Malaria is still causing significant mortality and morbidity in the global community [
Compared to adults, pregnant women and children below five years of age are more affected [
Among the challenges to confront the disease, development of resistant strains of the parasite to the conventional antimalarial drugs is a common phenomenon. In other words, the plasmodial parasites that were initially responding to the antimalarial drugs are now losing sensitivity [
As history shows, plants were a major source for the antimalarial drugs. This further hints at investigating medicinal plants to discover novel antimalarial drugs [
For the purpose of authentication, first, the branch of
In order to remove dust from the fruits, the fruits were first properly washed with distilled water and cleaned with a muslin cloth. To make a coarse powder and facilitate an extraction process, the plant fruits were air dried under shade at room temperature and pulverized with the aid of a mortar and pestle. The cold maceration technique as per the method described by Bello et al. [
At the end of the maceration, the flask was removed out of the shaker, put on a table, and left for 30 minutes to form a layer of distillate and marc. Then, separation of the marc from the resulting distillate containing the 80% methanol crude extract was done first using muslin cloth, followed by filtration with the aid of a Whatman filter paper number 1 (Whatman®, England) under suction filtration. This cold maceration procedure, in the same solvent system, was repeated twice using the marc left. The filtrate was then transferred to a round bottom flask and concentrated using a rotary evaporator (Buchi Rotavapor R-200, Switzerland) under reduced pressure. Then, the resulting extract was frozen in a deep freezer overnight and freeze dried with a lyophilizer (Operon, Korea Vacuum Limited, Korea) to remove water at -50°C and vacuum pressure of 200 mbar. The concentrated extract was transferred into an amber glass bottle and kept at -20°C until use. The percentage yield was calculated using the following formula:
The experiment was conducted on Swiss albino mice whose weights were in a range between 25 and 31 g and 6 to 8 weeks old. The animals were obtained from the Ethiopian Public Health Institute. Before conducting the experiment, acclimatization of the animals for a week to the experimental condition was done. The experiment was conducted in an animal house at Addis Ababa University, College of Health Sciences, School of Pharmacy. Polypropylene cages were utilized to house the animals where they stayed in a 12-hour light-dark cycle. A standard pellet food was given regularly to the mice while water was provided
To determine the safety of the plant extract, the Organization for Economic Cooperation and Development number 425 guideline [
As mentioned in Section
Chloroquine sensitive
The method described by Peter et al. [
At D4, the blood was collected from the tail of each mouse using clean and nongreasy slides to prepare thin blood films. The slides were stained with freshly prepared 10% Giemsa for 15 minutes. The stain was then washed off with distilled water. After allowing the slides to air-dry, the slides were viewed microscopically using the ×100 objective. By counting the number of PRBCs out of erythrocytes in random fields of the microscope, the percentage parasitemia (PP) was obtained. For each mouse, two stained slides were examined. Three fields with approximately 200-500 cells were counted for each slide, and PP for each mouse was determined as described below [
The following formula was used to calculate the mean percentage parasitemia suppression (PPS) [
For each group, the mean survival time (MST) was calculated as follows [
The weight and rectal temperature of each mouse were recorded just before treatment and after treatment on D4. The mean percentage changes were then calculated and analyzed for each group [
The phytochemical constituents of the 80% methanolic crude fruit extract of
After organizing, the data were fed into SPSS version 22 and then analyzed. To compare the mean PPS, MST, changes in mean body weight, PCV, and rectal temperature of the
The physical nature of the extract was found to be dry brownish powder. A total of 97.9 g (19.58% yield) of the extract was harvested from the 80% methanolic crude extract of the fruits of
The test for acute toxicity showed that no mortality was observed within the first day and the next 2-week period of observation. Furthermore, the plant caused no visible signs of acute toxicity as evidenced by the gross behavioral and physical observations of the experimental mice.
The chemosuppressive effect of the plant extract is summarized in Table
The 80% methanolic crude fruit extract of
Group | % parasitemia | % suppression | Survival time (day) |
---|---|---|---|
CON | 0.00 | ||
CFE100 | 72.65v^,x^,y^,z^ | ||
CFE200 | 87.88v^,w^y^,z^ | ||
CFE400 | 93.29v^,w^,x^,z^ | ||
CQ10 | 100.00v^ |
Values are described as
As compared to the vehicle-treated group, the three doses of the extract significantly (
Changes in weight and rectal temperature of mice infected with
Group | Weight (g) | Temperature (°C) | ||||
---|---|---|---|---|---|---|
D0 | D4 | Change | D0 | D4 | Change | |
CON | ||||||
CFE100 | ||||||
CFE200 | ||||||
CFE400 | ||||||
CQ10 |
Values are described as
As compared to the vehicle-treated mice, the mice in the extract-treated group exhibited a dose-dependent and statistically significant (
Changes in packed cell volume of mice infected with
Preliminary phytochemical screening test done on the 80% methanolic crude fruit extract of
Preliminary qualitative phytochemical screening of the 80% methanolic fruit crude extract of
Phytochemicals | Test result |
---|---|
Alkaloids | |
Tannins | |
Saponins | |
Flavonoids | |
Terpenoids | |
Steroids | x |
Glycosides |
x: shows absence of phytoconstituents;
The study to determine the amount of each phytochemical in the 80% methanolic fruit crude extract of
Quantitative determination of phytochemicals in the 80% methanolic crude fruits extract of
More than 80 percent of the African population rely on traditional herbal medicine to meet their primary healthcare demand. Nonetheless, pharmacological investigations to standardize and set the antimalarial efficacy and safety of such plants were not largely done to this indigenous gift [
In the present study, crude fruit extract of
Phytoconstituents such as alkaloids, abundantly localized in the plant extract, could be responsible for the antiplasmodial activity as plant-derived alkaloids such as quinine are evidenced to possess a potent antimalarial activity. This plant also contains terpenoids, phenolic compounds, and flavonoids. These bioactive principles have been reported to possess a range of therapeutic activities including antimalarial activity in the literature [
In discovering antimalarials from plants, the extract is ideally expected to prevent reduction in PCV, body weight, and body temperature due to the development of parasitemia [
The three doses of methanolic crude extract significantly prevented rectal temperature drop due to parasitemia as compared to the vehicle. These activities probably indicate that the extract ameliorates some pathological processes that cause reduction in internal body temperature and metabolic rates. In a 4-day suppressive test, a candidate antimalarial agent should prolong survival time to be an active antimalarial agent. The extract significantly prolonged the survival time of mice as compared to the control. However, unlike CQ, the mice were not cured. This might be due to the incomplete clearance of the parasite or short half-life profile of active constituents [
A candidate antimalarial agent should elicit parasite suppression of 30% or greater [
It can be deduced from the acute toxicity test that the oral medial lethal dose (LD50) of the extract could be greater than 2000 mg/kg of the extract as per OECD guideline no. 425 [
Due to the lack of genetically modified immunocompromised mice, the study was not conducted on
The findings of the present study indicated that the fruits of
Vouchers and specimens of the investigated plant are deposited at the national herbarium of Addis Ababa University, College of Natural Sciences. The datasets used to support the finding of this study are available from the corresponding author upon request.
Ethical clearance was granted by the ethical review committee of the School of Pharmacy, College of Health Sciences, Addis Ababa University.
The authors declare that they have no conflicts of interest.
GH conceived the proposal and SA reviewed it. GH performed the experiment and analyzed and interpreted the data. Both authors participated in the write-up of the manuscript. Both authors have read and approved the final manuscript.
The financial support of Addis Ababa University and Mettu University is gratefully acknowledged. The research was funded by the School of Graduate Studies of Addis Ababa University, Research Grant no. GSR/2597/09.