Sain, Amyra Amat
(2016)
Optimisation Of In Vitro Intracellular Antileishmanial Assay And Evaluation Of Senna Spectabilis (DC.) H. S. Irwin Barneby Methanolic Leaf Extract And Its Constituents Against Leishmania Major Using Bioassay-Guided Isolation Approach.
Masters thesis, Universiti Sains Malaysia.
Abstract
Leishmaniasis affects millions of people each year. Alternative approaches are needed
because majority of those infected live in countries which could not afford financially in marketdriven
dmg discovery. Current treatments faced side effects such as cardiotoxicity and
nephrotoxocity, and require a long course of treatment which may develop resistance of
parasites. Senna spp have been known for antiparasitic characteristics and recently reported for
its antileishmanial activity against extracellular L. major. There are two assays used to test in
vitro antileishmanial activities in this work which are leishmanicidal and intracellular amastigote
assays. The main aim of this study was to establish and optimise the in vitro antileishmanial
intracellular amastigote assay, while maintained the leishmanicidal assay as the preliminary
screening tool to obtain the hits. The optimised intracellular amastigote assay was then used to
evaluate antileishmanial activities of active constituents from a medicinal plant, S. spectabilis via
bioassay-guided isolation approach. At first, the evaluation of antileishmanial activity of
methanolic leaves extract from S. spectabilis were carried out using the leishmanicidal assay
which was also known as in vitro extracellular promastigote assay with promastigote stage of L.
major as the parasite. This assay was done based on Alamar Blue® assay with little
modification. As a result, ethyl acetate (CS-EA) extract of the plant was found to be active with
IC50 = 70.29 ± 0.38 1-ig/mL and was further fractionated. For further antileishmanial evaluation of
fractions, the in vitro intracellular amastigote assay was used, and this assay was first to be
established in Malaysia. In this assay, amastigote stage of L. major was used to infect THP-1 cell
(macrophage) stably before treating the infection with substituents from S. spectabilis in 16-well
chamber slide format. The seeding number of THP-1 cells was optimized at 2.0x 104 cells/well
and ratio of infection was optimised at 5: 1 of parasite to host. The assay was then validated using
Amp-Bas the standard drug with EC50 = 0.437 ± 1.06 11M.
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