Indian Journal of Biochemistry & Biophysics Vol. 46, April 2009, pp 154-160

Inhibition of local effects of Indian Daboia/ 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 , 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 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- 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 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. Fibrinogenolytic each were injected intradermally with twice the activity was assayed according to the previously minimum hemorrhagic dose (MHD) of venom sample described method 31 . The human plasma fibrinogen (10 µg) in 50 µl saline. After 3 h, the mice were (50 µg) was incubated with the venom sample (10 µg) anaesthetized by diethyl ether inhalation, a dorsal in 40 µl of 5 m M Tris HCl buffer ( pH 7.4) containing patch of the skin was removed and the inner surface 10 m M NaCl. The reaction was terminated after 6 h was observed for the hemorrhage. The MHD was by adding 20 µl denaturing buffer containing 1 M calculated as the amount of venom sample that urea, 4% SDS and 4% β-mercaptoethanol and produced a hemorrhagic spot of 10 mm. samples were analyzed on 10% SDS-PAGE as previously described method 32 . The protein estimation Edema-inducing activity was carried out according to the previously described The edema inducing activity was assayed 33 method using bovine serum albumin (BSA) as a according to the previously described method 28 . standard. Groups of 5 mice were injected in the right footpads with six-times the minimum edema dose (MED) of Statistical analysis venom sample (6 µg) in 20 µl saline. The left All the experiments were repeated for five footpads received saline and served as controls. The independent observations. The data were presented as mice were anaesthetized by diethylether inhalation; mean ± S.E.M. the legs were removed at the ankle joint after 1 h. Increase in weight due to edema was calculated as Results edema ratio, which equals the weight of edematous The toxicity studies D. russelli venom in mouse leg x 100/weight of control leg. The MED was models revealed the minimum hemorrhagic dose defined as the amount of venom sample required to (MHD) to be 5 ± 0.03 µg, and the 2 x MHD caused cause an edema ratio of 120%. the hemorrhagic area of 20 ± 0.05 mm, whereas the minimum edema dose (MED) was 1 µg and the 6 x Myonecrotic activity MED caused an edema ratio of 170 ± 2%. The venom The myonecrotic activity was assayed according to was pro-coagulant in nature and caused the rapid 29 the previously described method . Half the LD 50 clotting of human plasma when added directly. It value of venom sample (1.25 mg/kg body wt) in 50 µl degraded the human fibrinogen with A α-chain saline was injected intramuscularly into the right thigh degraded preferentially over the B β-chain, while the of three groups of mice (n = 5). The group receiving γ-chain was resistant and remained intact, irrespective 50 µl saline alone served as a control experiment. The of incubation period and concentrations of venom mice were anaesthetized after 3 h by diethyl ether sample used. The venom caused an extensive dermo- inhalation. The thigh muscle tissues dissected out and myonecrosis at the injected site. It hydrolyzed from the site of venom sample injection were fixed in casein and also hyaluronic acid with the specific Bouin’s solution and dehydrated by processing activity of 1.1 ± 0.05 and 1.25 ± 0.04 units/min/mg, through different grades of alcohol and chloroform: respectively. alcohol mixture. The processed tissues were The methanolic extract of grape seeds inhibited the embedded in paraffin wax, cut in to a 4 µm thick proteolytic activity of the venom in a dose-dependent sections, which were stained with hematoxylin-eosin manner. At the venom: extract ratio of 1: 8, complete for microscopic observations (Leitz Wetzlar Germany inhibition of proteolytic activity was observed type-307-148.002 microscope) and photographs were (Fig. 1a). Inhibition was further confirmed by the casein MAHADESWARASWAMY et al : ANTI-VENOM POTENTIAL OF GRAPE SEEDS 157

Fig. 1 ―(a):Inhibition of proteolytic activity [ D. russelli venom (100 µg) was pre-incubated with the increased amounts of the extract for 30 min at 37 °C. The reaction was initiated by adding 0.4 ml of 2% casein in 0.2 M Tris HCl buffer ( pH 8.5) and adjusted the reaction volume to 1 ml with the buffer and incubated for 2 h at 37 °C. The activity was determined as described in the ‘Materials and Methods’. Values represent as mean ± S.E.M (n = 5); and (b): Inhibition of proteolytic activity in casein zymogram [ D. russelli venom (50 µg) was pre-incubated with the extract for 30 min at 37 °C and resolved by SDS-PAGE (10%) containing 2% casein under non-reduced condition and processed as described in the ‘Materials and Methods’. Lane 1, 50 µg of venom sample alone; lane 2, venom: extract ratio of 1: 8; lane M, molecular weight markers from top to bottom: myosin-H-chain (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (31.0 kDa), soyabean trypsin inhibitor (21.5 kDa) and lysozome (14.4 kDa)]

Fig. 2 ―(a): Inhibition of hyaluronidase activity [ D. russelli venom (100 µg) was pre-incubated with the increased amounts of the extract for 30 min at 37 °C and incubated with hyaluronic acid in a final reaction volume of 0.3 ml of 0.2 M sodium acetate buffer containing 0.15 M NaCl for 2 h at 37 °C. The hyaluronidase activity was carried out as described in the ‘Materials and Methods’. Values represent mean ± S.E.M (n = 5); and (b): Inhibition of hyaluronidase activity in zymogram [D. russelli venom (50 µg) was pre-incubated with the extract for 30 min at 37 °C and resolved by SDS-PAGE (10%) containing hyaluronic acid (0.17 mg/ml) under non-reduced condition and processed as described in the ‘Materials and Methods’. Lane 1, 50 µg of venom sample alone; lane 2, venom: extract ratio of 1: 2; lane M; molecular weight markers from top to bottom: myosin-H-chain (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (31.0 kDa), soyabean trypsin inhibitor (21.5) and lysozome (14.4 kDa)] zymogram assay. Lack of translucent activity band in caused due to 2 x MHD (Fig. 3). Further, the extract zymogram assay in presence of the extract and completely abolished the hemorrhagic edema induced appearance in its absence against a blue background by the venom dose-dependently and at the venom: suggested the inhibition (Fig. 1b). Further, the extract extract ratio of 1: 20, the edema ratio due to 6 x MED also inhibited the hyaluronidase activity of the venom of 170 ± 2% neutralized to 117 ± 3.1% (Fig. 4). in a dose-dependent manner and the venom: extract The extract also inhibited the myonecrotic activity ratio of 1: 2 caused the complete inhibition of the induced at the site of venom injection and caused activity (Fig. 2a). Inhibition was also confirmed in the complete inhibition of the myonecrosis at the venom: hyaluronidase zymogram assay (Fig. 2b). The extract extract ratio of 1: 8. The longitudinal section of effectively inhibited the hemorrhage induced by the muscle tissues revealed extensive destruction of venom in a dose-dependent manner and at the venom: myocytes and disorganized myofibrils with the extract ratio of 1: 25 neutralized the hemorrhage accumulation of polymorphonucelar leucocytes at the 158 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, APRIL 2009

Fig. 3 ―Inhibition of hemorrhagic activity [ D. russelli venom (10 µg) was pre-incubated with the increased amounts of the extract for 30 min at 37 °C and injected intradermally into groups of mice in a total volume of 50 µl saline. After 3 h, mice were anaesthetized and sacrificed, dorsal patch of the skin surface was removed and the diameter of hemorrhagic spot on the inner surface of the skin was measured in mm. Saline ( A), venom alone ( B), venom: extract ratios of (1: 5, 1: 10, 1: 15, 1: 20 and 1: 25) ( C-G) respectively, and extract (250 µg) alone ( H]

Fig. 5 ―Inhibition of muscle tissue necrosis [ D. russelli venom (1.25 mg/kg body wt.) was pre-incubated with the extract at 37 °C for 30 min and injected intramuscularly into the right thigh muscle of mice in a total volume of 50 µl saline. The mice were sacrificed after 3 h and the muscle tissues dissected out from the injected site Fig. 4 ―Inhibition of edema inducing activity [ D. russelli venom and processed for the histopahological studies as described in the (6 µg) was pre-incubated with increased amounts of the extract for ‘Materials and Methods’. Saline injected control section ( A), 30 min at 37 °C and injected into the right footpads of mice in a total venom injected section ( B), venom pre-incubated with the extract volume of 20 µl saline. The mice were anaesthetized and sacrificed at venom: extract ratio of 1: 8 ( C)] after 1 h and the edema ratio was calculated. Values represent mean blistering, hemorrhage, edema, dermonecrosis, ± S.E.M (n = 5)] myonecrosis and inflammation. The bite causes the venom injected site (Fig. 5b), while in presence of the hemostatic disturbances, leading to bleeding disorders extract, the tissue sections revealed normal muscular and damage of the vital organs. Although anti-venom striations (Fig. 5c), similar to the control section therapy is effective in neutralizing the systemic (Fig. 5a). toxicity, if administered in time,but it is less effective The D. russelli venom at 1 µg concentration or ineffective against local tissue destruction, revealed the plasma clotting time of 31 ± 1.5 s, as resulting in the continuous tissue destruction at the compared to the control value of 190 ± 4 s. The seed bite site even after the neutralization of systemic extract prolonged the clotting time of the venom dose- toxicity 14,15 . Thus, there is a great demand for new dependently from 31 ± 1.5 s to a maximum of 87 ± alternative therapy. 3.5 s (Fig. 6) at the venom: extract ratio of 1: 20. The methanolic extract of grape seeds abolished Further, the extract inhibited the fibrinogenolytic both proteolytic and hyaluronidase activities, with the activity of the venom dose-dependently. At the later inhibited more effectively. The hemorrhagic venom: extract ratio of 1: 10, the preferential activity of the venom was also inhibited. The tissue inhibition of B β chain degradation over A α chain was destruction and hemorrhage is primarily due to the observed, while at venom: extract ratio of 1: 30, degradation of structural and adhesive components inhibition of both A α and B β chains degradation was such as collagen, elastin, fibronectin and achieved (Fig. 7). glycosaminoglycans of the extracellular matrix Discussion (ECM) of connective tissue, surrounding the blood Envenomation by D. russelli venom is well vessels and capillaries by matrix-degrading venom characterized by its prominent local effects involving hyaluronidases and metalloproteses. These two MAHADESWARASWAMY et al : ANTI-VENOM POTENTIAL OF GRAPE SEEDS 159

Fig. 6 ―Inhibition of pro-coagulant activity [ D. russelli venom (1 µg) was pre-incubated with the increased amounts of the Fig. 7 ―Inhibition of fibrinogen degradation [D. russelli venom extract at 37 °C for 30 min and was added independently to 0.2 ml (1 µg) was preincubated with the increased amounts of the extract at of citrated human plasma and the clotting was initiated by adding 37 °C for 30 min and the mixture was then incubated with the human fibrinogen (50 µg) at 37 °C for 6 h in presence of 10 m M 20 µl CaCl 2, (0.25 M) as described in ‘Materials and Methods’. Values represent mean ± S.E.M (n = 5)] Tris-HCl buffer ( pH 7.4) and analyzed on 10% SDS-PAGE under reduced condition as described in ‘Materials and Methods’. Lane 1, fibrinogen alone; lane 2, fibrinogen + venom; lanes 3-6, fibrinogen groups of hydrolytic enzymes appear to be the + venom: extract ratios of (1: 10, 1: 20, 1: 30 and 1: 40) respectively principal agents responsible for tissue degradation at and lane M, molecular weight markers from top to bottom: myosin- the site of bite. H-chain (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (43.0 kDa), carbonic anhydrase The snake venom metalloproteases (SVMPs) are +2 (31 kDa), soyabean trypsin inhibitor (21.5 kDa)] the Zn -dependent hemorrhagic metalloproteses of the metzincin family enzymes which degrade the activity of the venom. Inhibition of myonecrotic structural protein scaffold of the ECM. While activity is undoubtedly strengthened by the restoration hyaluronidases degrade the hyaluronic acid into small of normal striated musculature in histopathological fragments, hyaluronic acid is a megadalton polymer studies. Earlier, similar neutralization of myonecrotic and holds aggrecan monomers, metal ions and water activity by various plants extracts and their bioactive molecules through ionic interaction. Thus, the component(s) has been reported 18-22 . degradation of ECM components not only destroys The D. russellii venom is rich in components that the mechanical support of the tissue, but also makes interact with the coagulation cascade and make the the ECM smooth and fragile, resulting in easy venom highly pro-coagulant. It is likely that inhibition diffusion of systemic toxins into the circulating of fibrinogenolytic activity by the extract is blood 34,35 . responsible for the observed inhibition of pro- As the hemorrhagic metalloproteses are usually coagulant activity of the venom. Snake venom dependent on divalent metal ions for their activity, the fibrinogenases releasing fibrinopeptides A and or B metal ions appear to play role both in the catalysis and from the N-terminal disulfide knot of A α or B β chains as well as offer structural stability for the enzymes. are thrombin-like enzymes and are found to be The seed extract might chelate the metal ion or responsible for the pro-coagulant activity while, those interact with a specific domain to inactivate the acting from the C-terminal end are responsible for the 37 hemorrhagic protease and that could have led to anti-coagulant activity . inhibition of the hemorrhage. Similar results were Conclusion also observed by different plant extracts and bioactive 18-22 The methanolic extract of grape seeds inhibited the component(s) . Although inhibitory mechanisms local toxicity and as well as pro-coagulant activity of are not well understood for hyaluronidases, there are D. russelli venom. However, further studies are reports suggesting the inhibition of the activity by the 36 needed for the isolation of specific inhibitors for the plant components . Thus, it appears that inhibition of efficient management of threatening local toxicity and hemorrhagic metalloproteases and hyaluronidases the fatal systemic toxicity of D. russelli bite. could offer effective protection against tissue destruction at the bitten region. Inhibition of edema- Acknowledgements inducing activity suggests the inhibition of Authors thank Dr. K S Girish and S Nagaraju, inflammatory reactions, most likely phospholipase A 2 Department of Biochemistry, University of Mysore, 160 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, APRIL 2009

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