Tuğrul Çağrı Akman, Samed Şimşek, Zeynep Akşit, Hüseyin Akşit, Ali Aydın, Şevki Adem, Ali Rıza Tüfekçi, İlyas Yıldız
Abstract BACKGROUND Ebenus macrophylla Jaub. & Spach (EM), an endemic Turkish plant, is a rich source of bioactive compounds with potential therapeutic applications. This study aimed to analyze the phytochemistry of its leaf, stalk, and root extracts and evaluate their antioxidant, antimicrobial, enzyme inhibitory, and antiproliferative effects. RESULTS Liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) analysis identified 39 phenolic compounds. Among the major phenolics identified, hesperidin (in the leaf and stalk), isoquercitrin (in the leaf), catechin (in all parts), and vanillic acid (in the root) were particularly abundant. The leaf extract showed the strongest antioxidant activity (2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH) Half maximal inhibitory concentration (IC 50 ): 14.08 μg mL −1 ) and the highest phenolic content (62.27 mg GAE g −1 ), which is attributed to its high levels of hesperidin and shikimic acid. Antimicrobial assays showed significant activity by the leaf extract against Clostridium perfringens and Pseudomonas aeruginosa (Minimum Inhibitory Concentration (MIC): 31.3 μg mL −1 ). In enzyme inhibition studies, the leaf extract showed stronger effects than standard inhibitors on α ‐glucosidase (IC 50 : 1.56 μg mL −1 −1 ) and tyrosinase (IC 50 : 18.01 μg mL −1 ). Molecular docking analyses confirmed that hesperidin forms multiple stabilizing interactions with α ‐glucosidase and blocks substrate access. Antiproliferative studies showed that the root extract was significantly active against A549, A2780, and HeLa cancer cell lines (Growth Inhibition 50% (GI 50 ): 1.00–3.22 μg mL −1 ) and minimal toxicity toward normal BEAS‐2B cells. CONCLUSION The distribution of phenolic compounds detected in different plant parts is the main factor determining the strong antioxidant, antimicrobial, antidiabetic, and antiproliferative potential of EM. This study demonstrates that the species is a pharmaceutically promising natural bioactive source and provides the scientific basis for further in vivo research. © 2025 Society of Chemical Industry.