Purification and characterization of an anti-hemorrhagic protein from Naja naja (Indian cobra) venom.

Snake venom Kunitz-type proteins are well known to inhibit serine proteases but a few studies have also shown matrix metalloproteases (MMPs) inhibition. In view of the fact that MMPs and snake venom metalloproteases (SVMPs) have similar catalytic site, inhibition of SVMP activity by Kunitz-type proteins remains to be studied. Recent proteomic studies of Naja naja (N. naja) venom revealed the abundance of Kunitz-type proteins. In this regard, present study aimed at purification of a protease inhibitor from N. naja venom that inhibits the toxicity of SVMPs rich Echis carinatus (E. carinatus) venom. N. naja venom effectively inhibited E. carinatus venom-induced hemorrhage. Purification of the active principle responsible for anti-hemorrhagic effect was achieved by fractionation of N. naja venom in three successive chromatographic steps. SDS-PAGE revealed that purified anti-hemorrhagic protein (NNAh) has an apparent molecular mass of ∼44 kDa and single peak in RP-HPLC demonstrated its homogeneity. NNAh also inhibited myonecrosis induced by E. carinatus venom and reduced activity of creatine kinase in NNAh treated animal sera substantiated the anti-myonecrotic effect. Hemorrhage and myonecrosis inhibitory effects of NNAh were further supported by inhibition of E. carinatus venom-mediated gelatinolysis and collagenolysis. NNAh falls into the category of Kunitz-type serine protease inhibitor as determined by peptide mass fingerprinting and shown to be a strong inhibitor of chymotrypsin. Collectively our data signify that NNAh is a Kunitz-type chymotrypsin inhibitor which also inhibited metalloprotease activities of E. carinatus venom. In future, complete sequence of NNAh and peptide region(s) responsible for inhibition will assist to deduce the mechanism of action.

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.

Differential action of medically important Indian BIG FOUR snake venoms on rodent blood coagulation.

Snakebite is a global health problem affecting millions of people. According to WHO, India has the highest mortality and/or morbidity due to snakebite. In spite of commendable research on Indian BIG FOUR venomous species; Naja naja and Bungarus caeruleus (elapid); Daboia russelii and Echis carinatus (viperid), no significant progress has been achieved in terms of diagnosis and management of biting species with appropriate anti-snake venom. Major hurdle is identification of offending species. Present study aims at differentiation of Indian BIG FOUR snake venoms based on their distinguish action on rodent blood coagulation. Assessment of coagulation alterations by elapid venoms showed negligible effect on re-calcification time, prothrombin time, activated partial thromboplastin time and factors assay (I, II, V, VIII and X) both in vitro and in vivo. However, viperid venoms demonstrated significant anticoagulant status due to their remarkable fibrinogen degradation potentials as supported by fibrinogenolytic activity, fibrinogen zymography and rotational thromboelastometry. Though results provide hint on probable alterations of Indian BIG FOUR snake venoms on blood coagulation, the study however needs validation from human victim's samples to ascertain its reliability for identification of biting snake species.

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.

Topical application of serine proteases from Wrightia tinctoria R. Br. (Apocyanaceae) latex augments healing of experimentally induced excision wound in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Wrightia tinctoria R. Br. (Apocyanaceae) is a folk medicinal plant known to have immunomodulatory, anti-inflammatory and antihemorrhagic potential. Wrightia tinctoria latex is used for treatment of various clinical conditions including psoriasis, blisters, mouth ulcers, and extensively for topical application on fresh wounds to promote accelerated healing. AIMS OF THE STUDY: To investigate the wound healing potential of Wrightia tinctoria latex proteases using a mouse model. MATERIALS AND METHODS: Proteolytic activity of Wrightia tinctoria latex proteases (WTLP) was determined on various substrates (casein, gelatin and collagen (type-I and IV)). The thermal stability and the class of proteases present in WTLP were determined using heat treatment and specific protease inhibitors, respectively. Excision wound model in mice was used to evaluate the healing potential of WTLP application (twice daily, 10mg/kg). Neosporin, a standard drug, was used for comparison. The progression of healing was monitored using physical (wound contraction), biochemical (collagen content, catalase and MMP activity) and histological examinations. RESULTS: WTLP contains thermostable serine proteases, which are completely inhibited by PMSF. WTLP showed strong caseinolytic, gelatinolytic and collagenolytic activity. The excision wound healing rate upon WTLP treatment was significantly higher than (>2-fold) the control group (49% vs. 18%, (**)p<0.01) on day 3 and throughout the study. PMSF pre-treated and heat denatured WTLP failed to promote wound healing. In addition, serial biochemical analysis of the granulation tissue demonstrated 1.5-fold more (2444 ± 100 vs. 1579 ± 121 µg/100mg tissue) hydroxyproline content and 5.6-fold higher catalase activity (16.7 ± 1.3 vs. 3 ± 0.3 units/mg) compared to controls. Further, the enhanced collagen content and matrix metalloproteinase activity correlated with wound contraction rate following WTLP and Neosporin treatment. Histological analysis on day 9 confirmed complete epithelialization, re-establishment of skin structure and accelerated wound healing following WTLP treatment. CONCLUSIONS: The thermostable serine proteases of Wrightia tinctoria latex are directly involved in the wound healing process. Our findings provide a biochemical basis for the role of WTLP in the enhancement of wound healing. The study supports traditional topical application of Wrightia tinctoria latex on fresh wounds to promote accelerated healing.