Indian Journal of Biochemistry & Biophysics Vol. 46, April 2009, pp 154-160 Inhibition of local effects of Indian Daboia/Vipera russelli venom by the methanolic extract of grape ( Vitis vinifera L.) seeds Y H Mahadeswaraswamy, S Devaraja, M S Kumar, Y N J Goutham and K Kemparaju* Department of Studies in Biochemistry, University of Mysore, Mysore 570006, India Received 01 May; revised 21 January 2009 Although anti-venom therapy is available for the treatment of fatal bite by snakes, it offers less or no protection against the local effects such as dermo- and myonecrosis, edema, hemorrhage and inflammation at the bitten region. The viper species are known for their violent local effects and such effects have been commonly treated with plant extracts without any scientific validation in rural India. In this investigation, the methanolic extract of grapes ( Vitis vinifera L.) seed was studied against the Indian Daboia/Vipera russelli venom-induced local effects. The extract abolished the proteolytic and hyaluronidase activities and also efficiently neutralized the hemorrhage, edema-inducing and myonecrotic properties of the venom. In addition, the extract also inhibited partially the pro-coagulant activity of the venom and abolished the degradation of A α and B β chains of human fibrinogen. Thus, the extract possesses potent anti-snake venom property, especially against the local effects of viper bites. Keywords: Hyaluronidase, Local effects, Metalloprotease, Neutralization, Snake venom, Proteolytic activity , Hemorrhagic activity, Edema-inducing activity, Myonecrotic activity, Fibrinogenolytic activity, Pro-coagulant activity Daboia russelli, Vipera russelli, Grape seed, Vitis vinifera L. Daboia russelli is one of the predominant poisonous and acetylcholine 3. The envenomation involves snakes of Indian subcontinent and is endemic in subcutaneous or intramuscular injection of venom different states of India. The problem is particularly into the prey/human victims. The pathology of severe in the eastern zone of India. The death toll snakebite includes both local and systemic effects from D. russelli bite is highest in the Burdwan district such as neurotoxicity (pre/post synaptic), myotoxicity, of West Bengal, where about 1300 deaths occur every cardiotoxicity, coagulant (pro/anti), hemostatic 1 year . In humans, D. russelli bite causes severe local (activating/inhibiting), hemorrhagic, hemolytic and tissue destruction, more frequently the necrosis results edema forming activities 4. The venom has been in an irreversible loss of tissue and requires studied extensively for various active principles such amputation of the affected limb. In addition, systemic as pro-coagulant factors 5, ATPase 6, phospholipase 7 8 9 alterations such as coagulopathy and effects on A2 , trypsin inhibitors , daboiatoxin-like factor , multiple organs give rise to serious complications platelet aggregation inhibitors 10 , hemorrhagins 11 , such as cardiovascular shock, pulmonary bleeding and neurotoxic peptide 12 and a heat stable protein drct-113 . 2 hemorrhage in the central nervous system . The most effective and accepted therapy for Hemorrhagic syndrome is one of the most serious snakebite patient is immediate administration of consequences of D. russelli bite. specific/polyvalent anti-venoms fallowing envenom- The venom comprises a complex mixture of ation. Generally, the anti-venoms do not provide enzymatic and non-enzymatic proteins, peptides and enough protection against venom-induced local tissue small organic compounds, such as citrate, nucleosides damage and often associated with risk of anaphylaxis and serum reactions. Further, the anti-venom _________________ development is time-consuming, expensive and *Corresponding author 14,15 E-mail: [email protected] requires ideal storage condition , and therefore, Tel.: +91-821-2419625(O) alternative therapies are in demand. Over the years, Abbrevia tions: BSA, bovine serum albumin; DMSO, dimethyl several attempts have been made for the development sulfoxide; ECM, extracellular matrix; MED, minimum edema of the snake venom antagonists, especially from plant dose; MHD, minimum hemorrhagic dose; OD, optical density; PDMAB, p-dimethyl aminobenzaldehyde; SVMPs, snake venom sources and the anti-venom activity has been confirmed 16-22 metalloproteases. in some plant extracts and their isolates . MAHADESWARASWAMY et al : ANTI-VENOM POTENTIAL OF GRAPE SEEDS 155 Earlier, we have reported that the methanolic casein was precipitated by the addition of 1.5 ml of extract of grape fruit ( Vitis vinifera L) seeds inhibits 0.44 M trichloroacetic acid. The digested casein in the the enzymatic (casenolytic, hyaluronolytic and supernatant (1 ml) was determined using Folin- fibrinogenolytic) and pharmacological (edema- Ciocalteu’s reagent. One unit of activity was defined inducing, hemorrhage and myonecrosis and pro- as the amount of venom sample required to cause an coagulation) activities of the Indian saw-scaled viper increase in optical density (OD) by 0.01 at 22 (Echis carinatus ) venom . The present study has 660 nm/min. examined the effectiveness of methanolic extract of The proteolytic activity was also performed in 24 grape fruit seed against the Indian D. russelli venom. SDS-PAGE zymogram as described previously . Briefly, casein was incorporated at a final Materials and Methods concentration of 2% in the SDS-PAGE matrix (10%) Hyaluronic acid, casein and fibrinogen were from and the venom sample (50 µg) prepared under non- Sigma Chemicals Co, St. Louis, MO, USA. All other reduced condition. The gel was rinsed in 2.7% Triton chemicals used were of analytical grade. D. russelli X-100 for 1 h after electrophoresis and incubated in a venom was purchased from Hindustan Snake Park, developing buffer (50 m M Tris, 40 m M NaCl and 200 Kolkatta, India. mM CaCl 2. 2H 2O) for 18 h and stained with 0.1% Swiss albino mice weighing 20-25 g were obtained Coomassie brilliant blue R-250. The gel was analyzed from the Central Animal House facility, Department for clear zones of substrate hydrolysis against a blue of Zoology, University of Mysore, Mysore, India. The background. animal care and handling were conducted in Hyaluronidase activity compliance with the National Regulations for Animal The hyaluoronidase activity was assayed according Research. The animal experiments were carried out 25 to the previously described method . The venom after reviewing the protocols by the Animal Ethical sample (100 µg) was incubated with 50 µg of Committee of the University of Mysore. hyaluronic acid in 300 µl 0.2 M sodium acetate Preparation of seed methanolic extract buffer, pH 5.5 containing 0.15 M NaCl at 37 °C for Fruit seeds ( Vitis vinifera L.) collected from the 2 h. The reaction was terminated by adding 50 µl of local market, Mysore were washed thoroughly with potassium tetraborate and boiled for 3 min. The water, shade-dried, grounded into coarse powder. The coloring reagent p-dimethyl aminobenzaldehyde powder (5 g) was soaked in 20 ml of methanol for (10% PDMAB) 1.5 ml (acetic acid: hydrochloric acid; 30 min with constant stirring and the extract was 9: 1; v/v) was added and incubated for 30 min at 0 filtered through Whatman filter paper grade I. The 37 C. The absorbance was measured at 585 nm. filtrate was dried at room temperature to evaporate Activity was expressed as n moles of N-acetyl methanol and dried sample was weighed (20 mg) and glucosamine released/min/mg venom sample. dissolved in minimum volume of dimethyl sulfoxide The SDS-PAGE zymogram was also performed as 26 (DMSO) and diluted in distilled water (1: 9, v/v) 22 . described previously . Briefly, hyaluronic acid was DMSO and distilled water (1: 9, v/v) mixture did not incorporated at a final concentration of 0.17 mg/ml in induce any of the toxic effects in mice and served as the SDS-PAGE matrix (10%). The venom sample control experiments. For inhibition studies, the venom (50 µg) prepared under non-reduced condition was sample was pre-incubated with various amounts of electrophoresed. Thereafter, the gel was soaked methanolic extract for 30 min at 37 °C and the venom: consecutively 3-times in 50 ml of 0.15 M NaCl in extract ratio (w/w) was used unless otherwise sodium phosphate buffer (0.1 M, pH 5.5) containing indicated. 5%, 0.05% and 0% Tritan X-100 for 1 h. This was followed by equilibrating the gel in 0.1 M sodium Proteolytic activity formate buffer, pH 5.5 containing 0.15 M NaCl for The proteolytic activity was determined according 18 h at 37 °C with constant agitation. The gel was to the previously described method 23 using casein washed in 0.015 M Tris-HCl buffer ( pH 7.9) and (2% in 0.2 M Tris-HCl buffer, pH 8.5) as substrate. placed in 0.1% stains-all solutions for 2 h and kept in The venom sample (100 µg) was incubated with the dark until photographed. The appearance of a 0.4 ml casein in a total volume of 1 ml for 2 h at translucent activity band against a dark blue 37 °C. The reaction was terminated and the undigested background indicated the enzyme activity. The 156 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, APRIL 2009 working stain was prepared just before use by taken using Photometrics colorsnap CF camera (Leitz combining 1 ml of stock with 1 ml formamide, 4 ml Diaplan Germany, Roper Scientific Photometrics isopropanol, 0.37 ml 1 M Tris-HCl buffer, pH 7.9 and type- A014872002 ) attached to the microscope. de-ionized water to a volume of 20 ml. The molecular weight marker proteins run on one edge were stained Coagulant and fibrinogenolytic activities with Coomassie brilliant blue R-250. The plasma coagulation activity was assayed according to the previously described method 30 . The Hemorrhagic activity normal human citrated plasma (200 µl) was incubated The hemorrhagic activity was assayed according to with venom sample (1 µg) and the clotting time was 27 the previously described method . Groups of 5 mice recorded against a light source.