Effects of Ethanolic Leaf Extract of Euphorbia hirta on Snake Venom Induced Toxicity in Mice

nakebite is considered a neglected tropical trauma that affects thousands of people worldwide. The number of snakebites that occur each year may be as high as five million. They result in about 2.5 million poisonings and 20,000 to 125,000 deaths (World Health Organization, 2015). The frequency of bites varies greatly in different parts of the world. They occur most commonly in Africa, Asia and Latin America with rural areas more greatly affected.

The World Health Organization (WHO) has estimated that up to 80 percent of people in the developing world are dependent on traditional system of medicines primarily because of their easy accessibility, wide affordability and cultural familiarity.
Over the years, many attempts have been made for the development of snake venom antagonists from plant sources.Some works have been done in other to find out the phytochemicals in some plants that have ethno botanical usage for snake envenomation (Ukachukwu, 2015).Some plants have shown to possess anti-snake venom potentials.Plants like Annonasenegalensis, Moringaoleifera, Alluimcepa, Allium sativum have been used by the Fulani Herdsmen in Taraba State for the management of snakebite (Ameen et al., 2015).Extracts of Uvaria chamae has also shown to neutralize some biological effects of Naja nigricollis snake venom in rats (Omale et al., 2013).
Recently, work has been carried out in the department of Biochemistry, ANSU, Uli on phytochemical composition of Euphorbia hirta, in silico work has been carried out which revealed that some of the phytochemicals obtained from this plant have strong affinity for snake venom PLA2 compared with the control ligand used (Ukachukwu, 2015).The phytochemical constituents of Euphorbia hirta that have been isolated include; flavonoids (Xu andChen, 2006), alkaloids (Xiang et al., 2005), fatty acids, terpenoids, polysaccharides, vitamins, sterols, proteins, and minerals.Research has shown that this plant is a good source of omega-3 fatty acid.Its pharmacological effects include; antibacterial (Zhang et al., 2002), antiinflammatory (Chan et al., 2000), antioxidant and woundhealing (Rashed et al., 2003) properties.Aside from in silico work done on this plant, few work have been done to ascertain its snake venom neutralizing potentials.The aim of this study therefore is to carry out an in vivo assessment of the anti-snake venom neutralizing potentials of Euphorbia hirta ethanolic extract in mice.

MATERIALS AND METHODS
Chemicals, solutions and equipment: All chemicals used in the present study were of analytical grade and purchased from QULIKEM, India.Centrifuge (Heraeus Christ GMBH Estrode), Analytical balance, measuring cylinder, micropipette, mortar, pestle, beakers, retord stand, burette, syringes and deep freezer.

COLLECTION, IDENTIFICATION AND EXTRACTION PROCEDURE OF THE PLANT SAMPLE
The leaves of Euphorbia hirta was harvested from a farm behind the department of Biochemistry Chukwuemeka Odumegwu Ojukwu University in Anambra State and was identified by Mrs. Emezie, A. U., a pharmacologist in the department of pharmacology, school of pharmacy, Nnamdi Azikiwe University, Agulu campus.Its voucher number is PCG 474/A/022.These leaves were dried at room temperature (25 0 C) for a couple of days and later blended into fine powder using dry blender.250g of the powder was macerated in 1litre of ethanol in an air tight plastic container and allowed to stand for 48hrs.The extract was filtered successively using muslin cloth and Whatman filter paper No. 42.The extract was then concentrated to dryness over a water bath at 50 o C

LABORATORY ANIMAL
Albino mice were purchased from the animal house of the Department of Pharmacology, School of Pharmacy, Nnamdi Azikiwe University, Agulu Campus, Anambra State, Nigeria.These animals were used as approved in the study of snake venom toxicity.The mice were maintained under normal laboratory condition of humidity, temperature (25±1°C) and light (12 hours night/day cycle) with access to clean water.An ethical clearance for animal use was obtained for this research work.

SNAKE VENOM
Lyophilized Naja nigricollis venom was purchased from the Department of Pharmacognosy and Drug Development, Ahmadu Bello University, Zaria Nigeria.

Determination of LD50 of plant extract:
The LD50 of the ethanolic leaf extract of Euphorbia hirta was carried out according to the method described by Dietrich Lorke (1983).

Procedure:
This method has two phases.

Phase 1
This phase required nine mice.The nine animals were divided into three groups of three animals each.Each group of animals were orally administered different doses (10, 100 and 1000 mg/kg) of test substance (leaf extract).The animals were placed under observation for 24 hours to monitor their behavior as well as any mortality.

Phase 2
This phase involved the use of four animals, which were distributed into four groups of one animal each.The animals were administered higher doses (2000, 3000 4000 and 5000 mg/kg) of test substance (leaf extract) and then observed for 24 hours for behavior as well as mortality.
Then the LD50 was calculated with the formula:  50 √ 0   100 D0 = Highest dose that gave no mortality, D100 = Lowest dose that produced mortality The formula below was used to determine the dose volume of extract and snake venom administered to each mouse.

Determination of LD50 of snake venom:
The LD50 of Naja nigricollis venom was carried out according to the method described by Dietrich Lorke (1983).

BLEEDING TIME:
For the determination of the bleeding time, modified procedure of Mohammed et al. (1969) was used.Four hours after envenoming the mice and treating them with plant extract, the tail of each mouse was gently pieced with lancet.A piece of white filter paper was used to blot the blood gently from the punctured surface of the body.The readings were taken every 15 sec.The end result occurs when the paper was no longer stained with blood.

CLOTTING TIME:
For the determination of the clotting time, the modified method of Igboechi and Anuforo (1986) was used.Clotting time is the time required for a firm clot to be formed in fresh blood on glass slides.The blood sample was collected from, the rats via tail bleeding and a drop was placed on a clean plain glass slide and every 15 sec, a tip of office pin was passed through the blood until a thread-like structure was observed between the drop of blood and tip of the pin.The thread-like structure was an indication of a fibrin clot.The time was recorded.

In vivo snake venom toxicity neutralizing potential of Euphorbia hirta plant extract on envenomed mice:
Thirty albino mice were randomly divided into six groups of five mice each.
Group 1: Control group that received snake venom and normal saline.
Group 2: Envenomed mice that received plant extract at zero minute delay.
Group 3: Envenomed mice that received plant extract at five minutes delay.
Group 4: Envenomed mice that received plant extract at ten minutes delay.
Group 5: Envenomed mice that received plant extract at fifteen minutes delay.
Group 6: Envenomed mice that received combined extracts of Euphorbia hirta and Portulaca oleracea at ten minute delay.
The venom was administered intraperitoneally at a dose of 1414 µg/kg body weight of mice and the extract was administered orally at a dose of 250 mg/kg body weight of mice at different time interval.

Twenty-four hour acute toxicity (LD50) result of the ethanolic leaf extract of Euphorbia hirta.
The different groups for the LD50 of plant extract were given the different doses (ml) of extract above and the result presented in In the first phase of the experiment, the first three groups of mice that received a dose range of 10-1000mg/kg of the extract had no mortality.The four groups of mice in the second phase of the test that received a higher dose range of 2000-5000mg/kg of the extract had no mortality as well.
LD50value is >5000mg/kg since there was no mortality.
The different groups for the LD50 of Naja nigricollis venom were given the different doses (µg/ml) of extract above and the result presented in In the first phase of the experiment, the first three groups of mice that received a dose range of 10-1000 mg/kg of the venom had no mortality.The four groups of mice in the second phase of the test that received a higher dose range of 2000-5000 mg/kg of the venom all died within 24 hours.
This result shows that the venom of Naja nigricollis administered is very toxic and harmful in animal model.
According to Lorke's method, the LD50 is calculated as follows LD50= √1000  2000 Clotting time: The result of the effects of Euphorbia hirta against Naja nigricollis venom on blood clotting time is as presented in Table 3: The result of the effect of the administration of the snake venom in addition with normal saline in mice is presented in Table 5: The result of the effect of the administration of the snake venom in addition with the plant extract at zero minute delay in mice is presented in Table 6: The bar chat shows a 60% survival rate in the control of all the groups and a 80% survival rate in the 15min and 100% in the other entire group.

DISCUSSION
Snakebites are considered a neglected tropical trauma that affects thousands of people worldwide.Although anti-venom (which is prepared from animal sera) immunotherapy is the only treatment available against snake envenomation, it is associated with many side effects which include; anaphylactic shock, pyrogen reaction and serum sickness.These are possible outcomes of the action of antigenic proteins present in higher concentrations in anti-venom (Assafim et al., 2006).Also, antivenom (anti sera) do not neutralize the local tissue damage (Gutierrez et al., 2009), are not available in remote areas and are quite expensive.
Although, the use of plants against the effects of snakebites has been recognized, more scientific attention has been given to it since last 2 decades (Alam and Gomes, 2003).
Several plants have been used in folk medicine throughout the world as treatment against snakebites (Houghton and Osibogun., 1993;Melo et al., 1994;Maiorano et al., 2005;Oliveira et al., 2005;Cavalcante et al., 2007;Lomonte et al., 2009;De Paula et al., 2010;Omale et al., 2013).Till date few plants materials have been evaluated in well controlled assays and only a few of them have been found to be effective against Snake envenomation.Snakebite is an important cause of morbidity and mortality and is one of the major health problems in areas in Nigeria.
The search for bioactive molecules in plants used in folk medicine has been growing in the past few years.This study shows that N. nigricollis venom can inhibit or induce metabolism in mice and has also shown that E. hirta medicinal plant, neutralized some biological effects induced by N. nigricollis venom.
The phytochemical constituent of E. hirta has shown the presence of saponin, alkaloid, tannin, flavonoid, cardiac glycoside, terpenoids, protein and starch as its active phytoconstituents with saponin as the major constituent.The presence of these constituents in most plants has been reported to have proven its medicinal usage in health issues (Okafor and Ezejindu, 2014).
Pharmacologically, it is a good antibiotic, antioxidant, anticancer, antimicrobial, and anti-inflammatory.
The LD50 test for the ethanolic leaf extract of Euphorbia hirta plant showed no mortality or abnormal behavior on the groups of mice that received both the lower and higher dose after 24hrs (Table 1), hence, its phytochemical studies that showed that it is good for consumption.Its LD50 value is > 5000 mg/kg.
The LD50 of N. nigricollis venom showed no mortality on the groups of mice that were envenomed with a lower dose of 10-1000 mg/ml but the experimental animals in the groups that received a higher dose range of 2000-5000 mg/ml of N. nigricollis venom all died within 24 hrs (Table 2) which tells us how toxic this venom can be when it gets in contact with one's biological system.
Regardless of the precise mechanism, E. hirta appear to be a promising chemical agent for use as first aid treatment, or in combination with antiserum.Many snake venoms are known to cause pathological properties associated with hematological disturbances leading to in coagulability of blood.Some local tissue necrosis always accompanies envenomation from this snake species.Spontaneous bleeding and coagulation disturbances are some of the hematological effects of N. nigricollis in patients (Warrell et al., 1976).
A prolonged blood clotting time is a function of clotting factors deficiency.The result of clotting time shows that Naja nigricollis venom is an anticoagulant.In the envenomated animals (group 2) that were not treated with extract there was significant increase in clotting time due to the presence of venom.In groups 3 and 4 treated with E. hirta and P. oleracea + E. hirta extract respectively, the extract neutralized this effect of the venom and the clotting time was maintained at the normal level when compared with the control group 1.The decrease in clotting time level observed in Table 3 establishes the fact that treatment of animals with venom/extract mixture abolished the blood incoagulability.
The capacity of plasma to form thrombin is also relevant in the blood coagulation system.These entire blood characteristic are affected by the toxic components of N. nigricollis venom (Denson et al., 1992).
Bleeding time is associated with integrity of blood vessels and is known to cause pathological disturbances leading to incoagulability of blood.5).This shows how effective the used N. nigricollis venom is.
The result of the zero minutes delay shows the effectiveness of the plant extract used because there was 100% survival (table 6).
Euphorbia hirta plant extract neutralized the toxic effects of the venom at the five minutes delay.There was a 100%s urvival (Table 7).
Though one death was encountered in the ten minutes delay, the plant extract still showed its effectiveness because there was an 80% survival (table 8).
The result of the fifteen minutes delay also showed a 100% survival.This shows that the neutralizing potentials of Euphorbia hirta plant extract was still intact fifteen minutes after envenomation (Table 9).
Portulaca oleracea+ Euphorbia hirta plant extracts neutralized the toxic effects of the venom at the fifteen minutes delay as shown in Table 10.

CONCLUSION
From this study, it can be concluded that the extract of Euphorbia hirta is effective in neutralizing the toxic effects of Naja nigricollis venom.Time of treatment is of essence because the longer the time taken after the envenomation, the more disastrous the toxins in the venom damages the body cells.
Further experiment could address the fractioning of the Euphorbia hirta extract in order to identify the bioactive compounds responsible for these observations, their efficacy, safety and the mechanism of action which could possibly lead to the development of pharmaceutical formulations for treating snakebite accidents-victims.

ACKNOWLEDGEMENT
First and foremost, I am grateful to God Almighty who guided and saw me through this phase in my academic pursuit.Encouragement on academic research work, guidelines and the financial aspect was not accomplished by me alone.Many people contributed to the success of this work.
My appreciation goes to my supervisor Mrs. Anene, I.N for her professional and motherly guidance at various stages of this study and I am also grateful to my lecturers for their moral support.
Special thanks to my parents Mr. and Mrs. Agusi for their financial and moral support.
I extend my gratitude to my colleague Ogbunachi Oge who helped in getting materials and information related to this work.

Table 1 Table 1 :
Result for the LD50 experiment for plant extract.

Table 2 Table 2 :
Result for the LD50 experiment for Naja nigricollis venom.

Table 3 :
Effect of Euphorbia hirta extract on clotting time after envenomationThe result of the effects of Euphorbia hirta plant extracts against Naja nigricollis envenomation on blood clotting time shows that Naja nigricollis venom increased the clotting time in mice and the plant extract decreased the clotting time.

Table 4 :
Effect of Euphorbia hirta extract on bleeding time after envenomation.
Values are Mean ±S.E.M (n=4)The result of the bleeding time of Euphorbia hirta plant extracts against Naja nigricollis envenomation shows that group 2 that was administered snake venom has a higher value compared to the control group indicating a deleterious effect of the snake venom.The result of groups

Table 5 :
Effect of the administration of snake venom and normal saline(Control)

Table 6 :
Effect of the administration of snake venom and plant extract at zero minute delay.
In groups 3 and 4 treated with Euphorbia hirta and P. oleracea + E. hirta extract respectively, the extract neutralized this effect of the venom and the bleeding time returned to the normal level when compared with the control group 1.The decrease in bleeding time level observed in Table4establishes the fact that treatment of animals with venom/extract mixture proved effective.
The result for the bleeding time of this study shows that N. nigricollis venom caused severe external hemorrhage.The level of bleeding time increased significantly (p<0.05) in the envenomated animals in group 2 that were not treated with