NMR HetCA, HPTLC-DPPH• bioautography and LC-MS methodologies for the direct identification of bioactive compounds from Juglans regia L. and Sambucus nigra L.

The present study was carried out within the European project EthnoHERBS in which, Juglans regia L. and Sambucus nigra L. species were selected for further investigation as there were extensive references in ethnopharmacological studies for their use in the treatment of skin ailments [ 1 ] [ 2 ]. Initially, LC-MS dereplication facilitated the annotation of 61 compounds from the methanolic extract of J. regia (flavonoids and naphthoquinone derivatives), while from the hydroalcoholic extract of S. nigra were annotated 36 metabolites (phenolic derivatives and flavonoids). In parallel, both extracts were fractionated by Fast Centrifugal Partition Chromatography (FCPC) and then, High Performance Thin Layer Chromatography (HPTLC) was performed to investigate their chemical content, while simultaneously the antioxidant capacity of their ingredients was evaluated using HPTLC bioautography (HPTLC-DPPH•). In addition, NMR spectra of the fractions were acquired while their DPPH free radical scavenging capacity and inhibitory activity against collagenase enzyme was evaluated in vitro and the Heterocovariance Approach (HetCA) was employed in MATLAB. From the correlation of the spectral data and results of bioactivity assays , high correlation in HetCA plots (pseudospectra) indicated responsible for the activities quercetin derivatives and 1,4,8-trihydroxynaphthalene 1- O -glucopyranoside, in the case of J. regia , while for the hydroalcoholic extract of S. nigra the caffeoylquinic acid and quercetin derivatives were directly identified as active components. In conclusion, HetCA approach and HPTLC bioautography, facilitated the identification of bioactive metabolites prior to their isolation, accelerated the experimental process and reduced the cost of the analysis required by conventional approaches for the study of complex natural preparations. Publication History Article published online: 05 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany