Abstract
Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) represent the dominant etiological agents of pyometra in cattle, and their strong biofilm-forming ability critically contributes to therapeutic failure and escalating antimicrobial resistance. This study aimed to develop and evaluate an eco-friendly hydrogel chitosan formulation containing Moringa oleifera (M. oleifera) leaf extract as an alternative antibacterial and antibiofilm strategy. The ethanolic extract of M. oleifera was characterized using GC–MS. Hydrogel formulations (10, 20, 30, and, 40%) were prepared and assessed for bacterial growth inhibition and biofilm degradation over 24, 48, and, 72 hours using microplate spectrophotometry. The formulations exhibited clear concentration- and time-dependent activities. The 10% formulation produced the strongest inhibition against E. coli, whereas S. aureus responded most effectively to the 30% formulation, which maintained stable inhibitory effects across all incubation periods. Biofilm assays revealed transient suppression in E. coli, with maximal degradation at 48 hours, while S. aureus showed more sustained inhibition, particularly under the 30% formulation. Correlation analysis demonstrated a stronger alignment between growth inhibition and biofilm reduction in E. coli, indicating more synchronized antibacterial and antibiofilm effects (p < 0.05). The M. oleifera–chitosan hydrogel formulation exhibited promising dual activity against planktonic and biofilm-associated bacteria, highlighting its potential as an eco-friendly therapeutic candidate for the management of bovine uterine infections and for reducing reliance on conventional antibiotics.
References
Adeyemo, R.O., Famuyide, I.M., Dzoyem, J.P., Lyndy Joy, M. 2022. Anti-Biofilm, Antibacterial, and Anti-Quorum Sensing Activities of Selected South African Plants Traditionally Used to Treat Diarrhoea. Evidence-Based Complementary and Alternative Medicine 2022 1, 1307801. https://doi.org/10.1155/2022/1307801.
Al-Marzoqi, A.H., Hadi, M.Y., Hameed, I.H. 2016. Determination of metabolites products by Cassia angustifolia and evaluate antimicobial activity. Journal of Pharmacognosy and Phytotherapy 8 2, 25-48. https://doi.org/10.5897/JPP2015.0367.
Almeida-Bezerra, J.W., Menezes, S.A., Silva, J.T.d.C., de Sousa, S.G., Alves, D.S., Alencar, G.G., Araújo, I.M., Rodrigues, E.Y.d.S., Oliveira-Tintino, C.D.d.M., da Cruz, R.P., Rocha, J.E., Tintino, S.R., Barbosa-Filho, J.M., Morais-Braga, M.F.B., de Menezes, I.R.A., Raposo, A., Coutinho, H.D.M. 2024. Analysis of the antibiotic-potentiating activity, absorption, distribution, metabolism, and excretion (ADME) and the Molecular docking properties of phytol against multi-drug-resistant (MDR) strains. Antibiotics 13 12, 1171. https://doi.org/10.3390/antibiotics13121171.
Amin, Y.A., Ali, R.A., Fouad, S.S., Ibrahim, R.M. 2021. The deleterious effect of postpartum pyometra on the reproductive indices, the metabolic profile, and oxidant/antioxidant parameters of dairy cows. Veterinary World 14 2, 329. www.doi.org/10.14202/vetworld.2021.329-338.
Ardean, C., Davidescu, C.M., Nemeş, N.S., Negrea, A., Ciopec, M., Duteanu, N., Negrea, P., Duda-Seiman, D., Musta, V. 2021. Factors influencing the antibacterial activity of chitosan and chitosan modified by functionalization. International Journal of Molecular Sciences 22 14, 7449. https://doi.org/10.3390/ijms22147449.
Armansyah, T., Siregar, T., Sutriana, A., Suhartono, S., Panjaitan, B., Agustine, R., Saputri, N., Nurlita, U. 2023. Endometritis control and enhanced reproductive performance in Aceh cow using medicinal plants. American Journal of Animal and Veterinary Sciences 18 3, 235-242. https://doi.org/10.3844/ajavsp.2023.235.242.
Bessalah, S., Faraz, A., Dbara, M., Khorcheni, T., Hammadi, M., Ajose, D.J., Saeed, S.I. 2024. Antibacterial, anti-biofilm, and anti-inflammatory properties of Gelatin–Chitosan–Moringa-Biopolymer-Based wound dressings towards Staphylococcus aureus and Escherichia coli. Pharmaceuticals 17 5, 545. https://doi.org/10.3390/ph17050545.
Casillas-Vargas, G., Ocasio-Malavé, C., Medina, S., Morales-Guzmán, C., Del Valle, R.G., Carballeira, N.M., Sanabria-Ríos, D.J. 2021. Antibacterial fatty acids: An update of possible mechanisms of action and implications in the development of the next-generation of antibacterial agents. Progress in Lipid Research 82, 101093. https://doi.org/10.1016/j.plipres.2021.101093.
Djunaidy, V.P., Putri, D.K.T. 2020. The effect of snakehead fish (Channa striata) scale chitosan on colony counts in the interaction between Streptococcus sanguinis and Streptococcus mutans in vitro (in Indonesian: Pengaruh kitosan sisik ikan haruan (Channa striata) terhadap jumlah koloni interaksi Streptococcus sanguinis dan Streptococcus mutans secara in vitro) Dentin 4 3, 100-110. https://ppjp.ulm.ac.id/journals/index.php/dnt/article/view/2598. .
Dzuvor, C.K.O., Pan, S., Amanze, C., Amuzu, P., Asakiya, C., Kubi, F. 2022. Bioactive components from Moringa oleifera seeds: production, functionalities and applications – a critical review. Critical Reviews in Biotechnology 42 2, 271-293. https://doi.org/10.1080/07388551.2021.1931804.
El-Sherbiny, G.M., Alluqmani, A.J., Elsehemy, I.A., Kalaba, M.H. 2024. Antibacterial, antioxidant, cytotoxicity, and phytochemical screening of Moringa oleifera leaves. Scientific Reports 14 1, 30485. https://doi.org/10.1038/s41598-024-80700-y.
Gani, B.A., Asmah, N., Soraya, C., Syafriza, D., Rezeki, S., Nazar, M., Jakfar, S., Soedarsono, N. 2023. Characteristics and antibacterial properties of film membrane of chitosan-resveratrol for wound dressing. Emerging Science Journal 7 3, 821-842. https://doi.org/10.28991/ESJ-2023-07-03-012.
Garcia, L.G.S., Rocha, M.G.d., Freire, R.S., Nunes, P.I.G., Nunes, J.V.S., Fernandes, M.R., Pereira-Neto, W.A., Sidrim, J.J.C., Santos, F.A., Rocha, M.F.G. 2023. Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans. Journal of Applied Oral Science 31, e20230146. https://doi.org/10.1590/1678-7757-2023-0146.
Ghareeb, M.A., Hamdi, S.A.H., Fol, M.F., Ibrahim, A.M. 2022. Chemical characterization, antibacterial, antibiofilm, and antioxidant activities of the methanolic extract of Paratapes undulatus clams (Born, 1778). J Appl Pharm Sci 12 5, 219-228. https://doi.org/10.7324/JAPS.2022.120521.
González-Romero, J., Guerra-Hernández, E.J., Rodríguez-Pérez, C. 2022. Chapter 19 - Bioactive compounds from Moringa oleifera as promising protectors of in vivo inflammation and oxidative stress processes. In Hernández-Ledesma, B. & C. Martínez-Villaluenga (Eds.), Current Advances for Development of Functional Foods Modulating Inflammation and Oxidative Stress (19). Academic Press. 379-399. https://doi.org/10.1016/B978-0-12-823482-2.00011-X.
Haniastuti, T., Puspasari, T.A., Hakim, E.R., Tandelilin, R.T. 2023. Potential effect of giant freshwater prawn shell nano chitosan in inhibiting the development of Streptococcus mutans and Streptococcus sanguinis biofilm in vitro. International journal of dentistry 2023 1, 8890750. https://doi.org/10.1155/2023/8890750.
Haris, Z., Ahmad, I. 2024. Green synthesis of silver nanoparticles using Moringa oleifera and its efficacy against gram-negative bacteria targeting quorum sensing and biofilms. Journal of Umm Al-Qura University for Applied Sciences 10 1, 156-167. https://doi.org/10.1007/s43994-023-00089-8.
Herdiana, Y., Wathoni, N., Shamsuddin, S., Joni, I.M., Muchtaridi, M. 2021. Chitosan-based nanoparticles of targeted drug delivery system in breast cancer treatment. Polymers 13 11, 1717. https://doi.org/10.3390/polym13111717.
Iancu, I., Popa, S.A., Degi, J., Gligor, A., Popa, I., Iorgoni, V., Nistor, P., Imre, K., Nichita, I., Herman, V. 2025. Aerobic uterine pathogens in dairy cattle: Surveillance and antimicrobial resistance profiles in postpartum endometritis. Antibiotics 14 7, 650. https://doi.org/10.3390/antibiotics14070650.
Idris, N., Johannes, E., Dwyana, Z. 2022. Potential of hexadecanoic acid as antimicrobials in bacteria and fungi that cause decay in mustard greens Brassica juncea L. International Journal of Applied Biology 6 2, 36-42. https://journal.unhas.ac.id/index.php/ijoab/article/view/20198.
Jadhav, V., Bhagare, A., Ali, I.H., Dhayagude, A., Lokhande, D., Aher, J., Jameel, M., Dutta, M. 2022. Role of Moringa oleifera on green synthesis of metal/metal oxide nanomaterials. Journal of Nanomaterials 2022 1, 2147393. https://doi.org/10.1155/2022/2147393.
Jafernik, K., Ładniak, A., Blicharska, E., Czarnek, K., Ekiert, H., Wiącek, A.E., Szopa, A. 2023. Chitosan-based nanoparticles as effective drug delivery systems—A review. Molecules 28 4, 1963. https://doi.org/10.3390/molecules28041963.
Japheth, K.P., Kumaresan, A., Patbandha, T.K., Baithalu, R.K., Selvan, A.S., Nag, P., Manimaran, A., Oberoi, P.S. 2021. Supplementation of a combination of herbs improves immunity, uterine cleansing and facilitate early resumption of ovarian cyclicity: A study on post-partum dairy buffaloes. Journal of Ethnopharmacology 272, 113931.
Jayathilaka, E.H.T.T., Nikapitiya, C., De Zoysa, M., Whang, I. 2022. Antimicrobial peptide Octominin-encapsulated chitosan nanoparticles enhanced antifungal and antibacterial activities. International Journal of Molecular Sciences 23 24, 15882. https://doi.org/10.3390/ijms232415882.
Kasimanickam, R., Bhowmik, P., Kastelic, J., Ferreira, J., Kasimanickam, V. 2025. From infection to infertility: Diagnostic, therapeutic, and molecular perspectives on postpartum metritis and endometritis in dairy cows. Animals : an open access journal from MDPI 15 19. https://doi.org/10.3390/ani15192841.
Kim, S., Kim, T.-J. 2025. Inhibitory effect of Moringa oleifera seed extract and its behenic acid component on Staphylococcus aureus biofilm formation. Antibiotics 14 1, 19. https://doi.org/10.3390/antibiotics14010019.
Liu, Y., Jiang, Y., Zhu, J., Huang, J., Zhang, H. 2019. Inhibition of bacterial adhesion and biofilm formation of sulfonated chitosan against Pseudomonas aeruginosa. Carbohydrate polymers 206, 412-419. https://doi.org/10.1016/j.carbpol.2018.11.015.
Mekibib, B., Belachew, M., Asrade, B., Badada, G., Abebe, R. 2024. Incidence of uterine infections, major bacteria and antimicrobial resistance in postpartum dairy cows in southern Ethiopia. BMC Microbiology 24 1, 4. https://doi.org/10.1186/s12866-023-03160-w.
Menon, L.S., Ghosh, N., Biswas, S. 2026. Effect of Moringa oleifera protein-coated gold nanoparticles as naturally derived disruptors of biofilms. International journal of biomaterials 2026, 1381604. https://doi.org/10.1155/ijbm/1381604.
Mugayi, L.C., Mukanganyama, S. 2024. Antimycobacterial and antifungal activities of leaf extracts from Trichilia emetica. Scientifica 2024 1, 8784390. https://doi.org/10.1155/sci5/8784390.
Mulat, M., Banicod, R.J.S., Tabassum, N., Javaid, A., Karthikeyan, A., Jeong, G.J., Kim, Y.M., Jung, W.K., Khan, F. 2025. Multiple strategies for the application of medicinal plant-derived bioactive compounds in controlling microbial biofilm and virulence properties. Antibiotics (Basel) 14 6, 555. https://doi.org/10.3390/antibiotics14060555.
Najah, Z., Alshawish, M. 2023. GC-MS analysis of Rhus tripartita roots extract. Journal Humanitarian and Applied Sciences 8 16, 331-339. https://doi.org/10.65137/jhas.v8i16.458.
Neto, J.X.S., Pereira, M.L., Oliveira, J.T.A., Rocha-Bezerra, L.C.B., Lopes, T.D.P., Costa, H.P.S., Sousa, D.O.B., Rocha, B.A.M., Grangeiro, T.B., Freire, J.E.C., Monteiro-Moreira, A.C.O., Lobo, M.D.P., Brilhante, R.S.N., Vasconcelos, I.M. 2017. A Chitin-binding protein purified from Moringa oleifera seeds presents anticandidal activity by increasing cell membrane permeability and reactive oxygen species production. Frontiers in Microbiology 8, 980. https://doi.org/10.3389/fmicb.2017.00980.
Ojo, O., Fonkui, T.Y., Mmutlane, E.M., Ndinteh, D.T. 2021. Antibacterial effects and phytochemical profiling of ethnomedicinal plant Lecaniodiscus cupanioides Planch. ex Benth from South-Western Nigeria. Pharmacologyonline 3, 914-926. https://pharmacologyonline.silae.it/files/archives/2021/vol3/PhOL_2021_3_A101_Ojo.pdf.
Pérez-Flores, J.G., García-Curiel, L., Pérez-Escalante, E., Contreras-López, E., Aguilar-Lira, G.Y., Ángel-Jijón, C., González-Olivares, L.G., Baena-Santillán, E.S., Ocampo-Salinas, I.O., Guerrero-Solano, J.A., Portillo-Torres, L.A. 2025. Plant antimicrobial compounds and their mechanisms of action on spoilage and pathogenic bacteria: A bibliometric study and literature review. Applied Sciences 15 7, 3516. https://doi.org/10.3390/app15073516.
Permadi, A., Sutanto, S., Wardatun, S. 2018. Comparison of dractionated and non-fractionated extraction methods on the total flavonoid content of Physalis angulata herb using colorimetry. Student Online Journal of Pharmacy 1 1, 1-10. https://jom.unpak.ac.id/index.php/farmasi/article/view/706.
Pitjamit, S., Vichiansan, N., Leksakul, K., Boonyawan, D. 2024. Optimization of Ultrasonic and Microbubble Disinfection for Escherichia coli and Staphylococcus aureus: Experimental Design and Effectiveness Evaluation. J Eng Technol Sci 56 6, 742-755. https://doi.org/10.5614/j.eng.technol.sci.2024.56.6.6.
Qadir, A., Singh, S.P., Akhtar, J., Ali, A., Arif, M. 2018. Chemical composition of Saudi Arabian Sukkari variety of date seed oil and extracts obtained by slow pyrolysis. Indian Journal of Pharmaceutical Sciences 80 5, 940-946. https://doi.org/10.4172/pharmaceutical-sciences.1000441.
Rosmaidar, R., Handayani, M., Fadillah, F., Armansyah, T., Siregar, T.N., Hafizuddin, H., Husnurrizal, H. 2021. The Effect of Red Betel Leaf (Piper crocatum) and Moringa Leaf Extracts on Endometritis Levels in Aceh Cows. Traditional Medicine Journal 26 3, 161-168. https://doi.org/10.22146/mot.64626.
Subash, P., Srinivasa Rao, K. 2021. In silico molecular docking analysis for potential anti-Alzheimer's compounds from the methanolic leaf extract of Erythroxylum monogynum using Gas chromatography–mass spectrometry. Journal of Saudi Chemical Society 25 8, 101285. https://doi.org/10.1016/j.jscs.2021.101285.
Suhartono, S., Ismail, Y.S., Muhayya, S.R. 2019. The interference of Moringa oleifera leaf extracts to modulate quorum sensing-facilitated virulence factors. Biodiversitas Journal of Biological Diversity 20 10, 3000-3004. https://doi.org/10.13057/biodiv/d201031.
Sundarajan, S., Abdurahman, N.H. 2019. Chemical compound characterizations of patchouli leaf extract via GC-MS, LC-QTOF-MS, FTIR, and 1H NMR. International Journal of Engineering Research & Technology 8 6, 1054-1059. https://doi.org/10.17577/IJERTV8IS060541.
Talib, M., Nabeel, M.A., Haq, S.U., Waqas, M.S., Jamil, H., Aqib, A.I., Muneer, A., Fouad, D., Ataya, F.S. 2023. Recent trends in S. aureus and E. coli-based endometritis, and the therapeutic evaluation of sodium alginate-based antibiotics and nanoparticles. Gels 9 12, 955. https://doi.org/10.3390/gels9120955.
Thasmi, C.N., Hafizuddin, H., Husnurrizal, H., Dasrul, D., Sutriana, A., Gani, B.A., Nazar, M. 2024. Moringa oleifera as a potential bioactive agent against Gram-positive and negative bacteria: In-silico analysis of 1YN5 and 3RG1 receptor binding. Biodiversitas Journal of Biological Diversity 25 8. https://doi.org/10.13057/biodiv/d250812.
Thasmi, C.N., Husnurrizal, H., Wahyuni, S., Hafizuddin, H., Siregar, T.N. 2021. Identification of bacteria in Aceh cattle with repeat breeding. Veterinarija ir zootechnika 79 2, 32-36. https://vetzoo.lsmuni.lt/data/vols/2021/7902/pdf/thasmi.pdf.
Wen, Y., Li, W., Su, R., Yang, M., Zhang, N., Li, X., Li, L., Sheng, J., Tian, Y. 2022. Multi-target antibacterial mechanism of Moringin from Moringa oleifera seeds against Listeria monocytogenes. Frontiers in Microbiology 13, 925291. https://doi.org/10.3389/fmicb.2022.925291.
Yang, M., Tao, L., Kang, X.-R., Li, L.-F., Zhao, C.-C., Wang, Z.-L., Sheng, J., Tian, Y. 2022. Recent developments in Moringa oleifera Lam. polysaccharides: A review of the relationship between extraction methods, structural characteristics and functional activities. Food Chemistry: X 14, 100322. https://doi.org/10.1016/j.fochx.2022.100322.

This work is licensed under a Creative Commons Attribution 4.0 International License.
Copyright (c) 2026 Cut Nila Thasmi, Husnurrizal Husnurrizal, Tongku Nizwan Siregar, Hafizuddin Hafizuddin, Amalia Sutriana, Herrialfian Herrialfian, Faisal Jamin, Darniati Darniati, Shakira Ramadhani, Naila Nuha Najdha, Alya Fadilah, Syeicha Le Adzra