Clove leaf oil: Phytochemical profiling and antimicrobial potential against mastitis-associated microorganisms

Abstract

Clove (Syzygium aromaticum) leaf represents a valuable underutilised plant resource with significant potential for essential oil production. This study characterised clove leaf oil (CLO) from fallen leaves using a standard physicochemical analysis and GC-MS evaluation of phytochemicals. Subsequently, the study evaluated antimicrobial efficacy against four mastitis-associated microorganisms (Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC 25922, Candida albicans ATCC 14053, and Saccharomyces cerevisiae ATCC 9763) using agar well diffusion and broth microdilution methods. Steam distillation yielded 2.05% essential oil from fallen leaves. Phytochemical analysis identified eugenol as the dominant compound (58.59%), alongside β-caryophyllene, eugenyl acetate, and other bioactive constituents (30 identified compounds). The optimal emulsion formulation at a 1:4 ratio (CLO: Tween 80) resulted in a visually clear emulsion with a transmittance exceeding 90%. At 15% concentration, CLO produced inhibition zones comparable to 10% povidone iodine against C. albicans (12.93 vs 13.42 mm), S. cerevisiae (16.20 vs 16.11 mm), greater against S. aureus (14.88 vs 13.49 mm), but smaller against E. coli (12.28 vs 14.14 mm), with MIC values of 3.75-5% and MBC values of 7.5-15%, indicating predominantly bacteriostatic activity. These findings demonstrate that CLO represents a promising natural alternative for routine teat disinfection in sustainable dairy farming operations.

Keywords

• Antimicrobial activity
• Clove leaf oil
• Bovine mastitis
• Essential oil
• Eugenol

Ethical Statement

This in vitro study used commercially available American Type Culture Collection (ATCC) reference strains and did not involve live animals, clinical samples, or human subjects. Therefore, ethical approval was not needed.

Thanks

The author expresses sincere gratitude to the Centre for Higher Education Funding and Assessment under the Ministry of Higher Education, Science, and Technology of the Republic of Indonesia and to the Endowment Fund for Education (LPDP) under the Ministry of Finance of the Republic of Indonesia for fully funding this research through the Indonesian Education Scholarship program (BPI). The author also sincerely thanks Prof. Puguh Surjowardojo and Prof. Osfar Sjofjan (Faculty of Animal Science, Universitas Brawijaya) and Prof. Hermin Sulistyarti (Faculty of Mathematics and Natural Sciences, Universitas Brawijaya) for their valuable insights, constructive feedback, and expert guidance during the dissertation examination, which greatly contributed to the improvement of this manuscript.

References

1. Akinwale, A., Tiwari, R., Yamaki, J., & Parang, K. (2024). Mechanistic study of antimicrobial effectiveness of cyclic amphipathic peptide [R4W4] against methicillin-resistant Staphylococcus aureus clinical isolates. Antibiotics, 13(6), 555. https://doi.org/10.3390/antibiotics13060555
2. Alighiri, D., Eden, W.T., Cahyono, E., & Supardi, K.I. (2018). Quality improvement by batch vacuum distillation and physicochemical characterization of clove leaf oil in Central Java, Indonesia. Journal of Physics: Conference Series, 983(1), 012163. https://doi.org/10.1088/1742-6596/983/1/012163
3. Artanti, A.N., Prihapsara, F., Mas' ud, N., & Ermawati, D.E. (2021). Optimization of the proportion of surfactant, co-surfactant, and candlenut oil for self-nanoemulsifying drug delivery system (SNEDDS) of secang heartwood (Caesalpinia sappan L.) methanolic extract. ALCHEMY, 17(1), 124-131. https://doi.org/10.20961/alchemy.17.1.39054.124-131
4. Barraqui, N.S.G., Garcia, E.F., Lopes, G.A., Fernandes, C.P., Silva, Q.M., & França, H.S. (2025). Antibacterial activity of nanoemulsion of essential oil from Brazilian pepper tree (Schinus terebinthifolia Raddi) against sensitive and multidrug-resistant strains. Brazilian Journal of Biology, 85, e286942. https://doi.org/10.1590/1519-6984.286942
5. Batiha, G., Alkazmi, L., Wasef, L., Beshbishy, A., Nadwa, E., & Rashwan, E. (2020). Syzygium aromaticum L. (Myrtaceae): Traditional uses, bioactive chemical constituents, pharmacological and toxicological activities. Biomolecules, 10(2), 202. https://doi.org/10.3390/biom10020202
6. Benhamada, O., Benhamada, N., Boussouf, L., & Leghouchi, E. (2024). Chemical profile by GC-MS and protective effect of Algerian cloves (Syzygium aromaticum) against Lactobacillus spp. and Streptococcus spp. isolated from dental caries. International Journal of Secondary Metabolite, 11(4), 833-852. https://doi.org/10.21448/ijsm.1438721
7. Bigliardi, P.L., Alsagoff, S.A.L., El-Kamel, M.A., Phua, K.K., & Wu, Y. (2017). Povidone-iodine in wound healing: A review of current concepts and practices. International Journal of Surgery, 44, 260-268. https://doi.org/10.1016/j.ijsu.2017.06.073
8. Braja, I.W.R.S., Permana, I.D.G.M., & Suhendra, L. (2022). Formulation and stability of clove leaf (Syzygium aromaticum L.) essential oil microemulsion. International Journal of Current Research, 11, 197-211. https://doi.org/10.20546/ijcmas.2022.1109.023

9. Cerezo, S., Hernández, M.L., Palomo-Ríos, E., Gouffi, N., García-Vico, L., Sicardo, M.D., Sanz, C., Mercado, J.A., Alfaro, F.P., & Martínez-Rivas, J.M. (2021). Modification of 13-hydroperoxide lyase expression in olive affects plant growth and results in altered volatile profile. Plant Science, 313, 111083. https://doi.org/10.1016/j.plantsci.2021.111083
10. Clinical and Laboratory Standards Institute (CLSI). (2020). Performance standards for antimicrobial susceptibility testing (30th ed.): CLSI supplement M100. Clinical and Laboratory Standards Institute.
11. Cobirka, M., Tancin, V., & Slama, P. (2020). Epidemiology and classification of mastitis. Animals, 10(12), 2212. https://doi.org/10.3390/ani10122212
12. Currie, C., Zimmerman, S., Kuhns, L., Herrera, C., Fox, B., Crofts, A., Horn, H., Chevrette, M., Stankey, R., & Boll, J. (2016). The power of asymmetry: Architecture and assembly of the gram-negative outer membrane lipid bilayer. Annual Review of Microbiology, 70, 255 78. https://doi.org/10.1146/annurev-micro-102215-095308.
13. Dewanti, R., Ariyadi, B., Martien, R., & Zuprizal, Z. (2024). Self-nanoemulsifying drug delivery system of black soldier fly (Hermetia illucens) oil: Optimization, formulation, and characterization. Journal of Applied Pharmaceutical Science, 14(02), 192–199. https://doi.org/10.7324/JAPS.2024.153961
14. Fagere, Z.O., & Al Magbou, A.Z. (2016). Antibacterial activity of clove oil against some microorganisms at Khartoum state, Sudan. Advancement in Medicinal Plant Research, 4(4), 122-128. https://www.netjournals.org/pdf/AMPR/2016/4/16-026.pdf
15. Flynn, J., Gleeson, D., Fitzpatrick, S., Garvey, M., & O'Brien, B. (2021). The effect of disinfectant ingredients on teat skin bacteria associated with mastitis in Irish dairy herds. Irish Veterinary Journal, 74(1):1. https://doi.org/10.1186/s13620-020-00179-7.
16. Gopinath, N. (2023). Antimicrobial potential of lemon and onion extracts against gram-positive and-negative bacteria. International Journal of Secondary Metabolite, 10(4), 626 637. https://doi.org/10.21448/ijsm.1290157
17. Halaji, M., Graham, J., & Nasrollahian, S. (2024). A review of the mechanisms that confer antibiotic resistance in pathotypes of E. coli. Frontiers in Cellular and Infection Microbiology, 4(14), 1387497. https://doi.org/10.3389/fcimb.2024.1387497.
18. Haro-González, J.N., Castillo-Herrera, G.A., Martínez-Velázquez, M., & Espinosa-Andrews, H. (2021). Clove essential oil (Syzygium aromaticum L. Myrtaceae): Extraction, chemical composition, food applications, and essential bioactivity for human health. Molecules, 26(21), 6387. https://doi.org/10.3390/molecules26216387.
19. Haxhiaj, K., Wishart, D.S., & Ametaj, B.N. (2022). Mastitis: What it is, current diagnostics, and the potential of metabolomics to identify new predictive biomarkers. Dairy, 3(4), 722–746. https://doi.org/10.3390/dairy3040050
20. Humphries, R., Bobenchik, A. M., Hindler, J. A., & Schuetz, A. N. (2021). Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing, M100. Journal of Clinical Microbiology, 59(12), 10 1128. https://doi.org/10.1128/jcm.01623-23.
21. Indriyanti, E., & Dinurrosifa, R. (2022). A green synthesis of acetyl eugenol by sonochemical method and potential as anti-inflammatory in-vitro. Jurnal Kimia dan Pendidikan Kimia, 7(3), 324-332. https://doi.org/10.20961/jkpk.v7i3.66630.
22. International Organization for Standardization (ISO). (1997). ISO 3141:1997 – Oil of clove leaf [Syzygium aromaticum (L.) Merr. et Perry, syn. Eugenia caryophyllus (Sprengel) Bullock et S. Harrison]. International Organization for Standardization.
23. International Organization for Standardization (ISO). (1998). ISO 11024-1:1998 – Essential oils – General guidance on chromatographic profiles – Part 1: Preparation of chromatographic profiles for presentation in standards. International Organization for Standardization.
24. Lagarón, J., Torres‐Giner, S., Cabedo, L., & Figueroa-López, K. (2020). Development of electrospun Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) monolayers containing eugenol and their application in multilayer antimicrobial food packaging. Frontiers in Nutrition, 7, 140. https://doi.org/10.3389/fnut.2020.00140.
25. Nguyen, T.K., Tran, L.T.T., Viet, D.H., Thai, P.H., Ha, T.P., Ty, P.V., Duc, L.P., Huu, D.T.T., & Cuong, L.C.V. (2023). Xanthine oxidase, α-glucosidase and α-amylase inhibitory activities of the essential oil from Piper lolot: In vitro and in silico studies. Heliyon, 9, e19148. https://doi.org/10.1016/j.heliyon.2023.e19148
26. Nurmitasari, Y., & Mahfud, M. (2021). Optimization of essential oil extraction from dried clove leaves (Syzygium aromaticum) using solvent-free microwave extraction by face-centered central composite design. IOP Conference Series: Materials Science and Engineering, 1053(1), 012121. https://doi.org/10.1088/1757-899X/1053/1/012121
27. Preeti, Sambhakar, S., Malik, R., Bhatia, S., Al Harrasi, A., Rani, C., Saharan, R., Kumar, S., Geeta, & Sehrawat, R. (2023). Nanoemulsion: an emerging novel technology for improving the bioavailability of drugs. Scientifica, 2023(1), 6640103. https://doi.org/10.1155/2023/6640103
28. Pujiarti, R., Humairoh, A., Wibisono, M.G., & Hardiyanto, E.B. (2020). Effect of chopping on yield, physico-chemical properties, and chemical composition of Clove (Syzygium aromaticum L.) leaf essential from three varieties. In IOP Conference Series: Earth and Environmental Science, 449(1), 012020. https://doi.org/10.1088/1755-1315/449/1/012020.
29. Qian, W., Sun, Z., Wang, T., Yang, M., Liu, M., Zhang, J., & Li, Y. (2019). Antimicrobial activity of eugenol against carbapenem-resistant Klebsiella pneumoniae and its effect on biofilms. Microbial Pathogenesis, 139, 103924. https://doi.org/10.1016/j.micpath.2019.103924.
30. Raharjo, R., Caroko, N., & Sutopo, T.A. (2024). The influence of clove leaf size variations on the characteristics of clove leaf essential oil production. BIO Web of Conferences, 137, 03017. https://doi.org/10.1051/bioconf/202413703017
31. Schreiner, T.B., Santamaria-Echart, A., Ribeiro, A., Peres, A.M., Dias, M.M., Pinho, S.P., & Barreiro, M.F. (2020). Formulation and optimization of nanoemulsions using the natural surfactant saponin from quillaja bark. Molecules, 25(7), 1538. https://doi.org/10.3390/molecules25071538
32. Shunmugaperumal, T., Chakraborthy, P., Rahman, S., & Putta, C. (2023). Development, systematic optimisation and biofilm disruption activity of eugenol-based nanosized emulsions stabilised with Tween 80. Journal of Microencapsulation, 40, 517–533. https://doi.org/10.1080/02652048.2023. 2244094.
33. Suttiarporn, P., Taithaisong, T., Namkhot, S., & Luangkamin, S. (2025). Enhanced eugenol composition in clove essential oil by deep eutectic solvent-based ultrasonic extraction and microwave-assisted hydrodistillation. Molecules, 30(3), 504. https://doi.org/10.3390/molecules30030504
34. Syamsi, A.N., Pratiwi, M., & Nugroho, A.P. (2019). Inhibition activity of garlic (Allium sativum) skin aqueous extract on mastitis causing microorganisms. Animal Production, 21(1), 38 42. https://dx.doi.org/10.20884/1.jap.2019.21.1.673
35. Ullah, M.A., Hassan, A., & Hamza, A. (2023). Role of clove in human medical history. SAR Journal of Anatomy and Physiology, 4(2), 10-19. http://doi.org/10-19. 10.36346/sarjap.2023.v04i02.001
36. Vincenti, S., Mariani, M., Alberti, J.C., Jacopini, S., Brunini-Bronzini de Caraffa, V., Berti, L., & Maury, J. (2019). Biocatalytic synthesis of natural green leaf volatiles using the lipoxygenase metabolic pathway. Catalysts, 9(10), 873. http://dx.doi.org/10.3390/catal9100873