Antimalarial Activity of Ethanolic Leaf Extract of Maesobotrya barteri and Effects on Some Hematological and Biochemical Parameters of Mice

Aim: This study was conducted to determine the antimalarial activity of ethanolic leaf extract of Maesobotrya barteri, and effects on some hematological and biochemical parameters. techniques in order to generate permanently stained sections. Results: Maesobotrya barteri showed a significant parasitemia reduction. For all three doses of the extract administered to infected mice, there was a significant reduction in the mean number of parasitized red blood cells during the four days of treatment. The concentration of 1000 mg/kg body weight of the plant extract had the highest antimalarial activity with a parasite inhibition of 97.2%. There was a significant increase in PCV level and haemoglobin concentration. The WBC count showed an increase which was not statistically significant. The ESR level was lowered for the treated groups when compared to the negative control. The liver markers assay revealed a significant lowering in the levels of ALT, AST, ALP and total bilirubin for all treated groups when compared to the negative control. The levels of the liver enzymes for the positive control was similar to the values obtained for the extract treated groups. The histology of the infected, untreated group showed endothelial cell derangement in the central vein (CV) and hepatic necrosis. The histological section of groups treated with standard drugs and 1000 mg/kg bwt extract showed the impression of normal histology. Conclusion: This study has established the antiplasmodial activity of Maesobotrya barteri . The study also confirmed that the plant has hematopoietic potential and hepato-enhancing property. These findings support the folkloric use of the plant in the treatment of malaria.


INTRODUCTION
Phytochemicals are naturally occurring bioactive substances. They are found in different parts of plants in different concentrations. They are deemed valuable for their various functions [1] amongst which are: They play important protective and disease preventive role in plants and other organisms including humans, some of them attract pollinating agents, whereas others prevent predators. Due to the therapeutic properties of phytochemicals, most are now employed for culinary uses [2]. Recently, phytochemicals have attracted much interest from researchers in various fields because of their various applications [1]. They are valuable biosource of drugs used frequently in traditional systems of medicine, modern medicine, pharmaceutical intermediates and as leads in drug synthesis [3]; They have been reported to be responsible for the antimicrobial, antibiotic, anticancer, antihelminthic and antisickling properties of many agents [4]. Significant bioactive components found in plants include; alkaloids, flavonoids, tannins, phenolic compounds and others [5]. The extraction of these phytochemicals and their application has been the focus of advances in science of late [4].
The knowledge derived from their application is being harnessed daily in health care provision.
Malaria is a mosquito transmitted infectious disease caused by plasmodium species with symptoms such as fever, headache, shivering, nausea, anemia, hepatomegaly etc [6]. The most severe form of the disease is caused by Plasmodium falciparum [7]. This disease is common in the tropics, being prominent in regions like the Central and Latin America, sub Saharan Africa and some parts of Asia. Parham et al. [8] reported that they are a major cause of morbidity and contribute significantly to the total recorded death in these areas. The socioeconomic impact of malaria endemicity has cost implication which include money value of lost workdays, reduced productivity owing to absenteeism and morbidity and underperformance at the job [9]. There is a wide gap in economic prosperity between malarious and nonmalarious nations citing the disparity in purchasing power as an example [10]. There has been concerted efforts towards the control and prevention of malaria. These have come in various forms like the use of mosquito nets, indoor residual spraying and the development of vaccines [11][12][13][14].
Despite these efforts, a number of factors are greatly facilitating the spread and persistence of malaria. The high cost of orthodox medicine and particularly, the current resistance to available antimalarial drugs by the parasite are among the major factors [15] . Drugs like chloroquine, quinine, primaquine and mefloquine have become ineffective in the treatment of malaria and there has been reported cases of emergence of resistance to certain artemisinin class of drugs in some areas [16].
Plasmodium berghei is a protozoan parasite that causes malaria in certain rodents. It is commonly used in the study of human malaria due to its ability to infect rodents and relative ease of genetic engineering. It is similar to other species of plasmodium which causes human malaria, has similar life-cycle to these species and causes disease in mice with signs alike to that seen in human malaria [17].
Synthetic compounds are used in the treatment of malaria [18]. Some of these drugs are effective both as curative and preventive agents. Various agents are effective against different forms of the parasite and at different life stages of the parasite [19]. The common drugs used in malaria therapy like chloroquine, mefloquine, halofantrine, lumefantrine, primaquine and artesunate belong to various drug classes like 4aminoquinolines, arylaminoalcohols, 8aminoqunoline and artemisinin [20][21][22][23][24]. Traditional plants have been used to treat malaria for thousands of years and are the source of the two main groups of modern antimalarial drugs, artemisinin and quinine derivatives, both from Artemisinia annua and Cinchona spp respectively [25]. A research finding reported that about 160 families of plants consisting of 1200 plant species have been documented as possessing both antiplasmodial and antipyretic activities and are extensively used in three continents. Among these families were those of Annonaceae, Anacardiaceae, Crassulaceae, Fabaceae and Euphorbiaceae [25]. One of such plants that form part of African rich medicinal flora, is Maesobotrya barteri. It is a member of the Euphorbiaceae family [26] and is widely spread. In Nigeria, it is well distributed and known by various names such as mmiriogu among the Ibos, Uvune in Etche land and Oruru in Benin. As of its pharmacological relevance, various communities use the plant to treat several health conditions like diarrhea, stomach ache, venereal infections, malaria etc [27,28]. It was reported that the plant contained the following bioactive compounds-tannins, saponins, cardiac glycosides, deoxysugar and terpenes [29]. Anemia is a common symptom associated with malaria. In anemic conditions, the total amount of red blood cells or the hemoglobin concentration in whole blood is decreased, hence oxygen carrying capacity of the blood is lowered [30]. Hemolytic anemia is witnessed in malaria infection due to the destruction of red blood cells. Following this, there is increased level of bilirubin in the blood and also liver enzymes like aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels are elevated [31].

Sample Preparation
The leaves of the plant were obtained and dried under shade. They were ground into fine powder using a pulverizer. The powder (2.4 kg) was macerated in absolute ethanol (99.9%) at room temperature for 72 hours. It was then filtered using a filter paper and the filtrate was condensed in a rotary evaporator. The filtrate was further evaporated to dryness in a water bath at 50°C. A greenish residue weighing 36 g was obtained. The extract was kept in air tight sample bottles in a refrigerator until needed.

Phytochemical Screening
The crude leaf extract was phytochemically screened for the presence of alkaloids, tannins, glycosides, flavonoids, anthraquinones, saponins and cardenolides employing standard procedures and tests [32].
Quantification of the phytochemical constituents was done using a BUCK M910 Gas chromatography equipped with a flame ionization detector (FID).

Acute Toxicity Test
The safety of the leaf extract of M. barteri was determined using a standard method [33]. The animals were administered orally with doses of the leaf extract ranging from 1000 mg/kg to 5000mg/kg body weight. The animals were all kept under the same conditions and observed for toxicity signs and mortality for 24 hours for a seven (7) days period. LD 50 values were calculated as geometric mean of the dose that resulted in 0 and 100% lethality.

Invivo antimalarial studies
Twenty-eight (28) albino mice weighing 20-25 g were selected, and used for this study. The study was carried out after approval by the Animal Welfare Research Ethics Committee of the University of Port Harcourt, Rivers State Nigeria. Animal experiments were conducted in accordance with the internationally accepted principle for laboratory animal use and care [34]. Chloroquine sensitive strain of Plasmodium berghei (NK 65) was obtained from the Malaria Research and Phytotherapy Centre, University of Port Harcourt, Port Harcourt.
Twenty four (24) of the mice were injected intraperitoneally with 1x10 6 Plasmodium berghei (NK 65 strain) parasitized red blood cells in 0.5ml of water. After 72 hours, the infection was confirmed by viewing the Giemsa stained thin blood films made with drops of blood from the tail of the infected mice and studied under the microscope (magnification ×100) [35]. The animals were distributed into seven groups and received oral treatments as given below: Group 1: Normal mice fed ad libitum with normal feed and distilled water. Group 2: Infected mice treated with 10 ml/kg bwt distilled water daily. Group 3: Infected mice treated with 25 mg/kg bwt chloroquine. Group 4: Infected mice treated with 4 mg/kg bwt artesunate. Group 5: Infected mice treated with 500 mg/kg bwt M. barteri. Groups6: Infected mice treated with 800 mg/kg bwt M. barteri Group 7: Infected mice treated with 1000 mg/kg bwt M. barteri.
Group 2 served as the negative control, and group 3 was the positive control. Thin blood films were made daily for the 4 days of administration with blood obtained from the tail of the animals to determine the number of parasitized red blood cells (parasite density). On the fifth day, the animals were sacrificied after subjecting to mild anaesthesia using chloroform. Blood was collected by cardiac puncture and transferred to EDTA and heparin bottles which were used for hematological and biochemical tests respectively. The liver was harvested for histological studies.
The percentage parasitemia was determined by microscopic examination using the formula: The average percentage parasite inhibition was determined using the formula: Av % inhibition = (Av. Parasitemia in Negative Control-Av. Parasitemia in treated group/ Av. parasitemia in Negative control)×100]

Hematological Investigations
The haematocrit was determined using the method of Green and Ezilo [36]. Haemocytometer method was used to estimate the number of white blood cells. Leukocyte differential count was determined using the formula of Osim et al. [37]. The haemoglobin concentration was determined by haemometric method. The erythrocyte sedimentation rate (ESR) was determined by Westergreen method.

Liver enzymes
The activity of the enzymes, aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) were determined by kinetic methods with Randox Kits [36].

Total bilirubin (T.Bil) concentration
To determine the total bilirubin level, the method of Jendrassik and Grof [37] was used.

Histological Techniques
To generate permanently stained sections, small portions of the liver were fixated in formolsaline, dehydrated in various concentrations of alcohol, cleared with xylene and embedded. They were sectioned using the microtome and stained with haematoxylin and eosin [38].

Statistical Analysis
The data obtained from the experiment were expressed as mean + SEM. One way analysis of variance (ANOVA) was used to analyze the data. Multiple comparison was done using post-Hoc. The values were considered to be statistically significant at p <0.05 [39]. Table 1 shows the preliminary phytochemical screening of the ethanolic extract of Maesobotrya barteri. The result reveals that the plant contained alkaloids, flavonoids, steroids, carbohydrates, cardenolides and saponins. Flavonoids was the most abundant with kaempferol having a concentration of 45.5ug/g, rutin, a concentration of 27.ug/g, and epicatechin and catechin present in the concentrations of 7.4 ug/g and 25.2 ug/g respectively. (Table 2). This finding is supported by the report of Ogwuche et al. [40] which stated that the aerial parts of M.barteri had these secondary metabolites in addition to tannins. According to Ojewole [41], the pharmacological relevance of ethnomedicinal plants is due to their phytochemical composition. As such, the active ingredients present in the plant under study is highly implicated in its pharmacological property. For instance, there has been reports of the antiplasmodial property of alkaloids [42]. The presence of triterpenoids, saponins and glycosides was reported to be responsible for the antiplasmodial activity of the root extract of Haematostaphis barteri [43]. The steroids tumacone and tumacoside have been shown to inhibit the growth of P. falciparium [44]. The result of the acute toxicity studies conducted is contained in Table 3. The result shows that the plant extract did not cause mortality when administered orally at concentrations of 5,000 mg/kg, 3000 mg/kg, 2000 mg/kg and 1000 mg/kg body weight with the calculated median lethal dose of about 2250 mg/kg body weight. The plant could be deemed safe for administration without concern for hepatic or cellular damage. It is in keeping with the findings of Hodge and sterner [45] and Ghosh [46] who reported that with such concentration as median lethal dose, a plant could be considered relatively safe.

RESULTS AND DISCUSSION
The mean number of parasitized red blood cells for each group of animals is presented in Table  4. The result revealed that there was a significant parasitemia reduction (P<0.05) when compared to the negative control. The reduction in parasitemia level at the three doses of the extract were similar to those observed for the reference drugs-25mg/kg chloroquine and 4mg/kg artesunate. The 1000 mg/kg bwt extract had the highest parasitemia reduction by the fifth day post administration. The average percentage parasite inhibition/clearance were calculated for the three doses of the extract to be 95.5%, 95.3% and 97.2% (Table 5).The treatment with the plant extract showed a lowering of the parasite density in all extract treated groups. M.barteri belonging to the family Euphorbiaceae have been previously reported to have antimicrobial activity [28]. The research finding of the study conducted by Ogwuche et al. [39] revealed that the triterpene B-amyrin isolate from the aerial parts of M.barteri exhibited biological activity against various microbial culture growth. Its use in the traditional treatment of malaria has been reported although the scientific basis has not been established. From the results of the present study, M. barteri showed a significant antimalarial activity against Plasmodium berghei in vivo. The antimalarial action is dose dependent since the highest dose of the plant extract has the greatest parasite clearance. ( Table 5). The lower and median doses had similar percentage parasite inhibition. Deharo et al. [47] states that in vivo antimalarial activity of plant extracts can be categorized as moderate, good and very good if the plant extract showed 50% or more chemosuppression/ inhibition at 500,250 and 100 mg/kg/day extract dose respectively. This suggests that the plant's antimalarial property can be rated as being moderate. Similar reports have rated other plants having antimalarial potential based on this scale [48,49]. Languas galangal and Agelanthus dodeneifolius have been shown to have similar suppressive action as M. barteri [50]. The standard drugs showed a chemosuppression/inhibition that could be deemed very good and this is in keeping with previous reports [51]. Chloroquine is reported to act by interfering with the parasites iron metabolic machinery [52]. Artemisia annua, Annona senegalensis and Addhathoda schimperiana have been suggested to follow same mechanism of action, owing to the fact that their bioactive compounds like alkaloids are the parent compound for synthetic products like chloroquine [53,54]. Flavonoids have been detected in the artemisia species and have been reported to show significant anti-material activity against P. falciparium [53]. These set of compounds (alkaloids and flavonoids) were identified in the plant, hence the allusion that the presence of these secondary metabolites could be the reason for the plant's therapeutic action.  The effect of the plant extract on some hematological indices is shown in Table 6. The effect of different concentrations of the plant on PCV shows a significant increase (P<0.05) for the treated groups when compared to the negative control; the PCV levels for the treated groups were similar to the value for the positive control. Comparing the ESR of the treated groups to the negative control showed a decrease which was not statistically significant (P>0.05). The haemoglobin concentration for the treated groups showed a significant (P<0.05) increase relative to the negative control; the WBC count for the treated groups showed an increase when compared to the negative control; the levels of eosinophils and basophils were elevated in the treated groups whereas the Neutrophil level was significantly (p<0.05)

Fig. 1. Chromatogram of the phytochemical composition of M. barteri
lowered relative to the negative control. The decreased WBC count in the infected, untreated group has been attributed to the damaging effect of the parasite in hematopoietic stem cells and hence the progression of the disease [55], this is because white cells are first line of defence in the incidence of infection [56]. The extract increased WBC count suggestive of the ability of the extract to increase the production of the cells of the immune system, thereby increasing the ability of the mice to combat the infection. The increase in levels of eosinophil and basophil is consistent with the findings of Bakhubaira [57]. The lowering of the neutrophil level on day 5 post administration could be suggestive of normalization of immune response following recovery; this is noteworthy because leukocytosis is indicative of pathological condition [58]. Upon administration of plant extract, PCV level was elevated, implying the amelioration of the hemolytic condition occasioned by infection. This is indicative of the hematopoietic potential of the plant extract. This is in line with findings of Ovuakporaye [59] which stated that plants with antimalarial property always increased the levels of the blood elements. The mechanism of action has been suggested as being by a direct effect on the reticuloendothelial system or by directly eliminating parasites via radical generation [60]. In the infected and untreated group, the haemoglobin concentration was lowered; this is consistent with the finding of George and Ewelike-Ezeani [61]. The reason has been given as due to the destruction of the red blood cells. Extract administration elevated the concentration of haemoglobin, suggesting that the extract may have had stimulatory effect on red cell production (erythropoiesis). The findings of this study agrees with the report of a previous study stating that ESR level increase during attack with malaria [62]. In a bid to effect cure, the plant extract caused alteration in haematological indices, an observation which has been made in a previous report [63].
The level of the liver enzymes (AST, ALT and ALP) were significantly lowered (p<0.05) when compared to the negative control. The total bilirubin concentration was significantly (p<0.05) elevated in the negative control when compared to the treated groups. The concentration of total bilirubin for the treated groups were similar to the value observed for the positive control ( Table 7). The biochemical tests can be checked to follow hepatocellular integrity and liver injury, especially in cases of infection. The levels of these enzymes provide valuable confirmatory and suggestive basis for assessing liver status [64], [65]. The administration of the plant extract caused a decrease in the activities of AST, ALT and ALP. There is a liver stage in the life cycle of the parasite in the course of the progression of the disease. The elevated levels of these enzymes in the infected, untreated group may be attributed to damage to the liver via oxidative stress upon infection; the lowering of the activity of these enzymes could be said to be as a result of the healing of the tissue damage by the extract. Hillwell [66] explained that plants containing flavonoids have good anti-oxidant property. It's reasoned that the healing was achieved via mopping up of the free radicals by flavonoids contained in the plant extract. The total bilirubin concentration was lowered upon extract administration, further establishing the positive effect of the extract on the liver. This is important, recalling that the lysis of red cells in malaria is largely responsible for increased plasma concentration of total bilirubin. The histopathology results are shown in Figs. 2-7.  Fig. 7 is the histology of mice treated with 1000 mg/kg bwt extract. The features include central vein which is congested and normal hepatocytes. The impression is a normal histology. The endothelial cell derangement in the central vein (CV) and hepatic necrosis evidenced in the negative control can be attributed to the exoerythrocytic stage of the parasite's life cycle in the liver. This is in agreement with the report by Prudencio [67] which states that motile sporozoites migrate and infect hepatocytes forming parasitophorous vacuoles. The disruption in the plasma membrane of the hepatocytes induces spontaneous necrosis of the affected liver cells. Partial or total blockage of hepatic arteries and lactic acid generation also contribute to the cellular derangement. For the extract treated groups, the group treated with 1000 mg/kg bwt of the extract showed no evidence of hepatic necrosis. The histology was similar to that of the standard drug treated groups. The parasitophorous vacuoles closing up indicates the healing of the liver by the plant extract [68]. It can be inferred that the extract mopped up most free radicals generated by the presence of Plasmodium berghei and helped stabilize the plasma membrane of the hepatocytes thereby reducing any possible necrotic effect caused by the parasite.
Haematoxylin and Eosin stained

CONCLUSION AND RECOMMENDA-TION
This study has shown that Maesobotrya barteri possesses antimalarial activity. The plant also increased the levels of haemoglobin, packed cell volume and white blood cells while lowering the levels of AST, ALT, ALP and total bilirubin, implying that the plant has both hematopoietic and hepatic-enhancing functions. These effects further establishes its antimalarial potential. However, further studies should be carried out to understand how the combination of the plant with other plants with known antimalarial activity can boost its therapeutic potency.