Background
Malaria is one of the major infectious diseases responsible for the high rate of mortality and morbidity in developing countries. It is caused by Apicomplexan protozoan parasites of the genus
Plasmodium [
1]. The disease burden is very alarming in the sub-Saharan countries and mainly affects children less than five years of age, which accounts for about 78% of all global deaths [
2]. Malaria also causes series adverse effects during pregnancy for both mother and infant, such as maternal anaemia, low birth weight, and increased perinatal and infant mortality [
3]. This infectious disease is highly fatal unless diagnosed and treated in a timely fashion. It may also predisposes the patient to opportunistic infections [
4].
Ajuga remota belongs to a genus of about 40–50 species of annual and perennial herbaceous flowering plants in the Mint (
Lamiaceae) family [
5,
6]. The aqueous or alcohol extract of fresh or dried leaves of
A. remota have been traditionally used for the treatment of diabetes, malaria, pain and fevers, toothache, skin disease, hypertension, stomach ache, pneumonia, liver problem and swelling of legs [
7,
8]. Various compounds have been isolated from the leaves of
Ajuga remota, including five different neo-clerodane diterpenes [
8,
9]. Sterols (ajugalactone), triterpenoid (ergosterol-5, 8-endoperoxide and iridoid glycoside (8-O-acetylharpagide) were also isolated from the plant, as well as phenolic acids, flavonoids and saponins [
8,
10]. The plant has wide traditional medicinal applications, and the present study was designed to confirm its anti-malarial efficacy and safety of its use.
Discussion
Ajuga remota is widely applied in the folkloric medicine of Ethiopia for treating various types of disorders [
17]. Plant medicines has been utilized by majority of the world population for primary health care [
18]. However, lack of validated information has been a major concern with respect to the use of plant medicines [
19].
The results of the acute toxicity of
A. remota revealed that there was no mortality observed up to the maximum dose level of 2000 mg/kg body weight of the extract administered orally, where single high dose is recommended for testing acute toxicity [
12]. No behavioural changes were observed either viz apathy and reduced locomotors activity. Accordingly, 2000 mg/kg of each plant extract was found safe,
i.e., the approximate median lethal dose (LD
50) of the extracts in the experimental mice was higher than 2000 mg/kg. The results observed in the acute toxicity study with
A. remota is in agreement with those of the previous study [
20]. The reduction in body weight gain is a simple and sensitive index of toxicity after exposure to toxic substances [
21]. In sub-acute toxicity study, all the groups treated with 500, 750 and 1000 mg/kg doses of the crude extract did not show significant change in weight on day 4 as compared to day 0 except the group received 500 mg/kg of the extract which showed significant (P < 0.05) body weight gain on day 4. This observation indicates that the crude extracts may not contain appetite stimulant compounds [
22]. Study on haematological parameters is important to discriminate the toxic effects of exogenous compounds on animals [
23]. In this study, PCV, an index of anaemia, did not show significant difference on day 4 as compared to day 0 of either treated or untreated mice. This might indicate the safety nature of the extract.
In vivo models are usually applied in antimalarial studies for they allow the possible prodrug effect and likely boosting of the immune system in eradication of the infectious agent [
24]. In the four-day parasitaemia suppression study, treatment of mice with crude extract of
A. remota reduced the erythrocytic stage development of
P. berghei. Parasitaemia was suppressed in a dose dependent manner indicating that the plant has antimalarial activity. Similar results were obtained in studies reported from the same species of
A. remota [
25]. The parasite suppressive effect of plant extract might be through indirect boosting of the immune system or by inhibition of other target pathways which are not fully realized [
26]. Likewise, the parasite inhibitory effect of the extract with unknown compounds might be attributed to the antiplasmodial activity of specific compound or group of compounds [
27].
Ajuga remota possesses compounds such as, terpenoids, sterols, flavonoids and saponins that might be responsible for its antiplasmodial activity [
8‐
10].
The mean survival time is important to evaluate the anti-malarial activity of plant extracts [
28]. The extract prolonged survival time of mice at all dose levels which is associated with suppression of parasitaemia. Haematological abnormalities like anaemia, body weight loss and temperature reductions are common characteristics of
P. berghei infected mice [
29]. Plants with anti-malarial activity are expected to prevent body weight loss in infected mice resulting from rise in parasitaemia. The crude extract of
A. remota significantly prevented body weight loss in a dose dependent manner on day 4 as compared to day 0.
Plasmodium berghei infected mice showed decrease in metabolic rate before death which as a result caused drop in internal body temperature [
30]. Thus, plant extracts with anti-malarial activity must prevent rapid falling of body temperature [
29]. Crude extract of
A. remota prevented body temperature drop at 50 and 100 mg/kg doses on day 4 as compared to day 0.
Rodent malaria causes parasite-induced fall of PCV, which occurred approximately 48 h post-infection [
29].
Plasmodium berghei infected mice suffer from anaemia because of erythrocyte destruction, either by parasite multiplication or by spleen reticuloendotelial cell action as the presence of many abnormal erythrocytes stimulates the spleen to produce many phagocytes [
22]. All of these mechanisms are accountable to malaria induced anaemia both in mice and men [
31]. It was noted that crude extract of
A. remota prevented reduction in PCV at all dose levels in between day 0 and day 4. Though
A. remota contains saponins [
8,
10] responsible for haemolytic effects, the sub-acute toxicity study done in healthy non-infected mice confirmed that the crude extract did not cause haematological abnormalities (PCV reduction). PCV reduction and body weight loss preventive effect of the hydroethanolic crude extract of
A. remota in infected mice is consistent with the sub-acute toxicity study carried out in healthy non-infected mice. This confirms that the plant extract has no impact on the measured parameters. The preventive effect of haematological abnormalities (PCV reduction), body weights lose and rectal temperature drop by the crude extract of
A. remota infer its antimalarial activity.
In the curative test, crude extract of
A. remota did not eradicate parasites completely on day 7. However, it showed parasite suppressive effects. Complicated syndromes of malaria comprise of many inflammatory mediators which may enhance cell to cell interaction (cytoadherence), cell stimulation through malaria-derived antigens and host-derived factors like cytokines. Cytokines are also responsible to cause fever in the host [
32]. The curative antiplasmodial properties of
A. remota may be due to the inhibition of the production and/or release of these inflammatory mediators associated with malaria. Surprisingly,
A. remota is traditionally used for its pain and fever reduction ability [
7,
8,
33].
In the prophylactic assay,
A. remota resulted in better efficacy than the negative control on day 8. The probable mechanism to produce prophylactic activity on
P. berghei infection might be inhibiting proliferation of parasites due to direct cytotoxic effect [
34] and modulation of the membrane of the erythrocytes preventing parasite invasion [
35].
In vivo antiplasmodial activity can be classified as moderate, good, and very good if an extract displayed percentage parasitaemia suppression equal to or greater than 50% at a dose of 500, 250 and 100 mg/kg body weight per day, respectively [
36]. Based on this classification, the crude extract of
A. remota showed very good antiplasmodial activity below 100 mg/kg/day dose level. Similarly, four day suppressive in vivo evaluation of the wet and dried leaf aqueous extracts of
A. remota showed 90.35 and 82.82% suppression of parasitaemia, respectively [
25].