4.2. Determination of In Vitro Anti-Trypanosomal Activity of Compounds

All tested compounds were isolated from Holarrhena africana A. DC. (a synonym of H. floribunda (G. Don) T. Durand & Schinz) (Apocynaceae) and identified as described in our previous communication [14]; ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectra to assess the identity and purity can be found in the supplementary information of [14]. The in vitro activities of 1–10 against BSF trypanosomes were determined using a resazurin-based assay protocol as described [24,27,42] using cell densities adjusted to 2 × 10⁵ cells/mL for T. b. brucei and 5 × 10⁵ cells/mL for Tc-IL3000. Cells were exposed to at least 11 doubling dilutions of test compound starting at 100 µL, with the last well in the row receiving medium without test compound as a drug-free control. The trypanosome culture and test compounds were incubated for a period of 48 h at 37 °C for T. b. brucei and 32 °C for Tc-IL3000 under a 5% CO₂ atmosphere. In all the assays, pentamidine and diminazene aceturate were used as positive controls. Fluorescence was measured in the 96-well plates with a FLUOstar Optima (BMG Labtech, Aylesbury, Bucks, UK) at wavelengths of 544 nm for excitation and 590 nm for emission. IC₅₀ values were calculated by non-linear regression using an equation for a sigmoidal dose-response curve with variable slope (Prism 5.0, GraphPad Software, San Diego, CA, USA).