Wojciech Rogóż, Aleksandra Owczarzy, Karolina Kulig, Małgorzata Maciążek-Jurczyk

Designing experimental human serum antioxidant models (EHSAMs) and optimizing DPPH and ABTS assays to evaluate their antioxidant activity

DOI: 10.32383/farmpol/235952

2026-10-02

Research’s study. External (exogenous) factors may influence the antioxidant activity of endogenous antioxidants. Therefore, appropriately designed research models are needed to evaluate their impact accurately. They should include more components than single-antioxidant solutions, and they should also be more available for scientific research than biological samples, such as serum. This could create a more realistic representation of in vivo conditions in in vitro analysis. Experimental human serum antioxidant models (EHSAMs) are an example of this type of multi-component system.

Purpose. The aim of the study was to adapt the protocols for selected colorimetric assays to study the antioxidant activity of EHSAMs models and to confirm the validity of the obtained results.

Materials and methods. In order to achieve these goals, spectrophotometric measurements were performed using the DPPH and ABTS assays. The antioxidant activity of human serum albumin (HSA) and normal human serum (S1) samples at various dilutions was analyzed. In the second step of the study, a laboratory-prepared model of normal human serum, labeled as sample 7 (EHSAM N7), was checked.

Results. The antioxidant activity of HSA was lower than that of S1 in both DPPH and ABTS assays, regardless of the incubation time. It was determined that the optimal dilutions for the DPPH and ABTS assays were 15-fold and 200-fold, respectively. The optimal incubation times were 30 and 90 minutes for the DPPH and ABTS assays, respectively. Furthermore, it was concluded that the DPPH assay should be used only as a supplementary tool for the ABTS assay. These results made it possible to evaluate the antioxidant activity of EHSAM N7 and to compare it with that of HSA and S1. Based on this, it was concluded that the antioxidant activity of the tested samples could be described by the following order: HSA=EHSAM N7

Conclusions. Spectrophotometric colorimetric assays, especially the ABTS assay, can be used to reliably evaluate the antioxidant activity of HSA, normal human serum, and EHSAMs models. The initial hypothesis that there is no significant difference in the antioxidant activity of HSA and EHSAM N7 was confirmed. The concept of using EHSAMs models in pharmaceutical research is justified, and its implementation could be beneficial for scientists, patients, and industry representatives.

Keywords: EHSAMs, antioxidants, DPPH, ABTS, serum, albumin.

© Farm Pol, 2026, 82(4): 171–182