Antitrypanosomal assay

A 2 days old culture of T. brucei brucei in the exponential phase was diluted with IMDM to 5000 parasites/mL. Maximum permissible limit of DMSO in the assay was 0.5 %. The assays were set up in clear 96 well microplates. For primary screening (Single concentration of 20 μg/mL in duplicate) extract dilutions (1 mg/mL) were prepared from the stock extracts (20 mg/mL) in IMDM medium. Each well received 4 μL of diluted extract sample and 196 μL of the culture volume (total culture volume 200 μl). The plates were incubated at 37 °C in 5 % CO₂ for 48 h. Alamar blue (10 μl) (AbD Serotec, catalog number BUF012B) was added to each well and the plates were incubated further for overnight. Standard fluorescence was measured on a Fluostar Galaxy fluorometer (BMG LabTechnologies) at 544 nm excitation, 590 nm emission. Pentamidine and α-difluoromethylornithine (DFMO) were tested as standard (Table 1). The extracts that have shown more than 90 % inhibition of T. brucei growth in primary screening were subjected to secondary screening for dose–response analysis. Active extracts were screened at concentrations ranging from 10 – 0.4 μg/mL. IC₅₀ and IC₉₀ values were computed from dose response growth inhibition curve by XLfit version 5.2.2.

In vitro antitrypanosomal activity of the most active plant extracts against Trypanosoma brucei

(A complete list of plants screened is presented as supplement material –Additional file 1: Table S1 and Table S2) NPID- Natural Product Identification Details (accession number); Plant parts- BK- stem bark; LF- leaves; FL- flowers; ST- stem; FR- fruit; IC₅₀ and IC₉₀ values are mean ± SD