ENHANCING ACCURACY IN ASSESSING POLYSACCHARIDE (FUCOIDAN, DEXTRAN, OR XYLAN) ANTIOXIDANT ACTIVITY: ADDRESSING PRECIPITATION EFFECTS IN THE DPPH ASSAY
antioxidant methods; stress oxidative; sugar; carbohydrate; hydrocolloids
The DPPH assay is one of the most widely used antioxidant tests, with various published protocols for its implementation. The DPPH reagent is typically dissolved in organic solvents, which are also used to precipitate polysaccharides. To investigate whether polysaccharide precipitation occurred during the DPPH test, the antioxidant activity of ten different polysaccharides, polysaccharide-rich (PRE) or polysaccharide-poor extracts (PPE), and small sugars (SS) was evaluated. Regardless of the protocol used, a precipitate was observed after centrifugation, except with SS or PPE. Sugar quantification, electrophoresis, and NMR analyses confirmed the presence of polysaccharides in the precipitated. This phenomenon led to an underestimation of the antioxidant activity of samples. To address this issue, a "sample blank" (sample solution + solvent used to dilute DPPH) was added to the protocol. As a result, the antioxidant activity values of the samples increased by up to 2.5 times. Overall, the data suggest that the currently used DPPH assays may not be the most suitable for accurately evaluating the antioxidant activity of polysaccharides or PRE. The incorporation of a sample blank and a centrifugation step in the DPPH assay protocols is necessary to avoid underestimation of the antioxidant activity of these samples.