Title of Invention	"A PROCESS OF PREPARATION OF ANTICOAGULANT AND ANTIPLATELET AGGREGATION AGENT FROM THE EPICARP, PULP AND SEED WITH PULP OF POMEGRANATE (PUNICA GRANATUM).
Abstract	Preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum). Separating the epicarp, pulp and seed with pulp from the ripe fruits of pomegranate. Drying the epicarp, pulp and seed with pulp separately at room temperature (25±2°C) and crushing them in a pastle and mortor to make powder. Dipping powders of all the three parts separately in a sterile distilled water in the ratio of I: 10 (W/V) and incubating at room temperature (25± 2°C) for 24 h and shaking the flasks containing the powder in distilled water several times. Filtering the aqueous extracts through cotton and evaporating the filtrate from each part on water bath at 80° C. Powdering the solid precipitate in a pastle and mortor for testing anticoagulant and antiplatelet aggregation activities.

Title of Invention

"A PROCESS OF PREPARATION OF ANTICOAGULANT AND ANTIPLATELET AGGREGATION AGENT FROM THE EPICARP, PULP AND SEED WITH PULP OF POMEGRANATE (PUNICA GRANATUM).

Abstract

Preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum). Separating the epicarp, pulp and seed with pulp from the ripe fruits of pomegranate. Drying the epicarp, pulp and seed with pulp separately at room temperature (25±2°C) and crushing them in a pastle and mortor to make powder. Dipping powders of all the three parts separately in a sterile distilled water in the ratio of I: 10 (W/V) and incubating at room temperature (25± 2°C) for 24 h and shaking the flasks containing the powder in distilled water several times. Filtering the aqueous extracts through cotton and evaporating the filtrate from each part on water bath at 80° C. Powdering the solid precipitate in a pastle and mortor for testing anticoagulant and antiplatelet aggregation activities.

Full Text	Field of invention A Process of preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum). Acute myocardial infarction (AMI) is one of the most common diseases resulting into death. In United States alone, 1.1 million AMIs occur each year. The mortality rate with AMI is approximately 30% with more than half of these deaths occurring before the stricken individual reaches the hospital. Although the mortality rate after admission for AMI has declined by about 30% over the last two decades, approximately 1 of every 25 patients who survives initial hospitalization dies in the first year after AMI. Survival is markedly reduced in elderly patients (Over 75), whose mortality rate is 20% at one month and 30% at one year after AMI. The pathogenesis of cardiovascular diseases is responsible for 30% of all deaths in developing and developed countries. Besides, thrombus formation in the arteries of brain can also cause stoke and death. Post operative formation of thrombus in the injured arteries can produce pulmonary embolism or embolism in the coronary arteries and thus produce AMI and death. Generally AMI occurs when coronary blood flow decreases abruptly after thrombotic occlusion of a coronary artery previously narrowed by atherosclerosis; The thrombus develops rapidly at the site of vascular injury. This may be multifactorial, but in most cases, infarction occurs when an atherosclerotic plaque fissures, ruptures or ulcerates and when conditions (Local or systemic) favor thrombogenesis, so that a mural thrombus forms at the site of rupture and leads to coronary artery occlusion. After an initial platelet monolayer forms at the site of the ruptured plaque, various agonists (Collagen, ADP, epinephrine, serotonin) promote platelet activities. After agonist stimulation of platelets, there is production and release of thromboxane A2 (a potent local vasoconstrictor), further platelet activation and potential resistance to thrombolysis. The coagulation cascade is activated on exposure of tissue factor in damaged endothelial cells at the site of the ruptured plaque. Factors VII and X are activated, ultimately leading to the conversion of prothrombin to thrombin, which then converts fibrinogen to fibrin. Fluid phase and clot-bound thrombin participate in an auto amplification reaction that leads to further activation of the coagulation cascade. The culprit coronary artery eventually becomes occluded by a thrombus containing platelet aggregates and fibrin strands. In order to avoid and break this vast chain of mechanisms leading to coronary artery blockade which results into acute myocardial infarction and hence death in quite a high percentage of cases, thrombolytic and antithrombotic agents are required to prevent thrombus formation to stop extension of the thrombus. Thrombolysis The principal goal of thrombolysis is prompt restoration of coronary arterial patency. The thrombolytic agents such as tissue plasminogen activator (tPA), streptokinase, anisoxylated plasminogen streptokinase activator complex (APSAC) and reteplase (rPA), all act by promoting the conversion of plasminogen to plasmin, which subsequently lyses fibrin thrombi. This therapy can reduce the relative risk of in hospital death by up to 50% when administered within the first hour of the onset of symptoms of AMI. Antithrombotic Agents: Thrombosis plays an important role in the pathogenesis of acute Myocardial Infarction and strokes due to vascular insufficiency. The primary goal of treatment with antiplatelet and antithrombin agents is to establish and maintain patency of the infarct- related artery. A secondary goal is to reduce the patient's tendency to thrombosis and thus the likelihood of mural formation or deep venous thrombosis, either of which can result in pulmonary embolisation. The degree to which antiplatelet and antithrombin therapy achieves these goals, partly determines how effectively it reduces the risk of mortality from AMI. At present aspirin is the standard antiplatelet agent tor patients with AMI and for the prevention of stroke and death due to cardiovascular diseases. This is now considered an essential element in the management of patients with suspected AMI and is effective across the entire spectrum of acute coronary syndromes. Buccal absorption of chewed aspirin causes rapid inhibition of cyclooxygenase in platelets followed by a reduction of thromboxane A2 levels. This is followed by regular daily oral administration of the drug, though it has many side effects. Hepain is a mucopolysaccharide which is prepared commercially from a variety of animal tissues. Unfractionated heparin (UFH) is the standard antithrombin agent used in clinical practice, though its precise role in patients treated with thrombolytic agents remains uncertain. Though the available data does not show any convincing benefit of UFH with respect to either coronary arterial patency or mortality rate when added to a regimen of aspirin and a non-fibrin specific thrombolytic agent such as streptokinase, it appears that the immediate administration of intravenous UFH, in addition to a regimen of aspirin and tPA, helps to facilitate thrombolysis and to establish and maintain patency of the infarct- related artery. Though it is regularly used post-operatively or after myocardial infarction to prevent thrombus formation or to stop further extension of thrombus, it has many side effects such as thrombocytopenia, osteoporosis, hyper-sensitivity and transient alopecia. Low-molecular weight heparin preparations (LMWHS) are being used with frequency as alternative to UFH for anticoagulation in patients with AMI. Thus, these are the drugs used in clinical practice as thrombolytic and antithrombotic agents. But all these drugs are prescribed only after the problem of thrombosis starts in coronary arteries. Post problems, aspirin is continued for an extended period as a prophylactic measure but not heparin. Thus, till todate there is no drug available which can be given as a prophylactic measure to the public without causing any side effects. Recently, this antiplatelet aggregation activity has been observed in some fruits and vegetables also which are still not in therapeutic use. Since the present proposed drug is prepared from a highly nutritious edible fruit pomegranate (Punica granatum) it can very safely be given as a prophylactic measure even without deciding probability of the disease. It will prevent thrombus formation in the arteries by its antiplatelet aggregation and anticoagulant properties and protect from acute myocardial infarction caused by thrombus formation in the coronary arteries. 4. Description A process of preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum). The objective of the present invention is to prepare an antithrombotic drug which can be safely given by oral route, hence making the application easier and safer. For this preparation a variety of pomegranate known as "Ganesh" was selected. This is cultivated in tropical and subtropical parts of the world and belongs to the family Punicaceae and is supposed to be highly nutritious. Accordingly the present invention provides a process of preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum), comprising the steps of a. Separating the epicarp, pulp and seed with pulp from the ripe fruits of pomegranate. b. Drying the epicarp. pulp and seed with pulp separately at room temperature (25±2°C) and crushing them in a pastle and mortar to make powder. c. Dipping powders of all the three parts separately in a sterile distilled water in the ratio of 1 :10 (W/N) and incubating at room temperature (25± 2°C) for 24h and shaking the flasks containing the powder in distilled water several times. d. Filtering the aqueous extracts through cotton and evaporating the filtrate from each part on water bath at 800 c. e. Powdering the solid precipitate in a pastle and mortor for testing anticoagulant and antiplatelet aggregation activities. These extracts were weighed and added in glass viais (5 ml capacity) in the amount adjusted to make 0.25, 0.5, 1.5, 2.0, 2.5, 3.0 and 4% in 2 ml human blood. Empty vials were used as controls for coagulation of blood and vials containing EDTA (Ethylene Diamine Tetrachlor Acetic Acid) to make 0.5% in 2 ml blood were used as control for anticoagulant activity. In each vial containing different concentrations of dehydrated aqueous extract of epicarp, pulp and seed with pulp, 2 ml human blood from volunteers was added. It was also added in empty vials and vials containing EDTA. In empty vials the blood coagulated immediately. In vials containing epicarp extract, anticoagulation activity was observed even at 0.25%, which was the lowest percentage used. It was comparable to the vials containing EDTA. The epicarp showed its complete anticoagulant activity in all the percentages used. The dehydrated aqueous extract of the pulp started showing its anticoagulation activity from 2% onwards. In lower concentrations clots were formed. The dehydrated aqueous extract of seed with pulp started showing anticoagulant activity from 3.5 % onwards. In 3% only a few clots were formed whereas in 2 and 2.5% there was complete clotting of blood comparable to that in empty vials. Similar results were obtained with rat and rabbit blood also. Antiplatelet aggregation activity The antiplatelet aggregation activity of all the three samples (epicarp, pulp and seed with pulp) of pomegranate was tested on human blood as follows: Venous blood was collected from volunteers who had not taken any medication for at least fifteen days before donation. Blood was collected in plastic syringes through sterilized needles. The coagulation of blood, was prevented by mixing 9 volumes of blood with 1 volume of 3.8% sodium citrate. These samples were centrifuged for 15 minutes at 180 x g and the supernatant platelet rich plasma (PRP) was collected. The PRP (40 I) Was aggregated with epinephrine (10 MO in an aggregometer (Chronologue Model, 600) at 37°C at the stirring speed of 1200 rpm keeping platelet poor plasma (PPP) in the blank cuvet. For experiments with drugs, the PRP (40 µl) was preincubated with aqueous extract of epicarp (10 µl) in 2%, pulp (10 pi) in 3% and seed with pulp (10 µl) in 3% for 5 minutes in 37°C for each prior to addition of the aggregating agent (epinephrine). In these concentrations of the samples, complete inhibition of platelet aggregation was observed. (Fig.1). These experiments have been repeated several times with similar results. Hence, it can be developed pharmaceutically as a medicine for preventing and treating thrombotic diseases in clinical practice. As it is derived from an edible fruit, it can be safely given to patients orally to prevent or treat thrombotic diseases. It can also be consumed by general public as a prophylactic measure with benefits without being under observation of a doctor. We claim 1. A process of preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate Punica grnatum), comprising the steps of: a. Separating the epicarp, pulp and seed with pulp from the ripe fruits of pomegranate. b. Drying the epicarp, pulp and seed with pulp separately at room temperature (25±2° C) and crushing them in a pastle and mortor to make powder. c. Dipping powders of all the three parts separately in a sterile distilled water in the ratio of 1:10 (W/V) and incubating at room temperature (25±2°C) for 24 h and shaking the flasks containing the powder in distilled water several times. d. Filtering the aqueous extracts through cotton and evaporating the filtrate from each part on water bath at 80° C. e. Powdering the solid precipitate in a pastle and mortor for testing anticoagulant and antiplatelet aggregation activities. 2. A process for the preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Tunica granatum), substantially as herein described with the specified figures and drawings.

Full Text

Field of invention
A Process of preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum).
Acute myocardial infarction (AMI) is one of the most common diseases resulting into death. In United States alone, 1.1 million AMIs occur each year. The mortality rate with AMI is approximately 30% with more than half of these deaths occurring before the stricken individual reaches the hospital. Although the mortality rate after admission for AMI has declined by about 30% over the last two decades, approximately 1 of every 25 patients who survives initial hospitalization dies in the first year after AMI. Survival is markedly reduced in elderly patients (Over 75), whose mortality rate is 20% at one month and 30% at one year after AMI. The pathogenesis of cardiovascular diseases is responsible for 30% of all deaths in developing and developed countries.
Besides, thrombus formation in the arteries of brain can also cause stoke and death. Post operative formation of thrombus in the injured arteries can produce pulmonary embolism or embolism in the coronary arteries and thus produce AMI and death.
Generally AMI occurs when coronary blood flow decreases abruptly after thrombotic occlusion of a coronary artery previously narrowed by atherosclerosis; The thrombus develops rapidly at the site of vascular injury. This may be multifactorial, but in most cases, infarction occurs when an atherosclerotic plaque fissures, ruptures or ulcerates and when conditions (Local or systemic) favor thrombogenesis, so that a mural
thrombus forms at the site of rupture and leads to coronary artery
occlusion. After an initial platelet monolayer forms at the site of the ruptured plaque, various agonists (Collagen, ADP, epinephrine, serotonin) promote platelet activities. After agonist stimulation of platelets, there is production and release of thromboxane A2 (a potent local vasoconstrictor), further platelet activation and potential resistance to thrombolysis.
The coagulation cascade is activated on exposure of tissue factor in damaged endothelial cells at the site of the ruptured plaque. Factors VII and X are activated, ultimately leading to the conversion of prothrombin to thrombin, which then converts fibrinogen to fibrin. Fluid phase and clot-bound thrombin participate in an auto amplification reaction that leads to further activation of the coagulation cascade. The culprit coronary artery eventually becomes occluded by a thrombus containing platelet aggregates and fibrin strands.
In order to avoid and break this vast chain of mechanisms leading to coronary artery blockade which results into acute myocardial infarction and hence death in quite a high percentage of cases, thrombolytic and antithrombotic agents are required to prevent thrombus formation to stop extension of the thrombus.
Thrombolysis
The principal goal of thrombolysis is prompt restoration of coronary arterial patency. The thrombolytic agents such as tissue plasminogen activator (tPA), streptokinase, anisoxylated plasminogen streptokinase activator complex (APSAC) and reteplase (rPA), all act by promoting the conversion of plasminogen to plasmin, which subsequently lyses fibrin thrombi. This therapy can reduce the relative risk of in hospital death by up to 50% when administered within the first hour of the onset of symptoms of AMI.
Antithrombotic Agents:
Thrombosis plays an important role in the pathogenesis of acute Myocardial Infarction and strokes due to vascular insufficiency. The primary goal of treatment with antiplatelet and antithrombin agents is to establish and maintain patency of the infarct- related artery. A secondary goal is to reduce the patient's tendency to thrombosis and thus the likelihood of mural formation or deep venous thrombosis, either of which can result in pulmonary embolisation. The degree to which antiplatelet and antithrombin therapy achieves these goals, partly determines how effectively it reduces the risk of mortality from AMI. At present aspirin is the standard antiplatelet agent tor patients with AMI and for the prevention of stroke and death due to cardiovascular diseases. This is now considered an essential element in the management of patients with suspected AMI and is effective across the entire spectrum of acute coronary syndromes. Buccal absorption of chewed aspirin causes rapid inhibition of cyclooxygenase in platelets followed by a reduction of thromboxane A2 levels. This is followed by regular daily oral administration of the drug, though it has many side effects.
Hepain is a mucopolysaccharide which is prepared commercially from a variety of animal tissues. Unfractionated heparin (UFH) is the standard antithrombin agent used in clinical practice, though its precise role in patients treated with thrombolytic agents remains uncertain. Though the available data does not show any convincing benefit of UFH with respect to either coronary arterial patency or mortality rate when added to a regimen of aspirin and a non-fibrin specific thrombolytic agent such as streptokinase, it appears that the immediate administration of intravenous UFH, in addition to a regimen of aspirin and tPA, helps to facilitate thrombolysis and to establish and maintain patency of the infarct- related artery.
Though it is regularly used post-operatively or after myocardial infarction to prevent thrombus formation or to stop further extension of thrombus, it has many side effects such as thrombocytopenia, osteoporosis, hyper-sensitivity and transient alopecia.
Low-molecular weight heparin preparations (LMWHS) are being used with frequency as alternative to UFH for anticoagulation in patients with AMI.
Thus, these are the drugs used in clinical practice as thrombolytic and antithrombotic agents. But all these drugs are prescribed only after the problem of thrombosis starts in coronary arteries. Post problems, aspirin is continued for an extended period as a prophylactic measure but not heparin. Thus, till todate there is no drug available which can be given as a prophylactic measure to the public without causing any side effects.
Recently, this antiplatelet aggregation activity has been observed in some fruits and vegetables also which are still not in therapeutic use.
Since the present proposed drug is prepared from a highly nutritious edible fruit pomegranate (Punica granatum) it can very safely be given as a prophylactic measure even without deciding probability of the disease. It will prevent thrombus formation in the arteries by its antiplatelet aggregation and anticoagulant properties and protect from acute myocardial infarction caused by thrombus formation in the coronary arteries.
4. Description
A process of preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum).
The objective of the present invention is to prepare an antithrombotic drug which can be safely given by oral route, hence making the application easier and safer. For this preparation a variety of pomegranate known as "Ganesh" was selected. This is cultivated in tropical and subtropical parts of the world and belongs to the family Punicaceae and is supposed to be highly nutritious.
Accordingly the present invention provides a process of preparation of anticoagulant and antiplatelet aggregation agent from the epicarp, pulp and seed with pulp of pomegranate (Punica granatum), comprising the steps of
a. Separating the epicarp, pulp and seed with pulp from the ripe fruits
of pomegranate.
b. Drying the epicarp. pulp and seed with pulp separately at room
temperature (25±2°C) and crushing them in a pastle and mortar to
make powder.
c. Dipping powders of all the three parts separately in a sterile distilled
water in the ratio of 1 :10 (W/N) and incubating at room temperature
(25± 2°C) for 24h and shaking the flasks containing the powder in
distilled water several times.
d. Filtering the aqueous extracts through cotton and evaporating the
filtrate from each part on water bath at 800 c.
e. Powdering the solid precipitate in a pastle and mortor for testing
anticoagulant and antiplatelet aggregation activities.
These extracts were weighed and added in glass viais (5 ml capacity) in the amount adjusted to make 0.25, 0.5, 1.5, 2.0, 2.5, 3.0 and 4% in 2 ml
human blood. Empty vials were used as controls for coagulation of blood and vials containing EDTA (Ethylene Diamine Tetrachlor Acetic Acid) to make 0.5% in 2 ml blood were used as control for anticoagulant activity.
In each vial containing different concentrations of dehydrated aqueous extract of epicarp, pulp and seed with pulp, 2 ml human blood from volunteers was added. It was also added in empty vials and vials containing EDTA. In empty vials the blood coagulated immediately. In vials containing epicarp extract, anticoagulation activity was observed even at 0.25%, which was the lowest percentage used. It was comparable to the vials containing EDTA.
The epicarp showed its complete anticoagulant activity in all the percentages used. The dehydrated aqueous extract of the pulp started showing its anticoagulation activity from 2% onwards. In lower concentrations clots were formed. The dehydrated aqueous extract of seed with pulp started showing anticoagulant activity from 3.5 % onwards. In 3% only a few clots were formed whereas in 2 and 2.5% there was complete clotting of blood comparable to that in empty vials. Similar results were obtained with rat and rabbit blood also.
Antiplatelet aggregation activity
The antiplatelet aggregation activity of all the three samples (epicarp, pulp and seed with pulp) of pomegranate was tested on human blood as follows:
Venous blood was collected from volunteers who had not taken any medication for at least fifteen days before donation. Blood was collected in plastic syringes through sterilized needles. The coagulation of blood, was prevented by mixing 9 volumes of blood with 1 volume of 3.8% sodium
citrate. These samples were centrifuged for 15 minutes at 180 x g and the supernatant platelet rich plasma (PRP) was collected. The PRP (40 I) Was aggregated with epinephrine (10 MO in an aggregometer (Chronologue Model, 600) at 37°C at the stirring speed of 1200 rpm keeping platelet poor plasma (PPP) in the blank cuvet. For experiments with drugs, the PRP (40 µl) was preincubated with aqueous extract of epicarp (10 µl) in 2%, pulp (10 pi) in 3% and seed with pulp (10 µl) in 3% for 5 minutes in 37°C for each prior to addition of the aggregating agent (epinephrine). In these concentrations of the samples, complete inhibition of platelet aggregation was observed. (Fig.1).
These experiments have been repeated several times with similar results. Hence, it can be developed pharmaceutically as a medicine for preventing and treating thrombotic diseases in clinical practice. As it is derived from an edible fruit, it can be safely given to patients orally to prevent or treat thrombotic diseases. It can also be consumed by general public as a prophylactic measure with benefits without being under observation of a doctor.

We claim
1. A process of preparation of anticoagulant and antiplatelet aggregation agent
from the epicarp, pulp and seed with pulp of pomegranate Punica grnatum),
comprising the steps of:
a. Separating the epicarp, pulp and seed with pulp from the ripe fruits of
pomegranate.
b. Drying the epicarp, pulp and seed with pulp separately at room temperature
(25±2° C) and crushing them in a pastle and mortor to make powder.
c. Dipping powders of all the three parts separately in a sterile distilled water in
the ratio of 1:10 (W/V) and incubating at room temperature (25±2°C) for 24 h
and shaking the flasks containing the powder in distilled water several times.
d. Filtering the aqueous extracts through cotton and evaporating the filtrate from
each part on water bath at 80° C.
e. Powdering the solid precipitate in a pastle and mortor for testing anticoagulant
and antiplatelet aggregation activities.
2. A process for the preparation of anticoagulant and antiplatelet aggregation
agent from the epicarp, pulp and seed with pulp of pomegranate (Tunica
granatum), substantially as herein described with the specified figures and
drawings.

Documents:

896-del-2003-abstract.pdf

896-del-2003-claims.pdf

896-del-2003-correspondence-others.pdf

896-del-2003-correspondence-po.pdf

896-del-2003-description (complete).pdf

896-del-2003-drawings.pdf

896-del-2003-form-1.pdf

896-del-2003-form-13.pdf

896-del-2003-form-19.pdf

896-del-2003-form-2.pdf

896-del-2003-form-3.pdf

896-del-2003-form-4.pdf

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Patent Number

220096

Indian Patent Application Number

896/DEL/2003

PG Journal Number

28/2008

Publication Date

11-Jul-2008

Grant Date

15-May-2008

Date of Filing

14-Jul-2003

Name of Patentee

SINGH UDAI PRATAP

Applicant Address

A-2, NEW MEDICAL ENCLAVE, BANARAS HINDU UNIVERSITY, VARANASI-221005, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SINGH UDAI PRATAP
2	SINGH MANDAVI

PCT International Classification Number

A61P 7/02

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA