Место издания:IB FRC Komi SC UB RAS Syktyvkar, Russia
Аннотация:Fulvic acids (FA), a subclass of humic substances, are structurally complex organic macroligands known for their selective interactions with biological macromolecules and potential use as biostimulants. However, standardized protocols for isolating highly active FA components and quality control criteria remain underdeveloped. In this study, preparative reverse-phase high-performance liquid chromatography (RP-HPLC) with a water/isopropanol eluent system was employed to fractionate a commercial FA product (Fulvagra, Humintech GmbH, Germany). Sixteen fractions were collected and characterized using Fourier transform infrared spectroscopy, UV-vis and fluorescence spectroscopy, high-resolution mass spectrometry (HRMS), and size exclusion chromatography (SEC).Antioxidant capacity (AOC) was assessed using the ABTS assay. Hydrophobic fractions, comprising over 50% of the eluted material, showed markedly enhanced AOC values (0.9–1.4 mmol TE/g), compared to hydrophilic fractions and the parent FA sample (0.65 µmol ABTS•+/mg). The increase in antioxidant activity correlated strongly with molecular parameters indicative of aromatic and conjugated structures, particularly condensed tannins and lignin-like compounds, as identified by HRMS. A strong correlation (R² = 0.96) was also observed between AOC values and the fluorescence asymmetry parameter (ASM350), indicating the utility of optical methods for rapid AOC estimation.To deepen the interpretation of the relationship between chemical structure and antioxidant activity, the same dataset was used to construct a predictive linear regression model based on the contribution of condensed tannins to the FA molecular profile. The resulting equation, AOC (mmol TE/g) = 2.6 × condensed tannins + 0.29 (R² = 0.92), accurately described the AOC response based on the Van Krevelen diagram-derived abundance of condensed tannin-like structures. Model validation using HRMS data for four selected FA fractions demonstrated strong agreement between predicted and experimental AOC values, underscoring the robustness of the approach.Given the sensitivity of optical properties to conjugated aromatic structures, further correlation analysis was conducted between AOC and fluorescence parameters. A particularly strong inverse relationship was found with ASM350 (R² = 0.96), reflecting a systematic increase in both red-band fluorescence intensity and antioxidant capacity as the content of conjugated phenolic structures increased. This finding supports the use of one-dimensional fluorescence spectroscopy as a rapid, non-destructive proxy for antioxidant activity in FA-based materials. The consistency of the ASM350–AOC correlation with direct FT-ICR MS data further confirms its applicability for quality control of commercial FA products.
