Edema-inducing Activity Research Articles

Toxicological analyses of the venoms of Nigerian vipers Echis ocellatus and Bitis arietans

BackgroundAmong the medically important snakes in Nigeria, Echis ocellatus and Bitis arietans have the most lethal venom. These venoms were classified according to the presence of snake venom metalloproteinases (SVMPs), snake venom phospholipase A2 (PLA2s), and snake venom serine proteases (SVSPs). Toxicological analyzes were performed to understand the significance of different protein families in venoms.MethodsProteins were separated from venom using column chromatography. The skin and footpad of mice were used to determine hemorrhagic and edematogenic activities. Caprine blood plasma was used to test fibrinolytic activity in vitro.ResultsThe results showed that, compared to the crude venom, the SVMP fraction induced hemorrhagic effects with a diameter of 26.00 ± 1.00 mm in E. ocellatus and 21.33 ± 1.52 mm in B. arietans. Both SVSP and SVMP had anticoagulant effects; however, the SVSP fraction had a stronger effect, with a longer anticoagulation time of 30.00 ± 3.00 min in E. ocellatus and 26.00 ± 2.00 min in B. arietans. These main venom toxins, SVMPs, SVSPs, and PLA2, were found to have edema-forming effects that were optimal at 2 h after envenomation. PLA2s had the highest edema-inducing activity, with onset 30 min after envenomation.ConclusionsGiven the importance of SVMPs in altering the integrity of the membrane structure and impairing the blood coagulation system, an antivenom that can specifically neutralize its activity could inhibit the hemorrhage effects of the venoms.

In-Vivo Antivenom Effect of Annona senegalensis (Pers) Against Biological Activities of Naja nigricollis (Hallowell) Envenomation

Snake bites are a common, serious medical issue often neglected in developing countries. This study aims to investigate the effectiveness of methanolic extract from the root bark of Annona senegalensis in neutralizing the biological activities of Naja nigricollis envenomation in vivo. The methanolic extract was fractionated using a liquid-liquid fractionating method. The inhibition of venom-induced edematogenic and hemorrhagic effects was evaluated in albino rats using a pre-incubation protocol. The aqueous-methanol fraction (AM) of A. senegalensis AM root methanolic extract significantly inhibited the hemorrhagic and edema-inducing activities of the venom. The methanolic extract derived from the root bark of A. senegalensis has exhibited promising potential as an effective anti-snake venom agent.

Evaluation of the properties of Bungarus caeruleus venom and checking the efficacy of antivenom used in Bangladesh for its bite treatment.

As a disaster-prone country with unique geographical features, snake biting is a major public health concern in Bangladesh. The primary reasons of mortality from snakebite include late presentation to the hospital, low efficacy of antivenom, and a lack of adequate management facilities. Because snake venom characteristics vary depending on geographical location, antivenom should be manufactured from snakes native to the region in which it would be administered. Bungarus caeruleus is a highly venomous snake contributing to the major snakebite issue in Bangladesh. Therefore, the neutralization efficacy of the antivenom against B. caeruleus venom was evaluated in the current study along with the characterization of venom. For biological characterization of venom, RP-HPLC and SDS-PAGE profiling, hemolytic activity, hemorrhagic activity, phospholipases A2 (PLA2) activity, edema inducing activity and histopathological observations were carried out following standard protocol. LD50 of the venom was calculated along with neutralization potency of Incepta antivenom through probit analysis. Results showed that venom possesses phospholipase A2 activity, hemolytic activity and edema inducing activity while hemorrhagic activity was absent in the skin of envenomed mice. Histopathological alterations including necrosis, congestion and infiltrations were observed in envenomed mice organs after hematoxylin and eosin staining. Neutralization study showed that Incepta polyvalent antivenom could neutralize (potency 0.53mg/ml) the lethal effect in in vitro study on mice. Further investigation on snakebite epidemiology and clinical observations of the envenomed patients will help in combating the snakebite problem more efficiently.

Evaluation of anti-inflammatory activity of Garcinia indica, a wild edible fruit by inhibiting secretory phospholipase A2 group IIA enzyme from human pleural fluid

Introduction and Aim: Garcinia indica is a wild edible fruit plant distributed in tropical regions of India. Fruits of G. indica were traditionally used to treat chronic inflammatory diseases like rheumatoid arthritis, gastrointestinal disorders, etc., But the basis of anti-inflammatory function of the fruit is not understood. Therefore, this study aims to evaluate the anti-inflammatory function of G. indica. Initially phytochemical screening of G. indica was carried out, tested antioxidant potency of G. indica fruit and evaluated its anti-inflammatory function by inhibition of secretory phospholipase A2 IIA (sPLA2IIA) enzyme. Methodology: The Soxhlet extraction method was used for the preparation of extracts of G. indica fruit. DPPH and phosphomolybdenum assays were carried out to estimate the antioxidant activities of G. indica fruit. The inhibition of sPLA2IIA, modulating indirect hemolytic and edema inducing activity and protein denaturation assays were done to evaluate the anti-inflammatory function. Results: Aqueous and solvent extracts of G. indica fruit showed a wide variety of phytochemicals. The ethanol extract of G. indica fruit showed greater antioxidant activity (79.12 % ±1.2) and reduction power (68.14% ± 4.4). The extract showed sPLA2IIA inhibition to a greater extent (50%), neutralized sPLA2IIA induced indirect hemolysis (IC50 45.518 µg/mL) and mouse paw edema (119.35% ± 2.74) with the IC50 value of 45.12 ±1.36µg. Conclusion: The extracts of G. indica contain a wide variety of phytochemicals. The ethanol extract of G. indica fruit exhibited a greater antioxidant activity and anti-inflammatory activity. Thus, the results defended the traditional usage of the G. indica fruit in the indigenous system of medicine.

In vivo and in vitro prospection of the anti-ophidic properties exercised by the extracts of Jacaranda decurrens L.

The research and development of alternative treatments for snakebites (e.g., medicinal plants) is necessary due to the high costs of the existing ones. The effects of the aqueous extracts from Jacaranda decurrens leaves, roots, and xylopodium were analyzed upon the venom-induced (Bothrops spp. and Crotalus spp.) systemic and local toxicity. The extracts were able to partially inhibit the phospholipase activity of the venoms from Bothrops jararacussu and Crotalus durissus terrificus. The myotoxic, edema-inducing, coagulant, and hemorrhagic activities were also inhibited. The SDS-PAGE showed that the venom proteins were intact after their incubation with the extracts. This suggests that the possible mechanism of inhibition is not related to the degradation of the protein but rather to their binding to specific sites of the enzymes. The extracts significantly prolonged the survival time of animals in the lethality assay performed with Crotalus durissus terrificus venom and its toxin (crotoxin). The anti-ophidic activity of medicinal plants may aid in the management of snakebites in distant locations by reducing the victim’s local effects and time to heal.

Individual variations in the protein profiles and functional activities of the eyelash palm pit-viper (Bothriechis schlegelii) venom from the Colombian southwest region

Bothriechis schlegelii is a venomous snake found in Central and South America, mainly sighted in regions devoted to agriculture. However, in Colombia, little is known about its contribution to the total envenoming cases. Furthermore, there are no reports of the biochemical and functional activities of venoms from the southwest populations, and the differences respecting other populations are unknown. This study analyzed the protein profiles of venom samples obtained from three specimens originating from this region of Colombia using electrophoresis and chromatography. The lethality, edema-induction, hemorrhagic, defibrinating, coagulant, and indirect hemolytic activities were also evaluated. As a result, venoms were composed of proteins with a wide range of molecular weights, most of them below <37 kDa, with differences between male and female electrophoretic and chromatographic profiles. These variations were also observed in the evaluation of venom functional activities such as pro-coagulant, indirect hemolytic, and edema-inducing activities, whereas neither hemorrhagic nor defibrinating activities were detected. These results are also different considering reports with venom samples from other geographical locations, restating the existence of high intraspecific variability in B. schlegelii venoms, which could have relevant pathophysiological and therapeutic implications.

Thrombin- and plasmin-like and platelet-aggregation-inducing activities of Plumeria alba L. latex: Action of cysteine protease

Ethnopharmacological relevanceIn folk medicine, parts of Plumeria alba L. are used for the treatment of many diseases, with its latex being used for curing skin diseases and promoting wound healing. Aim of the studyThis study aimed to study the role of P. alba L. latex in hemostasis and platelet aggregation. Materials and methodsThe latex of P. alba L. was processed to remove waxes and enrich protein content, and the final extract was named Plumeria alba L. natant latex (PaNL). PaNL was analyzed for protease activity against casein. The type of protease in PaNL was identified by using protease inhibitors such as E−64, phenylmethylsulfonyl fluoride, ethylenediaminetetraacetic acid, and pepstatin A. Human fibrinogen, fibrin, and collagen types I and IV were subjected to hydrolysis with different concentrations of PaNL. The thrombin-like activity of PaNL was determined by analyzing its fibrinogen-clotting and procoagulant activities. The role of PaNL in platelet aggregation was also investigated. Its hemorrhagic and edema-inducing activities were evaluated in a mouse model. Phytochemical compounds were identified by gas chromatography–mass spectroscopy. ResultsThe findings of casein/gelatin zymography confirmed that PaNL possesses protease activity. The results of the protease inhibition study indicated the presence of a cysteine-type protease(s) in PaNL. PaNL hydrolyzed the subunits of fibrinogen, fibrin, and collagen types I and IV. Its fibrin-degradation activity indicated that PaNL possesses plasmin-like activity. PaNL induced clotting of citrated human plasma within 3 min of incubation in the absence of CaCl2, indicating the presence of thrombin-like activity, which was further confirmed by the results of the fibrinogen-clotting assay. PaNL induced platelet aggregation in the absence of agonists. There was no hemolytic activity. Mice injected with PaNL did not show edema/ hemorrhagic activity. ConclusionPaNL possesses procoagulant, fibrino(geno)lytic, thrombin- and plasmin-like activities and induces platelet aggregation, which could explain its usage for wound treatment in folk medicine.

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.

Potent procoagulant and platelet aggregation inducing serine protease from Tridax procumbens extract

Background: Tridax procumbens extract (TPE) has been widely used in tribal/folk medicine to stop bleeding and to enhance wound healing process. Based on its traditional medicinal importance, the TPE is evaluated for its possible role in blood coagulation cascade. Objective: This study has been conducted to evaluate the TPE for the presence of protease associated with fibrino (geno) lytic, collagenolytic properties, and its action on blood coagulation cascade and platelet aggregation to substantiate its procoagulant nature. Materials and Methods: The TPE was analyzed in vitro for proteolytic, fibrinogenolytic, and collagenolytic activity. It was also analyzed for its effect on blood coagulation and platelet aggregation. In vivo studies have been conducted for hemorrhagic and edema inducing activity. Results: A non-toxic serine protease having procoagulant property associated with fibrino (geno) lytic, collagenolytic (both type I and type IV), and platelet aggregation inducing was identified and evaluated from TPE. The TPE decreased the clotting time of human plasma as evaluated by recalcification time and partial thromboplastin time by 19.8 and 1.53 folds, respectively. This suggests its procoagulant nature. TPE also enhanced the adenosine diphosphate-/epinephrine-induced platelet aggregation by 1.35 and 1.38 folds, respectively. Conclusion: The TPE serine protease is a non-toxic procoagulant with fibrino (geno) lytic and collagenolytic activities and induces platelet aggregation. Further, isolation and characterization of active molecule in TPE will allow us to exploit the pharmacological potential of TPE on coagulation cascade.

Betulinic, oleanolic and ursolic acids inhibit the enzymatic and biological effects induced by a P-I snake venom metalloproteinase

Betulinic acid (BA), Oleanolic acid (OA) and Ursolic acid (UA), are pentacyclic triterpenoids with widespread occurrence throughout the plant kingdom, these compounds are widely recognized by their pharmacological and biological properties, such as, anti-tumoral, anti-inflammatory, anti-microbial and hepatoprotective activity. In this work we determined the inhibitory ability of these compounds on the enzymatic, hemorrhagic, myotoxic and edema-inducing activities of Batx-I, a P-I metalloproteinase isolated from Bothrops atrox venom. BA, UA and OA inhibited the proteolytic activity of Batx-I on gelatin with IC50 values of 115.3, 223.0 and 357.3 μM, respectively. Additionally, these compounds showed inhibition of the hemorrhagic activity of Batx-I in skin with IC50 345.7, 643.5 and 1077.0 μM for BA, UA and OA in preincubation experiments. In studies with independent-injection, in which Batx-I was injected and then, at the same site, a concentration of 600 μM of each compound were administered at either 0, 5 or 10 min, BA showed a significant reduction of hemorrhage at 0 and 5 min. In addition, these compounds inhibited myotoxicity and edema-forming activity of Batx-I at 600 μM concentration. Molecular docking studies suggested that these compounds could occupy part of the substrate binding cleft of the enzyme affecting its catalytic cycle. In this manner, triterpenic acids are candidates for the development of inhibitors for the prevention of local tissue damage in snakebite envenomation.

Local and systemic effects of BdipTX-I, a Lys-49 phospholipase A2 isolated from Bothrops diporus snake venom

The present work aimed to isolate a basic phospholipase A2 (PLA2) from Bothrops diporus snake venom (BdV), evaluate and compare the myotoxic and oedema-inducing activities, as well as the systemic effects caused by both the isolated PLA2 and BdV on Swiss mice. A Lys-49 PLA2 (BdipTX-I) was obtained through two chromatographic steps: an ion-exchange and a reverse phase. The local (oedema and myotoxicity) and systemic (hepatic and renal functions) effects were then assessed for BdipTX-I and BdV. Results showed that the oedema-inducing activity was significant in all tested doses (5 and 20 μg/paw) for both BdipTX-I and BdV. Myotoxicity was evaluated by the increase of serum CK, CK-MB and LDH, and results showed that BdV effect is more prominent than BdipTX-I effect. The systemic effects were evaluated by determining specific laboratory markers: AST, ALT, GGT, ALP, urea, creatinine, protein and calcium. BdipTX-I and BdV were able to induce renal changes in the experimental model, leading to proteinuria (induced both by BdipTX-I and by BdV) and uremia (induced only by BdV). Thus, it is concluded that the systemic effects of BdV and BdipTX-I occur differently.

P.3.b.028 - Aberrant salience inventory and first rank symptoms: assessing proneness to psychosis beyond diagnosis

Betulinic acid (BA), Oleanolic acid (OA) and Ursolic acid (UA), are pentacyclic triterpenoids with widespread occurrence throughout the plant kingdom, these compounds are widely recognized by their pharmacological and biological properties, such as, anti-tumoral, anti-inflammatory, anti-microbial and hepatoprotective activity. In this work we determined the inhibitory ability of these compounds on the enzymatic, hemorrhagic, myotoxic and edema-inducing activities of Batx-I, a P-I metalloproteinase isolated from Bothrops atrox venom. BA, UA and OA inhibited the proteolytic activity of Batx-I on gelatin with IC50 values of 115.3, 223.0 and 357.3 μM, respectively. Additionally, these compounds showed inhibition of the hemorrhagic activity of Batx-I in skin with IC50 345.7, 643.5 and 1077.0 μM for BA, UA and OA in preincubation experiments. In studies with independent-injection, in which Batx-I was injected and then, at the same site, a concentration of 600 μM of each compound were administered at either 0, 5 or 10 min, BA showed a significant reduction of hemorrhage at 0 and 5 min. In addition, these compounds inhibited myotoxicity and edema-forming activity of Batx-I at 600 μM concentration. Molecular docking studies suggested that these compounds could occupy part of the substrate binding cleft of the enzyme affecting its catalytic cycle. In this manner, triterpenic acids are candidates for the development of inhibitors for the prevention of local tissue damage in snakebite envenomation.

The potential of aqueous extracts of Bellucia dichotoma Cogn. (Melastomataceae) to inhibit the biological activities of Bothrops atrox venom: A comparison of specimens collected in the states of Pará and Amazonas, Brazil

Ethnopharmacological importanceThe effectiveness of aqueous extract of Bellucia dichotoma Cogn. (Melastomataceae) specimems collected in Santarém, PA, against some biological activities of Bothrops atrox venom (BaV) has been scientifically proven. Here, we analyzed the components and assessed the anti-snakebite potential of aqueous extracts of bark of B. dichotoma collected in Manaus, AM, (AEBd-MAO) and Santarém, PA, (AEBd-STM), both in Brazil. Materials and methodsThe phytochemical profiles of the aqueous extracts were identified using thin layer chromatography (TLC), and the concentrations of phenolics were determined by colorimetric assay. The inhibitory potential of the extracts was tested against the phospholipase A2, coagulant and gelatinolytic activities of BaV in vitro and its defibrinating and edema-inducing activities in vivo. Interaction between BaV and the extracts was investigated using SDS-Page electrophoresis and Western blotting. Extract cytotoxicity and antioxidant potential were assessed using the human fibroblast cell line MRC-5 and the DPPH assay in cell culture, respectively. ResultsWhile there was no difference between the phytochemical profiles of the extracts, AEBd-MAO had higher concentrations of total phenolics, total tannins and hydrolysable tannins. The extracts inhibited 100% of the phospholipase and coagulant activity of BaV when pre-incubated. Without pre-incubation, however, there was no reduction in phospholipase activity, although significant inhibition of coagulant activity was observed. In the doses used in folk medicine, without pre-incubation, both extracts inhibited 100% of the coagulant activity of BaV. In vivo, the extracts were unable to inhibit the defibrinating activity of the venom but were effective in inhibiting its edema-inducing activity. In the profiles of the extracts pre-incubated with BaV, not all the protein bands revealed by SDS-PAGE and Western blot were observed. Both extracts had a high antioxidant potential and neither had a cytotoxic effect. ConclusionAlthough the concentrations of phenolics in each extract were different, the anti-snakebite potential was similar for the concentrations of extract tested. Our findings are of importance for the quality control of this raw material, which, once tested in accordance with Brazilian National Health Surveillance Agency recommendations, may be suitable for use as a phytomedicine to complement treatment of the local effects induced by Bothrops venoms.

Inhibitory effect of pinostrobin from Renealmia alpinia, on the enzymatic and biological activities of a PLA2

Pinostrobin is a flavanone isolated from Renealmia alpinia, a plant used in folk medicine to treat snakebites. We tested the inhibitory ability of pinostrobin on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The compound displayed IC50 values of 1.76mM and 1.85mM (95% Confidence intervals: 1.34–2.18 and 1.21–2.45) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. When mice were injected with PLA2 preincubated with 0.4, 2.0 and 4.0mM of pinostrobin, myotoxic activity induced by the PLA2 was inhibited up to 87%. Nevertheless, these values decreased up to 56% when the pinostrobin was injected into muscle after PLA2. Pinostrobin inhibited edema-forming and anticoagulant activities of the PLA2. In order to have insights on the mode of action of pinostrobin, intrinsic fluorescence and ultraviolet studies were performed. Results suggest that pinostrobin interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that pinostrobin forms hydrogen bonds with residues His48 and Asp49 of PLA2, besides, a π-π stacking interactions with those of residues Phe5 and Trp31, and rings C of flavanone and Tyr52 of the toxin.

The biflavonoid morelloflavone inhibits the enzymatic and biological activities of a snake venom phospholipase A2

The biflavonoid morelloflavone has been reported as inhibitor of secretory PLA2s (phospholipases A2 from human synovial and bee venom sources); however, its capacity to interact and inhibit snake venom PLA2 activities has not been described. In this work we tested the inhibitory ability of morelloflavone on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The biflavonoid displayed IC50 values of 0.48mM (95% Confidence intervals: 0.45–0.51) and 0.38mM (95% Confidence intervals: 0.36–0.40) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. In addition, morelloflavone inhibited in a time-dependent manner and irreversibly the PLA2 enzymatic activity. When mice were injected with PLA2 preincubated (preincubation assay) with 0.13, 0.63 and 1.26mM of the biflavonoid, the myotoxic activity induced by the PLA2 was inhibited up to 63%. Nevertheless, these values decreased up to 38% when the morelloflavone was injected into muscle after PLA2. Moreover, morelloflavone inhibited, in a concentration-dependent manner, edema-forming activity of the PLA2 in the footpad. Morelloflavone also inhibited the anticoagulant activities of the PLA2 in concentration-dependent mode. In order to have insights on the mode of action of morelloflavone, intrinsic fluorescence studies were performed. Results of these assays suggest that morelloflavone interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that morelloflavone forms hydrogen bonds with residues Gly33, Asp49, Gly53 and Thr68 of the enzyme. In addition, our results suggested a π–π stacking interaction between rings A of morelloflavone with that of the residue Tyr52, and Van der Waals interactions with Gly32, His48 and Ala56. Our molecular modeling results suggest that morelloflavone may occupy part of substrate binding cleft of the PLA2. Morelloflavone is a candidate for the development of inhibitors to be used in snakebite envenomation.

Alkylation of histidine residues of Bothrops jararacussu venom proteins and isolated phospholipases A2: a biotechnological tool to improve the production of antibodies.

Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

Comparison of venom composition and biological activities of the subspecies Crotalus lepidus lepidus, Crotalus lepidus klauberi and Crotalus lepidus morulus from Mexico

The rock rattlesnakes Crotalus lepidus comprise a group (lepidus, klauberi, morulus and maculosus) of poorly known mountain cold-tolerant snakes in Mexico. In particular, Crotalus lepidus morulus is a snake endemic of the northeast of Mexico, whereas Crotalus lepidus klauberi and C. l. lepidus are distributed in some regions of the north and central Mexico and southern U. S. Until now very little data are available from C. lepidus subspecies from Mexico, as the terrain inhabited by these snakes is generally steep and rugged. In this work, we have determined some biochemical and biological properties of C. l. morulus, C. l. klauberi and C. l. lepidus crude venoms. Some minor differences in venoms were noted in SDS-PAGE, HPLC profile and MALDI-TOF mass spectrometry analysis. Partial sequences of metalloproteinases, phospholipases A2 (PLA2) and galactose-specific lectins were identified in the venoms. Venoms of C. l. klauberi and C. l. lepidus had significantly higher hemorrhagic and lethal activities than C. l. morulus venom. Proteolytic activity in azocasein was higher in C. l. morulus venom, whereas gelatin hydrolysis was higher in C. l. klauberi. Fibrinogenolytic and PLA2 activities were very similar in all venoms tested. The histological observations in the gastrocnemius muscle damaged by venoms from all the subspecies confirmed myonecrotic and hemorrhagic activities (at 3 and 24 h), which resulted in a poor regenerative response after 14 days. However, C. l. lepidus and C. l. klauberi venom induced a higher increase in the plasma activity of creatine kinase (CK), evidencing higher myotoxicity, whereas paw edema-inducing activity was higher in C. l. lepidus venom. The results indicate that the venoms from the three subspecies have similar protein profiles in electrophoresis, HPLC and molecular weight determinations. However, differences were found in the biological activities in mice. Notably, the venoms of C. l. lepidus and C. l. klauberi present higher toxicity (lower LD50) and hemorrhagic activity than C. l. morulus venom.

Glycolic acid inhibits enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P–I metalloproteinase from Bothrops asper snake venom: Insights from docking and molecular modeling

Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P–I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC50 of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV–vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn2+ ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn2+, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation.

Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Abstract Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.

Egyptian horned viper Cerastes cerastes venom hyaluronidase: Purification, partial characterization and evidence for its action as a spreading factor

Novel Hyaluronidase CcHaseII (33 kDa) of the most dangerous horned viper Cerastes cerastes (Cc) was purified and partial characterized in a set of biochemical assays. CcHaseII was purified by applying a protocol of two successive chromatographic steps; gel filtration on a Sephacryl S-200 and cation exchange chromatography on CM-Sepharose columns. It has specific activity 4000 units/mg protein against 154 units/mg protein for the whole venom with 26-purification fold. The enzymatic activity of the purified Hyaluronidase stimulated by Na+ and inhibited by entire tested cations, metalloproteinase inhibitors and heparin. CcHaseII (5–10 μg) enhanced one hundred percent of hemorrhagic activity of the potent purified hemorrhagic SVMP of corresponding venom (CcHTI) and enhanced edema-inducing activity of Cc venom in a dose-dependent manner. Furthermore, the described purification procedure allows simple preparation of appreciable quantities of the CcHaseII for further studies. Eventually, exploration of snake venom antigenic parts is the most crucial factor for establishing good immunogens and specific diagnostic reagents.