Investigation of Hypericum species of greek, bulgarian and serbian flora. NMR-Heterocovariance (NMR-HetCA) chemometrics approach for the identification of bioactive compounds of H. empetrifolium

In EthnoHERBS EU project, following thorough ethnopharmacological research in the Balkan Peninsula, the Hypericaceae plant family showed substantial potential for discovering dermatologically active therapeutic agents [ 1 ]. This study aimed to evaluate the phytochemical and biological properties of selected Hypericaceae species ( Hypericum empetrifolium, H. maculatum, H. perforatum, H. barbatum, H. triquetrifolium ), traditionally used against various skin disorders. The dichloromethanic, methanolic, and hydromethanolic (50:50, v/v) extracts were analysed via High-Performance Thin-Layer Chromatography (HPTLC), while total phenolic (TPC) and total flavonoid (TFC) content were quantified using the Folin-Ciocalteu and aluminum chloride methods, respectively. Antioxidant activity was assessed using the DPPH assay (200 μg/mL), and inhibitory effects on collagenase and tyrosinase were also evaluated. The methanolic extract of H. empetrifolium aerial parts showed notable antioxidant and anti-collagenase activity and was selected for further fractionation using Centrifugal Partition Chromatography (CPC). Fractions were evaluated for their free radical scavenging using the HPTLC-DPPH bioautography method. Moreover, the methanolic extract showed significant in vitro collagenase inhibition, an enzyme relevant to wound healing and anti-aging, that comes in agreement with the traditional use of the plant species in Southeastern Europe to speed the healing process of wounds and scabs. To identify bioactive metabolites prior to isolation, the NMR-Heterocovariance (NMR-HetCA) chemometrics approach was employed, correlating NMR spectra with biological data using SHY and STOCSY tools within the MATLAB environment. This method proved effective for detecting minor bioactive metabolites. In parallel, LC-MS dereplication led to the identification of 40 compounds, including flavonoids, phenolic acids, catechin derivatives, and their glucosides. Publication History Article published online: 05 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany