Naja Naja Venom Research Articles

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.

Phytochemical screening and evaluation of the anti-venom effect of leaves extracts of Vitex negundo

Phytochemical screening and evaluation of the anti-venom effect of leaves extracts of Vitex negundo

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.

ASSESSMENT OF SNAKE ANTIVENOM ACTIVITY OF TAMARINDUS INDICA SEED COAT EXTRACT

Snakebite is a life-threatening medical emergency, and globally responsible for millions of deaths. In snakebites accidents only deaths are not a concern, it leads to more morbidities. Due to scanty healthcare facilities in rural areas of India, many people seek alternative treatment available in ethnic practices. Tamarindus Indica (TI) plant is rich in medicinal value and used to treat many diseases including snakebite treatment traditionally. In view of this TI seed coat extract (TISCE) was evaluated for antivenom activity. The phytochemical screening of TISCE was performed to understand its chemical composition. TISCE was evaluated for antivenom activity against Indian cobra venom (ICV), common krait venom (CKV), Russells viper venom (RVV), and saw-scaled viper venom (SCV) for phospholipase A2 (PLA-2), haemorrhagic in vitro and in vivo, procoagulant, proteolytic activity, and lethality studies. TISCE majorly contains saponins, glycosides, alkaloids, and phenolic compounds. Minimum indirect haemorrhagic dose (MIHD) observed for ICV (12.5 µg), CKV (5.0 µg),RVV (10.0 µg), and SVV (12.5 µg). TISCE inhibits the procoagulant activity of all venoms at a concentration of 18.0 µg. It also shows the neutralization of proteolytic enzymes of venom in a dose-dependent manner. A pre-incubated mixture containing five lethal dose 50 (LD50) of venom and TISCE was injected intravenously, all mice survived as venom neutralized by TISCE. The present study demonstrates the ability of TISCE to neutralize snake venom using suitable in vivo and in vitro methods. Further studies required to unravelling the specific active chemical constituent of TISCE that may used as novel alternative snakebite treatment. TISCE was able to prolong the deaths during the simulation study and may be used in the topical pharmaceutical formulation that will reduce local venom reactions causing much morbidity, which will collectively with Anti-snake venom (ASV), used to treat envenomed patients more effectively.

Appraisal of Antiophidian Properties of Cardiospermum halicacabum L. Leaf and Callus Extracts against Naja Naja Venom

Considering the limitations of antivenom therapy towards toxicity following envenomation, investigators are probing for effective phytochemicals with promising antivenom effects. In the current study, Cardiospermum halicacabum leaf and callus methanol extracts were investigated for their ability to neutralize Indian cobra (Najanaja) venom. Soxhlet extraction using methanol was performed since leaf and callus extracts demonstrated significant neutralization of N. najavemon in preliminary studies. In vitro neutralizing potential of both extracts were evaluated against phospholipase A2, preoteases and hyaluronidases. In addition, alleviation of N. naja-induced characteristic edema inducing activity, myotoxicity and coagulation, hemolytic activity were performed in mouse models and human blood samples. Leaf and callus methanol extract contained high concentrations of phenols, alkaloids, steroids and saponins and demonstrated significant in vitro inhibition of N. naja PLA2, proteases and hyaluronidase activities at 30-40 µg concentrations. Significant neutralization of N. naja-induced indirect hemolysis, edema, anticoagulant effect and myotoxicity was attained using both extracts. Overall leaf and callus extracts (1mg/kg) postponed animal survival by 33 ± 9.3 and 50 ± 12.4 mins, respectively. C. halicacabum leaf and callus methanol extracts demonstrated significant neutralization of N. naja enzymes that contribute to grievous effects on various tissues/organs. The study scientifically supports the anecdotal use of C. halicacabum in complementary medicine and demonstrates that callus extract is more potent antidote than leaf. Further, callus extract is a potent candidate for isolation of phytochemicals towards management of snakebite.

Serine protease from Indian Cobra venom: its anticoagulant property and effect on human fibrinogen

An unusual low molecular weight serine protease (NnP28) has been purified from Indian Cobra (Naja naja) venom from the western region of the Indian sub-continent. We reported the purification and characterization of low molecular NnP28, emphasizing its role on human fibrinogen and anticoagulant property. NnP28 was purified using gel filtration column chromatography followed by ion exchange chromatography. Protein gel electrophoresis revealed its molecular weight ≈28 kDa. The exact molecular mass of NnP28 was found to be 27.12 kDa by mass spectrometry, hydrolyzing casein specifically, inhibited by PMSF suggesting it has a serine protease. NnP28 prolonged the clotting time of re-calcified human citrated plasma and activated partial thromboplastin time (APTT) exhibiting anticoagulant property. NnP28 exhibited fibrinogenolytic activity. Thus, the present study demonstrates the presence of unusual low molecular weight serine protease, emphasizing its importance of region-specific Indian cobra species.

Omics and organoids - a route to improved anti-venom.

Two new studies on snake venom glands have implications for improved anti-venom development. One describes the de novo assembly of an Indian cobra reference genome and venom gland transcriptome; the other describes the derivation of snake venom gland organoids.

Metabolites from the citrus extracts inhibit the activity of selected proteins in Indian Cobra (Naja naja) venom

Ethnopharmacological relevanceSnakebite is a severe problem in many parts of the world, specifically in tropical and subtropical regions. A range of medicinal plant extracts are administered for treating snake bite. Of the many common plants, extracts of Citrus species have been documented to be used for treating snake bite and have been shown to decrease the snake venom toxicity. AimThe aim of the current work is to evaluate the utility of citrus peel extracts (Citrus aurantium L. and Citrus reticulate Blanco) in the management of Indian cobra envenomation. Materials and methodsPeels of citrus species were evaluated for their phospholipase A2, protease and haemolytic inhibition properties. The phytochemicals present in the extract were inferred using GC-MS. In-vivo studies, using mice model, were done to confirm the inhibitory effect of the extracts. Molecular docking was used to understand the possible binding modes of selected phytochemicals to snake venom phospholipase. ResultsCitrus peel extracts are rich in polyphenols, flavonoids and tannins. The methanolic extract of Citrus aurantium L. and Citrus reticulate Blanco inhibits phospholipase (75%), protease (71%) and hemolysis (80%) activity of the venom. GC-MS analyses indicate the presence of β-sitosterol, n-hexadecanoic acid, eicosanoic acid, and flavone in both the extracts. In addition, C. reticulate extract contains α-tocopherol and squalene. Molecular docking revealed that α-tocopherol, spiro [androst-5-ene-17,1′-cyclobutan]-2′-one,3-hydroxy-(3β,17β)- and β-sitosterol acetate bind with moderate affinity to the catalytic site of phospholipase A2. ConclusionThe present study provides new molecular insight and scientific evidence on the utility of the methanolic extracts of citrus peels to neutralize the venom toxins of Naja naja.

Study on Snake Venom Protein-Antibody Interaction by Surface Plasmon Resonance Spectroscopy

The development of a portable and inexpensive surface plasmon resonance (SPR) measurement device with the integrated biosensor for the detection of snake venom protein is presented in this paper. For the construction of the sensing element, amine coupling chemistry is used to bio-functionalize silver coated glass slide with antibodies like immunoglobulin (IgG). The immobilization of the antibody is confirmed by spectroscopic measurements like ultraviolet-visible spectroscopy (UV-Vis) and Fourier-transforms infrared spectroscopy (FTIR). The device is calibrated with the standard solution of sodium chloride and ethanol before testing venom protein samples. To investigate the bio-molecular interactions, crude venom of Indian cobra (concentration range: 0.1 mg/ml‒1.0 mg/ml) in the phosphate buffer solution (PBS) are exposed to the biosensor. The experimentally measured data indicate the shift in the plasmon resonance angle from its initial value (52°) to 54° for 0.1 mg/ml and 60° for 1.0 mg/ml protein solution.

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.

Anti-snake Venom Activities of Ethanol and Aqueous Extract of <i>Cassia hirsute</i> against Indian Cobra (<i>Naja naja</i>)Venom Induced Toxicity

Snake bite leads to medical emergencies and sometimes death. It is clinically managed by administration of monovalent/polyvalent antisera and it exhibit early or late adverse reactions and sometimes these adverse effects lead to fatalities. Cassia hirsute has been used against snake bite by the traditional healers; however, there is paucity of scientific data in support of this claim. To examine the anti-snake venom activities of ethanolic and aqueous extracts of Cassia hirsute against Indian cobra (Naja naja) venom induced toxicity. Anti-snake venom activities of ethanolic and aqueous extracts of Cassia hirsute against Indian cobra (Naja naja) venom induced toxicity was studied in mice, rats and sheep by using various models as follows: Phospholipase A2 induced hemolysis on sheep RBC, Naja naja induced anticoagulation[Coagulation activity], Naja naja induced bleeding, In- vivo Neutralization activity against lethality induced by Naja naja venom (2mg/kg) in rats, Naja naja venom induced odema, liver function of venomized rats. The Minimum lethal dose (MLD) was found as 2mg/kg and both ACH and ECH shown significant neutralization of Naja naja venom induced lethality. Both extracts inhibited phospholipase A2 dependent hemolysis of sheep RBC's and showed a high significant reduction (P<0.001) in the anti-coagulant activity of venom with 80.51% inhibition of anti-coagulation of ECH at 350 ;g and 79.84% of ACH at 300 ;g. ECH and ACH 300, 600 and 900 mg/kg shows significant (P<0.001) decrease in bleeding time also. These findings suggest a potential antivenom role of Cassia hirsute against Naja naja venom.

Isolation and characterization of bioactive compounds of Clematis gouriana Roxb. ex DC against snake venom phospholipase A2 (PLA2) computational and in vitro insights

Bioactive compounds were isolated from Clematis gouriana Roxb. ex DC. The compounds were separated, characterized, the structures elucidated and submitted to the PubChem Database. The PubChem Ids SID 249494134 and SID 249494135 were tested against phospholipases A2 (PLA2) of Naja naja (Indian cobra) venom for PLA2 activity. Both the compounds showed promising inhibitory activity; computational data also substantiated the results. The two compounds underwent density functional theory calculation to observe the chemical stability and electrostatic potential profile. Molecular interactions between the compounds and PLA2 were observed at the binding pocket of the PLA2 protein. Further, this protein–ligand complexes were simulated for a timescale of 100 ns of molecular dynamics simulation. Experimental and computational results showed significant PLA2 inhibition activity.

Rapid and selective detection of experimental snake envenomation – Use of gold nanoparticle based lateral flow assay

In this study, we have developed a gold nanoparticle based simple, rapid lateral flow assay (LFA) for detection of Indian Cobra venom (CV) and Russell’s viper venom (RV). Presently, there is no rapid, reliable, and field diagnostic test available in India, where snake bite cases are rampant. Therefore, this test has an immense potential from the public health point of view. The test is based on the principle of the paper immunochromatography assay for detection of two snake venom species using polyvalent antisnake venom antibodies (ASVA) raised in equines and species-specific antibodies (SSAbs) against venoms raised in rabbits for conjugation and impregnation respectively. The developed, snake envenomation detection immunoassay (SEDIA) was rapid, selective, and sensitive to detect venom concentrations up to 0.1 ng/ml. The functionality of SEDIA strips was confirmed by experimental envenomation in mice and the results obtained were specific for the corresponding venom. The SEDIA has a potential to be a field diagnostic test to detect snake envenomation and assist in saving lives of snakebite victims.

The standard aqueous stem bark extract of Mangifera indica L. inhibits toxic PLA2 – NN-XIb-PLA2 of Indian cobra venom

The aqueous extract of Mangifera indica is known to possess diverse medicinal properties, which also includes anti-snake venom activities. However, its inhibitory potency and mechanism of action on multi-toxic snake venom phospholipases A2s are still unknown. Therefore, the objective of this study was to evaluate the modulatory effect of standard aqueous bark extract of M. indica on NN-XIb-PLA2 of Indian cobra venom. The in vitro sPLA2, in situ hemolytic and in vivo edema inhibition effect were carried out as described. Also the effect of substrate and calcium concentration was carried out. M. indica extract dose dependently inhibited the GIA sPLA2 (NN-XIb-PLA2) activity with an IC50 value of 7.6μg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ∼40μg/ml concentration. Further, M. indica extract (0–50μg/ml) inhibited the edema formed in a dose dependent manner. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect of M. indica extract on the NN-XIb-PLA2. Further, the inhibition was irreversible as evident from binding studies. The in vitro inhibition is well correlated with in situ and in vivo edema inhibiting activities of M. indica. As the inhibition is independent of substrate and calcium and was irreversible, it can be concluded that M. indica extract mode of inhibition could be due to direct interaction of components present in the extract with the PLA2 enzyme. The aqueous extract of M. indica effectively inhibits svPLA2 enzymatic and its associated toxic activities, which substantiate their anti-snake venom properties. Further in-depth studies on the role and mechanism of the principal constituents present in the extract, responsible for the anti-PLA2 activity will be interesting to develop them into potent antisnake component and also as an anti-inflammatory agent.

Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization.

Geographical variations of snake venoms can result in suboptimal effectiveness of Indian antivenoms that are currently used in most South Asian countries. This study investigated the toxicity and neutralization profile of the venom and toxins from Pakistani spectacled cobra, Naja naja, using VINS polyvalent antivenom (VPAV, India), Naja kaouthia monovalent antivenom (NKMAV, Thailand), and neuro bivalent antivenom (NBAV, Taiwan). Cation-exchange and reverse-phase high-performance liquid chromatography fractionations followed by toxin identification through liquid chromatography-mass spectrometry (MS)/MS indicated that the venom comprised mainly of postsynaptic neurotoxins (NTXs) (long neurotoxins [LNTXs], 28.3%; short neurotoxins [SNTXs], 8%), cytotoxins (CTXs) (31.2%), and acidic phospholipases A2 (12.3%). NKMAV is the most effective in neutralizing the lethal effect of the venom (potency = 1.1 mg venom/mL) and its LNTX (potency = 0.5 mg toxin/mL), consistent with the high content of LNTX in N. kaouthia venom. VPAV was effective in neutralizing the CTX (potency = 0.4 mg toxin/mL), in agreement with the higher CTX abundance in Indian cobra venom. All the three antivenoms were weak in neutralizing the SNTX (potency = 0.03-0.04 mg toxin/mL), including NBAV that was raised from the SNTX-rich Taiwanese cobra venom. In a challenge-rescue experiment, envenomed mice were prevented from death by a maximal dose of VPAV (intravenous 200 μL) but the recovery from paralysis was slow, indicating the need for higher or repeated doses of VPAV. Our results suggest that optimal neutralization for Pakistani N. naja venom may be achieved by improving the formulation of antivenom production to enhance antivenom immunoreactivity against long and SNTXs.

Comparison of the primary structures, cytotoxicities, and affinities to phospholipids of five kinds of cytotoxins from the venom of Indian cobra, Naja naja

The molecular mechanism underlying the hemolytic and cytolytic processes of cobra cytotoxins (CTXs) is not yet fully elucidated. To examine this, we analyzed the amino acid sequences, hemolytic and cytotoxic activities, and affinities to phospholipids of the five major CTXs purified from the venom of Indian cobra, Naja naja. CTX2, CTX7, and CTX8 belonged to S-type, and CTX9 and CTX10 to P-type. Comparisons of CTX7 with CTX8 and CTX9 with CTX10 revealed similar primary structures and hemolytic and cytolytic activities. CTX2, whose primary structure was rather different from the others, showed several times weaker hemolytic and cytolytic biological activities than the others. The comparison of CTX2 with CTX7 suggested the importance of Lys30 in loop II for the strong hemolytic and cytolytic activities of S-type CTXs. Cloning of 12 CTX cDNAs from the Naja naja venom cDNA library revealed that 18 out of 23 substitutions found in CTX cDNAs were nonsynonymous. This clearly indicated the accelerated evolution of CTX genes. Multiple sequence alignment of 51 kinds of CTX cDNAs and calculations of nonsynonymous and synonymous substitutions indicated that the codons coding the three loops' regions, which may interact with the hydrophobic tails of phospholipids, have undergone an accelerated evolution. In contrast, the codons coding for amino acid residues considered to participate in the recognition and binding of the hydrophilic head groups of phospholipids, eight Cys residues, and those likely stabilizing β core structure, were all conserved.

Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin.

In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells, bactericidal or hemolytic activity. The future therapeutic application of NnPLA2-I for treatment and prevention of cardiovascular disorders is therefore suggested.

Detection and neutralization of cobra venom using rabbit antiserum in experimental envenomated mice

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the venom of Indian cobra (Naja naja naja) in various tissues (brain, heart, lungs, liver, spleen, blood, kidneys, and tissue at the site of injection) of mice after cobra venom injected at different time intervals (0, 2, 4, 6, 8, and 12 h intervals up to 24 h). Whole venom antiserum or individual venom protein antiserum (14, 29, 65, 72, and 99 kDa) could recognize N. n. naja venom by Western blotting and ELISA, and antibody titer was also assayed by ELISA. Antiserum raised against cobra venom in rabbit significantly neutralized the toxicity of venom-injected mice at different time intervals after treatment. The assay could detect N. n. naja venom levels up to 2.5 ng/ml of tissue homogenate, and the venom was detected up to 24 h after venom injection. Venom was detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area, and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart, and lungs. Development of a simple, rapid, and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Antileukemic potential of PEGylated gold nanoparticle conjugated with protein toxin (NKCT1) isolated from Indian cobra (Naja kaouthia) venom.

Limited efficacy of current first-line treatment for leukemia calls attention for further development of efficient strategies. Recently, much attention has been given to nanoparticle-based drug delivery systems loaded with dual drugs to improve current disease therapies by overcoming toxicity. In the present study, we document to explore an approach to conjugate gold nanoparticles (GNPs) with protein toxin (NKCT1), a protein toxin from the Indian cobra (Naja kaouthia) venom, and to establish its antileukemic activity. GNP was prepared by NaBH4 reduction method. UV–vis spectroscopy of GNP showed the absorbance at 530 nm for plasma resonance. Dynamic light scattering (DLS) size of GNPs was 2–8 nm and the GNP-NKCT1 was 68–122 nm. CD spectra of GNP-NKCT1 showed change in percentage of β-turn as compared with NKCT1. GNP-NKCT1 significantly inhibited leukemic cell growth in dose- and time-dependent manner by two- to threefold more than NKCT1. For human leukemic lymphoma cell line and human myelogenous leukemic cell line, the IC50 dose was found to be 1.2 and 0.75 μg/ml, respectively, observed by trypan blue exclusion method and tetrazolium bromide reduction assay. Flow cytometric analysis showed appreciable number of both cell lines in early and late apoptotic stages and arrested cell cycle in the G1 phase by GNP-NKCT1. Resilient power of leukemic cell line after wound healing and migration or invasive power of the cell line was significantly low in GNP-NKCT1-treated plate than the control plate. These analyses reveal that GNP-NKCT1 possesses significant and selective anticancer activity, likely by inducing programmed cell death through mitochondrial and/or lysosomal pathway.

Detection and neutralization of venom of Indian cobra by rabbit antiserum in experimental envenomation using mice

A sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Indian cobra ( Naja naja naja ) venom in various organs (brain, heart, lungs, liver, spleen, blood, site of injection and kidneys) as well as tissue at the site of injection of mice, at various time intervals (0, 2, 4, 6, 8 and 12 h intervals up to 24 h) after venom injection. Antiserum significantly neutralized venom levels in serum and tissue samples. Whole venom antiserum or individual venom protein anti serum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize Naja naja venom by Western blotting and ELISA, and Naja naja naja venom presented antibody titer when assayed by ELISA. The assay could detect Naja naja naja venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 24 h after venom injection. A highly sensitive and species-specifc microtitre ELISA was also developed to detect venoms of four medically important Indian snakes ( Naja naja naja ) in autopsy specimens of mice. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specifc diagnostic kit based on this ELISA technique useful to clinicians is discussed.