SYSTEMATIC AND COMPARATIVE STUDY OF THE INFLUENCE OF SOLVENTS AND CONCENTRATIONS ON THE QUANTIFICATION OF BIOACTIVE COMPOUNDS OF SPIRULINA AND CHLORELLA

The determination of bioactive compounds in microalgae requires standardized methods, which makes it difficult to compare studies and consolidate data in the literature. Different protocols for the preparation of extracts, solvents and concentrations result in significant variations in the values of total phenolics, proteins and antioxidant activity, compromising the reproducibility and interpretation of the results. This work sought to systematically evaluate the effect of different solvents (methanol, ethanol and acetone) and concentrations (0, 25, 50, 75 and 100%) on the quantification of total phenolics, proteins and antioxidant activity (DPPH, ABTS and FRAP) in Chlorella and Spirulina biomasses. For each condition, the ratio of 1:20 (w/v) was used, followed by stirring, ultrasonication, centrifugation and filtration. The data were submitted to two-factor ANOVA, considering solvent and concentration as fixed factors. In all cases, significant effects of solvent, concentration and interaction between both were observed (p < 0.0001). In Chlorella, the highest value of total phenolics was obtained in 75% acetone (~3.5 mg EAG/g), accompanied by higher protein content. The correlation with proteins was 0.73 (p < 0.0001), indicating its contribution to the overestimation of phenolics, although it does not fully explain the variation. DPPH peaked at 75% ethanol and acetone (~7), ABTS at 100% methanol (~19) and FRAP at 75% acetone (~30) (values expressed in μmol Trolox/g). For Spirulina, the highest value of total phenolics was observed at 0%, with approximately 11.76±0.07 mg GAE/g, coinciding with higher protein contents in the same extracts (5.29±0.03 mg/mL). The correlation was very strong (r = 0.95; p < 0.0001), showing that the apparent increase in phenolics is directly associated with the presence of soluble proteins, such as phycobiliproteins. Although they interfere with quantification, proteins also contributed to antioxidant activity, as evidenced by the high DPPH values already at 0% (15.14±0.28 µmol Trolox/g). The ABTS and FRAP radicals showed their highest values in 100% methanol, reaching 32.52±0.23 and 57.00±2.96 µmol Trolox/g, respectively. It is concluded that the quantification of total phenolics alone may lead to misleading interpretations, and the protein contribution should always be considered. Moreover, for antioxidant activity, different solvents and concentrations favor distinct classes of compounds, meaning that no single protocol can fully represent the antioxidant activity of microalgae. Therefore, combined or sequential extraction strategies are recommended to broaden the spectrum of recovered compounds and advance methodological standardization.