Plant collection and authentication
Roots of E. kebericho were collected in November 2015 in Jimma Arjo Woreda of Eastern Wollega, Ethiopia. Leaves with flower specimen of the plant were identified and authenticated at Aklilu Lema Institute of Pathobiology (ALIPB), and the vouchers were deposited at the National Herbarium of Addis Ababa University with number DA 01.
Preparation of plant extracts
Air dried powdered plant material was macerated in an Erlenmeyer flask with 80% methanol at room temperature for a period of 72 hours. It was then filtered with gauze followed by Whatman filter paper (No.1). The residue was re-macerated once again to increase the yield. The filtrate was concentrated using rotary evaporator to remove methanol. Then the concentrated filtrate was lyophilized to remove water.
Test organism and experimental animals
Trypanosoma congolense was obtained from Department of Veterinary Parasitology, Addis Ababa University by infecting Swiss albino mice via intraperitoneal inoculation.
Swiss albino mice of either sex, weighing 30-35 g (age 10-12 weeks) were purchased from Ambo University (Ethiopia). They were fed with standard pellet and provided water ad libitum; maintained at room temperature of 23-25°C with relative humidity of 60-65%. The care and handling of animals were in accordance with internationally accepted guidelines for use of animals [9].
Experimental procedures
Evaluation of in vitro antitrypanosomal activity
In vitro test was performed in triplicates to detect any motile trypanosomes in a 96 well microplate. Twenty microliter of blood containing about 16-32 organisms per field were mixed with 5 μL of the test substance at concentrations of 2.5, 5, 10, 20mg/mL to produce test concentrations of 0.5, 1, 2, 4.0 mg/mL, respectively.
Phosphate buffer saline (pH 7.2) and standard trypanocidal drug, diminazene aceturate (DA) were used to serve as untreated control and treated controls, respectively. The mixtures were incubated at 37°C for up to 3 h. During the period, motility of the parasites was checked in 20 min interval under microscopy (X40 objective lens). Briefly, about 2 μL of test mixtures was placed microscope slide and covered with cover slips and the parasites observed for reduced motility or complete cessation of motility.
Evaluation of in vivo antitrypanosomal activity
Thirty mice of either sex were randomly grouped into five (I- V) groups of 6 animals per group. They were intraperitoneally infected with 0.2 mL of T. congolense (5*105 parasites/mL) suspension. Groups I and II were administered 0.3 mL distilled water per orally and DA (3.35mg/kg) per orally respectively to serve as untreated and treated controls, while groups III, IV, and V were administered with the extract at daily doses of 100, 200 and 400mg/kg body weight respectively for 7 consecutive days per orally from 10th days of parasite inoculation. Parasitemia and packed cell volume (PCV) were observed every 4 days for 21 days while body weight and rectal temperature was monitored every 2 days [10].
Determination of parasitemia
On the tenth day post infection and every four days, the parasitemia level of mice was checked. Parasitemia was monitored by examining blood drawn from the tail of mice under microscopy at × 400 magnifications using the “Rapid Matching” method of Herbert and Lumsden [11]. Monitoring of parasitemia was performed every four days until the 21th day post-treatment initiation [12,13].
Determination of packed cell volume
PCV was determined using microhematocrit centrifuge and microhematocrit tube reader. PCV was monitored on day of treatment initiation and every 4 days until 21th day post treatment initiation [14,15].
Determination of body weight
Body weight of experimental animals were recorded on the day of parasite challenge, day of treatment initiation and every other day for 21days [16].
Determination of rectal temperature
Rectal temperature was measured using digital rectal thermometer (Mettler Toledo, Switzerland) on the day of parasite inoculation, day of treatment commencement and every other day thereafter for 21days [14].
Phytochemical screening for secondary metabolites
Standard screening tests of the extract was carried out for secondary metabolites according to the methods described in the literature [17–22].
Statistical analysis
Data were presented as mean ± SEM and analyzed using Statistical Package for Social Science version 20. Analysis of variance was employed to test statistical difference within all groups followed by Tukey test for significance test between two groups. P values less than 0.05 were considered statistically significant.