Chemicals and Equipment
Chemicals and solvents
Dimethyl sulfoxide (DMSO), Methanol, distilled water, and reference drugs (Diminazene aceturate), phosphate glucose buffered saline solution (PBSG)
Aluminium foils, cover slide, microscopic slide, mortar and pestle, digital weighing balance, diamond pencil, desiccator, EDTA coated syringe, heparinized capillary tube, Whatman No.1 filter paper, glove, haematocrit reader, microhematocrit centrifuge, oven, refrigerator, petridish, microscope, micropipettes, Rotary evaporator, sterile lancet, syringe 1ml, cristaseal, spatula and flasks were employed in this study.
The experiment was conducted in Veterinary pharmacology and Toxicology Laboratory of the College of Veterinary Medicine and Agricuture, Addis Ababa University, Bishoftu Ethiopia.
Samples of the plant were authenticated by the national herbarium of Addis Ababa University College of Natural Science. The identification was done by Mr. Melaku Wondaferash (Botanist), and the report contained local name, botanical name and its family; Teji sar- Cymbopogon citratus (DC.) Stapf- Poaceae and Fetto- Lepidium Sativum L.- Brassicaceae, and samples were deposited with specified voucher numbers (AY1 and AY3) respectively.
The plants were planted in the Garden of toxic and medicinal plants established by the author of this thesis in 2016 at the College of Veterinary Medicine and Agriculure, Addis Ababa University, Bishoftu for future use.
After collection, the plant materials were washed with tap water to remove unnecessary particles, dried under shade, and grounded mechanically. The materials were sieved and weighed before subjected to extraction procedures.
Crude extract preparation
The plant materials were extracted by maceration technique using 80%Methanol (LOBA CHEMIE PVT LTD) through mixing the grinded and weighted plant material with 80% Methanol in 1:7 ratios. After 72 hrs maceration with regular shaking, the mixtures were strained using strainer to remove solids and further filtered with whatman filter paper No 1. The filtered solutions were evaporated using Rota vapour (BUCHI Rotavapor R-200) to remove the solvent to the acceptable level. Remnants from the Rota vapour were poured in to petridishes and put in a dry oven at a temperature of 40oC to remove the remained solvent. The prepared solid extracts were stored in a desiccator until the experimental procedures were conducted. The dried solid extracts were weighted to determine the percent yield as indicated in Table 1.
Acute toxicity test
The acute toxicity study was conducted according to the Organization for Economic Co-operation and Development (OECD), 2001 guideline fixed dose (2000mg/kg) toxicity test. A group of six female albino mice were fasted from food but not water for 4hrs before administration and 2hrs after oral administration of the test extract. The mice were critically followed continuously for 1 hr after administration of the extracts; intermittently for 4 hrs over a period of 24hrs for death, gross behavioural changes and other signs of toxicity. The follow up continues for 14days post treatment (15).
Experimental mice inoculation
The donor mice were infected with field isolates of T.congolense directly from trypanosome infected cattle at the Ghibe valley. After 12 days of infection, the donor mice reached at its peak level of parasitemia. Blood was collected from donor mice by cardiac puncture and/or drawing through tail veins after mice were anaesthetised using chloroform. Collected blood was diluted with phosphate buffered saline (PBS) to increase the volume of the inoculum. Healthy mice were injected intraperitonneally with 0.2ml of the inoculum containing 107 parasites per millilitre approximately (16). After 9–11 days post infection the experimental mice were tested for the development of parasitemia, and only positive mice were drawn in to the experimental groups.
Dose adjustment, grouping of mice and administration of the plant extracts
Three test doses were adjusted as 100mg/kg, 200mg/kg and 400mg/kg based on the toxicity profile of the extracts. The dried and weighted plant extracts were reconstituted with 10% Dimethyl sulfoxide (DMSO) to have intended concentrations. A total of 60 Mice were then grouped into nine groups having six mice per group (CC100, CC200, CC400, LS100, LS200, LS400, DA3.5, DA28, NC and UU). CC100, CC200 and CC400 are groups of mice treated with 100, 200 and 400mg/kg of C.citratus extract, respectively. LS100, LS200 and LS400 were 100, 200 and 400 mg/kg L.sativum extract treated mice groups. DA3.5 and DA28 were positive controls took 3.5mg/kg and 28mg/kg standard drug Diminazene aceturate (DA), respectively. Whereas the NC group was the negative control infected with the parasite but treated only with 1ml of 10%DMSO (vehicle). The UU group was uninfected untreated healthy mice used as a reference. The treatment was started on day 12 post infection. The extracts were administered every morning for seven consecutive days. The control groups (DA3.5, DA28 and NC) were treated once on the first day of the above mentioned doses. All treatments were administered via intraperitoneal route.
Determination of parasitaemia and body weight
Parasitaemia was monitored every other day starting from the first day of treatment and continues until the 14th day. These were done by microscopic examination of blood obtained from the tail of each mouse. The tail was cut to extrude blood, and a drop of blood was placed on a microscope slide and a wet smear was prepared by covering the drop by cover slips. The smears were examined microscopically at 400X total magnification (camera aided Olympus microscope). The degree of parasitaemia was determined using the “Rapid Matching” method of Herbert and Lumsden. Smears were prepared in triplicates from each animal and the mean value of slide counts was taken per sample examined microscopically. Logarithm values of these counts were obtained by matching with the table and the charts given by Herbert and Lumsden (17).
The body weight (in gram) of each mouse in all groups was taken on the day of treatment was commenced (day 0) and every other day (on Days 2, 4, 6, 8, 10, 12 and 14) up to the 14th day.
Determination of packed cell volume (PCV) and differential white blood cell count
PCV was measured to predict the effectiveness of the test extracts in preventing hemolysis resulting from increasing parasitaemia associated with trypanosomosis. It was monitored on Days 0, 7 and 14. Blood was collected from tail of each mouse in heparinized microhaematocrit capillary tubes filled up to 3/4th of their length. The tubes were then sealed immediately with crystal seal and centrifuged in a micro haematocrit centrifuge (Hawksley micro haematocrit centrifuge, England) for 5 min. After centrifugation the packed cell volume was measured using haematocrit reader (Hawksley micro haematocrit reader, England). The effect of extracts in improving PCV of treated animals was compared with the controls.
The white blood cell count (WBC) was determined on thin blood films stained with Giemsa stain obtained from each mouse on the 14th day post extract administration.