Antiplasmodial Activity of 80% Methanolic Extract and Solvent Fractions of Stem Bark of Acacia tortilis in Swiss Albino Mice

Background Malarial infection has significant negative impact on the health of the world population. It is treated by modern and traditional medicines. Among traditional medicinal plants, Acacia tortilis is used by different communities as antimalarial agent. Therefore, the objective of this study is to validate antimalarial activity of the stem bark of Acacia tortilis in mice. Methods To evaluate antimalarial activity of the plant, 4-day suppressive, curative, and prophylactic antimalarial test models were used. Parasitemia, packed cell volume (PCV), survival time, rectal temperature, and body weight were used to evaluate the effect of the plant extracts. Data were analyzed using SPSS version 26 followed by Tukey's post hoc multiple comparison test. Results The crude extract and dichloromethane fraction significantly suppressed the level of parasitemia (p < 0.001) and increased mean survival time (p < 0.01) at all tested doses. Similarly, significant effects were observed in mean survival time, % change of PCV, weight, and temperature in both curative and prophylactic antimalarial test models. Conclusions The methanolic extract and solvent fractions of the stem bark of Acacia tortilis has shown antimalarial activity, and the finding supports the traditional use and the in vitro studies. Thus, this study can be used as an initiation for researchers to find the most active phytochemical entity and to conduct additional safety and efficacy tests.


Introduction
Malarial infection has a signifcant negative impact on the health of the world population [1]. Higher deaths were enumerated in under-fve children in sub-Saharan Africa where infectious diseases are still the primary public health concern [2,3]. Children and pregnant women are selectively afected by malaria. In 2019, malaria was responsible for the death of 409,000 people. Of these 94% deaths occurred in Africa and the death of young children accounts for a total of 274,000 [4]. Children who recovered from cerebral malaria (2%) develop several disabilities and impairments [5]. In 2020, it is estimated that 215.2 million cases and 386,400 deaths in malaria-endemic countries in Africa [6].
According to 2019 World Health Organization estimation, the incidence of malaria was 229 million of which 409, 000 deaths were registered in the world while the most (94%) was in the African region [7]. Apart from death, the disease results serious complications like cerebral malaria, severe anemia, hypoglycemia, and acute renal failure [8,9].
In 2020, a year after the COVID-19 pandemic, the number cases of malaria rose to 241 million, an increment of 12 million cases as of 2019. In the African region, between 2019 and 2020, the cases of malaria had grown from 213 to 228 million and deaths from 534 000 to 602, 000 between 2019 and 2020. Te region has valued 95% and 96% global cases and deaths, respectively, among which the deaths of under-fve children accounted 80% [10].
A single antimalarial drug is not efective for both stages (liver and intra-erythrocytic) of malaria parasite. Due to this many drugs may be used to have complete elimination of a parasite from the already established infection [11].
Acacia tortilis (Fabaceae family) is a slow growing tree having an umbrella-shaped canopy [12]. Traditionally, the plant has inspiring medicinal uses for mouth infections and dental problems [13], dry cough, and diphtheria [14]. It has been proved that it contains antidiabetic [15], antifungal [16], antidiarrheal [17], antihyperlipidemic [18], antiinfammatory [19], antimalarial, and antileshmanial [20] activities. Similarly, the whole plant [21] and the stem bark [22] of Acacia tortilis showed very active and active in vitro antiplasmodial activities, respectively. Resistance to artemisinin antimalarial drugs was reported in murine malaria models [23] and in patients on the Cambodia-Tailand border [24]. So, there is a need to validate the in vitro antimalarial activity in mice model and innovate new drugs to fll the resistance problem. Terefore, this study was aimed to investigate antimalarial activity of the plant in the rodent model and ensure which solvent fraction(s) is/are more efective so that a clue about the nature of the efective phytochemical constituents can be obtained.

Plant Material.
Te stem bark of the plant was collected from Makisegnit Woreda, Central Gondar, Amhara, Ethiopia in November 2021. Identifcation and authentication was made by a botanist and a voucher specimen number was given (MA03).

Experimental Animals and Parasite.
Swiss albino mice weighing 20 to 30 g, aged 6 to 8 weeks (males for antimalarial test and females for acute oral toxicity test) were selected and used. Te mice were given free access to pelleted food and water. Tey were kept in a standard plastic cage at room temperature and light having a cycle of 12 h light and 12 h dark. Te mice were acclimatized to the laboratory class 7 days prior to the start of the experiment. Plasmodium berghei strain which is chloroquine sensitive was used for the antimalarial test. Te continuity of the parasite was ensured by transferring blood from infected to noninfected mice weekly.
Animals were handled based on the internationally accepted guidelines for care and use of animals. Ethical issues and the study protocol were approved by the ethical committee.

Extraction and Fractionation.
Te stem bark was frst cleaned and then dried under shade. Te dried bark was grounded into small pieces using mortar and pestle. About 1.5 kg coarse powder of the bark was weighed by Wensar analytical balance (Swastic Systems and Services, India) and then extracted with the cold maceration technique. Ten, No 1 Whatman flter paper was used to flter the extract. Te mark was re-extracted two times by adding similar volume of the fresh solvent. Te fltrates added together and were allowed to be concentrated at a temperature less than 40°C. Te concentrated extract was then frozen and dried using a lyophilizer. Te dried extract was then fractionated using hexane, dichloromethane, and water. Initially, the crude extract was mixed with water and then shaken using a separatory funnel. Hexane was added three times separately to get a hexane fraction. Ten, dichloromethane was added to the residue three times and then dichloromethane fltrate was obtained. Te extracts were concentrated using a rotary evaporator. Te aqueous residue was dried using a lyophilizer. Finally, the crude extract and the fractions were stored at −20°C until being used for the experiment.

Phytochemical
Screening of the Stem Bark of Acacia tortilis. Both the crude extract fractions were screened for the presence or absence of secondary metabolites such as tannins, favonoids, anthraquinones, glycosides, phenols, steroids, terpenoids, alkaloids, and saponins using standard screening tests [25].

Acute Oral Toxicity
Test. Acute toxicity test for the crude extract was performed based on the guideline 420 developed by the Organization for Economic Co-operation and Development (OECD) [26]. Female mice with the age 6 to 8 weeks were used for the test. Tey were fasted 4 h before and 2 h after administration of the crude extract. Initially, sighting study was performed to determine the starting dose by administering 2000 mg/kg of the extract to a single mouse. Since no sign of toxicity and death was observed in 24 h, the same dose was given to 4 mice through oral gavage. Te presence of toxicity, death, and food intake was strictly followed for 4 h and then for 14 days.
2.6. Grouping and Dosing. Animals were randomly assigned into 5 groups (6 animals per group) for each model. Group I (negative control) received 10 ml/kg of the dissolving vehicle (2% tween 80 for hexane and dichloromethane fractions and 10 ml/kg distilled water for the crude extract and distilled water fraction). Group II received the positive control, and the groups from III to V received 100 mg/kg, 200 mg/kg, and 400 mg/kg of the crude extract and fractions.

Inoculation.
First, the level of parasitemia for the donor mice was determined (20%-30%). After ether anesthesia, mice were sacrifced through cervical dislocation and then blood was taken by cardiac puncture and collected in a heparinized tube. Te blood was diluted with normal saline (0.9%) to the level of 5 × 10 7 of infected red blood cell (RBC) in 1 ml. Each mouse was given 0.2 ml of blood (containing 1 × 10 7 infected RBCs) intraperitoneally.

Four-Day Suppressive Test.
Peter's suppressive test method was used to assess the chemo-suppressive efect of the plant extracts against chloroquine sensitive P. berghei [27]. Prior to infection, the weight of mice, packed cell 2 Evidence-Based Complementary and Alternative Medicine volume (PCV), and temperature were measured. Ten, thirty mice for the crude extract and each of solvent fractions were parasitized on the frst day (day 0). Two hour later, mice were randomly grouped into 5 groups and given doses as indicated in the grouping and dosing section. Treatment doses were continued being given at 24, 48, and 72 h (until the third day). On the fourth day of infection (96 h later), blood was taken from the tail of each mouse and then the parasitemia level and percentage chemosuppression was determined by preparing thin smears on the microscope slides. At the end of the experiment, the weight of mice, packed cell volume (PCV), and temperature were measured. Ten, the mean survival time was evaluated by following the mice for 30 days (day 0 to day 29).

Curative Test.
Te curative test was conducted for the crude extract and dichloromethane fraction, which have shown relatively higher parasitemia suppression in a fourday suppression test. Te curative potential of the plant in an established infection was conducted using the method indicated by Raley and Peters [28]. For each test extract, thirty mice were infected on the frst day (day 0). After day 3 (72 h), mice were grouped into fve groups (six per group) and treated with respective doses of the crude extract and dichloromethane fraction as indicated in the grouping and dosing section. Treatment doses were continued to be given at 96, 120, and 144 h. Te level of parasitemia was recorded daily from day 3 to day 6. Te weight of mice, packed cell volume (PCV), temperature, and survival time were also recorded.

Prophylactic Test.
Te prophylactic efect of the crude extract and dichloromethane fraction was done as indicated by Peters et al. [27]. For both of the extracts, mice (thirty for each) were randomly assigned to fve groups and treated as pointed in the grouping and dosing section. Treatment was consecutively given daily for four days and all mice were intraperitoneally infected with the parasite (1 × 10 7 P. berghei) on the 5th day. Blood smears were prepared 72 h after infection and the parasitemia level was determined. In addition, the weight of mice, temperature, packed cell volume (PCV), and survival time were also recorded.

Determination of Parasitemia and Survival Time.
Blood smears from each mouse were applied on diferent microscope slides and then were fxed with methanol. Ten, the slides were stained with 10% Geimsa stain for 15 min and were washed with water and then dried at room temperature. Finally, parasite-infected RBCs were counted using microscope having a magnifcation power of 100x. Te level of parasitemia was calculated by the experiment blinded laboratory technician. % Parasitemia was computed by enumerating the infected RBC and total RBC from the blood flms while parasitemia suppression was calculated by comparing parasitemia in the negative control with parasitemia in the treated group with the following formulas [29]: (1) At last, mice were followed for 30 days (from day 0 to day 29) and their mean survival time (MST) was determined as indicated in the following formula [29]: MST � Total number of da ys mice survive d Total number of mice .
3.5. Determination of Packed Cell Volume, Rectal Temperature, and Body Weight. Blood was taken from the tail of each mouse and was collected in heparinized microhaematocrit capillary tubes to 75% of their height and then was sealed. Te tubes were then placed on a centrifuge and were rotated at 12,000 rpm for 5 min. Packed cell volume (PCV) was computed through the following formula [30]: PCV � Volume of erythrocytes in a given volume of bloo d Total bloo d volume × 100.
Te weight of each mouse was measured using the weighing balance, and rectal temperature was tested using the rectal thermometer. Te changes before and after treatment were then calculated.
Evidence-Based Complementary and Alternative Medicine 3.6. Data Analysis. Te data were analyzed using SPSS version 25. Te results were expressed as mean ± SEM (standard error of the mean). One-way ANOVA and Tukey's post hoc test for comparisons were used to compare differences in the groups. Results were considered signifcant at 95% confdence level at P value < 0.05.

Yields of the Crude Extract and Solvent Fractions.
After extracting 1.5 kg of the stem bark with 80% methanol, 121 g of the crude extract was obtained. Upon fractionation of 90 g of the crude extract, 65 g, 15.5 g, and 9.5 g of water, dichloromethane, and hexane fractions were obtained, respectively.

Phytochemical
Screening. Te extract of the stem bark was screened for the availability of diferent phytochemicals. Based on this, many phytochemicals were present in the crude extract and were proved to be attracted to dichloromethane as indicated in Table 1.

Acute Oral Toxicity Test.
Te crude extract of the stem bark of Acacia tortilis at 2000 mg/kg dose did not show any sign of toxicity or death in the 14-day follow-up period. No visible adverse efects like changes in feeding, body weight, hair erection, urination, lacrimation, salivation, and movement were observed, indicating the extract is safe.

Efects of the Crude Extract and Solvent Fractions of the Stem Bark of Acacia tortilis in the 4-Day Suppressive Test.
Te crude extract and dichloromethane fraction produced signifcant diferences on the parasitemia level and mean survival time at all tested doses as compared to the negative control. In addition, the aqueous fraction showed a meaningful diference on the parasitemia level (at 400 mg/kg dose)  Data are expressed as mean ± SEM; n � 6, a � compared to the negative control, b � compared to the positive control, c � compared to 100 mg/kg, d � compared to 200 mg/kg, e � compared to 400 mg/kg, 1 p < 0.05, 2 p < 0.01, 3 p < 0.001, * � negative and positive controls for dichloromethane and hexane fractions, SEM � standard error of the mean, D0 � day 0, D4 � day 4, DW � distilled water, CE � crude extract, AF � aqueous fraction, DF � dichloromethane fraction, HF � hexane fraction, T80 � tween-80, and CHQ � chloroquine.    (Table 2). Te activity of both the crude extract and solvent fractions increased as the dose increases. As indicated in Table 3, the crude extract produced a meaningful efect on the % change of packed cell volume (PCV) (400 mg/kg), temperature change (200 and 400 mg/ kg), and weight change at all tested doses. Aqueous fraction produced signifcant diferences on temperature and weight change at 400 mg/kg dose. In addition, dichloromethane fraction (at all tested doses) produced signifcant efects on % changes of PCV, temperature, and weight; but hexane fraction showed signifcant activities only on temperature change at 200 and 400 mg/kg doses. Te activities for all the extracts increase as the dose increases indicating the efects are dose dependent.

Efects of the Crude Extract and Dichloromethane Fraction of the Stem Bark of Acacia tortilis in the Curative Test Model.
Te crude extract and dichloromethane fraction has shown a relatively greater antimalarial activity in the 4-day suppressive test. Terefore, these extracts are selected for further evaluation in curative and prophylactic antimalarial test models.
Both the crude extract and the dichloromethane fraction revealed signifcant efects on % parasitemia from day 5 to day 7 (Table 4). Similarly, both the crude extract and the fraction showed a meaningful diference on mean survival time as compared to the negative control at all tested doses (p < 0.001). Te activities of each extract increased as the dose increases.
As shown in Table 5, the crude extract produced signifcant diference on the % change of PCV and rectal temperature (at 200 and 400 mg/kg) as well as body weight at 400 mg/kg dose. In addition, dichloromethane fraction produced a meaningful diference on % change of the weight and rectal temperature at all tried doses in a dose-dependent manner (p < 0.001), while a signifcant efect on the % change of body weight was observed at 400 mg/kg dose (p < 0.05).

Efects of the Crude Extract and Dichloromethane Fraction of the Stem Bark of Acacia tortilis in the Prophylactic Test
Model. In comparison with the negative control, both the crude extract and dichloromethane fraction showed signifcant efects on both the % parasitemia (p < 0.001) and mean survival time (p < 0.01) at all tested doses (100, 200, and 400 mg/kg). Both of the extracts showed comparable efects on % suppression and mean survival time ( Table 6).
As compared to the negative control, signifcant differences on the % change of PCV, rectal temperature, and body weight were seen at 400 mg/kg dose of both the crude extract and dichloromethane fraction. Dichloromethane fraction produced relatively higher activities than the crude extract on the tested parameters (Table 7).

Discussion
Te current treatment of malaria gets serious challenges due to the emergence of resistance to the available drugs and unavailability of vaccines [31,32]. Malaria caused by P. falciparum is a serious disease, if untreated, it may progress to being life threatening and then result in death [33]. Terefore, there is a need to fnd new medicines from diferent sources.
Te extracts of Acacia tortilis were evaluated for their acute oral toxicity and antimalarial activities in three rodent test models. Using rodents for testing antimalarial activity of the compounds is important since it can show the activity of prodrugs that need activation in living systems unlike in vitro studies [34]. Terefore, the rodent malaria model was used to test the antimalarial activity of the plant extract. Te crude extract of the plant did not show any toxicity signs at a dose of 2000 mg/kg. Accordingly, this extract can be considered good for further studies since the LD 50 is above 20 times the minimum tried efective dose (100 mg/kg) [35].
Te antimalarial activities of the crude extract and solvent fractions of Acacia tortilis were evaluated using standardized models. Accordingly, the 4-day suppressive test was conducted for evaluating schizontocidal activity at the start of the infection while the curative test was employed to assess curative potential of the extracts on an  Evidence-Based Complementary and Alternative Medicine already established infection, and the prophylactic test was done to assess the infection preventive activity of the plant [36]. According to the category of biological substances, the study result showed the extract is endowed with antimalarial activity and the result was in line with the previous very active and active in vitro antiplasmodial activities of the whole plant and the bark, respectively [21,22,37]. An extract with greater than 30% suppressive efect (as compared to the negative control) on the level of parasitemia is considered as efective [38]. As shown in Table 2, in the 4-day suppressive test, both the crude extract and dichloromethane fraction showed parasitemia suppression at all tested doses (p < 0.001) confrming the probable schizontocidal efect. All the tested doses of the extract revealed an increase in the mean survival time explaining the associated decrease in the parasitemia level. Tis result is in line with the study conducted on Croton macrostachys [39]. In addition, the better activity of the dichloromethane fraction on the % change of PCV, temperature, and weight was in line with the very active in vitro antiplasmodial activity of the dichloromethane extract of the plant. Te diference in the activity may be due to the variation in the presence of secondary metabolites in the fractionating solvents. In addition, variation in the concentration of secondary metabolites in the fractionating solvents may account for the activity diference.
In the curative test, signifcant suppression on the parasitemia level was observed at all tested doses of both the crude extract and dichloromethane fraction, indicating the efect of the extract on the established infection. In this model, antimalarial activity was tested for the crude extract and dichloromethane fraction, since they showed better activity in the 4-day suppressive test model in a dosedependent manner.
After confrming the positive curative efect, the evaluation was continued to validate the prophylactic efect of the plant. In the study, the crude extract and dichloromethane fraction had shown a chemoprophylactic efect in a dose-dependent manner. Several secondary metabolites like alkaloids and favonoids were screened in both the crude extract and dichloromethane fraction. Secondary metabolites are implicated in antiplasmodial activities through diferent mechanisms. Alkaloids are known to possess antimalarial activity [40]. Saponins, favonoids, and terpenoids may be responsible for the observed antimalarial activity [41]. In addition, secondary metabolites are involved in several functions including endoperoxidation by terpenoids [42], DNA intercalation by anthraquinones [43], disruption of detoxifcation of heme by alkaloids [44], inhibition of protein synthesis by alkaloids and disruption of nucleic acids by favonoids [45], inhibition of superoxide dismutase and inhibition of DNA synthesis by coumarins [46], and free radical scavenging by tannins [47]. Furthermore, glycosides are known to have a direct antiplasmodial efect [48]. Te observed antimalarial efect may be due to the in concert efect of these secondary metabolites.

Conclusions
Te methanolic extract and solvent fractions of the stem bark of Acacia tortilis has shown antimalarial activity, and the fnding supports the traditional use and the in vitro studies. Tus, this study can be used as an initiation for researchers to fnd the most active phytochemical entity and to conduct additional safety and efcacy tests.

Data Availability
Te datasets are available from the corresponding author upon reasonable request.

Ethical Approval
Te investigation protocol and ethical issues were approved by the research and ethics committee of the Department of Pharmacology with approval number SOP4/290.