Phytochemical characterization and biological activities of wild Ceratonia siliqua L. leaves: antioxidant, antibacterial, and cytotoxic effects

Abstract

Carob (Ceratonia siliqua L.) is a Mediterranean plant traditionally valued for its nutritional and therapeutic properties. Despite the recognized biological potential of its various parts, the pharmacological value of carob leaves remains underexplored. This study aimed to evaluate the phytochemical content and assess the antioxidant, antibacterial, and cytotoxic properties of wild carob leaves ethanolic extract (CLEE). The total polyphenol, flavonoid, and condensed tannin contents of CLEE were quantified as 163.50 ± 9.25 mg gallic acid equivalent/g dry matter (DM), 22.23 ± 8.61 mg quercetin equivalent/g DM, and 34.35 ± 9.70 mg catechin equivalent/g DM, respectively. HPLC-DAD analysis revealed cinnamic, ferulic, sinapic, rutin, and succinic acids as the dominant phenolic compounds. CLEE exhibited strong antioxidant activity, with IC₅₀ values of 0.293 ± 0.034 mg/mL for DPPH and 19.48 ± 2.47 mg/mL for ABTS assays, alongside a total antioxidant capacity of 189.01 ± 7.18 µg ascorbic acid equivalent/mg. The extract also demonstrated notable antibacterial effects against a range of bacterial strains, as evaluated by disc diffusion and broth microdilution methods. Cytotoxicity assays revealed that CLEE exerted a selective antiproliferative effect against the MCF-7 breast cancer cell line, with an IC₅₀ value of 28.26 ± 3.15 µg/mL. Molecular docking highlighted rutin as a key compound contributing to the antioxidant activity. In silico absorption, distribution, metabolism, and excretion profiling showed that most detected bioactive compounds possess good oral bioavailability and comply with Lipinski's rule of five. These findings suggest that CLEE contains pharmacologically relevant constituents and holds promise as a source of natural antioxidants and antimicrobial and anticancer agents. The study supports the valorisation of wild carob leaves, often considered an agricultural by-product, for potential applications in nutraceutical and pharmaceutical development.