Until now biochemical effects of Mesobuthus tamulus venom, which is accountable for a number of deaths of children in India, is not studied. In our study we collected more than 30 scorpions from the fields and hilly regions of Rajampet, Kadapa district, Andhra Pradesh. We studied the effect of chemical agents like alcohol, formaldehyde and iso-propanol on scorpion venom at various concentrations. After 120 hours it was exhibiting lesser effectiveness (venom activity) and is found to be functioning as anti-venom for scorpion bite and involving in prophylaxis within 2 minutes. The extract from scorpion glands appeared to be of proteoid nature which is less soluble in pure water and is soluble in saline solution, but insoluble in absolute alcohol and ether. The activity of the venom is neutralized by the prolonged action in alcohol majorly. The various screening methodologies, characterization of venom and invitro analysis of scorpion venom will be discussed.
Scorpions are eight-legged predatory animals, members of the order Scorpions and class Arachnida belonging to the phylum Arthropoda. These are abundantly found in temperate parts of the globe, predominantly in semi-arid regions and especially in sub-tropical countries like India, Malaysia, China etc. Though the Scorpions normally available in dry climates they drink water as they need access to the moisture (Benton, T.G. 2008). Their habit of being nocturnal and inhabiting shady crevices reduces the deficit of their body fluids (Bastawade, D. B. 1992.). Scorpions are able to Regulate the quantity of venom by the scorpions is done by injecting with each sting by striated muscles. The typical amount of venom injected varies between 0.1 and 0.6mg. In addition to this information, the data propose that these animals confine the use of their venom by using it only to pin down large prey. Generation of venom by scorpion takes several days to refill the supply once it has been used, and is costly with respect to the energy needed to produce venom. So, they have two kinds of venom: opaque, more powerful venom intended to kill heavier threats, and translucent, weaker venom intended to stun only.
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Primarily, Prey is caught by the use of their chelae (Pincers). Depending on the strength of the prey, they will then either infuse it with neurotoxic venom or crush the prey. They feed on the prey by killing or paralyzing it (Casper, G. S. 1985.). Every species of scorpion possess venom. In common, venom of scorpion is illustrated as neurotoxic in nature. There are omissions to this generality, for example Hemiscorpious leprutus possesses cytotoxic venom (Bin Gao et al. 2008.). A diversity of small proteins as well as K+ and Na+ ions present in neurotoxins of scorpions intervene with neurological responses of the victim. The prey will be successfully captured as the action is rapid and the results of the sting can be harsh. Scorpion venoms are raised to act upon other member of phylum Arthropoda for their survival and hence most scorpions are comparatively innocuous to humans. Their stings cause only local effects (like swelling, numbness or pain) in human. However some scorpion species, mostly members of family Buthidae, can be precarious to humans. The scorpion species Mesobuthus tamulus belongs to this family, and is harmful to humans.
MATERIALS AND METHODS
Collection of Scorpions
Scorpions: The scorpions (Mesobuthus tamulus gangeticus) were originally collected at Kadapa Province in the Andhra Pradesh state, India. They were kept in large plastic boxes and glass bottles at room temperature of 35ÂÂ±2oC, at 65ÂÂ±10% humidity, in the laboratory. Daily, they were fed with cockroaches or crickets and supplied with water (Hadley, Neil F. 1970.).
Fig.1 Mesobuthus tamulus
Fig.2 Catching the scorpion
Fig.3 Scorpions are kept in an aerated bottle.
SCREENING OF VENOM
Venom was obtained from mature Mesobuthus tamulus scorpions by sacrificing the animal. Then the venom is collected from the sting.
Venom is treated with ethanol, formaldehyde and Iso-propanol separately. The absorbance readings are noted @ 510nm using calorimeter at different time intervals.
Fig.4 Laboratory set up for experimentation on scorpion venom
Assay of venom
The assay of venom was done by using ethanol, iso-propanol and formaldehyde at different concentrations. Semi-quantitative PLA2 assay was done with egg yolk as substrate. A suspension of 20 ml was made by using 9 ml chicken egg yolk, 1.49 ml 1.34 mM EDTA, 2.51 ml 34.18 mM NaCl, 2.0 ml 50 mM Tris-HCl, 4.44 ml 6.8 mM CaCl2, 0.56 ml 14.52 mM saline and pH 7.5. Venom was added to 2 ml egg yolk suspension, mixed well and incubated at 37ÂÂ°C (room temp.) for 1 hour. Incubates were positioned on a boiling water bath and the time needed to coagulate was noted. Positive and negative controls are Pancreatic PLA2 and 14.52 mM Saline respectively (chakrabarty et al., 2000).
CHARACTERISATION OF VENOM
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Every species of scorpion have venom. Commonly, their venom is expressed as neurotoxic in nature. A better way of classification is in dividing the toxin's effect on Na+ ion channels (Chakrabarty, D. et al. 1993.)
Type I: Deactivates the Na+ ion channels and extends the action potential of nerve;
TypeÂÂ II: Stimulates the Na+ ion channels and cause impulsive and recurring excitation of nerve;
Type III: Decreases the exchange of Na+ ions and K+ ions between the intracellular (ICF) and extracellular (ECF) fluid.
INVITRO ANALYSIS OF VENOM
Generally, many people hold the notion that scorpion venom is a single compound. The facts revealed few things. In common the venom of scorpion contains many toxins, enzymes, salts, biogenic amines, undisclosed substances and water (Ricardo C. Rodriguez de la Vega et al. 2003.).
To make things easier, toxins are known to affect Na+ ion, K+ ion and Ca+ ion channels with regard to electrolyte balance. Interruption in the electrolyte balance can disturb the following.
Na+ and Ca+ ions permeability disturbs the heart.
Na+ ions disturb the homeostasis by kidney.
Na+ and K+ ions disturb the cell membrane integrity and nerve transmission.
Absorbance values @ 510nm
0.023Calcium disturbs the muscles and is a vital secondary messenger.
Effect of Ethanol, Formaldehyde and Iso-propanol on Scorpion Venom
The action of ethanol, formaldehyde and iso-propanol on scorpion venom at different concentrations was carried out. The venom of 0.1mg is taken in each 50ml volume of 50% and 99% concentrations of all three chemicals (C2H5OH, HCHO & C3H7OH).
Fig.5 M.tamulus scorpions in ethanol
Fig.6 Bar Diagram showing changes in absorbance at various time intervals in 50% and 99% Iso-propanol
Through our study, we have obtained the following statistical data regarding the effects showed by 50% and 99% ethanol, formaldehyde and iso-propanol each on 0.1mg of venom.
Table 1: Absorbance values showing effect of 50ml each of 50% and 100% ethanol on 0.1mg scorpion venom
Table 2: Absorbance values showing effect of 50ml each of 50% and 100% iso-propanol on 0.1mg scorpion venom.
Absorbance values @ 510nm
Fig.7 Bar Diagram showing changes in absorbance at various time intervals in 50% and 99% Ethanol
Table 3: Absorbance values showing effect of 50ml each of 50% and 100% formaldehyde on 0.1mg scorpion venom.
Absorbance values @ 510nm
Fig.8 Bar Diagram showing changes in absorbance at various time intervals in 50% and 99% Formaldehyde
By analyzing the absorbance values obtained at different time intervals with respective to different chemicals, we draw the following conclusions. All chemicals are showing the highest peak at 30 min of time. The absorbance values are becoming constant at and after 45min, which means that the proteins and enzymes are degraded so that the values are going down. In the case of all chemicals the final absorbance values is low, but in case of ethanol the value is still lower comparative to iso-propanol and formaldehyde. So ethanol is very efficient in neutralizing the venom action. The chemical reaction which took place in 99% ethanol (absolute ethanol) with 0.1mg venom shows less absorbance values implying that it neutralizes efficiently comparative to the same amount of venom in 50% ethanol.
We are grateful to Savana Venkata Subba Naidu, Savana Hymavathy and workers who have helped us to collect live Scorpions in our farm and in the forest.