Ligustrum vulgare (LV), widely cultivated in Europe and often used in hedges, has been historically recognized in folk medicine for its potential health benefits. This study focused on exploring the untargeted identification of secondary metabolites in ethanol extracts (70% v/v) from different morphological parts (young shoots, leaves, flowers and fruits) of LV at various stages of plant development, using ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS). Additionally, the selected biological activities (antioxidant activity, cyclooxygenase-2 inhibition (COX-2), α-amylase inhibition and cytotoxicity) of the tested extracts were determined. Untargeted metabolomics showed that LV extracts were a rich source of phenylethanoid compounds, flavonoids, iridoids and their derivatives. The flowers of LV had the highest content of oleuropein (33.43 ± 2.48 mg/g d.w.). The lowest antioxidant activity was obtained for ripe and post-seasonal fruits, while in the case of other samples, the activity was at a similar level. All tested extracts showed α-amylase and COX-2 inhibitory activity. In addition, LV extracts showed strong antiproliferative properties in colorectal (HT29) and liver (HepG2) cancer cell lines. The obtained results show the difference in the content of bioactive compounds in various morphological parts of Ligustrum vulgare. These differences may influence the multifaceted medicinal potential of this plant.

High-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) coupled with radical scavenging assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) can be both used for the detection of the antioxidants in plant extracts. In this study, the ethanolic (70% v/v) extracts from different morphological parts of Ligustrum vulgare collected at different stages of maturity were used as the source of antioxidants. The final identification of antioxidants was performed using high-resolution mass spectroscopy (HRMS). As a result, 19 compounds with antioxidant properties detected with HPLC-ABTS assay and 10 compounds detected with HPTLC-DPPH/ABTS assay were identified, mostly from the group of iridoids, phenylethanoids, and flavonoids. When comparing different L. vulgare samples, it was found that the extracts obtained from leaves contained the greatest number of antioxidants. The results of this study suggest that HPTLC-DPPH/ABTS as well as HPLC-ABTS derivatization coupled with the HRMS can be successfully used for profiling and identification of antioxidants from natural sources. Planar chromatography is more suitable for screening multiple samples because of its simplicity, whereas more challenging liquid chromatography provides more detailed information and is therefore better for a selected set of samples.

This study introduces a High-Performance Thin-Layer Chromatography (HPTLC)-based method for assessing α-amylase activity and inhibition. The method was designed as an alternative to the conventional 3,5-dinitrosalicylic acid (DNS) assay, which reports only total inhibition and lacks information on specific hydrolysis products. Starch hydrolysis was examined using α-amylase from porcine pancreas, human saliva, and Aspergillus oryzae. Coupling HPTLC with mass spectrometry enabled the identification of six products formed during enzymatic breakdown. For validation, three key markers of activity (maltose, maltotriose, maltotetraose) were quantified after 30 min of incubation, confirming accuracy and reproducibility. The validated approach was applied to twelve edible flowers tested as hydroalcoholic extracts and infusions. Heather, peony, and rose showed the highest inhibitory activity, corresponding to 7.68 ± 0.37, 2.37 ± 0.11, and 1.77 ± 0.07 mg acarbose equivalents per gram of dry weight, respectively. Compared with the DNS assay, the HPTLC method demonstrated greater precision, minimized matrix interference, and, uniquely, allowed direct visualization of how inhibitors altered both total activity and the distribution of hydrolysis products. These advantages highlight the value of HPTLC as a screening tool for complex plant materials and support its potential in food research, nutraceutical development, and quality control.