Introduction

Snake envenoming is a major public health issue in the rural tropics with large numbers of envenoming and deaths. In India there are about 216 different species are found, of which 53 species are reported to be poisonous. The common poisonous snakes found in India are Cobra (Naja naja), Krait (Bangarus Caeruleus), Russell’s viper (Daboia russelli) and Saw Scaled Viper (Echis Carinatus) (Bawaskar, 2004). About 35,000 to 50,000 people die of snakebite every year in India. Echis carinatus (Saw-scaled viper) occurs in both India and Pakistan, where it is responsible for a large number of snakebite case, reaching 95% of envenomations in the state of Jammu (Chippaux, 1998). Antivenom immunotherapy is the only specific treatment against snake venom envenomation. Antivenoms are usually hyper immune sera collected from animals which bind and inactivate venom components. There are various side effects of antivenom such as anaphylactic shock, pyrogen reaction and serum sickness. Most of these symptoms may be due to the action of high concentrations of nonimmunoglobulin proteins present in commercially available hyper immune antivenom (Maya Devi et al, 2002). Antiserum development in animal is time consuming, expensive and requires ideal storage condition. Over the years many attempts have been made for the development of snake venom antagonists especially from plants sources. Extracts from plants have bee used among traditional healers, especially in tropical areas where here are plentiful sources, as therapy for snakebite for a long time (Saka Daduang et al., 2005). Several medicinal plants, which appear in old drug recipes or which have been passed on by oral tradition, are believed to be snakebite antidotes. In modern science, there have been many attempts to study these plants to clarify their effectiveness (Houghton et al., 1993 and Otero et al., 2000). India has a rich tradition of the usage of medicinal plants. Many Indian medicinal plants are recommended for the treatment of snakebite (Alam et al., 2003). Some of these plant extracts has shown antivenom property on land snakes and their possible mechanism of action have been suggested. In almost any part of the world, where venomous snakes occur, numerous plant species are used as folk medicine to treat snakebite. The present investigation explored the Echis carinatus venom neutralizing activity of Andrographis paniculata and Aristolochia indica plant extracts by in vivo and in vitro methods.

Materials and Methods

Venom and Experimental animals

Medicinal Plans

Preparation of Extracts

Tests for anti-snake venom activities

Statistical Analysis

Results

The antivenom potential of Andrographis paniculata and Aristolochia indica plant extracts were tested against Echis carinatus venom by in vivo and in vitro methods. The lethal toxicity (LD₅₀) of Echis carinatus venom was assessed using 18g, Balb/c strain mice. About 12μg of Echis carinnatus venom was found to be LD₅₀ for 18g of mice. The neutralization of lethality was done by mixing constant amount of venom with various dilutions of Andrographis paniculata and Aristolochia indica Plant extracts and incubated at 37°C for 30 minutes prior to injection. We found that 0.19mg of Andrographis paniculata and 0.17mg of Aristolochia indica plant extracts were able to completely neutralize the lethal activity of 2LD₅₀ of Echis carinatus venom (Table 1). When comparing to Andrographis paniculata , Aristolochia indica effectively neutralize the lethal toxicity (Fig 1).

Figure 1

Table 1: Neutralization of venom induced lethality by and Plant extracts

Figure 2

Figure 1: Dose Response curve for Neutralization of Lethality by a) and

In edema forming activity, the mice immunized with Echis carinatus venoms showed increase in footpad thickness. About 7μg of Saw-scaled viper venom induced edema formation within 3h which is considered as 100% activity. The edema was reduced up to 30% when 3.5mg of plant extracts /mg venom was given. There was no further reduction in the percentage of edema even when there was an increase in antivenom dose (Fig 2).

Figure 3

Figure 2: Neutralization of edema induced by Echis carrinatus venom by a) and

In the case of hemorrhagic activity, Saw-scaled viper venom produced visible hemorrhagic spot. About 8μg of venom produced a hemorrhagic spot of 10mm diameter (MHD). Both Plant extracts were able to neutralize the hemorrhage induced by the venom. In phospholipase activity (PLA2), Echis carinatus venom able to produce hemolytic haloes in agarose-sheep erythrocytes gels. About 10μg of Saw scaled viper venom produced 11mm diameter hemolytic halo, which is considered to be 1U (U/10μg). This shows that Echis carinatus venoms have the enzymes (PLA2) that has the ability to lyse sheep RBC's. Andrographis paniculata and Aristolochia indica Plant extracts were capable of inhibiting PLA2 dependent hemolysis of sheep RBC's induced by Echis carinatus venom in a dose dependent manner (Fig 3). We found that that 0.17mg of Andrographis paniculata and 0.14mg of Aristolochia indica plant extracts were able to completely inhibit PLA2 dependent hemolysis of sheep RBC's induced by Echis carinatus venom

Figure 4

Figure 3: Inhibition of Phospholipase activity by a) and

The minimum coagulant dose (MCD) was determined as the venom dose inducing clotting of plasma in 60s. About 120μg of Saw-scaled viper venom clotted human citrated plasma within 60s. In the neutralization assay, the absence of clot formation shows the neutralizing ability of both Plant extracts. We found that that 2.1mg of Andrographis paniculata and 1.8mg of Aristolochia indica plant extracts were able to completely neutralize coagulant activity. High dose of venom caused rapid clotting that required very high dose of antivenom to neutralize.

Discussion

Snakebite is a common medical emergency encountered in the tropics and estimated 35,000 to 50,000 people die of snakebite every year in India (Sharma et al., 2004). Saw scale vipers or Echis carinatus are commonly found in the semi-arid deserts of Western Rajasthan with predominant nocturnal habitus. Snake venom is a highly complex mixture of a variety of biological substances including 90% of water, 20-25 enzymes, and a large number of low molecular weight peptides. Envenomation by Echis carinatus is associated with a mortality rate of 10-20%, if effective treatment is not initiated early. The major cause of mortality is due to increased bleeding tendency caused by the venom. Venom of viper is a mixture of multipleenzymes and low molecular weight peptides, some of which are responsible for the bleedingmanifestations. Multiple mechanisms have beensuggested for the bleeding occuring afterenvenomation. These include disseminatedintravascular coagulation (DIC) due to spontaneous activation of factor V and factor Xby pro-coagulants present in the venom. It has also been suggested that the small dose of venom,as typically injected in humans, leads to continuousactivation of fibrinogen, producing a fragile fibrinmore susceptible to lysis than ordinary fibrin,leading to bleeding manifestations. Vascular endothelial damage caused by the haemorrhagin present in the venom also contributes to bleeding manifestations. (Manoj Lakhotia et al., 2002). Antivenom is the specific antidote for snakebite envenomation. Antivenom against snakes bites are lacking in the rural areas of coastal region. Antiserum; being the only therapeutic agent, its development from animal source is time consuming and expensive. Although, use of plants against the effects of snakes bite has been long recognized, more scientific attention has been given since last 20 years (Santosh et al., 2004). Many Indian medicinal plants are recommended for the treatment of snakebites (Alam et al., 2003). In our present study we check the antivenom potential of Andrographis paniculata and Aristolochia indica plant extracts against Echis carinatus venom. It is essential to understand the pharmacological action of snake venom in order to device a rational treatment for snakebite. An important initial step in finding antivenom potential of plant extracts is the pre-clinical testing using in vivo and in vitro methods to assess the neutralizing potential of the antivenom (plant extracts) against a range of different venom effects. The neutralization ability of snake antivenoms is still assessed by the traditional in vivo lethality assay (minimum effective dose ED₅₀), comparable to those used for bacterial antitoxins, usually performed in mice (WHO, 1981). Thus various pharmacological activities like lethality, edema forming activity, hemorrhagic activity, phospholipase activity (PLA2), procoagulant activity caused by Echis carinatus venom were carried out. Neutralization of these pharmacological effects was carried out using Andrographis paniculata and Aristolochia indica plant extracts. Neutralization studies can be performed by incubating of venom and plant extracts prior to testing (pre-incubation method). The results showed that the both plant extracts were capable of neutralizing the lethality induced by the venom. The Echis carinatus venom showed the presence of PLA2 enzymes by means of producing hemolytic haloes in indirect hemolytic assays. Both plant extracts were capable of inhibiting PLA2 dependent hemolysis of sheep RBCs in a dose dependent manner. The medicinal plants Thea sinensis Linn and Cordia verbenacea were effectively neutralized the phospholipase A2 activity induced by snake venoms (Yao-Ching Hung et al., 2004 and Ticli et al., 2005). Edema-forming activity was assessed for Echis carinatus venom and both plant extracts were found to be effective in neutralization of edema induced by venoms. There was a significant decrease in the edema (footpad thickness) when there was an increase in the antivenom (plant extract) dose. Procoagulant activity induced by Echis carinatus venom was studied using human citrated plasma and Andrographis paniculata and Aristolochia indica plant extracts were found to be effective in the neutralization of procoagulant activity. The present experimental results indicate that Andrographis paniculata and Aristolochia indica plant extracts were effective in neutralizing the main toxic and enzymatic effects of Echis carinatus venom. The antivenom properties of both plant extracts were potent enough to neutralize the lethality and various pharmacological activities of venom. The result from this preliminary study indicates that both plant extracts could be used for therapy in patients with snakebite envenomation. Further investigations are needed for identification and purification of the active components involved in the neutralization of the snake venom.