MATERIALS AND METHODS

Plant material: The stem of Adansonia digitata (Bombacaceae) was collected from Ido-Ekiti, Ekiti State Nigeria. The plant was identified and authenticated by Mr. K.A. Adeniyi and Mr. L.T. Soyewo in the herbarium unit of Forest Research Institute of Nigeria (FRIN) with identification number (No. FHI 109806).

Extraction of plant material: The stem bark peels were air-dried at room temperature to avoid possible degradation or denaturation of their putative compounds. The air-dried stem bark of Adansonia digitata was blended to powder using an electric blender. This was stored in a glass container. Blended air-dried stem bark was soaked in sufficient volume of methanol for 72 h at room temperature. It was continually stirred after each 24 h. After 72 h, the mixture was then filtered and the filtrate was concentrated using rotary evaporator at 40°C. The concentrate was heated over a water bath to obtain a solvent free extract which was stored in a refrigerator at 4°C.

Experimental animals: Forty eight albino mice weighing between 18-20 g were obtained from the animal house, Institute of Advanced Medical Research and Training (IAMRAT), College of Medicine, University of Ibadan, Nigeria. The animals were acclimatized for two weeks in the animal house and fed ad libitum on rat chow and water throughout the period of the experiment.

Parasites: The Plasmodium berghei was obtained from the Institute of Advanced Medical Research and Training (IAMRAT), College of Medicine, University of Ibadan, Nigeria. A standard inoculum of 1×107 of parasitized erythrocytes from a donor mouse in volumes of 0.2 mL was used to infect the experimental animals intra-peritoneally.

Transfection and treatment: Estimation of the therapeutic effects of the aqueous and methanolic extract of Adansonia digitata stem bark on established infection was carried out according to the method described by Ryley and Peters (1970). The mice were transfected intraperitonially with an inoculums size of 1×10⁷ of chloroquine sensitive strain of Plasmodium berghei infected erythrocytes. Parasitemia was confirmed after 72 h. The animals were divided into six groups of eight mice each and they were treated for five days, 72 h after transfection using two different concentrations (200 and 400 mg kg^–1 b.wt. day^–1). The chloroquine group received 5 mg kg^–1 b.wt. day^–1 while the negative control received the vehicle (5% v/v tween 80) only. Blood samples were collected from the mice tails each day and thin films were made. The thin film was first fixed in 100% methanol and air-dried prior to staining with Giemsa stain. Percentage parasitemia and percentage clearance/chemosuppression were estimated.

Statistical analysis: Results were expressed as Mean±Standard error of mean. The Duncan multiple range test and student t-test were used to analyze and compared the results at 95% confidence level. Values of p<0.05 were considered significant.

RESULTS

There was a daily increase in parasitemia levels in the negative control group (Fig. 1) and significant reduction in the extract treated groups was observed. Reduction in the parasitemia level at 400 mg kg^–1 b.wt. day^–1 was significantly (p<0.05) higher than 200 mg kg^–1 b.wt. day^–1.

Aqueous extract at 400 mg kg^–1 b.wt. had (76.14±0.06) chemosuppression while the methanolic extract had (90.18±0.04) chemosuppression on the fifth day of treatment. The stem bark extract of Adansonia digitata produced significant (p<0.05) dose dependent reduction in parasitemia levels in the two doses relative to negative control group. Chloroquine showed highest chemosuppression/clearance and zero parasitemia was observed on the third day of treatment which was maintained throughout the study (Fig. 2).

In Fig. 3, the Packed Cell Volume (PCV) in the negative control was significantly lowered (20.00±0.00). In the extract treated groups, the PCV improves in a dose dependent manner. Methanolic extract at 400 mg kg^–1 b.wt. was found to be higher (35.00±0.00) than the aqueous extract at 400 mg kg^–1 b.wt. (30.00±0.00) after the fifth day treatment. Chloroquine treated group had the highest value (44.67±2.60) when compared with all the other extract treated groups.

A significant (p<0.05) increase in mean survival time was recorded in all the extract treated groups when compared to negative control. Aqueous extract at 400 mg kg^–1 b.wt. had mean survival time of (10.9) days while methanolic extract at 400 mg kg^–1 b.wt. had mean survival time of (15.8) days. Chloroquine treated group had the highest mean survival time of 21 days (Fig. 4).

DISCUSSION

Presently, there is no single drug that is potent against malaria infection and effective combination therapy includes artemisinin derivatives (Fidock et al., 2004) or mixtures of the older drugs such as combination malarone (Taylor and White, 2004; Winter et al., 2006). The use of artemisinin combination therapy is however limited due to its high cost and accessibility. Plants are usually considered to be possible candidates as alternative and rich source of new drugs. Majority of the population in many tropical countries depend on traditional medical remedies using herbs (Zirihi et al., 2005). The trends of parasitemia among the extracts of Adansonia digitata stem bark treatment groups and negative control appeared to demonstrate the antimalarial potential of the plant. Parasitemia in the negative control was higher than all the treatment groups.

Fig. 1:	Percentage parasitemia in Plasmodium berghei infected mice treated with extract of Adansonia digitata stem bark, results are expressed as mean of 8 determinations±Standard Error of Mean (SEM)

Fig. 2:	Percentage clearance/chemosuppression in Plasmodium berghei infected mice treated with extract of Adansonia digitata stem bark, results are expressed as mean of 8 determinations±Standard Error of Mean (SEM)

Fig. 3:	Packed cell volume of Plasmodium berghei infected mice treated with extract of Adansonia digitata stem bark, results are expressed as mean of 8 determinations±Standard Error of Mean (SEM)

Parasitemia in the negative control was higher than all the treatment groups. This showed that all the treatment had effect on the growth of Plasmodium berghei parasites in mice. Results showed a significant (p<0.05) dose dependent increase in percentage chemosuppression/clearance and a significant decrease in percentage parasitemia of the extracts at the two doses. From the study, methanolic extract of Adansonia digitata stem bark exhibited the highest antimalaria (Fidock et al., 2004) activity.

Fig. 4:	Mean survival time of Plasmodium berghei infected mice treated with extract of Adansonia digitata stem bark, results are expressed as mean of 8 determinations±Standard Error of Mean (SEM)

This observation agrees with the earlier work by Ajaiyeoba (2005) and Musila et al. (2013), who reported that methanolic extract of the stem bark of Adansonia digitata were able to reduce the number of Plasmodium berghei parasites in mice. The significant (p<0.05) activity of methanolic extract of Adansonia digitata stem bark at 400 mg kg^–1 b.wt. observed during established infection, though lowered was comparable to the standard drug chloroquine (5 mg kg^–1 b.wt.). The observed antimalarial activity is consistent with the traditional use of the plant as a herbal medication against the disease in Nigeria. The lower activity of the extract could be as a result of the crude nature of the extract which can be improved by further purification. The observed higher efficacy of the standard drug chloroquine which was higher than the extract treated groups may in part be due to non selectivity of the extract or slow absorption and poor bioavailability of the extract. Although, the mechanism of action of this extract has not yet been elucidated, suggested mechanisms of action for some antimalarial compounds isolated from plants include intercalation with the parasite DNA (Kirby et al., 1995), inhibition of hemozoin polymerization in the parasite (Banzouzi et al., 2004; Onyeibor et al., 2005; Karou et al., 2007a, b), inhibition of Plasmodium falciparum lactate dehydrogenase (pfLDH), an essential enzyme for energy generation within the parasite through glycolysis (Royer et al., 1986; Gomez et al., 1997), inhibition of protein synthesis (Kirby et al., 1989), interference with the formation of mitotic spindle and the assembly of microtubules into typical axonemes in gametes, thus inhibiting the formation of mobile microgametes (Jones et al., 1994; Billker et al., 2002), enhancing elevation of red blood cell oxidation (Etkin, 1997), or inhibition of proteolytic processing of circumsporozoite protein by a parasite-derived cysteine protease, thereby preventing sporozoite invasion of host cells (Coppi et al., 2006). The extract could have elicited its action through either of these mechanisms or by some other unknown mechanism.

Also, a direct proportionality between parasite clearance and packed cell volume increase was observed. Plasmodium parasite is well known to cause red blood cell haemolysis resulting in anaemic state. There was a dose dependent increase in the packed cell volume of all the treatment groups. The low packed cell volume in the negative control is an indication of hemolytic anaemia.

A previous work by Ramadan et al. (1994) reported that aqueous extract of the fruit pulp of Adansonia digitata had a LD₅₀>8000 μg mL^–1 which was categorized as non toxic to mice. The non-toxicity of the fruit pulp of Adansonia digitata explains why most of the plant parts: Fruit pulps, leaves and seeds are consumed by many communities (Kamatou et al., 2011; Nguta et al., 2011). The observed antimalarial potential in the extract treated group may be attributed to the presence of various secondary metabolites. Previous studies have also shown the antimalarial activity of alkaloids and flavonoids in plants (Okokon et al., 2005; Balogun et al., 2009). The presence of the various chemical compounds in high concentration in the extracts may be responsible for their high antimalarial potential. There was significant (p<0.05) increase in the mean survival time in extract treated group when compared to the control. The chloroquine treated group had the highest Mean Survival Time (MST) as there was no death recorded.

CONCLUSION

The stem bark extract of Adansonia digitata has therapeutic effects on Plasmodium berghei infected mice at higher concentration. The results of this study showed that methanolic extract possess the highest activity suggesting the presence of certain bioactive compounds in it. Based on the findings of this study, it is evident that Adansonia digitata possess promising and potent antimalarial effect which justifies its usage in folk medicine for the management of malaria.