Progressive hemorrhage and myotoxicity induced by echis carinatus venom in murine model: neutralization by inhibitor cocktail of n,n,n `,n `-tetrakis (2-pyridylmethyl) ethane-1,2-diamine and silymarin

Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn(2+)MPs), phospholipase A(2)s (PLA(2)s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N', N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn(2+)MPs of ECV (IC50: 6.7 mu M). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA(2)s (IC50: 12.5 mu M) and HYs (IC50: 8 mu M) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity

EC-PIII, a novel non-hemorrhagic procoagulant metalloproteinase: Purification and characterization from Indian Echis carinatus venom

Procoagulant snake venom toxins find extensive use as reagents in laboratory tests and diagnostic kits. In the present study we report a novel P-III class procoagulant SVMP, EC-PIII from Echis carinatus venom. EC-PIII was purified using a combination of gel-filtration and anion-exchange chromatography. It has a molecular mass of 110kDa and is a dimeric protein as determined by SDS-PAGE. DLS results show that the protein is homogenous and stable in solution. Peptide mass fingerprinting revealed that the peptides obtained show high homology to the other members of SVMP family. The enzymatic studies revealed that EC-PIII shows protease activity and is inhibited by metalloproteinase inhibitors such as EDTA. EC-PIII exhibits procoagulant effect under in-vitro conditions. Local toxicity studies revealed that EC-PIII is devoid of hemorrhagic as well as myotoxic activities. This is the first report of a non-hemorrhagic SVMP to be identified from Indian Echis carinatus venom. EC-PIII can find potential use in diagnostic and other therapeutic uses owing to its biochemical and pharmacological properties

Progressive hemorrhage and myotoxicity induced by echis carinatus venom in murine model: neutralization by inhibitor cocktail of n,n,n `,n `-tetrakis (2-pyridylmethyl) ethane-1,2-diamine and silymarin

Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn(2+)MPs), phospholipase A(2)s (PLA(2)s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N', N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn(2+)MPs of ECV (IC50: 6.7 mu M). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA(2)s (IC50: 12.5 mu M) and HYs (IC50: 8 mu M) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity

Local and systemic toxicity of echis carinatus venom: neutralization by cassia auriculata l. leaf methanol extract

Viper bites cause high morbidity and mortality especially in tropical and subtropical regions, affecting a large number of the rural population in these areas. Even though anti-venoms are available, in most cases they fail to tackle viper venom-induced local manifestations that persist even after anti-venom administration. Several studies have been reported the use of plant products and approved drugs along side anti-venom therapy for efficient management of local tissue damage. In this regard, the present study focuses on the protective efficacy of Cassia auriculata L. (Leguminosae) against Echis carinatus venom (ECV) induced toxicity. C. auriculata is a traditional medicinal plant, much valued in alternative medicine for its wide usage in ayurveda, naturopathy, and herbal therapy. Further, it has been used widely by traditional healers for treatment of snake and scorpion bites in the Western Ghats of Karnataka, India. In the present study, C. auriculata leaf methanol extract (CAME) significantly inhibited enzymatic activities of ECV proteases (96 +/- A 1 %; P = 0.001), PLA(2) (45 +/- A 5 %; P = 0.01) and hyaluronidases (100 %; P = 0.0003) in vitro and hemorrhage, edema and myotoxicity in vivo. Further, CAME effectively reduced the lethal potency of ECV and increased the survival time of mice by similar to 6 times (17 vs 3 h). These inhibitory potentials of CAME towards hydrolytic enzymes, mortal and morbid symptoms of ECV toxins clearly substantiates the use by traditional healers of C. auriculata as a folk medicinal remedy for snakebite

Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom

Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn2+) chelating agents; N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn2+ and revealed potent inhibition of ECV rnetalloproteases (ECVMPs) in vitro (IC50: 6.7 mu M) compared to DTPA and TTD. The specificity of TPEN towards Zn2+ was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific zn(2+) chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage. (C) 2014 Elsevier Ltd. All rights reserved

Differential action of Indian Big Four snake venom toxins on blood coagulation

Snake venom toxins affect hemostasis by modulating blood coagulation factors resulting in pro/anti-coagulant status of blood. Most of the reported effects are in vitro which do not reflect in-vivo coagulation status. The specific interference of venom toxins on coagulation factor(s) in vivo can be used as a marker to identify the snake species responsible for envenomation and administration of species-specific anti-venom thereafter. The current review attempts to highlight specific alterations induced by BIG FOUR venomous snakes of India towards blood coagulation factors. Future insights in this regard will be valuable in identifying the snake species responsible for bite which in most cases is unknown

Purification and characterization of an anti-hemorrhagic protein from Naja naja (Indian cobra) venom

Snake venom Kunitz-type proteins are well known to inhibit serine proteases but a few studies have also shown matrix metalloproteases (MMPs) inhibition. In view of the fact that MMPs and snake venom metalloproteases (SVMPs) have similar catalytic site, inhibition of SVMP activity by Kunitz-type proteins remains to be studied. Recent proteomic studies of Naja naja (N. naja) venom revealed the abundance of Kunitz-type proteins. In this regard, present study aimed at purification of a protease inhibitor from N. naja venom that inhibits the toxicity of SVMPs rich Echis carinatus (E. carinatus) venom. N. naja venom effectively inhibited E. carinatus venom-induced hemorrhage. Purification of the active principle responsible for anti-hemorrhagic effect was achieved by fractionation of N. naja venom in three successive chromatographic steps. SDS-PAGE revealed that purified anti-hemorrhagic protein (NNAh) has an apparent molecular mass of ∼44 kDa and single peak in RP-HPLC demonstrated its homogeneity. NNAh also inhibited myonecrosis induced by E. carinatus venom and reduced activity of creatine kinase in NNAh treated animal sera substantiated the anti-myonecrotic effect. Hemorrhage and myonecrosis inhibitory effects of NNAh were further supported by inhibition of E. carinatus venom-mediated gelatinolysis and collagenolysis. NNAh falls into the category of Kunitz-type serine protease inhibitor as determined by peptide mass fingerprinting and shown to be a strong inhibitor of chymotrypsin. Collectively our data signify that NNAh is a Kunitz-type chymotrypsin inhibitor which also inhibited metalloprotease activities of E. carinatus venom. In future, complete sequence of NNAh and peptide region(s) responsible for inhibition will assist to deduce the mechanism of action