Alkaline Extraction in Air Enhances Antioxidant and Biological Activities of Humic Acids

Humic acids (HAs) possess diverse functionalities, endowing them with multiple applications as bioactive compounds in agriculture. Alkaline extraction is key to obtaining HAs from their source material. The presence of oxygen during extraction can lead to oxidative changes in the humic structure. The extent of HA transformation depending on their origin remains poorly understood, and the effect of alkaline extraction on the HA biological activities is yet to be estimated. Here, we compare the physicochemical properties of HAs extracted from fresh organic material, compost, in air (HA-O2) and under nitrogen (HA-N2). We also assess the antioxidant properties of HAs-O2 and HAs-N2 from compost (HAC), Retisol (HAR), and Chernozem (HACh) and relate them to the HA biological activities. Changes in the HAC properties were analyzed using the following techniques: elemental composition, ultraviolet–visible and infrared spectroscopy, 13C nuclear magnetic resonance (13C-NMR), electron paramagnetic resonance (EPR), gel filtration using Sephadex G-75 gel, and potentiometric titration. The HA antioxidant properties were explored using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay (antiradical activity) and phosphomolybdenum assay (total antioxidant capacity). The HA biological activity was estimated by priming radish and wheat seeds (0.5 g L−1 HAs, 25 °C, 5 h for radish and 14 h for wheat), followed by germination tests. Alkaline extraction of HAC in air vs. nitrogen resulted in a 1.2-fold increase in the O/C ratio and optical density at E465, oxidation of aliphatic fragments, a 2-fold increase in the contents of functional groups, and a 1.2-fold increase in the number of paramagnetic centers. All HA-O2 preparations have demonstrated an enhanced antiradical activity (1.3–1.6 times) and total antioxidant capacity (1.1–1.3 times) compared to HA-N2. The Vigor Index of seeds primed with HA-O2 was 1.1-to-1.8-fold higher than those treated with HA-N2, depending on the HA origin. We demonstrate that alkaline treatment in air benefits the antiradical and biological activities of HAs, making such preparations more attractive for use as natural antioxidants and priming agents. This opens up new perspectives for using O2-modified HAs as innovative plant stimulants in agriculture.