Identification and analgesic activity study of analgesic protein â ¦-2 from Naja naja atra venom.

Background: Acid-sensing ion channel 1a (ASIC1a) plays a critical role in physiological and pathological processes. To further elucidate the biological functions of ASICs and their relationships with disease occurrence and development, it is advantageous to investigate and develop additional regulatory factors for ASICs. Methods: In this study, cation exchange chromatography was used to separate seven chromatographic components from Naja naja atra venom. Capillary electrophoresis was employed to detect that Ⅶ peak component containing a main protein Ⅶ-2, which could bind to ASIC1a. The analgesic effects of Ⅶ-2 protein were determined using hot plate methods, and ASIC1a expression in spinal cord tissue from rats with inflammatory pain was detected using western blot. Results: The purified Ⅶ-2 protein named Naja naja atra venom-Ⅶ-2 (NNAV-Ⅶ-2) was obtained by Sephadex G-50 gel filtration, which exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 6.7 kD. Remarkably, the NNAV-Ⅶ-2 protein demonstrated a significant analgesic effect and downregulated ASIC1a expression in the spinal cord tissue of rats with inflammatory pain. Conclusions: The analgesic mechanism of the NNAV-Ⅶ-2 protein may be associated with its binding to ASIC1a, consequently downregulating ASIC1a expression in neural tissues.

Development and characterization of nanobody against envenomation by Naja naja oxiana.

Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 µg of recombinant nanobody could neutralize the toxic effects of 20 µg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.

Dimethyl ester of bilirubin ameliorates Naja naja snake venom-induced lung toxicity in mice via inhibiting NLRP3 inflammasome and MAPKs activation.

Naja naja snake bite causes thousands of deaths worldwide in a year. N. naja envenomed victims exhibit both local and systemic reactions that potentially lead to death. In clinical practice, pulmonary complications in N. naja envenomation are commonly encountered. However, the molecular mechanisms underlying N. naja venom-induced lung toxicity remain unknown. Here, we reasoned that N. naja venom-induced lung toxicity is prompted by NLRP3 inflammasome and MAPKs activation in mice. Treatment with dimethyl ester of bilirubin (BD1), significantly inhibited the N. naja venom-induced activation of NLRP3 inflammasome and MAPKs both in vivo and in vitro (p < 0.05). Further, BD1 reduced N. naja venom-induced recruitment of inflammatory cells, and hemorrhage in the lung toxicity examined by histopathology. BD1 also diminished N. naja venom-induced local toxicities in paw edema and myotoxicity in mice. Furthermore, BD1 was able to enhance the survival time against N. naja venom-induced mortality in mice. In conclusion, the present data showed that BD1 alleviated N. naja venom-induced lung toxicity by inhibiting NLRP3 inflammasome and MAPKs activation. Small molecule inhibitors that intervene in venom-induced toxicities may have therapeutic applications complementing anti-snake venom.

Inhibition of the Naja naja venom toxicity by polymeric nanoparticles loaded with Leucas aspera methanolic extract.

Background: Snakebite is a neglected tropical disease that affects millions of people worldwide. Developing effective treatments can make a significant contribution to global health efforts and public health initiatives. To reduce mortality due to snakebite, there is an immediate need to explore novel and effective treatment methodologies. In that context, nanoparticle-based drug delivery is gaining a lot of attention. Hydrophilic nanoparticles are suitable for the delivery of therapeutic peptides, proteins, and antigens. Methods: The present investigation is aimed at evaluating the anti-ophidian potential of the methanolic extract of the ethno-medicinal herb Leucas aspera (Willd.) loaded within chitosan nanoparticles (CNP-LA), against the Indian cobra (Naja naja) venom enzymes. For this purpose, nanoparticles were prepared using the ionic gelation method to enhance the efficacy of the extract. The physicochemical and structural features of nanoparticles were investigated using dynamic light scattering (DLS), Fourier-transform Infrared (FTIR), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD) techniques. Results: It was found that CNP-LA has an average size of 260 nm with a polydispersity index of 0.132 (PDI) and zeta potential of 34.7 mV, with an encapsulation efficiency of 92.46%. The in vitro release study was performed at pH 5.0 and 7.4. Furthermore, in vitro studies indicated that CNP-LA inhibited the phospholipase A2, hemolytic, and caseinolytic activities of Naja naja venom with the percentage inhibition of 92.5%, 83.9%, and 94.5%, respectively. Conclusion: This is the first report on the application of herbal methanolic extract loaded within chitosan nanoparticles for neutralizing snake venom enzymes with increased efficiency.

Phytochemical characterization and phospholipase A2 inhibitory effect of Vitex negundo L. root extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Snake bites are a critical health issue in many parts of the world particularly in Asian countries lacking efficient health facilities in rural areas. Cobra is the most common snake type in Asia and is responsible for a large number of mortalities particularly in rural areas. Plants are usually considered the most effective and easy-to-approach treatment for snake bites in rural areas of various countries. Vitex negundo L. is an important medicinal plant traditionally used to treat snake bite envenomation in many countries of Asia. AIM OF THE STUDY: From literature survey of plants traditionally used in the treatment of snake bites in Asian countries including India, Pakistan and Sri Lanka, roots of V. negundo were selected for the present study. Anti-snake venom potential of its roots was assessed through various in vitro assays targeting the phospholipase A2 enzyme. MATERIALS AND METHODS: V. negundo roots were sequentially extracted in different organic solvents to get fractions and in methanol to get total extract. The extracts were evaluated for phospholipase A2 (PLA2) inhibitory potential through inhibition of venom-induced hemolysis, ADP-induced platelet aggregation, PLA2-induced fatty acid hydrolysis and anticoagulant effect of cobra venom. Antioxidant power was determined using DPPH and superoxide radical scavenging assays. GC-MS and HPLC analysis was performed for the total methanol extract. RESULTS: Strong PLA2 inhibitory effect was observed for all the extracts. The ethyl acetate, acetone and methanol fractions significantly inhibited toxic effects of cobra venom under in vitro conditions. Radical scavenging potential of these fractions was also significantly high as compared to non-polar fractions in both DPPH and superoxide scavenging assays. Phytochemical analysis indicated high phenolic and flavonoid contents in these fractions. GC-MS and HPLC analysis of total methanol extract confirmed the presence of bis(2-ethylhexyl) phthalate, phenol, o-Guaiacol, palmitic acid-methyl ester, methyl stearate, quercetin and kaempferol in the plant. CONCLUSION: The study concluded that the roots of V. negundo, particularly their polar extracts, have strong PLA2 inhibitory effect against cobra venom confirming their traditional use to manage snake bites. The roots of this plant can be further studied for isolation of plant-based antisera.

CTXP, The Major Cobra Toxin Peptide from Naja Naja Oxiana Venom; A Promising Target for Antivenom Agent Development.

BACKGROUND AND OBJECTIVE: Snakebite envenoming is a serious public health issue causing more than 135,000 annual deaths worldwide. Naja Naja Oxiana is one of the most clinically important venomous snakes in Iran and Central Asia. Conventional animal-derived polyclonal antibodies are the major treatment of snakebite envenoming. Characterization of venom components helps to pinpoint the toxic protein responsible for clinical manifestations in victims, which aids us in developing efficient antivenoms with minimal side effects. Therefore, the present study aimed to identify the major lethal protein of Naja Naja Oxiana by top-down proteomics. METHODS: Venom proteomic profiling was performed using gel filtration (GF), reversed-phase (RP) chromatography, and intact mass spectrometry. The toxicity of GF-, and RP-eluted fractions was analyzed in BALB/c mice. The rabbit polyclonal antisera were produced against crude venom, GF fraction V (FV), and RP peak 1 (CTXP) and applied in neutralization assays. RESULTS: Toxicity studies in BALB/c identified FV as the major toxic fraction of venom. Subsequently, RP separation of FV resulted in eight peaks, of which peak 1, referred to as "CTXP" (cobra toxin peptide), was identified as the major lethal protein. In vivo neutralization assays using rabbit antisera showed that polyclonal antibodies raised against FV and CTXP are capable of neutralizing at least 2-LD50s of crude venom, FV, and CTXP in all tested mice. CONCLUSION: Surprisingly, the Anti-CTXP antibody could neutralize 8-LD50 of the CTXP peptide. These results identified CTXP (a 7 kDa peptide) as a potential target for the development of novel efficient antivenom agents.

Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights.

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

The protective mechanism of Naja Naja atra venom on diabetic kidney disease.

Background: Diabetic kidney disease (DKD) is a serious microvascular complication of diabetes that affects both type 1 and type 2 diabetes patients at a high incidence rate. Naja Naja atra venom (NNAV) has been shown to have protective effects and improved renal function in diabetic rats. However, its mechanism of action is still unclear. This study aims to unravel the effectiveness and mechanisms of NNAV on DKD. Methods: We conducted in vitro experiments in which Human Kidney-2 (HK-2) cells were stimulated with high glucose, and exposed to varying concentrations of NNAV. Cell morphology, as well as α-SMA, TGF-ß1, and E-cadherin levels, were analyzed using immunofluorescence and western blot. In vivo experiments involved a diabetic rat model, where varying concentrations of cobra α-neurotoxin (CTX) were administrated via gastric treatment. We observed and noted pathomorphological changes, measured biochemical and oxidative stress indices, and used western blot to assess podocin and nephrin levels. Results: High glucose levels can induce a decrease in E-cadherin expression and an increase in α-SMA and transforming growth factor-ß1 (TGF-ß1) expression in HK-2 cells. NNAV can inhibit the transdifferentiation of HK-2 cells to myofibroblast (MyoF) in a high glucose environment and reduce the expression of TGF-ß1. Cobra α-neurotoxin (CTX) can reduce urine protein in diabetes model rats at an early stage, which is dose-independent and has a time application range. CTX can regulate the expression of nephrin and podocin. Conclusion: The present study indicates that CTX and NNAV attenuate STZ and high glucose-induced DKD. Its mechanisms of action are associated with inhibiting oxidative stress and TEMT. The study suggests that NNAV and CTX might be a potential therapeutic drug for treating DKD.

Evaluation of Neutralization Potential of Naja naja and Daboiarusselii Snake Venom by Root Extract of Cyanthillium cinereum.

Aim: One of the main reasons for the death due to snake bites is the non-availability of antivenoms in the regions where they are needed. The use of medicinal plants and plant-based natural products as an alternative to antivenom will become a milestone in snake bite envenomation. The present study investigates the in vitro antivenom properties of Cyanthillium cinereum root extracts. Materials and methods: The C. cinereum root's aqueous extract was prepared by the Soxhlet extraction method, and phytochemical screening was performed to detect the presence of various bioactive compounds. Thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) analysis were performed for the detection and identification of phytochemical constituents. In this study, an in vitro model is used to assess the antivenom capability of aqueous extract. Venom toxicity and neutralization assays were as follows: An in vitro pharmacological evaluation was performed by direct hemolysis assay, indirect hemolytic assay, proteolytic activity, neutralization of procoagulant activity, and gelatin liquefaction method. Results: Qualitative analysis of phytochemicals by the standard method showed the presence of various phytochemical constituents. Also, GC-MS analysis showed the presence of three major compounds that possess antivenom activity from the obtained 60 bioactive compounds, and their chemical structures were also determined. Venom protein profiling was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The plant extract was able to neutralize the Naja naja (N. naja) and Daboia russelii (D. russelii) venom induced hemolysis and it was reduced below 50 and 40%, respectively and the extract was also able to reduce the hemolytic halo produced by venoms. Procoagulant activity and gelatin liquefaction assay showed that venom-induced clotting was neutralized by increasing the root extract concentration sufficiently. Conclusion: The aqueous extract of the root of C. cinereum showed potent in vitro venom-neutralizing activity, and it can be used as a formidable therapeutic agent against N. naja and D. russelii envenomation. How to cite this article: Suji S, Dinesh MD, Keerthi KU, Anagha KP, Arya J, Anju KV. Evaluation of Neutralization Potential of Naja naja and Daboia russelii Snake Venom by Root Extract of Cyanthillium cinereum. Indian J Crit Care Med 2023;27(11):821-829.

Multilevel Comparison of Indian Naja Venoms and Their Cross-Reactivity with Indian Polyvalent Antivenoms.

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA2 activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA2s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable.

Augmented rescue of macroglobulins by supplementation of anti-snake venom with methanolic extract of Andrographis paniculata in Naja naja envenomation.

Proteins of the macroglobulin family are prime targets of venom enzymes in snake bite. A massive reduction in the active concentration of these multifunctional proteins in snake bite, makes the living system vulnerable to dysregulation. This study investigates the ability of Indian polyvalent anti-snake venom (ASV), methanolic extract of Andrographis paniculata (MAP) and their combination in rescuing human alpha 2-macroglobulin (A2MG) and its homologues in rat plasma, from inactivation by Naja naja (N.N) venom enzymes. In-vitro experiments were conducted with heparinized human plasma and in-vivo experiments with female Wistar rats. Along with appropriate controls, there were 3 test groups in in-vitro and 8 test groups in in-vivo experiments. The in-vitro test groups were exposed to N.N venom for zero, 30 or 90 min prior to incubation with ASV or MAP or reduced ASV supplemented with MAP and incubated for 16 h at 37 °C. Chymotrypsin-bound esterase (CTBE) activity of A2MG was estimated. Rats were administered the venom intramuscularly and treated with ASV/MAP/ASV + MAP. CTBE activity of macroglobulin homologues was measured on day 1, 7 and 14. Survival of animals was noted. In human plasma, addition of ASV or MAP or ASV + MAP prevented loss of A2MG activity maximally to the extent of 88-100% (p = 0.001). In rats, reduced concentration of ASV supplemented with MAP showed complete rescue of macroglobulin homologues and 90% survival. The compulsive evidence from this study, underscores the merits of using this multipronged strategy in rescuing the macroglobulins and improving survival in envenomation due to N.N.

A Contemporary Exploration of Traditional Indian Snake Envenomation Therapies.

Snakebite being a quick progressing serious situation needs immediate and aggressive therapy. Snake venom antiserum is the only approved and effective treatment available, but for selected snake species only. The requirement of trained staff for administration and serum reactions make the therapy complicated. In tropical countries where snakebite incidence is high and healthcare facilities are limited, mortality and morbidities associated with snake envenomation are proportionately high. Traditional compilations of medical practitioners' personal journals have wealth of plant-based snake venom antidotes. Relatively, very few plants or their extractives have been scientifically investigated for neutralization of snake venom or its components. None of these investigations presents enough evidence to initiate clinical testing of the agents. This review focuses on curating Indian traditional snake envenomation therapies, identifying plants involved and finding relevant evidence across modern literature to neutralize snake venom components. Traditional formulations, their method of preparation and dosing have been discussed along with the investigational approach in modern research and their possible outcomes. A safe and easily administrable small molecule of plant origin that would protect or limit the spread of venom and provide valuable time for the victim to reach the healthcare centre would be a great lifesaver.

Isolation and Characterization of Two Postsynaptic Neurotoxins From Indian Cobra (Naja Naja) Venom.

The Indian Cobra (Naja naja) is among the "Big Four" responsible for most of the snakebite envenoming cases in India. Although recent proteomic studies suggest the presence of postsynaptic neurotoxins in N. naja venom, little is known about the pharmacology of these toxins. We isolated and characterized α-Elapitoxin-Nn2a (α-EPTX-Nn2a; 7020 Da) and α-Elapitoxin-Nn3a (α-EPTX-Nn3a; 7807 Da), a short-chain and long-chain postsynaptic neurotoxin, respectively, which constitute 1 and 3% of N. naja venom. α-EPTX-Nn2a (100-300 nM) and α-EPTX-Nn3a (100-300 nM) both induced concentration-dependent inhibition of indirect twitches and abolished contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Indian polyvalent antivenom (1 ml/0.6 mg) prevented the in vitro neurotoxic effects of α-EPTX-Nn2a (100 nM) and α-EPTX-Nn3a (100 nM). The addition of Indian polyvalent antivenom (1 ml/0.6 mg), at the t90 time point, could not reverse the in vitro neurotoxicity of α-EPTX-Nn2a (100 nM). The in vitro neurotoxicity of α-EPTX-Nn3a (100 nM) was partially reversed by the addition of Indian polyvalent antivenom (1 ml/0.6 mg), as well as repeated washing of the tissue. α-EPTX-Nn2a displayed non-competitive antagonism of concentration-response curves to carbachol, with a pA2 of 8.01. In contrast, α-EPTX-Nn3a showed reversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.17. De novo sequencing of α-EPTX-Nn2a and α-EPTX-Nn3a showed a short-chain and long-chain postsynaptic neurotoxin, respectively, with 62 and 71 amino acids. The important observation made in this study is that antivenom can reverse the neurotoxicity of the clinically important long-chain neurotoxin, but not the short-chain neurotoxin, from N. naja venom.

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling.

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both "water" and "membrane" conformations of the central loop (loop-2) were determined by X-ray crystallography. The "water" conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The "membrane" conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step-the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their "water" NMR models. We found that the both toxins transform their "water" conformation of loop-2 into the "membrane" one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes.

Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats.

Venom proteome profiling of Naja naja from the Western Ghats region in Kerala was achieved through SDS-PAGE and RP-HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 115 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified through conventional and 39 peptides through homology-driven proteomics approaches. Fourteen peptides derived through de novo complements the Mascot data indicating the importance of homology-driven approaches in improving protein sequence information. Among the protein families identified, glutathione peroxidase and endonuclease were reported for the first time in the Indian cobra venom. Immunological cross-reactivity assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 µg/mL) value than that of PSAV (6.04 µg/mL) and Virchow (6.03 µg/mL) antivenoms. Western blotting experiments indicated that all the antivenoms elicited poor binding specificities, especially towards low molecular mass proteins. Second-generation antivenomics studies revealed that VINS antivenom was less efficient to detect many low molecular mass proteins such as three-finger toxins and Kunitz-type serine protease Inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja from the Western Ghats and emphasized the need for developing more efficient antivenoms.

Venomics of the Enigmatic Andaman Cobra (Naja sagittifera) and the Preclinical Failure of Indian Antivenoms in Andaman and Nicobar Islands.

The Andaman and Nicobar Islands are an abode to a diversity of flora and fauna, including the many endemic species of snakes, such as the elusive Andaman cobra (Naja sagittifera). However, the ecology and evolution of venomous snakes inhabiting these islands have remained entirely uninvestigated. This study aims to bridge this knowledge gap by investigating the evolutionary history of N. sagittifera and its venom proteomic, biochemical and toxicity profile. Phylogenetic reconstructions confirmed the close relationship between N. sagittifera and the Southeast Asian monocellate cobra (N. kaouthia). Overlooking this evolutionary history, a polyvalent antivenom manufactured using the venom of the spectacled cobra (N. naja) from mainland India is used for treating N. sagittifera envenomations. Comparative evaluation of venoms of these congeners revealed significant differences in their composition, functions and potencies. Given the close phylogenetic relatedness between N. sagittifera and N. kaouthia, we further assessed the cross-neutralising efficacy of Thai monovalent N. kaouthia antivenom against N. sagittifera venoms. Our findings revealed the inadequate preclinical performance of the Indian polyvalent and Thai monovalent antivenoms in neutralising N. sagittifera venoms. Moreover, the poor efficacy of the polyvalent antivenom against N. naja venom from southern India further revealed the critical need to manufacture region-specific Indian antivenoms.

Potential of herbal cocktail of medicinal plant extracts against 'big four' snake venoms from India.

BACKGROUND: Venomous snake bites cause acute medical emergencies and are fatal. India accounts for large proportion of snake-bite deaths globally. Medically important 'BIG FOUR' snakes of India are Bungarus caeruleus (krait), Naja naja (cobra), Echis carinatus (saw-scaled viper) and Daboia russelii (Russell's viper). Polyherbal formulations have been proved to be effective in treatment of diseases than a single formulation. OBJECTIVE(S): To evaluate aqueous ethanolic extract cocktail of Azadirachata indica, Butea monosperma, Citrus limon, Clerodendrum serratum and Areca catechu for antidote potential against BIG FOUR venoms in ex vivo and in vivo model. MATERIALS AND METHODS: Anti-hemorrhagic and venom neutralization studies were performed in seven-day old chick embryo model for ex vivo studies. In vivo studies were performed using male Swiss albino mice for antivenom potential of herbal cocktail by performing anti-edematic, anti-hemorrhagic, anti-myotoxic activity, and venom neutralization. RESULTS: Herbal cocktail exhibited differential venom inhibition potential against four venoms tested. Hemorrhagic activity was completely neutralized by the herbal cocktail; myotoxic activities of krait and Russell's viper venom were neutralized; while anti-edematic activity was observed for krait and cobra venom. Herbal cocktail completely neutralized venom lethality (3∗LD50) of krait and saw-scaled viper venom. CONCLUSION: Inhibitions of various venom components of all four venoms suggests presence of phytochemicals in herbal cocktail with therapeutic properties. Further studies would help in the development of a formulation as a first-aid towards treatment of snake bite victims.

The Metabolomic Profiles of Sera of Mice Infected with Plasmodium berghei and Treated by Effective Fraction of Naja naja oxiana Using 1H Nuclear Magnetic Resonance Spectroscopy.

BACKGROUND: The use of venom fractions from the Iranian cobra could be useful adjunct treatments of malaria with chloroquine. A metabolomic investigation with 1HNMR spectroscopy was conducted on an effective fraction tested earlier using Plasmodium berghei as an experimental murine model. PURPOSE: We sought to ascertain both safety and anti-parasitic effects of experimental therapies. METHODS: After purification of the venom fractions, 25 mice were infected, then treated for 4 days with 0.2 ml of 5 mg/kg, 2.5 mg/kg and 1 mg/kg of the effective fraction, chloroquine, and a drug vehicle. An ED50 was obtained using Giemsa staining and real-time PCR analysis. The toxicity tests inspecting both liver and kidney tissues were performed. RESULTS: A clear inhibitory effect on parasitaemia was observed (with 75% inhibition with 5 mg/kg and 50% reduction when 2.5 mg/kg dosage used). ED50 obtained 2.5 mg/kg. The metabolomics were identified as differentiation of aminoacyl-t-RNA biosynthesis, valine, leucine, isoleucine biosynthesis and degradation pathways were observed. CONCLUSION: Upon therapeutic effects of cobra venom fraction, further optimization of dose-dependent response of pharmacokinetics would be worthwhile for further exploration in adjunct experimental venom therapies.

Evaluation of the merit of the methanolic extract of Andrographis paniculata to supplement anti-snake venom in reversing secondary hemostatic abnormalities induced by Naja naja venom.

Increasing evidence suggests a sizable involvement of hemotoxins in the morbidity associated with envenomation by the Indian spectacled cobra, Naja naja (N.N). This study investigates the ability of Indian polyvalent anti-snake venom (ASV), methanolic extract of Andrographis paniculata (MAP) and their combination in reversing the hemostatic abnormalities, viz. activated partial thromboplastin time(aPTT), prothrombin time(PT) and thrombin time(TT) in citrated plasma. These parameters were assessed in 2 groups of experiments. Group 1: Without the prior incubation of plasma with venom and Group 2: With prior incubation of plasma with venom for 90 min at 37°C. Venom caused significant (p < 0.001) prolongation in aPTT (175%), PT (49%) and TT (34%) in Group 1 and ASV could completely bring them back to normal. MAP showed a concentration-dependent reversal in aPTT, normalization of PT and prolongation of TT. When low concentration of ASV was supplemented with MAP, their combined effect in normalizing aPTT and PT improved by 37% and 26% respectively when compared to ASV alone. In Group 2, venom caused significant (p < 0.001) prolongation in aPTT (231%), PT (312%) and TT (245%). ASV had limited effect in reversing aPTT (52%), TT (31%) but completely normalized PT. MAP was marginally effective in reversing the prolonged aPTT and PT but caused further prolongation of TT. Combination of ASV and MAP was more effective than ASV alone in reversing venom-induced increase in aPTT (52%) and PT (29%). The study proved that, a drastic reduction of ASV by 70%, could be effectively supplemented by MAP in combating hemostatic abnormalities induced by NN venom.

Proteome Analysis of Toxic Fractions of Iranian Cobra (Naja naja Oxiana) Snake Venom Using Two-Dimensional Electrophoresis and Mass Spectrometry.

Snake venoms are mostly composed of various proteins and peptides with toxicity and pharmacological effects depending on their geographical sources. Naja naja oxiana is one of the most medically important venomous snakes in Iran and Central Asia. The bite of this type of snake can cause severe pain and swelling, as well as neurotoxicity. Without medical treatment, symptoms quickly worsen and death can occur soon. A detailed understanding of venom components can provide new insight into the production of antivenom against toxic agents instead of crude venom. Specific antibodies against toxic fractions are of utmost importance in neutralizing crude venom. Therefore, the proteome profile of these fractions of Naja naja oxidana venom was analyzed using fractionation by gel filtration, two-dimensional electrophoresis, mass spectrometry, and data mining. Base on the results, in total, 32 spots were detected and categorized into three protein families, namely three-finger toxin (3FTx), phospholipase, and Cysteine-rich secretory proteins (CRISP). These proteins consist of more than 70% crude venom all with a molecular weight below 25 kDa. The 3FTx as a highly diverse constituent in the venom of Naja species was in large quantity in this district. Short-chain neurotoxins, including short neurotoxin, cytotoxin, and muscarinic toxin-like protein, were in abundance, respectively. In conclusion, the recognition of toxic fractions of Naja naja oxiana in this region could be of great help in the production of an effective antivenom against similar compositions. It can also help the medical care department to find out the clinical sign of cobra venom. To the best of our knowledge, this was the first study to report the proteomic of toxic fractions of Naja naja oxiana in Iran.