Wound Infections from Taiwan Cobra (Naja atra) Bites: Determining Bacteriology, Antibiotic Susceptibility, and the Use of Antibiotics-A Cobra BITE Study.

Wound necrosis and secondary infection are common complications after Naja atra bites. Clinical tools to evaluate the infection risk after Taiwan cobra bites are lacking. In this Cobra BITE study, we investigated the prevalence of wound infection, bacteriology, and corresponding antibiotic usage in patients presenting with Taiwan cobra snakebites. Patients with wound infection lacking tissue necrosis were included in developing Cobra BITE score utilizing univariate and multiple logistic regression, as patients with wound necrosis require antibiotics for infection treatment. 8,295,497 emergency department visits occurred in the span of this study, with 195 of those patients being diagnosed as having cobra bites. Of these patients, 23 had wound necrosis, and 30 had wound infection, resulting in a wound infection rate of 27.2% (53/195). Enterococcus faecalis and Morganella morganii were the main bacteria identified in the culture report regardless of whether patients' wounds had necrosis. As per our Cobra BITE score, the three factors predicting secondary wound infection after cobra bites are hospital admission, a white blood cell count (in 103/µL) × by neu-trophil-lymphocyte ratio value of ≥114.23, and the use of antivenin medication. The area under the receiver operating characteristic curve for the Cobra BITE score system was 0.88; ideal sensitivity and specificity were 0.89 and 0.76. This scoring system enables the assessment of wound infections after N. atra bites, and it could be modified and improved in the future for other Naja spp. bites.

In vitro anti-venom potential of various solvent based leaf extracts of Andrographis serpyllifolia (Rottler ex Vahl) Wight against Naja naja and Daboia russelli.

ETHNOPHARMACOLOGICAL RELEVANCE: Snake bite is a major occupational hazard in tropical and subtropical countries including India as per the World Health Organization. Naja naja (Indian cobra) and Daboia russelli (Russell's viper) are the two poisonous snakes commonly associated with human mortality in India. Andrographis serpyllifolia (Rottler ex Vahl) Wight has been documented in ethnobotanical records as a plant possessing potent anti-snake venom activity. AIM OF THE STUDY: The present study is aimed for systematic evaluation of in vitro anti-venom potential of various solvent based leaf extracts of A. serpyllifolia against toxic venom enzymes of Naja naja and Daboia russelli. MATERIALS AND METHODS: Different solvent based leaf extracts of A. serpyllifolia were tested against the snake venoms of Naja naja and Daboia russelli obtained from Irula Snake Catchers Industrial Co-operative Society Limited, Kancheepuram, Tamil nadu, India. Three different in vitro neutralization assays such as indirect hemolysis, procoagulent and lytic activities and seven in vitro enzyme inhibition assays such as protease, acetylcholinesterase, phosphomonoesterase, phosphodiesterase, 5'nucleotidase, phospholipase A2, hyaluronidase and post synaptic acetylcholine receptor binding activity were carried out according to standard protocols. The results were analyzed using the standard ANOVA procedures. RESULTS: Among various solvent based leaf extracts of A. serpyllifolia tested, aqueous extract showed maximum neutralizing and inhibitory activities against Naja naja and Daboia russelli venoms. CONCLUSIONS: The various in vitro enzymatic studies reveal that the aqueous leaf extract of A. serpyllifolia plant could inhibit most of the toxic enzymes of the Naja naja and Daboia russelli venoms which could be further confirmed by in vivo studies.

In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.

Antitoxin activity of aqueous extract of Cyclea peltata root against Naja naja venom.

OBJECTIVES: Snakebites are a significant and severe global health problem. Till date, anti-snake venom serum is the only beneficial remedy existing on treating the snakebite victims. As antivenom was reported to induce early or late adverse reactions to human beings, snake venom neutralizing potential for Cyclea peltata root extract was tested for the present research by ex vivo and in vivo approaches on Naja naja toxin. MATERIALS AND METHODS: Ex vivo evaluation of venom toxicity and neutralization assays was carried out. The root extracts from C. peltata were used to evaluate the Ex vivo neutralization tests such as acetylcholinesterase, protease, direct hemolysis assay, phospholipase activity, and procoagulant activity. Gas chromatography-mass spectrometry (GC-MS) analysis from root extracts of C. peltata was done to investigate the bioactive compounds. RESULTS: The in vivo calculation of venom toxicity (LD50) of N. naja venom remained to be 0.301 µg. C. peltata root extracts were efficiently deactivated the venom lethality, and effective dose (ED50) remained to be 7.24 mg/3LD50 of N. naja venom. C. peltata root extract was found effective in counteracting all the lethal effects of venom. GC-MS analysis of the plant extract revealed the presence of antivenom compounds such as tetradecanoic and octadecadienoic acid which have neutralizing properties on N. naja venom. CONCLUSION: The result from the ex vivo and in vivo analysis indicates that C. peltata plant root extract possesses significant compounds such as tetradecanoic acid hexadecanoic acid, heptadecanoic acid, and octadecadienoic acid which can counteract the toxins present in N. naja.

Successful use of camelid (alpaca) antivenom to treat a potentially lethal tiger snake (Notechis scutatus) envenomation in a dog.

This report describes a confirmed clinical case of tiger snake (Notechis scutatus) envenomation in a domestic dog that was successfully treated with a novel polyvalent camelid (alpaca; Llama pacos) antivenom. Samples collected from the dog were assayed for tiger snake venom (TSV) using a highly sensitive and specific ELISA. The TSV concentration in serum and urine at initial presentation was 365 ng/mL and 11,640 ng/mL respectively. At the time of initial presentation whole blood collected from the dog did not clot and the Prothrombin Time was abnormally increased (>300 s). Serum was also visibly hemolysed. The dog was administered antihistamine, dexamethasone and 4000 Units (sufficient to neutralise 40 mg of TSV) of a novel polyvalent alpaca antivenom diluted in 0.9% NaCl. At 4 h post-antivenom treatment the dog's clinical condition had improved markedly with serum TSV concentrations below the limit of detection (<0.015 ng/mL), consistent with complete binding of venom antigens by the alpaca antivenom. Coagulation parameters had begun to improve by 4 h and had fully normalised by 16 h post-antivenom. Venom concentrations in both serum and urine remained undetectable at 16 h post-antivenom. The dog made a complete recovery, without complications, suggesting that the alpaca-based antivenom is both clinically safe and effective.

Protective effect of Euphorbia hirta and its components against snake venom induced lethality.

ETHNOPHARMACOLOGICAL RELEVANCE: Despite the use of snake anti-venom therapy, herbal medicine is still in practice to treat snakebites. Euphorbia hirta is traditionally used as antidote for snakebites and also for numerous other ailments. However, the scientific evidence for its anti-snake venom property is still lacking. MATERIALS AND METHODS: Methanolic extract of E. hirta was evaluated for anti-venom activity under in vitro and ex vivo conditions. Histopathological changes in the vital organs of the mice were also monitored. UHPLC-SRM/MS was used to estimate the phenolic constituents whereas GC-MS analysis was performed to analyze the volatile metabolites present. The major compound was further evaluated for its contribution to the overall inhibitory potential of the extract. RESULTS: Methanolic extract of E. hirta completely inhibited the venom enzymes under in vitro and reduced the edema ratio. The extract increased the survival time (>24h) of mice which was further evidenced by histopathological analysis of vital organs. Phytochemical analysis revealed higher content of phenolic (144 mg/g extract) compounds in the extract. UHPLC-SRM/MS demonstrated that ellagic acid, gallic acid and quinic acid are the major phenolics whereas GC-MS analysis revealed pyrogallol as the major constituent (60.07%) among the volatile components of the extract. It was also shown that pyrogallol has the ability to differentially inhibit venom protease but not phospholipase A2. CONCLUSION: The present study confirmed that E. hirta methanolic extract was able to completely inhibit Naja naja venom induced toxicity under in vitro as well as ex vivo conditions, thus providing scientific evidence to its traditional use.

Protective activity of medicinal plants and their isolated compounds against the toxic effects from the venom of Naja (cobra) species.

ETHNOPHARMACOLOGICAL RELEVANCE: Various medicinal plants have protective properties against the toxicities of the venom of cobra snake (Naja species). They may be used as local first aid for the treatment of snakebite victims, and can significantly inhibit lethality, cardio-, neuro-, nephro- and myotoxicity, hemorrhage, and respiratory paralysis induced by the cobra snake venom. The plants or their extracts may also complement the benefits of conventional anti-serum treatment. AIM OF THE REVIEW: This review provides information on the protective, anti-venom, properties of medicinal plants against snakebites from cobras. In addition, it identifies knowledge gaps and suggests further research opportunities. METHODS: The literature was searched using databases including Google Scholar, PubMed, ScienceDirect, Scopus and Web of Science. The searches were limited to peer-reviewed journals written in English with the exception of some books and a few articles in foreign languages. RESULTS: The plants possess neutralization properties against different cobra venom enzymes, such as hyaluronidase, acetylcholinesterase, phospholipase A2 and plasma proteases. Different active constituents that show promising activity against the effects of cobra venom include lupeol acetate, ß-sitosterol, stigmasterol, rediocides A and G, quercertin, aristolochic acid, and curcumin, as well as the broad chemical groups of tannins, glycoproteins, and flavones. The medicinal plants can increase snakebite victim survival time, decrease the severity of toxic signs, enhance diaphragm muscle contraction, block antibody attachment to venom, and inhibit protein destruction. In particular, the cardiovascular system is protected, with inhibition of blood pressure decline and depressed atrial contractility and rate, and prevention of damage to heart and vessels. The designs of experimental studies that show benefits, or otherwise, of use of medicinal plants have some limitations: deficiency in identification and isolation of active constituents responsible for therapeutic activity; lack of comparison with reference drugs; and little investigation of the mechanism of anti-venom activity. CONCLUSION: Despite some current deficiencies in experimental or clinical analysis, medicinal plants with anti-venom characteristics are effective and so are candidates for future therapeutic agents. We suggest that emphasis on identification and testing of active ingredients in research in the future will assist better understanding of their anti-venom activity.

Effect of externally applied jidesheng anti-venom on skin and soft-tissue necrosis after Chinese cobra bite: a retrospective study.

OBJECTIVE: To evaluate the effects of Jidesheng anti-venom used externally for skin and soft-tissue necrosis from Chinese cobra bite. METHODS: A retrospective review was performed according to the clinical data recorded from January 2002 to December 2012. A total of 126 patients (116 females and 10 males) with skin and soft-tissue necrosis due to Chinese cobra bite were divided into two groups. The control group was treated externally with 40% glyceride magnesium sulfate (n = 52), and the treatment group was given Jidesheng anti-venom externally (n = 74). The data collected included maximum local necrotic area of skin and soft tissues, de-tumescence time, healing time, and skin-grafting rate. RESULTS: There were no significant differences in gender, age, and disease condition between the control and treatment groups (P > 0.05). No statistically significant difference was found in de-tumescence time between the two groups (P > 0.05). The maximum local necrotic area of skin and soft tissues was (19.9 +/- 7.3) cm2 in the treatment group, while it was (23.3 +/- 6.4) cm2 in the control group. The healing time of the treatment group was shorter than that of the control group [(32.1 +/- 3.7) vs (34.4 +/- 4.5) days)]. The skin-grafting rate in the treatment group was lower than that of the control group (10.81% vs 25.00%). There were statistically significant differences in maximum local necrotic area of skin and soft tissues, healing time, and skin-grafting rate between the control and treatment groups (all P < 0.05). CONCLUSION: External application of Jidesheng anti-venom may help to promote wound healing and reduce the skin-grafting rate in cases of skin and soft-tissue necrosis due to Chinese cobra bite.

Antivenom activity of triterpenoid (C34H68O2) from Leucas aspera Linn. against Naja naja naja venom induced toxicity: antioxidant and histological study in mice.

The isolated and identified triterpenoid, 1-hydroxytetratriacontane-4-one (C34H68O2), obtained from the methanolic leaf extract of Leucas aspera Linn. was explored for the first time for antisnake venom activity. The plant (L. aspera Linn.) extract significantly antagonized the spectacled cobra (Naja naja naja) venom induced lethal activity in a mouse model. It was compared with commercial antiserum obtained from King Institute of Preventive Medicine (Chennai, Tamil Nadu, India). N. naja naja venom induced a significant decrease in antioxidant superoxide dismutase, glutathione (GSH) peroxidase, catalase, reduced GSH and glutathione-S-transferase activities and increased lipid peroxidase (LPO) activity in different organs such as heart, liver, kidney and lungs. The histological changes following the antivenom treatment were also evaluated in all these organs. There were significant alterations in the histology. Triterpenoid from methanol extract of L. aspera Linn. at a dose level of 75 mg per mouse significantly attenuated (neutralized) the venom-induced antioxidant status and also the LPO activity in different organs.

Identification and evaluation of agents isolated from traditionally used herbs against Ophiophagus hannah venom.

The aim of this study was firstly to identify active molecules in herbs, that are traditionally used for the treatment of snake bite, such as Curcuma antinaia, Curcuma contravenenum, Andrographis paniculata, and Tanacetum parthenium; secondly to test similar structurally related molecules and finally to prepare and evaluate an efficient formulation against Ophiophagus hannah venom intoxification. Three labdane based compounds, including labdane dialdehyde, labdane lactone, and labdane trialdehyde and two lactones including 14-deoxy-11,12-didehydroandrographolide and parthenolide were isolated by column chromatography and characterised. Using the isolated rat phrenic nerve-hemidiaphragm preparation, the antagonistic effect of crude extracts, isolated compounds and prepared formulations were measured in vitro on the inhibition of the neuromuscular transmission. Inhibition on muscle contraction, produced by the 5 µg/mL venom, was reversed by test agents in organ bath preparations. A labdane trialdehyde, isolated from C. contravenenum, was identified as the best antagonising agent in the low micromolar range. Tests on formulations of the most potent C. contravenenum extract showed, that the suppository with witepsol H15 was an effective medicine against O. hannah venom. This study elucidated the active compounds, accounting for the antivenin activity of traditionally used herbs and suggested the most suitable formulation, which may help to develop potent medicines for the treatment of snake bite in the future.

Mucuna pruriens Linn. seed extract pretreatment protects against cardiorespiratory and neuromuscular depressant effects of Naja sputatrix (Javan spitting cobra) venom in rats.

Mucuna pruriens has been used by native Nigerians as a prophylactic for snakebite. The protective effects of M. pruriens seed extract (MPE) were investigated against the pharmacological actions of N. sputatrix (Javan spitting cobra) venom in rats. The results showed that MPE-pretreatment protected against cardiorespiratory and, to a lesser extent, neuromuscular depressant effects of N. sputatrix venom. These may be explained at least in part by the neutralisation of the cobra venom toxins by anti-MPE antibodies elicited by the MPE pretreatment.

In-vitro and in-vivo antivenin activity of 2-[2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde against Ophiophagus hannah venom.

OBJECTIVES: Curcuma zedoaroides A. Chaveerach & T. Tanee, locally known as Wan-Paya-Ngoo-Tua-Mia, is commonly used in the North-Eastern part of Thailand as a 'snakebite antidote'. The aim of this study was to isolate the active compound from the rhizome of C. zedoaroides, to determine its structure and to assess its antagonistic activity in vitro and in vivo against King cobra venom. METHODS: The active compound was obtained from C. zedoaroides by extraction with acetone followed by purification using column chromatography; its X-ray structure was determined. Its inhibition of venom lethality was studied in vitro in rat phrenic nerve-hemidiaphragms and in vivo in mice. KEY FINDINGS: The acetone extract of the Curcuma rhizomes contained a C20 dialdehyde, [2-(5,5,8a-trimethyl-2-methylene-decahydro-naphthalen-1-yl)-ethylidene]-succinaldehyde, as the major component. The isolated curcuma dialdehyde was found active in vitro and in vivo for antivenin activity against the King cobra venom. Using isolated rat phrenic nerve-hemidiaphragm preparations, a significant antagonistic effect on the inhibition of neuromuscular transmission was observed in vitro. Inhibition on muscle contraction, produced by the 4 microg/ml venom, was reversed by 2-16 microg/ml of Curcuma dialdehyde in organ bath preparations over a period of 2 h. Mice intraperitoneally injected with 0.75 mg/kg venom and dialdehyde at 100 mg/kg had a significantly increased survival time. Injection of Curcuma dialdehyde (100 mg/kg) 30 min before the subcutaneous injection of the venom resulted in a 100% survival time after 2 h compared with 0% for the control group. CONCLUSIONS: The in vitro and in vivo evaluation confirmed the medicinal use of traditional snake plants against snakebites. The bioactivity is linked to an isolated molecule and not a result of synergistic effects of a mixture. The active compound was isolated and the structure fully elucidated, including its stereochemistry. This dialdehyde is a versatile chemical building block and can be easily obtained from this plant source.

Anti-necrosis potential of polyphenols against snake venoms.

Polyphenols from the extracts of Areca catechu L. and Quercus infectoria Oliv. inhibited phospholipase A(2), proteases, hyaluronidase and L-amino acid oxidase of Naja naja kaouthia Lesson (NK) and Calloselasma rhodostoma Kuhl (CR) venoms by in vitro tests. Both extracts inhibited the hemorrhagic activity of CR venom and the dermonecrotic activity of NK venom by in vivo tests. The inhibitory activity of plant polyphenols against local tissue necrosis induced by snake venoms may be caused by inhibition of inflammatory reactions, hemorrhage, and necrosis. The result implies the therapeutic potential of plant polyphenols against necrosis in snakebite victims.

The protective effects of Mucuna pruriens seed extract against histopathological changes induced by Malayan cobra (Naja sputatrix) venom in rats.

Seed of Mucuna pruriens (Velvet beans) has been prescribed by traditional medicine practitioners in Nigeria as a prophylactic oral antisnake remedy. In the present studies, we investigated the protective effects of M. pruriens seed extract (MPE) against histopathological changes induced by intravenous injection of Naja sputatrix (Malayan cobra) venom in rats pretreated with the seed extract. Examination by light microscope revealed that the venom induced histopathological changes in heart and blood vessels in liver, but no effect on brain, lung, kidney and spleen. The induced changes were prevented by pretreatment of the rats with MPE. Our results suggest that MPE pretreatment protects rat heart and liver blood vessels against cobra venom-induced damages.

The protective effect of Mucuna pruriens seeds against snake venom poisoning.

ETHNOPHARMACOLOGICAL RELEVANCE: The seed, leaf and root of Mucuna pruriens have been used in traditional medicine for treatments of various diseases. In Nigeria, the seed is used as oral prophylactics for snakebite. AIM OF THE STUDY: To study the protective effects of Mucuna pruriens seed extract against the lethalities of various snake venoms. MATERIALS AND METHODS: Rats were pre-treated with Mucuna pruriens seed extract and challenged with various snake venoms. The effectiveness of anti-Mucuna pruriens (anti-MPE) antibody to neutralize the lethalities of snake venoms was investigated by in vitro neutralization. RESULTS: In rats, MPE pre-treatment conferred effective protection against lethality of Naja sputatrix venom and moderate protection against Calloselasma rhodostoma venom. Indirect ELISA and immunoblotting studies showed that there were extensive cross-reactions between anti-MPE IgG and venoms from many different genera of poisonous snakes, suggesting the involvement of immunological neutralization in the protective effect of MPE pre-treatment against snake venom poisoning. In vitro neutralization experiments showed that the anti-MPE antibodies effectively neutralized the lethalities of Asiatic cobra (Naja) venoms, but were not very effective against other venoms tested. CONCLUSIONS: The anti-MPE antibodies could be used in the antiserum therapy of Asiatic cobra (Naja) bites.

Daboia russellii and Naja kaouthia venom neutralization by lupeol acetate isolated from the root extract of Indian sarsaparilla Hemidesmus indicus R.Br.

The present study reports the isolation and purification of lupeol acetate from the methanolic root extract of Indian medicinal plant Hemidesmus indicus (L.) R.Br. (family: Asclepiadaceae) which could neutralize venom induced action of Daboia russellii and Naja kaouthia on experimental animals. Lupeol acetate could significantly neutralize lethality, haemorrhage, defibrinogenation, edema, PLA(2) activity induced by Daboia russellii venom. It also neutralized Naja kaouthia venom induced lethality, cardiotoxicity, neurotoxicity and respiratory changes in experimental animals. Lupeol acetate potentiated the protection by snake venom antiserum action against Daboia russellii venom induced lethality in male albino mice. Venom induced changes in lipid peroxidation and super oxide dismutase activity was antagonized by lupeol acetate. Snake venom neutralization by lupeol acetate and its possible mechanism of action has been discussed.

Screening of plants containing Naja naja siamensis cobra venom inhibitory activity using modified ELISA technique.

Enzyme-linked immunosorbent assay (ELISA) has been modified for screening plants with antagonistic activity to Naja naja siamensis cobra venom. Aqueous extracts from plants were investigated for their inhibitory effects on the binding of anti-cobra venom antibody to antigen, cobra venom, fixed onto 96-well microtiter plates. Ingredients in extracts were allowed to react with immobilized venom before the subsequent addition of antivenom antibody. Venom components affected by exposure to the extracts, unable to interact with their specific antibody, were predicted to be unable to bind to their native destinations or natural receptors. Curcuma cf. zedoaria, an old Thai medicinal plant, showed clear inhibitory activity in the ELISA test. Neurotoxin and protein degradative enzymes, major components in venom, were identified as targets of this extract in Western immunoblotting analysis. Ingredients in the extract showed high affinity to the toxin in competition assay by immunoprecipitation. The extract attenuated toxin activity by extending contraction time of diaphragm muscle after envenomation and had a potency to protect cellular proteins from venom degradative enzymes. Curcuma parviflora, with less activity in ELISA, exhibited acceptable results in two experiments but negative results in two experiments, whereas Curcuma longa, having low activity in the ELISA test, never showed any favorable results. Screening of 36 samples could classify plants into an inhibition range of 0 to 86%. This modified ELISA is recommended as a preliminary screening method for inhibitors with a large number of samples.

Inhibition of Naja kaouthia venom activities by plant polyphenols.

Plant polyphenols from the aqueous extracts of Pentace burmanica, Pithecellobium dulce, Areca catechu and Quercus infectoria were tested for their inhibitory activities against Naja kaouthia (NK) venom by in vitro neutralization method. The first three extracts could completely inhibit the lethality of the venom at 4 LD50 concentration and the venom necrotizing activity at the minimum necrotizing dose while also inhibited up to 90% of the acetylcholinesterase activity of NK venom at much lower tannin concentrations than that of Quercus infectoria. The ED50 of plant tannins in inhibiting NK venom activities varied according to condensed tannins and their content in the extracts. Molecular docking of the complexes between alpha-cobratoxin and either hydrolysable or condensed tannins at their lowest energetic conformations were proposed. The anti-venom activities of these plant polyphenols by selectively blocking the nicotinic acetylcholine receptor and non-selectively by precipitation of the venom proteins were suggested.

In vitro snake venom detoxifying action of some marine algae of Gulf of Mannar, south-east coast of India.

The extract of the brown seaweed Padina boergesenii and the red seaweed Hypnea valentiae was found to detoxify (in vitro) the venom of Naja nigricollis. There was a remarkable reduction in the mortality of albino mice after intraperitoneal (i.p.) administration of reconstituted venom with the extract compared to those challenged with the venom only. The survival of the animals exposed to the venom incubated with the different concentrations of the extract was used as the in vitro detoxification parameter.