Background
Though malaria mortality rates have fallen by 47 % globally and by 54 % in the World Health Organization (WHO) African Region since 2000, about 198 million cases and 584 000 deaths occurred in 2013 showing that malaria is still among the leading human infectious diseases with 90 % of the cases and deaths occurring in Africa [
1]. In Ethiopia, although evidences suggest a substantial decline in malaria cases and deaths in recent years compared to the baseline year of 2004, the occurrence of several isolated outbreaks was reported nationwide and the disease remains a major cause of outpatient consultation in the country [
2].
Due to climatic changes and coupled-human-natural factors malaria epidemiology is progressively changing putting more people at-risk. Moreover, there is a rapid emergence of drug-resistant
Plasmodium strains. For instance, resistance has already been developed against the latest first-line anti-malaria drug, artemisinin, in Asia [
3]. Malaria control efforts are further complicated by the increased resistance of mosquito vectors to insecticides [
4] together with challenges of having effective anti-malaria vaccines. Thus, there is urgent need to search for effective, easily available, affordable and safe alternative anti-malaria drugs that can be integrated into the existing malaria control interventions to successfully curtail the disease and for its eventual elimination or eradication.
It is well-known that plants have been and are still the mainstay of traditional medicine against malaria and other diseases in resource-limited settings as over one-third of the population in such countries lack access to essential medicines [
5]. However, the claimed potency of medicinal plants has to be scientifically evaluated and toxicological studies should be done. Rigorous
in vitro and
in vivo toxicological investigations are required to determine the type and degree of toxicity, safety and efficacy of plant products in malaria drug research and ultimate discovery as well as to recommend or discourage a plants’ traditional medicinal use.
To this end, various studies have been conducted to evaluate the safety and anti-malarial efficacy of traditionally used plants in Asia and Africa. For example, Ramazani
et al. [
6] worked on ten Iranian plant species but only -
Boerhavia elegans,
Solanum surattense and
Prosopis juliflora – showed a promising anti-plasmodial activity
in vitro and
in vivo with no toxicity. Verma
et al. [
7] reported that
Holarrhena antidysenterica and
Viola canescens exhibited
in vitro anti-plasmodial activity in Himalaya. From sub-Saharan Africa in Burkina Faso [
8], promising
in vitro anti-plasmodial results were obtained for the plant
Dicoma tomentosa with the dichloromethane, diethylether, ethylacetate and methanol extracts demonstrating a high activity. In the same study hot water and hydroethanolic extracts also showed a good activity, which was also confirmed
in vivo for all tested extracts.
Despite their wide use in the traditional healthcare, the work that has been done to evaluate the safety and efficacy of Ethiopian traditional medicinal plants is relatively less extensive. A number of traditional medicinal plants remain unevaluated.
Osyris quadripartita which is commonly used in Ethiopian traditional medicinal practices [
9‐
12] and elsewhere in Africa [
13] is a case in point. Specifically, the leaf of this plant is used to traditionally treat malaria [
14,
15] in Ethiopia and cancer in Algeria [
16]. The anti-inflammatory property of a methanol extract of
O. quadripartita was demonstrated in effectively reducing increased capillary permeability induced in rats by various chemical mediators [
17]. The antioxidant effect of this plant species has also been evaluated and was found to be good [
18]. The plant has anti-bacterial and anti-fungal activities [
19]. According to this same work, the plant was active against Gram-negative bacteria such as
Escherichia coli and
Pseudomonas aeruginosa as well as Gram-positives like
Staphylococcus aureus. Though its effect was relatively lesser compared to other parallel test extracts the plant crude extract had a comparable efficacy to that of the positive control (gentamicin sulphate). The anti-bacterial and anti-fungal effects of
O. quadripartita were confirmed by a latter more comprehensive study [
20]. A different species within the genus,
Osyris alba, which was tested against
Entameba histolytica,
Giardia intestinalis and
Candida albicans has shown a reasonable effect [
21].
Phytochemical studies on the genus
Osyris has yielded hexyl and hexenyl derivatives, sesquiterpenes, phenolic acids, flavoinoids, pyrrolizidine and quinolizidine alkaloids, long chain hydrocarbons and fattyacids, triterpenes, dihydro-β-agarofuran sesquiterpenes, phenolics phenyl propanoids and lignans [
17,
21‐
26]. However, the possible anti-plasmodial effect of the genus was not tested
in vivo and
in vitro either. The aim of this study was, therefore, to assess the anti-plasmodial activity and safety threshold of
O. quadripartita in Swiss albino mice in an attempt to contribute towards screening traditional medicinal plants for malaria control.
Discussion
While methanol yielded significantly higher extraction efficiency, the chloroform extract was the lowest. The probable reason for this variation could be due to high concentration of polar compounds in the leaf of the plant species that better dissolve in methanol which is a polar solvent. The quality and quantity of phytochemicals extracted from plant materials differ depending on, among other factors, the solvent type used. The comparative ability of extraction solvents of penetrating the cellular membrane to extract the intracellular ingredients from the plant material may impact extract yield. Some reports show that methanol extracts more number and types of compounds in plant materials than other extraction solvents such as aceton, chloroform, ether, water and ethanol [
41]. While chloroform is preferred to extract terpenoids and flavonoids water is employed to solubilize a wide range of plant metabolites like anthocyanins, starches, tannins, saponins, terpenoids, polypeptides and lectins. Methanol releases more diverse phytochemicals such as anthocyanins, tannins, saponins, terpenoids, xanthoxylines, totarol, quassinoids, lectones, flavones, phenols and polyphenols [
42].
The finding that the 3 % T-80, which was used as a NC (for the chloroform extract) in the present study and in which the extract was dissolved to deliver to the mice, neither suppressed malaria parasitaemia in the infected mice nor prevented weight loss and PCV depletion indicated lack of possible T-related effect on the malaria parasites.
Oral administration of the methanol, aqueous and chloroform extracts of
O. quadripartita did not show changes in the general appearance or behavioral pattern of the experimental mice until the end of 14 days. Further, no death was observed in the animals receiving the extract up to a dose of 2000 mg/kg BW, which is about 10 times the minimum effective dose tested (200 mg/kg). If a test substance has a lethal dose (LD
50) higher than 3 times the minimum effective dose, it can be a good candidate for further studies [
43]. Therefore, absence of mortality up to an oral dose of 2000 mg/kg could indicate that the test extracts were safe and this could explain the routine use of the plant by the local people for traditional management of malaria.
Significant BW increase among
P. berghei-infected mice four days after ingesting 400 and 600 mg chloroform crude leaf extract of
O. quadripartita compared to the untreated controls suggests the effect of the extract in preventing malaria-related weight loss. It is well-established that BW loss is one feature of murine malaria [
44]. The present result is in agreement with other similar studies that reported mice BW loss using different plant products and extraction solvents [
45‐
47]. Lack of significant reduction in the mean BW among aqueous and methanol extract-fed infected mice four days post-treatment further signifies the positive effect of the plant material on mice BW.
The absence of significant PCV reduction among extract-treated mice at the doses of 200 and 400 mg/kg of the aqueous extract may indicate the protective activity of the crude extract. Furthermore, observing a significantly lower PCV reduction among the same groups of mice at the highest dose (600 mg/kg) shows the presence of anti-malarial chemicals in the dose administered. But it appears that the activity of the methanol extract was not strong enough to significantly prevent PCV reduction among P. berghei-infected mice. On the contrary, the PCV of chloroform extract-treated mice remained not significantly changed on D4, irrespective of dose, underlining the protective role of the extract against malaria.
The influence of malaria on hematological parameters is extensively investigated and PCV reduction is considered a hallmark of both human and rodent malaria (reviewed in Lamikanra
et al. [
48]. Infected mice may suffer from severe malarial anemia because of rapid erythrocyte destruction, either by parasitaemia and/or spleen reticulo-endothelial cells. For instance, in one study it was noted that within an estimated 48 hrs of post-infection rodent PCV was depleted to 43-44 % [
49]. Further,
P. berghei increased erythrocyte fragility and led to subsequent reduction of PCV in infected-mice [
50].
Multiple other
in vivo studies on rodent malaria using diverse plant species from Ethiopia as well as abroad have reported similar results [
6,
45‐
47,
51,
52]. Scarcity of previous reports pertaining to anti-malarial activity of
O. quadripartita including the relative composition and predominance of its leaf chemicals could not permit a discussion from comparative perspective. Although the antimicrobial [
19,
20] and antiparasitic activities [
21] of
O. quadripartita were investigated to some extent, its plasmodial effect was little explored.
The 4-day suppressive test is a standard test commonly used for
in vivo anti-malarial phytochemical screening in which ≥30 % reduction in parasitaemia following treatment makes a product to be considered active [
36,
43]. Accordingly, the chloroform extract of
O. quadripartita which showed 31.7 % suppression at the lowest, 37.5 % at the medium and 41.26 % at the highest doses can be classified as active. The dose-dependent variation in chemosuppression could be attributed to the low concentration of schizocidal compounds in natural products and as such their activity may be undetectable in lower doses. This increased percent suppression of parasitaemia with increased dose was observed by other studies on different plant species [
45,
47,
53].
However, both the aqueous and methanol extract of O. quadripartita didn’t display comparable suppressive activity on P. berghei even at the highest dose delivered (600 mg/kg). This at least undetectable level of anti-malarial activity of aqueous and methanol crude leaf extract of O. quadripartita may be an indication that the active ingredients extracted by these solvents might have less potent anti-malarial property. This can be explained by the fact that some plants may contain chemicals that are more soluble in polar solvents such as water, ethanol and methanol while others contain chemicals that are more soluble in non-polar solvents such as chloroform. Thus, the crude aqueous or methanol extracts in the current study were higher in terms of yield but they contained little compounds that were efficacious at least against P. berghei.
A prolonged MST with significant difference, compared to the NC, was observed for mice treated with all the three extracts regardless of dose except for 200 mg/kg of the aqueous extract implying the role of the plant material in the control of murine malaria. Particularly, the chloroform extract was highly associated with prolonged MST even at the minimum dose implicating the dominant presence of antimalarial bioactive compounds of the plant tissue in this extract.
Nevertheless, the chloroform extract itself was less effective compared to CQ, the standard drug against P. berghei. CQ treatment (25 mg/kg) during the infection seemed radically cleared parasitaemia or at least there was no microscopically detectable parasitaemia. Rodent malaria clinical manifestation like diarrhea, lethargy, piloerection, reduced locomotor activity, etc. were non-existent among the CQ-treated showing that the parasitological cure was clinical as well. Mice were appearing and acting healthy on day 28. The undetectable level clearance of parasitaemia following the CQ chemotherapy indicates that the P. berghei strain used in the study was highly sensitive to the drug and lends support that this rodent malaria model system remains effective for in vivo anti-malarial testing.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SG, MG, BE, HM conceived the research idea and participated in the design of the study. SG collected the experimental plant and performed the laboratory work, acquired and analyzed the data. HM drafted the manuscript. MG and BE revised the manuscript. All authors have read and approved the final version of the manuscript.