References
purposes. The roots of the plant were washed with clean water to get rid of soil and debris. It was then sliced into minute sizes and allowed to air dry in open air at room temperature (approx. 25– 28°C) for 14 days. The dried rhizome was then pounded into a coarse powder using a mortar and pestle for size reduction, and then finely blended to a fine size. 200g of the pulverized material was soaked in 2000ml of 70% v/v ethanol in a conical flask for 72 hours at room temperature. To enhance the efficiency of extraction, the mixture was intermittently vibrated using a GFL shaker (No. 3017 MBH, Germany). Whatman No. 1 filter paper was used to vacuum filter the macerate. Hereafter, the filtrate was concentrated at a low pressure and a temperature not exceeding 40°C using a rotary evaporator (Heidolph, Germany). To remove all forms of residual extracting solvent, the extract was further heated over a water bath until a semi solid concentrate emerged. Of the 200g used, the extract weighed 7.5 g, representing an extraction efficiency of 3.75%. The resulting extract was tightly stored in an airtight container at 4°C untill needed. 2.2. Phyto-chemical Screening Qualitative phytochemical analysis was carried out on the extract using standard protocols as a means of determining the presence of secondary metabolites. Parameters checked include: carbohydrates, flavonoids, tannins, sterols, terpenes, alkaloids and oils 2.3. Experimental Animals Swiss albino mice of about four weeks were obtained from the National Institute of Pharmaceutical Research and Development , Idu, Abuja. The mice obtained from the animal facility centre comprised mice of both male and female sexes, weighing between 16-22g. The mice were properly housed in neat plastic standard cages with beddings made of wood shavings under laboratory standard conditions (12-hour light/dark cycle, temperature of 25 ± 2°C). The animals were fed ad libitum with standard feed for rodents and water administered using a water bottle bottle inserted with a canula. Strict adherence to procedure was religiously followed as enshrined by the NIH guideline for the use and management of laboratory animals (NIH Publication No. 85-23, revised 1985) as well as the NIPRD Standard Operating Procedures . The study protocol was approved by the institutional ethics committee. 2.4. Parasite Inoculation The parasites (Trypanosoma brucei brucei) were obtained from the National Institute for Medical Research , Lagos, Nigeria, and maintained at the animal facility in National Institute of Pharceutical Research and Development by serial intra-peritoneal inoculation in donor mice every 4–5 days. A mouse whose parasitamia level had been determined was euthanized under mild anesthesia, and blood was sourced by cardiac puncture into a heparinized tube. Blood was diluted with phosphate-buffered saline to achieve a standard inoculum of approximately 1 x 106 trypanosomes per mouse. The mice were all inoculated with the standard inoculums intra-peritoneally. 2.5. Acute Toxicity Test The acute oral toxicity of the ginger extract was assessed using Lorke’s method [9]. The study was conducted in two phases. In phase one, nine mice were randomly divided into three groups (n=3) and administered single oral doses of 10, 100, and 1000 mg/kg body weight of the extract, respectively. The mice were assessed for the first 4 hours for toxicity signs that may include (e.g., paw licking, salivation, stretching, weakness, lethargy, respiratory distress) and then daily for seven days for possible cases of mortality. From the results of the first phase, a second phase was done using another group of nine mice, also divided into three groups (n=3). They were given higher doses of 1350, 2500, and 5000 mg/kg body weight, with the same standard procedure of observation strictly followed. The median lethal dose (LD50) was calculated as the geometric mean of the lowest dose that caused mortality (Dm) and the highest dose that resulted in no mortality (D0), using the formula: LD50 = ?(D0 x Dm) 2.6. Anti-Trypanosomal Activity Evaluation 2.6.1. Four-Day Suppressive Test The ability of ginger extract to suppress already established infection was checked using the standard Peter’s 4-day test. Mice were randomly divided into five groups (n=6). Three hours post-infection (Day 0), treatment was initiated as follows: • Group I (Negative Control).Received 5 ml/kg of normal saline. • Group II (Positive Control). Received 5 mg/kg of diminazene aceturate. • Groups III, IV, V:Received 100, 200, and 400 mg/kg of the Z. officinale extract, respectively. • All treatments were given orally once daily for 4 days consecutively (Day 0 to Day 3). On the fourth day, thin smears of blood were made from the mouse’s tail veins, they were fixed with methanol, stained with Giemsa, and examined microscopically to evaluate the level of parasitemia. The percentage chemosuppression was calculated for each group using the formula: % Chemosuppression = [(A-B)/A] x 100 where A is the mean parasitemia in the negative control group and B is the mean parasitemia in the test group. 2.6.2. Curative Test The test mice were first inoculated with T. b. brucei and left untreated until parasitemia was ascertained microscopically (i.e, approximately 72 hours post-inoculation, Day 3). The mice were then put into five groups (n=6) as described for the suppressive test. Treatments were given orally once daily for 7 days, starting from Day 3. The level of parasitemia was evaluated daily from Day 4 onwards via blood smears sourced from the tail. The percentage reduction in parasitemia was calculated on Day 7 post-inoculation. 2.6.3. Prophylactic (Repository) Test The prophylactic potential was evaluated using the method described by Peters and Robinson [1992]. Mice were out into five groups (n=6) and treated beforehand for four days (Day 0 to Day 3). On Day 3, the mice were innoculated withT. b. brucei. Seventy-two hours after inoculation (Day 6), blood smears were prepared, and the level of parasitemia was evaluated. The repository effect of the extract was determined by calculating the percentage suppression of parasitemia. 2.7. Statistical Analysis All retrieved data are presented as Mean ± Standard Error of the Mean . Data analysis was done using one-way Analysis of Variance followed by a post-hoc test with GraphPad Prism software (version 5.0). Values of p < 0.05 were considered statistically significant. 3. Results 3.1. Phytochemical Screening The results obtained from the qualitative phytochemical evaluation of the ethanolic root extract of Zingiber officinale are synoptically presented in Table 1. The extract was found to be positive for carbohydrates, flavonoids, tannins, and alkaloids. However, others, like sterols, terpenes, and oils, were found to be absent. Table 1: Phytochemical Composition of Ethanolic Root Extract of Z. officinale Carbohydrates Present Flavonoids Present Tannins Present Alkaloids Present Steroids Absent Terpenes Absent Oils Absent 3.2. Acute Toxicity (LD50) In the first phase of the toxicity test (10, 100, 1000 mg/kg), no death or marked behavioral alterations were noticed. In the second phase, at higher doses of 1350 and 2500 mg/kg, the mice displayed signs of toxicity, including paw licking, salivation, body stretching, and reduced locomotor activity; however, no death or mortality was recorded. At the highest dose of 5000 mg/kg, within 24 hours, 100% mortality was recorded. The LD50 was calculated as follows: D0 (highest dose with no mortality) = 2500 mg/kg Dm (lowest dose with 100% mortality) = 5000 mg/kg LD50 = ?(2500 x 5000) = ?(12,500,000) ? 3,535.5 mg/kg 3.3. Anti Trypanosomal Activity 3.3.1. Suppressive Effect The ethanolic extract of Z. officinale (Tab 2) depicts a dose dependent chemosuppressive effect against T. b. brucei. At varying doses of 100, 200, and 400 mg/kg, the root extract brought about a significant (p < 0.05) suppression of parasitemia by 58.3%, 62.3%,
