Antiplasmodial activity of Vernonia adoensis aqueous, methanol and chloroform leaf extracts against chloroquine sensitive strain of Plasmodium berghei in vivo in mice

Objective The aim of this study was to investigate the antiplasmodial effects of the crude aqueous, methanol and chloroform extracts of the leaves of Vernonia adoensis in Plasmodium berghei infected Swiss albino mice using Peters’ 4-day suppressive test. Results The number of mice used for the toxicity test was 20 (5/group) and for each extract and control groups 5 mice per group was used. The aqueous, methanol and chloroform extracts of V. adoensis leaves indicated statistically significant (P < 0.05) suppression of parasitaemia in the treated mice. The highest inhibition was that of the methanol extract treated mice (83.36%) followed by aqueous (72.26%) and chloroform (54.34%) at an oral dose of 600 mg/kg b.wt. Each extract prevented body weight loss and packed cell volume (PCV) reduction as compared to the negative control groups. The survival time of the mice treated with chloroform based on Kaplan–Meir analysis was 12.53 ± 0.37 at 600 mg/kg b.wt, while the negative control was 7.93 ± 0.37 days. The LD50 of the extracts was greater than 3000 mg/kg body weight. In conclusion, the crude leaves extract of V. adoensis have demonstrated antiplasmodial effect in vivo. P. berghei infection is suppressed in a dose-dependent manner showing relevance of the traditional use of the plant. Electronic supplementary material The online version of this article (10.1186/s13104-018-3835-2) contains supplementary material, which is available to authorized users.


Introduction
Malaria is still a public health problem in many parts of the tropics. Plasomodium falciparum and Plasomodium vivax are the most fatal species [1][2][3][4][5]. Currently quinine and artemisinin are the two effective drugs obtained from two traditional medicinal plants. Quinine was obtained from the bark of the Cinchona tree [6,7] and artemisinin from the plant Artemisia annua [8,9]. However, in the recent years these drugs show some degree of resistance [10,11].
Vernonia adoensis (V. adoensis) is among the numerous traditionally used medicinal plants in Ethiopia. It is used for malaria, gastro-intestinal complaints, muscle spasm and for healing wounds [12][13][14][15]. In Tanzania a rootinfusion is taken for stomach-pain and as anti-tuberculosis and fresh roots sliced and cooked is taken with milk against gonorrhoea [12]. Previously the antimicrobial [9], antioxidant and antipyretic [14] property of V. adoensis is reported.
In this study the antiplasmodial effect of the leaf crude extract of V. adoensis against chloroquine sensitive P. berghei in Swiss albino mice is tested. and any plant material collection took place upon official authorization in accordance with country's laws and following international guidelines. The identification and authentication of the plant specimen was carried out by Professors Ensermu Kelbessa and Sebsebe Demissew at the National Herbarium of the Addis Ababa University (AAU). A voucher specimen number GZ 02/2012 of the plant sample was placed at the Herbarium.

Preparation of crude plant extracts
The preparation of crude extracts was done based on our previous published works [16,17]. In summary the collected plant leaves of V. adoensis were cleaned and air dried at room temperature under a shade in the Biomedical Science Laboratory of AAU. An electrical grinding mill (IEC, 158 VDE 0660, Germany) was used to grind the leaves. Sensitive digital weighing balance (AND: FX-320, Japan) was used to weigh the powdered plant material. Each extract was prepared in 1:10 ratio (w/v). Measurement of the percentage yield of each extract was done.

Experimental animals
We obtained the Swiss albino mice (25-34 g) from the Animal House of the College of Natural Sciences of AAU. Standard pellet diet and tap water ad libitum are fed to the mice housed in standard cages. All experiments were done three times and the tables represent the mean of the three experiments in each case. The Ethics Committee of the College of Natural Sciences of Addis Ababa University gave approval to run the experiment.

Toxicity test
Acute toxicity test (single dose exposure) of aqueous, methanol and chloroform extracts from the leaves of V. adoensis were evaluated in 3-h fasted Swiss albino mice through an oral administration of 2000, 2500 and 3000 mg/kg body weight of mice. The Organization for Economic Cooperation and Development (OECD) guidelines 425 procedure was followed to test the toxicity.

Evaluation of the antiplasmodial activity
In the in vivo evaluation of the antiplasmodial activity of the plant extracts was employed against chloroquine (CQ) sensitive P. berghei in mice using the standard 4-day suppressive method [18]. Gentle heart puncture from donor mouse and anesthetized with chloroform afforded 1 ml blood the rising parasitaemia being about 33%. Then, the 1 ml blood was diluted with 4 ml of physiological saline and thus, one ml blood contains about 5 × 10 7 infected red blood cells. The procedure we followed is previously given in Tekalegn et al. [16] and Zerihun et al. [17]. Twenty-five male mice were infected with P. berghei and randomly divided into five groups of five mice per group. Three test groups and two control groups (dH 2 O or 3% Tween 80 as a negative control CQ as positive control). Each mouse was inoculated on day 0, (D 0 ) (intraperitoneally) with 0.2 ml of infected blood having approximately 1 × 10 7 P. berghei parasitized red blood cells as standard inoculum. The different doses given were 200 mg/kg, 400 mg/kg and 600 mg/kg of body weight and 0.2 ml CQ at 25 mg/kg. Standard intragastric tube was used to give the extracts and the controls. Treatment was started after 3 h of infection on D 0 and then following four consecutive days in 24 h schedule.
On the 5th day Blood sample was collected from tail snip of each mouse on the 5th day. Thin smears preparation is as given previously [16,17]. The percentage parasitaemia and suppression was calculated as:

Determination of body weight and packed cell volume (PCV)
The body weight and PCV of each mouse in all the groups was recorded before infection and after infection. The average body weights were calculated as: Heparinized microhematocrit capillary tubes up to 3/4th of their length was used to collect blood from tail of each mouse for PCV measurement. The tubes were sealed by crystal seal and placed in the microhematocrit centrifuge (Microheamatocrit Centrifuge, 583298, Hawksley & Sons Ltd, England) with the sealed ends out wards. The sample was centrifuged at 12,000 rpm for 4 min. The volume of the total blood and the volume of red blood cells were measured and PCV was calculated as:

Determination of mean survival time
Mortality was observed daily and the number of days from the time of inoculation of the parasite up to death was recorded for each mouse in the treatment and Volume of erythrocytes in a given volume of blood Total blood volume × 100 control groups throughout the follow up period. The mean survival time (MST) for each group was calculated using the SPSS version 20 applying Kaplan-Meir statistical analysis.

Data analysis
Results of the study were expressed as mean ± standard error of the mean (M ± SEM). Data obtained from the parasitaemia, body weight, PCV and survival times were analyzed using Windows SPSS Version 15. The one-way analysis of variance (ANOVA) and paired-samples Student's t test were used to compare results among and within groups for differences between initial and final results. The result was considered statistically significant at 95% confidence level and P-value < 0.05.

Toxicity test
No death occurred in any of the groups and dose levels during the entire period of 24 h of observation. Furthermore, during the gross physical and behavioral observation of the experimental mice, for mice treated groups with the aqueous and methanol (99.5%) extracts at the dose of 3000 mg/kg showed some reduction in feeding activity, hair erection and rigidity after the administration of the doses. In general, the oral administrations of the aqueous, methanol and chloroform extracts of V. adoensis in each doses of 2000, 2500 and 3000 mg/kg did not produce any significant changes in usual behavior and also no mortality. Therefore, the LD50 is greater than 3000 mg/kg body weight.

Antiplasmodial activity
The results of this study indicated that the in vivo aqueous, methanol and chloroform leaf extracts of V. adoensis exhibited a potent activity against P. berghei malaria parasite ( Table 1). The Parasitaemia suppressive effects produced by all the test extracts were significant (P < 0.05) as compared with their respective negative control groups. The highest percent suppression at 600 mg/kg body weight was 83.36%. The mice treated with the standard drug (chloroquine 25 mg/kg) were completely free from the parasites in all the experiments using the aqueous, methanol and chloroform leaf extracts of V. adoensis (Table 1).

Body weight and packed cell volume (PCV)
The plant extracts prevented body weight loss and the mean body weight on D 0 and day 4 (D 4 ) did not show statistically significant difference (P > 0.05) except at lower dose level of 200 mg/kg in both aqueous and chloroform extracts. Therefore, the prevention of body weight reduction by each extracts was dose dependent. Body weight of the mice in the extract treated groups on D 4 is significantly higher (P < 0.05) than that of the mice in the negative control group (Table 2). Table 3 shows that the test extract doses of V. adoensis prevented PCV reduction due to parasitaemia infection. Each result showed that PCV in the respective negative control groups were significantly (P < 0.05) reduced in D 4 while in the extracts treated groups significant change was not observed (P > 0.05). Furthermore, the analysis of variance performed between the extract treated groups in comparison with the corresponding negative control groups showed highly significant variation.

Mean survival time
All the experimental mice treated with the different extracts had increased dose dependent mean survival days (e.g. the chloroform extract exhibited the maximum MST of 12.53 at 600 mg/kg b.wt) as compared to the respective negative control groups (the maximum MST was 7.93). But all the mice treated with CQ survived for 3 months (Additional file 1).

Discussion
All the test leaf extracts of aqueous, methanol and chloroform of V. adoensis have shown different degrees of parasitaemia inhibition in dose-related fashion. The highest suppression was recorded in the methanol extract    [7,19]. The parasitaemia inhibition could be attributed to the presence of these metabolites. Furthermore, the present result is in agreement with the result of previous in vivo study by Abosi and Raseroka [20] that reported suppressive effect of ethanolic extracts from the leaves and root bark of Vernonia amygdalina, where the leaf extract at 500 mg/kg resulted in 67% suppression of parasitaemia while the root-bark extract exerted 53.5% suppression at the same dose.
Similarly, a study carried out by Melariri and co-workers [21] showed a marked growth inhibition of parasites with values of 85% and 95% by the combination of dichloromethane extracts of leaves of Cymbopogon citrates and V. amygdalina at dose levels of 400 and 600 mg/kg b.wt against chloroquine sensitive strains of P. berghei in mice respectively. V. amygdalina leaves dichloromethane extract alone exerted 95.8% parasitaemia suppression at a higher dose of 800 mg/kg. In addition, the aqueous crude extract of the aerial part of V. ambigua had significant (P < 0.05) parasitaemia inhibition in a dose dependent suppression of parasite growth [10].
The statistical multiple comparison of the effect of each extracts on the body weight and PCV among groups on the 5th day of post-treatment and between D 0 and D 4 have shown that the two parameters to be within the normal range of values established for mice by Flecknell [15], adult body weight of 25-40 g and PCV of 32-54%. Therefore, the absence of any significant differences in the body weight and PCV parameters provides a support for the safety (non-toxic) of V. adoensis at all doses administered to the experimental mice. Previous studies on different species of Vernonia that also includes the present work have justified the potential of this genus as an antiplasmodial agent.
In conclusion, the crude leaves extract of V. adoensis have demonstrated antiplasmodial effect in Swiss albino mice. P. berghei infection is suppressed in a dose-dependent manner. The traditional use of the plant has thus some relevance based on this study.

Limitations
The study was done only on the leaves of the plant and also only on crude extracts. It will be good if sub-fractions of the different extracts are tried. In addition using other parts of the plant such as the roots and flowers would have been more useful to come up with strong conclusion.