Drugs, chemicals and Materials
Chloroquine (Julphar), DPPH (97% Japan Tokyo), normal isotonic saline, sodium citrate, pentobarbitone, dimethyl sulfoxide, 5% sorbitol, 10% Giemsa stain, distilled water, tween-80, 1 ml syringes with needles, feeding tube, vials, gloves, 70% ethanol, ferric chloride, sodium hydroxide, hydrochloric acid, concentrated H2SO4 and Wagner’s reagent (Iodine in Potassium Iodide) were used for the experiment.
Plant collection and preparation of the leaf latex of Aloe weloensis
The leaf of Aloe weloensis was collected from Gubalafto (North East, Ethiopia) in February, 2019. Identification of the plant was carried out by a professor Sebsebe Demisew and a specimen of the plant material was deposited in the National Herbarium of Addis Ababa University (AAU) with voucher specimen number of SD010/11. The leaf of Aloe weloensis was cut transversely close to the stem and then, leaf was inclined towards collecting plate to obtain yellowish exudate. The latex was dried under shade at room temperature with optimal ventilation. The dried latex was kept in clean vial and stored a desiccator until used for the experiment
Experimental animals and parasite
Healthy Swiss albino mice either sex (20-35 gram) and 2–3monthes) were used in the study. The mice were gained from Wollo University and kept in the animal house of pharmacy department in 12 h light -12 h dark cycle and permitted free to diet and water ad libitum (22). Animals were acclimatized to the laboratory conditions for one week before the initiation of the experiment. Plasmodium berghei ANKA strain was obtained from EPHI. The parasite was maintained by serial passage of blood from infected mice to uninfected ones on seven days basis. This study was carried out based on the guide for the care and use of laboratory animals (23-25).
Phytochemical screening
Standard screening tests on the leaf latex was carried out to determine various plant constituents. The latex was screened for the presence of alkaloids, anthraquinones, flavonoids, glycosides, phenols, saponins and tannins compounds by using standard procedures (26, 27).
Acute oral toxicity study
Acute oral toxicity study was carried out based on OECD guidelines 425 (28). One female Swiss albino mouse was fasted for 4 hours and the fasting body weight of the animal was measured. Then, the leaf latex was administered to mouse at a dose of 2000 mg/kg. Then, mouse was kept under strict observation of physical and behavioral changes for one day, with special attention during the first four hours. Following the result from the first mouse, another four mice were fasted for 4 hours and then, the latex was administered to each mouse at the dose of 2000 mg/kg and were observed in the same manner. The observation was continued for fourteen days for any signs of overt toxicity and based on toxicity result; the dose of the latex was determined for experimental study.
In vitro antioxidant activity of the leaf latex Aloe weloensis
Antioxidant activity of the latex of Aloe weloensis leaf was evaluated by using DPPH free radical scavenging assay (29). A weight of 4 mg of DPPH was dissolved in 100 ml methanol in the dark and 3.9 mL of a 0.1 mM methanolic solution of DPPH was mixed with a 0.1 ml methanolic solution of different concentrations (12.5–400 μg/ml) of the latex and incubated in the dark for thirty minutes at room temperature. Ascorbic acid used as standard antioxidant. After thirty minutes, the absorbance of the mixture and the control at 517nm were read by using a UV spectrophotometer. The test was carryout in triplicate and the percent of scavenging of inhibition of was calculated as:
% free radical scavenging= A – B x 100
A
A; the absorbance of the control, B absorbance of the latex or ascorbic acid
In vitro antimalarial evaluation of the leaf latex of Aloe weloensis
Chloroquine sensitive P. falciparum (3D7 strain) was used in vitro blood stage culture to determine antimalarial efficacy of Aloe weloensis. Plasmodium falciparum culture was maintained in the method describe with some modification (30, 31). Plasmodium falciparum (suspension of 3D7) synchronized in 5% sorbitol to ring stage was seeded (200 μl/well with 2% ring stages and ORh+ red blood cells at 2% hematocrit) in 96-well tissue culture plates. Then, latex of A. weloensis leaf in different concentrations (10 - 320 μg/ml) was added to these wells. Chloroquine at the same concentration was used as the standard control and dimethyl sulfoxide without the tested samples were used as the negative control. The parasites were cultured for 30h in the desiccators and then, incubated at 37°C for 72h in 2% O2, 5% CO2 and 93% N2 (22, 31). The infected RBCs were transferred into freshly prepared complete medium to propagate the culture. After 72h incubation, the cultures were preserved at - 20 °C and the parasites were harvested. The thin blood smears were prepared and fixed with 100%methanol and stained with 10% Giemsa for 30munite to evaluate the growth stage of the parasites. The parasitemia was examined under the microscope and IC50 was determined by plotting concentration of the latex on percentage of growth inhibition. Percentage growth inhibition of the parasites was determined by using the following formula (22, 30).
% of growth inhibition = Mean parasitemia of the control – Mean parasitemia of the sample x 100
Mean parasitemia of control
Parasite inoculation
Plasmodium berghei ANKA strain was used for induction of malaria in experimental mice. The parasites were maintained by intraperitoneal serial passage of blood and parasitemia level (30-37%) of P. berghei infected donor mice were determined (32, 33). Donor mouse was anaesthetized by pentobarbitone at 150 mg/kg i.p. and infected blood was collected by cardiac puncture into heparinized vacutainer tube containing trisodium citrate (0.5%). The blood was then diluted in normal saline (0.9%) and RBC count of normal mice so that the final suspension would contain about 1×107 parasitized red blood cells (PRBCs) in 0.2ml suspension (22, 23). Each mouse used in the study was infected intraperitoneally with 0.2ml containing 1×107P. berghei parasitized RBCs.
Dosing and grouping of the animals
The mice were divided to five groups randomly (n=6). Group I (negative control) was treated with 10 mg/kg 2% Tween-80 in distilled water (TW80); Group II, III and IV were treated with 200 ,400 and 600 mg/kg doses of the leaf latex respectively and Group V was treated with the standard drug, chloroquine (25 mg/kg) (23, 24).
Antimalarial activity of the leaf latex of A. weloensis in 4-days suppressive test (Peter’s test)
Randomly assigned mice were inoculated with parasite and after three hours inoculation, infected mice were treated according to their respective grouping once daily for four days. On day five, blood samples were collected from tip tail of each mouse and slides were prepared. Then % inhibition, parasitemia level and survival time was determined.
Antimalarial activity of the leaf latex of A. weloensis in curative test (Rane’s test)
On the first day (day 0), the mice were injected intraperitonially with standard inoculum of 1x 107 P. berghei infected erythrocytes. After seventy-two hours, mice were randomly assigned into five group (n=6). Group I was treated with vehicle; group II, III and IV were treated three doses of the latex of A. weloensis respectively; group V was treated with chloroquine daily for 5 days. Thin blood films were prepared from tail blood of each mouse daily for five days to determine the levels of parasitemia and Mean survival time for each group (22, 23, 33).
Peripheral blood smears preparation
Thin smears of blood were made from the tail of each mouse on the fifth day (D4). The smears were applied on microscopic slides and the blood was drawn evenly across a second slide to make thin blood films and allowed to dry at room temperature. Then they were fixed with 100 % methanol and stained with 10 % Giemsa stain (pH = 7.2) for 15 minutes.
Parasitemia determination
Each stained slide for each mouse was examined under microscope. The parasitemia level was determined by counting the number of parasitized erythrocytes in random fields of the microscope. Percent parasitemia and percent suppression were calculated by using the following formula, respectively.
% Parasitemia = (number of parasitized RBC) x 100
(total number of RBC)
% Suppression = (mean parasitemia of negative control-mean parasitemia of treated group) x100
(mean parasitemia of negative control)
Determination of mean survival time
Mean survival time (MST) is another parameter that is commonly used to evaluate the efficacy of antimalarial plant materials. Mortality was monitored every day and the number of the days from the time of infection up to death was recorded for each mouse in the treatment and control groups throughout the follow-up period and the MST was calculated for each group by using the following formula.
MST = Sum of survival time of all mice in a group (days)
Total number of mice in that group
Packed Cell Volume Measurement
The packed cell volume (PCV) was measured to predict the effectiveness of the test latex in preventing hemolysis resulting from increasing parasitemia associated with malaria. Blood was collected from the tail of each mouse in heparinized micro hematocrit capillary tubes by filling three-quarters of its volume. The tubes were sealed by sealant and placed in a micro hematocrit centrifuge with the sealed ends outwards.
The blood was then have centrifuged at 12,000 rpm for 15 min. The tubes were then taken out of the centrifuge and PCV were determined using a standard Micro-Hematocrit Reader, the PCV of each mouse was then measure before infection and on day four after infection using the formula (23, 24, 32).
PCV = (Volume of erythrocytes in a given volume of blood)
(Total blood volume)
Determination of body weight and temperature changes
The body weights of the mice were determined to observe whether the leaf latex was prevented weight loss for Peter’s test, the body weight of each mouse was measured before infection (day 0) and on day 4 using a sensitive digital weighing balance. Rectal temperature was also measured by a digital thermometer before infection, and four hours after infection and then daily.
Statistical analysis
The results of the study were expressed as the mean ± standard error of the mean. Statistical analysis of the data was carried out with one-way analysis of variance followed by Tukey post hoc multiple comparison test. Significant differences were set at p values lower than 0.05.