Characterization of anti-plasmodial, analgesic and anti-inflammatory fraction of Maytenus senegalensis (lam.) Exell leaf extract in mice

The treatment inadequacy and toxicity associated with conventional anti-malarial, anti-inflammatory and analgesic drugs has called for the search of alternatives from medicinal plants, particularly, their phytochemicals with inherent pharmacological properties. In the present study, purified fraction of M. senegalensis leaf was evaluated for antimalarial, anti-inflammatory and analgesic properties. Antimalarial study was conducted against Plasmodium chabaudi and Plasmodium berghei using 4 days suppressive test, while anti-inflammatory and analgesic studies were conducted using egg albumin induced paw oedema and acetic acid induced pain model respectively. Sub-acute toxicity was assessed using serum biochemical parameters following 3 weeks administrations of the purified fraction. The purified fraction of M. senegalensis leaf shows dose dependent antiplasmodial activity with percentage curative effects of 15.24 ± 0.89, 45.70 ± 3.43 and 48.50 ± 4.56 at 75, 150 and 300 mg/kg bw against Plasmodium chabaudi and % curative effects of 44.25 ± 3.21, 72.74 ± 6.54 and 76.30 ± 8.32 respectively against Plasmodium berghei. The purified fraction exhibited 53.16 ± 4.09 and 60.76 ± 7.54 anti-inflammatory effect, 43.35 ± 4.98% and 44.83 ± 3.86% analgesic effect at 75 and 150 mg/kg bw respectively. GC-MS analysis confirmed the presence of 20α)-3-hydroxy-2-oxo-24-nor-friedela-1(10),3,5,7-tetraen-carboxylic acid-(29)-methylester, 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro- and 3-hydroxy-20(29)-lupen-28-ol and a terpenes (phytol) as the major antimalarial compounds in the fraction. The purified fraction increases the serum total proteins and transaminases concentrations but had no effect on serum levels of sodium, potassium, chloride, alkaline phosphatase, triglyceride and glucose in the mice. The purified fraction of M. senegalensis leaf exhibited promising antimalarial, analgesic and anti-inflammatory activities. Thus, could serve as a template for the synthesis of new drug.


Introduction
Malaria is an infectious protozoic and parasitic disease caused by five Plasmodium parasites: vivax, falciparum, malariae, Knowlesi and ovale [1]. More than half of the world's population are at risk of malaria, with about 212 million new case and 429, 000 death annually [2]. Sub-Saharan Africa accounts for over 90% of the malaria cases and deaths predominantly in children of age below five years and pregnant women [2]. Poor rural dwellers in in tropical and subtropical areas are highly vulnerable to this attacked owing to the favorable and ideal climatic condition for the reproduction and development of vectors, and parasites [3]. In addition, drug resistance is one of the major challenges facing malarial eradication program word wide [4].
Inflammation and pains are gaining research popularity owing to the etiologic role they play in various human diseases [5]. Dexamethasone, opioids, morphine and aspirin and other drugs have been established for the management of pain and inflammation; however, these drugs have recorded limited success due to unintended effects such as gastric lesions caused by nonsteroidal anti-inflammatory drugs [6,7]. Thus, the search for drugs alternative from natural product is recommended.
Natural products contain metabolite that has therapeutic values for uses in the managements of several diseases [8,9]. The therapeutic effect plants are however, associated with their secondary metabolites they contain, particularly the alkaloids, terpenoids, and flavonoids, which are known to play defensive role in plants but exihibited different pharmacological effects in human/ animals [10].
Maytenus senegalensis (Lam.) Exell is an African medicinal plant, commonly used traditionally for the treatment of a number of ailments, including rheumatism, snakebites, diarrhoea, eye infection, and dyspepsia [11]. Previous study has demonstrated that the extracts from various parts of M. senegalensis possess in vitro antiplasmodial, anti-leishmanial, and antibacterial activities [12]. However, literature survey revealed dearth of scientific information on the pharmacological activities of purified fraction. The present study, therefore evaluated the antiplasmodial analgesic and anti-inflammatory effects of purified fractions from Maytenus senegalensis (Lam.) Exell leaf extract in mice.

Experimental animals
A total of ninety (90) adult swiss albino mice weighing 25.34 ± 0.98 g were obtained from National Veterinary Research Institute (NVRI), Vom, Plateau State of Nigeria. The mice handling and experimentation was in concordance with the guidelines for laboratory animal use and care as contained in the European Convention on Animal Care Guidelines and Protocol.

Parasite
Plasmodium chabaudi and Plasmodium berghei NK65 chloroquine-sensitive strain was obtained from National Institute of Pharmaceutical Research and Development (NIPRD) Abuja, Nigeria and maintained in the laboratory by serial passage in mice.

Sample preparation
The plant Maytenus senegalensis (Lam.) Exell was collected from Bida, Niger state. The plant was authenticated by a botanist, from Department of Biological sciences, Federal university of Technology Minna, Nigeria. The leave was cleaned and air-dried at room temperature. The dried leave was pulverized into a coarse powder using mortar and pestle. The pulverized sample was stored in air-tight container.

Extraction and purification of Maytenus senegalensis (lam.) Exell fraction
Maytenus senegalensis (Lam.) Exell leaf (50 g) powder was moistened with 200 mL of 95% ethanol, alkalinified with 200 mL of ammonia solution and macerated for 24 h followed by extraction with ethanol. The ethanol extract was filtered, concentrated and treated with 1.0 N hydrochloric acid. The filtrate was further alkalinified with ammonia solution and the extract was obtained by fractionation in separating funnel using chloroform [13]. The fraction was purified and subjected to thin layer chromatography to obtained the pure fraction (0.6 g) for structural elucidation [13].
Anti-Plasmodial screening of the purified fraction of Maytenus senegalensis (lam.) Exell Four days (4) suppressive test were used to evaluate the antimalarial properties of the purified fraction of Maytenus senegalensis (Lam.) Exell as described by Jigam et al. [14]. A total of 15 P. berghei infected mice were randomly grouped into five (I-V) of 3 mice each. Groups I -III animals were treated with 75, 150 and 300 mg/kg body weight each of purified fraction. Groups IV and V received normal saline (2 ml/kg body weight) and chloroquine (5 mg/kg body weight) to serve as negative and positive controls respectively. The same procedures (Four days (4) suppressive test) were repeated for Plasmodium chabaudi All the treatments were done orally for 4 consecutive days. Daily parasitaemia count was carried out by preparing a Giemsa stained-thin film and viewed under microscope as described by Jigam et al. [14].

Anti-inflammatory study
Anti-inflammatory activity of the purified fraction was tested using egg albumin induced paw oedema in mice according to the methods of Winter et al. [15]. A total of twelve (12) mice were randomly grouped into four (A-D) of 3 mice each and were administered a single dose of 75 and 150 mg/kg bw of the purified fraction, 150 mg/kg.bw acetylsalicylic acid and 2 ml/kg bw normal saline respectively 30 min before the injection of the albumin into the right hind limb. The percentage inhibition of oedema was calculated for each dose using the formula:

Analgesic study
Analgesic effect was assessed according to the method described by Nwafor et al. [16]. A total of twelve (12) mice were randomly grouped into four (A-D) of 3 mice each and were administered a single dose of 75 and 150 mg/kg bw of the purified fraction, 150 mg/kg.bw sodium diclofenac and 2 ml/kg bw normal saline respectively for 60 min before they were challenged with 0.75% v/v acetic acid. Group D (control group) received 2 mL/kg body weight of normal saline. The number of abdominal constrictions induced by acetic acid were counted after 5 min. Observations were made over 10 min and mean value for each group was calculated. Percentage inhibition of abdominal constriction by the purified fraction and sodium diclofenac were determined in relation to the control. Toxicological study Animals (5 each) were dosed 0 (control), 75 mg/kg and 150 mg/kg bwt of purified fraction of Maytenus senegalensis (Lam.) Exell orally for 3 wks. Procedures described by Shittu et al. [17] was followed during blood sample collection and serum preparation for biochemical analysis. Serum biochemical parameters including alkaline phosphatase (ALP), Aspartate transaminase (AST) and alanine transaminase (ALT) were determined as described previously [18]. The concentrations of serum total proteins [19], sodium, potassium, and chloride [20] were determine using standard methods.

Gas chromatography and mass spectroscopy (GC-MS) analysis of bioactive compounds
The purified fraction of Maytenus senegalensis (Lam.) Exell was subjected to Gas Chromatography and Mass Spectroscopy for the determination of bioactive volatile compounds as described previously [21].

Data analysis
Data analysis was performed using Statistical Package for Social Sciences (SPSS) One-way Analysis of Variance (ANOVA) followed by Duncan Multiple Range Test (DMRT). Data were expressed as means ± SEM of triplicate determinations. Significant was considered at p < 0.05.

Anti-inflammatory
The purified fraction of M. senegalensis leaf exhibited dose dependent inhibition egg albumin-induced paw oedema with percentage inhibition of 53.16 ± 4.09 and 60.76 ± 7.54 at 75 and 150 mg/kg bw respectively while ASA exhibited 63.29 ± 5.98 inhibition of paw oedema (Table 3).

Analgesic effect
The purified fraction of M. senegalensis leaf exhibited dose dependent abdominal constrictions with percentage inhibition of 43.35 ± 4.98and 44.83 ± 3.86 at 75 and 150 mg/kg bw respectively while sodium diclofenac (SD) exhibited 74.88 ± 6.87 inhibition of paw oedema (Table 4).

Biochemical parameters
Sub-chronic administration of the purified fraction of M. senegalensis significantly (p < 0.05) increase the concentrations of transaminases (aspartate transaminase and alanine transaminase), and proteins when compared with the untreated control. However, sodium, potassium, chloride, alkaline phosphatase, triglyceride and glucose concentrations were not (p < 0.05) significantly altered by treatment with purified fraction of M. senegalensis (Table 5).

Discussion
The anti-plasmodial potency of some plants has been associated with the presence of some secondary metabolites such as alkaloids [9]. Findings presented in Table 3 shows that the purified fraction of M. senegalensis demonstrated a good antimalarial activities, in concordance with the classification of Munoz et al. [22], which stated that the antiplasmodial agent are classified on the basis of the percentages parasite inhibition as moderate", "good", and "very good when there is percentage inhibition of above 50% at metabolite concentration of 500, 250 and 100 mg/kg bwt. However, the antiplasmodial effects of the purified fraction at 150 and 300 mg/kg bw not significantly difference (p > 0.05), this may suggest that the maximum anti malaria effect of the purified fraction was achieved at 150 mg/kg. The proposed mechanism of antiplasmodial effect of the purified fraction could be by the elevation of erythrocytes oxidation and inhibition of the plasmodium protein synthesis, a mechanism that has been attributed to antimalaria activities of some phytoconstituents [23].
Evidence for the anti-inflammatory properties of flavonoids and alkaloids have been reported by several studies using different models of inflammation [24][25][26]. The significant anti-inflammatory effects demonstrated by the purified fraction of M. senegalensis leaf could be mechanistically explained by the fact that phyochemicals are known to inhibit the enzymes involved in the production of inflammatory mediator including cyclooxygenase and 5-lipoxygenase pathways [27].  The present study revealed that the purified fraction of M. senegalensis leaf significantly decreased the acetic acid induced pain in mice. This study showed that the purified fraction of M. senegalensis leaf contains active analgesic component [28]. This finding is in concordance with previous studies on morphine alkaloid fraction of Stephia glabra known as Gindarudine, which showed significant analgesic effect when tested by the same method [29]. The significant analgesic and anti-inflammatory effects of the purified fractions of M. senegalensis leaf extracts in vivo is noteworthy. Plants with these added pharmacological phenomena in conjunction with antiplasmodial effects are better antimalarials than plants with the later potential only [30,31].
Biochemical parameters have been widely used as an indicator of pathological condition, toxicology or safety of a test substance, treatment outcome and general health status of animals [32][33][34][35]. Among these biochemical parameters, transaminases, alkaline phosphatases, proteins, lipid profile and electrolyte are the most widely employed in assessing the liver and kidney integrity following plant extract administration to animals [33]. Alterations in the normal activities or concentrations of these biochemical parameters are conventional indicators of any of the following conditions; renal or nephrotic impairments, hepatocellular injury, cellular leakage, loss of functional integrity of cell membrane, biliary cirrhosis or liver hepatitis [32]. Consequently, the concentrations of triglyceride, sodium, potassium, chloride, alkaline phosphatase and glucose concentrations were not significantly (p < 0.05) altered by treatment with 75 and 150 mg/kg bw M senegalensis purified fraction. This is an indication that the functional integrity of kidney is well preserved and that the purified fraction of M. senegalensis does not induced any form of pathological conditions to the kidney. The increases in transaminases (aspartate transaminase and alanine transaminase), and proteins concentration is an indication that the liver integrity is not well preserved. The purified fraction might have interfered with the equilibrium in protein metabolism in favor of anabolism. Such drastic increase in protein levels could, negatively affect cellular homeostasis and consequently effect the health of the animals [36,37].

Conclusion
The purified fraction of M. senegalensis leaf exhibited promising antimalarial, analgesic and anti-inflammatory activities. Thus, could serve as a template for the synthesis of new drug.