Background
Half of the world population is at risk of malaria [
1], the most common, and severe parasitic mosquito-borne disease [
2]. Moreover, malaria claims more lives in Africa than in any other continent, as more than 90% of worldwide malaria related deaths occur in this region, which makes it the second cause of death after HIV/AIDS [
3].
No single antimalarial drug is effective against all liver and intra-erythrocytic forms of the parasite, which could co-exist in the same patient. As a result, complete elimination of the parasite infection may require more than one drug during treatment of an established infection [
4]. Besides, efforts to develop an effective blood stage vaccine have not met with much success primarily because of antigenic diversity and a poor understanding of protective host immune responses [
5,
6].
The genomic plasticity of the mosquito and the plasmodium parasite has added another dimension for the problem through increasing resistance to drugs, demanding an investment in research and development of newer agents for malaria control [
6]. Hence, traditional medicinal plants could be considered as an alternative source of new drugs, as some antimalarial drugs (quinine, artemisinin) in use today were of plant origin [
7].
Olea europaea Linn (family: Oleaceae) is an extensively used plant for different kind of ailments in traditional medicine of various countries. Its bark, fruits, leaves, wood, seeds, and oil are used in different forms, alone or sometimes in combination with other herbs [
8‐
10]. In east Africa (including Ethiopia), root, bark and leaf extracts are used to treat malaria and other infections [
11‐
13]. In vitro studies also reported that the dichloromethane/methanol (1:1) leaf extract of
O. europaea possessed anti-plasmodial activity on
P. falciparum with an IC50 of 12 μg/ml [
14]. Moreover, extracts from different parts of the plant have been reported to exhibit various pharmacological activities, including antidiabetic [
15], anticancer [
16], antioxidant and antimicrobial [
17], antihypertensive [
18], antiviral [
19] and anti-inflammatory [
18].
Although ethno botanical and in vitro studies [
11,
12,
14] showed that
O. europaea has anti-malarial activity, no report is available in the literature whether the plant also possesses in vivo activity. This study therefore was initiated to investigate the anti-plasmodial activity of the plant in rodent model of malaria and as well as to further ascertain in which fraction (s) the constituents responsible for anti-malarial activity are concentrated so as to provide a clue about the nature of the phytochemical constituents responsible for its action.
Discussion
The 80ME of the plant was investigated for its antimalarial effect using three models. Peter’s 4-day suppressive test was used to evaluate schizontocidal activity, during early infection while the Rane’s test was used to study curative ability during established infection and the repository test was used to study the prophylactic activity of the plant. In all methods, determinations of percent inhibition of parasitemia and survival time were the most reliable parameters [
29] and compounds are considered active when reduction in parasitemia is ≥30% [
30]. Accordingly, all the test doses of the crude extract as well as the chloroform and n-butanol fractions presumed to be active in the four-day suppressive test.
In the 4-day suppressive activity, the crude extract inhibited the level of parasitemia in a dose dependent manner, confirming the potential schizontocidal activity of the plant extract in early infection whereby the primary attack due to malaria can be prevented or mitigated [
21]. Along with it, all the three doses of the extract improved survival time of infected mice, indicating its parasite suppressive activity and thereby reduction of the overall pathogenic effect of the parasite on test groups [
31].
In the second model (Rane’s test), there was significant parasitemia suppression in all the doses used during the course of treatment, suggesting the curative potential of the extract. Justifying the probable rapid action of the extract [
32], the relative suppression of parasitemia in extract treated mice started after the first dose as compared to the negative control group (Fig.
1). The overall lower curative than suppressive effect could possibly be due to short duration of action of the constituent(s) to cover the exponentially growing parasites in established infection [
31]. This could be supported by the observation that oleuropein and its metabolite (hydroxytyrosol), chief constituents of the leaf of
O. europaea, are rapidly absorbed after oral administration with a maximum plasma concentration occurring 2 h after administration and rapidly distributed and excreted in urine [
33,
34]. This is in agreement with other studies where crude extracts had less effect on established infection than early infection [
35,
36].
In the prophylactic test, the extract produced the lowest percentage suppression of parasitemia as compared to its effect in the 4-day suppressive and curative tests; however, all the doses of the extract demonstrated significant suppressive effect on the level of parasitemia compared to negative control group. The lower chemo-suppressive effect of the crude extract in prophylactic test might have arisen from rapid metabolism that inactivates the active component of the extract responsible for antimalarial activity [
37]. Another possibility would be that the extract might have acted through metabolic activation of the immune system [
38] and hence parasite clearance could not be total. This finding is in agreement with other studies in which the chemo-suppressive effect of prophylactic test is lower than that of 4-day suppressive and curative effects [
39].
To further concentrate active principle responsible for the antimalarial activity, the powder of the 80ME was successively fractionated by solvents of differing polarity and their antimalarial activity were evaluated [
29].
The results revealed that the n-butanol fraction showed the highest parasitemia suppression followed by the chloroform fraction and then the aqueous fraction, with maximum percentage suppression about 51, 39 and 21 at their higher dose, respectively. Likewise, survival time was better prolonged in n-butanol and chloroform fractions than aqueous fraction which could be ascribed to the relative higher parasitemia clearance (reduced parasite burden) observed for these fractions [
40]. Besides, the highest and least chemo-suppressive effects of n-butanol and aqueous fractions, respectively, are in agreement with reports of other studies [
26,
41]. This indicates the difference in the type and concentration of the bioactive secondary metabolites in the fractions, the most active subgroups being localized in the n-butanol and chloroform fractions (Table
9).
The 4-day chemo-suppressive effect of the 80ME exhibited more suppression than the fractions. Besides, the crude extract treated mice displayed improved survival time than mice treated with the fractions. The reduction in activity of the crude extract upon fractionation could be explained by the loss of additive or synergistic action among the chemical compounds in the extract and/or less concentration of bioactive compounds in the fractions [
26]. This finding is in agreement with other studies in which the fractions reported to show less in vivo antimalarial activity than the crude extracts [
26,
42].
A potent antimalarial is expected to ameliorate anemia, prevent body weight loss, and stabilize temperature in infected mice with parasite [
43]. PCV was measured to determine the effectiveness of 80ME and solvent fractions of the leaf of
O. europaea in preventing malaria-induced hemolysis alongside its antimalarial activity. Accordingly, the crude extract (in all the three models) as well as the three fractions of the plant prevented the reduction in PCV of parasite infected mice when compared with their respective negative control groups, implying that the extract could avert anemia due to malaria infection which might be due to destruction (clearance) and/or sequestration of infected erythrocytes [
43].
Although the aqueous fraction displayed parasitemia reduction of < 30%, it showed significant protective effect on PCV. This could be probably due to the presence of phenols and other metabolites which have anti-oxidant and membrane protecting effects [
17,
44]. Hydroxyl groups of phenolic compounds displays acidic characteristics, which makes them excellent antioxidants due to the electron donating activity [
45]. The result of PCV, protective effect, in this study is concordant with the findings of Wannang et al [
46] and Saba et al [
47], however it is not in agreement with others [
25]. The inconsistency may be due to the absence of detectable concentration of phytodetergents like saponins which destroy cell membrane by prompting cholesterol liberation that results erythrocyte hemolysis and PCV reduction [
48].
The ability of the plant extract to prevent PCV reduction may be due to clearance of parasites from infected erythrocytes before hemolysis and/or by improving erythropoiesis [
49] through differentiation inducing effects or generation of RBCs of olive leaf compounds such as apigenin 7-glucoside and luteolin 7-glucoside on hematopoietic stem cells [
50]. Apart from this, decreased invasion and impaired intra-erythrocytic development of the parasites could be also responsible for the protective effect of RBC abnormalities [
51]. Moreover, malaria infection activates the immune system and thereby causing the release of free radicals and reactive oxygen species that results in degradation of haemoglobin and development of anemia [
52,
53]. Yet, the antioxidant properties of
O. europaea leaf extract [
17] especially polyphenolic compounds may protect RBCs from oxidative stress and help prolong the survival of both normal and infected RBCs during malaria infection.
In rodents, infection with parasites (increased parasitemia) results in decreased metabolic rates and severe hypothermia that could lead to death [
54]. However, the extract showed the temperature stabilizing effect in all cases of 80ME and the solvent fraction test groups, with the effect of n-butanol being the highest among the fractions. This may, in addition to parasite suppression, probably indicate that the extract controlled the immune system of infected mice as well as adjusted some pathological processes and offset the reduction in metabolic rate that caused drop in internal body temperature. The effect seen in the aqueous fraction, despite the low parasitemia suppression, indicates the presence of secondary metabolites that could stabilize body temperature in the presence of infection (parasite) [
55]. Moreover, ethno-botanical studies described that the plant is helpful in regulating body temperature in humans [
56]. Therefore, the plant has a promising effect in stabilization of body temperature for malaria infection together with its antimalarial effect.
The decrement of body weight in malaria has been associated with decreased food intake, disturbed metabolic function and hypoglycemia [
31]. Accordingly, prevention of body weight reduction was observed at the middle and higher doses of 80ME treated mice of all models. Besides, body weight loss was less in mice treated with n-butanol fraction followed by chloroform fraction, which could be explained by their relative parasitemia suppression effect. This protective effect is concordant with the findings from some studies [
57] and discordant with others [
55]. The inconsistency of the results might be due to variation in nutrient content and concentration of appetite suppressing components such as saponins and tannins which were not detected in this study, except tannins in aqueous fraction [
58]. The higher effect of crude extract on PCV, rectal temperature and body weight relative to fractions could result mainly due to its, besides to synergistic effect of constituents, higher parasitemia suppression effect [
42].
The results of phytochemical screening in both the crude and fractions indicated that the leaf of
O. europaea is rich in many secondary metabolites including polyphenols, flavonoids, terpenoids, steroids, alkaloids and glycosides. The result is in agreement with previous phytochemical studies done on this plant [
59,
60]. There are a number of anti-plasmodial secondary plant metabolites that have shown antimalarial activities belonging to the classes of alkaloids, terpenes, flavonoids, xanthones, anthraquinones, phenolic compounds, sesquiterpenes and other related compounds [
61,
62].
Phytochemicals found in 80ME and solvent fractions could have an individual or synergistic effect to exert their antimalarial activity through different proposed mechanisms. Thus, one way of the effect of the plant on
P. berghei infection may be due to inhibiting the growth and multiplication of the parasite [
63]. In view of that, flavonoids have been found to exert their effect by inhibition of the influx of L-glutamine and myoinositol into infected RBCs that are important for parasite growth [
64], while steroidal compounds were found to exert their antimalarial activity by changing the membrane of infected RBC and hence block entry of essential nutrients into the RBCs and thereby into the parasite [
65]. On the other hand, these phytochemicals could have exerted their action by cytotoxic effect on the parasites [
63]. Sesquiterpenes (like artemisinin) and alkaloids (like chloroquine) exert their antimalarial effect by formation of potentially toxic heme-adducts [
66,
67]. Similarly, polyphenols may also contribute to the antiplasmodial activity by inhibiting haem polymerization so that the unpolymerized haem is toxic for the parasite [
68]. Secondary metabolites may also modulate membrane properties of the erythrocytes, thereby preventing parasite invasion [
69]. Besides, phytochemicals like steroids, flavonoids and others may also exert their anti-malarial effects not only by directly affecting the pathogen, but also by indirectly stimulating natural and adaptive defense mechanisms of the host [
70,
71].
What is more, studies revealed that the leaf of
O.europaea possesses potent anti-inflammatory property [
56,
60]. The inflammatory condition of malaria is characterized by free radical generation, activation of phospholipase activity resulting in generation of eicosanoids (prostaglandins) and other cytokines such as tumor necrosis factor (TNF), interferon-
γ (IFN-
γ) and interlekun-1
β (IL-1
β), which up regulate expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) that is involved in the binding of the parasitized red blood cells to the vascular endothelium [
69]. Accordingly, the anti-inflammatory effect of the plant could possibly augment the reduction in overall pathogenic effect of the parasite, in addition to the aforementioned mechanisms, through inhibition of the production and/or release of cytokines.
Consequently, the observed antimalarial activity of the 80ME, in the three models, could be attributed to the presence of secondary metabolites like terpenoids, flavonoids, phenols, alkaloids and other compounds. Likewise, alkaloids, terpenoids, phenols, flavonoids and steroids detected in butanol and chloroform fractions could have contributed to their antimalarial activities, while the low chemo-suppressive effect observed in aqueous fraction might be attributed to a differential distribution of these secondary metabolites in this fraction. Therefore, based on the aforementioned observations it is plausible to assume that the leaf extract of O. europaea is a potential antimalarial agent, justifying the claimed use of the plant for malaria control.