Antiplasmodial Efficacy of Anacardium occidentale in Albino Mice Infected with Plasmodium berghei

Introduction: Resistance of malaria parasites by most malarial drugs prompted the search for other drugs that are effective against the parasite. In endemic nations of the world, medicinal plants are often used to treat malaria. Among such plants is Anacardium occidentale which in addition to treating malaria, the plant has traditionally been used to treat diarrhoea, dysentery, colonic pains, genital problems, venereal diseases, impotence, bronchitis, cough and syphilis-related skin disorder. This research aimed at exploring the efficacy of A. occidentale in the treatment of malaria. Methods: The efficacy of A. occidentale was evaluated in-vivo in Swiss albino mice infected with Plasmodium berghei NK65. Blood samples of mice infected with P. berghei were tested against the plant extract for four consecutive days. Results: The results revealed a significant reduction in the parasitaemia of the mice after treated with varying doses (400 mg/kg, 600 mg/kg and 800 mg/kg) of A. occidentale relative to the control groups. This revealed that tested doses of the plant extract produced various curative effects. A. occidentale exhibited high antimalarial properties of 80.66% and 80.69% curative at 600 mg/kg and 800 mg/kg doses respectively. However, low antimalarial property (54.20%) was observed at 400 mg/kg treatment. Conclusions: This result shows that A. occidentale extract possess promising antimalarial activities which can be explored for malaria therapy.


Introduction
Malaria is a vector-borne disease transmitted by Anopheles mosquito in both humans and animals [1]. Plasmodium species, the aetiological agents include: P. falciparum, P. vivax, P. malariae and P. ovale. Malaria is vectored by female mosquitoes of the species An. funestus, An. moucheti, An. gambiae, and An. arabiensis [2,3]. In 2016, there were 216 million cases of malaria worldwide resulting in an estimated 731,000 deaths, approximately 90% of these cases and deaths occurred in Africa [4]. The disease is prevalent in tropical and some subtropical regions of Africa, Central and South America, Asia, and Oceania. The intensity and risk of malaria transmission in endemic areas vary significantly. For instance, more than 90% of clinical malaria and mortality occur mostly in sub-Saharan Africa [5]. In addition, highland (> 1,500 m) and arid areas (< 1,000 mm rainfall/year) typically have less malaria, although, these areas are prone to epidemic malaria if climatic conditions become favorable to mosquito development [4]. It has been estimated that about 40% of all fever episodes in sub-Saharan Africa are caused by malaria. In Nigeria, malaria is a major public health concern where it results in more morbidity and deaths than any country in the world [6]. About 97% of Nigeria population is at risk for malaria while only 3% of Nigeria's population that live in areas that are free of malaria [7]. The epi-• Page 2 of 6 • dirachta indica, Khaya senegalensis and Khaya grandifoliola. Azadirachta indica is called "neem tree" and is also used in other African countries as a decoction against fever and/or malaria [11].
A research into the antimalarial plants may be a lead way for new affordable phytotherapies in malaria treatment especially among the less privilege who are mostly at risk of the devastating effects of malaria [11]. Therefore, the present research evaluates the antimalarial efficacy of A. occidentale in malaria treatment using P. berghei as an animal parasite model.

Plant collection and identification
Fresh green leaves were obtained from the stem of A. occidentale from the Federal University of Akure campus. The plant was identified by a plant taxonomist in the department of Biology, Federal University of Technology Akure, Ondo State.

Extraction of plant material
The leaves were air dried under the shade in the laboratory. The dry leaves were pound to a coarse powder in mortar and then to fine powder in blender. Extraction was carried out by dispersing 250 g of ground plant material in 2 liters of 96% ethanol for 72 hours and the preparation was stirred every 2 hours. The preparation was filtered with muslin cloth and the extract concentrated using a rotary evaporator at a temperature not more than 40 °C. The solvent free extract was concentrated using water bath.

Experimental animals
Thirty-five (35) Swiss albino mice with average weight 16-25 g obtained from Institute of Advanced Medical Research and Training (IAMRAT), University College Hospital, University of Ibadan were used for the study. The mice were distributed into 7 groups of five mice each (Table 1) and kept in plastic cages with sawdust as beddings and given food and water every day. demiological situation of malaria is worsening with the spread of drug resistance in the parasite and insecticide resistance in the vector. This significantly account for increasing malaria morbidity in sub-Saharan Africa by P. falciparum to the existing first line drugs such as chloroquine and sulfadoxine/pyrimethamine. Currently, the biggest concern all over the globe is to treat patients with safe and effective medications and to avoid the parasites developing resistant against such medications [8]. Resistance to currently available antimalarial drugs has been confirmed in most of the human malaria parasite species especially P. falciparum and P. vivax. This resistance poses a threat to control and prevention of malaria, this necessitated the need to develop new drugs that are effective and affordable [9]. One of the drugs in which P. falciparum resistance has been reported in 2001 is Artemisinin [10]. This was observed in the border between Thailand and Myanmar, Artemisinin resistance became a major concern in 2008. Also, in 2009, P. falciparum resistant to ACTs was reported in patients in five regions and states in south-eastern Myanmar [10].
As a result of resistant malaria parasite strains and chemotherapy treatment failure, ethnomedicine has proven to be a veritable source of antimalarial treatment regimens or a template for the synthesis of novel antimalarial molecules [9]. Nigeria has rich flora diversity and many of the plant species are used by some indigenous people for medicinal purposes. A larger number of medicinal plants are used to treat malaria in the Southern part of the country where rain forests exist and originate a humid tropical climate, with ideal conditions for persistent malaria transmission all year round [11]. Some plant species are used for malaria treatment across all ethnic and cultural groups in the country, for example, Alstonia boonei (Apocynaceae), Azadirachta indica (Meliaceae) and Cymbopogon citratus (Poaceae).
Plants of the Meliaceae family are commonly used for malaria treatment in Nigeria, like the species Aza-

Groups
Experimental plan Remarks

Groups 1, 2, 3
Were infected with Plasmodium berghei and administered with Anacardium occidentale plant extract at 400, 600 and 800 mg/kg body weight respectively for 4 consecutive days.

Group 4
Were infected with Plasmodium berghei and treated with 10 mg/kg of chloroquine phosphate solution for 4 consecutive days.

Group 5
Were infected with Plasmodium berghei and only given 5 ml/kg of distilled water.

Group 6
Were administered with 800 mg/kg body weight of Anacardium occidentale plant extract for 4 consecutive days.
Plant extract control

Group 7
Were neither infected nor treated. Positive control

Antiplasmodial activity of the plant extracts and chloroquine
The antiplasmodial activity of the plant extracts at graded doses of 400, 600 and 800 mg/kg body weight; the chloroquine at 10 mg/kg and negative control was presented in Table 2. On day 1 (24 hrs) and 2 (48 hrs), the percentage parasitaemia decreased in mice treated with A. occidentale at 400, 600 and 800 mg/kg and mice treated with 10 mg/kg of chloroquine. However, optimum activity of A. occidentale was recorded in all of the treatment groups in day 3 (72 hrs). At day 5 the curative test showed that there was no significant difference in the percentage parasitaemia at different doses of 400, 600, 800 mg/kg for the plant extract, and chloroquine at 10 mg/kg.
The mean parasitemia was highest in the infected untreated mice (negative control) while there was no parasitaemia in the red blood cells of mice treated with chloroquine, which is the standard control ( Table 2). The plant extract cleared the parasitaemia to some extent in the various dosages at different intervals.

Percentage weight loss/gain of albino mice after treatment
The result showed significant weight loss in all the treated mice except mice treated with 800 mg/kg of A. occidentale, 10 mg/kg of chlorine and the positive control (mice neither infected nor treated). It was further observed that highest percentage weight loss (18.66%) was noted in mice treated with 800 mg/kg of A. occidentale for 24 hours while the least percentage weight loss (1.57%) was observed in mice treated with 10 mg/kg for 48 and 72 hours. Generally, it was noted that parasitaemia do not correlate with percentage weight loss. Carl Pearson's correlation showed no association between parasitaemia and weight loss (r = 0.295, n = 24 and p = 0.161). For instance mice infected with P. berghei but untreated showed the highest parasitaemia (10.39%) but has a low weight loss of 8.59%, while mice treated with 600 mg/kg of A. occidentale has parasitaemia of 1.05 and weight loss of 11.11% (Table 3)

Inoculation of mice
A Swiss albino mouse (which served as the donor) was administered a standard inoculum of P. berghei intraperitoneally on day one. On the 7 th day (after the parasite has stabilized in the host mouse) the mouse with a rising parasitaemia of about 20-35% was sacrificed and 0.1 ml of Acid Citrate Dextrose (ACD) was drawn into the syringe before blood was taken from the heart through cardiac puncture. The blood was diluted with isotonic saline to make inoculum for infecting experimental mice. The experimental mice were inoculated with 0.2 ml of diluted parasitized red blood cells specimen.

Determination of parasitaemia
Blood was obtained from the mice by cutting the tail from which thick and thin blood smears were prepared on sterile slides. The smears were fixed with methanol for 5 minutes with 10% Giemsa stain. The slides were observed under oil immersion objective lens of the compound microscope. The infected red blood cells and total number of red blood cells for each smear was recorded. The data obtained were used to determine the percentage parasitaemia and percentage curative were calculated using the formula below.

Data analysis
The data obtained were analyzed and expressed as mean ± standard error. The mean and the level of significance for the differences between means of the data obtained were computed using Duncan's New Multiple Range Test (DNMRT) at P < 0.05 ally observed that the parasitaemia reduced while the percentage curative increased as the dose and time of treatment increased.

Discussion
Malaria is the major cause of health problem in tropical and developing countries of sub-Saharan Africa and South East Asia including India [12]. The emergence of widespread resistance of Plasmodium species to most antimalarial drugs has led to a more vigorous and concerted research in traditional use of plants for malaria treatment [13]. The results of this research revealed that A. occidentale exhibited potent antimalarial activity against P. berghei. This was noticeable as the parasitaemia reduced at different doses and time interval. This result agrees with the findings of [14] who reported that A. occidentale has the potential to relieve fever and cure malaria. This study provides a scientific evidence observed that some of the mice regained their weight after treatment. For instance, mice treated with 800 mg of A. occidentale regained their weight from 16 g to 21 g at 24 to 96 hours of treatment respectively. Similar result was observed in mice treated with 10 mg of chloroquine, where the mice regained their weight from 18.67 to 21.67 g as the parasitaemia reduced from 0.03% to 0% at 96 hours treatment time (Table 3).

Percentage curative of A. occidentale at 24 hours interval
The curative result showed that chloroquine achieved 100% percentage curative at 48 hours of treatment (Table 4). Meanwhile, 600 mg/kg and 800 mg/kg of A. occidentale achieved 80.66% and 80.69% percentage curatives respectively at 96 hours of treatment. The least percentage curative (54.20%) was observed at 400 mg/kg for 96 hours of treatment. It was gener- for the claims.
The findings of the present research also conforms to the work of [13] who reported that the ethanolic leaf extract of A. occidentale were effective against malaria parasite. The in vivo study of the ethanolic extract of A. occidentale showed that the plant was effective at different dosage levels. The curative antiplasmodial tests showed that mice treated with A. occidentale at graded 400, 600, 800 mg/kg resulted in a decrease in parasitic load compared to the negative control.
The plant extract showed no significant difference (p > 0.05) against P. berghei in mice tested at all dosage levels from 24 hours to 96 hours of treatment compared to the mice in the untreated group. The percentage curative antiplasmodial activity of A. occidentale and chloroquine (standard control) at 10 mg/kg body weight from this study showed that chloroquine, the standard drug cleared the parasites by 100% compared to the percentage of clearance by plant extract that varies at graded doses of 400, 600 and 800 mg/kg against P. berghei infection. This agrees with the findings of [15] who reported total clearance of parasitaemia in the experimental mice treated with chloroquine, the author affirmed that the drug is still effective against malaria parasites that have not developed resistance yet. A. occidentale demonstrated curative effect of 54.20%, 80.66% and 80.69% at 400 mg, 600 mg and 800 mg respectively. This result showed that the drug is more effective at higher doses. However, the development of new drugs from the highly active natural products, which have already been discovered, is crucial in order to overcome the increasing resistance of Plasmodium to available antimalarial drugs. Therefore, there is a need to further phytoresearch especially on plants which have already been shown to have antimalarial activities.

Conclusion
The results of this study show that the leaf of A. occidentale is efficacious against P. berghei infection. This result has established the underlying principle for the traditional use of the A. occidentale in the malaria treatment, and demonstrated that medicinal plants which have reputation for antimalarial properties can be screened in order to ascertain their efficacy and determine their potentials as sources of new antimalarial drugs. However, the development of new drugs from the highly active natural products, which have already been discovered, is crucial in order to overcome the increasing resistance of Plasmodium to available antimalarial drugs. Therefore, there is a need to further phytoresearch especially on plants which have already been shown to have antimalarial activities.