In vivo antimalarial activity of crude aqueous leaf extract of Pyrenacantha staudtii against Plasmodium berghei ( NK 65 ) in infected mice

Antimalarial activity of the crude aqueous leaf extract of Pyrenacantha staudtii was evaluated using chloroquine-sensitive Plasmodium berghei infection in mice with an objective to finding scientific evidence for the use of the plant as traditional antimalarial remedy in Ido/Osi LGA of Ekiti state, Nigeria. The crude aqueous extract of P. staudtii Engl. (Icacinaceae) (100, 200 and 500 mg/kg) was administered orally to mice infected with P. berghei in 4 days suppressive test. The antiplasmodial effect during the test of the plant in blood was determined and the extract at these doses induced 58.0 to 63.4% activity in comparison with untreated (negative) control group. Chloroquine produced 100% activity. The antimalarial activity showed by P. staudtii during the test justifies its use in traditional medicine for treating of malaria in the area.


INTRODUCTION
Malaria continues to be a devastating disease, affecting millions of people living in the endemic areas in the developing world (Hopkins et al., 2007).Numerous attempts have been made to control the disease by using vector control measures and/or chemoprophylaxis, but they have had limited success (Trigg and Kondrachine, 1998).Immunoprophylaxis holds a promise, but effective vaccines are still not available.Presently, the most effective way of dealing with malaria is the administration of chemotherapeutic agents.Control of the main causative agents of malaria; Plasmodium falciparum and Plasmodium vivax, by use of the classical drugs of chloroquine and primaquine has been frustrated by the resistance of the malarial parasites to these drugs (Foote and Cowman, 1994;Borst and Ouellete, 1995;Garg et al., 1995;Collins and Jefferey, 1996).There is therefore a need to consistently searching for drug with novel modes of action to treat the disease.
Plants have been a great source of medicine useful in the treatment of various diseases (Bako et al., 2005).Therefore, to search for antimalarial drug from plant origin cannot be neglected, more especially, that the antimalarial drugs in use today (quinine and artemisinin) were isolated from plants (Gessler et al., 1994).Pyrenacantha staudtii is an annual herb found in the light tropical rain forest and farmland bushes.It is a woody climber with green influorescent flowers (Falodun and Usifoh, 2006).The plant is widely distributed in south Nigeria and West Cameroons, and across central Africa to Uganda and Angola and it belongs to the family Icacinaceae (Burkill, 1985).The leaves are intensively bitter and the aqueous extract of the plant has been claimed by many traditional medicine practitioners to effectively treat many ailments including malaria, ulcer, gastrointestinal tract infections and threatened abortion (Anosike et al., 2008), dysmenorrheal and intestinal colic (Falodun and Usifoh, 2006).
P. staudtii is traditionally used for the treatment of blemnorrhea, hernia, insomnia, intestinal pain and diarrhea in Nigeria (Awe et al., 2011).The plant was among the plants mentioned to cure malaria among the people of Ido/Osi LGA of Ekiti State, Nigeria (Olorunniyi and Morenikeji, 2013).Since this plant is commonly used in traditional medicine to treat malaria, this experiment was initiated with an objective of investigating its antimalarial potential against Plasmodium berghei (NK65) in infected mice.

Plant collection, authentication and preparation
The fresh leaves of P. staudtii were collected from Ajowa Farm at Ido-Ekiti in Ido/Osi LGA of Ekiti State, Nigeria.The plant was identified and authenticated at the Forestry Research Institute of Nigeria (FRIN), Ibadan where voucher specimen was deposited with number FHI No 108805.The leaves of the plants were airdried inside a room and then grounded into a coarse powder.The coarse powder (200 g) was extracted using distilled water for 48 h at room temperature.The extract was filtered to obtain a filtrate which was concentrated to dryness over a water bath.Appropriate concentrations of the extract were made by serial dilution with Olorunniyi and Morenikeji 343 distilled water for further experimentation.

Malaria parasites inoculation
Chloroquine sensitive P. berghei (Nk65) was obtained from Malaria Drug Research Laboratory, Institute for Advance Medical Research and Training (IMRAT), College of Medicine, University of Ibadan, Ibadan, Nigeria.Parasites were maintained through serial passage in mice.

In vivo antimalarial test in early infection (4-day suppressive test)
Evaluation of suppressive potential of the extract was done using Knight and Peters 4-day suppressive test against P. berghei beighei infection in mice (Knight and Peters, 1980;David et al., 2004).Adult Swiss albino mice weighing 22 to 25 g were injected with 0.2 ml of aliquot 10 6 parasitized erythrocytes, P. berghei beighei NK65 intraperitoneally (i.p.).Food and water were provided ad libitum.The mice were divided into groups of five per cage.On day 0 (that is, the day of infection), the crude aqueous leaf extract of the plant with the following concentrations (100, 200, 500 mg/kg/day) body weight were administered through oral route 3 h post-infection to every mouse in group 1 to 3, respectively.An initial toxicity test was conducted using the plant extract in which concentration at 500 mg/kg body weight was observed to be saved for the animals.Two control groups were set up which were groups 4 and 5. Mice in group 4 were treated with 10 mg/kg/day chloroquine body weight (Akuodor et al., 2010;Olorunniyi, 2013) to serve as positive control and mice in group 5 were kept untreated but only given water as placebo to serve as negative control.On day 1, 2 and 3, all the animals were treated accordingly (with the same dose and same route) as on day 0. Thin blood smears were prepared on day 11 post-infection.Blood films were fixed in absolute methanol, stained with Giesma stain for 25 mins at pH 7 and then microscopically examined (1000× magnification).Parasitaemia was determined microscopically by counting at least a total number of 1000 uninfected and infected erythrocytes from different fields.Percentage parasitemia was calculated as follows: No of infected erythrocytes Percentage parasiteamia = × 100% Total No of erythrocytes The percentage suppression of parasitaemia was expressed as mean chemosuppression and this was calculated for each dose level by comparing the mean parasitaemia in infected untreated (negative) control with those of treated mice.The difference between the mean value of the control group (taken as 100%) and those of the experimental groups were calculated and expressed as percent reduction or activity using the following equation: Mean parasitaemia treated Activity = 100 -× 100 % Mean parasitaemia (-ve) control

RESULTS
The suppressive activity of the crude aqueous leaf extract of P. staudtii against P. berghei berghei NK65 in infected mice was examined in early infection (4-day suppressive test).The crude aqueous extract at 100 mg/kg body weight of mice gave 61.6% chemosuppression when compared with the untreated (negative) control group on  1).Percentage chemosuppression was observed not to be related with increasing the concentrations of the extract.
The results showed mean parasitaemia in mice from the ranges of 2.99% ± 0.05 to 2.61% ± 0.5.The mean parasitaemia in chloroquine group was 0 and the mean parasitaemia of untreated control was 7.12% ± 1.3.

DISCUSSION
Crude aqueous leaf extract of P. staudtii was observed to show intrinsic antimalarial activity considering its percentage chemosuppression in comparison with the untreated control group in 4-day suppressive test (Knight and Peters, 1980;David et al., 2004).Treatment of mice infected with P. staudtii showed no dose-dependent chemosupression in comparison with the untreated control group unlike the results of Ajaiyeoba et al. (2006) in which the activity of methanol extract of Annona senegalensis depended on the doses of the extract.The highest chemosupression observed in P. staudtii was 200 mg/kg/day treated group of mice.It can be deduced that increasing the concentration of the extract above 100 mg/kg body weight produced no additional suppressive effect against malarial infection.The antimalarial activity showed by P. staudtii could be attributable to the presence of alkaloids which was one of its constituents (Anosike et al., 2008).However, the active compound(s) known to give this activity need to be identified.The antimalarial activity showed by P. staudtii justifies its use in traditional medicine for treating malaria among the people of Ido/Osi LGA of Ekiti State, Nigeria (Olorunniyi and Morenikeji, 2013), where the plant was collected for the experiment.

Table 1 .
Antimalarial activity of crude aqueous leaf extract of Pyrenacantha staudtii and chloroquine in mice infected with Plasmodium berghei berghei NK65 in early infection (4-day suppressive test).