Anticancer activity of eugenol from clove (Syzygium aromaticum L.) against MCF-7 breast cancer cells: in silico and in vitro evaluation
Abstract
Breast cancer is a major cause of cancer-related deaths among women globally. Despite notable progress in the treatment options, the pursuit of effective natural-based therapeutic alternatives continues to be essential. Syzygium aromaticum L. (cloves) is rich in eugenol, a phenolic compound known for its antimicrobial, anti-inflammatory, and anticancer effects. This study aimed to assess the anticancer activity of eugenol extracted from cloves against MCF-7 breast cancer cells using in silico molecular docking and in vitro cytotoxicity assays. The cloves collected from Natuna District and Magetan District (Indonesia) were extracted through Soxhlet extraction with n-hexane and maceration with ethanol. Gas Chromatography-Mass Spectrometry (GC-MS) analysis identified eugenol as the primary component, making up 53.6% of the Soxhlet extract. The molecular docking results indicated that eugenol has significant binding affinities for AKT (-5.1 kcal/mol) and ESR1 (-5.9 kcal/mol), two essential proteins involved in the proliferation and survival of breast cancer cells. Cytotoxicity assessments using the MTT assay revealed a dose-dependent decrease in MCF-7 cell viability, with notable inhibition occurring at concentrations between 15.625 and 2.000 ppm. These results suggest that the eugenol-rich clove extract demonstrates promising anticancer potential by exerting direct cytotoxic effects and influencing key molecular targets within the breast cancer signalling pathway.
References
Alfehaid M. Male Breast Cancer (MBC) - A Review. Pol Przeglad Chir Pol J Surg. 2023;95(6):24–30.
Antonio Pérez J, Salinas P, Poblete MT, Cardemil B. Breast cancer in males. Report of 4 cases. Rev Med Chil. 1995;123(10):1270–4.
Bardhan P, Bui MM, Minton S, Loftus L, Carter WB, Laronga C, et al. HER2-positive male breast cancer with thyroid cancer: An institutional report and review of literature. Ann Clin Lab Sci. 2012;42(2):135–9.
Li X, Zhang H, Yang H, Song Y, Zhang F, Wang A. Modifiable Risk Factors for Breast Cancer: Insights From Systematic Reviews. Public Health Nurs. 2025;42(2):1060–71.
Ang BH, Teo SH, Ho WK. Systematic Review and Meta-Analysis of Lifestyle and Reproductive Factors Associated with Risk of Breast Cancer in Asian Women. Cancer Epidemiol Biomarkers Prev. 2024;33(10):1273–85.
Vishwakarma G, Mehta A, Saifi M, Garg D, Paliwal D. Modifiable (Sleeping Pattern and Stress) and Non-Modifiable Risk Factors Associated with Breast Cancer: A Matched Case-Control Study in Delhi, India. Asian Pac J Cancer Prev. 2022;23(7):2469–76.
Budhwar M, Sharma M, Mehra S, Chopra M. Diagnostic-prognostic biomarkers and their clinical implication in breast cancer. In 2024. p. 493–511. Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85203484391&doi=10.1007%2F978-981-99-6263-1_19&partnerID=40&md5=363e35fd5eb8bda2687be4e930df2e32
Park KR, Nam D, Yun HM, Lee SG, Jang HJ, Sethi G, et al. β-Caryophyllene oxide inhibits growth and induces apoptosis through the suppression of PI3K/AKT/mTOR/S6K1 pathways and ROS-mediated MAPKs activation. Cancer Lett. 2011;312(2):178–88.
Hellmann SS, Thygesen LC, Tolstrup JS, Grønbaek M. Modifiable risk factors and survival in women diagnosed with primary breast cancer: Results from a prospective cohort study. Eur J Cancer Prev. 2010;19(5):366–73.
Abrahao R, Ruddy KJ, Laurent CA, Chubak J, Haupt EC, Brunson AM, et al. Trastuzumab therapy and new-onset hypertension in adolescents and young adults with breast cancer. Breast Cancer Res Treat. 2025;213(1):81–92.
Modan B. Epidemiology of breast cancer. Preventive aspects. Isr J Med Sci. 1981;17(9–10):804–9.
Daraei A, Izadi P, Khorasani G, Nafissi N, Naghizadeh MM, Younosi N, et al. Epigenetic Changes of the ESR1 Gene in Breast Tissue of Healthy Women: A Missing Link with Breast Cancer Risk Factors? Genet Test Mol Biomark. 2017;21(8):464–70.
Ma H, Luo J, Press MF, Wang Y, Bernstein L, Ursin G. Is there a difference in the association between percent mammographic density and subtypes of breast cancer? luminal a and triple-negative breast cancer. Cancer Epidemiol Biomarkers Prev. 2009;18(2):479–85.
Atkinson RL, El-Zein R, Valero V, Lucci A, Bevers TB, Fouad T, et al. Epidemiological risk factors associated with inflammatory breast cancer subtypes. Cancer Causes Control. 2016;27(3):359–66.
John EM, Koo J, Phipps AI, Longacre TA, Kurian AW, Ingles SA, et al. Reproductive characteristics, menopausal status, race and ethnicity, and risk of breast cancer subtypes defined by ER, PR and HER2 status: the Breast Cancer Etiology in Minorities study. Breast Cancer Res [Internet]. 2024;26(1). Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85194884973&doi=10.1186%2Fs13058-024-01834-5&partnerID=40&md5=f9ce679cfcc0a265621ae730e96f527a
Derviş E, Yurt Kilcar A, Medine EI, Tekin V, Cetkin B, Uygur E, et al. In Vitro Incorporation of Radioiodinated Eugenol on Adenocarcinoma Cell Lines (Caco2, MCF7, and PC3). Cancer Biother Radiopharm. 2017;32(3):75–81.
Gupta N, Cohen JL, Rosenbaum C, Raam S. Estrogen receptors in male breast cancer. Cancer. 1980;46(8):1781–4.
Ma M, Ma Y, Zhang GJ, Liao R, Jiang XF, Yan XX, et al. Eugenol alleviated breast precancerous lesions through HER2/PI3K-AKT pathway-induced cell apoptosis and S-phase arrest. Oncotarget. 2017;8(34):56296–310.
Dwivedil V, Shrivastaval R, Hussain S, Ganguly C, Bharadwaj M. Comparative anticancer potential of clove (Syzygium aromaticum) - An Indian spice - Against cancer cell lines of various anatomical origin. Asian Pac J Cancer Prev. 2011;12(8):1989–93.
Kaur K, Kaushal S, Rani R. Chemical Composition, Antioxidant and Antifungal Potential of Clove (Syzygium aromaticum) Essential Oil, its Major Compound and its Derivatives. J Essent Oil-Bear Plants. 2019;22(5):1195–217.
Khan FA, Akhtar S, Almohazey D, Alomari M, Almofty SA. Extracts of Clove (Syzygium aromaticum) Potentiate FMSP-Nanoparticles Induced Cell Death in MCF-7 Cells. Int J Biomater [Internet]. 2018;2018. Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85053042587&doi=10.1155%2F2018%2F8479439&partnerID=40&md5=3d8118c86fcbf59261ce3cb758b79fb7
Atmoko HD, Pangestuti IR, Maulina E, Kusuma TM, Wardani AK, Kaltsum U. In silico study to molecular docking study testing the activity of clove flower essential oil components (Syzigium aromaticum (L)) as anti-breast cancer on EGFR receptors. In Padang, Indonesia; 2023 [cited 2024 Feb 15]. p. 020128. Available from: http://aip.scitation.org/doi/abs/10.1063/5.0120359
Alonso H, Bliznyuk AA, Gready JE. Combining docking and molecular dynamic simulations in drug design. Med Res Rev. 2006;26(5):531–68.
Forli S, Olson AJ. A Force Field with Discrete Displaceable Waters and Desolvation Entropy for Hydrated Ligand Docking. J Med Chem. 2012;55:623–38.
Jendele L, Krivak R, Skoda P, Novotny M, Hoksza D. PrankWeb: a web server for ligand binding site prediction and visualization. Nucleic Acids Res. 2019 July 2;47(W1):W345–9.
Dwivedil V, Shrivastaval R, Hussain S, Ganguly C, Bharadwaj M. Comparative anticancer potential of clove (Syzygium aromaticum) - An Indian spice - Against cancer cell lines of various anatomical origin. Asian Pac J Cancer Prev. 2011;12(8):1989–93.
Idris H, Suryani E, Gustia H, Ramadhan AI. The effect of various essential oil and solvent additives on the botanical pesticide of Piper Aduncum essential oil on formulation antifungal activity. Results Eng [Internet]. 2022;16. Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85138452619&doi=10.1016%2fj.rineng.2022.100644&partnerID=40&md5=3c3cfcf4a55a87f2875ed689fedc91a7
Akoko LO, Rutashobya AK, Lutainulwa EW, Mwanga AH, Kivuyo SL. The effect of reproductive, hormonal, nutritional and lifestyle on breast cancer risk among black Tanzanian women: A case control study. PLOS ONE [Internet]. 2022;17(2 February). Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85124327724&doi=10.1371%2Fjournal.pone.0263374&partnerID=40&md5=43a07f0850fa77c7e0b62931c0dc5c9b
Dierssen-Soto T, Gómez-Acebo I, Palazuelos C, Fernández-Navarro P, Altzibar JM, Gonzalez-Donquiles C, et al. Validating a breast cancer score in Spanish women. The MCC-Spain study. Sci Rep [Internet]. 2018;8(1). Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85042113618&doi=10.1038%2Fs41598-018-20832-0&partnerID=40&md5=f250e7d3fdc3e927b8d75d01d6305562
Mirabelli CK, Nusse R, Tuveson DA, Williams BO. Perspectives on the role of Wnt biology in cancer. Sci Signal [Internet]. 2019;12(588). Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85068912557&doi=10.1126%2fscisignal.aay4494&partnerID=40&md5=54a9d5771af3c146d9a7c3f1e41f1c87
Mukai K, Urai T, Asano K, Nakajima Y, Nakatani T. Evaluation of Effects of Topical Estradiol Benzoate Application on Cutaneous Wound Healing in Ovariectomized Female Mice. Plos One. 2016;11(9):1–15.
De-Sá-Júnior PLDS, Câmara DAD, Costa AS, Ruiz JLM, Levy D, De Azevedo RA, et al. Apoptotic effect of eugenol envolves G2/M phase abrogation accompanied by mitochondrial damage and clastogenic effect on cancer cell in vitro. Phytomedicine. 2016;23(7):725–35.
Debnath A, Lo YH, Bhattacharya M, Wen ZH, Chakraborty C, Das A. Eugenol’s anti-cancer properties, its modulation of signalling pathways, and cascades across various cancers: A review. Curr Res Biotechnol [Internet]. 2025;10. Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-105015035814&doi=10.1016%2Fj.crbiot.2025.100330&partnerID=40&md5=6c6b2a388f9ba6950a47f2daff3ddd3a
Fadhil ZA, Amin EEM, Mahmood SM, Hussein AH. Preparation of a nanoemulsion from clove oil extract containing phenol and studying its effect on a human MCF-7 breast cancer cell line. Salud Cienc Tecnol [Internet]. 2025;5. Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-105006602104&doi=10.56294%2Fsaludcyt20251535&partnerID=40&md5=9078b75ef74cae321c69d822bb1dfd37
Debnath A, Lo YH, Bhattacharya M, Wen ZH, Chakraborty C, Das A. Eugenol’s anti-cancer properties, its modulation of signalling pathways, and cascades across various cancers: A review. Curr Res Biotechnol [Internet]. 2025;10. Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-105015035814&doi=10.1016%2Fj.crbiot.2025.100330&partnerID=40&md5=6c6b2a388f9ba6950a47f2daff3ddd3a
Sihombing INN, Arsianti A. Network pharmacology prediction and molecular docking analysis on the mechanism of eugenol as a candidate against estrogen receptor-positive breast cancer. J Pharm Pharmacogn Res. 2024;12(5):837–51.
Gonzales MC, Grayson J, Lie A, Yu CH, Shiao SYPK. Gene-environment interactions and predictors of breast cancer in family-based multi-ethnic groups. Oncotarget. 2018;9(49):29019–35.
Yan X, Zhang G, Bie F, Lv Y, Ma Y, Ma M, et al. Eugenol inhibits oxidative phosphorylation and fatty acid oxidation via downregulation of c-Myc/PGC-1β/ERRα signaling pathway in MCF10A-ras cells. Sci Rep [Internet]. 2017;7(1). Available from: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85031046402&doi=10.1038%2Fs41598-017-13505-x&partnerID=40&md5=c0aade6313798cc7ce90268bc67db46a
