Albizia lebbeck seed methanolic extract as a complementary therapy to manage local toxicity of Echis carinatus venom in a murine model.

CONTEXT AND OBJECTIVE: Viperid venom-induced chronic local-toxicity continues even after anti-snake venom treatment. Therefore, traditional antidote Albizia lebbeck L. (Fabaceae) seed extract was tested against Echis carinatus S. (Viperidae) venom (ECV)-induced local toxicity to evaluate its complementary remedy. MATERIALS AND METHODS: Soxhlet extraction of A. lebbeck seeds was performed with the increasing polarity of solvents (n-hexane to water); the extract was screened for phytochemicals (alkaloids, anthraquinones, flavonoids, glycosides, phenolics, saponins, steroids and tannins). In preliminary in vitro analysis, A. lebbeck methanolic extract (ALME) demonstrated significant inhibition of ECV proteases, the major enzyme-toxin responsible for local- toxicity. Therefore, in vitro neutralizing potential of ALME was further evaluated against hyaluronidases and phospholipase A2 (1:1-1:100 w/w). In addition, alleviation of ECV induced characteristic local- toxicity [haemorrhage (i.d.) and myotoxicity (i.m.)] was determined in mice. RESULTS: ALME contained high concentrations of phenolics and flavonoids and demonstrated significant in vitro inhibition of ECV protease (IC50 = 36.32 µg, p < 0.0001) and hyaluronidase (IC50 = 91.95 µg, p < 0.0001) at 1:100 w/w. ALME significantly neutralized ECV induced haemorrhage (ED50 = 26.37 µg, p < 0.0001) and myotoxicity by significantly reducing serum creatinine kinase (ED50 = 37.5 µg, p < 0.0001) and lactate dehydrogenase (ED50 = 31.44 µg, p = 0.0021) levels at 1:50 w/w. DISCUSSION AND CONCLUSION: ALME demonstrated significant neutralization of ECV enzymes that contribute in local tissue damage and haemostatic alterations. The study scientifically supports the anecdotal use of A. lebbeck in complementary medicine and identifies ALME as principle fraction responsible for antivenom properties.

Virtual analysis of structurally diverse synthetic analogs as inhibitors of snake venom secretory phospholipase A2.

Due to the toxic pathophysiological role of snake venom phospholipase A2 (PLA2 ), its compelling limitations to anti-venom therapy in humans and the need for alternative therapy foster considerable pharmacological interest towards search of PLA2 specific inhibitors. In this study, an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on VRV-PL-V (Vipera russellii venom phospholipase A2 fraction-V) belonging to Group II-B secretory PLA2 from Daboia russelli pulchella is carried out in order to study the structure-based inhibitor design. The accuracy of the model was validated using multiple computational approaches. The molecular docking study of this protein was undertaken using different classes of experimentally proven, structurally diverse synthetic inhibitors of secretory PLA2 whose selection is based on IC50 value that ranges from 25 µM to 100 µM. Estimation of protein-ligand contacts by docking analysis sheds light on the importance of His 47 and Asp 48 within the VRV-PL-V binding pocket as key residue for hydrogen bond interaction with ligands. Our virtual analysis revealed that compounds with different scaffold binds to the same active site region. ADME analysis was also further performed to filter and identify the best potential specific inhibitor against VRV-PL-V. Additionally, the e-pharmacophore was generated for the best potential specific inhibitor against VRV-PL-V and reported here. The present study should therefore play a guiding role in the experimental design of VRV-PL-V inhibitors that may provide better therapeutic molecular models for PLA2 recognition and anti-ophidian activity.

Evidence for existence of venom 5' nucleotidase in multiple forms through inhibition of concanavalin A.

Pharmacologically active 5' nucleotidase is a ubiquitously distributed enzyme in snake venoms. In this study the effect of concanavalin A (Con-A) on different snake venoms 5' nucleotidase activity is tested in order to know the protein nature which will ultimately help in purification of the enzyme with high yield. Con-A inhibited Naja naja, Naja kauthia, Naja melanoleuca, Naja naja sputatrix, Agistrodon halys blomhoffii, Bothrops asper and Oxyranus scutellas venom 5' nucleotidase activity at different concentrations. This indicates the presence of glycopart in the protein, thus glycoprotein in nature. Vipera russellii, Vipera plaestenae, Agistrodon contratrix, Bitis orientis, Echis carinatus and Trimeresures malabaricus was not inhibited by Con-A, indicating absence of glycopart in the protein. This study for the first time shows existence of 5' nucleotidase in multimeric forms.

The anti-snake venom properties of Tamarindus indica (leguminosae) seed extract.

In Indian traditional medicine, various plants have been used widely as a remedy for treating snake bites. The aim of this study was to evaluate the effect of Tamarindus indica seed extract on the pharmacological as well as the enzymatic effects induced by V. russelli venom. Tamarind seed extract inhibited the PLA(2), protease, hyaluronidase, l-amino acid oxidase and 5'-nucleotidase enzyme activities of venom in a dose-dependent manner. These are the major hydrolytic enzymes responsible for the early effects of envenomation, such as local tissue damage, inflammation and hypotension. Furthermore, the extract neutralized the degradation of the Bbeta chain of human fibrinogen and indirect hemolysis caused by venom. It was also observed that the extract exerted a moderate effect on the clotting time, prolonging it only to a small extent. Edema, hemorrhage and myotoxic effects including lethality, induced by venom were neutralized significantly when different doses of the extract were preincubated with venom before the assays. On the other hand, animals that received extract 10 min after the injection of venom were protected from venom induced toxicity. Since it inhibits hydrolytic enzymes and pharmacological effects, it may be used as an alternative treatment to serum therapy and, in addition, as a rich source of potential inhibitors of PLA(2), metalloproteinases, serine proteases, hyaluronidases and 5 cent-nucleotidases, the enzymes involved in several physiopathological human and animal diseases.

Strong myotoxic activity of Trimeresurus malabaricus venom: role of metalloproteases.

Trimeresurus malabaricus is an endemic snake found in the Southern region of Western Ghats section of India along with the more widely distributed species like Naja naja and Daboia russelii. T. malabaricus venom is not lethal when injected (i.p.) up to 20 mg/kg body weight in mice, but causes extensive local tissue degeneration. N. naja and D. russelii are highly toxic (i.p.) with minimum local tissue damage in experimental mice. In this study a comparative analysis of local tissue damage of T. malabaricus venom is made with N. naja and D. russelii snake venoms of the Southern regions of Western Ghats. T. malabaricus venom exhibits caseinolytic activity 16 and 24 times more than N. naja and D. russelii venom. Inhibition studies with specific protease inhibitors reveal that the major proteases belong to metalloproteases. T. malabaricus venom hydrolyses gelatin and induces strong hemorrhagic activity in mice. Both N. naja and D. russelii fail to hydrolyze gelatin even at very high concentration and did not induce any hemorrhagic activity. With D. russelii venom small hemorrhagic spot was observed at the site of injection. The hemorrhagic activity of T. malabaricus venom is completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor. The i.m. injection of T. malabaricus venom causes extensive degradation of muscle tissue within 24 h. The light microscopic observation of muscle tissue showed congestion of blood vessels and hemorrhage at the early stage followed by extensive necrosis of muscle fibers. The elevated levels of serum CK and LDH activity further supported the muscle degeneration. Such pathological symptoms were not seen with N. naja and D. russelii snake venom. The hemorrhagic and the muscle necrosis was completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor strongly suggests that the major toxin component in the T. malabaricus venom is metalloprotease and its activity can be easily neutralized using chelating agents and its use in the first aid as chelation therapy is beneficial.

Isolation and characterization of hyaluronidase a "spreading factor" from Indian cobra (Naja naja) venom.

Hyaluronidase, ubiquitous enzyme in snake venoms, known originally as "spreading factor", has not been well studied. The present study describes the purification and characterization of hyaluronidase from Indian cobra (Naja naja) venom and provides systematic evaluation of the spreading property of the enzyme. Hyaluronidase (NNH1) has been purified through gel permeation and ion exchange chromatography. The molecular mass was found to be 70.406 kDa by MALDI-TOF mass spectrometry and with the (p)i pI of 9.2. The amino acid sequence of the N-terminus was found to be NEQSTHGAYV. The enzyme shows absolute specificity for hyaluronan and belongs to the group of neutral active enzymes. Tetrasaccharides are the final product of hyaluronan digestion. The enzyme cleaves beta 1,4-glycosidic linkage and belongs to a group of endo-beta-N-acetyl hexosaminidases. Hyaluronidase indirectly potentiates the myotoxicity of VRV-PL-VIII, a phospholipolytic myotoxin, and also the hemorrhagic potency of a hemorrhagic complex-I. Localization of hyaluronan in human skin section and selective degradation by venom hyaluronidase (NNH1) corroborate the plausible in vivo degradation of hyaluronan in the extracellular matrix (ECM) resulting in easy dissemination of VRV-PL-VIII myotoxin and hemorrhagic complex-I.

Crystal structures of the free and anisic acid bound triple mutant of phospholipase A2.

Phospholipase A2 catalyses the hydrolysis of the ester bond of 3-sn-phosphoglycerides. Here, we report the crystal structures of the free and anisic acid-bound triple mutant (K53,56,120M) of bovine pancreatic phospholipase A2. In the bound triple mutant structure, the small organic molecule p-anisic acid is found in the active site, and one of the carboxylate oxygen atoms is coordinated to the functionally important primary calcium ion. The other carboxylate oxygen atom is hydrogen bonded to the phenolic hydroxyl group of Tyr69. In addition, the bound anisic acid molecule replaces one of the functionally important water molecules in the active site. The residues 60-70, which are in a loop (surface loop), are disordered in most of the bovine pancreatic phospholipase A2 structures. It is interesting to note that these residues are ordered in the bound triple mutant structure but are disordered in the free triple mutant structure. The organic crystallization ingredient 2-methyl-2,4-pentanediol is found near the active site of the free triple mutant structure. The overall tertiary folding and stereochemical parameters for the final models of the free and anisic acid-bound triple mutant are virtually identical.

Isolation and characterization of a novel postsynaptic/cytotoxic neurotoxin from Daboia russelli russelli venom.

A postsynaptic neurotoxin was purified from Daboia russelli russelli venom using gel filtration, ion-exchange chromatography and reverse-phase high-performance liquid chromatography. The N-terminal sequence, molecular mass and pharmacological activities of the neurotoxin/cytotoxin indicate that it is a short-chain neurotoxin like that found in Elapid venom. This is the first report on the presence of such a postsynaptic neurotoxin from D. r. russelli venom.

Role of catalytic function in the antiplatelet activity of phospholipase A2 cobra (Naja naja naja) venom.

Three acidic phospholipases A2 from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 microM). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A2 with platelet rich plasma. Parabromophenacyl bromide modified PLA2 enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity.

Purification and characterization of three acidic, cytotoxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Three acidic phospholipases A2 (NN-I2c-PLA2, NN-I2d-PLA2 and NN-I2c-PLA2) were purified by successive chromatography of Indian cobra (Naja naja naja) venom on CM-Sephadex C-25, Sephadex G-50 and QAE Sephadex A-25 columns. They have molecular weights of 13,000-14,500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis. They showed tryptophan specific fluorescence emission spectra (approximately 345 nm). All the three phospholipases A2 were enzymatically highly active with specific activities 9-17 micromol min(-1) mg(-1). They were non-lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight. They induced edema in mouse foot pads and were cytotoxic to Ehrlich ascites tumour cells. They did not exhibit direct haemolytic and anticoagulant activities.

Isolation and characterization of an endogenous inhibitor of phospholipase A2 from Indian cobra (Naja naja naja) venom.

A PLA2-inhibitor has been purified from Indian cobra (Naja naja naja) venom by the combination of ion-exchange and gel-filtration chromatography. The inhibitor, NN-I3 was a peptide with mol.wt 6500 and has a fluorescence emission maxima ca 340 nm. NN-I3 specifically inhibited the enzyme activity of the three acidic PLA2 from the same venom. The inhibition of NN-I2d-PLA2 and NN-I2c-PLA2 by NN-I3 was of mixed type and NN-I2c-PLA2 was of uncompetitive type. Neither the inhibitor nor the individual mixtures of acidic PLA2 with the inhibitor (1:1 w/w or 1:2 mol:mol) were lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight.

A platelet aggregation inhibitor phospholipase A2 from Russell's viper (Vipera russelli) venom: isolation and characterization.

Vipera russelli venom is reported to contain multiple forms of phospholipase A2 (PLA2). Three PLA2s (VRV-PL-V, VRV-PL-VI and VRV-PL-VIIIa) have been purified and characterized in this laboratory. A PLA2(VRV-PL-IIIb) inhibiting platelet aggregation was purified from the same venom in two steps involving CM-Sephadex C-25 column chromatography followed by gel filtration on Sephadex G-50. VRV-PL-IIIb is a basic (pI 7.3-7.7) isoenzyme, with a mol. wt between 14,000 and 15,000. It induced neurotoxic symptoms in experimental mice with an i.p. LD50 of 5.2 mg/kg body weight. VRV-PL-IIIb inhibits ADP-induced platelet aggregation in a dose-dependent manner. It induced oedema in the foot pads of mice and is devoid of anticoagulant, myotoxic and direct haemolytic activities. Antibodies to VRV-PL-IIIb showed a single precipitin line against the antigen as well as whole venom.

Effects of chlorpromazine on the enzymatic and toxic properties of the most basic phospholipase A2 from Vipera russelli snake venom.

The effect of chlorpromazine on various biological activities of phospholipase A2 VRV-PL-VIIIa from Vipera russelli snake venom was investigated. The drug inhibited the in vitro phospholipase A2 activity of the enzyme by 55%. The ID50 was found to be 1.15 microM. Increasing substrate concentration relieved the inhibition of phospholipase A2 activity by the drug indicating probable interaction with the substrate. The drug totally quenched the fluorescence intensity of the enzyme. Chlorpromazine increased the LD50 of the enzyme by 1.6 fold. The drug also inhibited hemolytic and anticoagulant potencies but failed to inhibit edema inducing activity and myotoxicity of the enzyme.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian cobra) venom.

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue.

Monoclonal antibodies to VRV-PL-VIIIa, a basic multitoxic phospholipase A2 from Vipera russelli venom.

VRV-PL-VIIIa, the most basic phospholipase A2 (PLA2) from the venom of Vipera russelli, induces multiple toxic effects, including neurotoxicity, myotoxicity, edema and hemorrhage. Rabbit polyclonal anti-serum was raised against VRV-PL-VIIIa. The antiserum cross-reacted in enzyme-linked immunosorbant assay (ELISA) with two other PLA2 from the same venom, VRV-PL-V and VRV-PL-VI, and with ammodytoxin A, caudoxin and crotoxin. Twenty-two hybridoma cell lines secreting monoclonal antibodies against VRV-PL-VIIIa were isolated. The monoclonal antibodies exhibited apparent binding affinities in ELISA with VRV-PL-VIIIa ranging over two orders of magnitude. Most of the monoclonal antibodies cross-reacted moderately with VRV-PL-V and weakly with VRV-PL-VI. None of the antibodies cross-reacted with ammodytoxin, caudoxin or crotoxin. Reducing the disulfide bonds of VRV-PL-VIIIa lowered the ELISA signals of each monoclonal antibody to nonspecific levels, suggesting that all the antibodies recognize conformational epitopes. Four of the 22 antibodies neutralized the enzymatic activity of VRV-PL-VIIIa. Interestingly, two of the four exhibited the lowest affinities of the monoclonal antibody library for VRV-PL-VIIIa in ELISA, while the other two exhibited the highest. Each of the monoclonal antibodies was biotinylated and spatial binding relationships were evaluated by competition ELISA.

Dissociation of enzymatic activity from toxic properties of the most basic phospholipase A2 from Vipera russelli snake venom by guanidination of lysine residues.

The most basic phospholipase A2 VRV-PL-VIIIa purified from Russell's viper venom is a toxic enzyme. It induced neurotoxicity, myotoxicity, and oedema and was lethal to mice at 5.3 micrograms/g body weight. It also inhibited the coagulation of the human plasma. The epsilon-amino groups of lysine residues of the toxic enzyme VRV-PL-VIIIa were guanidinated with o-methylisourea. Guanidination of the enzyme did not alter the enzymatic activity markedly. The guanidinated enzyme became non-lethal in doses up to 16 micrograms/g body weight, and failed to elicit neurotoxic symptoms in experimental animals and oedema in the foot pads of mice. Also, its myotoxic and anticoagulant potencies were decreased significantly.

Effects of myonecrotic snake venom phospholipase A2 toxins on cultured muscle cells.

We have attempted to establish a cell culture model suitable for molecular mechanism of action studies of necrotic phospholipases A2 (PLA2). Three myonecrotic PLA2 were purified, one basic PLA2 from Naja nigricollis venom and two basic PLA2 (VRV-PL-V and VRV-PL-VIIIa) from Vipera russelli venom. The effects of these PLA2 on several established muscle cell lines were evaluated. As judged by light microscopy, some, but not all, cell lines detached from the culture plate in a time- and concentration-related fashion. Naja nigricollis PLA2 was the most potent at eliciting this effect, followed by VRV-PL-V and VRV-PL-VIIIa. The two most sensitive cell lines, 1447 and 1456, were chosen for further study using N. nigricollis PLA2. Cellular protein and nucleic acid syntheses were inhibited by the toxin in a time- and dose-related manner. However, it appeared that most, if not all, of the inhibition was due to toxin-induced reduction of precursor uptake, suggesting effects at the plasma membrane level. The putative membrane effects were specific, in that uptake of calcium, choline or glucose was not inhibited by the toxin. Moreover, treating the cells with toxin failed to significantly increase lactate dehydrogenase release into the medium. Polyclonal antiserum prepared against N. nigricollis basic PLA2 neutralized the toxicity completely with 1456 cells, but only partially with the 1447 cell line. Both the 1447 and 1456 lines appear to be suitable as cell culture models for necrotizing PLA2 molecular mechanism of action studies.

Immunochemical cross-reactivity of neurotoxic phospholipase A2 enzymes from Indian cobra (Naja naja naja) venom using polyclonal antibodies.

Rabbit antibodies were prepared against purified phospholipase A2 (NN-XIa-PLA2) from Indian cobra (Naja naja naja) venom. The PLA2 has haemolytic, neurotoxic, myotoxic, cytotoxic and oedema-inducing activities apart from the catalytic activity. The immunological cross-reactivity of structurally similar neurotoxic PLA2s was investigated using enzyme-linked immunosorbent assay (ELISA) and immunodiffusion. Anti-NN-XIa-PLA2 IgG cross-reacted with other purified neurotoxic PLA2s from the same venom. Immunochemical cross-reactions of anti-NN-XIa-PLA2 IgG with NN-XIa-PLA2, NN-XIb-PLA2, NN-XIII-PLA2, NN-IVb1-PLA2 and NN-Vb-PLA2 were shown by a very high ELISA titre and a single precipitin band on double immunodiffusion agarose plates. The catalytic activity of these PLA2s was inhibited dose-dependently by anti-NN-XIa-PLA2 IgG but was unable to neutralize lethality and neurotoxic symptoms in experimental animals injected with neurotoxic PLA2. Anti-NN-XIa-PLA2 IgG fails to neutralize myotoxicity and oedema-inducing activities of NN-XIa-PLA2 and NN-XIII-PLA2. Anti-NN-XIa-PLA2 IgG inhibited cytotoxic effects of NN-XIa-PLA2 dose-dependently, but failed to inhibit NN-XIII-PLA2-induced cytotoxicity. Direct haemolytic activity of NN-XIa-PLA2 and NN-XIII-PLA2 was inhibited dose-dependently by these antibodies. The results indicate the presence of separate catalytic and pharmacologic site(s).

Comparative characterization of two toxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Indian cobra venom contains many phospholipase A2 (PLA2) toxins. In the present study two toxic PLA2s have been purified from the Indian cobra (Naja naja naja) venom by column chromatography. The NN-XIa-and NN-XIb-PLA2s have mol. wts between 10,700 and 15,000. The NN-XIa-PLA2 induces myotoxic effects, oedema and neurotoxicity in mice and has an i.p. LD50 of 8.5 mg/kg body weight. The NN-XIa-PLA2 is also cytotoxic to Ehrlich ascites tumour cells. The other PLA2, NN-XIb, in contrast has an i.p. LD50 of 0.22 mg/kg body weight, and it induces acute neurotoxicity. The NN-XIb-PLA2 is devoid of the other biological activities which are exhibited by NN-XIa-PLA2.

Analysis of Vipera russelli venom using polyclonal antibodies prepared against its purified toxic phospholipase A2 VRV PL-V.

Vipera russelli venom induces predominantly neurotoxic, myotoxic necrotic and hemorrhagic symptoms in experimental animals and has several hydrolytic enzyme activities. In this study, V. russelli venom is characterized both as a PLA2 and as a toxin. Anti PL-V Ig (antibodies to a toxic phospholipase A2 VRV PL-V of V. russelli venom) nullifies the toxicity of whole V. russelli venom to a great extent. The neurotoxic symptoms vanish completely in the presence of anti PL-V Ig. The cross reacting components of whole V. russelli venom were removed by precipitating them from whole venom by the addition of anti PL-V Ig. The non-cross reacting components present in the supernatant were checked for toxicity. There was a significant reduction in toxicity. The LD50 value of the supernatant had increased from 4.1 mg/kg body weight to 11.7 mg/kg body weight and it showed about 34% of the total venom phospholipase A2 activity. It had edema forming, hemorrhagic and hemolytic activity but failed to induce neurotoxic, anticoagulant and myotoxic effects.