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Phytochemical Composition and Antimalarial Activity of Methanol Leaf Extract of Crateva adansonii in Plasmodium berghei Infected Mice

The need for new compounds active against malaria parasites is made more urgent by the rapid spread of drug-resistance to available antimalarial drugs. The crude methanolic leaf extract of Crateva adansonii was investigated for its antimalarial activity against Plasmodium berghei (NK65) infected mice. A total of 15 mice were intraperitoneally infected with chloroquine sensitive P. berghei strain and divided into 5 equal groups, group 1 served as negative control (untreated), groups 2,3 and 4 were given 200, 400 and 600 mg/kg Crateva adansonii methanolic leaves extract respectively while group 5 served as positive control and was given 5 mg\kg chloroquine for five days. The phytochemical constituents of the plant extract were evaluated to elucidate the possibilities of their antimalarial effects. The extract produced a significant dose dependent decrease in the level of parasitaemia when compared to infected untreated group. Also, the extract at dose of 400 mg/kg and 600 mg/kg produced significant increase in body weight and PCV of the infected mice as compare to mice treated with 200 mg/kg of extract and infected untreated group. Phytochemical screening showed that the leaves extract contains alkaloids, anthraquinones, Original Research Article Tsado et al.; BBJ, 6(4): 165-173, 2015; Article no.BBJ.2015.039 166 tannins, flavonoids, saponins cardiac glycosides and steroids. It is concluded that Crateva adansonii could serve as a possible source of antimalarial compounds.


INTRODUCTION
Malaria is a chronic endemic disease that obstructs social and economic development. It is the leading cause of mortality and morbidity around the word with an estimated 225 million malaria case and 781,000 death being reported globally in 2009 [1]. Most of the reported malaria cases occur in tropical and subtropical regions where the atmospheric condition (temperature and rainfall) are favourable for the development of vectors and parasite [2], the mortality occur mostly in young children and pregnant woman [3]. The attack of malaria during pregnancy stage usually results into severe anemia and impairment of fetal nutrition which contribute to the low birth weight, premature delivery mental retardation and 60% miscarriages [4]. Malaria is caused by protozoan of genus Plasmodium transmitted to the vertebrate by female Anopheles mosquitoes in the vertebrate host, the sexual blood forms of the parasite are the life cycle stage that are responsible for the morbidity and mortality of plasmodia infections [5]. Four species of malaria parasite cause disease in human: Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax and Plasmodium ovale where as 3 species give rise to considerable natural morbidity, only P. falciparum result in high mortality [6], as a result of prevalence, virulence and drug resistance. The continuous spread of the Plasmodium falciparum resistance to the commonly use anti-malarial drug including the newly introduce Artemisinin Combination Therapy (ACT) has resulted in resurgence in treatment failure [7], and hence the need to intensify research in the area of development of new anti malarial drugs especially from medicinal plant with traditional antimalarial reputation.
Crateva adansonii DC, also known as Crateva religiosa or sacred garlic pear, belongs to family Capparaceae. The plant is in high demand, especially its leaves for the treatment of ear infections. The bark is widely used for stomach troubles and held to have tonic properties. In Senegal the roots figure in several treatments for syphyilis, jaundice and fever. Agboke and coworkers claimed the antimicrobial properties of its leaves [8]. Two phytoconstituents had also been isolated and identified as oleanolic acid and 4-epi-hederagenin [9].
In order to scientifically validate the medicinal claimed of Crateva adansonii and determine its effectiveness as a potential source of new antimalarial agent, the present study was undertaken to evaluate its phytochemical composition and in vivo anti-malarial properties against Plasmodium berghei infected mice.

Experimental Animal
Swiss albino mice weighing between 20-25 g were used in this study. The animals were obtained from the National Institute for Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria. They were housed in plastic cages with saw dust bed and given standard laboratory diet and water ad-libitum. They were then allowed to acclimatize for two weeks to their new environment before the initiation of the experiments

Parasite
A chloroquine-sensitive strain of Plasmodium berghei (NK-65) was obtained from the National Institute for Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria and maintained by re-infestation via intraperitoneal with infected blood suspension (0.2 ml) containing about 1 x 10 7 suspension of P. berghei parasitized red blood cells.

Sample Preparation and Extraction Procedure
The collected fresh leaves of Crateva adansonii was washed with clean-water and air dried. The dried sample was grounded using a grinder mill. Extraction of plant material was performed by soxhlet extraction using methanol. The resulting methanol extract was concentrated in a water bath and stored in a refrigerator until required.

Determination of Yield of Extract
The percentage yield of methanol leaf extract of Crateva adansonii was determined by weighing the coarse sample before extraction and the methanol leaf extract of Crateva adansonii after concentration and then calculated using the formula.
Percentage yield (%) = Weight (g) of the concentrated extract X 100 Weight (g) of the Crateva adansonii leaf

Curative test
Evaluation of the curative potential of the crude extract in Peter's test was carried out according to the method described by Ryley and Peters [12]. On Day 0, standard inocula of 1 × 10 7 infected erythrocytes were inoculated in mice intraperitoneally. Seventy-two hours later, mice were randomly divided into their respective groups and dosed accordingly once daily for five days. The extract was dissolve in normal saline.
Group I Mice were given 200 mg/kg b.w of methanol leaves extract of C. Adansonii. Group II Mice were given 400 mg/kg b.w of methanol leaves extract of C. Adansonii. Group III Mice were given 600 mg/kg b.w of methanol leaves extract of C.
Adansonii. Group IV Mice received 5 mg chloroquine /kg body weight. Group V Mice were given normal saline/kg body weight.

Daily parasitaemia count
On each day a drop of blood were collected from the tail of each rat, smeared unto a microscopic slide to make thin films, stained with 10% Giemsa stain and examined microscopically to monitor the parasitaemia level.

Determination of packed cell volume (PCV)
The capillary tubes were filled with blood to about 1cm or two-third (2/3) of its length and the vacant end of each of the capillary tubes was sealed by plastic seal or sealer to protect the blood level from spilling. The tubes were placed in haematocrit centrifuge with seal side towards the periphery and then centrifuge for 5-6 minutes.
The percentage of packed cell volume or haematocrit was read directly from haematocrit reader [13].

Statistical Analysis
Data were analyzed using statistical package for social science (SPSS) version 16 and presented as means±SEM. Comparisons between different groups was done using Analysis of Variance (ANOVA) and Duncan's Multiple Range Test (DMRT). Values of P<0.05 were considered as statistically significant as described by Mahajan, [14].

Extract Yield
The percentage yield of leaves extract of Crateva adansonii is shown in Table 1. The yield of methanol leaves extract of Crateva adansonii was 25.09%. Table 2 shows the result of qualitative phytochemical composition of methanol leaf extract of Crateva adansonii. The results revealed the present of alkaloids, anthraquinones, tannins, flavonoids, saponins cardiac glycosides and steroids, however, phlobatannins were absent.

Parasitaemia count
The average daily parasitaemia level of the Plasmodium berghei infected mice treated with methanol leaves extract of Crateva adansonii are shown in Fig. 1 and

Body weight changes
Effect of methanol leaves extract of Crateva adansonii on body weight of Plasmodium berghei infected mice is shown in Fig. 2 the body weight of the infected untreated mice and infected treated with 200 mg/kg of Crateva adansonii show significant decrease in body weight after 5 days of treatment. However the infected mice treated with 400 mg/kg, 600 mg/kg of Crateva adansonii as well as those treated with 5 mg/kg chloroquine show significant increase in body weight after 5 days of treatment.

Packed cell volume
Effect of methanol leaves extract of Crateva adansonii on PCV of Plasmodium berghei infected mice are shown in Fig. 3 the PCV of P. berghei infected untreated mice and infected treated with 200 mg/kg of Crateva adansonii show significant decrease in PCV after 5 days of treatment. However the infected mice treated with 400 mg/kg, 600 mg/kg of Crateva adansonii as well as those treated with 5 mg/kg chloroquine show significant increase in PCV after 5 days of treatment.  Saponins are used as adjuvants in the production of vaccins [17]. Steroids are used in the stimulation of bone marrow and growth. They stimulate lean body mass and also play vital roles in the prevention of bone loss in elderly men [18]. Alkaloids have been used as CNS stimulant, topical anesthetic in ophthalmology, powerful painkillers, and antipyretic action among other use [19]. The cardiac glycoside has been used for over two centuries as stimulant in cases of cardiac failure and diseases [20]. The presence of tannins in the leaves extract of Crateva adansonii suggests the ability of these plants to play major roles as antifungal, antidiarrheal, antioxidant and antihemorrhoidal agent [21]. Tannins also have astringent properties, plants containing tannins have been reported to be used for healing of wounds, varicose ulcers, hemorrhoids, frostbile and burn in herbal medicine [22].
The presence of all these phytochemcials in the leaves extract of Crateva adansonii is an indication that this plant, if properly screened, could yield a drug of pharmacological significance. However the absence of phlobatannins agree with early studies which also found that not all phytochemicals are present in all plants and those present differ with the solvent used in the extraction process [23].
The in vivo antimalarial effect of Crateva adansonii against Plasmodium berghei infected mice was evaluated. The extract showed a significant dose dependent and progressive reduction in parasitaemia with time, this is a very promising feature in the potentially of Crateva adansonii as an antimalarial drug. However, the antilamalarial effect demonstrated by Crateva adansonii leaves extract was lower when compared to chloroquine. Chloroquine has been used as the standard antimalarial drug because of its established activities on P. berghei [24]. The P. berghei, a rodent malarial parasite although not able to infect man and other primate has been used in antimalarial assays because of its sensitivity to chloroquine [25]. Also the insignificant difference in the level of parasite count of infected mice treated with 200 mg/kg of the extract when compared with the control group reflect the inactivity of the extract which could be attributed to low concentration of bioactive agents at that doses.
Anemia, body weight loss and body temperature reduction are the general features of malariainfected mice [26]. So, an ideal antimalarial agents obtained from plants are expected to prevent body weight loss in infected mice due to the rise in parasitemia. In the present study, extract of C. adansoni significantly prevented weight loss associated with increase in parasitemia level.
Blood parameters including packed cell volume (PCV) is used to assess anemia, erythrocytosis, hemodilution and hemoconcentration due to disease condition [13]. One of the major reasons for the development of anemia is oxidative stress [27], the immune system of the body is activated by malarial infection thereby causing the release of free radicals. In addition to this the malaria parasite also stimulates certain cells to produce reactive oxygen species (R.O.S) their by resulting in haemoglobin degradation [28]. Thus, the significant decrease level of PCV and body weight of the untreated mice is an indication of anemic condition caused by the malarial infection. The significant increase in level of PCV and body weight in mice treated with Crateva adansonii at 400 and 600 mg/kg when compare with the control group is an indication of ameliorating potentials of the plant extract on the anaemia induced by the malarial infection. Though the rodent malaria model, P. berghei is not similar to that of human Plasmodium parasite the antiparasitic activities demonstrated by the extract especially at doses of 600mg/kg against P. berghei mice in this study could be an indication that the extract could possibly be effective against human malarial parasite [29].
The antimalarial activity demonstrated by this plant might be attributed to the presence of alkaloids or flavonoids which have been identified presently in this work; or even a combined action of more than one metabolite. However, the active compound(s) known to give this observed activity need to be identified.

CONCLUSION
The study suggests that C. adansonii leaf contains important phytoconstituents that could be implicated in the observed antimalarial effect of the plant. The study also represents an important preliminary demonstration of the potential of this antimalarial herbal medicine to treat human malaria. However, a bioguided fractionation of the methanolic extract of C. adansonii leaves aiming to isolating active compound(s) is reccomended.

CONSENT
It is not applicable.

ETHICAL CLEARANCE
Ethical Clearance was given by Federal University of Technology, Minna/Nigerian Ethical Review Board (CUERB) in accordance with International standard on care and use of experimental animals.