Anti-Venom Studies on Olax viridis and Syzygium guineense Extracts

Olax viridis (Olacaceae) and Syzygium guineense (Myrtaceae) are shrubs commonly found in the tropics. They are traditional folkloric medicine for a great number of sicknesses. Olax viridis has a wide range of applications in ethnomedicine which include treatment for ulcers, veneral diseases, ringworm, sleeping sickness, diarrhea, fever. Syzygium guineense has been reported as antidiarrheal agent. Liquid from the bark and roots have been reported to act as a purgative when mixed with water. Both plants have been claimed to have antivenom properties. However, there are no scientific reports on snake venom neutralizing activities of these plants. The plant samples were collected from Olowa in Dekina Local Government Area in Kogi State, Nigeria. The chemicals and reagents used were of analytical grade. Wistar albino rats (male) weighing between 180-200 g were randomly divided into seven groups of three (3). Groups 1-7 received water, normal saline, venom, venom and Olax viridis, venom and Syzygium guineense, Olax viridis and Syzygium guineense respectively. The extracts were administered orally at the dose of 400 mg kg −1 b.w of rats and 1 h later, the venom (0.08 mk kg −1 ) was administered. Pulse rate, blood glucose, rectal temperature, plasma cholesterol, triacylglycerol, creatine kinase activity and edema were measured. Significant neutralization of the effects of Naja katiensis venom was observed in the groups of rats that received the extracts. Blood glucose, pulse rate, rectal temperature and creatine kinase activity were elevated in the untreated envenomated groups. These results suggest that oral administration of Olax viridis and Syzygium guineense extracts possess antivenom property, thus, providing the rationale for their use in treatment of sake envenomation.


INTRODUCTION
Snake bites pose a major health risk in many countries, with the global incidence of snake bites exceeding 5,000,000 per year (Williams et al., 2010). This problem is more profound in the developing countries, particularly in areas where the access to medical service and to the antiophidic treatment is challenging (Mendes et al., 2008). Although, the majority of snake species are non-venomous and typically kill their prey with constriction, venomous snakes can be found on every continent except Antarctica (Kasturiratne et al., 2008). The outcome of snake bite depends on numerous factors including species of snake, the area of the body bitten, the amount of venom injected and the condition of the victim. Bites from non-venomous snakes can also cause injury, often due to lacerations caused by the teeth or from a resulting infection. A bite may also trigger an anaphylactic reaction, which is potentially fatal.
In many parts of the world, regular treatment for snake venom accident is serum therapy, which involves the parentheral administration of antiophidian serum (antivenoms). This therapy efficiently neutralizes the systemic toxic effects, preventing death of victims. However, antivenoms have some disadvantages, thus limiting their efficient use (Chippaux and Goyffon, 1998; Science Publications AJPT Heard et al., 1999;Silva et al., 2007): For example, they can induce adverse reactions ranging from mild symptoms to serious (anaphylaxis) and in addition they do not neutralize the local tissue damage (Gutierrez et al., 2009). Thus, complementary therapeutics needs to be investigated, with plants being considered as a major source (Soares et al., 2005).
In many countries, plant extracts have long been in use traditionally to treat envenomation (Mors et al., 2000). The exact mechanisms of action of the plant extracts remain largely illusive, however, a number of previous reports indicate that plant-derived compounds such as rosmarinic acid Aung et al., 2010), quercetin (Nishijima et al., 2009) and glycyrrhizin (Assafim et al., 2006) can inhibit biological activities of some snake venoms in vivo and in vitro.
Olax viridis (O. viridis) has a wide range of application in ethnomedicine. In West Africa, the pulverized bark and root are used as dressing for ulcers and treatment of veneral diseases, ring worm (Watt and Bregar-Brandiwik, 1962). In the northern part of Nigeria, the root is used in the treatment of sleeping sickness, as anti-diarrheal agent and the treatment of febrile headache. The leaves are also used as remedy for cough, fever and wound (Ajali and Okoye, 2009).
Syzygium guineense (S. guineense) fruits are used as remedy for dysentery. In traditional medicine, liquid from the pounded bark and roots has been reported to act as purgative when mixed with water. The present study was carried out to determine the venom neutralizing effects of O. viridis and S. guineense in rats.

Plant Material
Fresh leaves of Olax viridis and Syzygium guineense were collected from farms located in Olowa in Dekina Local Government Area of Kogi State, Nigeria. The fresh leaves were rinsed with clean water to remove dirt and were air-dried in the laboratory for three weeks and pulverized into fine powders using mortar and pestle. Prior to air-drying, the plant samples were identified in the Department of Biological Sciences (Botany unit), Kogi State University, Anyigba, Nigeria. A voucher specimen has been kept.

Preparation of Plant Extracts
Powdered samples (200 g) each were extracted using cold maceration for 48 h in 1000 mL −1 of methanol. The mixtures were there after filtered. The solvent from the total extract was distilled off and the concentrate was evaporated on a water bath to a syrupy consistency. The percentage yields of the extracts were 14.5 and 3.55% for O. viridis and S. guineense respectively.

Animals
Wistar albino rats (male) weighing between 180-300 g was obtained from Mr. Friday Emmanuel, Department of Biochemistry, Kogi State University, Anyigba, Nigeria. This study was approved by the Department of Biochemistry according to the Institutional ethics. These animals were used as approved in the study of snake venom toxicity. Rats were allowed to acclimatize for two weeks with access to clean water and animal feeds (supplied by Top feeds, Anyigba) in the experimental site. They were maintained in standard conditions at room temperature, 60±5% relative humidity and 12 h light dark cycle.

Animal Grouping and Treatment
The wistar albino rats were randomly divided into seven groups of three rats.

Group:
Control group that received only water (2 mL) Group 2: Control group that received normal saline (2 mL) Group 3: Envenomed rats that did not receive any treatment Group 4: Envenomed rat treated with Olax viridis Group 5: Envenomed rats treated with Syzygium guineense Group: Control group that received only Olax viridis Group: Control group that received only Syzygium guineense The extracts were administered orally at the dose of 400mg kg −1 body weight of rats and 1 h later, the venom was administered intraperitoneally at a dose of 0.08 mg kg −1 body weight of rats.

Antiedematogenic Activity Evaluation Design
Antiedematogenic property of the extracts was measured in the right hind paw edema model (Bispo et al., 2001). The rats were divided into four groups of three rats each. The extracts were administered orally at the dose of 400 mg kg −1 body weight of rats. 1 h later, the animals Science Publications AJPT were injected subcutaneously in the right hind paw with venom (0.08 mg kg −1 body weight). The paw volumes were measured 1, 2, 4, 6 and 24 h after venom injection. Group 4 rats were treated with indomethacin (100 mg kg −1 body weight, I.P) as control for anti inflammatory activity. Group 3, negative control was injected with venom only in the right hind paw and with normal saline in the left hind paw. Edema was expressed as percentage of the difference between the left and right paw volumes and compared with venom control.

Determination of Pulse Rate
The pulse rate was determined using the femoral artery in the groin of the femur of the hind leg. The rats were restrained and once settled, the pulse rate was taken by placing finger over the femoral artery. The pulse was counted for one min using a stop watch.

Blood Glucose Determination
The blood glucose level was determined according to the method described by (Nelson et al., 2012). ACCU Check glucose test meter was used for the determination of the blood glucose in the experimental rats before and after envenomation. A drop of blood from 2 mL collected via tail bleeding of rats was applied to the strip area containing the chemical leading to glucose dye oxidoreductase reaction, causing colour change to occur. The strip was inserted into the meter and the blood glucose concentration was displayed. Before the determination the rats were fasted overnight.

Antipyretic Activity Determination
The method of (Laura and Dorian, 2008) was used to evaluate the antipyretic activity of the extracts. The rats were fasted overnight and their rectal temperature was recorded using digital thermometer with a rectal probe. The rectal temperature was recorded before and after envenomation.

Creatine Kinase Activity Assay
The activity of serum creatine kinase was determined according to the method described by Szasz et al. (1976). Randox CK 110 kit was used for the quantitative in vitro determination of the enzyme activity. The creatine activity was calculated using the formula: U/I = 8095 X ∆A at 340nm/min where ∆A = Change in absorbance.

Plasma Triglyceride Level Measurement
The plasma triglyceride level was determined according to the method described by Tietz et al. (1990).
Randox TR 210 kit was used for the quantitative in vitro determination of triglyceride in plasma.
Triglyceride concentration was calculated using this formula: Absorbanceof sample Concentration of s tan dard(mmol / L) Absorbanceof s tan dard ×

Determination of Plasma Cholesterol
The plasma cholesterol was measured by the method of Richmond, 1973. Randox CH 200 kit was used for the quantitative in vitro determination of cholesterol in plasma. Using a standard, the concentration of cholesterol in the sample was calculated by the formula:

Absorbanceof sample
Concentration of s tan dard(mmol / L) Absorbanceof s tan dard ×

Statistical Analysis
The mean values ± S.E.M was calculated for each parameter. Results were statistically analyzed by oneway Analysis-of-Variance (ANOVA) followed by Benferonis multiple comparisons. P<0.05 was considered as significant.

Pulse Rate
In the pulse rate study, there was a significant (P<0.05) increase in the pulse rate of the group (3) administered Naja katiensis venom compared with the control groups (Table 1). Reduction in the pulse rate of the extract treated groups following envenomation was observed, thus, indicating hypotensive effect of the extracts and this reductions were statistically significant (P<0.05) when compared with group 3.

Blood Glucose
The effects of the two plant extracts on the blood glucose level of rats following envenomation is presented in Table 2. The plant extracts significantly (P<0.005) reduced hyperglycemia induced by the snake venom (Group 3). As seen in Table 2 for group 6 and 7, the extracts reduced blood glucose level indicative of hypoglycemic properties of the plants.

Antipyretic Activity
In the antipyretic study, the rectal temperature of group 3 animals after envenomation is 40.00±0.404°C and 34.06±0.493°C before envenomation. The plant extract treated groups showed significant (P<0.05)

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AJPT reduction in rectal temperature (Table 3). S. guineense showed more antipyretic activity than O. viridis.

Creatine Kinase Activity
There was elevated creatine kinase activity in group 3 rats ( Table 4). The extract treated groups 4 and 5 showed reduced activities of the enzyme significantly (P<0.05). S. guineense had more protective effect than O. viridis.

Lipid Profile
The lipid profiles were reduced by the venom, 1.157±0.078 and 1.217±0.110 mmol/L for triglyceride and cholesterol respectively in group 3. The extract treated groups ( Table 5) offered some protection for both lipids even though this is not statistically significant when compared with the control (group 3) for triglyceride but significant for cholesterol. Values in the same column with the same superscripts are considered not significant (P>0.05). Values in the same column with different superscripts are statistically significant (P<0.05), when compared with control (group 3).

Antiedematogenic Effect of the Plant Extracts
The snake venom (Naja katiensis) produced a rapid on set in paw edema in group 3 but not in group 1, 2 and 4 ( Table 6). The plant extracts reduced edema formation in the treated groups and the reduction is comparable to the standard drug used (indomethacin).

DISCUSSION
Snake bites being a major public health problem claim a large number of lives in the African continent and the world at large. Anti-snake venom remains the specific (antidote) for snake venom poisoning. This anti-snake venom are usually derived from horse sera. They contain horse immunoglobulins, which frequently causes complement medicated side effects and other proteins that cause serum sickness and occasionally, anaphylactic shock. Although, the use of plants against the effects of snake bites has been long recognized, more scientific attention has been given to since last 20 years (Alam and Gomes, 2003).
In this investigation, venom neutralizing potential of O. viridis and S. guineense plant extracts were studied against Naja katiensis venom rats. Many biochemical parameters such as blood glucose, lipid profile, creatine kinase activity, pulse rate were measured. The measurement of these parameters in plasma is of importance in the assessment of the pathophysiological state of snake bite victims. The results suggest that Naja katiensis venom can disturb rat metabolism and the plant extracts were capable of neutralizing the lethality induced by the venom.
The result of the effect of the plant extracts on pulse rate of rats after envenomation is presented in Table 1. There was a significant (P<0.05) increase in the pulse rate of group 3 rats that were administered the snake venom only. This increase in pulse rate might be due to increased metabolic activity or the heart disease (Pangana and Pangana, 2010). In other groups and groups treated with the plant extracts pulse rate was reduced. This reduction in the extract treated groups revealed the hypotensive effect of the plant extracts. In blood glucose level measurement ( Table 2) there was significant increase in blood glucose in group 3 envenomed rats. Many snake venoms are known to cause hyperglycemia in rats and mice (Al-jammaz et al., 1999;Pung et al., 2005;Sleat et al., 2006). A few venoms induced hypoglycemia.
In the present study, the levels of blood glucose were significantly increased in the envenomated animals in group 3 that were not treated with the extracts. This increase in blood glucose level could be attributed to the effects of venom in glycogen metabolism in the hepatocytes, muscle fibres and medullary catecholamines that stimulate glycogenolysis and gluconeogensis in the tissues (Ohhira et al., 1991;Marsh et al., 1997). In group 4 and 5 animals that received extracts of Olax viridis and Syzygium guineense, there was no significant increase in their blood glucose before and after envenomation. Furthermore; there was no significant change in blood glucose level in group 6 and 7 animals which only received the extracts of O. viridis and S. guineense without envenomation.
These results indicate hypoglycemic activity of the plants. This might be due to insulin-like mechanism most probably through the peripheral glucose consumption. Table 3 presents the result obtained from the evaluation of the antipyretic activity of the plant extracts. There was a significant increase in the rectal temperature of group 3 rats that were injected intraperitoneally with Naja katiensis venom but were not treated with extracts when compared with the values obtained before envenomation. The drastic reduction in rectal temperature in extract treated groups is indicative of antipyretic activity of the two plant extracts.   There was a significant(P<0.05) increase in the activity of Creatine kinase enzyme assayed for in group 3 rats when compared with group 4 and 5 that received oral doses of the plant extracts ( Table 4). The plant extracts showed protective effects, the activity of the enzyme was reduced in the extract treated groups. The increase in activity obtained in group 3 might be due to muscle necrosis causing the enzyme to leak out of the muscle into the plasma; however, the plant extracts were able to render protection against this.

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The results of the effects of the plant extracts for the plasma lipid profiles in rats after Naja katiensis envenomation is as presented in Table 5. There are few reports on the effects of snake venom on the rate of lipid metabolism. Decreased plasma cholesterol and triglyceride levels were observed in group 3 rats. This result suggests that the snake venom might have mobilized lipids from adipose and other tissues. Lipolytic enzymes, which are present in many snake venoms, could have splited tissue lipid with the liberation of free fatty acids (Dev and Papasani, 2006). It has also been reported that increased total plasma lipid levels caused by administration of snake venom and the disturbance of lipid metabolism, could be attributed to liver damage and destruction of cell membranes of animal tissues (Al-Sadoon et al., 2011). However, plasma cholesterol and triglycerides have been shown to decrease following some other venoms injection in rats (Salman, 2011). In this study, the plant extracts offered some protection against the lipolytic activity of the venom. Cholesterol is more in the extract treated groups than the control (group 3).
In this study, the antiedematogenic effects of the two plant extracts were demonstrated ( Table 6). The extracts of the plants were able to neutralize the edema induced by Naja katiensis venom. One of the consequences of snake bite is local inflammation. The snake venom induces a striking dose-dependednt edema. This snake bite may lead to shock, because of loss of fluid and tissue compression (Garfin et al., 1985) which could contribute to the development cardiovascular disturbances. There are many inflammatory mediators which participate in the production of edema in a variety of inflammatory conditions. Among others, histamine, prostaglandins, kinins and leukotrienes could be implicated in the resulting edema in the case of snake venoms. Edema seems to be clearly related with prostaglandin production, because an important reduction of the inflammatory effects is induced by indomethacin, a known inhibitor of cyclooxygenase.
Olax viridis and Syzygium guineense extracts significantly (P<0.05) reduced venom induced edema. Olax viridis has already been reported as a plant that inhibits inflammation (Ajali and Okoye, 2009). This study therefore confirms the anti-inflammatory property of this plant. Although, this study was not designed to investigate the mechanism of inhibition, it might be said that the two plant extracts are capable of inhibiting the production of mediators involved in the inflammation induced by Naja katiensis venom, effect that has been found in studies made with plant extracts (Kiuchi et al., 1983). Plant extracts constitute a rich source of novel compounds of potential therapeutic interest in the inhibition of venom toxins. This result suggests that the plant extracts investigated contain anti-inflammatory agents that reduced the Naja katiensis venom-induced edema.

CONCLUSION
In, the results of the present study indicate the potent snake venom neutralizing capacity of these plant extracts against Naja katiensis venom and have the potential of an alternative or complementary treatment strategy of envenomation by Naja katiensis. However, further specific studies need to be conducted to discover the exact compounds responsible for these observations,

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AJPT their efficacy, safety and the antiophidian mechanism of action which could possibly lead to the development of a new chemical antidote for snake envenoming.

ACKNOWLEDGEMENT
The researcher are grateful to Mr. Friday T. Emmanuel for his technical assistance on this study.