Naja Naja Research Articles

From birth to bite: the evolutionary ecology of India's medically most important snake venoms

BackgroundSnake venoms can exhibit remarkable inter- and intraspecific variation. While diverse ecological and environmental factors are theorised to explain this variation, only a handful of studies have attempted to unravel their precise roles. This knowledge gap not only impedes our understanding of venom evolution but may also have dire consequences on snakebite treatment. To address this shortcoming, we investigated the evolutionary ecology of venoms of Russell’s viper (Daboia russelii) and spectacled cobra (Naja naja), India’s two clinically most important snakes responsible for an alarming number of human deaths and disabilities.MethodologySeveral individuals (n = 226) of D. russelii and N. naja belonging to multiple clutches (n = 9) and their mothers were maintained in captivity to source ontogenetic stage-specific venoms. Using various in vitro and in vivo assays, we assessed the significance of prey, ontogeny and sex in driving venom composition, function, and potency.ResultsConsiderable ontogenetic shifts in venom profiles were observed in D. russelii, with the venoms of newborns being many times as potent as juveniles and adults against mammalian (2.3–2.5 ×) and reptilian (2–10 ×) prey. This is the first documentation of the ontogenetic shift in viperine snakes. In stark contrast, N. naja, which shares a biogeographic distribution similar to D. russelii, deployed identical biochemical cocktails across development. Furthermore, the binding kinetics of cobra venom toxins against synthetic target receptors from various prey and predators shed light on the evolutionary arms race.ConclusionsOur findings, therefore, provide fascinating insights into the roles of ecology and life history traits in shaping snake venoms.

Phage display-derived alpaca nanobodies as potential therapeutics for Naja atra snake envenomation.

Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified VHH genes from isolated peripheral blood mononuclear cells and constructed a phage display VHH library of 1.0 × 107 transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

Development of chicken egg yolk antibodies (IgY) against venom of cobra (Naja naja) and krait (Bungarus caeruleus) and a study on its neutralization potential

The Big Four snakes are the leading causes of snakebite mortality in India. Among them, The Indian spectacled cobra (Naja naja) and common krait (Bungarus caeruleus) contribute about one-third of the deaths. The conventional anti-snake venoms (ASV) produced from equine serum are found to be expensive, with a high frequency of clinical side effects, low specificity, and production drawbacks. The current study, therefore, attempted to develop chicken egg yolk immunoglobulins (IgY) as antivenoms against the venoms of cobra and krait venoms. The major pathophysiological properties of the venoms and the efficacy of monovalent ASV IgY were evaluated. The titer of anti-cobra and anti-krait IgY was found to be 1:64000 on the 145th day post-immunization. The results showed that venoms (lethal doses (LD50): cobra:0.375 mg/kg, krait:0.0375 mg/kg of mice) had hemolytic, pro-coagulant, inflammatory, hemorrhagic, and edematic activities that were effectively neutralized by ASV-IgY. 1 mL of monovalent ASV IgY was able to neutralize 0.22 mg of cobra venom and 0.028 mg of krait venom whereas 1 mL of commercial polyvalent antivenom was able to neutralize 0.28 mg of cobra venom and 0.035 mg of krait venom. The study results suggest that using chicken IgY as an monovalent antivenom could effectively treat snakebites.

Perceptions on Indigenous Medicine, First Aid and Treatment of Snakebite among Sri Lankan Farmers

Since snakebite is a serious public health concern in Asia, one of the most affected countries is Sri Lanka. The incidence, species variety, clinical symptoms, and management of snakebite in Asia, with a focus on Sri Lanka, are all covered in this review paper. Many of the venomous snake species found in Asia are abundant and diverse. Snake bites are a typical occurrence, especially in rural areas where farming and encounters between humans and snakes are common. Numerous snake species, including the dangerous Russell’s viper and the spectacled cobra, are found in Sri Lanka specifically. Depending on the type of snake bitten and the amount of venom administered, different clinical symptoms might result from a snake bite. Pain, bruising, swelling, and systemic symptoms like nausea, vomiting, and paralysis are typical ones. Treatment that is given too slowly or not at all can result in fatal consequences. Antivenom delivery, wound care, and supportive measures are all essential components of managing snake bite cases effectively. However, obstacles including a lack of access to medical facilities and less availability of antivenom sometimes make it difficult to get treatment quickly, particularly in isolated places and concern about indigenous treatment as well. Public awareness campaigns, better healthcare facilities, and epidemiology research are only a few of the measures taken against snakebite in Sri Lanka. To lessen the number of snakebites in, government institutions, medical professionals, and international organizations must work together. Better antivenom medicines and improved preventive and treatment plans for snakebite in this area require more study and funding.International Journal of Human and Health Sciences Vol. 08 No. 02 Apr’24 Page: 116-125

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

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. 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. It was found that CNP-LA has an average size of 260nm 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. This is the first report on the application of herbal methanolic extract loaded within chitosan nanoparticles for neutralizing snake venom enzymes with increased efficiency.

Clinico-Epidemiological Profile, Trends, and Health-Related Outcomes of Snakebite Victims: A One-Year Prospective Study from Eastern India.

Snakebite envenomation is a significant life-threatening public health problem in Southeast Asia (SEA). In this region, India reported the largest number of snakebite deaths from 2000 to 2019 (1.2 million), with an average of 58,000 deaths yearly. This prospective observational study was carried out among snakebite victims at the emergency department (ED) of a tertiary care public sector hospital in eastern India. A total of 145 cases of venomous snakebite were investigated. More than half (n = 81, 56%) of the snakebite victims were between 17 to 45 years. Most of the snakebite victims were male (68%) and were farmers (53%) by occupation. The majority of snakebites occurred during the daytime (76%) and while outdoors (67%). Most victims sustained a bite on the lower extremity (71%). The peak incidence of snakebites occurred from June to September (69%). Three-quarters of all patients were unaware of the required first aid measures following a snakebite. Among the 145 venomous snakebites, 48 were presumptively identified as the Indian cobra, 32 by the Indian krait, 56 by the Russel's viper, and 9 by saw-scaled viper. The mean duration from the snakebite to the onset of systemic effects in the Indian cobra was 52 ± 14.28 min, 66 ± 18.35 min in the Indian krait, 42 ± 13.47 min in Russel's viper, and 48 ± 16.38 min in saw-scaled viper. Respiratory failure was the commonly observed complication following an elapid envenomation. The mortality rate was 2.1% among the patients treated with antivenom. Snakebite is considered an occupational hazard in India, commonly affecting the young population in their productive period. The peak incidence was during monsoon season, and the majority had neurotoxic envenomation following an elapid bite (55%) that contributed to the increased mortality and morbidity among young adults. Of the 145 patients, the majority (84%) recovered fully with treatment; 16% of the victims developed morbidity viz cellulitis, respiratory failure, acute renal failure, compartment syndrome, local tissue necrosis, intracerebral hemorrhage, and disseminated intravascular coagulation. Appropriate first aid measures and timely medical intervention can significantly improve the treatment outcome following snakebites.

Reply to Jalink, M.B. Comment on "Senthilkumaran et al. Bilateral Simultaneous Optic Neuritis Following Envenomations by Indian Cobra and Common Krait. Toxins 2022, 14, 805".

We thank the author for showing interest in our article [...].

Differential effects of the venoms of Russell’s viper and Indian cobra on human myoblasts

Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell’s viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy. Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian ‘Big Four’ snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell’s viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell’s viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.

Surgical management of traumatic evisceration of abdominal contents in a common cobra

Surgical management of traumatic evisceration of abdominal contents in a common cobra

Biodiversity of Snakes During Monsoon in Bhadrawati, District Chandrapur (M.S.) India

Abstract: The snakes are the most feared reptiles on the earth, and inhabit the most inhospitable terrains of the world including dry lands, wetland and all the possible areas. The Bhadrawati region is having a large number snakes since ancient times and the name itself suggest that there are snakes in this region. So in order to assess biodiversity of snakes in the Bhadrawati region snakes were studied during monsoon season. Snakes plays important ecological role in food chain. Depletion of these animals throughout the globe and their extinction is causing a conscientious and diligent task to the people of all spheres of the society to conserve them. Species wise concentration of snakes was estimated to predict proportion of particular snake species in the region. In all near about 300 snakes were captured in the rainy season which include 246 non poisonous 53 poisonous and 01 semi poisonous type during monsoon season of 2022. Common Indian Cobra Naja naja is poisonous and Indian rat snakes among non poisonous and only one species of semi poisonous i.e. common cat snakes were found to be predominant in the region. The studies indicate prevalence of snakes in the region

Systematic review and meta-analysis on the efficacy of Indian polyvalent antivenom against the Indian snakes of clinical significance.

Snakebite in India is a severe problem as it causes a mortality rate of 58,000 and a disability rate of 140,000 every year which is the highest among any other country. Antivenom is the primary therapy for snakebite, and its manufacturing techniques have essentially stayed unaltered for over a century. Indian polyvalent antivenom, a scientifically validated medicine for treating the toxic effects of snakebites, is available against the venom of the so-called Big Four snakes namely Spectacled cobra (Naja naja), Saw-scaled viper (Echis carinatus), Russell's viper (Daboia russelli) and the Common krait (Bungarus caeruleus), responsible for majority of the deaths in India. India hosts many other species of snakes, including cobras, kraits, saw-scaled vipers, sea snakes, and pit vipers, responsible for clinically severe envenomation. Neutralization strategy has been applied to access the efficacy of antivenoms, crucial for reducing snake bite deaths and disabilities. This review aims to conduct a systematic review and meta-analysis on the neutralization efficiency of the Polyvalent Antivenom (PAV) and focus on the factors that may contribute to the poor recognition of the antivenom towards the venom toxins. Reports focusing on the investigation of antivenom efficacy were searched and collected from several databases. Preclinical studies that reported the neutralization efficacy of the commercial antivenom against the medically important snakes of India were included. The articles were screened based on the inclusion criteria and 8 studies were shortlisted for meta-analysis. Pooled proportion was calculated for the antivenom efficacy reported by the studies and was found to be statistically significant with a 95% confidence interval. The heterogenicity in the venom toxicity and neutralization potency of the antivenom was evident in the overall estimate (proportion) and individual data. We provide comprehensive evidence on antivenom efficacy against medically important snakes from various parts of India which may aid in identifying the gaps in snake envenomation therapy and the need for novel potentially improved treatment of snakebites.

Effect of Microgravity on the Crystallization of Cardiotoxin from the Venom of Spectacled Cobra Naja naja

Effect of Microgravity on the Crystallization of Cardiotoxin from the Venom of Spectacled Cobra Naja naja

Genetic insights: mapping sex-specific loci in Siamese cobra (Naja kaouthia) sheds light on the putative sex determining region.

The location of female-specific/linked loci identified in Siamese cobra (Naja kaouthia) previously has been determined through in silico chromosome mapping of the Indian cobra genome (N. naja) as a reference genome. In the present study, we used in silico chromosome mapping to identify sex-specific and linked loci in Siamese cobra. Many sex-specific and sex-linked loci were successfully mapped on the Z sex chromosome, with 227 of the 475 specific loci frequently mapped in a region covering 57Mb and positioned at 38,992,675-95,561,177bp of the Indian cobra genome (N. naja). This suggested the existence of a putative sex-determining region (SDR), with one specific locus (PA100000600) homologous to the TOPBP1 gene. The involvement of TOPBP1 gene may lead to abnormal synaptonemal complexes and meiotic chromosomal defects, resulting in male infertility. These findings offer valuable insights into the genetic basis and functional aspects of sex-specific traits in the Siamese cobra, which will contribute to our understanding of snake genetics and evolutionary biology.

Effect of Microgravity on the Crystallization of Cardiotoxin from the Venom of Spectacled Cobra Naja naja

Cardiotoxins, which belong to the family of three-finger toxins, are the main components of cobra venom. They exhibit various types of biological activity, including antimicrobial and cytotoxic against cancer cells. Data on the minimal structural differences between individual toxins are necessary for understanding the molecular mechanisms of their action. This information can be obtained by high-resolution X-ray diffraction analysis. The influence of microgravity on the crystal packing and diffraction quality of crystals of cardiotoxin from cobra Naja naja has been investigated. Cardiotoxin crystals, which were grown on the International Space Station, provided maximally high resolution for the structure of this toxin. Protein crystallized extremely in the hexagonal space group, whereas more than half of crystals grown under laboratory conditions belonged to the orthorhombic system.

Identifying key factors contributing to treatment costs for snakebite envenoming in private tertiary healthcare settings in Tamil Nadu, India.

India suffers ~58,000 annual deaths due to snakebites. The 'Big Four' snakes (Russell's viper, Indian cobra, common krait, and saw-scaled viper) that are responsible for most bites cause diverse clinical effects. Delayed treatment increases the risk of serious complications and treatment costs. Although government hospitals offer free treatment for snakebites in India, most patients opt for private healthcare, which is an out-of-pocket expense as they often lack health insurance coverage. This study aims to analyse snakebite treatment costs in private tertiary care hospitals in Tamil Nadu, India and identifies the key factors contributing to treatment costs. The treatment cost details for 913 snakebite victims were collected from 10 private tertiary care hospitals across Tamil Nadu. The data were classified into hospital, pharmacy, investigation, and laboratory costs, and analysed to determine various factors that contribute to the costs. The results demonstrate that the average treatment costs vary widely for different snakes. The hospital and pharmacy costs are higher than investigation and laboratory costs for all snakebites. Notably, Russell's viper bites cost significantly more than the bites from other snakes. Overall, the type of snake, nature of complications, specialist treatments required, and arrival time to hospitals were identified as some of the key factors for higher treatment costs. These data demonstrate that ~80% of snakebite patients can be treated with INR 100,000 (~GBP 1000 or USD 1200) or less. This study emphasises the urgent need to improve rural medical care by providing appropriate training for healthcare professionals and essential resources to facilitate early assessment of patients, administer the initial dose of antivenom and refer the patients to tertiary care only when needed. Moreover, the outcome of this study forms a basis for developing appropriate policies to regulate snakebite treatment costs and provide affordable medical insurance for vulnerable communities.

The Royal Armoury: Venomics and antivenomics of king cobra (Ophiophagus hannah) from the Indian Western Ghats

Despite being famous as ‘the king’ of the snake world, the king cobra (Ophiophagus hannah) has remained a mysterious species, particularly with respect to its venom ecology. In contrast, venom research has largely focussed on the ‘big four’ snakes that are greatly responsible for the burden of snakebite in the Indian subcontinent. This study aims to bridge the current void in our understanding of the O. hannah venom by investigating its proteomic, biochemical, pharmacological, and toxinological profiles via interdisciplinary approaches. Considering their physical resemblance, the king cobra is often compared to the spectacled cobra (Naja naja). Comparative venomics of O. hannah and N. naja in this study provided interesting insights into their venom compositions, activities, and potencies. Our findings suggest that the O. hannah venom, despite being relatively less complex than the N. naja venom, is equally potent. Finally, our in vitro and in vivo assays revealed that both Indian polyvalent and Thai Red Cross monovalent antivenoms completely fail to neutralise the O. hannah venom. Our findings provide guidelines for the management of bites from this clinically important yet neglected snake species in India.

Redescription and new record of Paracapillaria (Ophidiocapillaria) najae (Nematoda: Trichuroidea) in the monocled cobra Naja kaouthia from central Thailand: morphological and molecular insights.

The parasitic nematode Paracapillaria (Ophidiocapillaria) najae De, 1998, found in the Indian cobra Naja naja is redescribed and re-illustrated in the present study. The monocled cobra Naja kaouthia was discovered to be a new host for this parasite in central Thailand. A comprehensive description extending the morphological and molecular characteristics of the parasites is provided to aid species recognition in future studies. The morphometric characters of 41 parasites collected from 5 cobra specimens are compared with those described in the original studies. Phylogenetic analyses using mitochondrial cytochrome c oxidase subunit 1 and nuclear 18S ribosomal RNA genes were performed to provide novel information on the systematics of P. najae. Similar characteristics were observed in the examined nematode samples, despite being found in different hosts, confirming their identity as P. najae. The molecular genetic results support the species status of P. najae, indicating P. najae is well defined and separated from other related nematode species in the family Capillariidae. Morphological descriptions, genetic sequences, evolutionary relationships among capillariids and new host and distribution records of P. najae are discussed. Paracapillaria najae specimens found in the Thai cobra had some morphological variation, and sexual size dimorphism was also indicated. Paracapillaria najae was found to infect various cobra host species and appeared to be common throughout the Oriental regions, consistent with its hosts' distribution.

1760-P: Mining Animal Venoms as a Source of Insulinotropic Peptides for Potential Diabetes Therapy

T2D development and progression is widely known to be caused by pancreatic beta cell dysfunction, whether in the presence or absence of insulin resistance. There are however limited diabetes drugs that directly target the beta cell. Hence, there is a need to discover yet untapped pathways to increase or rescue beta cell function. Animal venoms provide a rich resource of bioactive compounds, many of which are highly potent and selective. Venom peptides have been harnessed for therapeutic drug development, such as the blockbuster diabetes drug, exenatide, a GLP-1 receptor agonist, being a synthetic form of exendin-4 that was first isolated from the saliva of the Gila monster. We hypothesize that we can further discover and develop new therapeutic agents that promote pancreatic beta cell function based on venom-derived compounds. We studied up to seven animal venoms from snakes, bees and scorpions. Pancreatic beta cells (including EndoC-βH1 cells, human islets, and MIN6 cells) were treated with crude animal venoms from each species and evaluated for insulin secretion capacity. We used a range of doses that elicit insulin secretion without causing cellular damage. Venoms from Apis mellifera (honey bee) and Naja naja (Indian cobra) were shown to robustly stimulate insulin secretion in a dose-dependent manner without affecting cell viability. Treatment with these venoms were also able to rescue diabetic cell models. We prioritized these two venoms for fractionation via gel filtration and reverse phase HPLC. After subsequent screening of the venom sub-fractions in insulin secretion assays, we pinpointed specific sub-fractions responsible for stimulating insulin release. Ongoing work is in progress to identify and validate selected peptides for insulinotropic activity, safety and stability. The insulinotropic peptides (and any modifications) may represent new therapeutic strategies for tackling T2D in future. Disclosure N.Ng: Stock/Shareholder; BetaLife. C.Koh: None. C.Ching: None. P.Gopalakrishnakone: None. A.Teo: None. Funding A*STAR Career Development Fund (202D800020)

Perceptions and awareness of snakebite envenoming in hilly areas of Madikeri: A cross sectional study

Abstract Background: Snake bite is one of the neglected public health problems in many tropical and subtropical countries. Wrong perception and practice of unproven traditional methods about snakes and snakebites may increase mortality due to snakebite envenoming. Objective: The objective is to assess the knowledge, attitude and practice regarding snakes and snakebites among the rural population in the Kodagu district. Materials and Methods: A community-based cross-sectional study was conducted between August-2021 and October-2021 in the rural field-practice area of Bhagamandala Primary Health Centre. Systematic random sampling was incorporated into the study to select the study participants. Data were collected using a predesigned and pretested questionnaire. Data entry was done in Microsoft Excel and was analyzed using STATA (16.1), StataCorp, College Station, Texas, USA. Results: Out of 1284 participants, the majority 964 (75.07%) of participants identified spectacled cobra correctly and 1142 (88.94%) correctly identified it as venomous. About 1228 (95.64%) participants had correct knowledge regarding Anti-Snake venom being an effective treatment for snake bites and the overall knowledge regarding snake bites was adequate among only 380 (29.60%) of the study population. Whereas, 188 (14.64%) of them even considered visiting a local quack or faith healer after a Snakebite. Conclusions: In our study, the overall knowledge regarding snake bites was adequate among 29.60% of the study population. The knowledge regarding the venemosity of snakes was good, but identification was poor. The participants had correct knowledge about ASV and its availability. The harmful practices such as visiting traditional healers and applying native medicine are still seen in 14.64% of participants.

Identification of Potential Insulinotropic Cytotoxins from Indian Cobra Snake Venom Using High-Resolution Mass Spectrometry and Analyzing Their Possible Interactions with Potassium Channel Receptors by In Silico Studies.

Snake venoms are a potential source of bioactive peptides, which have multiple therapeutic properties in treating diseases such as diabetes, cancer, and neurological disorders. Among bioactive peptides, cytotoxins (CTXs) and neurotoxins are low molecular weight proteins belonging to the three-finger-fold toxins (3FTxs) family composed of two β sheets that are stabilized by four to five conserved disulfide bonds containing 58-72 amino acid residues. These are highly abundant in snake venom and are predicted to have insulinotropic activities. In this study, the CTXs were purified from Indian cobra snake venom using preparative HPLC and characterized using high-resolution mass spectrometry (HRMS) TOF-MS/MS. Further SDS-PAGE analysis confirmed the presence of low molecular weight cytotoxic proteins. The CTXs in fractions A and B exhibited dose-dependent insulinotropic activity from 0.001 to 10µM using rat pancreatic beta-cell lines (RIN-5F) in the ELISA. Nateglinide and repaglinide are synthetic small-molecule drugs that control sugar levels in the blood in type 2 diabetes, which were used as a positive control in ELISA. Concluded that purified CTXs have insulinotropic activity, and there is a scope to use these proteins as small molecules to stimulate insulinotropic activities. At this stage, the focus is on the efficiency of the cytotoxins to induce insulin. Additional work is ongoing on animal models to see the extent of the beneficial effects and efficiency to cure diabetes using streptozotocin-induced models.