Malaria is a life threatening protozoal infection caused by Plasmodium species and a major public health problem in more than 100 countries in the world (1). In Africa, Democratic Republic of the Congo and Nigeria account for over 40% of the estimated total of malaria deaths globally. Sub-Saharan Africa carries the bulk of the global malaria burden, 71% of cases and 86% of global deaths (2). The problem of malaria is very severe in Ethiopia where it has been the major cause of illness and death for many years. According to records from the Ethiopian Federal Ministry of Health, 75% of the country is malarious with about 68% of the total population living in areas at risk of malaria (3). Multi-drug resistant strains of the parasite to antimalarial drugs proved to be a challenging problem in malaria control in most parts of the world (4). Unfortunately, the rapid spread of drug-resistant to P.falciparum parasites, development of insecticide resistance, lack of reliable effective vaccine and unaffordable antimalarial drugs compromise the efficacy of available antimalarial drugs and have imposed a serious negative impact on malaria control interventions (5, 6). Absence of clinically proven vaccine, resistance by the parasite against first line and increase in transmission which has drawn attention to develop new drugs to alleviate these challenges (7).
According to studies there are 1200 plant species from 160 families that are used to treat malaria globally(8). Traditional medicines are the most important and sometimes the solitary source of medicine for about 80% of the Ethiopian population and more than 95% of the medicinal preparation are plant origin as they are easily accessible and affordable (9). However, scientific studies on the status of use of phyto-medicine preparation of crude extracts and isolation of active principles is very minimal (10). Schinus molle L (S.molle) is one of the plants that are traditionally used for malarial treatment in Ethiopia. An ethnobotanical survey showed that in Sasiga, Karsa Mojo and Mada Jalala district, Western Ethiopia seeds of S.molle used for treatment of malaria (11-13). It is a tree belonging to the Anacardiaceae family which is native to subtropical regions of South America (14). In traditional medicine, the plant is used against coughs, colds, tuberculosis, bronchitis, and fever (15). In Ethiopia, S.molle traditionally used for used for treatment of different type of diseases such as; wound around rectal area (16), eye infection, allergy, hemorrhoids, respiratory infection (17), Jaundice, diarrheal and tonsillitis (18).
The previous report showed that the extract obtained from S.molle fruit had antimicrobial activity (15). The petroleum ether extract of fruit of S.molle exhibited a high level of antifungal activity where caused full suppression for B.cinereaat dose, 1000 ppm The methanolic crude extracts of pepper tree (S. molle) were evaluated through in vitro test had good growth suppression activity for Botrytis fabae and Vicia faba L (19) and the same extract of the aerial parts also had promising adulticidal and egg hatching inhibitory effects against H. contortus (20). On the other hand, reports showed that the extract of S.molle seeds also possess immunomodulatory, antioxidant (21), anti-inflammatory, analgesic effects (22), repellent activity (23), larvicidal property (24), antiviral, topical antiseptic as well as antifungal activities (20). Thus, based on ethnobotanical studies mentioned above, present study evaluated the in vivo antimalarial activity of the crude extract and solvent fractions of S. molle seeds.
Material and method
Plant material collection and preparation
The fresh seeds of S.molle were collected from Maraki campus, University of Gondar, 728 km from Addis Ababa, Northwest Ethiopia. The plant was identified and authenticated with a voucher number AB015/2010 and deposited at biology department, university of Gondar, Ethiopia. The seeds were cleaned and air-dried in the shade at room temperature. The dried seeds were coarsely powdered using electric grinder. Then the powdered plant materials were stored in a plastic container and they were kept at room temperature until extraction.
Crude extract preparation and fractionation of the crude extract
The crude extract was prepared by cold maceration technique, by refluxing 1000 g of plant material in methanol (80%). After 72 hrs the mixture was filtered using Whatman filter paper no 1 (Whatman, England). The extract was concentrated in a rota vapor. Then placed in a deep freeze overnight and dried using a lyophilizer (Operan, Korea vacuum limited, Korea) and stored in a refrigerator until fractionation. Portion of the methanol crude extract was suspended in a reparatory funnel with distilled water. Then the suspension was shaken by adding chloroform each time 3 times and the chloroform fraction was obtained. The aqueous residue was then shaken with ethyl acetate 3 times to obtain the ethyl acetate fraction. The chloroform and ethyl acetate fractions were concentrated in Rota vapor. The aqueous residue was also lyophilized to obtain the aqueous fraction. Then the fractionations were kept in an amber color glass bottle and stored in a refrigerator.
Phytochemical analysis of the crude extract and solvent fraction
Standard screening tests of the extract were carry out for various plant chemical constituents; for the presence or absence of secondary metabolites such as terpenes, alkaloids, steroidal compounds, phenolic compounds, tannins, diterpeniod, sesquterpeniods, tritereniods, saponins and flavonoids using standard procedures (25).
Acute toxicity testing
Five female Swiss albino mice were randomly selected. After being fasted for 3 hrs, a fixed dose of 2000 mg/kg of S.molle seeds crude extract was administered to a single mouse via the oral route by oral gavage. Similarly, food was withheld for 1hrs after extract administration. Animals were observed for gross changes such as loss of appetite, hair erection, lacrimation, tremors, convulsions, salivation, diarrhea, mortality and other signs of toxicity for next 24hrs. In view of the fact that no deaths of the study mice at the test dose of 2000 mg/kg were observed, additionally 4 mice were sequentially dosed. Then the mice were observed for any signs of toxicity daily for 14 days to assess safety of the extract (26).
In vivo antimalarial tests
Experimental animals grouping and dosing
Male Swiss albino mice, weighing 24 to 35 gm and 6 to 8 weeks old obtained from Ethiopian public health institution, Addis Ababa. They were housed in plastic cages with softwood shavings and chips as beddings. They were exposed to a 12:12 dark-to-light cycle. They had free access to pellet diet and clean drinking water. All mice were acclimatized to the working environment one week before the beginning of the experiment (27). Group I received 0.5 ml distilled water per kg body weight. Groups II, III and IV received 100, 200 & 400 mg/kg body weight of S.molle seeds extract orally respectively in all antimalarial testing models. The standard drug, (25 mg/kg/day chloroquine phosphate), was administered to Group V as positive control group.
Parasite and parasite inoculation
In vivo antimalarial testing in mice was done using chloroquine sensitive strain of Plasmodium berghei (ANKA strain) obtained from Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Ethiopia. Albino mice previously infected with P.berghei and having parasitemia level of 3037% were used as donor. After anaesthetizing with ketamine, the blood from these mice was collected via cardiac puncture into a test tube having 0.5 % trisodium citrate. The blood was then diluted with physiological saline (0.9%) based on parasitemia level of the donor mice and the red blood cell (RBC) count of normal mice in such a way that 1 ml blood contains 5 ? 107 infected RBCs. Each mouse was then given 0.2 ml of this diluted blood intraperitoneally, which contained 1 ? 107P.berghei infected RBCs (28, 29).The amount of normal saline used in the dilution was determined by the level of parasitaemia (%) of the infected donor mice. If 0.5 ml of blood was removed then dilution was done with (X-0.5) ml of saline, where X was the % parasitaemia (30).
Four-day suppressive antimalarial test (Peters test)
The residual infection protocol described by Peter was employed to evaluate the four day suppression activity of the extract. This test was used to evaluate the schizontocidal activity of the extract and the fractions against P.berghei infected mice (31). Female Swiss albino mice weighing 2435 grams were randomly divided into five groups (five mice per cage) for each extract and treated as above mentioned in grouping of animals. Treatment was started after 3 hrs of infection with P. berghei on day 0 (D0, inoculation day) and was continued daily for four days (i.e. from D0 to D3). Treatment continued for three successive days (from D0 – D2). On the 4th day (D3), mice were infected with 1 ? 107 P. berghei infected red blood cells. After treatment was completed, thin blood film was prepared from the tail of each animal on day 4 to determine parasitemia and percentage inhibition. In addition, each mouse was observed daily for determination of survival time.
Curative antimalarial activity test (Ranes test)
Evaluation of the curative potential of the crude extract and the most active fraction in Peters test was carried out according to the method described by Ryley and Peters (32). On Day 0, standard inocula of 1 ? 107 infected erythrocytes were inoculated in mice intraperitoneally. Seventy-two hours later, following confirmation of parasitaemia, the mice were randomly assigned into two control and three test groups, each group treated accordingly as described above. Treatment continued for 5 consecutive days at a single dose per day. At day seven, 10% of Geimsa stained (Science Lab, USA) thin blood film was prepared and examined microscopically to determine the percentage of parasitaemia. Mean survival time for each group was determined by calculating the average survival time (days) of mice starting from date of inoculation over a period of 30 days (D0-D29).
Prophylactic antimalarial test (repository test)
Compounds identified as being active in four-day suppressive assays can subsequently be further examined through the use of several secondary tests in mice from which prophylactic test are one of them. Evaluation of prophylactic potential of the extract was done according to Fidock et al (28). Twenty five mice were randomly grouped into five groups as mentioned above accordingly and then treated for four consecutive days (D0 to D3). On the fifth day (D4), a standard inoculum (2 ? 107P. berghei IRBCs, 0.2 ml) was administered by IP to each mouse. After 72 hrs of infection (D7), thin blood smears were prepared from tail of each mouse on a microscopic slide.
Packed cell volume measurement
Packed cell volume (PCV) was measured to predict the effectiveness of the test extract and fractions in preventing hemolysis resulting from increasing parasitemia associated with malaria. Heparinized capillary tubes were used for collection of blood from tail of each mouse. The capillary tubes were filled with blood up to ?th of their volume and sealed at the dry end with sealing clay. The tubes were then placed in a micro-hematocrit centrifuge with the sealed end outwards and centrifuged for 5 min at 12,000 rpm. The tubes were then taken out of the centrifuge and PCV was determined using a standard Micro-Hematocrit Reader. PCV is a measure of the proportion of RBCs to plasma and measured before inoculating the parasite and after treatment and calculated by using the formula described by Gilmour and Sykes (33):
PCV = “Volume of erythrocytes in a given volume of blood” /”Total blood volume”
Parasitemia level measurement
Thin smears of blood were made from the tail of each mouse on day 4 for Peters test and on day 37 for Ranes test. The smears were applied on microscope slides (76 ? 26 mm), fixed with absolute methanol for 15 min and stained with 10% Geimsa stain at pH 7.2 for 15 min. The stained slides were then washed gently using distilled water and air dried at room temperature. The stained slides for each mouse were examined under Olympus microscope with an oil immersion nosepiece of 100 ? magnifications. Then, the percent suppression of each extract with respect to the control groups and the parasitaemia was determined by counting a minimum of six fields per slide to calculate the average parasitemia as shown below(34).
%Parasitemia =”Number of parasitized RBC” /”Total number of RBC” x 100
Finally, percent parasitemia suppression of the extract was compared with respect to the controls and parasitemia suppression was calculated using the following formula (29).
% suppression = (mean parasitemia of negative control-mean parasitemia of treated group)/(mean parasitemia of negative control) x 100
Determination of mean survival time
Mean survival time (MST) is another parameter that is commonly used to evaluate the efficacy of antimalarial plant extracts. An extract that results in survival time greater than that of infected non-treated mice was considered as active. Mortality was monitored daily 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 (29).
“MTS=” “Sum of survival time of all mice in group(days)” /”Total number of mice in the group”
Monitoring of body weight and temperature changes
Body weight change is one of parameter used to assess the effectiveness of plant extracts (28). For Peters test, 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 another parameter and measured by a digital thermometer before infection, four hours after infection and then daily. For Ranes test, body weight and temperature were measured before infection and from day 37 after infection. In order to rule out the effect of the extract on body weight, temperature and PCV; the crude extract was administered to healthy mice at the doses used for four days suppressive test. The body weight of each mouse in all groups was taken before treatment (D1) and after infection at day seven (D7) for prophylactic tests.
The data of the study were expressed as mean ± SEM (standard error of mean) for each group of experiments. Data on the levels of parasitemia, variations in body weight and survival times were analyzed using windows SPSS version 21.0. The differences between means of measured parameters were compared using one-way ANOVA followed by Tukeys HSD multiple comparison test. The P values < 0.05 were regarded as statistically significant.
During experimental procedures, experimental animals were handled and cared according to the internationally accepted laboratory animals use, care and welfare guideline (35). Ethical clearance was requested to and obtained from animal ethics committee of Department of Pharmacology, School of Pharmacy and University of Gondar.