## 4 Discussion
Since we are working with crude extracts that present a mixture of compounds with synergistic/antagonistic effects, it is possible that the best solution for the desired function may be related to specific concentrations where the dilution of certain compounds would be beneficial. Another interesting fact is that not all the MAAs have the same ability to act as photoprotectors [37]. At this point, further chemical characterization of the extracts could help to discriminate the presence of MAAs and which of them are present/at a relative quantity. Regarding also the production of MAAs, it is known that their production is affected by several abiotic factors, preferring the summer period and moderate depth [29, 39]. A recent study also focused on the fact that \(G\). \(turturu\) tends to reduce the production of MAAs in the presence of intense UV radiation [40], suggesting that the production of such compounds may be highly influenced by different factors that are not totally controlled when the macroalga is grown in a natural environment. The anti-inflammatory potential of seaweed has also been explored in an attempt to find potential sustainable and safer solutions with less side effects, especially for treatments of chronic inflammation [2]. During inflammation, oxidative stress increases and the cellular antioxidant capacity decreases, leading to large quantities of produced free radicals that will interact with fatty acids, cell membranes, proteins, and other components, promoting permanent alterations in cellular functions [41]. This process is mediated by a system of soluble factors that differ in their source and composition, one of them being the production of nitric oxide by macrophages. This compound is responsible for inducing vasodilatation, acting as a cytotoxic agent for pathogens [41]. Therefore, the discovery of novel compounds able to act as anti-inflammatory could be a new insight in this field. A dose-response evaluation of each extract was performed in a macrophage cell line due to the direct implication of these cells in inflammatory processes, using the same range of concentrations used for 3T3 cells (Figure S2). Similar patterns were found in both cases: extracts with a higher concentration of ethanol in the extraction procedure (E1 and E2) showed higher cytotoxic effects when compared with the other four extracts. For each extract, all the concentrations above 90% cell viability were used to analyze the nitric oxide production (Figure 3). Comparing with the control (cells subjected only to LPS solution), two extracts showed significant differences: E3 at 0.25 mg\(\cdot\)mL\({}^{-1}\) (\(p<0.05\)), reducing the NO production to 80%, and E6 at 0.01 (\(p<0.01\)) and 0.25 mg\(\cdot\)mL\({}^{-1}\) (\(p<0.0001\)), reducing 27% and 38.3%, respectively. However, higher concentrations stimulated NO production on macrophages cells. In macroalgae, several types of bioactive compounds have already been described for their anti-inflammatory potential, namely, pigments (such as carotenoids), sulfated polysaccharides, proteins and their derivatives (such as phycobiliproteins), fatty acids (such as polyunsaturated fatty acids) and other compounds such as halogenated compounds or terpen [10, 29]. Some of them are known to be produced by red macroalgae and specifically by \(G\). \(turturu\), such as carotenoids, phycobiliproteins, and sulfated polysaccharides, among others, which could help to explain the reduction in NO production in those extracts. However, different methods for anti-inflammatory evaluation, such as Western blot quantification of inflammatory markers' expression (e.g., TNF-\(\alpha\), interleukins, among others), should be implemented, since different pathways may be activated and the specificity of the technique is higher.