Identification of the fractions responsible for plant root elongation-promoting and antioxidant activities of humic product extracted from composted wood thinnings

Abstract Background HS-2 ® Pro, a humic product, is produced from composted thinnings of coniferous trees and is commercially available as a biostimulant. In this study, it was fractionated into humic acid (HA), hydrophobic fulvic acid (FA), hydrophilic FA, and highly hydrophilic FA to identify humic fractions with biostimulant effects. Each fraction was characterized using diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, high-performance size-exclusion chromatography (HPSEC), and reversed-phase high-performance liquid chromatography (RP-HPLC) with π–π interactions, and their root elongation-promoting and antioxidant activities were evaluated. Results The humic product comprised approximately 70% of its weight on an ash-free basis in the HA fraction, 15% in the hydrophobic FA fraction, 2.5% in the hydrophilic FA fraction, and 4.7% in the highly hydrophilic FA fraction. DRIFT spectroscopy showed that the composition of functional groups varied considerably among the fractions. The estimation of the size distribution of the molecular associations in each fraction by HPSEC indicated that the constituents of the HA fraction are forming larger molecular associations, and the constituents of the FA fractions occur as relatively small aggregates and unbound molecules. The RP-HPLC analysis revealed that each humic fraction was composed of many constituents with different degrees of hydrophilicity/hydrophobicity. The root elongation-promoting activity evaluated from the elongation of the primary roots of komatsuna seedlings was highest in the hydrophilic and highly hydrophilic FA fractions, followed by the HA fraction, whereas the hydrophobic FA fraction showed no significant activity. The antioxidant activity evaluated based on the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity was higher in the HA and hydrophobic FA fractions and lower in the hydrophilic and highly hydrophilic FA fractions. Conclusion The present study revealed that the constituents of the hydrophilic FA, highly hydrophilic FA, and HA fractions were responsible for the root elongation-promoting activity of the humic product, whereas the constituents of the HA and hydrophobic FA fractions were responsible for the antioxidant activity. Graphical Abstract