In Vivo Anti-Plasmodial Effect of Ethanol and Aqueous Extracts of Alchornea cordifolia

Malaria is one of the most important tropical diseases and the greatest cause of hospitalization and death among children aged 6 months to 5 years [1]. Malaria has great morbidity and mortality than any other infectious disease of the world [2]. The World Health Organisation reported that there were an estimated 246 million malaria cases distributed among 3.3 billion people at risk in 2006, causing at least a million deaths. Data from Nigeria further reveals that 92% of pregnant women and children under 5 years of age are very susceptible to malaria because of their low immunity and resistance [3]. Approximately 80% of malaria cases in the world are estimated to be in Africa where the disease is endemic [4].


Introduction
Malaria is one of the most important tropical diseases and the greatest cause of hospitalization and death among children aged 6 months to 5 years [1]. Malaria has great morbidity and mortality than any other infectious disease of the world [2]. The World Health Organisation reported that there were an estimated 246 million malaria cases distributed among 3.3 billion people at risk in 2006, causing at least a million deaths. Data from Nigeria further reveals that 92% of pregnant women and children under 5 years of age are very susceptible to malaria because of their low immunity and resistance [3]. Approximately 80% of malaria cases in the world are estimated to be in Africa where the disease is endemic [4].
Five species of the plasmodium parasites can infect humans. However, the most wide spread and virulent form of the disease is caused by Plasmodium falciparum, and is responsible for about 80% of all malaria cases, and about 90% of the deaths [5]. Plasmodium vivax, P. ovale and P. malariae have milder symptoms in humans and not generally fatal. A fifth specie, P. knowlesi, is a zoonosis that causes malaria in macaques and also in humans. The female anopheles mosquito transmits these parasites to humans. In Africa and other countries where malaria is endemic, traditional medicinal plants are frequently used to treat or cure malaria [6]. It is a fact that conventional anti-malarial drugs such as chloroquine, quinine and artemisinin derivatives originated from plants [7]. It is therefore important to investigate the anti-malarial activity of medicinal plants in order to determine their potentials as source of new anti-malarial agents [8].
Alchornea cordifolia known as Christmas Bush is a shrub or small tree found abundantly along the coastal area of the West African Subregion. Alchornea cordifolia is used for treatment of a variety of diseases by traditional medical practitioners in Nigeria. Its different parts had been used to treat diarrhoea, wounds, sores, and cuts [9]. A. cordifolia is also reported to possess a multiplicity of biological effects. It is anti-*Corresponding author: Ene A.C, Department of Biochemistry, Federal University of Technology, Owerri, Nigeria, Tel: +234 (0) 803 854 4994; E-mail: chineduene@gmail.com Page 2 of 4 bark) were harvested in large quantities and air-dried for about 3 weeks to a constant weight under shade in the laboratory. The dried samples were ground into powdered form using an electric grinder (Saisho 200W) and stored separately.
Using maceration method, 100 g of each powdered sample was soaked separately in 600 ml each of distilled water and ethanol of analytical grade respectively, for 72 hour. Each sample solution was filtered using Whatman No.1 filter paper. The aqueous and ethanol filtrates were separately concentrated using water-bath at 45°C. All the extracts were weighed and then stored in well stoppered containers and kept in a refrigerator at 4°C until used.
Animals: Twenty-seven swiss albino mice weighing 15-20 g used for this study were obtained from University of Nigeria, Nsukka. The mice were acclimatized to laboratory conditions for a period of 14 days before the commencement of this study. The animals were fed with standard mouse feed (Vital finisher, Nigeria) and clean drinking water ad libitum. The mice were weighed divided into 9 groups of 3 mice each. The groups consisted of 3 ethanol extract/treatment groups, 3 aqueous extract/treatment groups, artesunate standard control, chloroquine standard control and untreated control groups.

Culture of the chloroquine resistant Plasmodium berghei parasites
The chloroquine resistant Plasmodium berghei (NK 65) used was obtained from Department of Veterinary Pathology, University Of Nigeria, Nsukka. The parasite was maintained by sub-passaging into healthy mice via an intraperitoneal route as earlier described [5,16,17]. Briefly, one millilitre of P. berghei infected blood was diluted with 10 ml of phosphate buffer saline (PBS) pH 7.2. The dilution was such that each 0.2 ml had approximately 10x10 7 infected red cells/parasites per kg of body weight. Infection of each mouse was effected with a single intraperitoneal inoculum of 0.1 ml of diluted infected blood. Parasitemia was confirmed in the animals after 24 h of infection by making a smear of the blood on a microscopic glass slide, staining with Giemsa and viewing under the microscope.
Twenty four hours (24 h) after infecting the mice with the malaria parasites and parasitemia confirmed, the plant's ethanol and aqueous extracts were administered to the test groups at a dose level of 100 mg/ kg body weight (b.w.t) for a total period of 4 days. The dose level of 100 mg/kg b.w.t of the extract was adopted based on a preliminary study carried out in mice [19]. Artesunate and chloroquine standards were administered once a day to the standard control groups at the standard dose of 1.6 and 10 mg/kg b.w.t respectively for 4 days [19]. The untreated control group mice were not treated but given the vehicle (normal saline). All drug administration was done through the intraperitoneal route [5].

Estimation of parasitemia
Parasitemia was monitored in all the groups starting from day 0 to day 14 using thick and thin blood films made from blood obtained from the tail vein of mice [5,18]. The smear was air-dried, stained with 10% Giemsa at pH 7.2 for 15 mins, washed under running tap and allowed to dry. After which it was examined under the microscope with x100 objective lens to access the level of parasitemia. Percentage parasitemia was calculated according to the method outlined by Iwalewa et al. [20,21]

Phytochemical studies
Various parts of Alchornea cordifolia extracted with ethanol and water were screened for phytochemicals using standard procedures [7,22].

Statistical analysis
Data generated were expressed as mean ± standard deviation. Chisquare, One-way analysis of variance (ANOVA) and post-hoc Tukey tests were used to analyze the datawith the aid of Graph Pad Prism version 5.3. Differences between means at p≤0.05 were considered statistically significant.

Yield of extracts
The yield of ethanol showed that more extracts were obtained from leaves followed by stem bark and root respectively (Table 1). For aqueous extract, same applies, more extracts were from the leaves followed by stem bark and root respectively (Table 1). Generally, ethanol yielded more extracts than the aqueous solvent, except for the root where aqueous solvent yielded more.

Phytochemistry
Phytochemical analysis of the ethanol and aqueous extracts of different parts of A. chordifolia showed the presence of some active ingredients like tannins, saponins, flavonoids, terpenoids, steroids and alkaloids (Table 2).

Body weight of experimental animals
The body weight of the animals infected with P. berghei in all the groups and treated with ethanol and aqueous extracts including the artesunate standard control group showed an increase in their body weights ( Figure 1). The untreated and chloroquine control groups showed a decrease in their body weights (Figure 1).

Level of parasitemia
The negative untreated control group infected with P. berghei and the group treated with chloroquine became weak after day 6 and later died. For the infected mice treated with artesunate (1.6 mg/kg body weight), the parasite cleared on the day 6 of treatment and the animals survived. However, when the infected animals treated with crude ethanol extract of the root and leaf extract of Alchornea cordifolia were compared on day 5, there was a great reduction in parasitemia level from (1.20 ± 0.16 to 1.00 ± 0.16) and (1.86 ± 0.80 to 0.70 ± 0.10)
The comparisons were made between days 0, day 3, day 7 and day 14. It showed that the parasitemia level of the infected animals treated with the ethanol extracts of the root and leaf of A. cordifolia showed a very significant difference (p<0.05) when compared with the other treatment groups, untreated infected animals and the chloroquine standard control group ( Figure 3). However, there was no significant difference (p>0.05) observed when the infected mice treated with chloroquine were compared with untreated infected mice (Figures 2 &  3). It should be noted though that the other extracts of A. cordifolia showed anti-malarial activity, but was not as significant as the activity exhibited by the ethanol extracts of leaf and root, with the leaf extract having the greater activity (Figures 2 and 3).
The parasitemia was considerably reduced but not completely cleared in the groups treated with ethanol and aqueous extracts, as noted in Figures 2 and 3. However, this did not lead to the survival of the experimental animals as some of the mice started dying as the parasite load increased after treatment was withdrawn as opposed to the group treated with artesunate which survived.

Discussion
The use of ethanol and water as solvents for the extraction of active metabolites in plant is in consonance with folkloric procedure of the use of decoctions and alcohol extracts. From Table 1, it shows that ethanol as solvent generally but non-significantly (χ2 = 1.201; p = 0.5485) yielded more extracts from the leaves and stem bark than water. For both ethanol and water, the trend of extract yield was leaves > stem bark > root. The percentage (%) yield of the extracts varied, probably due to the solvent medium. The methods employed required no heat, thus preserving most of the thermo-labile metabolites in their active forms.

Page 4 of 4
Results on Table 2 show that tannins, flavonoids, anthraquinones, alkaloids and glycosides were widely distributed in both ethanol and aqueous extracts of the leaves, stem bark and roots of A. cordifolia. On the other hand, saponins and terpenes were found present in the aqueous extracts, but absent in the ethanol extracts, while phlobatannins were detected only in the aqueous root extract. The high preponderance of these phytochemicals may be responsible for the antimalarial activity exhibited by the plant [16,19].
Administration of both the ethanol and aqueous extracts of A. cordifolia elicited percentage increases in the animals' body weights with only the ethanol leaves extract causing significantly (p<0.05) increased gain in total body weight ( Figure 1). On the other hand, there were observed significant (p<0.05) decreases in percentage body weights of the animals administered chloroquine standard drugs and the untreated control animals. The observed increases in body weights of the animals administered the extracts may not be attributed to the presence of phytochemicals because such secondary metabolites are known to be non-nutritional. Furthermore, there were observed increases in body weights of animals administered Artersunate standard drug which is a purified chemical substance. This indicates that the body weight increases in these groups of animals can directly be associated with their anti-malaria activities, helping the animals overcome the infection and hence giving them leverage to continue to metabolize and grow without serious hindrances.
In this study, the ethanol extract demonstrated higher anti-malarial activity than the crude aqueous extract. Ethanol is less dense than water and might possess greater diffusibility in the same medium than water. This might account for the greater efficacy exhibited by ethanol extract over the aqueous extract. It might also be possible that the active metabolites were more soluble in ethanol than water conferring this advantage on the ethanol extract. It can be clearly seen from the results that percentage parasitemia was reduced by the plant's crude extracts in Plasmodium berghei infected mice, pointing to the fact that the plant is endowed with antimalarial activity. Evidence comes for this assertion from studies that reported antimalarial activity of other species of the same genus such as Phyllantus amarus, as can be seen in the study 'In vivo antimalarial effects of ethanol and crude aqueous extracts of Phyllantus amarus'.
However, complete eradication of parasitemia was not achieved with any of the extracts when compared with the use of artesunate, but the observation of overall high percentage antimalarial activity of the plant, especially with ethanol leaves and stem bark extracts, indicates the need for further studies towards identification, isolation and purification of the active components apparently present in the ethanol extract.

Conclusion
The results of this study have shown that the ethanol leaves and stem bark extracts of Alchonea cordifolia exhibited high anti-malarial activities than the aqueous extracts. It is noteworthy that the other extracts of A. cordifolia showed mild anti-malarial activity. The study therefore justifies the traditional use of the plant leaves in the treatment of malaria. Nevertheless, further work is encouraged.