Background

Neglected tropical diseases (NTDs) are a group of about 20 diseases, typically endemic in 149 tropical and subtropical countries that represent a significant public health impact worldwide by affecting more than 1 billion people and being responsible for over 500,000 deaths per year (Cheuka et al., 2016; Mitra and Mawson, 2017). Until nowadays, drug discovery against NTDs exhibited limited success in translating potential drug candidates into effective therapies. Indeed, the current clinically used drugs against NTDs are characterized by various disadvantages including severe adverse effects, complicated administration procedures, lengthy treatment duration and emergence of resistance (Cheuka et al., 2016). Natural products represent a valuable alternative source of novel and structurally diverse compounds that deserves attention in drug discovery against NTDs. Numerous metabolites isolated from plant, microbial and marine sources such as alkaloids, phenolic compounds, quinones, terpenes, saponins, lignans, toxoids and anthranoids have been investigated (Cheuka et al., 2016) and it has been estimated that approximately at least 25% of the presently used drugs in modern medicine are derived from plants (Lahlou, 2007).

Among NTDs, leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania spp., causing a range of manifestations and exhibiting progressively increasing frequency in non-endemic western developed countries (Burza et al., 2018). Numerous of agents of natural origin, plant derived bioactive compounds and their secondary metabolites have been tested as antileishmanial drugs, such as flavonoids, sterols, chalcones, coumarins, tannins and aurones, iridoids, quinones and quinolone alkaloids (Singh et al., 2014). The assessment of the bioactive potential of biological extracts or molecules involves numerous assays performed at animal, cell-based or molecular levels (Lage et al., 2018) while the first steps of the biological screening and evaluation of plant extracts usually involve the screening of compounds in cell-based in vitro assays (Griffiths and Sundaram, 2011).

The half maximal inhibitory concentration (IC₅₀) is a representative quantitative measure of the potency of a substance to inhibit in vitro a specific biological process by 50%, (Lahlou, 2007; Aykul and Martinez-Hackert, 2016) and is primarily defined through in vitro test models. Since Leishmania spp. have a digenetic life cycle, the antileishmanial effect of drugs is tested on both the promastigote (infective form in the intermediate host) and the amastigote form (intracellular form in the tissue of the mammalian host) (Bates and Rogers, 2004). In this regard, IC₅₀ is determined for both promastigotes and intracellular amastigotes and is defined as the concentration of a compound that causes inhibition of growth in the 50% of Leishmania promastigotes and of intracellular amastigotes, respectively. To this end, the in vitro screening of the antileishmanial activity of Total Phenolic Fraction (TPF) is performed on L. infantum and L. major promastigotes and on Leishmania-infected J774A.1 macrophages in which amastigotes survive and multiply (Bilbao-Ramos et al., 2012). Moreover, the cytotoxicity of TPF in vitro against J774A.1 macrophages is also tested through the determination of the CC₅₀ (cytotoxicity concentration 50%) that is the compound’s concentration that causes cytotoxicity in the 50% of the cells (Kyriazis et al., 2013). In addition, we employ the selectivity index (SI) that is defined as the ratio of the 50% cytotoxic concentration of J774A.1 macrophages to the 50% inhibitory concentration of Leishmania spp. amastigotes (CC₅₀/IC₅₀). A compound with SI value greater than 1 is considered to be more selective against Leishmania spp. parasites and is regarded as a promising potential agent in the treatment of leishmaniasis (Makwali et al., 2015). All the above indices are crucial in order to conclude whether or not a tested compound is appropriate as an antileishmanial agent.