Comparative Evaluation of Aqueous, Methanolic, and Ethanolic Extracts of Clinopodium vulgare L. subsp. vulgare: Anticancer and Antimicrobial Properties

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Abstract
Clinopodium vulgare L. subsp. vulgare (Lamiaceae) is a medicinal plant rich in bioactive phytochemicals with potential therapeutic properties. The present study aimed to comparatively evaluate the anticancer and antimicrobial activities of aqueous, methanolic, and ethanolic extracts of C. vulgare L. subsp. vulgare. Antiproliferative effects were investigated on PC-3 prostate cancer cells using the WST-1 assay, while morphological alterations were assessed by inverted microscopy. The mechanisms of apoptosis have been elucidated by measuring caspase-3, -8, and -9 protein levels using the ELISA method. In addition, antimicrobial activity was determined against a broad panel of bacterial and yeast strains using the disk diffusion method. All extracts significantly inhibited PC-3 cell proliferation in a dose- and time-dependent manner. Among the tested extracts, the ethanolic extract exhibited the lowest IC50 values and the highest antiproliferative activity. Morphological analysis of PC-3 cells treated with 200 µg/mL extracts revealed characteristic apoptotic features, whereas HUVEC cells showed comparatively milder morphological alterations, indicating selective cytotoxicity toward cancer cells. Caspase-3, -8, and -9 levels were significantly elevated in treated PC-3 cells, suggesting activation of both intrinsic and extrinsic apoptotic pathways. Furthermore, the extracts demonstrated moderate antimicrobial activity, with inhibition zones ranging from 8.2 to 15.8 mm, and the ethanolic extract showing the strongest antibacterial and antifungal effects. Overall, the findings indicate that C. vulgare L. subsp. vulgare extracts, particularly the ethanolic fraction, possess multifunctional bioactive properties characterized by selective antiproliferative, pro-apoptotic, and moderate antimicrobial activities, supporting their potential as a natural source of therapeutic agents.

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