Structural and functional studies of a snake venom phospholipase A2-like protein complexed to an inhibitor from Tabernaemontana catharinensis.

Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A2 (PLA2) and PLA2-like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58-71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA2-like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors.

A First Look at the Inhibitory Potential of Urospatha sagittifolia (Araceae) Ethanolic Extract for Bothrops atrox Snakebite Envenomation.

Bothrops atrox snakebites are a relevant problem in the Amazon basin. In this biodiverse region, the ethnomedicinal approach plays an important role as an alternative to antivenom therapy. Urospatha sagittifolia (Araceae) is a plant used for this purpose; however, its neutralizing properties have not been scientifically accessed. To fill this gap, we investigated the ability of U. sagittifolia to modulate the catalytic activity of Bothrops atrox venom, and their toxic consequences, such as local damage and lethality. The venom profile of B. atrox was assessed by chromatography and electrophoresis. Inhibition of the three main enzymatic and medically important toxins from the venom was evaluated using synthetic substrates and quantified by chromogenic activity assays. Additionally, the neutralization of lethality, hemorrhage and edema were investigated by in vivo assays. The possible interactions between venom proteins and plant molecules were visualized by polyacrylamide gel electrophoresis. Finally, the phytochemical constituents present in the ethanolic extract were determined by qualitative and quantitative analyses. The ethanolic extract reduced the activity of the three main enzymes of venom target, achieving ranges from 19% to 81% of inhibition. Our in vivo venom neuralizations assays showed a significant inhibition of edema (38.72%) and hemorrhage (42.90%). Additionally, lethality was remarkably counteracted. The highest extract ratio evaluated had a 75% survival rate. Our data support the biomedical value of U. sagittifolia as a source of natural enzyme inhibitors able to neutralize catalytically active B. atrox venom toxins and their toxic effects.

Pharmacological re-assessment of traditional medicinal plants-derived inhibitors as antidotes against snakebite envenoming: A critical review.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional healers have used medicinal plants to treat snakebite envenomation worldwide; however, mostly without scientific validation. There have been many studies on the therapeutic potential of the natural products against snake envenomation. AIM OF THE STUDY: This review has highlighted snake venom inhibitory activity of bioactive compounds and peptides from plants that have found a traditional use in treating snakebite envenomation. We have systematically reviewed the scenario of different phases of natural snake venom inhibitors characterization covering a period from 1994 until the present and critically analysed the lacuna of the studies if any, and further scope for their translation from bench to bedside. MATERIALS AND METHODS: The medicinal plant-derived compounds used against snakebite therapy were reviewed from the available literature in public databases (Scopus, MEDLINE) from 1994 till 2020. The search words used were 'natural inhibitors against snakebite,' 'natural products as therapeutics against snakebite,' 'natural products as antidote against snake envenomation,' ' snake venom toxin natural inhibitors,' 'snake venom herbal inhibitors'. However, the scope of this review does not include computational (in silico) predictions without any wet laboratory validation and snake venom inhibitory activity of the crude plant extracts. In addition, we have also predicted the ADMET properties of the identified snake venom inhibitors to highlight their valuable pharmacokinetics for future clinical studies. RESULTS: The therapeutic application of plant-derived natural inhibitors to treat snakebite envenomation as an auxiliary to antivenom therapy has been gaining significant momentum. Pharmacological reassessment of the natural compounds derived from traditional medicinal plants has demonstrated inhibition of the principal toxic enzymes of snake venoms at various extents to curb the lethal and/or deleterious effects of venomous snakebite. Nevertheless, such molecules are yet to be commercialized for clinical application in the treatment of snakebite. There are many obstacles in the marketability of the plant-derived natural products as snake envenomation antidote and strategies must be explored for the translation of these compounds from drug candidates to their clinical application. CONCLUSION: In order to minimize the adverse implications of snake envenomation, strategies must be developed for the smooth transition of these plant-derived small molecule inhibitors from bench to bedside. In this article we have presented an inclusive review and have critically analysed natural products for their therapeutic potential against snake envenomation, and have proposed a road map for use of natural products as antidote against snakebite.

Prospects and Challenges of Developing Plant-Derived Snake Antivenin Natural Products: A Focus on West Africa.

Snakebite envenomation (SBE) is an important public health issue that is now receiving renewed attention following its reclassification as a Neglected Tropical Disease (NTD). Most incidences occur in rural areas of resource-limited countries, as such, timely and appropriate medical care for SBE is often inaccessible. The administration of anti-snake venom serum (ASV) is the only effective definitive treatment of SBE, but treatment failure to available ASVs is not uncommon. Emerging evidence highlights the potential of small-molecule compounds as inhibitors against toxins of snake venom. This presents an encouraging prospect to develop an alternative therapeutic option for the treatment SBE, that may be amenable for use at the point of care in resource-constraint settings. In view of the pivotal role of natural products in modern drug discovery programmes, there is considerable interest in ethno-pharmacological mining of medicinal plants and plant-derived medicinal compounds toward developing novel snake venom-neutralising therapeutics. In this review, we compile a collection of medicinal plants used in the treatment of SBE in West Africa and highlight their promise as potential botanical drugs or as sources of novel small-molecule compounds for the treatment of SBE. The challenges that must be surmounted to bring this to fruition including the need for (sub) regional collaboration have been discussed.

Effects of photobiomodulation therapy on the local experimental envenoming by Bothrops leucurus snake.

Bothrops leucurus is the major causative agent of snakebites in Brazil's Northeast. The systemic effects of its venom are effectively neutralized by antivenom therapy, preventing bitten patients' death. However, antivenom fails in neutralizing local effects that include intense pain, edema, bleeding, and myonecrosis. Such effects can lead to irreversible sequels, representing a clinically relevant issue for which there is no current effective treatment. Herein, the effects of photobiomodulation therapy (PBMT) were tested in the local actions induced by B. leucurus venom (BLV) in mice (n = 123 animals in 20 experimental groups). A continuous emission AlGaAs semiconductor diode laser was used in two wavelengths (660 or 780 nm). Mechanical nociceptive thresholds were assessed with the electronic von Frey apparatus. Local edema was determined by measuring the increase in paw thickness. Hemorrhage was quantified by digital measurement of the bleeding area. Myotoxicity was evaluated by serum creatine kinase (CK) activity and histopathological analysis. PBMT promoted anti-hypernociception in BLV-injected mice; irradiation with the 660 nm laser resulted in faster effect onset than the 780 nm laser. Both laser protocols reduced paw edema formation, whether irradiation was performed immediately or half an hour after venom injection. BLV-induced hemorrhage was not altered by PBMT. Laser irradiation delayed, but did not prevent myotoxicity caused by BLV, as shown by a late increase in CK activity and histopathological alterations. PBMT was effective in the control of some of the major local effects of BLV refractory to antivenom. It is a potential complementary therapy that could be used in bothropic envenoming, minimizing the morbidity of these snakebite accidents.

Modulatory Activities of Plant Extracts on Jellyfish Cytotoxicity.

INTRODUCTION: The potential efficacy of selected plant extracts to counteract the dermal toxicity of jellyfish envenomation was investigated using an in vitro cell culture model. METHODS: We studied plant extracts from Carica papaya, Ananas comosus, and Bouvardia ternifolia, known for their antivenom properties, in pairwise combinations with tissue homogenates of the jellyfish Pelagia noctiluca, Phyllorhiza punctata, and Cassiopea andromeda, to evaluate modulations of jellyfish cytotoxic effects. L929 mouse fibroblasts were incubated with pairwise jellyfish/plant extract combinations and examined by MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). RESULTS: C papaya and A comosus significantly lowered the cytotoxicity of P noctiluca and P punctata but induced a slight worsening of C andromeda cytotoxicity. Conversely, B ternifolia was protective against P punctata, ineffective against P noctiluca, and worsened C andromeda cytotoxicity. CONCLUSIONS: Data showed species-specific and contrasting effects of plant extracts, suggesting that those containing protease activities, namely A comosus and C papaya, are more effective in lowering the cytotoxicity of jellyfish venom containing toxic peptidic factors such as phospholipase A. However, all examined plants require further investigation in vivo to evaluate their ability to counteract jellyfish injury to the skin.

Detoxifying Action of Aqueous Extracts of Mucuna pruriens Seed and Mimosa pudica Root Against Venoms of Naja nigricollis and Bitis arietans.

BACKGROUND: The World Health Organization included snakebite envenomation among Neglected Tropical Diseases in 2017. The importance of natural products from plants is enormous, given that most prescribed drugs originate from plants. Among this is Mucuna pruriens and Mimosa pudica, with many registered patents asserting their health benefits. OBJECTIVE: This study investigated the in vitro neutralizing effects of Mucuna pruriens seed and Mimosa pudica root extracts on venoms of Naja nigricollis and Bitis arietans. METHODS: In mice, the LD50 and phytochemical analysis of M. pruriens and M. pudica plant extracts were carried out prior to the evaluation of their haemolytic and fibrinolytic effect. Their effects on the activities of phospholipase A2 (PLA2) were also assessed. RESULTS: At a concentration of 50 mg/ml, both plant extracts were found to neutralize the fibrinolytic activity of N. nigricollis, but 400 mg/ml was required to neutralize the fibrinolytic activity of B. arietans. In haemolytic studies, 50 mg/ml concentration of M. pruriens extract suppressed haemolysis caused by N. nigricollis venom by 70% but at the same concentration, M. pudica extract reduced haemolysis by 49.4%. M. pruriens, at 50 mg/ml concentration, only inhibited phospholipase A2 activity by 7.7% but higher concentrations up to 400mg/ml had no effect against the venom of N. nigricollis; at 200 mg/ml. M. pudica extract inhibited PLA2 activity by 23%. CONCLUSION: The results suggest that M. pruriens and M. pudica may be considered as promising antivenom agents for people living in a snake-bite prone environment.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

BACKGROUND: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. OBJECTIVE: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. METHODS: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. RESULTS: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. CONCLUSION: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

Edematic and coagulant effects caused by the venom of Bothrops rhombeatus neutralized by the ethanolic extract of Piper auritum.

ETHNOPHARMACOLOGICAL RELEVANCE: In Colombia, the only authorized treatment to cure snakebite envenomation is with the use of antivenom. The antivenom neutralizes the systemic effects properly, but is not very effective at neutralizing local effects, thus several cases have lead to complications. On the other hand, rural communities turn to the use of plants that are easily accessible and available for basic health care. One of these plants is named Piper auritum (PA), which is traditionally highlighted in some indigenous communities of Antioquia and Chocó. AIM OF THE STUDY: The main objective of this work was to characterize the venom's toxicity by determining the Minimum Edema Dose (MED), the Minimum Coagulant Dose-Plasma (MCD-P), the Minimum Hemorrhagic Dose (MHD) and to determine the neutralizing power of the Total Ethanolic Extract (TEE) from leaves of PA on the localized and systemic effects caused by the Bothrops rhombeatus venom. MATERIALS AND METHODS: To begin, the minimum dose that causes edema-forming, coagulant and hemorrhagic activities was determined. The protocols investigated include coagulant and edematic activities caused by the venom of Bothrops rhombeatus which were neutralized by the TEE of PA. RESULTS: The MCD-P was found to be 0.206 ±â€¯0.026 µg, the MED is the same at 0.768 ±â€¯0.065 µg, and the MHD is 3.553 ±â€¯0.292 µg, which are different from the reports for Bothrops asper and Bothrops ayerbei. Next, a phytochemical screening was done to the TEE where mainly triterpenes, steroids, coumarins, saponins, and lignans were identified. Also present were 43,733 ±â€¯2106 mg AG/g ES of phenols, which are secondary metabolites that are probably responsible for the neutralization of coagulant and edematic activities at rates of 2363.870 µL and 1787.708 µL of extract/mg of venom, respectively. As a comparative parameter, the National Institute Health's (NHI) effective dose of the antivenom was used as a comparative parameter. In addition, we determined the toxicity of the TEE of PA on to Artemia salina, being moderately toxic at 6 and 24 h, while the essential oil of PA at the same observation hours is in the extremely toxic range. CONCLUSIONS: The results reflect that the extract of P. auritum has an anti-inflammatory effect similar to that of the NIH serum. It could be used as a complement of NIH antivenom, using them together so it contributes to effectively reduce inflammation and the socio-economic impact generated by the permanence of a patient victim of snakebite in health centers. CLASSIFICATIONS: Immunological products and vaccines.

Plants and Phytocompounds Active Against Bothrops Venoms.

Snakebite envenomation is an important health problem in tropical countries, with severe human and social consequences. In Latin America, the Bothrops species constitute the main threat to humans, and the envenomation caused by these species quickly develops into severe local tissue damage, including swelling, hemorrhaging, myonecrosis, skin ulceration, and pain. The systemic effects of envenomation are usually neutralized by antivenom serum therapy, despite its intrinsic risks. However, neutralization of local tissue damage remains a challenge. To improve actual therapy, two major alternatives are proposed: the rational design of new specific antibodies for most of the tissue damaging/ poor immunogenic toxins, or the search for new synthetic or natural compounds which are able to inhibit these toxins and complement the serum therapy. Natural compounds isolated from plants, mainly from those used in folk medicine to treat snakebite, are a good choice for finding new lead compounds to improve snakebite treatment and minimize its consequences for the victims. In this article, we reviewed the most promising plants and phytocompounds active against bothropic venoms.

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.

Natural Snake Venom Inhibitors and their Pharmaceutical Uses: Challenges and Possibilities.

Nowadays, treatment with specific antivenins is considered the only cure for snakebites accidents. However, access to antivenom obstructs the successful implementation of the World Health Organization international guidelines. In the last few years, natural organic compounds, peptides, and proteins with the ability to inhibit snake toxins and obtained from different sources such as plant extracts and animal blood have been proposed as antivenoms. In this work, we will focus on the inhibitors of the main venom toxins, phospholipases A2 and metalloproteinases, and their application as novel antivenoms.

Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa.

BACKGROUND: Antivenom is the treatment of choice for snakebite, which annually kills an estimated 32,000 people in sub-Saharan Africa and leaves approximately 100,000 survivors with permanent physical disabilities that exert a considerable socioeconomic burden. Over the past two decades, the high costs of the most polyspecifically-effective antivenoms have sequentially reduced demand, commercial manufacturing incentives and production volumes that have combined to create a continent-wide vacuum of effective snakebite therapy. This was quickly filled with new, less expensive antivenoms, many of which are of untested efficacy. Some of these successfully marketed antivenoms for Africa are inappropriately manufactured with venoms from non-African snakes and are dangerously ineffective. The uncertain efficacy of available antivenoms exacerbates the complexity of designing intervention measures to reduce the burden of snakebite in sub-Saharan Africa. The objective of this study was to preclinically determine the ability of antivenoms available in Kenya to neutralise the lethal effects of venoms from the most medically important snakes in East Africa. METHODS: We collected venom samples from the most medically important snakes in East Africa and determined their toxicity in a mouse model. Using a 'gold standard' comparison protocol, we preclinically tested the comparative venom-neutralising efficacy of four antivenoms available in Kenya with two antivenoms of clinically-proven efficacy. To explain the variant efficacies of these antivenoms we tested the IgG-venom binding characteristics of each antivenom using in vitro IgG titre, avidity and venom-protein specificity assays. We also measured the IgG concentration of each antivenom. FINDINGS: None of the six antivenoms are preclinically effective, at the doses tested, against all of the most medically important snakes of the region. The very limited snake polyspecific efficacy of two locally available antivenoms is of concern. In vitro assays of the abilities of 'test' antivenom IgGs to bind venom proteins were not substantially different from that of the 'gold standard' antivenoms. The least effective antivenoms had the lowest IgG content/vial. CONCLUSIONS: Manufacture-stated preclinical efficacy statements guide decision making by physicians and antivenom purchasers in sub-Saharan Africa. This is because of the lack of both clinical data on the efficacy of most of the many antivenoms used to treat patients and independent preclinical assessment. Our preclinical efficacy assessment of antivenoms available in Kenya identifies important limitations for two of the most commonly-used antivenoms, and that no antivenom is preclinically effective against all the regionally important snakes. The potential implication to snakebite treatment is of serious concern in Kenya and elsewhere in sub-Saharan Africa, and underscores the dilemma physicians face, the need for clinical data on antivenom efficacy and the medical and societal value of establishing independent preclinical antivenom-efficacy testing facilities throughout the continent.

Protective Effect of the Sulfated Agaran Isolated from the Red Seaweed Laurencia aldingensis Against Toxic Effects of the Venom of the Snake, Lachesis muta.

Snakebite is a serious occupational hazard affecting mainly rural populations of tropical and subtropical developing countries. Lachesis muta (Bushmaster) bites are extremely serious but are rarely reported in the literature. Bushmaster envenomings are characterized by intense local pain, edema, neurotoxicity, hypotension, local hemorrhage, and dramatic systemic alterations. Antivenom treatment has regularly been used for more than a century; however, it fails to neutralize local tissue damage and hemorrhage, leading to morbidity or disabilities in victims. Thus, the production and clinical use of antivenom must be improved. The present work characterizes, for the first time, a sulfated polysaccharide from the red seaweed, Laurencia aldingensis, including its neutralizing effect on some toxic activities of L. muta venom. Chemical and spectroscopic analyses showed that L. aldingensis produces sulfated agarans with the A-units partially C-2 sulfated or 6-O-methoxylated presetting the B-units in the cyclized (3,6-anhydro-α-L-galactose) or in the non-cyclized form (α-L-galactose). The latter is significantly substituted by sulfate groups on C-6. In vitro and in vivo assays showed that this sulfated agaran inhibited hemolysis, coagulation, proteolysis, edema, and hemorrhage of L. muta venom. Neutralization of hemorrhagic activity was also observed when the agaran was administered by different routes and after or before the venom injection. Furthermore, the agaran blocked the edema caused by a phospholipase A2 isolated from the L. muta venom. Experimental evidence therefore indicates that the sulfated agaran of L. aldingensis has potential to aid antivenom therapy of accidents caused by L. muta venom and may help to develop more effective antivenom treatments of snake bites in general.

Molecular modeling and snake venom phospholipase A2 inhibition by phenolic compounds: Structure-activity relationship.

In our earlier study, we have reported that a phenolic compound 2-hydroxy-4-methoxybenzaldehyde from Janakia arayalpatra root extract was active against Viper and Cobra envenomations. Based on the structure of this natural product, libraries of synthetic structurally variant phenolic compounds were studied through molecular docking on the venom protein. To validate the activity of eight selected compounds, we have tested them in in vivo and in vitro models. The compound 21 (2-hydroxy-3-methoxy benzaldehyde), 22 (2-hydroxy-4-methoxybenzaldehyde) and 35 (2-hydroxy-3-methoxybenzylalcohol) were found to be active against venom-induced pathophysiological changes. The compounds 20, 15 and 35 displayed maximum anti-hemorrhagic, anti-lethal and PLA2 inhibitory activity respectively. In terms of SAR, the presence of a formyl group in conjunction with a phenolic group was seen as a significant contributor towards increasing the antivenom activity. The above observations confirmed the anti-venom activity of the phenolic compounds which needs to be further investigated for the development of new anti-snake venom leads.

Connarus favosus Planch.: An inhibitor of the hemorrhagic activity of Bothrops atrox venom and a potential antioxidant and antibacterial agent.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant species Connarus favosus is used in folk medicine in the west of Pará state, Brazil, to treat snakebites. AIM OF THE STUDY: To investigate the potential of the aqueous extract of Connarus favosus (AECf) to inhibit hemorrhagic and phospholipase A2 activities induced by Bothrops atrox venom (BaV) and to determine the antioxidant and antimicrobial potentials of the extract. MATERIALS AND METHODS: AECf was analyzed phytochemically for phenolics (condensed tannins and hydrolyzable tannins) by colorimetry. Antioxidant activity was evaluated by quantitative assays using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Fe(3+)/phenanthroline. Antimicrobial activity was evaluated by the minimal inhibitory concentration test, and cytotoxicity was evaluated using human fibroblast cells (MRC-5). Inhibition of BaV-induced hemorrhagic activity was assessed after oral administration of the extract using pre-treatment, post-treatment and combined (BA plus AECf) treatment protocols. Inhibition of indirect hemolysis caused by phospholipase A2 (PLA2) was investigated in vitro. Interaction between AECf and BaV was investigated by SDS-PAGE electrophoresis, Western blot (Wb) and zymography. RESULTS: The phytochemical profile of AECf revealed ten secondary metabolite classes, and colorimetry showed high total phenolic and total (condensed and hydrolyzable) tannin content. AECf exhibited high antioxidant and antimicrobial potentials. The IC50 for the cytotoxic effect was 51.91 (46.86-57.50)µg/mL. Inhibition of BaV-induced hemorrhagic activity was significant in all the protocols, and inhibition of PLA2 activity was significant with the two highest concentrations. The BaV/AECf mixture produced the same bands as BaV by itself in SDS-PAGE and Wb although the bands were much fainter. Zymography confirmed the proteolytic activity of BaV, but when the venom was pre-incubated with AECf this activity was blocked. CONCLUSION: AECf was effective in reducing BaV-induced hemorrhagic activity when administered by the same route as that used in folk medicine and exhibited antioxidant and antimicrobial potentials.

The inhibitory potential of the condensed-tannin-rich fraction of Plathymenia reticulata Benth. (Fabaceae) against Bothrops atrox envenomation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnobotanical studies have shown that Plathymenia reticulata Benth. (Fabaceae) has been widely used in cases of snake envenomation, particularly in Northern Brazil. In light of this, the aim of this study was to evaluate the inhibitory potential of the condensed-tannin-rich fraction obtained from the bark of P. reticulata against the main biological activities induced by Bothrops atrox venom (BaV). MATERIALS AND METHODS: The chemical composition of the aqueous extract of P. reticulata (AEPr) was first investigated by thin-layer chromatography (TLC) and the extract was then fractionated by column chromatography on Sephadex LH-20. This yielded five main fractions (Pr1, Pr2, Pr3, Pr4 and Pr5), which were analyzed by colorimetry to determine their concentrations of total phenolics, total tannins and condensed tannins and to assess their potential for blocking the phospholipase activity of BaV. The Pr5 fraction was defined as the fraction rich in condensed tannins (CTPr), and its inhibitory potential against the activities of the venom was evaluated. CTPr was evaluated in different in vivo and in vitro experimental protocols. The in vivo protocols consisted of (1) pre-incubation (venom:CTPr, w/w), (2) pre-treatment (orally administered) and (3) post-treatment (orally administered) to evaluate the effect on the hemorrhagic and edematogenic activities of BaV; in the in vitro protocol the effect on phospholipase and coagulant activity using pre-incubation in both tests was evaluated. RESULTS: There was statistically significant inhibition (p<0.05) of hemorrhagic activity by CTPr when the pre-incubation protocol was used [55% (1:5, w/w) and 74% (1:10, w/w)] and when pre-treatment with doses of 50 and 100mg/kg was used (19% and 13%, respectively). However, for the concentrations tested, there was no statistically significant inhibition in the group subjected to post-treatment administered orally. CTPr blocked 100% of phospholipase activity and 63.3% (1:10, w/w) of coagulant activity when it was pre-incubated with BaV. There was a statistically significant reduction (p<0.05) in edema induced by BaV in the oral protocols. Maximum inhibition was 95% (pre-treatment). CONCLUSION: Our findings indicate that CTPr could be a good source of natural inhibitors of the components of snake venom responsible for inducing local inflammation.

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

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

Local and systemic toxicity of Echis carinatus venom: neutralization by Cassia auriculata L. leaf methanol extract.

Viper bites cause high morbidity and mortality especially in tropical and subtropical regions, affecting a large number of the rural population in these areas. Even though anti-venoms are available, in most cases they fail to tackle viper venom-induced local manifestations that persist even after anti-venom administration. Several studies have been reported the use of plant products and approved drugs along side anti-venom therapy for efficient management of local tissue damage. In this regard, the present study focuses on the protective efficacy of Cassia auriculata L. (Leguminosae) against Echis carinatus venom (ECV) induced toxicity. C. auriculata is a traditional medicinal plant, much valued in alternative medicine for its wide usage in ayurveda, naturopathy, and herbal therapy. Further, it has been used widely by traditional healers for treatment of snake and scorpion bites in the Western Ghats of Karnataka, India. In the present study, C. auriculata leaf methanol extract (CAME) significantly inhibited enzymatic activities of ECV proteases (96 ± 1 %; P = 0.001), PLA2 (45 ± 5 %; P = 0.01) and hyaluronidases (100 %; P = 0.0003) in vitro and hemorrhage, edema and myotoxicity in vivo. Further, CAME effectively reduced the lethal potency of ECV and increased the survival time of mice by ~6 times (17 vs 3 h). These inhibitory potentials of CAME towards hydrolytic enzymes, mortal and morbid symptoms of ECV toxins clearly substantiates the use by traditional healers of C. auriculata as a folk medicinal remedy for snakebite.

A comparison of the ability of Bellucia dichotoma Cogn. (Melastomataceae) extract to inhibit the local effects of Bothrops atrox venom when pre-incubated and when used according to traditional methods.

Bellucia dichotoma Cogn. (Melastomataceae) is one of various plant species used in folk medicine in the west of the state of Pará, Brazil, to treat snake bites. Many studies have been carried out to evaluate the effectiveness of anti-snake bite plants, but few of these use the same preparation methods and doses as those traditionally used by the local populations. This study therefore compared inhibition of the main local effects of B. atrox venom (BaV) by aqueous extract of B. dichotoma (AEBd) administered according to traditional methods and pre-incubated with BaV). The concentrations of phenolic compounds (tannins and flavonoids) in AEBd were determined by colorimetric assays. The effectiveness of AEBd in inhibiting the hemorrhagic and edematogenic activities of BaV was evaluated in mice in four different experimental in vivo protocols: (1) pre-incubation (venom:extract, w/w); (2) pre-treatment (p.o.); (3) post-treatment (p.o.); and (4) AEBd (p.o.) in combination with Bothrops antivenom (BA) (i.v.). To assess in vitro inhibition of BaV phospholipase A2 activity, the pre-incubation method or incorporation of AEBd or BA in agarose gels were used. The effect of AEBd on BaV was determined by SDS-PAGE, zymography and Western blot. Colorimetric assays revealed higher concentrations of (condensed and hydrolyzable) tannins than flavonoids in AEBd. Hemorrhagic activity was completely inhibited using the pre-incubation protocol. However, with pre-treatment there was no significant inhibition for the concentrations tested, and with the post-treatment only the 725 mg/kg dose of AEBd was able to inhibit 40.5% (p = 0.001) of the hemorrhagic activity of BaV. Phospholipase A2 activity was only inhibited when AEBd was pre-incubated with BaV. BaV-induced edema was completely inhibited with pre-incubation (p < 0.05) and significantly reduced (p < 0.05) with pre- and post-treatment (p.o.) for the concentrations tested. The reduction in local edema was even greater when AEBd was administered in combination with BA. The SDS-PAGE profiles showed that several of the BaV protein (SDS-PAGE) and enzyme (zymography) bands were not detected when the venom was pre-incubated, and Western blot revealed that this was not caused by the AEBd enzymes observed in the zymogram. The "pseudo inhibition" observed after pre-incubation in this study may be due to the presence of tannins in the extract, which could act as chelating agents, removing metalloproteins and Ca²âº ions and thus inhibiting hemorrhagin and PLA2 activity. However, when administered according to traditional methods, B. dichotoma extract was effective in blocking BaV-induced edematogenic activity and had an additional effect on inhibition of this activity by BA.