Bioassay Guided Fractionation of Marine Streptomyces sp. GMY01 and Antiplasmodial Assay using Microscopic and Flow Cytometry Method
Keywords:
drug discovery, malaria, antiplasmodial assay, Actinomycetes
Abstract
Genome mining study showed that marine-derived Streptomyces sp. GMY01 is a potential actinobacteria with abundant of secondary metabolite and anticancer activity. The study aimed to evaluate bioassay guided fractionation for antiplasmodial screening of GMY01 extract and to predict compound on active fractions. Ethyl acetate fraction was obtained from 11 days fermentation product of GMY01 and then it was fractionated using n-hexane and methanol solvent. Refractionated was applied using flash chromatography and column chromatography. Antiplasmodial assay was performed on Plasmodium falciparum FCR3 by microscopic method using thin blood smear + Giemsa stain, and flow cytometry method using SYBR Green I stain. Toxicity assay was performed on Vero cells line. Main constituent of active fraction was analyzed using LCMS/MS. The result of the study showed that ethyl acetate-methanol fraction has high antiplasmodial activity (IC50=3.96 µg/mL) with very low toxicity on Vero cells (IC50=30,072 µg/mL). Bioassay guided fractionation resulted F4.7 as highest Plasmodium inhibition (94.3% at 5 µg/mL) and was confirmed by microscopic and flow cytometry assay. Main constituent analysis showed C10H13NO (163.09971 Da) as mayor compound and predicted as nonribosomal polyketide synthetase (NRPS) secondary metabolite.
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Antony, H. A., & Parija, S. C. (2016). Antimalarial drug resistance: An overview. In Tropical Parasitology. https://doi.org/10.4103/2229-5070.175081
Bejon, P., Andrews, L., Hunt-Cooke, A., Sanderson, F., Gilbert, S. C., & Hill, A. V. S. (2006). Thick blood film examination for Plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density. Malaria Journal, 5, 5–8. https://doi.org/10.1186/1475-2875-5-104
Crespo-Ortiz, M. P., & Wei, M. Q. (2012). Antitumor activity of artemisinin and its derivatives: From a well-known antimalarial agent to a potential anticancer drug. Journal of Biomedicine and Biotechnology, 2012. https://doi.org/10.1155/2012/247597
Dharmaraj, S. (2010). Marine Streptomyces as a novel source of bioactive substances. In World Journal of Microbiology and Biotechnology. https://doi.org/10.1007/s11274-010-0415-6
Farida, Y., Widada, J., & Meiyanto, E. (2007). Combination Methods for Screening Marine Actinomycetes Producing Potential Compounds as Anticancer. Indonesian Journal of Biotechnology, 12(2), 988–997. https://doi.org/10.22146/ijbiotech.7772
Fong, K. Y., & Wright, D. W. (2013). Hemozoin and antimalarial drug discovery. Future Medicinal Chemistry, 5(12), 1437–1450. https://doi.org/10.4155/fmc.13.113
Ghanem, N. B., Sabry, S. A., El-Sherif, Z. M., & Abu El-Ela, G. A. (2000). Isolation and enumeration of marine actinomycetes from seawater and sediments in Alexandria. Journal of General and Applied Microbiology, 46(3), 105–111. https://doi.org/10.2323/jgam.46.105
Hall, B. F., & Fauci, A. S. (2009). Malaria control, elimination, and eradication: The role of the evolving biomedical research agenda. Journal of Infectious Diseases, 200(11), 1639–1643. https://doi.org/10.1086/646611
Hansen, M. B., Nielsen, S. E., & Berg, K. (1989). Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Journal of Immunological Methods. https://doi.org/10.1016/0022-1759(89)90397-9
Herdini, C., Hartanto, S., Mubarika, S., Hariwiyanto, B., Wijayanti, N., Hosoyama, A., Yamazoe, A., Nojiri, H., & Widada, J. (2016). Diversity of Nonribosomal Peptide Synthetase Genes in the AnticancerProducing Actinomycetes Isolated from Marine Sediment in Indonesia. Indonesian Journal of Biotechnology, 20(1), 34. https://doi.org/10.22146/ijbiotech.15266
Lambros, C., & Vanderberg, J. P. (1979). Synchronization of Plasmodium falciparum erythrocytic stages in culture. Journal of Parasitology. https://doi.org/10.2307/3280287
Machado, M., Murtinheira, F., Lobo, E., & Nogueira, F. (2016). Whole-Cell SYBR Green I Assay for Antimalarial Activity Assessment. Ann Clin Med Microbio, 2(1), 1010. https://www.jscimedcentral.com/MedicalMicrobiology/medicalmicrobiology-2-1010.pdf
Mustofa, Sholikhah, E. N., & Wahyuono, S. (2007). In vitro and in vivo antiplasmodial activity and cytotoxicity of extracts of Phyllanthus niruri L. herbs traditionally used to treat malaria in Indonesia. Southeast Asian Journal of Tropical Medicine and Public Health, 38(4), 609–615.
Rebelo, M., Sousa, C., Shapiro, H. M., Mota, M. M., Grobusch, M. P., & Hänscheid, T. (2013). A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum. PLoS ONE. https://doi.org/10.1371/journal.pone.0061606
Sholikhah, E. N., Wijayanti, M. A., Susidarti, R. A., Purwantini, I., Hestiyani, R. A. N., Yusuf, H., & Mustofa. (2016). Stage specificity of eurycomanone isolated from Eurycoma longifolia on Plasmodium falciparum cycles. American Journal of Pharmacology and Toxicology, 11(1), 1–7. https://doi.org/10.3844/ajptsp.2016.1.7
Tajuddeen, N., & Van Heerden, F. R. (2019). Antiplasmodial natural products: An update. Malaria Journal, 18(1), 1–62. https://doi.org/10.1186/s12936-019-3026-1
Trager, W., & Jensen, J. B. (2005). Human malaria parasites in continuous culture. Journal of Parasitology. https://doi.org/10.1645/0022-3395(2005)091[0484:HMPICC]2.0.CO;2
Undabarrena, A., Ugalde, J. A., Seeger, M., & Cámara, B. (2017). Genomic data mining of the marine actinobacteria Streptomyces sp. H-KF8 unveils insights into multi-stress related genes and metabolic pathways involved in antimicrobial synthesis. PeerJ, 2017(2). https://doi.org/10.7717/peerj.2912
Valdés, A. F. C., Martínez, J. M., Lizama, R. S., Gaitén, Y. G., Rodríguez, D. A., & Payrol, J. A. (2010). Actividad antimalárica in vitro y citotoxicidad de algunas plantas medicinales Cubanas seleccionadas. Revista Do Instituto de Medicina Tropical de Sao Paulo, 52(4), 197–201. https://doi.org/10.1590/S0036-46652010000400006
Vega-Rodríguez, J., Pastrana-Mena, R., Crespo-Lladó, K. N., Ortiz, J., Ferrer-Rodríguez, I., & Serrano, A. E. (2015). Implications of glutathione levels in the plasmodium berghei response to chloroquine and artemisinin. PLoS ONE, 10(5), 1–15. https://doi.org/10.1371/journal.pone.0128212
Vicente, J., Stewart, A., Song, B., Hill, R. T., & Wright, J. L. (2013). Biodiversity of Actinomycetes Associated with Caribbean Sponges and Their Potential for Natural Product Discovery. Marine Biotechnology. https://doi.org/10.1007/s10126-013-9493-4
Weber, T., Charusanti, P., Musiol-Kroll, E. M., Jiang, X., Tong, Y., Kim, H. U., & Lee, S. Y. (2015). Metabolic engineering of antibiotic factories: New tools for antibiotic production in actinomycetes. Trends in Biotechnology, 33(1), 15–26. https://doi.org/10.1016/j.tibtech.2014.10.009
Werdyani, S., Wijayanti, N., Fitria, A., & Rahmawati, S. (2017). Cytotoxic effects of ethyl acetate fractions from secondary metabolites of Streptomyces Sp. GMY01 on human breast cancer MCF7 cell lines. Asian Journal of Pharmaceutical and Clinical Research, 10(Special Issue August), 9–11. https://doi.org/10.22159/ajpcr.2017v10s3.21351
Zhang, L., Wang, J., Li, T., Li, P., Wang, Y., Yang, M., Liu, J., & Liu, J. (2019). Determination of the chemical components and phospholipids of velvet antler using UPLC/QTOF‑MS coupled with UNIFI software. Experimental and Therapeutic Medicine, 3789–3799. https://doi.org/10.3892/etm.2019.7372
Published
2020-12-07
How to Cite
Damayanti, E., Widada, J., Lotulung, P. D. N., Dinoto, A., & Mustofa, M. (2020). Bioassay Guided Fractionation of Marine Streptomyces sp. GMY01 and Antiplasmodial Assay using Microscopic and Flow Cytometry Method . Indonesian Journal of Pharmacy, 31(4), 281–289. https://doi.org/10.22146/ijp.809
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Research Article
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