Methods
The model used for the study was the w1118 (white) strain of Drosophila melanogaster, a gift from Dr. Isabel Palacios (UK) and maintained at the Institute of Biomedical Research, Kampala International University Western Campus, Ishaka-Bushenyi, Uganda. They were fed on cornmeal-yeast-agar medium (10) and maintained at 250C under a 12/12 hour light and dark cycle in a digital fly incubator. From the stock population, virgin females and young males were placed on fresh food and allowed to mate. Fresh eggs were collected, and dated to ensure that the dates of birth are synchronized for all flies. From these, 360 male adult flies were collected and divided into 3 groups to be fed on: normal food (NF), Amaranth extract, and ascorbic acid (ASA) groups. The NF (180 flies), was fed on the standard cornmeal diet, the Amaranth group (180 flies) fed on the Amaranth-fly food mixture and ASA group on ASA-fly food mixture. To determine the in vivo antioxidant activity, catalase enzyme activity and resistance to oxidative stress, groups of Drosophila flies were fed on the respective diets for 5 days (11).
Preparation of experimental food
Amaranth leaves used in the study were purchased from a vendor in the Central market of Ishaka town of Bushenyi Municipality of Western Uganda. Extraction was done as previously described (12). To prepare the fly food with Amaranth and ascorbic acid, 0.25mg and 0.5mg of Amaranth extract and ascorbic acid were each dissolved in 10mls of distilled water to obtain a concentration of 2.5mg/ml and 5mg/ml respectively. Each was separately added to 490mls of molten fly food and mixed thoroughly to obtain a mixture with Amaranth extract and Ascorbic acid each at a concentration of 0.05mg/ml and 0.1mg/ml. These mixtures were then poured in labeled vials and left to cool. The food was covered with cotton balls and stored at 40C.
Determination ofin vivoantioxidant activity
Ten flies each from the NF, Amaranth and ASA groups were separately anaesthetized on ice and homogenized in 100µl of cold 0.05% phosphate buffered saline tween (PBST) solution (pH 7.4). The homogenate was centrifuged at 4000g for 10 minutes and the supernatant collected and used for the determination of 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity. DPPH scavenging activity was determined as previously described (13). Briefly, 50µl of the fly homogenate was added to 5mls of 0.004% methanol solution of DPPH. This mixture was incubated in the dark at room temperature for 30 minutes. The absorbance of the mixture was then read at 517nm against a DPPH blank. The assay was carried out in triplicate and percentage of inhibition was calculated using the formula:
% Inhibition = [(AB-AA)/AB] *100
Where AB = Absorbance of blank; AA = Absorbance of test.
Determination of resistance to hydrogen peroxide induced oxidative stress
The resistance to hydrogen peroxide-induced oxidative stress was conducted as previously described with minor modifications (14). Briefly, ten fruit flies from each of the NF, Amaranth and ASA groups were transferred to empty vials after 5days of feeding. The flies were starved for 6 hours to stimulate the uptake of hydrogen peroxide. Afterwards, the flies were transferred to vials containing only filter paper soaked with 1% hydrogen peroxide in 5% sucrose solution. Flies alive/dead were recorded every day until the last one died.
Catalase enzyme activity
The assay was carried out on Drosophila flies fed on the NF, Amaranth and ASA food for five days. Ten flies per group anaesthetized by chilling on ice were homogenized in ice cold phosphate buffer saline (pH 7.4) and centrifuged at 2,500rpm for 10 minutes. The resulting supernatant was collected and used for the catalase assay. The catalase activity was measured as previously described (15).
Statistical Analysis
Data was analyzed using the free software Paleontological statistics software (PAST 3), expressed as mean and standard deviation and presented as graphs.
Data of DPPH scavenging and catalase activities were analyzed using factorial analysis of variance (ANOVA) followed by a Tukey’s multiple comparison test.
Oxidative stress resistance was determined using Kaplan-Meier survival analysis with significance set at P < 0.05.