Effectiveness of Black Cumin (Nigella Sativa) Seed Extract as an Antibacterial Agent Against Mycobacterium Tuberculosis Using The Resazurin Microtiter Assay (REMA) Method
Main Article Content
Tuberculosis remains a major global health problem caused by Mycobacterium tuberculosis. The rising prevalence of multidrug-resistant tuberculosis and the adverse effects associated with prolonged first-line therapy like rifampicin, highlight the need for alternative or adjunctive treatments. Nigella sativa, a medicinal plant rich in bioactive compounds, has attracted considerable attention because of its antimicrobial, antioxidant, anti-inflammatory, and immunomodulatory properties, which are primarily attributed to its major active compound, thymoquinone. This study aimed to determine the antimicrobial activity and minimum inhibitory concentration of Nigella sativa seed extract against Mycobacterium tuberculosis using the resazurin microtiter assay method. A true experimental study was conducted using the H37Rv strain of Mycobacterium tuberculosis. A methanolic extract of Nigella sativa seeds was prepared using the maceration method. Antibacterial activity was assessed using the resazurin microtiter assay (REMA) after 10 days of incubation. Bacterial growth inhibition was determined through visual observation of resazurin color change and confirmed using a microplate reader at 630 nm. Wells remaining blue indicated inhibition, whereas pink indicated growth. Rifampicin and sterile water were included as positive and negative controls respectively to validate experimental conditions and assay performance consistency throughout the study. Nigella sativa seed extract demonstrated antimicrobial activity with a minimum inhibitory concentration of approximately 0.125 mg/mL (31-62 µg/mL), indicated by the absence of color change and supported by lower absorbance values at 630 nm. Rifampicin showed inhibitory activity at significantly lower concentrations 0.125µg/mL, confirming its higher antimicrobial potency. Nigella sativa seed extract exhibits measurable antibacterial activity against Mycobacterium tuberculosis.
Keywords:
Nigella sativa, Mycobacterium tuberculosis, Resazurin Microtiter Assay, Minimum Inhibitory Concentration, Rifampicin, Tuberculosis
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Kabir, Y., Akasaka-Hashimoto, Y., Kubota, K., & Komai, M. (2020). Volatile compounds of black cumin (Nigella sativa L.) seeds cultivated in Bangladesh and India. Heliyon, 6(10), e05343. https://doi.org/10.1016/j.heliyon.2020.e05343
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Majdalawieh, A. F., & Fayyad, M. W. (2015). Immunomodulatory and anti-inflammatory action of Nigella sativa and thymoquinone: A comprehensive review. International Immunopharmacology, 28(1), 295–304. https://doi.org/10.1016/j.intimp.2015.06.023
Martin, A., Camacho, M., Portaels, F., & Palomino, J. C. (2003). Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: Rapid, simple, and inexpensive method. Antimicrobial Agents and Chemotherapy, 47(11), 3616–3619. https://doi.org/10.1128/AAC.47.11.3616-3619.2003
Nurchollifah, Y., Wijayatri, R., & Hidayat, I. W. (2021). Literature study of pharmacological effects of black seed (Nigella sativa) based on active seeds. Prosiding University Research Colloquium, 815–832. https://www.repository.urecol.org/index.php/proceeding/article/view/1647
Omollo, C., Singh, V., Kigondu, E., Wasuna, A., Agarwal, P., Moosa, A., Ioerger, T. R., Mizrahi, V., Chibale, K., & Warner, D. F. (2021). Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, 65(5). https://doi.org/10.1128/AAC.02554-20
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Randhawa, M. A. (2011). In vitro antituberculous activity of thymoquinone, an active principle of Nigella sativa. Journal of Ayub Medical College Abbottabad, 23(2), 78–81. https://pubmed.ncbi.nlm.nih.gov/24800349
Rutala, W. A., & Weber, D. J. (2010). Guideline for disinfection and sterilization of prion-contaminated medical instruments. Infection Control & Hospital Epidemiology, 31(2), 107–117. https://doi.org/10.1086/650197
Salem, M. L. (2005). Immunomodulatory and therapeutic properties of Nigella sativa L. seed. International Immunopharmacology, 5(13–14), 1749–1770. https://doi.org/10.1016/j.intimp.2005.06.008
Sarker, S. D., Nahar, L., & Kumarasamy, Y. (2007). Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods, 42(4), 321–324. https://doi.org/10.1016/j.ymeth.2007.01.006
Sawicki, R., Widelski, J., Oki?czyc, P., Truszkiewicz, W., Golus, J., & Sieniawska, E. (2022). Exposure to Nepalese propolis alters the metabolic state of Mycobacterium tuberculosis. Frontiers in Microbiology, 13. https://doi.org/10.3389/fmicb.2022.929476
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World Health Organization. (2023). Global tuberculosis report 2023. World Health Organization. https://iris.who.int/handle/10665/373828
Albakry, Z., Karrar, E., Ahmed, I. A. M., Oz, E., Proestos, C., El Sheikha, A. F., Oz, F., Wu, G., & Wang, X. (2022). Nutritional composition and volatile compounds of black cumin (Nigella sativa L.) seed, fatty acid composition and tocopherols, polyphenols, and antioxidant activity of its essential oil. Horticulturae, 8(7), 575. https://doi.org/10.3390/horticulturae8070575
Anderson, R. J., Groundwater, P. W., Todd, A., & Worsley, A. J. (2012). Antibacterial agents: Chemistry, mode of action, mechanisms of resistance and clinical applications. https://doi.org/10.1002/9781118325421.ch2
Daoudi, H., Bouafia, A., Meneceur, S., Laouini, S. E., Belkhalfa, H., Lebbihi, R., & Selmi, B. (2022). Secondary metabolite from Nigella sativa seeds mediated synthesis of silver oxide nanoparticles for efficient antioxidant and antibacterial activity. Journal of Inorganic and Organometallic Polymers and Materials, 32, 4223–4236. https://doi.org/10.1007/s10904-022-02393-y
Dhahir Mansour Al Sultani, T., Ayad Fakhri, S., & Al-Zuhairi, W. (2021). Comparison of the antimicrobial activity of Nigella sativa aqueous and oil extracts. Iranian Journal of War and Public Health, 13(4), 305–311. http://ijwph.ir/article-1-1093-en.html
Dookie, N., Rambaran, S., Padayatchi, N., Mahomed, S., & Naidoo, K. (2018). Evolution of drug resistance in Mycobacterium tuberculosis: A review on the molecular determinants of resistance and implications for personalized care. Journal of Antimicrobial Chemotherapy, 73(5), 1138–1151. https://doi.org/10.1093/jac/dkx506
Goel, S., & Mishra, P. (2018). Thymoquinone inhibits biofilm formation and has selective antibacterial activity due to ROS generation. Applied Microbiology and Biotechnology, 102(4), 1955–1967. https://doi.org/10.1007/s00253-018-8736-8
Hannan, M. A., Rahman, M. A., Sohag, A. A. M., Uddin, M. J., Dash, R., Sikder, M. H., Rahman, M. S., Timalsina, B., Munni, Y. A., Sarker, P. P., Alam, M., Mohibbullah, M., Haque, M. N., Jahan, I., Hossain, M. T., Afrin, T., Rahman, M. M., Tahjib-Ul-Arif, M., Mitra, S., ... Kim, B. (2021). Black cumin (Nigella sativa L.): A comprehensive review on phytochemistry, health benefits, molecular pharmacology, and safety. Nutrients, 13(6), 1784. https://doi.org/10.3390/nu13061784
Jantz, K., Lopez, G., & Milanzzo, N. (2025). Black seed: Health benefits, dosage, and side effects. Examine. https://examine.com/supplements/black-seed/
Kabir, Y., Akasaka-Hashimoto, Y., Kubota, K., & Komai, M. (2020). Volatile compounds of black cumin (Nigella sativa L.) seeds cultivated in Bangladesh and India. Heliyon, 6(10), e05343. https://doi.org/10.1016/j.heliyon.2020.e05343
Kapp, E., Joubert, J., Sampson, S. L., Warner, D. F., Seldon, R., Jordaan, A., de Vos, M., Sharma, R., & Malan, S. F. (2021). Antimycobacterial activity, synergism, and mechanism of action evaluation of novel polycyclic amines against Mycobacterium tuberculosis. Advances in Pharmacological and Pharmaceutical Sciences, 2021, 1–8. https://doi.org/10.1155/2021/5583342
Khalifa, R. A., Nasser, M. S., Gomaa, A. A., Osman, N. M., & Salem, H. M. (2013). Resazurin microtiter assay plate method for detection of susceptibility of multidrug-resistant Mycobacterium tuberculosis to second-line anti-tuberculous drugs. Egyptian Journal of Chest Diseases and Tuberculosis, 62(2), 241–247. https://doi.org/10.1016/j.ejcdt.2013.05.008
Mahfur, M. (2018). Profil metabolit sekunder senyawa aktif minyak atsiri jinten hitam (Nigella sativa L.) dari Habasyah dan India. PHARMACY: Jurnal Farmasi Indonesia, 15(1), 90. https://doi.org/10.30595/pharmacy.v15i1.2274
Mahmud, H. A., Seo, H., Kim, S., Islam, M. I., Nam, K. W., Cho, H. D., & Song, H. Y. (2017). Thymoquinone inhibits the replication of intracellular Mycobacterium tuberculosis in macrophages and modulates nitric oxide production. BMC Complementary and Alternative Medicine, 17(1). https://doi.org/10.1186/s12906-017-1786-0
Majdalawieh, A. F., & Fayyad, M. W. (2015). Immunomodulatory and anti-inflammatory action of Nigella sativa and thymoquinone: A comprehensive review. International Immunopharmacology, 28(1), 295–304. https://doi.org/10.1016/j.intimp.2015.06.023
Martin, A., Camacho, M., Portaels, F., & Palomino, J. C. (2003). Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: Rapid, simple, and inexpensive method. Antimicrobial Agents and Chemotherapy, 47(11), 3616–3619. https://doi.org/10.1128/AAC.47.11.3616-3619.2003
Nurchollifah, Y., Wijayatri, R., & Hidayat, I. W. (2021). Literature study of pharmacological effects of black seed (Nigella sativa) based on active seeds. Prosiding University Research Colloquium, 815–832. https://www.repository.urecol.org/index.php/proceeding/article/view/1647
Omollo, C., Singh, V., Kigondu, E., Wasuna, A., Agarwal, P., Moosa, A., Ioerger, T. R., Mizrahi, V., Chibale, K., & Warner, D. F. (2021). Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, 65(5). https://doi.org/10.1128/AAC.02554-20
Patoppoi, B. (2024). Surabaya catat 5800 kasus TBC hingga akhir Juni, Dinkes gencarkan screening di wilayah padat. Suara Surabaya. https://www.suarasurabaya.net/kelanakota/2024/surabaya-catat-5800-kasus-tbc-hingga-akhir-juni-dinkes-gencarkan-screening-di-wilayah-padat/
Ramadani, A. (2016). Manfaat habbatussauda, jinten hitam dari Timur Tengah yang memiliki banyak manfaat bagi kesehatan. Fakultas Keperawatan Universitas Airlangga. https://ners.unair.ac.id/site/index.php/news-fkp-unair/30-lihat/903-manfaat-habbatussauda-jinten-hitam-dari-timur-tengah-yang-memiliki-banyak-manfaat-bagi-kesehatan
Randhawa, M. A. (2011). In vitro antituberculous activity of thymoquinone, an active principle of Nigella sativa. Journal of Ayub Medical College Abbottabad, 23(2), 78–81. https://pubmed.ncbi.nlm.nih.gov/24800349
Rutala, W. A., & Weber, D. J. (2010). Guideline for disinfection and sterilization of prion-contaminated medical instruments. Infection Control & Hospital Epidemiology, 31(2), 107–117. https://doi.org/10.1086/650197
Salem, M. L. (2005). Immunomodulatory and therapeutic properties of Nigella sativa L. seed. International Immunopharmacology, 5(13–14), 1749–1770. https://doi.org/10.1016/j.intimp.2005.06.008
Sarker, S. D., Nahar, L., & Kumarasamy, Y. (2007). Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods, 42(4), 321–324. https://doi.org/10.1016/j.ymeth.2007.01.006
Sawicki, R., Widelski, J., Oki?czyc, P., Truszkiewicz, W., Golus, J., & Sieniawska, E. (2022). Exposure to Nepalese propolis alters the metabolic state of Mycobacterium tuberculosis. Frontiers in Microbiology, 13. https://doi.org/10.3389/fmicb.2022.929476
Webadmin. (2021). Kandungan dan manfaat jinten hitam. Poltekkespim. https://poltekkespim.ac.id/kandungan-dan-manfaat-jinten-hitam/
World Health Organization. (2022). Global tuberculosis report 2022. World Health Organization. https://iris.who.int/handle/10665/363752
World Health Organization. (2023). Global tuberculosis report 2023. World Health Organization. https://iris.who.int/handle/10665/373828
