**Characterisation of red clover (Trifolium pratense L.) isoflavones under different extraction conditions**

Marija Kovačević Babić ^1*, Mirela Kopjar², Karolina Sušac¹, Daniela Horvat¹

¹Agricultural Institute Osijek, Južno pregrađe 17, 31000 Osijek, Croatia
²Faculty of Food Technology, Franje Kuhača 18, 31000 Osijek, Croatia

ARTICLE INFO	ABSTRACT
TYPE: Original scientific paper https://doi.org/10.17508/CJFST.2025.17.2.10 PDF	Red clover (Trifolium pratense L.) is a widely used forage crop rich in isoflavones, bioactive compounds with diverse health-protective effects. In legumes, isoflavones occur in various chemical forms, but their health benefits are most pronounced in their free forms—aglycones. Although different extraction parametres (temperature, pH, sample-to-solvent ratio) can influence the efficiency of isoflavone extraction, this study specifically examined the effect of different HCl concentrations (2M, 4M, and 6M), with and without evaporation of the extract to dryness, on the hydrolysis of glycoside isoflavones into aglycones. The leaves and the whole plant of red clover were used as samples. High-Performance Liquid Chromatography (HPLC) was used for the identification and quantification of the isoflavones. The results revealed significant variations in isoflavone content among different plant parts. Red clover leaves had the highest total isoflavone content, with formononetin (3.739 mg/g DM) and biochanin A (2.484 mg/g DM) as the dominant compounds, whereas whole-plant extracts contained a higher content of daidzein (0.440 mg/g DM) and genistein (0.170 mg/g DM) compared to the leaves. Regarding the extraction method, the highest total isoflavone concentration (6.556 mg/g DM) was obtained from leaf extracts that were evaporated to dryness after hydrolysis with 2M HCl.
*CORRESPONDENCE Marija Kovačević Babić marija.kovacevic.babic@poljinos.hr
ARTICLE HISTORY Received: October 13, 2025 Accepted: November 14, 2025
CITATION Kovačević Babić M, Kopjar M, Sušac K, Horvat D. Characterisation of red clover (Trifolium pratense L.) isoflavones under different extraction conditions. Croatian Journal of Food Science and Technology (2025) 17 (2) 1 ― 8
COPYRIGHT: © 2025 by the author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).
	KEYWORDS red clover; leaves; whole plant; extraction; isoflavones; HPLC
	KEY CONTRIBUTION Compared to the whole red clover plant (Trifolium pratense L.), leaves are richer in isoflavones. Four isoflavones have been identified and quantified: daidzein, genistein, formononetin and biochanin A. The most dominant isoflavones are formononetin and biochanin A. The highest isoflavone content was obtained with 2 M HCl after evaporation to dryness.