## 4 Discussion
_Grateloujia_-specific SLE assumptions were made based on the literature: (1) dry biomass is mostly composed, in terms of massic contribution, of polysaccharides and proteins, which are almost exclusively soluble in water [21; 22; 23; 24]; (2) polysaccharides are mostly agaran-carrageenan hybrids (sulphated galactans) and thus have solubility/extractability highly conditioned by extraction variables such as time, LSR, temperature, and pH [22; 23; 25; 26; 27; 28; 29; 30]; (3) mycosporine-like amino acids can be extracted with all the solvents used (water, ethanol 25%, and 50% (_v/v_)), possibly varying the relative content in each MAA with the solvent [27; 31; 32]; (4) proteins and MAAs are the main compounds responsible for UV-absorbance [4; 27]; (5) ethanol inclusion in the solvent allows the recovery of compounds from highly polar (e.g., phenolic compounds) to medium polarity (carotenoids, sterols, fatty alcohols, etc. ), all of which might contribute to antioxidant activity [33; 34]; and (6) polysaccharides exhibit antioxidant activity, despite being below ethanol-extracted polar metabolites [22; 35]. Three \"slices\" of cubic data at ethanol percentages 0, 25, and 50 were chosen to be represented as 2D contour plots (Figures 1-10). Ethanol was chosen to be the variable seen in slices because the effects found were not expected to lead to smooth, gradual changes in the parameters, as ethanol inclusion in the solvent leads to compositional changes that are more drastic than the variation of physicochemical parameters. Thus, despite being successfully modelled, ethanol effect will be discussed mostly as the difference between 0, 25, and 50% (_v/v_), discretely. The first optimization addressed the effect of LSR, time, and ethanol percentage on the extraction of antioxidant and UV-absorbing molecules (Figures 1-5). Typically, LSR affects an extraction quadratically, having an optimal value, which decreases bilaterally [8; 36]. Concerning time, it is usual to observe a steep increase in yield and other parameters of the extraction initially, approaching a plateau as time increases further, or a peak followed by decrease, in the case of sensitive bioactivities or low-solubility compounds abundance [36]. Ethanol inclusion, in the case of seaweeds, in which biomass extractives are mostly composed of polysaccharides and proteins, is expected to decrease yield and impact bioactivities differentially. In the case of _G. tututuru_, in this work, most of these trends were confirmed, with some exceptions. Yield of _G. turturu_ hydroethanolic extractions (Figure 1) reached its maximum at 0% ethanol (water extraction), which was expected since water is capable of extracting the sulphated galactans of _G. turturu_, and ethanol decreases solubility [25; 26]. Moreover, water extracted the protein fraction from the biomass, which also had a significant massic contribution in its composition, where compounds (i.e., phycoerythrin) were present [4]. The effect pattern for LSR and time was maintained through different ethanol values; for the most part (the central range of values of time and LSR), yield increased linearly with LSR and time had almost no effect on extraction. Naturally, higher LSRs tended to favor the extraction of soluble compounds due to increased solvent availability. However, there were different LSR effects and time outside the mid-ranges. In extreme LSRs, time had a quadratic effect on extraction, with inverse trends depending on LSR. In low-range LSR (\"shortage\" of solvent), yield of short extraction times tended to decrease with an increase in time up to the 60 min of extraction and then increased again. It seems to be a counterintuitive phenomena, but it was repeatedly observed in previous works [37; 38; 39]. It may be that the initial contact of solvent with biomass promotd a rapid solubilization of certain extracellular biomass compounds and that the gradual extraction of intracellular compounds with time somehow promoted the precipitation of the formerly soluble, initally extracted ones.