levels compared to the LPS group in the activated macrophage cell line, RAW 264.7. As shown in Figure 3, the production of NO was lowered significantly (\(p<0.05\)) only in the cells incubated with ovine SW-D-P3 compared to the cells incubated with BL-D-P3. However, no statistically significant differences were observed between SW-D-P3 fractions of different animal origins (\(p>0.05\); Figure 3). The NO production of cells incubated in the presence of caprine, ovine, and bovine YAW-D-P3 are displayed in Figure 4. NO values were significantly reduced for all treatment groups compared to the LPS-stimulated cells (\(p<0.05\)), while ovine and caprine YAW-D-P3-treated cells exhibited lower NO production levels compared to both cells treated with bovine YAW-D-P3 and BL-D-P3 (\(p<0.05\)). Figure 4: Effect of YAW-D-P3 on LPS-induced NO production in RAW 264.7 cells. RAW 264.7 cells were treated as follows: LPS, cells stimulated only with 1 \(\mu\)g/mL LPS; D-P3, cells stimulated with LPS and subsequently incubated in the presence of YAW-D-P3 (exprine, ovine, and bovine; 0.011% \(w/v\) refers to starting YAW-D protein) or BL-D-P3 (blank) for 24 h. Data are presented as means \(\pm\) SEM of four independent experiments. Columns with different letters are significantly different (\(p<0.05\)). Figure 3: Effect of SW-D-P3 on LPS-induced NO production in RAW 264.7 cells. RAW 264.7 cells were treated as follows: LPS, cells stimulated only with 1 \(\mu\)g/mL LPS; D-P3, cells stimulated with LPS and subsequently incubated in the presence of SW-D-P3 (exprine, ovine, bovine, and mix; 0.038% \(w/v\) refers to starting SW-D protein) or BL-D-P3 (blank) for 24 h. Data are presented as means \(\pm\) SEM of four independent experiments. Columns with different letters are significantly different (\(p<0.05\)). Our results are in accordance with those previously reported by other groups using undigested and digested milk proteins from different animal sources. [PERSON] et al. [54] reported that the incubation of in vitro-digested skimmed milk in LPS-stimulated RAW 264.7 cells is able to reduce NO production. Similarly, the NO production was decreased in cells treated with digested \(\beta\)-lactoglobulin-lactose conjugate after a 1-day co-incubation with LPS [55]. [PERSON] et al. [56] reported that undigested caprine milk whey reduced NO production significantly in a dose-dependent manner in LPS-stimulated RAW 264.7 cells. Similarly, [PERSON] et al. reported reduced NO production in the same murine macrophage cell line in WPH-treated cells compared to the LPS-stimulated group [57]. Another study reported that a combination of whey protein isolate and galactose did not inhibit NO production in LPS-stimulated RAW 264.7 cells, while the corresponding glycated and fermented sample was able to reduce NO levels [58], which serves as proof of the major role of the fermentation process in releasing anti-inflammatory peptides from whey proteins. In line with this notion, [PERSON] et al. demonstrated a positive effect of camel milk fermentation on NO inhibition in RAW 264.7 [59]. In contrast to our results and those of the aforementioned studies, [PERSON] et al. reported that NO secretion levels were not affected by WPH treatment. It should be noted though that in this study, a different cell model and stimuli (Caco-2 and H2O2) were used, leading the authors to conclude that this intestinal epithelial cell line is not responsive to NO production [60].