Anti-venom Activity of Mucuna pruriens Leaves Extract Against Cobra Snake ( Naja hannah) Venom

work collaboration between all Author OOS designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. Authors PMN and ODO managed the analyses of the study. Author GOA managed the literature searches and wrote the second draft of the manuscript. All authors read and approved the final manuscript. ABSTRACT Aim: The study was done to investigate the anti-venom activity of Mucuna pruriens leaves extract against cobra snake ( Naja hannah) venom. Study Design: The mice were randomly grouped into six groups (A, B, C, D, E, and F) of five rats each. Group A served as the normal control (no induction), and the mice in the group were given normal saline (1ml/kg/body weight).Group B served as the test control (snake venom was induced but no treatment administered), Group C served as the standard control (snake venom was induced and treated with antivenin, a venom and thereafter the treatment with M. pruriens extract for Group D, E and F were done with 40 mg/ kg, 60 mg/ kg and 80 mg/ kg respectively intraperitoneally in the mice. Serum blood of the animals was used to assay for total cholesterol, bilirubin, AST, ALT, GSH and catalase levels after 14days. Result: The injection of crude venom of cobra snake ( Naja hannah) caused an increase in cholesterol, AST, ALT, bilirubin, catalase and glutathione in envenomated mice which significantly reduced (p<0.05) compared to all the controls after 14 days of treatment with the extract. Conclusion: The results suggests that 80 mg/ kg of the plant extract is more effective than the standard drug, therefore M. pruriens leave s has a greater anti-venom potential for curing snake bite, than antivenin.


INTRODUCTION
Each year in the world a lot of people receive venomous bites by snake and about 40, 000 of them die [1]. Echis carinatus and Naja hannah are the major causes of snakebite deaths in Plateau State, Nigeria. Snake venoms of Viperidae and Elapidae are known to consist of a complex mixtures of toxins and enzymes which are responsible for haemorrhage, myonecrosis, neurotoxicity and alteration of blood coagulation [2,3].
The only effective treatment of the modern medicine for serious snakebites is the use of the antidote (antivenin), derived from antibodies, produced in horse's blood serum after injecting the animal with snake venom. In humans, antivenin is administered either through the veins or injected into muscle and acts by neutralizing the snake venom which has entered the body. Because antivenin is obtained from horses, snakebite victims sensitive to horse products must be very carefully treated. The danger is that they could develop an adverse reaction or even an anaphylactic shock. Moreover the efficacy of the serums is dependent on the rapidity with which the specificity treatment is started and it is only specific for the venom used for the immunization. These represent an important limitation of this kind of therapy.
The genus Mucuna, belonging to the Fabaceae family, sub-family Papilionaceae, includes approximately 150 species of annual and perennial legumes. Among the various underutilized wild legumes, the velvet bean Mucuna pruriens ("Cowitch" and "cowhage" are the common English names) is widespread in tropical and sub-tropical regions of the world. It is considered a viable source of dietary proteins [4,5] due to its high protein concentration (23-35%) in addition its digestibility, which is comparable to that of other pulses such as soybean, rice bean, and lima bean [6]. It is therefore regarded as a good source of food.
M. pruriens is a popular Indian medicinal plant, which has long been used in traditional Ayurvedic Indian medicine, for diseases including Parkinsonism [7]. The beans have also been employed as a powerful aphrodisiac [8] and have been used to treat nervous disorders and arthritis [9]. The bean, if applied as a paste on scorpion stings, is thought to absorb the poison [9]. The non-protein amino acid-derived L-dopa (3, 4-dihydroxy phenylalanine) found in this underutilized legume leaves resists attack from insects, and thus controls biological infestation during storage. According to D'Mello, all anti-nutritional compounds confer insect and disease resistance to plants [10]. Further, L-dopa has been extracted from the leaves to provide commercial drugs for the treatment of Parkinson's disease. L-Dopa is a potent neurotransmitter precursor that is believed, in part, to be responsible for the toxicity of the Mucuna leaves [11]. Antiepileptic and anti-neoplastic activity of ethanolic extract from M. pruriens had been reported [12].
An ethanolic extract of M. pruriens leaves has demonstrated significant in vitro anti-oxidant activity, and there are also indications that ethanolic extracts of Mucuna puriens may be a potential source of natural anti-oxidants and anti-microbial agents [13]. All parts of M. pruriens possess valuable medicinal properties and it has been investigated in various contexts, for its anti-diabetic, aphrodisiac, anti-neoplastic, anti-epileptic, and anti-microbial activities [7]. Its anti-venom activities have been investigated and its anti-helminthic activity has been demonstrated [14,15]. M. pruriens has also been shown to be neuroprotective [16], and has demonstrated analgesic and anti-inflammatory activity [17]. M. pruriens has been reported to be a potential natural source of antioxidants with greater importance as a therapeutic agent in preventing or slowing oxidative stress related degenerative diseases [18]. In Nigeria, where the M. pruriens seeds are locally prescribed as an oral prophylactic for snake bite, it is claimed that when two seeds are swallowed, they protect the individual for a year against snake bites [19]. Therefore, the primary objective of the present study is to confirm the bioactivity of M. pruriens leaves extract against cobra snake (Naja hannah) venom.

Collection and Preparation of the Plant Sample
The fresh leaves of M. pruriens were collected from Kogi State, Nigeria. It was identified by Dr. S. M. Ayodele (Dept. of Botany, Kogi State University). The leaves were dried under room temperature for 5days. The dried sample was ground to powdery form, using the warring commercial blender.

Extraction of M. Pruriens Leaves
The plant material (500g) was defatted with 400ml hexane (C 6 H 14 ) by using a Soxhlet apparatus for 5h. The defatted powder plant material was air-dried. The air-dried defatted powdered plant material was then extracted with 400 ml ethanol (C 2 H 5 OH) by using a Soxhlet apparatus for 8h. The residue was dried over night and extracted with 250 ml water (H 2 O) by using a shaking water bath at 70ºC for 2h. The extraction with water was repeated three times. The water filtrates were mixed together. The ethanol and water extract were filtered and evaporated by using a rotary evaporator and freeze dryer to give the crude-dried extract. The dried extracts were stored at -20ºC until used.

Calculation:
Percentage yield (%) = weight of the plant extract Weight of the dried plant used × 100

Proximate Analysis of M. pruriens
The moisture content, ash content, carbohydrate content, crude fibre and crude protein were determined using methods as described by AOAC [20].

Phytochemical Screening
The ethanolic extract of M. pruriens leaves were screened for the presence of phytochemical compound as described by Treatise and Evans [21] and Sofowora [22].

Animals
Experimental mice were purchased from the animal house of NIPRID. The animals were housed in steel cages and kept at room temperature. The mice had no history of drug consumption that is; they had not been used for any investigation. The mice were put on standard mice pellet (feed) and pure drinking water and allowed to get acclimatized for 21 days before the start of the experiment. The study was done in accordance with the guidelines for animal use of Faculty of Sciences, Bingham University and had the project ID: BCH/02/012.

Induction of Anti-Snake Venom
The cobra snake venom was procured in a sample bottle from National Veterinary Research Institute, Vom, Plateau State, Nigeria. The cobra snake venom native preparations were given intraperitioneally (i.p) to the mice at a dose which were proportional to the weight of the animals. The volumes of preparation were identical and the same amounts were injected. The venom dose used in this study was estimated according to our preliminary experiments.

Experimental Design
White male albino mice of wistar strain of body weight ranging between 15-30g were used for the research study. The mice were randomly grouped into six groups (A, B, C, D, E, and F) of five rats each. Group A served as the normal control (no induction), and the mice in the group were given normal saline (1ml/kg body weight).Group B served as the test control (snake venom was induced but no treatment administered), Group C served as the standard control (snake venom was induced and treated with antivenin, a standard drug), Group D, E and F were all induced with the cobra snake venom and treated with ethanolic extracts of the leaves of M. pruriens (Table 1).

Determination of Serum Cholesterol Level
Total cholesterol was determined by the enzymatic endpoint method as described by Trinder [23]. In this method, cholesterol was determined after enzymatic hydrolysis and oxidation in a series of reaction. The indicator quinoneimine used in this method was formed from hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxidase.

Determination of Serum Bilirubin Level (Vander Bergh's Reaction)
The total bilirubin in serum was determined using the method of Jendrassik and Gróf [24] by coupling with diazotized sulfanilic acid after the addition of caffeine, sodium benzoate and sodium acetate. A blue azobilirubin was formed in alkaline Fehling solution II. This blue compound was determined selectively in the presence of yellow by products (green mixed coloration) by photometry at 578 nm.

Determination of Transaminases (ALT and AST)
Alanine Aminotransferase (ALT) was determined in a method as described by Reitman and Frankel [25]. In this method, ALT was measured by monitoring the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine in a reaction. Aspartate Aminotransferase (AST) was determined in a method as described by Reitman and Frankel [25]. In this method, AST was measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4-dinitrophenylhydrazine in a reaction.

Determination of Serum Glutathione Level
The principle was based on the reduction of 5,5 dithiobis (2-nitrobenzoic acid) (DTNB) with reduced glutathione (GSH) to produce a yellow compound. The reduced chromogen was directly proportional to GSH concentration and its absorbance was measured at 405 nm [26].

Determination of Serum Catalase Level
Catalase was determined by the method described by Cohen et al. [27]. In this method, catalase catalysed the conversion of hydrogen peroxide to oxygen and water in a reaction.

Statistical Analysis
Values are expressed as mean+S.E.M randomized complete block design analysis of variance was used for statistical analysis. P values less than 0.05 was considered significant.

Percentage Yield of Extract
The extract was thick and greenish in colour, with an ethanolic extraction of M. pruriens leaves which indicated the yield of 6.73%.

Proximate Analysis
The proximate analysis of the leaves in Table 3 has a moisture content of 11.37%, crude fiber 31.91%, crude fat 2.97%, carbohydrate 45.65%, Ash content 3.00%.

Effect of Venom Induction and Extract on Biochemical Parameters
The results revealed in Table 4a and 4b that, the injection of crude venom of cobra snake (Naja hannah) caused an increase in cholesterol, AST, ALT, bilirubin, catalase and glutathione in envenomated mice compared to the normal control mice. There was no significant difference in the levels of cholesterol, AST, ALT, bilirubin, catalase and glutathione of the treated groups when compared to the test control group. The levels of cholesterol, AST, ALT, bilirubin, catalase and glutathione were significantly reduced when compared to the test control after 14 days of treatment with the extract (Table  5a and 5b).

DISCUSSION
The phytochemical constituents in M. pruriens leaves are alkaloids, flavonoids, tannins, saponins, steroids, terpenoids, cardiac glycosides and anthraquinones ( Table 2). The antivenom activity observed in the mice treated with M. pruriens extract may be attributed to the presence of any of these compounds alkanoids, tannins, flavonoids, steroids and terpenoid [28]. The proximate analysis of the leaves in Table 3 has a moisture content of 11.37%, crude fiber 31.91%, crude fat 2.97%, carbohydrate 45.65%, Ash content 3.00%. The Ash content is known to enhance digestibility, slow down the release of glucose into the blood stream and reduces blood cholesterol level.
The present study revealed in Table 4a and 4b that, the injection of crude venom of cobra snake (Naja hannah) caused an increase in cholesterol, AST, ALT, bilirubin, catalase and glutathione in envenomated mice which significantly reduced after 14 days of treatment with the extract as shown in Table 5a and 5b. These findings are in agreement with other investigators who reported that the reduction in cholesterol, AST, ALT, bilirubin and catalase in envenomated mice was observed in laboratory animals treated with the extracts of M. pruriens leaves [29]. It might be assumed that, the increased levels of these serum constituents could be due to disturbance in renal functions as well as haemorrhages in some internal organs when challenged with a snake venom. In addition, the increasing in vascular permeability and haemorrhages in vital organs due to the toxic action of various snake venoms have been reported [30,31]. Also, the reduction in serum cholesterol, AST, ALT, catalase, glutathione albumin and total bilirubin levels in the envenomated mice could be attributed to the anti-venom potentials of the extract of M. pruriens administered.
Furthermore, acute renal damage together with glomerular, tubular and vascular lesions following various snake bites have been reported [32,33,34] with additional, increased vascular permeability and hemorrhages in various vital organs. Another factor is the increase vascular permeability due to toxic action of the venom which could contribute to the low level of protein from plasma and tissue [35]. An earlier study reported that injection of M. pruriens seed extract conferred protection against C. rhodostoma venom, as the pretreatment attenuated (albeit only partially) the cardio-respiratory depressant effects of C. rhodostoma venoms in anesthetized rats [36].
In Table 4a, elevation of ALT and AST in the mice administered with venom as observed in the serum have serious implication on health of the animals. Such elevations are found in cases of both liver damage and myocardial infarction [37]. The elevation of AST and ALT makes the liver a target of suspicion as this is usual in cases of hepatotoxicity caused by toxic agent (Rosalki, 1974). From the experiment it was observed that some of the mice died after 30 minute of induction with the snake venom except the (Normal control) and the ones treated with different dose of the venom/extract (Groups D-F) which survived till the end of the experiment.
There was significant difference between the time of death in the extract treated group and those treated with venom only showing that the plant extract had effect on the activity of the venom. Thus, it was obvious that M. pruriens leaves did show greater anti-venom activity, the extract of M. pruriens showed a better anti-snake activity compared with antivenin. This can be attributed to the fact that tannins are able to non-specifically bind to Naja hannah venom proteins and precipitate them, thus provoking the anti-lethal effects. However, a report suggested that the protective effect of M. pruriens extract pretreatment against cobra venom involved a direct action of M. pruriens extract on the heart, and, as such, immunological neutralization is not the only mechanism of protective effect of the M. pruriens extract pretreatment [38]. Also, the protective effect of M. pruriens seed against snake venom poisoning has been reported [39], which corroborates the results of this study that M. pruriens plant has great potential for use in the treatment of common cobra bites from the Naja subspecies.

CONCLUSION
In conclusion, this investigation revealed that the ethanolic extract of M. pruriens leaves has the following phytochemical components of saponin, terpenoid, flavonoids, steroids, alkaloids, anthraquinones. It was observed that 80 mg/ kg of the plant extract is more effective than the standard drug, therefore M. pruriens leaves has a greater medicinal plant for curing snake bite, than anti-venin.

CONSENT
Not applicable.

ETHICAL APPROVAL
All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.