Therapeutic Efficacy of Triple Regimen of Artemether, Lumefantrine and Hippocratea africana in the Treatment of Plasmodium berghei Infected Mice

Combination therapy is fast replacing monotherapy in the treatment of infectious diseases and Plasmodium resistance to artemisinin–based combination therapies (ACTs) is an emerging challenge. Our study aimed to evaluate the therapeutic efficacy of combining Artemether-Lumefantrine with crude root bark extract of Hippocratea africana, on mice infected with Plasmodium berghei. Forty-five albino mice which weighed 30 - 38g were grouped into five with seven mice in each. The mice were inoculated intraperitoneally with Plasmodium berghei and kept for seven days for the parasitaemia to develop. A daily single dose of 200mg/Kg body weight of extract of H. africana was administered orally for ten days, while therapeutic dose of Artemether-lumefantrine was administered as daily single dose to the relevant groups on the last six days of treatment. A non-parasitized and parasitize untreated groups served as controls. The weights of the animals were recorded before and after treatment. The animals were sacrificed and blood obtained for determination of percentage parasitaemia and the erythrocytes count of the parasitized mice using standard methods. The results showed the mean body weight and percentage body weight changes of parasitized mice treated with combination of ACT plus H. africana not statistically different from those of non-parasitized untreated mice. Parasitized mice treated with ACT plus Extract had a significantly (p < 0.05) reduced percentage parasitaemia when compared with those treated with ACT only. Treatment with ACT plus Extract also showed a significant increase in parasite clearance (100%) when compared to mice treated with either ACT only (93.10%) or Extract only (82.15%). We concluded that combining artemether, lumefantrine and H. africana root bark extract exhibited a good therapeutic efficacy as demonstrated by body weight recovery, parasite clearance and reversion of clinical signs induced by Plasmodium berghei parasitaemia. The triple regimen was more efficacious than ACT alone, and therefore, may be a useful regimen in addressing the emerging problem of resistance of plasmodium species to standards ACTs.


Introduction
Treatment of malaria continues to pose a big challenge, both to the sufferers and to all categories of health care providers [1]. Combination therapy, whether as polyherbal, synthetic agents or both, is becoming a commoner practice that is fast replacing monotherapeutic approaches in the management of malaria [2]. Different combinations and formulations of chemotherapeutic agents have been designed and employed in the treatment of clinical entities, especially in the Sub-Sahara Africa [2] WHO recommends Artemisinin-Based Combination Therapies (ACTs) for the treatment of uncomplicated malaria, which entails combining two or more active ingredients with different mechanisms of action, hence making ACTs the most effective antimalarial medicines available today [3]. The ACT artemether-lumefantrine has been shown to be very effective against malaria parasite through its haemolytic action [4]. Artemether interacts with blood components to generate free radicals which may destroy the malaria parasite, while lumefantrine eliminates residual parasites, reduces parasite burden, and resolves clinical symptoms of the disease [4,5].
The use of medicinal plants in the treatment of malaria is well reported. Concomitant use of WHO recommended artemisinin-based combination therapy (ACT) with medicinal plants extracts is a very common practice in the southern part of Nigeria.
In recent years, malaria has become more difficult to control and treat because Plasmodium falciparium has become resistant to available drugs, and mosquitoes that transmit the disease-causing parasites have also become resistant to insecticides [6]. This has led to intensification of the quest for effective treatment modality, especially in the face of challenges of co-infections and concurrent diseases [2]. The reported widespread resistance of Plasmodium species to the commonly available anti-malarial drugs has necessitated countries to review and deploy new antimalarial drug policies to ensure effective management of the disease (1,6). High costs, limited production of artemisinin derivatives, toxicity and other factors limit the use of ACT [7,8]. In view of the problems associated with antimalarial drug resistance, new drugs or drug combinations are required for effective treatment of malaria.
Plants have always been considered to be a possible alternative and rich source of new drugs. Most of the antimalarial drugs in use today such as quinine and artemisinin were either obtained directly from plants or developed using chemical structures of plant-derived compounds as templates [9]. There are reports of a renewed interest in indigenous medicine worldwide in the last decade, arising from the realization of the limitations of orthodox drugs [10]. In many developing countries, data available showed that one-fifth of patients use indigenous herbal remedies to treat malaria [11]. It has been observed by Adebayo and Krettli that some of these herbal remedies are used in combination with other medicines [10].
Hippocratea africana (Wild) Loess Hippocrateaceae, commonly known as African paddle-pod, inhabits the green forests and is a perennial climber with glaburous hairs and is widely distributed in tropical Africa, reproducing from seeds [12]. In West Africa, the plants is known by various names such as nnoto (Akan-Asante, Ghana), onchom (Mandyak, Guinea-Bissau), njabo (li) (Loko, Sierra Leone) Rdelbi (Fula-Pulaa, Senegal) and kesayso (Manding-Mandinka, The Gambia) [13]. In Nigeria, it is known. by the names godayi or gwaďayi (Hausa), balandibi (Fulfulde), ipungwa (Tiv), pọnju òwiwí (Yoruba) and mba (or eba) enang enang (Ibibio) [12,14 ] The root of the plant is used traditionally by the Ibibio of the Niger Delta region of Nigeria in the treatment of various ailments such as fever, malaria, body pains, diabetes and diarrhea [12,15]. The root is also used traditionally as an antipoison or antidote to treat liver diseases [16]. The plant has been reported to contain significant quantities of phytochemicals such as alkaloids, cardiac glycosides and flavonoids, tannins and flavonoids as the major constituents [17]. The root of H. Africana has been reported to possess in vivo antiplasmodial activity with LD 50 of 2.45 g kg -1 [16]. The blood schizontocidal activity and chemosuppressive effect, both in early and established infection in mice, were comparable to chloroquine at 5mg/kg [16)].
The aim of our study was to evaluate the therapeutic efficacy of combining Artemether-Lumefantrine, a WHO advocated artemisinin-based combination therapy (ACT), with crude root bark extract of H. africana, on Plasmodium berghei infected mice.

Collection and Identification of Plant Material
The roots of Hippocratea africana (Willd) Loes were harvested from its natural habitat and was identified and authenticated by a taxonomist in the Department of Botany, University of Uyo, Akwa Ibom State, Nigeria. A voucher specimen of the roots of Hippocratea Africana was deposited in the University of Uyo herbarium with voucher number. The roots of H. africana were washed with clean water and the bark scrapped with a sharp knife, sun dried and crushed with a mortar into pellets. The pellets were blended into powdered form using an electric blender. About 500g of the powdered H. africana root bark was blended in 1000ml of 80% ethanol. It was left overnight to achieve a good extraction. The mixture was filtered and the filtrate was concentrated in vacuo at 40°C to obtain a dry crude extract which could dissolve homogeneously in normal saline and distilled water.

Inoculation of Experimental Mice with Plasmodium Berghei
Forty-five albino mice which weighed between 30 -38g were divided into five groups of seven mice each. About 0.1ml of infected blood obtained from donor mouse was mixed with 10ml of normal saline and 0.2ml of the mixture, equivalent to 0.2ml of blood which containing about 1 x 10 7 Plasmodium berghei parasitized erythrocytes, was administered intraperitoneally to each animal. The inoculum consisted of 5 x 10 7 P. berghei infested erythrocytes per ml of blood from the donor mouse with a 66% parasitaemia. A nonparasitized group served as normal control. The animals were fed ad libitum and kept at room temperature of 28.0±2°C for the period which the experiment lasted [17,18]. The inoculated animals were kept for eight days for the parasite to develop. On the eighth day, thick films were prepared from blood collected through tail puncture of the parasitized Hippocratea africana in the Treatment of Plasmodium berghei Infected Mice animals to ascertain parasitaemia using the method described by Greenwood and Armstrong [19].

Preparation of Antimalarial Drugs
Coaterm brand of Artemether-lumefantrine containing 20mg of artemether and 120mg of lumefantrine was dissolved in a calculated amount of 0.9% saline in water, such that 0.08mg and 0.64mg of artemether and lumefantrine respectively were sustained in 0.5ml of solvent, equivalent to therapeutic doses of 3mg/Kg body weight of artemether and 18mg/Kg body weight of lumefantrine.

Experimental Design and Treatment of Experimental Animals
Based on already established safety dose of the crude root bark extract of Hippocratea africana reported [12,13,15], 200mg/Kg body weight of the plant extract was administered orally to respective groups of mice as shown on Table 1. The prepared solution of artemether-lumefantrine were administered orally to the respective group of mice (Table 1), depending on the group mean weight of the animals. The untreated control groups were administered normal saline. Pre-treatment and post-treatment weights of the mice were recorded.

Clinical Observation of Mice
All Non-parasitized and Parasitized mice were visually monitored for behavioral changes and signs of illness which include lethargy, piloerection, decreased locomotor activity and diarrhea. Any signs of illness observed were quantified using arbitrary scale and recorded as either absent (-), mild (+), moderate (++) or severe (+++), depending on severity. Pre-treatment and post-treatment weight were recorded.

Collection of Blood Sample and Parasitaemia Measurement
A drop of blood was collected from the mice by tail puncture and transferred onto the edge of a microscope slide (single, 76 × 26 mm thickness) and drawn evenly across a second slide to make a thin blood film and allowed to dry at room temperature. The smear was stained with Leishman stain. Slides were examined under light microscopy (Vickers Instruments) with oil immersion (x1000 magnification). Parasitaemia was counted based on the Leishman positive bodies which represent the parasitized red blood cells. The Leishman positive cells were counted with the aid of a graticule and hand counter. Five fields of approximately 200 cells each were counted and the parasitaemia was calculated as the percentage of the total red blood cells containing Leishman positive bodies.

Statistical Analysis
Standard computerized statistical tools were used in the analysis of the results obtained. All data were expressed as mean±standard deviation (SD). Analysis of Variance was used to analyze data, while Student's t-test was used for comparison. Any difference in mean was considered significant at p < 0.

Clinical Observations of Pretreatment Parasitized Mice
At the end of treatments, clinical examination of the parasitized untreated mice (group II) were severely lethargy with marked piloerection ( Table 2). The mice clustered together at the corner of their cages marked decrease in locomotor activity. The tail and pinnae were markedly paler compared to the normal animals (Group I). The remnant of food in the containers were markedly increased compared with the normal non-parasitized mice, with no evidence of passage of watery stool. Mice treated with combination of artemether-lumefantrine and H. africana (Group V) showed negative lethargy and piloerection. There was less decrease in locomotor activities in comparison with parasitized untreated group. Clinical features of treatment groups were as shown in Table 2. Table 3, the parasitized untreated mice (Group II) showed a significant (p < 0.05) decreases in the mean body weight and percentage reduction in mean body weight when compared with the mean body weight (MBW) and percentage body weight increases recorded for the normal control (Group I)). The mean body weight changes and the corresponding percentage changes in mean weight of treated groups (III, IV and V) were significantly (p < 0.05) increased when compared with the parasitized untreated group (Group II). Test groups treated with Artemetherlumefantrine only (Group III) and H. africana only (Group IV) showed significant (p < 0.05) decreases in mean body weight and percentage body weight changes when compared with the normal control (Group I). Mean body weight change and the percentage change in body weight recorded for test group treated with combination of ACT and H. africana (Group V) was not statistically different when compared with normal control group (Group I)..

Result of Treatments effect on Parasitaemia, Parasite Clearance and Mortality
The percentage of parasitaemia before commencement of treatments, after the various treatments and the percentage parasite clearance of the infected mice treated with the various treatments are as shown on Table 4.
There was a significant (p < 0.05) increase in the parasitaemia of the parasitized untreated mice at the end of the experiment when compared with the value recorded at the beginning of the treatment. Mean parasitaemia levels recorded for test groups III and IV were significantly reduced (p < .0.05) when compared to the value obtained for the parasitized untreated group. Test group V did not record any parasitaemia after the treatment.
The parasitized untreated mice recorded significant (P<0.05) percentage increase in parasitaemia at the end of experiment in comparison with normal group. The percentage parasitaemia recorded for test groups III, IV and V were significantly (p < 0.05) increased when compared with the untreated control group.. Test group V treated with ACT plus H. africana extract recorded a significantly (p < 0.05) reduced percentage parasitaemia when compared with group III treated with ACT only.
As seen in Table 4, the mean parasites clearance of test groups III, IV and V were significantly (p < 0.05) higher when compared to that obtained for parasitized untreated group, though parasite clearance for test group III was significantly higher than that of test group IV.. Test group V showed a significant increase in parasite clearance (100%) when compared to group treated with either ACT only (Group III) or H. africana only (Group IV).  e=Mean±Standard Deviation of 6 determinations, a=significantly different when compared with normal control (administered normal saline) at p < 0.05, b=significantly different when compared with test group II (parasitized untreated) at p < 0.05, ACT=Artemether-Lumefantrine, BW=Body weight, *=Negative change (a decrease).

Discussions
In Nigeria, several formulations of herbal medicines are used to treat malaria disease. The use of these herbal medicines alongside with the prescription drugs are well reported [20][21][22]. This is partly due to the challenge of parasite resistance to antimalarials, coupled with the complexity of the disease pathophysiology [23]. Recently resistance of Plasmodium falciparum to artemisinins, have documented in five countries, which is reported to manifest in the form of delayed parasite clearance [24].

Effects of the Treatments on Clinical Signs and Behavior
The pretreatment parasitized mice manifested physical signs consistent with Plasmodium berghei infection. The clumping together of the mice at the corner of the caged, piloerection, reduced locomotor activity and reductions in food intake were clinical manifestation of hypothermia, malaise and anorexia associated with Plasmodium berghei parasitaemia in mice [25]. Paleness of the pinnae and tail of the parasitized mice may be as result of reduced haemoglobin and other haematological imbalances, which correlates with the reports of other scholars [26,27]. Plasmodium berghei infection in mice is one of the well-employed animal models in malaria research, and this includes analyses on the severe pathology associated with malaria infections [25]. It was reported that Plasmodium berghei parasitaemia is associated with hypothermia and not fever [8]. Reduction in both food intake and body temperature was shown to be associated with an increased turnover in the brain of 5-hydroxytryptamine (5-HT, serotonin), a putative neurotransmitter [28]. Administration of ACT with the plant extract to the parasitized mice resulted in reversal of the clinical signs associated with the infection. Reversion of the clinical signs of parasitaemia by the triple regimen was better than observed for either ACT or the plant extract only. This implies that there nay be a synergic action between the ACT and the plant extract in either suppressing or reversing the impact of the parasites on body organs.

Impact of Treatments on Body Weights of Mice
Plasmodium berghei infected untreated mice exhibited significant loss in body weights probably due to diminished food intake evidenced by larger quantity of food remnant when compared with that of the normal control. This plasmodiuminduced weight loss is consistent with the earlier findings of other scholars [3,29,30]. Concomitant treatment of the infected mice ACT and H. africana root bark extract resulted in a significant weight recovery. The triple combination therapy yielded a better weight recovery than either ACT or H. africana extract alone. The observed weight recovery was likely due to reversal of the deleterious effects of parasitaemia on the animals (3). Some phytochemicals from the extract probably worked synergistically with the ACT to prevent body weight loss or induce body weight gain by unknown mechanisms.

Antiplasmodial Efficacy of Treatments
The root bark extract of H. africana alone demonstrated antiplasmodial activity that was comparable to artemetherlumefantrine. The root bark extract of H. africana was earlier reported to demonstrate significant antiplasmodial activity shown by higher parasite clearance and the dose-dependent suppression of parasitaemia that was greater than that of 5mg/kg body weight chloroquine prophylactic efficacy in P. berghei infected mice [31,15]. The observed therapeutic efficacy of H. africana may be due to the active phytoconstituents especially alkaloids and flavonoids demonstrated in the herb [31].
africana demonstrated 100% parasite clearance within the period of the experiment. Parasites clearance by administation of ACT and extract of H. africana was better either the ACT or plant extract alone. This implies that the ACT-herbs combination may be a better treatment modality for plasmodium infection in comparison to either standard ACT. This combinations form a triple regimen that may address the emerging problem of resistance to standard ACTs. Certain phytoconstituents in the extract may have potentiated the schizonticidal and chemosuppressive effects of the ACT [32]. Methoxylated flavones artemetin and casticin were reported to demonstrate synergistic action with Artemisinin, and flavanoids present in Artemisia annua was considered to probably contribute to the antimalarial action of extracts or herbal teas prepared from this species [32]. Hence, the observed increased antiplasmodial activity due to the added H. africana root bark extract may be due to synergistic action of the phytocontituents with ACT.

Conclusions
We concluded from our study that the triple regimen of artemether, lumefantrine and H. africana root bark extract exhibited a good therapeutic efficacy in the treatment of plasmodium berghei infection in experimental mice, as demonstrated by an excellent antiplasmodial activity, body weight recovery and reversion of clinical signs of the disease induced by Plasmodium berghei parasitaemia. From the data obtained from our study we concluded that the drug-herb combination therapy had a better cidal effect on plasmodium berghei and was more efficacious than the artemetherlumefantrine alone. The ACT-E. speciosa combination therapy, therefore, may be a useful regimen in addressing the emerging problem of resistance of plasmodium species to standards ACTs.