EP3585407A2 - Herbo-mineral formulation for the treatment of cardio vascular diseases and method of preparation thereof - Google Patents
Herbo-mineral formulation for the treatment of cardio vascular diseases and method of preparation thereofInfo
- Publication number
- EP3585407A2 EP3585407A2 EP18757495.9A EP18757495A EP3585407A2 EP 3585407 A2 EP3585407 A2 EP 3585407A2 EP 18757495 A EP18757495 A EP 18757495A EP 3585407 A2 EP3585407 A2 EP 3585407A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- formulation
- dry
- bhasma
- present
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/32—Burseraceae (Frankincense family)
- A61K36/328—Commiphora, e.g. mecca myrrh or balm of Gilead
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/59—Menispermaceae (Moonseed family), e.g. hyperbaena or coralbead
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Definitions
- Cardiovascular diseases have been observed to be one of the leading causes of death globally. It is a group of diseases that are associated with heart and blood vessels. It includes diseases such as Coronary artery disease, Cardiovascular disease, hypertensive heart disease, peripheral arterial disease, etc.
- Atherosclerosis a condition wherein plaque builds-up in the arteries, is a leading cause of CVD.
- the risk factors of CVD include high blood pressure, hypertension, stress, hyperlipidemia, Diabetes, physical inactivity, Obesity, etc. These are the risk factors that may be regulated to prevent CVDs. There also exists other risk factors such as old age, gender, family history, etc. that cannot be regulated.
- CVDs can be prevented by mitigating the established risk factors.
- Implementation of certain lifestyle modifications such as maintaining a healthy diet, limited alcohol consumption, tobacco cessation, reduced sugar consumption, stress management, etc alone can prove helpful in mitigating the risk of developing CVDs.
- lifestyle modifications prove to be inadequate in preventing CVD, medication or medical procedure may be necessary.
- Cardiovascular Diseases Many herbal formulations have been developed based on the knowledge of the healing properties of various herbs. However, the effectiveness of such formulations is arguable. There exists a need for an effective method of treating/managing Cardiovascular Diseases. OBJECTS OF THE DISCLOSED EMBODIMENTS
- the principal object of the embodiments disclosed herein is to provide a composition and method for treating Cardio vascular diseases.
- a second object of the embodiments disclosed herein is to provide a composition and method for preventing Cardio vascular diseases.
- Another object of the embodiments disclosed herein is to provide a herbo- mineral formulation and a method for its preparation.
- Fig.1(a) depicts a flowchart for the preparation of Swarna Makshika Bhasma; [007] Fig 1(b) depicts a flowchart for the preparation of Abhraka Bhasma;
- Fig 1(c) depicts a flowchart for the preparation of Loha Bhasma
- Fig 1(d) depicts a flowchart for the preparation of Mukta sukti Bhasma
- Fig 1(e) depicts a flowchart for the preparation of Pravala Bhasma
- Fig 2 depicts a flowchart for the preparation of fortified tablets
- Fig 3 depicts the effect of Test Drug on CK-MB activity
- Fig 4 depicts the effect of Test Drug on Na+K+ATPase
- Fig 5 depicts the effect of Test Drug on Mg2+ATPase
- Fig 6 depicts the effect of Test Drug on Ca2+ATPase
- Fig 7 depicts the effect of Test Drug on Lipid Peroxidation content
- Fig 8 depicts the effect of Test Drug on Superoxide dismutase activity
- Fig 9 depicts the effect of Test Drug on GSH activity
- Fig 10 depicts the effect of Test Drug on GPX activity
- Fig 11 depicts the effect of Test Drug on apoptotic markers
- Fig 12 depicts the effect of Test Drug Systolic Blood pressure
- Fig 13 depicts the effect of Test Drug Diastolic Blood pressure; according to embodiments as disclosed herein.
- cardiovascular Diseases may include any condition associated to heart and blood vessels such as cardiac arrhythmias, ischemic heart diseases, coronary artery disease, valve defects, etc.
- cardiovascular Diseases may include any condition associated to heart and blood vessels such as cardiac arrhythmias, ischemic heart diseases, coronary artery disease, valve defects, etc.
- the complications related to cardiovascular system may be any condition generally known to be related to or considered as risk factors of CVDs including hypertension, increased blood sugar, hyperlipidemia, etc.
- the disclosed formulation also find use as an anti- oxidating, anti-stress, hypolipidemic, atherogenic, antihypertensive, apoptotsis inhibiting and cardio-protective agent. Accordingly, the embodiments disclosed herein achieve a method for the treatment/prevention of Cardiovascular diseases. Also disclosed are embodiments of a method of reducing the risks of Cardiovascular diseases.
- the disclosed embodiments herein provide herbo-mineral formulation having herbs and minerals.
- the herbo-mineral formulation includes a herb component and a mineral component.
- the herbo-mineral formulation includes a herb component, a mineral component and a suitable excipient.
- the herb component includes the herbs Terminalia arjuna, Sida rombifolia, Withania somnifera, Tinospora cordifolia, Punica granatum, Embelia ribes, Rubia cordifolia, Nardostachys jatamansi, Emblica officinalis, Terminalia chebula, Terminalia bellerica, Piper longum, Piper nigrum, Zingiber officinalis, Boerhavia diffusa, Bamboo manna, Madhuka indica, Azhadirachta indica, Picrorhiza kurroa, Holy basil, Commiphora mukul, Steriospermum suaveolens, Premna mucronata, Gmelina arborea, Aegle marmelos, Oroxylum indicum, Desmodium gangeticum, Uraria picta, Solanum indicum and Solanum xanthocarpum or their extracts
- the herb component may include the herb as a whole or may include specific parts of the herb such as roots, fruits, stem, leaves, rhizome, etc.
- the herb component includes stem bark of Terminalia arjuna; root of Sida rombifolia, Withania somnifera, Rubia cordifolia, Nardostachys jatamansi, Boerhavia diffusa, Picrorhiza kurroa, Steriospermum suaveolens, Premna mucronata,Gmelina arborea, Aegle marmelos, Oroxylum indicum, Solanum indicum; fruit of Embelia ribes, Emblica officinalis, Terminalia chebula, Terminalia bellerica, Piper longum, Piper nigrum, Tribulus terrestris; bark of Azhadirachta indica; plant of Solanum xanthocarpum, Uraria picta, Des
- Herbs in whole or part, disclosed herein, maybe included in the formulation in any form that is generally known in the field.
- the herbs may be processed to form extracts, dried, powdered, pelleted, concentrated, etc.
- the herbs are dried and powdered which is further incorporated into the formulation.
- the herb component includes Terminalia arjuna in an amount in the range of 8 to 12 wt%, Sida rombifolia in an amount in the range of 2 to 6 wt%, Withania somnifera in an amount in the range of 2 to 6 wt%, Tinospora cordifolia in an amount in the range of 2 to 6 wt%, Punica granatum in an amount in the range of 2 to 6 wt%, Emblica officinalis in an amount in the range of 2 to 6 wt% and Commiphora mukul in an amount in the range of 2 to 6 wt%.
- the herb component includes atleast one of Embelia ribes, Rubia cordifolia, Nardostachys jatamansi, Terminalia chebula, Terminalia bellerica, Piper longum, Piper nigrum, Zingiber officinalis, Boerhavia diffusa, bamboo manna, Madhuka indica, Azhadirachta indica, Picrorhiza kurroa, Holy basil, Steriospermum suaveolens, Premna mucronata, Gmelina arborea, Aegle marmelos, Oroxylum indicum, Desmodium gangeticum, Uraria picta, Solanum indicum, Solanum xanthocarpum and Tribulus terrestris, preferably inan amount of ⁇ 4 wt%.
- the mineral component includes Bhasmas or calcined preparations such as Swarna Makshika bhasma, Abhraka bhasma, Loha bhasma, Muktasukti bhasma, and Pravala bhasma.
- the mineral component may also be selected from a group consisting of atleast one of Iron, Mica, Copper pyrite and Coral.
- the bhasmas along with the herb component form bioavailable herbo-mineral complexes which are useful in treating Cardio vascular Diseases and related complications.
- the mineral component further includes Shilajit.
- the herbo-mineral formulation it is also within the scope of claims provided herewith for the herbo-mineral formulation to include, as a substitute or additionally, other similar calcined preparations or minerals without otherwise deterring from the intended function of the herbo-mineral formulation.
- the mineral component includes shilajit in the range of 1 to 4 wt%.
- the mineral component includes Muktasukti bhasma in an amount of ⁇ 2 wt%, Loha bhasma in an amount of ⁇ 2 wt%, Abhraka bhasma in an amount of ⁇ 3 wt%, Swarnamaksika bhasma in an amount of ⁇ 2 wt% and Pravala bhasma in an amount of ⁇ 2 wt%.
- the disclosed formulation in the various embodiments herein, may further include a suitable excipient.
- suitable excipients includes solvents, binders, lubricants, herbal carriers, oils and salts that are generally known in the art.
- the excipient includes acacia gum.
- the amount of herb component and mineral component that may be included in the various embodiments of the disclosed formulation may be in the range of 0 to 12 wt%.
- the formulation includes Terminalia arjuna (8 to 12 wt%), Sida rombifolia (2 to 6 wt%), Withania somnifera (2 to 6 wt%), Tinospora cordifolia (2 to 6 wt%), Punica granatum (2 to 6 wt%), Emblica officinalis (2 to 6 wt%) and Commiphora mukul (2 to 6 wt%), Shilajit 1 to 4 wt%), Muktasukti bhasma ( ⁇ 2 wt%), Loha bhasma ( ⁇ 2 wt%), Abhraka bhasma ( ⁇ 3 wt%), Swarnamaksika bhasma ( ⁇ 2 wt%) and Pravala bhasma ( ⁇ 2 wt%).
- the formulation further includes atleast one of Embelia ribes, Rubia cordifolia, Nardostachys jatamansi, Terminalia chebula, Terminalia bellerica, Piper longum, Piper nigrum, Zingiber officinalis, Boerhavia diffusa, bamboo manna, Madhuka indica, Azhadirachta indica, Picrorhiza kurroa, Holy basil, Steriospermum suaveolens, Premna mucronata, Gmelina arborea, Aegle marmelos, Oroxylum indicum, Desmodium gangeticum, Uraria picta, Solanum indicum, Solanum xanthocarpum and Tribulus terrestris, preferably inan amount of ⁇ 4 wt%
- the amount of gum acacia may be any amount suitable to perform the activity of an excipient.
- the formulation may include gum acacia in the range of 0 to 50 mg per 500mg of the formulation, preferably 10 wt%.
- the herbo-mineral formulation disclosed herein may be formulated in various dosage forms such that it is suitable for oral administration.
- the herbo-mineral formulation may be in the form of powder, tablets, pellets, lozenges, granules, capsules, solutions, emulsions, suspensions, or any other form suitable for use.
- the herbo- mineral formulation is formulated in the form of powder suitable for oral administration.
- the herbo-mineral formulation is formulated in the form of tablets, preferably 500 mg tablets.
- Table 1 depicts the quantities of each ingredient in a 500 mg tablet.
- the tablet is a 500mg tablet having herb component, mineral component and an excipient as depicted in Table 1.
- Table 1 - Each 500 mg tablet includes:
- Embodiments of the disclosed herbo-mineral formulation (also referred to as 'drug' or 'test drug') in tablet form were analyzed for phyto constituents, physicochemical etc, by methods generally known in the field. The analysis and results obtained are included hereunder as examples by way of illustration only, and should not be construed to limit the scope of the claims provided herewith. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the claims.
- Phyto constituents study Tests were performed to screen the various phyto constituents such as glycosides, steroids, saponins, proteins, tannins etc.
- Table 3 depicts the results of Qualitative analysis performed for phytoconstituents. The test showed the presence of alkaloids, steroids, glycosides etc which could make the drug capable of curing diseases.
- (+) denotes presence and (-) denotes absence
- the method includes: levigating bhasmas and shilajit in a grinder; adding finely powdered herbs into the grinder; and adding grinding decoction while continuing grinding to obtain a ground mass.
- the bhasmas include atleast one of Muktasukti bhasma, Loha bhasma, Abhraka bhasma, Swarnamaksika bhasma and Pravala bhasma.
- the mixture of bhasmas and Shilajit may be in semi solid form.
- the levigation may be performed for a duration of around 3 hours.
- the finely powdered herbs include finely powdered stem bark of Terminalia arjuna, root of Sida rombifolia, root of Withania somnifera, root of Rubia cordifolia, root of Nardostachys jatamansi, root of Boerhavia diffusa, root of Picrorhiza kurroa, root of Steriospermum suaveolens, root of Premna mucronata, Gmelina arborea, root of Aegle marmelos, root of Oroxylum indicum, root of Solanum indicum, fruit of Embelia ribes, fruit of Emblica officinalis, fruit of Terminalia chebula, fruit of Terminalia bellerica, fruit of Piper longum, fruit of Piper nigrum, fruit of Tribulus terrestris, bark of Azhadirachta indica, plant of Solarium xanthocarpum, plant of Uraria picta, plant of
- the grinding decoction is a decoction of selected herbs (also referred as grinding herbs).
- the grinding decoction is a decoction of one or more grinding herbs selected from a group consisting of: Terminalia arjuna, Asparagus racemosus, Asafetida, Cuminum cyminum, Plumbago rosea, Baliospermum montanum, Ocimum sanctum, Aloevera, Plantago ovata, Steriospermum suaveolens, Premna mucronata, Gmelina arbor ea, Aegle marmelos, Oroxylum indicum, Desmodium gangeticum, Uraria picta, Solanum indicum, Solanum xanthocarpum and Tribulus terrestris.
- the decoction may be obtained by any method of decocting generally known in the field.
- the method of preparation of grinding decoction includes, soaking the grinding herbs; for example: soaking powdered dry bark of Terminalia arjuna, fresh root of Asparagus racemosus, resin Asafetida, dry fruit of Cuminum cyminum, dry root of Plumbago rosea, dry root of Basospermum montanum, dry leaves of Ocimum sanctum, fresh leaves of Aloevera, dry seeds of Plantago ovata, dry roots of Steriospermum suaveolens, dry roots of Premna mucronata, dry root of Gmelina arborea, dry root of Aegle marmelos, dry root of Oroxylum indicum, dry plant of Desmodium gangeticum, dry plant of Uraria picta, dry root of Solanum indicum, dry plant of Solanum xanthocarpum and dry fruit of Tribulus terres
- soaking may be performed by soaking the grinding herbs in 16 parts of water overnight.
- concentrating may be performed by boiling at high temperature, preferably about 80°C to 85°C, until l/8th of the liquid remains. Concentration may be confirmed with the help of Brix meter.
- the method of preparation may further include adding excipient to the ground mass, wherein gum acacia may be added to the ground mass by dissolving in the grinding decoction, while continuing grinding for 3 hours to obtain a semisolid mass.
- the method of preparation may further include drying at 50°C-60°C, preferably in a hot air oven; wet granulating; and punching to obtain 500mg tablets.
- Fig.2 depicts a flowchart for the preparation of fortified tablets.
- Table 4 depicts an embodiment of the Herbs required for grinding (grinding herbs).
- the bhasmas that are used in the various embodiments of the disclosed herbo- mineral formulation may be prepared by methods that are generally known in the field.
- Bhasmas may be prepared by selecting genuine standard minerals as starting material such as Swarnamakshika, Mica, Iron, pearl oyster etc; drying in a hot air oven; purifying the mineral by triturating, quenching, boiling, etc; triturating with herbal decoction; preparing into discs; drying of discs; preparing sharavasam puta, subjecting Sharavasam puta to Gaja puta, and powdering of discs once cooled.
- the powder may again be subjected to many repetitions of trituration with herbal decoction followed by preparing into discs; drying of discs; preparing sharavasam puta, subjecting Sharavasam puta to Gaja puta, and powdering, until bhasma is obtained.
- the number of repetitions may vary from 0 to 30 times. In an embodiment, the method is repeated as many as 30 times till bhasma is obtained.
- the starting materials used in the preparation of bhasmas may include standard minerals generally used in the field.
- the preparation of Swarna Makshika Bhasma includes swarna makshika as the starting material.
- Fig. 1(a) depicts a flowchart for the preparation of Swarna Makshika Bhasma using swarna makshika as the starting material.
- the preparation of Abhraka Bhasma includes Mica as the starting material.
- Fig. 1(b) depicts a flowchart for the preparation of Abhraka Bhasma using Mica as the starting material.
- the preparation of Loha Bhasma includes steel iron as the starting material.
- Fig. 1(c) depicts a flowchart for the preparation of Loha Bhasma using steel iron as the starting material.
- the preparation of Muktasukti Bhasma includes pearl oyster as the starting material.
- Fig. 1(d) depicts a flowchart for the preparation of Muktasukti Bhasma using alloys of Pearl oyster as the starting material.
- the preparation of Pravala Bhasma includes Coral as the starting material.
- Fig. 1(e) depicts a flowchart for the preparation of Pravala Bhasma using Coral as the starting material.
- purification of mineral includes mixing the mineral with rocksalt and lemon juice and heating strongly till partially oxidized into reddish powder which is further be used in the preparation of Swarna makshika Bhasma.
- purification of mineral includes quenching of mineral in Cow's milk wherein it is further used in the preparation of Abhraka Bhasma.
- purification of mineral includes quenching of mineral in Triphala decoction which is further used in the preparation of Loha Bhasma.
- purification of mineral includes quenching of mineral in Kanjika (sour medicated gruel) which is further used in the preparation of Mukta sukti Bhasma.
- purification of mineral includes boiling the mineral in alkaline solution of Barilla which is further used in the preparation of Pravala Bhasma.
- the herbal decoction used may be any herbal decoction that is generally used for triturating in the preparation of bhasmas.
- the herbal decoction includes atleast one of Nimbu Swarasa (Lemon juice) and Kulatha Kwatha (Decoction of Dolichos biflorus), wherein it is useful in the preparation of Swarna Makshika bhasma.
- the herbal decoction includes atleast one of Arka Ksheera (Latex of calotropes procera), Snuhi Ksheera (Latex of Euphorbia neriifolia), Vata Ksheera (Latex of Ficus bengalensis), Kakamachi Rasa (fresh juice of Solanum nigrum whole plant), Gokshura Kwatha (decoction of tribulus terrestris fruits), Apamarga Rasa (Juice of Achyranthus aspera plant),Vata Praroha Swarasa (juice of aerial root of Ficus bengalensis), Gomutra (Cow urine), Tulasi Swarasa (Fresh juice of Ocimum sanctum leaves), Kadali Shipha Jala (Juice of plantain rhizome), Eranda patra rasa (Juice of Ricinus communis leaves), and Guda (Jaggery), wherein it is useful in the preparation
- the herbal decoction includes Triphala Kashaya (decoction of fruits of Terminalia chebula, Terminalia bellerica and Emblica officinalis), wherein it is useful in the preparation of Loha Bhasma.
- the herbal decoction includes Aloe Vera juice, wherein it is useful in the preparation of Muktasukti Bhasma.
- the herbal decoction includes atleast one of Aloe vera (fresh juice of leaves), Sesbania seban (fresh juice of leaves), Asparagus racemosus (fresh juice of roots) and Cow milk, wherein it is useful in the preparation of Pravala Bhasma.
- the method includes administering to a patient a composition having a herb component, a mineral component and a suitable excipient, wherein the herb component includes the herbs Terminalia arjuna (8 to 12 wt%), Sida rombifolia (2 to 6 wt%), Withania somnifera (2 to 6 wt%), Tinospora cordifolia (2 to
- the mineral component includes Shilajit (1 to 4 wt%), and atleast on of Muktasukti bhasma ( ⁇ 2 wt%), Loha bhasma ( ⁇ 2 wt%), Abhraka bhasma ( ⁇ 3 wt%), Swarnamaksika bhasma ( ⁇ 2 wt%) and Pravala bhasma ( ⁇ 2 wt%).
- the patient may be any individual in need of such treatment including ones having/ suspected of having Cardiovascular diseases. Further, the patient may also be any individual having/ suspected of having any complication associated with heart and blood vessels.
- Cardio vascular diseases include cardiac arrhythmias, ischemic heart diseases, coronary artery disease, valve defects, mitral valve prolapse etc. Further, the patient may also be any individual prone to or having risks of Cardiovascular diseases, for example: individuals having hypertension, obesity, increased blood sugar, etc.
- the method of treating/preventing CVD includes administering the Disclosed formulationto a patient wherein the disclosed formulation acts as atleast one of anti-oxidating, anti-stress, hypolipidemic, atherogenic, antihypertensive, apoptotsis inhibiting and cardio-protective agent.
- the disclosed method of treatment may be used as a primary line of treatment or as an adjunct to other CVD treatment methods.
- the method may be instrumental in improving the health conditions of individuals having CVD.
- the dosage of the test drug and the treatment regimen may vary depending on the patient.
- the disclosed formulation was subjected to acute oral toxicity study, and a study to check its effect on behaviour and nervous system. The studies proved that Test drug is free from toxicity even at a dose of 6000mg/kg weight which was the maximum possible dose. It was also found to have no harmful effects on behaviour and nervous system.
- the Disclosed formulation also referred as Test drug or Test product
- Embodiments of the formulation disclosed herein is further described by reference to the following examples by way of illustration only, and should not be construed to limit the scope of the embodiments herein. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the claims.
- the aim of this study was to analyze the Cardioprotective activity of test product on Isoproterenol (ISP) induced experimental model of myocardial infarction.
- ISP Isoproterenol
- the effect of Test drug on the membrane bound enzymes like Na+K+ATPase, Ca2+ATPase and Mg2+ATPase were investigated.
- the action of Test drug on apoptotic and pre-apoptotic markers gene expression in ISP induced myocardial infarction was analyzed. Also, the effect of Test drug on CK-MB activity and oxidative stress markers was evaluated.
- Animals 35 male Sprague-Dawley rats of 150-200g body weight were selected for the study. Animals were housed in individual polycarbonate cages in a well- ventilated room under an ambient temperature of 23+2 degree C and 40-65% relative humidity, with artificial photoperiod 12-h light/ 12-h dark cycle. They were provided with standard rodent pellet diet (Nutrilab Rodent, Tetragon Chemie, India) and purified water ad libitum (RIOS, USA). Experimental animals were acclimatized for 7 days to the laboratory conditions prior to experimentation. The study protocol was approved by Institutional Animal Ethical Committee (IAEC).
- IAEC Institutional Animal Ethical Committee
- mice were randomized into 5 groups based on the body weight. ISP (120 mg/kg) was injected subcutaneously to rats on 19th and 21st day to induce experimental myocardial infarction. Test drug was orally administered throughout the study.
- Group IV Low dose:Test drug(50mg/kg/day,p.o)+ISP(120mg/kg,s.c.)
- Group V High dose:Test drug (100mg/kg/day,p.o)+ISP(120mg/kg, s.c.)
- CK-MB activity was measured by kit method (Spinreact, Spain) using Semi-automated biochemical analyzer.
- FIG. 3 depicts CK-MB activity. Rats induced with ISP showed significant elevation (P ⁇ 0.01) in CK-MB activity when compared to normal rats, whereas pre-treatment with test drug decreased (P ⁇ 0.01) its activity.
- the serum marker enzyme CK-MB in ISP induced rats serves as the index to assess the severity of myocardial injury. This increase in activity is accompanied by their concomitant increase in wet weight of myocardium confirms the onset of apoptotic pathway. Extent of cardio-protection offered by the drug is associated with the significant attenuation of CK-MB activity. Hence, pretreatment with Test Drug significantly possess cardioprotective effect and maintains myocardial membrane integrity.
- KC1 (50mM) 37.275mg in 10ml of distilled water
- ANSA 0.1%): 100mg in 39ml of 15% sodium meta bisulphate. Then 1ml of 20% sodium sulphite was added and the volume was made upto 100ml with distilled water.
- Protocol Na+K+ATPase was assayed by taking 250 ⁇ 1 of tris HC1 buffer followed by the addition of 50 ⁇ 1 of 600mM NaCl, 50 ⁇ 1 of 50mM KCL, along with 50 ⁇ 1 of ImM Na. EDTA, and 50 ⁇ 1 of 80mM ATP. The reaction mixture was pre- incubated at 37°C for lOmins.Then 25 ⁇ 1 of 10% homogenate was added to the test alone and further incubated at 37°C for lhr.The reaction was immediately arrested by the addition of 10% TCA. The control reaction rate was correspondingly assessed by adding 25 ⁇ 1 of 10% homogenate only after arresting the reaction.
- the precipitate was removed by centrifugation at 3500 rpm for 10 minutes.
- 1075 ⁇ 1 of distilled water 125 ⁇ 1 of Ammonium molybdate and 50 ⁇ 1 of ANSA were added and incubated for 10 mins at 37°C.
- the intensity of blue colour was read at 640 nm using spectrophotometer against a blank that contained all the reagents minus the supernatant. The results are expressed in ⁇ g of Pi liberated/min/mg of protein.
- Na + K + ATPase a membrane bound enzyme is responsible for sodium ion influx and potassium ion reflex during muscle contraction and relaxation. Induction of myocardial infarction with ISP inhibited the influx of sodium ions thereby hindering the contraction and relaxation of the heart muscle. Pretreatment with Test drug was comparable with the reference drug, carvedilol in enhancing the Na + K + ATPase activity
- Tris HC1 (0.1M) pH- 7.4 1.576g in 100ml of distilled water
- KC1 (0.1M) 74.55mg in 10ml of distilled water 3.
- MgC12 (0.1M) 200mg in 10ml of distilled water
- ANSA 0.1%) :100mg in 39ml of 15% sodium meta bisulphate. Then 1ml of 20% sodium sulphite was added and the volume was made upto 100ml with distilled water.
- Protocol Total ATPase was assayed by taking 0.75ml of tris HCL buffer followed by the addition of 50 ⁇ 1 of lOOmM KC1, along with 50 ⁇ 1 of lOOmM MgCl 2 , and 50 ⁇ 1 of 80mM ATP. The reaction mixture was pre- incubated at 37°C for 2mins.Then 50 ⁇ 1 of 10% homogenate was added to the test alone and further incubated at 37°C for 20mins. The reaction was immediately arrested by the addition of 500 ⁇ 1 of 10% TCA. Control reaction rate was correspondingly assessed by adding 50 ⁇ 1 of 10% homogenate only after arresting the reaction. The precipitate was removed by centrifugation at 3500 rpm for 10 minutes.
- Mg 2+ ATPase activity It was observed that ISP induced rats showed significant (P ⁇ 0.01) decrease in Mg 2+ ATPase activity when compared to the normal control rats. Whereas, pretreatment with Test drug (50 and lOOmg/kg) showed significant (P ⁇ 0.01) elevation in Mg 2+ ATPase activity irrespective of the doses. Mg 2+ ATPase regulates the intracellular Mg2+ levels. Pretreatment with Test drug was comparable with the reference drug, carvedilol in enhancing the Mg 2+ ATPase activity Example 3(d): Ca2+ ATPase
- ANSA (0.1 %) : 100mg in 39ml of 15% sodium meta bisulphate. Then 1ml of 20% sodium sulphite was added and the volume was made upto 100ml with distilled water.
- Protocol Ca 2+ ATPase was assayed by taking 0.75ml of tris HCL buffer followed by the addition of 50 ⁇ 1 of lOOmM KCl, 50 ⁇ 1 of lOOmM CaCl 2 and 50 ⁇ 1 of 80mM ATP. The reaction mixture was pre- incubated at 37°C for 2mins.Then 50 ⁇ 1 of 10% homogenate was added to the test alone and further incubated at 37°C for 20mins. The reaction was immediately arrested by the addition of 500 ⁇ 1 of 10% TCA. Control reaction rate was correspondingly assessed by adding 50 ⁇ 1 of 10% homogenate only after arresting the reaction. The precipitate was removed by centrifugation at 3500 rpm for 10 minutes.
- Intracellular calcium (Ca 2+ ) levels are maintained and regulated by Ca 2+ ATPase.
- Enhanced Ca 2+ ATPase and intracellular Ca 2+ overload in ISP-treated rats can be correlated to the action of adenylate cyclase.
- Phosphorylation of Ca 2+ channel protein by cAMP is expected to increase the Ca 2+ influx into the myocardium and thus burdening it.
- pre- supplementation with Test drug showed potent resistance to the perturbations in Ca 2+ ATPase caused due to ISP injection.
- TSA ThioBarbituric Acid
- Butylated Hydroxyl Toluene 0.05%):0.05gms in methanol.
- Protocol The method involved heating of 0.5ml of heart homogenate of experimental rats with 0.8ml saline, 0.5ml of BHT and 3.5ml TBA reagent for 11/2 min in a boiling water bath. After cooling, the solution was centrifuged at 2,000 rpm for 10 min and the precipitate obtained was removed. The absorbance of the supernatant was determined at 532 nm using spectrophotometer against a blank that contained all the reagents minus the biological sample. The values were expressed in mg/g tissue (Okhawa H et al., 1979).
- Lipid peroxidation (Thiobarbituric acid reactive substances): Figure 7 depicts LPO Content. ISP induced rats showed significant increase in the levels of heart TBARS when compared to normal control rats. Pretreatment with Test drug showed considerable decrease in the levels of heart TBARS in ISP-induced rats. The results were comparable with that of standard drug, Carvedilol. [0088] ISP treatment is known to produce free radical moieties via its quinine metabolites that react with oxygen ultimately resulting in enhanced generation of Reactive oxygen species (ROS). ROS, the highly toxic by-products of aerobic metabolism are known to react extensively with cell membranes and macromolecules enhancing formation of lipid peroxides thus leading to tissue damage.
- ROS Reactive oxygen species
- Lipid peroxidation is an important pathogenic event in myocardial necrosis and accumulation of lipid hydroperoxides reflects damage of the cardiac constituents.
- MDA a lipid peroxidation end-product
- isoproterenol administration might be due to free radical mediated membrane damage.
- TEST DRUG possess lipid peroxidation inhibitory activity.
- Phenazonium Metho Sulphate (PMS) (186 ⁇ ): 3mg in 10 ml of distilled water (930 ⁇ ). ⁇ 1 :5 dilutions were carried out to obtain 186 ⁇ .
- Tris HCL Buffer(0.4mM) 6.304g in 100ml of distilled water
- Glutathione peroxidase was assayed by taking 200 ⁇ of tris HCL buffer (0.4 M), 0.4 mM K.EDTA along with 100 ⁇ of sodium azide and 200 ⁇ of enzyme preparation (hemolysate) and mixed well. Thereafter, 200 ⁇ of reduced glutathione solution (2 mM) followed by 0.1ml H202 were added The overall reaction was arrested by adding 0.5ml of 10% TCA. The precipitate was removed by centrifugation at 4000rpm for 10 minutes. The absorbance was read at 412 nm using spectrophotometer. The non-enzymatic reaction rate was correspondingly assessed by replacing the enzyme sample by buffer. The results are expressed as mg of GSH consumed/min/mg protein (Rotruck, J et al., 1973). Reduced glutathione [GSH]
- DTNB (0.6mM): 12mg in 50ml P04 buffer.
- Protocol Glutathione content was estimated according to the method (Moren et al 1979). 0.25ml of serum was added to equal volume of ice cold 5% TCA. The precipitate was removed by centrifugation at 4000rpm for 10 minutes. To 1 ml aliquot of supernatant, 0.25ml of 0.2M phosphate buffer, pH 8.0 and 0.5ml of DTNB (0.6mM in 0.2M phosphate buffer, pH 8.0) was added and mixed well. The absorbance was read at 412 nm using spectrophotometer. The values were expressed in mg/g tissue.
- FIG. 9 depicts GSH activity
- Fig. 10 depicts GPX activity in the heart of normal and ISP induced rats.
- ISP induced in rats myocardial infarction showed significantly (p ⁇ 0.01) decreased in anti-oxidants level of positive control group and significantly increased in GSH ,GPX activities as compared to normal control group.
- Pretreatment with test drug dose dependency to ISP induced rats significantly increased the activities of these enzymes compared with positive control group.
- Glutathione is known to protect the myocardium against the free radicals mediated injury by the reduction of hydrogen peroxide, leads to decrease the reduced glutathione levels during the induction of cardiac necrosis (Kocak, H.,et al., 1992). Depressed GSH levels with enhanced protective mechanism to oxidative stress in myocardial infarction. ISP administration was found to reduce the level of GSH in plasma and cardiac tissue (Ji et al., 1988). GPX and GST activities are significantly depressed in ISP induced rats. Endogenous enzymes inactivation of GPX in the heart leads to accumulation of oxidized glutathione (Ferrari R et al 1985).
- Working standard Dilute 10ml of the stock solution to 50 ml with distilled water in a standard flask. One ml of this solution contains 200 ⁇ protein.
- Protocol Estimation of protein was assayed by taking 0.2ml of saline, 10% homogenate, followed by the addition of 1.25ml of working biuret reagent. It was incubated at room temperature for 15 minutes. The color intensity was read at 540nm.
- Isoproterenol (ISP)injected rats positive control
- ISP Isoproterenol
- Administration of test drug to ISP injected rats Low dose
- PCR was performed to determine the level of mRNA expression of Caspase, Bax, BC12 and p53. Briefly, total RNA was extracted from left ventricle (Heart) using TRIzol Reagent (Sigma, USA). After homogenization, the tubes were incubated for 10 minutes and centrifuged at 1000 rpm for 5 min. 200 ⁇ 1 of chloroform was added to the supernatant, allowed to incubate for 5 min at room temperature and centrifuged at 12000 rcf for 20 min. Then 500 ⁇ 1 of isopropyl alcohol was added to the supernatant to precipitate the total RNA and centrifuged at 12000 rcf for 15 min following the incubation period of 10 min.
- the formed cDNA was loaded in agarose gel, allowed to run the electrophoresis at 80V for 30 min and the gene expression was analyzed using the bands formed. 200 nanograms of RNA were used for reverse transcription polymerase chain reaction (RT-PCR) according to the manufacturer's instructions (Genet Bio, Korea).
- FIG. 11 illustrates the gene expression of apoptotic marker.
- ISP up-regulated the expression of apoptotic markers, Caspase 3, P53 and Bax and down regulated the expression of anti-apoptotic marker BCL-2.
- Caspase 3 results in the phosphorylation of other caspases
- P53 inhibits BCL-2 expression and conversely enhances Bax expression.
- Bax in turn inhibits the Cytochrome C thereby augmenting the reactive oxygen species generation.
- BCL-2 and Bax proteins are known to modulate the cell survival signals of various apoptotic stimuli (Sutton, et al., 1997).
- Test drug markedly up-regulated the expression of BCL-2, whereas down- regulated the expression of Caspase-3, P53 and Bax.
- Clinical observations No mortality was observed between the groups. The rats that were induced with isoproterenol showed increased heart rates and heart palpitation. Rigidity of the muscles was observed in the animals and there was decreased movement and activity in the animals. Exophthalmus, the protrusion of the eye balls, was very evident after the induction of isoproterenol. They exhibited increased respiration rate. Further, the symptoms of myocardial infarction were clearly visible in the ISP injected animals and the offset of these clinical signs prolonged in positive control.
- Table 6 depicts the body weight of animals for three weeks. No significant body weight changes were observed between the experimental groups on day 0, 7 and 14, whereas there was significant alteration in body weight on day 21.
- Table 6 Body weight of animals for three weeks.
- Table 7 depicts the organ weight of various groups. No significant difference in kidney and adrenals weight was observed between the treatment groups. The ISP induced MI rats showed increase in heart weight (24%) when compared to normal rats, whereas treatment with TEST DRUG altered the changes. Table 7: Organ weight of various groups
- Fig. 12 illustrates the effect of Test drug on Systolic B.P.
- Fig. 13 illustrates the effect of Test drug on Diastolic B.P.
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