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

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.

Keywords

• Medicinal plant
• prostate cancer
• antibacterial
• antifungal

Clinopodium vulgare L. subsp. vulgare' nin Sulu, Metanolik ve Etanolik Ekstraktlarının Karşılaştırmalı Değerlendirmesi: Antikanser ve Antimikrobiyal Özellikler

Abstract

Clinopodium vulgare L. subsp. vulgare (Lamiaceae), potansiyel terapötik özelliklere sahip biyoaktif fitokimyasallar açısından zengin bir tıbbi bitkidir. Bu çalışma, C. vulgare L. subsp. vulgare'nin sulu, metanolik ve etanolik özütlerinin antikanser ve antimikrobiyal aktivitelerini karşılaştırmalı olarak değerlendirmeyi amaçlamıştır. Antiproliferatif etkiler, WST-1 testi kullanılarak PC-3 prostat kanseri hücreleri üzerinde araştırılırken, morfolojik değişiklikler invert mikroskopi ile değerlendirilmiştir. Apoptotik mekanizmalar, ELISA yöntemi kullanılarak kaspaz-3, -8 ve -9 protein seviyelerinin araştırılması ile aydınlatılmıştır. Ek olarak, antimikrobiyal aktivite, disk difüzyon yöntemi kullanılarak geniş bir bakteri ve maya suşu paneline karşı belirlenmiştir. Tüm özütler, doz ve zamana bağlı olarak PC-3 hücre proliferasyonunu önemli ölçüde inhibe etmiştir. Test edilen özütler arasında, etanolik özüt en düşük IC50 değerlerini ve en yüksek antiproliferatif aktiviteyi göstermiştir. 200 µg/mL ekstraktlarla muamele edilen PC-3 hücrelerinin morfolojik analizi, karakteristik apoptotik özellikler ortaya koyarken, HUVEC hücreleri nispeten daha hafif morfolojik değişiklikler gösterdi; bu da kanser hücrelerine karşı seçici sitotoksisiteye işaret etmektedir. Ekstraklar ile muamele edilen PC-3 hücrelerinde kaspaz-3, -8 ve -9 seviyeleri önemli ölçüde yükselmiş olup, hem içsel hem de dışsal apoptotik yolların aktivasyonunu düşündürmektedir. Ayrıca, ekstraktlar 8,2 ila 15,8 mm arasında değişen inhibisyon bölgeleriyle orta düzeyde antimikrobiyal aktivite göstermiş ve etanolik ekstrakt en güçlü antibakteriyel ve antifungal etkileri sergilemiştir. Genel olarak, bulgular, özellikle etanolik fraksiyon olmak üzere C. vulgare L. subsp. vulgare ekstraktlarının, seçici antiproliferatif, pro-apoptotik ve orta düzeyde antimikrobiyal aktivitelerle karakterize edilen çok fonksiyonlu biyoaktif özelliklere sahip olduğunu ve doğal bir terapötik ajan kaynağı olarak potansiyellerini desteklediğini göstermektedir.

Keywords

• Tıbbi bitki
• prostat kanseri
• antibakteriyel
• antifungal

References

1. Burk, D.R., Sénéchal-Willis, P., Lopez, L.C., Hogue, B.G., & Daskalov, S.M. (2009). Suppression of LPS-induced inflammatory responses in RAW 264.7 murine macrophages by aqueous extract of Clinopodium vulgare L. (Lamiaceae). Journal of Ethnopharmacology, 126, 397–405. https://doi.org/10.1016/j.jep.2009.09.026
2. Chaves, J.O., de Souza, M.C., da Silva, L.C., Lachos-Perez, D., Torres-Mayanga, P.C., Machado, A.P.D.F., Forster-Carneiro, T., Vázquez-Espinosa, M., González-de-Peredo, A.V., Barbero, G.F., & Rostagno, M.A. (2020). Extraction of flavonoids from natural sources using modern techniques. Frontiers in Chemistry, 8, 507887. https://doi.org/10.3389/fchem.2020.507887
3. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disk susceptibility tests; approved standard. 11th ed. CLSI document M02-A11. Wayne (PA), Clinical and Laboratory Standards Institute, 2012.
4. Collins, C.H., Lyne, P.M., & Grange, J.M. (1989). Microbiological methods, 6th Butterworths.
5. De Pooter, H.L., & Schamp, N.M. 1986. Progress in Essential Oil Research, ed. E. J. Brunke, p. 139, Walter de Gruyter, New York.
6. Dai, J., & Mumper, R.J. (2010). Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules, 15(10), 7313-7352. https://doi.org/10.3390/molecules15107313
7. Dobrev, H.P. (2021). Treatment of numerous hand warts with Clinopodium vulgare tea. Wiener Medizinische Wochenschrift, 171(3), 82-83. https://doi.org/10.1007/s10354-020-00804-8
8. Georgieva, A., Sulikovska, I., Toshkova-Yotova, T., Djeliova, V., Amiri, S., Tsonevski, N., Petkova-Kirova, P., & Tasheva, K. (2025). Antitumor activity of whole-plant extracts from in vitro cultured and wild-growing Clinopodium vulgare plants on a panel of human tumor cell lines. Applied Sciences, 15(2), 925. https://doi.org/10.3390/app15020925

9. Hashim, G.M., Shahgolzari, M., Hefferon, K., Yavari, A., & Venkataraman, S. (2024). Plant-derived anti-cancer therapeutics and biopharmaceuticals. Bioengineering, 12(1), 7. https://doi.org/10.3390/bioengineering12010007
10. Hołota, M., & Posmyk, M.M. (2025). Nature-inspired strategies in cancer management: The potential of plant extracts in modulating tumour biology. International Journal of Molecular Sciences, 26(14), 6894. https://doi.org/10.3390/ijms26146894
11. Karakaş, F.P., Yildirim, A., & Türker, A. (2012). Biological screening of various medicinal plant extracts for antibacterial and antitumor activities. Turkish journal of biology, 36(6), 641-652. https://doi.org/10.3906/biy-1203-16
12. Khan, N.H., nur-E Kamal, M.S.A., & Rahman, M. (1988). Antibacterial activity of Euphorbia thymifolia Linn. Indian Journal of Medical Research, 87, 395–397.
13. Kokdil, G. (1998). Composition of the essential oil of Clinopodium vulgare L. ssp. arundanum (Boiss.) Nyman collected from two different localities in Turkey. Flavour and Fragrance Journal, 13, 470–472. https://doi.org/10.1002/(SICI)1099
14. Kroemer, G., El-Deiry, W.S., Golstein, P., Peter, M.E., Vaux, D., Vandenabeele, P., Zhivotovsky, B., Malorni, W., Knight R.A., Piacentini M., Nagata, S., & Melino, G. (2005). Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death & Differentiation, 12, 1463–1467. https://doi.org/10.1038/cdd.2008.150
15. Li, W., Pan, J., Chen, X., Guo, S., & Ouyang, X. (2025). The chemical composition, pharmacological activity, quality control, toxicity, and pharmacokinetics of the genus Clinopodium L. Molecules, 30(11), 2425. https://doi.org/10.3390/molecules30112425
16. Mallah, H., Diabasana, Z., Soultani, S., Idoux-Gillet, Y., & Massfelder, T. (2025). Prostate cancer: a journey through its history and recent developments. Cancers, 17(2), 194. https://doi.org/10.3390/cancers17020194
17. National Committee for Clinical Laboratory Standards (NCCLS). Methods for antifungal disk diffusion susceptibility testing of yeasts; proposed guideline M44-A. Wayne (PA), National Committee for Clinical Laboratory Standards, 2003.
18. Newman, D.J., & Cragg, G.M. (2020). Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. Journal of Natural Products, 83(3), 770-803. https://doi.org/10.1021/acs.jnatprod.9b01285
19. Opalkenova, G., & Obreskova, D. (1999). Antibacterial action of extracts of Clinopodium vulgare L. curative plant. Drug Development and Industrial Pharmacy, 25(3), 323–328. https://doi.org/10.1081/ddc-100102177
20. Petrova, M., Dimitrova, L., Dimitrova, M., Denev, P., Teneva, D., Georgieva, A., Petkova-Kirova, P., Lazarova, M., & Tasheva, K. (2023). Antitumor and antioxidant activities of in vitro cultivated and wild-growing Clinopodium vulgare L. plants. Plants, 12(8), 1591. https://doi.org/10.3390/plants12081591
21. Qureshi, K.A., Parvez, A., Uzzaman Khan M.M., Aspatwar, A., Atiya, A., Elhassan, G.O., Khan R.A, Erattil Ahammed S.Y, Khan W.U & Jaremko, M. (2024). Exploring nature's hidden treasure: Unraveling the untapped phytochemical and pharmacological potentials of Clinopodium vulgare L.–A hidden gem in the Lamiaceae family. Heliyon, 10(2), e24781. https://doi.org/10.1016/j.heliyon 2024.e24781
22. Sahare, P., Bravo-Vázquez, L.A., Veloz-Briones, D.A., Bernal-Vázquez, D., Bolaños-Fernández, I., Anguiano, B., Luna-Bárcenas, G. & Paul, S. (2025). Differential Expression of Key Oncogenic and Tumor Suppressor MicroRNAs Induced by Andrographolide in Androgen-Independent PC3 and Androgen-Dependent LNCaP Prostate Cancer Cells. Genes, 16(12), 1514. https://doi.org/10.3390/genes16121514
23. Sahoo, G., Samal, D., Khandayataray, P., & Murthy, M.K. (2023). A review on caspases: key regulators of biological activities and apoptosis. Molecular Neurobiology, 60(10), 5805-5837. https://doi.org/10.1007/s12035-023-03433-5
24. Sarikurkcu, C., Ozer, M.S., Tepe, B., Dilek, E., & Ceylan, O. (2015). Phenolic composition, antioxidant and enzyme inhibitory activities of acetone, methanol and water extracts of Clinopodium vulgare L. subsp. vulgare L. Industrial Crops and Products, 76, 961-966. https://doi.org/10.1016/j.indcrop.2015.08.011
25. Şener, S.Ö., Korkmaz, N., Akkaya, Ş., Badem, M., Aliyazıcıoğlu, R., Özgen, U., & Alpay Karaoğlu, Ş. (2018). Clinopodium vulgare L. subsp. vulgare Ekstresinin Antioksidan, Antimikrobial, Tirozinaz İnhibitor Aktiviteleri ve RP-HPLC ile Fenolik Bileşiklerinin Araştırılması. Gümüşhane Üniversitesi Fen Bilimleri Dergisi, 8(2), 230-238. https://doi.org/10.17714/gumusfenbil.306925
26. Stefanovic, O., Stankovic, M.S., & Comic, L. (2011). In vitro antibacterial efficacy of Clinopodium vulgare L. extracts and their synergistic interaction with antibiotics. Journal of Medicinal Plants Research, 5(17), 4074-4079.
27. Sun, S., Yu, Y., Jo, Y., Han, J.H., Xue, Y., Cho, M., Bae S.J., Ryu, D., Park, W., Ha, K.T. & Zhuang, S. (2025). Impact of extraction techniques on phytochemical composition and bioactivity of natural product mixtures. Frontiers in Pharmacology, 16, 1615338. https://doi.org/10.3389/fphar.2025.1615338
28. Taylor, R.C., Cullen, S.P., & Martin, S.J. (2008). Apoptosis: controlled demolition at the cellular level. Nature Reviews Molecular Cell Biology, 9(3), 231-241. https://doi.org/10.1038/nrm2312
29. Tourabi, M., Metouekel, A., Ghouizi, A.E.L., Jeddi, M., Nouioura, G., Laaroussi, H., Hosen, M.E., Benbrahim, K.F., Bourhia, M., Salamatullah, A.M., Nafidi, H.A., Wondmie, G.F., Lyoussi, B., & Derwich, E. (2023). Efficacy of various extracting solvents on phytochemical composition, and biological properties of Mentha longifolia L. leaf extracts. Scientific Reports, 13(1), 18028. https://doi.org/10.1038/s41598-023-45030-5
30. Turker, H., Yıldırım, A.B., & Karakaş, F.P. (2009). Sensitivity of Bacteria Isolated from Fish to Some Medicinal Plants. Turkish Journal of Fisheries and Aquatic Sciences, 9(2), 181-186. https://www.trjfas.org/uploads/pdf_711.pdf
31. Wani, A.K., Akhtar, N., Mir, T.U.G., Singh, R., Jha, P.K., Mallik, S.K., Sinha, S, Tripathi, S.K., Jain, A., Jha, A., Devkota, H.P., & Prakash, A. (2023). Targeting apoptotic pathway of cancer cells with phytochemicals and plant-based nanomaterials. Biomolecules, 13(2), 194. https://doi.org/10.3390/biom13020194
32. Xiang, Z., Liu, L., Xu, Z., Kong, Q., Feng, S., Chen, T., Zhou, L., Yang, H., Xiao, Y. & Ding, C. (2024) Solvent Effects on the Phenolic Compounds and Antioxidant Activity Associated with Camellia polyodonta Flower Extracts. ACS Omega, 9(25), 27192-27203. https://doi.org/10.1021/acsomega.4c01321
33. Yao, L., Li-Min, L., Yuan-Gen, X, Xue-Bin, S., Huan, L., & Si-Hui, N. (2020). Research progress on genus Clinopodium. China Journal of Chinese Materia Medica, 45(18), 4349-4357. https://doi.org/10.19540/j.cnki.cjcmm.20200604.601