Effect of Annickia polycarpa (Stten and Mass.) Annonaceae, leaf extract on chemo-suppression of hyperparasitemia, body weight, hematological indices and survivor time of Plasmodium berghei infested mice in the Peter’s test

Background: Malaria is an infectious disease that is spread through the bite of female anopheles mosquito resulting in the death of hundreds of thousands of people per year. Medicinal plants provide crude extracts and purified compounds for malaria treatment. Annickia polycarpa is one of such plants whose bark is used for this purpose. However, the antimalaria effect of its leaf is not known. We hereby report the investigation of antimalaria effect of A. polycarpa leaf. Methods: Antimalaria effect of the ethanol extract of A. polycarpa leaf (APLE) was investigated in P. berghei infested ICR mice in the Peter’s test. The effect of the extract on development and chemo-suppression of hyperparasitemia, reduction in body weight and mean survival time were evaluated. Full blood count analysis on the infected mice were performed to determine the effect of treatment with APLE on hematological indices such as red and white blood cells and platelets. Acute toxicity and phytochemical tests of the extract were also performed. Results: APLE administered orally at 50, 200 and 400 mg/kg produced profound dose-dependent chemo-suppressive effect of 89.37 – 95.50 % of P. berghei after 4 consecutive days of treatment which compares with 86.22 % obtained for Quinine 30 mg/kg i.m. under the same regimen. APLE also protected the mice against reduction in body weight associated with malaria which was P < 0.05 at 50 mg/kg. Furthermore, APLE promoted dose-dependent mean survival time in the Kaplan-Meier curve. Only 25.0 % of mice in the negative control group survived after 30 days compared to 100 % survival for mice in APLE 400 mg/kg and Quinine 30 mg/kg groups. The results from the full blood count shows that APLE caused (P < 0.05) dose dependent increase in RBC, HGB, HCT, WBCs, lymphocytes and platelets. The

LD50 of APLE was above 5000 mg/kg p.o. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi,have been identified as the causative agent of the disease in humans with the most pathogenic and deadliest specie is P. falciparum which also accounts for most of the malaria cases in Africa resulting in deaths of untreated and non-immune humans [3 -4]. The incidence of malaria and its associated mortalities have been controlled extensively in recent times through the use of insecticide-impregnated mosquito nets, mosquito insecticide sprays and the use of chemotherapeutic agents such as artemisinin and artemisinin derivatives to treat the disease. Even though artemisinin-based combination therapy is the first-line of treatment of malaria in Ghana, the increasing resistance of the plasmodium parasite to artemisinin and other available antimalarials remains a serious challenge to the whole world, which calls for public health attention [5]. As a result, there is a need for the development of new antimalaria drugs.
Medicinal plants have served as valuable sources of bioactive compounds for treatment of various diseases including parasitic infections. For example, it has been documented that, quinine and artemisinin which are predominantly used in the treatment of complicated malaria were obtained from Cinchona tree and Artemisia annua respectively [6 -7]. The African continent is densely populated with numerous medicinal plants whose potentials have not yet been explored [8][9][10]. polycarpa is known to treat malaria [11], there is no literature to show that the leaf has anti-malarial property. Hence, the aim of this study is to explore the antimalarial potential of 96 % ethanol extract of the leaf of A. polycarpa (APLE) in chloroquine sensitive Plasmodium berghei-infected mice. It was filtered after 4 days and re-extracted under the same conditions to ensure exhaustive extraction. The extracts were combined and evaporated to dryness in a rotary evaporator (Eyeler N1110, Tokyo-Japan). A dark green solid was obtained and labelled APLE. APLE was stored at 4 ºC in air-tight container until use.

Oral acute toxicity test
The acute toxicity test was carried out in order to estimate the median lethal dose (LD 50 ) of APLE as per the convention of the Organization for Economic Co-operation and Development, OECD, [14] with some modifications. A single oral dose of APLE 5000 mg/kg reconstituted with distilled water was administered to female ICR mice of body weight 21-28 g (n = 6). The animals were observed for signs of toxicity such as autonomic, neurological and / or behavioral changes and mortality at 2 h interval for 24 h post administration of the extract. The mice were observed further for 14 more days.
Maintenance of the parasite in the laboratory Chloroquine

Parasite inoculation
Blood from donor mice with rising parasitemia of 20 -30 % was diluted in physiological saline (Pharmanova Limited; Accra-Ghana) such that each 0.2 ml contained 1.0 x 10 7 P. berghei-infected RBCs. ICR mice (n = 30), were each injected with 0.2 ml (i.p.) of diluted blood with the P. berghei-infected RBCs [15]. The parasitemia levels of the mice was determined by microscopy after 72 h of infection.
In-vivo evaluation of anti-plasmodial activity in the Peter's Test.
Mice with parasitemia levels above 13 % were randomly selected into five different groups, with five animals (n = 5) in each group. Group I received sterile water, group II received 35 mg/kg quinine as a reference drug (i.m.), Group III, IV and V received 50, 200 and 400 mg/kg respectively of APLE (0.2 ml) orally for 4 more days post P. berghei-infection.

Parasite monitoring
The parasitemia levels in the blood of the P. berghei-infected mice were monitored using methods described previously [16]. Briefly, blood samples from each animal were taken onto a microscope slide by tail-bleeding on day 3, 5 and 8 post infection to prepare a thin film. The film of the blood was dried and fixed in methanol for 15 minutes, and stained in 10% Giemsa stain for 25 minutes. Excess stain was washed off and the slide was made to dry. The film was then immersed in oil and viewed at x100 magnification using the Olympus light microscope (Olympus CX21; Tokyo, Japan). The level of parasitemia was determined by counting the number of parasitized RBCs and normal RBCs per randomly selected fields.
The percentage parasitemia was calculated by: Hematological analysis Blood samples were collected from each animal by tail bleeding after the 8 th day post infection into Eppendorf tubes precoated with anti-coagulant (Na-EDTA). The blood samples, 50-80 µL, was diluted in 420 µL normal saline. Hematological parameters were determined by running a full blood count of the diluted blood using Abacus hem analyzer (Abacus 380; Budapest, Hungary). The dilution of the blood was accounted for by multiplying through by the dilution factor.

Body weight analysis
The body weight of the animals was monitored daily over the experimental period up to the 8 th day after inoculation.

Mean Survival Time (MST)
The number and date of death of animals from each group was recorded during the experimental period and for further 22 more days after inoculation. The mean survival time (MST) was determined over a period of 30 days.

Statistical Analysis
The data was analyzed using graph pad prism version 6 and presented as mean ± SEM. Variations were determined by comparative analysis using one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test to determine where the variation lies. Variations were considered significant when P ˂ 0.05.

Results
Yield of crude extract APLE yielded 15.78 % w/w of solid upon extraction.

Phytochemical screening
The result of the phytochemical screening is summarized in Table 1 below. ) also reduced to 6.15 ± 0.92 % (Fig.1). The overall chemo-suppression for the various treatment groups are indicated in Table 2. APLE had a dose dependent suppression of parasite especially on the 8 th day. The micrographs in Fig.   4 further confirmed that there was reduction of parasitized RBCs in all treatment groups compared to the negative control. Results are presented as percentage mean (n=5).  Table 3.  Results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to negative control.

Effect of treatment with APLE on WBCs and indices
The results obtained from the full blood count analysis of the effect of the extract on WBCs are below in Table 3. The APLE treatment groups had a dose dependent increase in WBCs with increasing lymphocytes levels. At the highest does, APLE caused a significant increase in the lymphocyte levels as compared to the negative controls. However, there were no significant difference in the WBC count in the treatment groups compared with the negative controls. Results are presented as mean ± SEM.
Effect of treatment with APLE on platelets and indices Effect of APLE on platelets and indices on day 8 after P. berghei infection is shown below in Table 6. APLE dose dependently increased platelets compared to the negative control. The effect on other platelet indices are also shown below (Table   6). Results are presented as mean ± SEM. *P < 0.05, compared to negative control.

Discussion
Amidst the various malaria treatment challenges, access to anti-malarial medications and emergence of drug resistance are critical obstacles to be considered in ensuring absolute control and eradication of the disease in the sub-Saharan region. The possibility of improving on the quality and management of malaria cases in these regions includes a home-based management approach, which is a common practice in African [17][18]. The practice is not only limited to the use of efficacious herbal medicine which are cost efficient and devoid of toxic effects but also ensures that all persons have access to health care due to its readily availability. Medicinal plants serve as natural reserves, where new anti-malarial agents could be obtained [19]. The stem bark of A. polycarpa has been established as an anti-malarial agent [11], but there is no literature to validate the use of the leaf as anti-malarial agent. The current study therefore sought to evaluate the antimalarial activity of the ethanolic leaf extract of A. polycarpa (APLE) and validate its traditional use.
Even though scientific validation of traditional remedies is rare, there are still evidence to indicate that it is constantly increasing especially in Africa [20]. A major consideration for validation according to WHO is the safety and effectiveness of the traditional medicine before its integration into the health care system [20]. Alterations in hematological parameters are considered as markers of malaria, which may be pronounced probably due to rising levels of parasites [21]. P. berghei infection may present pathological conditions including that of anemia, leukocytosis and thrombocytosis depending on the severity of infection.
Anemia is a common symptom of malaria which is evaluated by measuring the levels of RBC, HCT/PCV, HGB, MCV, MCH, MCHC and RDWc. The parasites attack RBCs and feed on the intracellular protein, hemoglobin (HGB), found in RBCs [22].
As the parasite population in the RBC increases, the cell raptures to release the parasites and their toxins into the blood stream. The released parasites attack and destroy new RBCs, constantly leading to the rapture and destruction of more cells especially in untreated cases resulting in reduced RBCs. Anemia due to malaria could be extremely complex and may involve a combination of factors not limited to accelerated hemolysis of only parasitized RBCs, but also accidental removal of unparasitized RBCs and ineffective erythropoiesis [21,23). The extracts dose dependently increased the RBC, HGB and HCT levels as compared to the negative control group and quinine treated group. This means that the extract protected against the rapid hemolysis of parasitized RBCs.
The size of the RBCs is a further definition of the type of the anemia; normacytic (normal MCV), macrocytic (increased MCV) and microcytic (decreased MCV) [24].
According to [25], MCV levels in healthy mice ranges from 48.9-50 U 3 . The current report indicates that increasing dose of APLE is likely to cause a reduction in MCV, MCH, MCHC and RDWc levels, leading to possible hypochromic microcytic anemia.
The insignificant WBC count with increased lymphocytes in the APLE (400 mg/kg) treated group as compared to the negative control is an indication of a possible antibody (B lymphocytes) production / secretion due to anti-plasmodial treatment with the extract. However, high WBC levels in the negative controls is as a result of increased inflammation due to high parasitemia.
Plasmodium parasite infection causes platelets levels to reduce, with decreased clotting factors. This increases the propensity to bleed heavily when there is an injury as a result of thrombocytopenia associated with malaria. There was no significant difference in the platelet count of the APLE treatment groups as compared to the negative control group, even though there seem to be an increase in platelet levels in the groups treated with the extract. APLE at increasing dose levels is likely to raise the levels of platelet close to normal to prevent thrombocytopenia associated with malaria.

Conclusion
The Effect of APLE or Quinine on the survival rate of P. berghei-infected mice after 30 days of inf Methods -formulas.docx