Original Article

Studies on antivenom activity of Andrographis paniculata and Aristolochia indica plant extracts against Echis carinatus venom

S Meenatchisundaram, Prajish, G Parameswari, T Subbraj, A Michael

Citation

S Meenatchisundaram, Prajish, G Parameswari, T Subbraj, A Michael. Studies on antivenom activity of Andrographis paniculata and Aristolochia indica plant extracts against Echis carinatus venom. The Internet Journal of Toxicology. 2008 Volume 6 Number 1.

Abstract


Andrographis paniculata and Aristolochia indica plant extracts were tested for antivenom activity against Echis carinatus venom. Both plant extracts were effectively neutralized the Echis carinatus venom induced lethal activity. About 0.19mg of Andrographis paniculata and 0.17mg of Aristolochia indica plant extracts were able to completely neutralize the lethal activity of 2LD2 of Echis carinatus venom. Various pharmacological activities like haemorrhagic, coagulant, edema and phospholipase activities were studied and these pharmacological activities were significantly neutralized by both plant extracts The above observations confirmed that Andrographis paniculata and Aristolochia indica plant extracts possess potent snake venom neutralizing capacity and could be used for therapeutic purposes in case of snakebite envenomations.

 

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

The free-dried snake venom powder of Echis carinatus was obtained from Irula’s Snake Catchers Industrial Co-operative Society Limited, Chennai and was stored at 4ºC. Male inbreed Swiss albino mice 18-20 gm were used for the studies of venom toxicity and in the experiments of venom neutralization. Institutional Animal Ethics Committee clearance at Institute of vector control and Zoonooses, Hosur, was obtained to conduct the experiment.

Medicinal Plans

Andrographis paniculata and Aristolochia indica plants were obtained from RVS Ayurveda College, Sulur.

Preparation of Extracts

The whole plant material was dried in shade and the air-dried plant was ground and extracted first by refluxing with petroleum ether (50–60 °C, for 72 h) and then with methanol (60–80 °C for 72 h). The methanol extract was concentrated in vaccuo and kept in a desiccator at room temperature for further use. Before use, it was dissolved in saline and centrifuged at 2000 rpm for 10 min at room temperature. The supernatant was used for further investigation and kept at 4 °C. The plant extracts were expressed in terms of dry weight.

Tests for anti-snake venom activities

Lethal toxicity

The median lethal dose (LD50) of Echis carinatus venom was determined according to the method developed by Theakston and Reid 1983. Various doses of venom in 0.2 ml of physiological saline was injected into the tail vein of mice (18-20gms), using groups of 3-5 mice at each venom dose. The LD50 was calculated with the confidence limit at 50% probability by the analysis of deaths occurring within 24 h of venom injection. The anti-lethal potentials of Andrographis paniculata and Aristolochia indica plant extracts were determined against 2LD50 of Echis carinatus venom. Various amount of Plant extracts (μl) were mixed with 2LD50 of venom sample and incubated at 37C for 30 minutes and then injected intravenously into mice. 3 – 5 mice were used at each antivenom dose. Control mice received same amount of venom without antivenom (Plant extracts). The median Effective Dose (ED50) calculated from the number of deaths within 24h of injection of the venom/antivenom mixture. The ED50 was expressed as μl antivenom/mouse and calculated by probit analysis.

Edema- forming Activity

The Minimum edema-forming dose (MED) of Echis carinatus venom was determined by the method of Lomonte et al. (1993) and Camey et al. (2002). Group of four mice were injected subcutaneously in the right footpad with various amounts of venom (0.25g - 10g) dissolved in 50l of Phosphate – Buffered Saline (PBS) pH 7.2. The left footpad received 50l of PBS alone (control). Edema was calculated as percentage of increase in the thickness of the right foot injected with venom compared to the left foot. The thickness of each footpad was measured every 30 min after venom injection with a low-pressure spring caliper (Rojas et al., 2005). Minimum edema-forming dose (MED) was the venom dose that induced 30% edema within 6 hours of venom injection when compared to control. The ability of both plant extracts in neutralizing the Edema- forming activity were carried out by pre-incubating the constant amount of venom and various dilutions Andrographis paniculata and Aristolochia indica plant extracts and incubated for 30 minutes at 37C. Then, groups of four mice (18 - 20g) were injected subcutaneously in the right footpad with 50l of the mixtures, containing venom/plant extracts, whereas the left footpad received 50l of PBS alone. Control mice were injected with venom in the right footpad and 50l of PBS in the left footpad. 1 hour after injection edema was evaluated as described by Yamakawa et al., 1976. Edema was expressed as the percentage increase in thickness of the right footpad compared to the right footpad of the control mice.

Haemorrhagic activity

The minimum haemorrhagic dose (MHD) of Echis carinatus venom was determined by the method described by Theakston and Reid, 1983. The minimum haemorrhagic dose was defined as the least amount of venom which when injected intradermaly (i.d.) into mice results in a haemorrhagic lesion of 10mm diameter in 24 hours. Neutralization of the haemorrhagic activity was estimated by mixing a fixed amount of venom with different amounts plant extracts. The Plant extract–venom mixture was incubated at 37°C for 1 h and 0.1 ml of the mixture was injected intradermaly into mice. The haemorrhagic lesion was estimated after 24 h.

Phospholipase activity

Phospholipase A2 activity was measured using an indirect hemolytic assay on agarose–erythrocyte–egg yolk gel plate by the methods described by Gutierrez et al., 1988. Increasing doses of Echis carinatus venom (µg) was added to 3mm wells in agarose gels (0.8% in PBS, pH 8.1) containing 1.2% sheep erythrocytes, 1.2% egg yolk as a source of lecithin and 10mM Cacl2. Slides were incubated at 37˚C overnight and the diameters of the hemolytic halos were measured. Control wells contained 15μl of saline. The minimum indirect hemolytic dose (MIHD) corresponds to a dosage of venom, which produced a hemolytic halo of 11mm diameter. The efficacy of antivenom (Plant extracts) in neutralizing the phospholipase activity was carried out by mixing constant amount of venom (µg) with different amount of plant extracts (µl) and incubated for 30 minutes at 37C. Then, aliquots of 10l of the mixtures were added to wells in agarose-egg yolk-sheep erythrocyte gels. Control samples contain venom without Plant extracts. Plates were incubated at 37C for 20 hours. Neutralization expressed as the ratio mg antibodies/mg venom able to reduce by 50% the diameter of the hemolytic halo when compared to the effect induced by venom alone.

Procoagulant activity

The procoagulant activity was done according to the method described by Theakston and Reid, 1983 modified by Laing et al., 1992. Various amounts of venom dissolved in 100µl PBS (pH 7.2) was added to human citrated plasma at 37C. Coagulation time was recorded and the Minimum Coagulant Dose (MCD) was determined as the venom dose, which induced clotting of plasma within 60 seconds. Plasma incubated with PBS alone served as control. In neutralization assays Constant amount of venom was mixed with various dilutions of plant extracts. The mixtures were incubated for 30 minutes at 37C. Then 0.1ml of mixture was added to 0.3ml of citrated plasma and the clotting times recorded. In control tubes plasma was incubated with either venom alone or plant extracts alone. Neutralization was expressed as effective dose (ED), defined as the ratio l antivenom (plant extracts)/mg venom at which the clotting time increased three times when compared with clotting time of plasma incubated with two MCD of venom alone.

Statistical Analysis

Statistical evaluation was performed using XL stat 2007 and SPSS 10 Softwares. P< 0.005 was considered statistically significant.

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.

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Author Information

Subramani Meenatchisundaram, Ph.D.
Department of Microbiology, RVS College of Arts and Science

Prajish
Department of Microbiology, RVS College of Arts and Science

Govindarajan Parameswari
Department of Microbiology, PSG College of Arts and Science

Thangappan Subbraj
Department of Microbiology, PSG College of Arts and Science

Antonysamy Michael
Department of Microbiology, PSG College of Arts and Science

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