In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC50, CC50 and SI Values

Abstract

Neglected tropical diseases gain the scientific interest of numerous research programs in an attempt to achieve their effective control or elimination. In this attempt, more cutting-edge public health policies and research are needed for the discovery of new, safer and effective drugs originated from natural products. Here, we describe protocols for the in vitro screening of a natural product-derived compound required for the determination of its antileishmanial potency. For this purpose, the Total Phenolic Fraction (TPF) derived from extra virgin olive oil is evaluated through the in vitro cell culture method against extracellular promastigote and intracellular amastigote Leishmania spp. forms. The aim of this article is to describe a step-by-step procedure that can be easily applied to accurately estimate the 50% inhibitory concentration (IC50), the 50% cytotoxic concentration (CC50) and the selectivity index (SI) via the resazurin reduction assay. These protocols are based on the ability of resazurin (oxidized blue form) to be irreversibly reduced by enzymes in viable cells and generate a red fluorescent resorufin product and can be easily expanded to the investigation of the antimicrobial activity in other microorganisms.

Exploring the Immunotherapeutic Potential of Oleocanthal against Murine Cutaneous Leishmaniasis

“…The in vitro antileishmanial effect of OLEO was evaluated on stationary-phase L. major promastigotes and intracellular amastigotes using a resazurin-based cell viability assay 40 . Promastigotes were seeded in 96-well tissue culture plates at a concentration of 1 × 10 6 parasites/well (Sarstedt) and increasing concentrations of OLEO (2.5 to 150 µg/mL) were added in triplicate.…”

“…The evaluation of the anti-amastigote activity of OLEO was performed in L. major infected-J774A.1 macrophages 40 . Macrophages were added in 96-well plates at a concentration of 2.5 × 10 5 cells/well.…”

Total Phenolic Fraction (TPF) from Extra Virgin Olive Oil: Induction of apoptotic-like cell death in Leishmania spp. promastigotes and in vivo potential of therapeutic immunomodulation

“…infantum and L . major promastigotes via the resazurin reduction assay, as we have previously described [ 14 , 21 ]. Briefly, various increasing concentrations of TPF (0–400 μg/ml) were added in triplicates, in separate 96-well flat bottom tissue culture plates containing the optimal concentration of L .…”

Antiproliferation Assay of Essential Oil of Curcuma Rhizoma (Curcuma xanthorrhiza Roxb.) Against P388 Leukemia Cell

“…Analisis data uji aktivitas antiproliferasi minyak atsiri rimpang temulawak dilakukan menggunakan perhitungan ANOVA dengan α-0,01 ditunjukkan pada Tabel 3, diketahui F hitung = 3 lebih kecil dibandingkan dengan F tabel (6,14). Nilai F hitung adalah 4,46, oleh karena itu uji aktivitas ini menunjukkan H 0 diterima, artinya tidak terdapat perbedaan yang nyata terhadap pengaruh peningkatan konsentrasi yang digunakan dari minyak atsiri rimpang temulawak terjadap inhibis sel leukemia P388.…”

“…13 Dalam bidang mikrobiologi optical density (OD) digunakan sebagai satuan hitungan, karena optical density (OD) sebanding dengan kepekatan sel didalam suspensi biakan. 14,15 IC 50 dihitung sebagai aktivitas antiproliferasi untuk melihat konsentrasi sampel yang dibutuhkan dalam menginhibisi 50% sel leukimia P388.…”