Background
Malaria has been a great challenge to humanity since time immemorial. An estimated 300–500 million people are affected by malaria throughout the world annually [
1]. This same world health organization (WHO) source indicates that 95 % of malaria-related deaths occur in sub-Saharan Africa, with children younger than five years of age and pregnant women being the most severely affected. In recent years, the burden of malaria has fallen in many parts of Africa following the introduction of more effective treatments and the scale-up of long-lasting insecticidal nets (LLIN) use [
2]. But, vector mosquito resistance to insecticides recommended for current use [
3] coupled with emergence of drug-resistant parasite strains to the latest antimalarials in use [
4‐
6] demand for new drugs with new modes of action.
Plants have been used in the traditional treatment of malaria for years in the past and present in various parts of the world [
7] apart from being source of various antimalarial drugs [
8]. In Ethiopia it is estimated that about 90 % of the population is dependent on traditional medicine, essentially plants [
9].
Ocimum lamiifloium Hochst. ex Benth and
Brucea antidysenterica Mill are among the most common traditional medicinal plants used in Ethiopia.
O. lamiifolium belongs to the family Lamiaceae. It is traditionally used to relieve pain, wound, fever, malaria and inflammatory disorders in Ethiopia [
10]. It is also used for treatment of intestinal disorders, eye disease and cough [
11].
Brucea antidysenterica (Simaroubaceae) is similarly used in traditional medicine for multiple purposes. Different parts of the plant are widely used against malaria, helminthic infections, fever, dysentery and other disorders [
11,
12]. Despite its wide application in the traditional healthcare domain only few organized and thorough scientific investigations have been undertaken to evaluate the safety and efficacy of Ethiopian traditional medicinal plants. In vivo and in vitro studies conducted on fruit extracts of
Brucea javanica, a species closely related to
Brucea antidysenterica, demonstrated antiplasmodial activity of the plant attributable to quassinoid constituents [
13]. Study also shows that the organic leaf extract of
Ocimum gratissimum, a species found under the genus where
Ocimum lamiifolium belongs, suppressed parasitaemia of
Plasmodium berghei in mice by 88.07 % at a dose of 100 mg/Kg body weight [
14]. Essential oil of
Ocimum gratissimum demonstrated significant antimalarial activities in the four-day suppressive in vivo test in mice [
15]. In vitro test conducted on three quinones isolated from
Ocimum basilicum, another related species, showed antiplamodial activity with an IC50 value of below 1 μg/ml [
16]. The present study, therefore, aimed at assessing the antimalarial activity of the crude extracts of the seed of
B. antidysenterica and leaf of
O. lamifolium against
P. berghei in Swiss albino mice.
Results
For seeds of Brucea antidysenterica, the highest yield was obtained for their methanol extract (8.69 g), followed by that for aqueous (5.64 g) and chloroform (4.79 g) extracts. For leaves of Ocimum lamiifolium, the highest yield was obtained for their methanol extract (7.79 g), followed by that for aqueous (6.20 g) and chloroform (5.21 g) extracts. However, there was no significant difference (p > 0.05) in yield among the different extracts.
In vivo acute toxicity test
The methanol extracts of the leaves of O. lamiifolium administered orally in a single dose of up to 2000 mg/kg body weight, showed no lethal effect within 24 h of observation. Gross physical and behavioral observation of the experimental mice revealed no visible signs of acute toxicity such as urination, hair erection, lacrimation, and reduction in feeding activity. In general, the mice were physically active. However, in case of the aqueous, chloroform and methanol extracts of the B. antidysenterica extract, lack of visible signs of acute toxicity and mice fatality were observed in the dose of 500 mg/kg body weight. At the level of 1000 mg/kg body weight, hair erection and sleepy activities were observed within 4 h although the mice became active from that time onwards. But, in the dose of 2000 mg/kg body weight all mice were dead within 24 h.
In vivo evaluation of the antimalarial activity of plant extracts
The result showed a significant (
p < 0.05) reduction in packed cell volume (PCV) values between D0 (inoculation day) and D4 (5 days after D0) in mice treated with chloroform and methnol extracts of the leaves of
O. lamiifolium at all the three dose levels (200 mg/kg, 400 mg/kg for chloroform and 600 mg/kg). Whereas, mice treated with aqueous extract did not show significant (
p > 0.05) PCV reduction at all the three dose levels. There was significant (
P < 0.05) change between D0 and D4 in body weight of mice treated with chloroform extracts at the three dose levels. The aqueous and methanol extracts treated mice had shown no significant (
P > 0.05) deviation in body weight between D4 and D0 (Table
1).
Table 1
Effect of aqueous, methanol and chloroform crude extracts of leaves Ocimum lamiifolium on body weight and PCV of P. berghei infected mice
NC | 0 | 52.80 ± 0.48 | 40.58 ± 0.64b
| 27.90 ± 0.64 | 23.98 ± 1.46 |
dH2O | 200 | 53.59 ± 2.88 | 44.75 ± 0. 96a
| 27.76 ± 0.20 | 26.88 ± 0.25a
|
| 400 | 55.09 ± 1.01 | 47.49 ± 2.22a
| 28.12 ± 0.13 | 29.56 ± 0.32a
|
| 600 | 51.98 ± 0.99 | 50.38 ± 0.52a
| 26.88 ± 0.15 | 27.20 ± 0.55a
|
CH3OH | 200 | 54.66 ± 1.15 | 42.67 ± 0.67b
| 26.50 ± 1.41 | 26.29 ± 2.53a
|
| 400 | 51.69 ± 1.11 | 40.13 ± 1.84b
| 27.56 ± 1.41 | 26.22 ± 1.40a
|
| 600 | 53.98 ± 0.99 | 56.09 ± 0.44b
| 29.10 ± 2.58 | 30.09 ± 2.16a
|
CHCl3
| 200 | 56.94 ± 0.50 | 46.36 ± 0.6b
| 29.38 ± 2.39 | 24.36 ± 1.44b
|
| 400 | 54.14 ± 1.56 | 42.30 ± 0.87b
| 33.70 ± 8.76 | 28.23 ± 6.90b
|
| 600 | 51.68 ± 0.74 | 43.23 ± 0.66b
| 28.66 ± 2.55 | 25.00 ± 0.51b
|
CQ | 25 | 54.73 ± 0.27 | 53.37 ± 0.21a
| 28.80 ± 0.23 | 30.97 ± 0.92b
|
Extracts of the seeds of
B. antidysenterica have significantly (
P < 0.05) prevented loss of body weight between D0 and D4 in mice treated with methanol extract at all the three dose levels (200 mg/kg, 400 mg/kg and 600 mg/kg) Mice treated with chloroform extract significantly (
p < 0.05) lost body weight only at two dose levels (200 mg/kg, 400 mg/kg) and those treated with aqueous extract demonstrated significant (
P < 0.05) body weight loss only at the dose level of 600 mg/kg. Significant (
P < 0.05) reduction in PCV was observed at all the three dose levels in mice treated with aqueous extract. Mice treated with methanol extract showed significant (
P < 0.05) reduction in PCV only at the dose level of 600 mg/kg. However, no significant (
P > 0.05) loss in PCV was shown between D0 and D4 in mice treated with chloroform extract at all the three dose levels (Table
2).
Table 2
Effect of aqueous, methanol and chloroform crude extracts of seeds of Brucea antidysentmerica on body weight and PCV of P. berghei infected mice
NC | | 53.37 ± 0.75 | 46.67 ± 1.79b
| 28.70 ± 3.24 | 27.60 ± 3.88 |
dH2O | 200 | 56.99 ± 2.02 | 45.10 ± 0.65b
| 32.25 ± 1.22 | 29.75 ± 0.09a
|
| 400 | 56.84 ± 2.83 | 43.53 ± 5.08b
| 31.50 ± 0.79 | 30.24 ± 0.52a
|
| 600 | 55.74 ± 1.00 | 48.62 ± 1.93b
| 25.80 ± 0.89 | 31.10 ± 1.37b
|
CH3OH | 200 | 50.78 ± 0.96 | 51.29 ± 0.29a
| 28.56 ± 1.41 | 31.22 ± 1.40b
|
| 400 | 53.19 ± 1.09 | 53.26 ± 2.20a
| 34.10 ± 2.58 | 36.09 ± 2.16b
|
| 600 | 56.62 ± 1.90 | 50.40 ± 3.55b
| 31.50 ± 2.30 | 33.35 ± 1.86b
|
CHCl3
| 200 | 55.67 ± 1.38 | 52.89 ± 1.30a
| 30.95 ± 2.25 | 32.36 ± 0.28b
|
| 400 | 52.09 ± 2.83 | 53.09 ± 0.95a
| 29.6 ± 1.83 | 31.25 ± 0.44b
|
| 600 | 51.8 ± 1.59 | 51.27 ± 1.95a
| 29.5 ± 1.41 | 29.39 ± 0.5a
|
CQ | 25 | 52.27 ± 0.74 | 51.82 ± 0.82a
| 30.54 ± 0.37 | 33 ± 0.37b
|
In the four day suppressive test, all the extracts resulted in reduction of parasitaemia level significantly (P < 0.05) as compared to the negative control for both plants. However, the extracts did not clear the parasite completely on D4 as opposed to the positive control group (treated with 25 mg/kg CQ) which had no detectable parasitaemia on the same post infection day.
The crude aqueous, methanol and chloroform extracts of
O. lamiifolium significantly suppressed the parasitaemia. The highest suppression was obtained for methanol (24.95, 24.57 and 26.06 %) and aqueous (22.16, 26.76 and 35.53 %) extracts treated mice at the dose levels of 200 mg/kg, 400 mg/kg and 600 mg/kg, respectively. The extracts also prolonged the mean survival time of infected mice compared to the non-treated ones (Table
3).
Table 3
Antimalarial activities of aqueous, methanol and chloroform extracts of leaves of O. lamiifolium and mean survival time of P. berghie infected mice
dH2O | 200 | 35.68 ± 1.42 | 22.16 | 10.95 ± 1.04*
|
400 | 33.57 ± 2.81 | 26.76 | 11.65 ± 0.65*
|
600 | 29.55 ± 0.89 | 35.53 | 12.50 ± 0.41*
|
CH3OH | 200 | 33.83 ± 3.43 | 24.95 | 10.00 ± 1.70*
|
400 | 34.00 ± 5.92 | 24.57 | 12.80 ± 0.76*
|
600 | 33.33 ± 11.69 | 26.06 | 11.00 ± 0.80*
|
CHCL3
| 200 | 38.00 ± 3.88 | 15.70 | 9.40 ± .67*
|
400 | 36.42 ± 2.01 | 19.21 | 9.80 ± 0.67*
|
600 | 35.75 ± 3.37 | 20.69 | 9.60 ± 1.51 |
Control | CQ (25 mg) | 0.00 ± 0.00 | 100.00 | ND |
T80 | 45.84 ± 2.11 | 0.00 | 7.00 ± 0.50 |
In the same way, the aqueous, methanol and chloroform extracts of
B. antdysenterica showed significant parasite suppression in all the doses administered and they exhibited significant suppression of the parasitaemia at a higher dose (600 mg/kg). The highest suppression was obtained for methanol (38.08, 43.33 and 46.44 %) and chloroform (31.23, 43.23 and 47.70 %) extracts treated mice at the dose levels of 200 mg/kg, 400 mg/kg and 600 mg/kg, respectively. The mean survival time (MST) of mice for a given extract was also longer compared to those in the negative control (Table
4).
Table 4
Antimalarial activities of aqueous, methanol and chloroform extracts of seeds of B. antidysenterica and mean survival time of P. berghie infected mice
dH2O | 200 | 39.69 ± 1.10 | 24.05 | 9.25 ± 0.69*
|
400 | 32.19 ± 0.62 | 38.40 | 10 ± 00.54*
|
600 | 28.39 ± 1.68 | 45.68 | 12.50 ± 0.41*
|
CH3OH | 200 | 34.36 ± 0.86 | 38.08 | 12.90 ± 1.04 |
400 | 28.57 ± 1.31 | 43.33 | 14.80 ± 1.20*
|
600 | 27.99 ± 0.89 | 46.44 | 15.00 ± 1.40*
|
CHCl3
| 200 | 35.94 ± 0.86 | 31.23 | 12.00 ± 1.70*
|
400 | 29.67 ± 1.31 | 43.23 | 13.00 ± 0.80*
|
600 | 27.33 ± 0.89 | 47.70 | 15.50 ± 0.41*
|
Controls | QC (25 mg) | 0.00 ± 0.00 | 100.00 | ND |
T80 | 52.26 ± 1.50 | 0.00 | 7.00 ± 0.50 |
Discussion
The methanol extracts of the two plants produced the highest yield and this could be due to high concentration of compounds in the plants that better dissolve in methanol. The highest yield of methanol extracts could also be attributed to relative ability of methanol to penetrate cellular membranes to extract ingredients in cells. The leaf extracts of
Ocimum lamiifolium did not cause mortality or any sign of behavioral change that is indicative of acute toxicity in the experimental mice after oral administration of extract (500 to 2000 mg/kg body weight). Therefore, the plant can be considered safe according to the Organization for Economic Cooperation and Development (OECD) guideline which recommends a maximum dose of 2000 mg/kg for acute toxicity [
19]. This could justify the common use of the plant in Ethiopian traditional medicine to treat malaria and other ailments.
In case of methanol extract of the seeds of Brucea antidysenterica, no visible signs of acute toxicity and death of the mice were observed at a dose of 500 mg/kg body weight while at the level of 1000 mg/kg body weight hair erection and sleepy activities were observed but no death was recorded. Meanwhile, at the dose of 2000 mg/kg body weight all mice were dead. This indicates that the lethal dose (LD50), a concentration of a chemical that kills 50 % of the test animals, of B. antidysenterica methanolic extract is less than 2000 mg/kg body weight.
All the three crude extracts (aqueous, methanol and chloroform) of B. antidysentrica and aqueous and methanol extracts of O. lamiifolium prevented weight loss. However, chloroform extract of O. lamiifilum did not prevent weight loss which could be attributed to the presence of compounds that have effect on appetite.
The PCV values of
P. berghei infected mice that were treated with the extracts of both plants were reduced as a result of malaria infection. The mean survival time of mice treated with all the extracts of
B. antidysenterica was longer compared to the respective negative control, confirming that the crude extracts of the study plant suppressed
P. berghei and probably reduced the overall pathogenic effect of the parasite in the study mice. However, mice treated with
O. lamiifolium extract lived shorter. This may be due to the suppressive effects of the plant. This suggests that the crude extracts of
B. antidysenterica were relatively more protective than that of
O. lamiifolium. It may be because of the relatively shorter half life of
O. lamiifolium [
23].
According to Krettli
et al. [
24] a compound is considered active when reduction in parasitaemia is ≥ 30 %. In this study, all test groups treated with methanol, aqueous and chloroform crude extracts of
B. antidesenterica induced more than 30 % suppression including the lower doses except the 200 mg/kg of the aqueous extract. Similar studies on other plant species such as
Acacia nilotica [
25],
Clerodendrum myricoides, Dodonea angustifolia and
Aloe debrana [
26] and
A. africanus [
27] reported significant parasitaemia suppression.
A remarkable suppression suggests the potency of the seed extracts of
B. antidyesenterica against malaria. The antimalarial activity of the plant might be attributed to the presence of bioactive compounds such as quassinoid and canthin alkaloids [
28]. However, the active compound(s) responsible for the observed activity need to be identified in future studies.
On the other hand, the lower doses (200 mg/kg and 400 mg/kg) of the methanol and aqueous extracts of
O. lamiifolium may be considered to have lower antimalarial activities. This could be due to the presence of active compounds at low levels in natural products whose activity may not be detected at lower doses [
24].
The difference between the antimalarial activities of extracts of both plants in all the three solvents may indicate differences in the level and composition of active compounds [
29]. The difference may also be due to differences between the solvents used for the extraction of the plant materials as the secondary metabolites/compounds could be different.
The observed low antimalarial activity of plant extracts tested could be partly explained by the fact that many antimalarial traditional medicinal plants may lack direct antiplasmodial activity to cure the disease but their beneficial role could be in their antipyretic, analgestic and immune stimulatory effect as demonstrated in other studies [
7,
10]. Furthermore, in traditional medicinal practice, the healers are known to prescribe concoctions made from different species of plants that might have high synergetic effects. According to Abdu
et al. [
30], biological actions can be due to these components in complicated concert of synergistic or antagonistic activities. Also, the effectiveness of a given extract can also be influenced by the rate of gastrointestinal uptake and the half life in plasma metabolism of the active compounds [
23].
The extracts of the two plant parts did not totally clear the parasitaemia up to the highest tested doses. Complete clearance of parasitaemia may be achieved within the toxic range of the extracts. These findings provide information on the antimalarial effect of the plant extracts tested and warrant carrying out of chronic toxicity studies.
The limitation of this study includes failure for not conducting additional antimalarial tests using curative and prophylactic methods.
Conclusion
This study has shown that aqueous, methanol and chloroform extracts of seeds of B. antiydesenterica and leaves O. lamiifolium exhibited a reasonable antiplasmodial activity. While O. lamiifolium was safe at the highest tested dose of the extracts, B. antidysenterica showed toxicity at 2000 mg/kg body weight. Further studies are recommended on antiplasmodial activities as well as safety profiles of the plants.
Ethical consideration
The study obtained ethical clearance from the Institutional Ethics Review Board of College of Natural Sciences, Addis Ababa University (No: GSR/1559/05). The mice were treated as per the guidelines on the care and use of animals for scientific purposes.
Availability of data and materials
Specimens of the two tested antimalarial plants were deposited at the National Herbarium of the Addis Ababa University with voucher numbers AK01 assigned for Ocimum lamiifolium and AK02 for Brucea antidysenterica. Data related to antimalarial in vivo tests of the two plants were deposited into a computer available at Aklilu Lemma Institute of Pathobiology, Addis Ababa University.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MG and BE conceived the research idea. AK conducted the experiment, collected the plant specimens, analyzed and interpreted the data as well as prepared the first draft. MG identified the plants. MG, BE, HM critically read and revised the paper. All authors read and approved the paper before its final submission.