In vitro Mechanistic Assays of Tetracyclic Iridoid Compounds Isolated from Morinda lucida Benth in Leishmania species
Faustus Akankperiwen Azerigyik
West African Centre for Cell Biology and Infectious Pathogens (WACCBIP), Department of Biochemistry, Cell and Molecular Biology (DBCMB), University of Ghana, Legon, Ghana and Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana
Michael Amoa-Bosompem
Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana and Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
Thelma Tetteh
Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana
Frederick Ayertey
Centre for Plant Medicine Research, P.O.Box 73, Mampong - Akuapem, Ghana
Ama Nyamekye Antwi
Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana
Kofi Baffour-Awuah Owusu
Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana
Kofi Kwofie Dadzie
Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
Georgina Isabella Djameh
Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana
Mark Tetteh-Tsifoanya
Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana
Shiro Iwanaga
Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
Alfred Ampomah Appiah
Centre for Plant Medicine Research, P.O.Box 73, Mampong - Akuapem, Ghana
Tomoe Ohta
Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan
Takuhiro Uto
Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan
Yukihiro Shoyama
Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan
Nobuo Ohta
Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
Theresa Manful Gwira
West African Centre for Cell Biology and Infectious Pathogens (WACCBIP), Department of Biochemistry, Cell and Molecular Biology (DBCMB), University of Ghana, Legon, Ghana
Mitsuko Ohashi *
Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, P.O.Box LG 581, Legon, Ghana and Section of Environmental Parasitology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
*Author to whom correspondence should be addressed.
Abstract
Aims: This study investigates the activity of tetracyclic iridoid compounds against Leishmania spp. and the mechanism(s) of action.
Study Design: An experimental study.
Place and Duration: Department of Parasitology, Noguchi Memorial Institute for Medical Research, between September 2017 and July 2018.
Methodology: The 50 % inhibitory concentration (IC50) of compounds against Leishmania donovani and L. major promastigotes were determined after 48 hours of incubation using the Alamar blue. Cytotoxicity of compounds was determined against cell lines using MTT assay. The anti-amastigote activity of compounds was further assessed by DAPI (4′,6-diamidino-2-phenylindole) staining. The mechanism of cell death induced by compounds was determined using nexin assay. Mitosis, cytokinesis and morphometry were monitored by DAPI and Kinetoplastid Membrane Protein (KMP) staining. Cell cycle arrest induced by compounds was analyzed by FACS.
Results: Molucidin and ML-F52 inhibited the growth of promastigote in L. donovani (Molucidin; IC50 = 2.94±0.60 µM, ML-F52; IC50 = 0.91±0.50 µM) and L. major (Molucidin; IC50 = 1.85± 0.20 µM, ML-F52; IC50 = 1.77± 0.20 µM). ML-F52 had a 10-fold cytotoxic effect on parasites relative to normal cell lines. Against intracellular forms, Molucidin and ML-F52 inhibited intracellular amastigote replication and infectivity. Amphotericin B, Molucidin and ML-F52, induced a dose-dependent apoptotic effect on promastigotes. Although no change in KMP-11 expression was observed, iridoids inhibited cell division and morphological changes in promastigote cultures. Molucidin and ML-F52 induced apoptotic mechanism of cell death, inhibited cytokinesis and induced phenotypic changes in promastigotes. Molucidin further induced ‘’nectomonad-like’’ forms and loss of kDNA, ML-F52 induced ‘cell-rounding’ with loss of flagellum. Molucidin also induced cell growth arrest at G2-M phase (54.5 %). A significant induction of apoptosis (P = .05) was shown by an enhanced peak in the sub-G1 confirming the apoptotic inducing properties of iridoids.
Conclusion: This study shows the anti-leishmania activity of tetracyclic iridoids which could be further investigated for the development of new chemotherapy against Leishmaniasis.
Keywords: Leishmania donovani, Leishmania major, In vitro screening, medicinal plants, Tetracyclic iridoids, Morinda lucida, Apoptosis