Title of Invention	A PROCESS FOR THE PREPARATION OF A NOVEL FIBRINOLYTIC PEPTIDE FROM THE VENOM OF INDIAN KING COBRA
Abstract	A novel fibrinolytic peptide which produces non clotted blood in whole animal and develops halos in fibrin plates has been prepared from venom of snake Indian king cobra ( Ophiophagus hannah ). The said peptide having a molecular weight of 610 daltons and amino acid sequence asparate - glutamate glycin - alanine- leucine, prepared by chromatographic method and purified by high performance liquid chromatography, reveals strong defibrinogenating activity, fibrinolytic activity, fibrinogenolytic activity and devoid of phospholipase activity, haemorrhagic activity, haemolytic activity showing its potential use for pharmacological purposes.

Title of Invention

A PROCESS FOR THE PREPARATION OF A NOVEL FIBRINOLYTIC PEPTIDE FROM THE VENOM OF INDIAN KING COBRA

Abstract

A novel fibrinolytic peptide which produces non clotted blood in whole animal and develops halos in fibrin plates has been prepared from venom of snake Indian king cobra ( Ophiophagus hannah ). The said peptide having a molecular weight of 610 daltons and amino acid sequence asparate - glutamate glycin - alanine- leucine, prepared by chromatographic method and purified by high performance liquid chromatography, reveals strong defibrinogenating activity, fibrinolytic activity, fibrinogenolytic activity and devoid of phospholipase activity, haemorrhagic activity, haemolytic activity showing its potential use for pharmacological purposes.

Full Text	The present invention relates to a process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king cobra ( Ophiophagus hannah ) having a molecular weight of 610 daltons and amino acid sequence asparate - glutamate glycin - alanine- leucine , devoid of phospholipase activity, haemorrhagic activity, haemolytic activity and useful for pharmacological purposes. The present invention particularly relates to a process for the isolation of a novel peptide having fibrinolytic activity from the venom of the snake Indian king cobra (Ophiophagus hannah). Thromobosis, or the occlusion of a blood vessel, is a major killer throughout the world. The classical use of heparin, urokinase, streptokinase in the management of thrombosis is very expensive and possess side effects. There are several snake venom components which show fibrinolytic activity and clot - lyric property. Reference may be made to : ATROXASE ; Willis T.W & Tu A.T, 1988, Purification and characterisation of atroxase, a non-hemorrhagic fibrinolytic protease from western diamond back rattle snake venom. Biochemistry, vol 27, pg 4769-4777), BASILASE (Datta G, Doug A, Will J and Tu A.T, 1995, Biochemical characterisation of Basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus, Arch. Biochem Biophys, Vol 317, Pg 365-373). LEBETASE (Sngur E and Sngur J, 1991, Purification and Characterisation of lebetase, a fibrinolvtic enzyme from vipera lebetina Snake venom, Biochem Biophys Acta, Vol 1074, Pg 223-224). The above said snake venom components showed fibrinolytic activity and clot lyric property. This initiated the possible application of these agents in the therapeutic management of thrombosis, in a much better way than the conventional existing thrombolytic agents. The major drawbacks of the prior art, with special reference to Heparin / urokinase /streptokinase can be summarised as: 1. High cost; 2. Not easily & widely available; 3. Possesses side effects (Haemorrhagic syndrome, bleeding). Reference may be made to Goodman & Oilman's The Pharmacological basis of therapeutics. 9th Edition, Editors Hardman J.G, Limbird LE, Malinoff P.B, Ruddun R.W, Goodman Oilman A. (1996) Pg 1341-1353. The snake Indian King Cobra (Ophiophagus hannah) is the largest venomous land snake available in the north-eastern hilly regions. Virtually, there is no information on King Cobra venom or venom constituents. We have for the first time isolated and identified a novel peptide from King Cobra venom which possessed strong fibrinolytic activity. It produces non clotted blood in whole animal and develops halos in fibrin plates. The peptide had a molecular weight of 610 daltons. The main objective of the present invention is to provide a process for the isolation of a novel fibrinolytic peptide useful for pharmacological purposes, from the venom of snake king cobra. Another object of the present invention is to provide a process for the isolation of a novel fibrinolytic agent useful for therapeutic application in cardiovascular disorders and as a biomedical research probe / tool. Stili another object of the present invention is to provide a process for the isolation of a cheaper fibrinolytic agent. Yet another object of the present invention is to provide a process for the isolation of a novel and cheaper fibrinolytic / thrombolytic agent from easily available resources such as the snake Indian king cobra, a natural product of Indian origin. Accordingly the present invention provides a process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king cobra ( Ophiophagus hannah ) having a molecular weight of 610 daltons and amino acid sequence asparate - glutamate glycin - alanine- leucine , devoid of phospholipase activity, haemorrhagic activity, haemolytic activity and useful for pharmacological purposes which comprises: (i) obtaining venom of shake king cobra by conventional methods; (ii) fractionating venom, so obtained, by conventional chromatographic methods using solvent system isopropanol: 0.1N HC1 (7 : 3 v/v), and obtaining spot on chromatographic plate . (iii) isolating an active compound with 0.04% yield from a spot obtained at step (ii) and visualized at 254nm in UV chamber such as herein described , (iv) purifying the said active compound obtained in step (iii) , using high performance liquid chromatography to get the desired novel fibrinolytic peptide. In an embodiment of the present invention the venom used may be that of snake king cobra such as Indian King Cobra (Ophiophagus hannah). In another embodiment of the present invention the fractionating and purifying may be effected using known chromatographic methods such as thin layer chromatography on silica gel followed by high performance liquid chromatography. By the process of the present invention a novel and potent fibrinolytic peptide has been purified from the venom of the Indian King Cobra (Ophiophagus hannah) by thin layer chromatography followed by high performance liquid chromatography. The novel peptide, so obtained, was devoid of phospholipase activity, haemorrhagic activity, haemolytic activity. The fallowings examples are given by way of illustration of the process and should not be construed to limit the scope of the present invention. Example 1 Purification of the novel fibrinolytic peptide: Thin layer chromatography of King Cobra Venom, was done in preactivated glass plates (20 + 10cm) coated with silica gel G (type 60), using solvent system isopropanol: 0.1N HC1 (7 : 3 v/v). Spots were visualized in (1) UV (254nm) chamber (2) iodine vapour (3) 0.1% ninhydrin in acetone and Rf value calculated. A spot observed having (1) yellow fluorescent at 254nm (2) yellow spot in iodine (3) purple colour with ninhydrin having Rf value of 0.48. Rechromatography of this spot in the same solvent/detection system produced a single spot of Rf 0.48. The purification process produced 0.04% yield of the active compound. The active compound was further purified on reverse phase high performance liquid chromatography (RP-HPLC;) Waters 486 system using a Nova pak C18 column (6OA0, 4um,3.9x 150mm) using solvent system methanol : water (60 : 40, v/v). The TLC purified active compound was passed through millipore filter (0.4 m) and applied in HPLC Column. The active compound produced a single sharp peak on RP-HPLC, with a retention of 2.05 mins, indicating the active compound was homogenous in nature. Structural/Spectroscopic Analysis: The ultraviolet spectra of the compound done in Perkin Elmer spectrophotometer 5503, using spectral methanol as solvent, produced a sharp peak at 260nm, when scanned from 200 to 400 nm. The fluorescence spectra of the compound was done in Hitachi fluorescence spectrophotometer (F4020) using Phosphate buffer system 20 mM, pH 7.4 as a solvent and also a base line fluorescence detector. Emission scanned from 300 nm to 450nm, when excited at 280 nm it showed an emision maxima (Emax) at 342. At 295 nm excitation, it showed an Emax of 348. Electron impact mass spectra (EIMS) of the compound was done in Jeol Ax 500 spectrometer using spectral methanol. The component showed M+ at m/z 610. Amino acid composition of the compound was done in PICO TAG Amino acid analysis System (Waters model 510 HPLC Pump) using Pico tag column. The peptide contained amino acids Aspartate, Glutamate Glycine, Alanine, Leucine. CD spectra of the compund done in Jeol 600 spectropoiarimeter (Tasco, Model 720) showed no secondary, tertiary structure and confirmed linear sequence of the peptide. The active compound isolated and purified from the Indian snake King Cobra venom as mentioned in example 1 was obtained in pure state, peptide in nature, with molecular weight of 610 daltons and having amino acids aspartate, glutamate, glycine, alanine, leucine. In the following examples the biological activity of me novel peptide were determined. Example 2 Defibrinogenating activity: Defibrinogenating activity of hannahpep was assayed in vivo according to Theakston R.D.G and Reid, H.A (1983). Development of simple standard assay procedure for the characterisation of snake venom. Bullentine of the World Health Organisation Geneva, Vol 61, Pg 949 - 956. The novel peptide (1 ug) in 0.9% saline (100 ml) was injected intravenously through caudal vein in male albino mice. (18-20 gm). After 1 hr, blood was collected from retroorbital plexus and defibrinogenating action was recorded. The minimum defibrinogenating dose (MDD) was defined as the minimum amount, of test compound which when injected (i.v) in male albmice produced noncoagulable blood after 1 hr. control animals (0.9% saline injected i.v) blood coagulate within 2 ± 0.2 mins as compared with ( l,14g) which produced non-coagulable blood observed upto 240 ± 0.5 min. MDD of the novel peptide w j found to be 100 ± 1.2 ng . Thus the novel peptide possesses defibrinogenatmg activity. Example 3 Fibrinolytic activity: Fibrinolytic activity of the novel fibrinolytic peptide was assayed in vitro after Astrup, T. and Muthertz, S (1952). The fibrin plate method for estimation of fibrinolytic activity. Archives of biochemistry and biophysics, Vol 40, pg 346-351. Fibrinogen (Fraction I, Sigma U.S.A) 166 mg was dissolved in 10 ml of 70 mM ammonium sulphate solution and poured in a 5 cm petridish. Thrombin (bovine, Sigma USA) 200 ml was added to the fibrinogen solution and mixed well. The solution was allowed to solidify at room temperature for 2 hours. Sample were applied on the surface of the gel and incubated at 37°C for 2 hours. The diameters of me lysed area (plaque) on the gel were men recorded. Saline (0.9%) was used as negative control, streptokinase (2 ug) as positive control the novel fibrinolytic peptide (100 ng) produced a plaque of 10 mm diameter. Streptokinase also produced a plaque of 10 mm diameter. Thus the novel fibrinolytic peptide possessed fibrinolytic activity. Example 4 Fibrinogenolytic activity : Fibrinogenolytic activity of the novel fibrinolytic peptide was assayed in vitro after Ware, A.G. Guest M.M. and Sergerd, W.H. (1947) Fibrinogen with special reference to its preparation and certain properties of the product. Archives of Biochemistry Biophysics, Vol 13, Pg 231-236. The novel fibrinolytic peptide (1 ug) roduced 67% fibrinogenolytic activity as compared to positive control (100%) streptokinase (2 ug). Thus the novel fibrinolytic peptide possessed fibrinogenolytic activity Example 5 Blood clot lytic activity : 0.5 ml fresh blood collected from albino mice (20 gm) was collected in different watch glass and allowed to clot for 20 rnin. Test samples were applied over die clot and incubated at 37°C for 2 hours. Lysis of clot was recorded. Saline (0.9%) was used as negative control, streptokinase (2 ug) as positive control. The novel fibrinolytic peptide (100 ng) produced 70% clot lysis as compared with positive control (100%) streptokinase. Thus the novel fibrinolytic peptide possessed clotlytic activity. Example 6 Plasma recaicification activity : Minimum clotting dose of plasma (MCDP) induced by the novel fibrinolytic peptide was assayed in vitro by the method of Theakston, R.D.G and Reid, H.A. (1983). Development of simple standard assay procedures for the characterisation of snake venom. Bullentine World Health Organisation, Geneva, Volume 61, pg 949-956.. Normal human plasma (0..2 ml) was incubated at 37°C for 15 minutes. Clotting was (25 mM, 0.1 ml) and cllotnng time was recorded in presence and absence of test substances. Saline (0.9%) was used as negative control. The novel fibrinolytic peptide (lag) increased the coagulation time by 3 fold, as compared with negative control. Thus, the novel fibrinolytic peptide showed plasma anticlotting activity. Example 7 Haemmorhagie activity : Cutaneous haemmorhagie activity of the novel fibrinolytic peptide was assayed in vitro in male swiss albino mice (20 gm) after the method of Kondo, H., Kondo, S., Ikezawa, H., Murata, R., and Ohsaka, A. (1969). Studies on the quantitative method of detennination of haemmorhagic activity of Habu Snake venom. Japanese journal of medical Science and Biology, Volume 13, pg 43-51. Minimum haemmorhagic dose (MHD) was defined as the amount to test substance when injected intradermally, produced a haemmorhagic spot of 10 mm diameter within 24 hours of observation. Saline (0.9%) was used as negative control, Russells viper venom (5 µg) was used as positive control. The novel fibrinolytic peptide (lµg) did not produced any haemorrhagic spot observed within 24 hours, as compared with positive control (10 mm diameter) Russell's viper venom. Thus the novel fibrinolytic peptide was found to be non haemorrhagie in nature. Example 8 Phospholipase A activity : Phospholipase A activity of the novel fibrinolytic peptide was assayed by the egg-yolk coagulation method of Neumann, W. and Habermann, E (1954). Beitrage zur charakteriseierung der wirkstoffe des bienengiftes. Archives of Experimental pathology and pharmacology, Volume 222, pg 367-370. 2 ml Egg-suspension (9 ml egg yolk, 2.51 ml 2% sodium chloride, 1.49 ml of 0.5% EDTA, 4.44 ml of 1% CaC12, 2.0 ml Tris-HCl buffer 50 mM, pH 7.5 and 0.56 ml of 0.85% NaCl.) and test sample were taken in test tube (12 + 75 mm), mixed and incubated at 37°C for 1 hour. The tubes were placed in a boiling water bath and time to coagulate the suspension was recorded. Saline (0.9%) was used as negative control, Russell's viper venom was used as positive control, of the novel fibrinolytic peptide (lug) induced coagulation time of the egg-yolk suspension was same as negative control (less than 1 min) indicating absence of phospholipase activity. Thus, the novel fibrinolytic peptide did not possess phospholipase activity. Example 9 Haemolytic activity : Haemolytic activity of the novel fibrinolytic peptide (l0ng - lµg) was assayed in vitro by incubating with 1 ml 1% human RBC suspension at 37°C for 30 min. RBC suspension was centrifuged 900 g for 30 mins and degree of lysis was measured at A540 nm against negative control (saline 0.9%) and positive control 100%(distilled water). The fibrinolytic peptide did not haemolyse human RBC. So, the novel fibrinolytic peptide was non-haemolytic in nature. It may be concluded from the above that a novel fibrinolytic peptide has been purified from the venom of the Indian snake King Cobra (Ophiophagus hannah). The peptide had a molecular weight of 610 daltons. This novel fibrinolytic peptide possesses strong defibrinogenating, fibrinolytic, fibrinogenolytic and plasma anticlotting activity. The novel fibrinolytic peptide is devoid of phospholipase activity, haemorrhagic and haemolytic activity. The main advantages of the present invention are : (1) The novel fibrinolytic peptide has been purified from King Cobra venom, the snake being distributed in India (Sunderbans, north east hilly regions), Bangladesh, Myannmar, Thailand, Combodia Vietnam. (Endoglyphs and other major venomous snakes of the World. A checklist Golay P., Smith H.M., Bloodley D.G., Dixon F.R., McCarthy C, Page J.C., Schatti B., Toriba I.M., Azemiops, 1993). (2) The novel fibrinolytic peptide agent has been purified using easy and cheaper conventional methods. (3) The novel fibrinolytic peptide posseses anticoagulant property. (4) The novel fibrinolytic peptide had no phospholipase activity, was non-haemorrhagic and non-haemolytic in nature. (5) The yield obtained was of the order of 0.04 +/- 0.5 %. We Claim: 1. A process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king cobra ( Ophiophagus hannah ) having a molecular weight of 610 daltons and amino acid sequence asparate - glutamate glycin - alanine- leucine , devoid of phospholipase activity, haemorrhagic activity, haetnolytic activity and useful for pharmacological purposes which comprises: (i) obtaining venom of snake king cobra by conventional methods; (ii) fractionating venom, so obtained, by conventional chromatographic methods using solvent system isopropanol: 0.1N HC1 (7 :3 v v) and obtaining spoion chromatographic plate . (iii) isolating an active compound with 0.04% yield from a spot obtained at step (ii) and visualized at 254nm in UV chamber such as herein described, (iv) purifying the said active compound obtained in step (iii) , using high performance liquid chromatography to get the desired novel fibrinolytic peptide. 2. A process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king cobra (Ophiophagus hannah) as described herein with reference to the example no. 1

Full Text

The present invention relates to a process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king cobra ( Ophiophagus hannah ) having a molecular weight of 610 daltons and amino acid sequence asparate - glutamate glycin - alanine- leucine , devoid of phospholipase activity, haemorrhagic activity, haemolytic activity and useful for pharmacological purposes.
The present invention particularly relates to a process for the isolation of a novel peptide having fibrinolytic activity from the venom of the snake Indian king cobra (Ophiophagus hannah).
Thromobosis, or the occlusion of a blood vessel, is a major killer throughout the world. The classical use of heparin, urokinase, streptokinase in the management of thrombosis is very expensive and possess side effects.
There are several snake venom components which show fibrinolytic activity and clot - lyric property. Reference may be made to :
ATROXASE ; Willis T.W & Tu A.T, 1988, Purification and characterisation of atroxase, a non-hemorrhagic fibrinolytic protease from western diamond back rattle snake venom. Biochemistry, vol 27, pg 4769-4777), BASILASE (Datta G, Doug A, Will J and Tu A.T, 1995, Biochemical characterisation of Basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus, Arch. Biochem Biophys, Vol 317, Pg 365-373).
LEBETASE (Sngur E and Sngur J, 1991, Purification and Characterisation of lebetase, a fibrinolvtic enzyme from vipera lebetina Snake venom, Biochem Biophys Acta, Vol 1074, Pg 223-224).
The above said snake venom components showed fibrinolytic activity and clot lyric property. This initiated the possible application of these agents in the therapeutic management of thrombosis, in a much better way than the conventional existing thrombolytic agents.
The major drawbacks of the prior art, with special reference to Heparin / urokinase /streptokinase can be summarised as:
1. High cost;
2. Not easily & widely available;
3. Possesses side effects (Haemorrhagic syndrome, bleeding).
Reference may be made to Goodman & Oilman's The Pharmacological basis of therapeutics. 9th Edition, Editors Hardman J.G, Limbird LE, Malinoff P.B, Ruddun R.W, Goodman Oilman A. (1996) Pg 1341-1353.
The snake Indian King Cobra (Ophiophagus hannah) is the largest venomous land snake available in the north-eastern hilly regions. Virtually, there is no information on King Cobra venom or venom constituents. We have for the first time isolated and identified a novel peptide from King Cobra venom which possessed strong fibrinolytic activity. It produces non clotted blood in whole animal and develops halos in fibrin plates. The peptide had a molecular weight of 610 daltons.
The main objective of the present invention is to provide a process for the isolation of a novel fibrinolytic peptide useful for pharmacological purposes, from the venom of snake king cobra.
Another object of the present invention is to provide a process for the isolation of a novel fibrinolytic agent useful for therapeutic application in cardiovascular disorders and as a biomedical research probe / tool.
Stili another object of the present invention is to provide a process for the isolation of a cheaper fibrinolytic agent.
Yet another object of the present invention is to provide a process for the isolation of a novel and cheaper fibrinolytic / thrombolytic agent from easily available resources such as the snake Indian king cobra, a natural product of Indian origin.
Accordingly the present invention provides a process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king cobra ( Ophiophagus hannah ) having a molecular weight of 610 daltons and amino acid sequence asparate - glutamate glycin - alanine- leucine , devoid of phospholipase activity, haemorrhagic activity, haemolytic activity and useful for pharmacological purposes which comprises:
(i) obtaining venom of shake king cobra by conventional methods;
(ii) fractionating venom, so obtained, by conventional chromatographic methods using solvent system isopropanol: 0.1N HC1 (7 : 3 v/v), and obtaining spot on chromatographic plate . (iii) isolating an active compound with 0.04% yield from a spot obtained at step (ii) and visualized at 254nm in UV chamber such as herein described ,
(iv) purifying the said active compound obtained in step (iii) , using high performance liquid chromatography to get the desired novel fibrinolytic peptide.
In an embodiment of the present invention the venom used may be that of snake king cobra such as Indian King Cobra (Ophiophagus hannah).
In another embodiment of the present invention the fractionating and purifying may be effected using known chromatographic methods such as thin layer chromatography on silica gel followed by high performance liquid chromatography.
By the process of the present invention a novel and potent fibrinolytic peptide has been purified from the venom of the Indian King Cobra (Ophiophagus hannah) by thin layer chromatography followed by high performance liquid chromatography. The novel peptide, so obtained, was devoid of phospholipase activity, haemorrhagic activity, haemolytic activity.
The fallowings examples are given by way of illustration of the process and should not be construed to limit the scope of the present invention.
Example 1 Purification of the novel fibrinolytic peptide:
Thin layer chromatography of King Cobra Venom, was done in preactivated glass plates (20 + 10cm) coated with silica gel G (type 60), using solvent system isopropanol: 0.1N HC1 (7 : 3 v/v). Spots were visualized in (1) UV (254nm) chamber (2) iodine vapour (3) 0.1% ninhydrin in acetone and Rf value calculated.
A spot observed having (1) yellow fluorescent at 254nm (2) yellow spot in iodine (3) purple colour with ninhydrin having Rf value of 0.48. Rechromatography of this spot in the same solvent/detection system produced a single spot of Rf 0.48. The purification process produced 0.04% yield of the active compound.
The active compound was further purified on reverse phase high performance liquid chromatography (RP-HPLC;) Waters 486 system using a Nova pak C18 column (6OA0, 4um,3.9x 150mm) using solvent system methanol : water (60 : 40, v/v). The TLC purified active compound was passed through millipore filter (0.4 m) and applied in HPLC Column.
The active compound produced a single sharp peak on RP-HPLC, with a retention of 2.05 mins, indicating the active compound was homogenous in nature. Structural/Spectroscopic Analysis:
The ultraviolet spectra of the compound done in Perkin Elmer spectrophotometer 5503, using spectral methanol as solvent, produced a sharp peak at 260nm, when scanned from 200 to 400 nm. The fluorescence spectra of the compound was done in Hitachi fluorescence spectrophotometer (F4020) using Phosphate buffer system 20 mM, pH 7.4 as a solvent and also a base line fluorescence detector. Emission scanned from 300 nm to 450nm, when excited at 280 nm it showed an emision maxima (Emax) at 342. At 295 nm excitation, it showed an Emax of 348.
Electron impact mass spectra (EIMS) of the compound was done in Jeol Ax 500 spectrometer using spectral methanol. The component showed M+ at m/z 610.
Amino acid composition of the compound was done in PICO TAG Amino acid analysis System (Waters model 510 HPLC Pump) using Pico tag column. The peptide contained amino acids Aspartate, Glutamate Glycine, Alanine, Leucine.
CD spectra of the compund done in Jeol 600 spectropoiarimeter (Tasco, Model 720) showed no secondary, tertiary structure and confirmed linear sequence of the peptide.
The active compound isolated and purified from the Indian snake King Cobra venom as mentioned in example 1 was obtained in pure state, peptide in nature, with molecular weight of 610 daltons and having amino acids aspartate, glutamate, glycine, alanine, leucine.
In the following examples the biological activity of me novel peptide were determined.
Example 2 Defibrinogenating activity:
Defibrinogenating activity of hannahpep was assayed in vivo according to Theakston R.D.G and Reid, H.A (1983). Development of simple standard assay procedure for the characterisation of snake venom. Bullentine of the World Health Organisation Geneva, Vol 61, Pg 949 - 956. The novel peptide (1 ug) in 0.9% saline (100 ml) was injected intravenously through caudal vein in male albino mice. (18-20 gm). After 1 hr, blood was collected from retroorbital plexus and defibrinogenating action was recorded. The minimum defibrinogenating dose (MDD) was defined as the minimum amount, of test compound which when injected (i.v) in male albmice produced noncoagulable blood after 1 hr. control animals (0.9% saline injected i.v) blood coagulate within 2 ± 0.2 mins as compared with ( l,14g) which produced non-coagulable blood observed upto 240 ± 0.5 min. MDD of
the novel peptide w j found to be 100 ± 1.2 ng . Thus the novel peptide possesses defibrinogenatmg activity.
Example 3
Fibrinolytic activity:
Fibrinolytic activity of the novel fibrinolytic peptide was assayed in vitro after Astrup, T. and Muthertz, S (1952). The fibrin plate method for estimation of fibrinolytic activity. Archives of biochemistry and biophysics, Vol 40, pg 346-351. Fibrinogen (Fraction I, Sigma U.S.A) 166 mg was dissolved in 10 ml of 70 mM ammonium sulphate solution and poured in a 5 cm petridish. Thrombin (bovine, Sigma USA) 200 ml was added to the fibrinogen solution and mixed well. The solution was allowed to solidify at room temperature for 2 hours. Sample were applied on the surface of the gel and incubated at 37°C for 2 hours. The diameters of me lysed area (plaque) on the gel were men recorded. Saline (0.9%) was used as negative control, streptokinase (2 ug) as positive control the novel fibrinolytic peptide (100 ng) produced a plaque of 10 mm diameter. Streptokinase also produced a plaque of 10 mm diameter. Thus the novel fibrinolytic peptide possessed fibrinolytic activity.
Example 4 Fibrinogenolytic activity :
Fibrinogenolytic activity of the novel fibrinolytic peptide was assayed in vitro after Ware, A.G. Guest M.M. and Sergerd, W.H. (1947) Fibrinogen with special reference to its preparation and certain properties of the product. Archives of Biochemistry Biophysics, Vol 13, Pg 231-236. The novel fibrinolytic peptide (1 ug) roduced 67% fibrinogenolytic activity as compared to positive control (100%) streptokinase (2 ug). Thus the novel fibrinolytic peptide possessed fibrinogenolytic activity
Example 5 Blood clot lytic activity :
0.5 ml fresh blood collected from albino mice (20 gm) was collected in different watch glass and allowed to clot for 20 rnin. Test samples were applied over die clot and incubated at 37°C for 2 hours. Lysis of clot was recorded. Saline (0.9%) was used as negative control, streptokinase (2 ug) as positive control. The novel fibrinolytic peptide (100 ng) produced 70% clot lysis as compared with positive control (100%) streptokinase. Thus the novel fibrinolytic peptide possessed clotlytic activity.
Example 6 Plasma recaicification activity :
Minimum clotting dose of plasma (MCDP) induced by the novel fibrinolytic peptide was assayed in vitro by the method of Theakston, R.D.G and Reid, H.A. (1983). Development of simple standard assay procedures for the characterisation of snake venom. Bullentine World Health Organisation, Geneva, Volume 61, pg 949-956.. Normal human plasma (0..2 ml) was incubated at 37°C for 15 minutes. Clotting was (25 mM, 0.1 ml) and cllotnng time was recorded in presence and absence of test substances. Saline (0.9%) was used as negative control. The novel fibrinolytic peptide (lag) increased
the coagulation time by 3 fold, as compared with negative control. Thus, the novel fibrinolytic peptide showed plasma anticlotting activity.
Example 7
Haemmorhagie activity :
Cutaneous haemmorhagie activity of the novel fibrinolytic peptide was assayed in vitro in male swiss albino mice (20 gm) after the method of Kondo, H., Kondo, S., Ikezawa, H., Murata, R., and Ohsaka, A. (1969). Studies on the quantitative method of detennination of haemmorhagic activity of Habu Snake venom. Japanese journal of medical Science and Biology, Volume 13, pg 43-51. Minimum haemmorhagic dose (MHD) was defined as the amount to test substance when injected intradermally, produced a haemmorhagic spot of 10 mm diameter within 24 hours of observation. Saline (0.9%) was used as negative control, Russells viper venom (5 µg) was used as positive control. The novel fibrinolytic peptide (lµg) did not produced any haemorrhagic spot observed within 24 hours, as compared with positive control (10 mm diameter) Russell's viper venom. Thus the novel fibrinolytic peptide was found to be non haemorrhagie in nature.
Example 8 Phospholipase A activity :
Phospholipase A activity of the novel fibrinolytic peptide was assayed by the egg-yolk coagulation method of Neumann, W. and Habermann, E (1954). Beitrage zur charakteriseierung der wirkstoffe des bienengiftes. Archives of Experimental pathology and pharmacology, Volume 222, pg 367-370. 2 ml Egg-suspension (9 ml egg yolk, 2.51 ml 2% sodium chloride, 1.49 ml of 0.5% EDTA, 4.44 ml of 1% CaC12, 2.0 ml Tris-HCl buffer 50 mM, pH 7.5 and 0.56 ml of 0.85% NaCl.) and test sample were taken in test tube (12 + 75 mm), mixed and incubated at 37°C for 1 hour. The tubes were placed in a boiling water bath and time to coagulate the suspension was recorded. Saline (0.9%) was used as negative control, Russell's viper venom was used as positive control, of the novel fibrinolytic peptide (lug) induced coagulation time of the egg-yolk suspension was same as negative control (less than 1 min) indicating absence of phospholipase activity. Thus, the novel fibrinolytic peptide did not possess phospholipase activity.
Example 9 Haemolytic activity :
Haemolytic activity of the novel fibrinolytic peptide (l0ng - lµg) was assayed in vitro by incubating with 1 ml 1% human RBC suspension at 37°C for 30 min. RBC suspension was centrifuged 900 g for 30 mins and degree of lysis was measured at A540 nm against negative control (saline 0.9%) and positive control 100%(distilled water). The fibrinolytic peptide did not haemolyse human RBC. So, the novel fibrinolytic peptide was non-haemolytic in nature.
It may be concluded from the above that a novel fibrinolytic peptide has been purified from the venom of the Indian snake King Cobra (Ophiophagus hannah). The peptide had a molecular weight of 610 daltons. This novel fibrinolytic peptide possesses strong defibrinogenating, fibrinolytic, fibrinogenolytic and plasma anticlotting activity. The novel fibrinolytic peptide is devoid of phospholipase activity, haemorrhagic and haemolytic activity. The main advantages of the present invention are :
(1) The novel fibrinolytic peptide has been purified from King Cobra venom, the snake being distributed in India (Sunderbans, north east hilly regions), Bangladesh, Myannmar, Thailand, Combodia Vietnam. (Endoglyphs and other major venomous snakes of the World. A checklist Golay P., Smith H.M., Bloodley D.G., Dixon F.R., McCarthy C, Page J.C., Schatti B., Toriba I.M., Azemiops, 1993).
(2) The novel fibrinolytic peptide agent has been purified using easy and cheaper conventional methods.
(3) The novel fibrinolytic peptide posseses anticoagulant property.

(4) The novel fibrinolytic peptide had no phospholipase activity, was non-haemorrhagic and non-haemolytic in nature.
(5) The yield obtained was of the order of 0.04 +/- 0.5 %.

We Claim:
1. A process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king
cobra ( Ophiophagus hannah ) having a molecular weight of 610 daltons and amino acid
sequence asparate - glutamate glycin - alanine- leucine , devoid of phospholipase activity,
haemorrhagic activity, haetnolytic activity and useful for pharmacological purposes which
comprises:
(i) obtaining venom of snake king cobra by conventional methods;
(ii) fractionating venom, so obtained, by conventional chromatographic methods using
solvent system isopropanol: 0.1N HC1 (7 :3 v v) and obtaining spoion chromatographic
plate . (iii) isolating an active compound with 0.04% yield from a spot obtained at step (ii) and
visualized at 254nm in UV chamber such as herein described, (iv) purifying the said active compound obtained in step (iii) , using high performance liquid
chromatography to get the desired novel fibrinolytic peptide.
2. A process for the preparation of a novel fibrinolytic peptide from venom of snake Indian king
cobra (Ophiophagus hannah) as described herein with reference to the example no. 1

Documents:

2384-del-1998-abstract.pdf

2384-del-1998-claims cancelled.pdf

2384-del-1998-claims.pdf

2384-del-1998-complete specification(granted).pdf

2384-del-1998-correspondence-others.pdf

2384-del-1998-correspondence-po.pdf

2384-del-1998-description (complete).pdf

2384-del-1998-form-1.pdf

2384-del-1998-form-2.pdf

2384-del-1998-form-3.pdf

2384-del-1998-form-9.pdf

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Patent Number

189277

Indian Patent Application Number

2384/DEL/1998

PG Journal Number

4/2003

Publication Date

25-Jan-2003

Grant Date

15-Jan-2004

Date of Filing

13-Aug-1998

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	PALLABI DE	PHYSIOLOGY, UNIVERSITY OF CALCUTTA, INDIA.
2	ANTONY GOMES	PHYSIOLOGY, UNIVERSITY OF CALCUTTA, INDIA.

PCT International Classification Number

A61K 31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA