Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice.

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed-phase high-performance liquid chromatography (RP-HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity.

Effects of photobiomodulation therapy on Bothrops moojeni snake-envenomed gastrocnemius of mice using enzymatic biomarkers.

Bothropic venom contains a range of biologically active substances capable of causing severe local and systemic envenoming symptomatology within its victims. The snake anti-venom is effective against systemic effects but has no neutralizing effect against the fast developing local effects. Herein, mice gastrocnemius injected with Bothrops moojeni venom (40 µg/kg) or saline solution were irradiated with HeNe (632.8 nm) and GaAs (904 nm) lasers (daily energy density of 4 J/cm2; 0.03/0.21 power density; 0.07/0.16 spot size; 1.2/0.04 total energy, 1 cm off contact, for HeNe and GaAs lasers, respectively) and euthanized in periods ranging from 3 h to 21 days. Blood biochemistry for creatine kinase (CK), alkaline phosphatase (ALP), acid phosphatase (AP), lactate dehydrogenase (LDH), aspartate transaminase (AST), and myoglobin and histopathological analysis, for assessing the degree of myonecrosis and regeneration of gastrocnemius, were done at every time interval. GaAs laser promoted faster photobiomodulation therapy (PBMT) effects, and the GaAs group exhibited a better clinical outcome than the HeNe group. Within the GaAs group, the serum levels of CK, LDH, AP, AST, and myoglobin, which were increased by the physiological effects of the venom, were reduced to initial baseline before snake envenomation in less time than those irradiated by the HeNe laser. However, the group receiving irradiation from the HeNe laser returned the levels of ALP activity to baseline faster than those of the GaAs group. Histopathological analysis revealed enhanced muscle regeneration in mice groups treated with both lasers. PBM promoted by GaAs and HeNe showed well-developed centrally nucleate regenerating cells and an increased number of newly formed blood vessels when compared to unirradiated muscle. We therefore suggest that GaAs had the best outcomes likely derived from a deeper penetrating longer wavelength. We conclude that PMBT is a promising, non-invasive approach to be further tested in pre-clinical studies with a goal to further its clinical use in skeletal muscle recovery in snakebite victims.

Pharmacological activity of Costus spicatus in experimental Bothrops atrox envenomation.

CONTEXT: Medicinal plants encompass a rich source of active compounds that can neutralize snake venoms or toxins. Costus spicatus (Jacq.) Sw. (Costaceae) is used by the Amazonian population to treat inflammation, pain and other pathological manifestations. OBJECTIVE: To evaluate the influence of C. spicatus aqueous extract on edema, peritonitis, nociception, coagulation, haemorrhage and indirect haemolytic activity induced by Bothrops atrox venom (BAV). MATERIALS AND METHODS: Dried and pulverized leaves were extracted with distilled water. Envenoming was induced by administration of B. atrox snake venom in Swiss Webster mice. The experimental groups consisted of BAV (at the minimum dose to induce measurable biological responses) and C. spicatus extract (CSE, 1.25, 2.5, 5.0, 7.5 and 10 mg/kg/25 µl phosphate-buffered saline) administered individually and in combination (BAVCSE). PBS was used as a control. In vitro assays were also conducted in order to evaluate phospholipase A2 coagulant activities (indirect haemolytic method). RESULTS: CSE significantly reduced the venom-induced edema and nociception at all concentrations tested and inhibited migration of inflammatory cells at the three least concentrations (5.0, 7.5 and 10 mg/kg/25 µl PBS). CSE was not effective in inhibiting coagulant, haemorrhagic and indirect haemolytic activities of the venom. DISCUSSION AND CONCLUSION: The data suggest that CSE could exhibit a central mechanism for pain inhibition, and may also inhibit prostaglandin synthesis. These findings corroborate the traditional administration of C. spicatus decoction to treat inflammatory disorders, including those caused by B. atrox envenomation.

Naja naja karachiensis envenomation: biochemical parameters for cardiac, liver, and renal damage along with their neutralization by medicinal plants.

Naja naja karachiensis envenomation was found to hit more drastically heart, liver, and kidneys. 400 µg/kg of venom-raised moderate serum levels of ALT (72 ± 4.70 U/L, 0.1 > P > 0.05), AST (157 ± 24.24 U/L, 0.1 > P > 0.05), urea (42 ± 3.08 mg/dL, 0.05 > P > 0.02), creatinine (1.74 ± 0.03 mg/dL, 0.01 > P > 0.001), CK-MB (21 ± 1.5 U/L, 0.05 > P > 0.02), and LDH (2064 ± 15.98 U/L, P < 0.001) were injected in experimental rabbits. However, lethality was enhanced with 800 µg/kg of venom in terms of significant release of ALT (86 ± 5.0 U/L, 0.05 > P > 0.02), AST (251 ± 18.2 U/L, 0.01 > P > 0.001), urea (57.6 ± 3.84 mg/dL, 0.02 > P > 0.01), creatinine (2.1 ± 0.10 mg/dL, 0.02 > P > 0.01), CK-MB (77 ± 11.22 U/L, 0.05 > P > 0.02), and LDH (2562 ± 25.14 U/L, P ≪ 0.001). Among twenty-eight tested medicinal plant extracts, only Stenolobium stans (L.) Seem was found the best antivenom (P > 0.5) compared to the efficacy of standard antidote (ALT = 52.5 ± 3.51 U/L, AST = 69.5 ± 18.55 U/L, urea = 31.5 ± 0.50 mg/dL, creatinine = 1.08 ± 0.02 mg/dL, CK-MB = 09 ± 0.85 U/L, and LDH = 763 ± 6.01 U/L). Other plant extracts were proved less beneficial and partly neutralized the toxicities posed by cobra venom. However, it is essential in future to isolate and characterize bioactive compound(s) from Stenolobium stans (L.) Seem extract to overcome the complications of snake bite.

An assessment of oxidant/antioxidant status in patients with snake envenomation.

OBJECTIVE: The aim of this study is to investigate the antioxidant status (TAS), oxidant status (TOS) and oxidative stress index (OSI) in patients with snake envenomation and to learn more about the pathophysiology of snake envenomation. METHOD: Between May 2009 and October 2010, 47 patients were admitted to our emergency department with snake bites, and as a control group 20 healthy volunteers were enrolled in this study. Serum, plasma, and erythrocyte components were prepared for all patients on admission and at the control after 1 month. Serum TOS/TAS levels were measured. RESULTS: No correlation was observed among age, gender and the levels of TAS, TOS and OSI. TAS, TOS and OSI levels were higher in snake envenomation patients upon arrival at the emergency department than in the healthy control group. Upon admission, all levels of patients with snake envenomation were higher than the control levels taken after 1 month. CONCLUSIONS: Serum TAS, TOS and OSI levels increase in snake envenomation patients. The results obtained in this study indicate that the snake bite was associated with a shift to an oxidative state, and oxidative stress plays an important role in the pathophysiology of snake envenomation.

[Study on clinical efficacy of combination of traditional Chinese medicine and western medicine in treatment of pit viper bites and peripheral blood inflammatory factors].

OBJECTIVE: To observe the clinical efficacy of combination of traditional Chinese medicine and western medicine in the treatment of patient bitten by agkistrodon halys pallas, and the changes in peripheral blood inflammatory factors (hs-PCR, IL-6, TNF-alpha). METHOD: Ninty-eight patients were divided into three groups according to their hospitalization dates: the western medicine group, in which 32 patients were treated with antivenom serum (6 000 U) for five days, once every day; the traditional Chinese medicine group, in which 32 patients were treated with anti pit viper No. 2 concentrated decoction (300 mL), twice to three times every day, for five days; and the combined traditional Chinese medicine and western medicine group, in which 35 patients were treated with the combination of Chinese and Western medicine treatment described above. Then blood samples of all of patients were obtained, and serum factors (hs-PCR, IL-6, TNF-alpha) in peripheral blood were measured by Elisa assay. Another 30 health volunteers were chosen as the normal control group. RESULT: The serum inflammatory factors were significantly higher in all patients of the three groups than that in healthy control before treatment (P < 0.01), and decreased significantly after treatment. In particular, the more remarkable reduction was found in the combined traditional Chinese medicine and western medicine group compared with the western medicine group and the traditional medicine group (P < 0.01). Symptom elimination in the three groups was superior to the western medicine group and the traditional medicine group at the first day and the third day of treatment (P < 0.05, P < 0.01). Total clinical effective rate was 100% in the combined traditional Chinese medicine and western medicine group, 84. 37% in the traditional medicine group and 65.62% in the western medicine group, the clinical effective rate of the combined traditional Chinese medicine and western medicine group was notably superior to that of the western medicine group and the traditional medicine group (P < 0.01). CONCLUSION: The serum inflammatory factors increased significantly in patients bitten by agkistrodon halys pallas. Treatment with the combined traditional Chinese medicine and western medicine can significantly decrease the serum inflammatory factors, and increase clinical effect, with more obvious clinical efficacy compared with the western medicine group and the traditional medicine group.

Vipera russelli venom-induced oxidative stress and hematological alterations: amelioration by crocin a dietary colorant.

Snakebite is a serious medical and socio-economic problem affecting the healthy individuals and agricultural and farming populations worldwide. In India, Vipera russelli snakebite is common, ensuing high morbidity and mortality. The venom components persuade multifactorial stress phenomenon and alter the physiological setting by causing disruption of the blood cells and vital organs. The present study demonstrates the anti-ophidian property of Crocin (Crocus sativus), a potent antioxidant against viper venom-induced oxidative stress. The in vivo oxidative damage induced by venom was clearly evidenced by the increased oxidative stress markers and antioxidant enzymes/molecules along with the proinflammatory cytokines including IL-1ß, TNF-α and IL-6. Furthermore, venom depleted the hemoglobin, hematocrit, mean corpuscular volume and platelet count in experimental animals. Crocin ameliorated the venom-induced oxidative stress, hematological alteration and proinflammatory cytokine levels. At present, administration of antivenom is an effective therapy against systemic toxicity, but it offers no protection against the rapidly spreading oxidative damage and infiltration of pro-inflammatory mediators. These pathologies will continue even after antivenom administration. Hence, a long-term auxiliary therapy is required to treat secondary as well as neglected complications of snakebite.

Efficacy evaluations of Mimosa pudica tannin isolate (MPT) for its anti-ophidian properties.

AIM OF THE STUDY: Evaluations of the anti-snake venom efficacy of Mimosa pudica tannin isolate (MPT) obtained from root of the plant. MATERIALS AND METHOD: MPT was investigated in vitro and in vivo for its efficacy against the venom of Naja kaouthia snake. RESULTS: In vitro: (1) mice injected i.p. with MPT pre-incubated with Naja kaouthia venom at concentrations as low as 0.625 mg/ml showed 100% survival after a 24-h observation period. (2) In the proteomics study, mice injected with MPT pre-incubated with the Naja kaouthia venom showed down-regulation of five serum proteins. (3) In the protein-dye-binding study, the percentage of Bradford dye-protein binding showed a reduction relative to the decrease in MPT concentration used to incubate with the venom. In vivo: the results from the animal studies showed that MPT had no in vivo protection against the Naja kaouthia venom (0.875 mg/kg) in four different rescue modes and in an oral pre-treatment experiment. CONCLUSION: The study indicated the promising ability of MPT to neutralize the Naja kaouthia venom in in vitro experiments but fell short in its in vivo potential. As such, the use of Mimosa pudica (Mimosaceae) as therapeutics for snake bites is questionable as all the possible in vivo rescue studies and pre-treatment of the active constituents showed no protection against the affected mice.

Tumor necrosis factor as a mediator of cardiac toxicity following snake envenomation.

OBJECTIVE: To investigate the possible role of tumor necrosis factor in mediating cardiotoxicity following venom injection in a rat. DESIGN: A randomized controlled experimental study using a Langendorff isolated heart model. SETTING: Animal laboratory. SUBJECTS: Adult male Wistar rats. INTERVENTIONS: The control group (n = 10) was injected with saline only. Each animal in the experimental groups 1-3 (n = 10 each) was injected with Vipera aspis venom 500 microg/kg intramuscularly. Group 1 animals received no additional substance beforehand, group 2 animals were injected intramuscularly with 250 microg of soluble tumor necrosis factor receptor (sTNF-R p55) 15 mins before the venom injection, and group 3 animals were injected intraperitoneally with 40 microg of anti-tumor necrosis factor 60 mins before the venom injection. MEASUREMENTS AND MAIN RESULTS: Cardiac performances were investigated following envenomation. Cardiac histology and myocardial tumor necrosis factor-RNA concentrations were assessed. Serum tumor necrosis factor concentrations rose and peaked 2 hrs following envenomation. A reduction in peak systolic pressures, maximum and minimum change in pressure over time, time-pressure integral, and coronary flow occurred in the venom-only-injected rats compared with controls, whereas blocking tumor necrosis factor activity prevented the deleterious cardiac effects of the envenomation. No histologic changes or increases in myocardial tumor necrosis factor-RNA concentrations were detected. CONCLUSION: These results strongly suggest that systemic release of tumor necrosis factor mediates cardiac toxicity following Vipera aspis envenomation.