Title of Invention

A SYNERGISTIC REJUVENATING AND REVITALISING PHARMACEUTICAL COMPOSITION

Abstract

ABSTRACT 1370/MAS/96 A synergistic, Rejuvenating and Revitalising pharmaceutical composition The synergistic, rejuvenating and revitalising pharmaceutical composition of this invention consists of a blend of eight vital amino acids, vitamins and compounds containing inorganic trace elements like zinc, calcium, iron, selinium iodine, magnesium and manganese. The composition enhances haemogloban synthesis and increases assimilation of trace elements by the system.

Full Text

This invention relates to a synergistic, rejuvenating and revitalising pharmaceutical composition. Proteins are essential constituents of living cells and play a very important role in the metabolisim of living organism. Proteins are nitrogenous substances consisting of amino acid residue, joined together by peptide linkages. A large number of proteins having different molecular configuration may be synthesised from a relatively small number of amino acid units. Individual proteins are characterised by the 'amino acid sequence in its structure and the molecular weight. Horns, hair, cartilage, tendons, and the like have different proteins as their basic building blocks. Evidently, a balanced diet should contain an adequate supply of proteins and amino acids to make good the losses that occur during tissue wastage, ageing and other metabolic changes taking place in living organism constantly, an adequate supply of proteins and amino acids to make good the losses that occur during tissue wastage, ageing and other metabolic changes that occur in living organisms constantly. As stated above, amino acids are the building blocks for proteins. Out of the twenty three vital amino acids that are required for making a large number of proteins required by human metabolism and growth, eight are considered essential. Human metabolism does not synthesise these amino acids at least in required level and hence they must form a part of our dietary suppliment in order to maintain metabolic activity, promoting growth, body building and resistance to diseases. Proteins from dietery intake are hydrolysed by various proteolytic enzymes in the stomach and small intestine to amino acids which are absorbed in the blood stream. These amino acids are metabolised in the body to body proteins and non proteinaceous nitrogenous compounds. They may also be converted into different amino acids which are subsequently metabolised to release energy, glycogen and fats. Therefore, having established the importance of amino acids in human metabolism and the inability of the human body to synthesise the eight essential amino acids, it is clear that a balanced diet must include adequate supply of these vital amino acids. Similarly, vitamins are organic compounds essential for the maintenance of normal health and growth. Like the vital amino acids, vitamins are also not synthesised by life forms, at least in sufficient quantity. Therefore, vitamins must also form a part of daily dietary intake. It is found that certain inorganic nutrients also play an essential and important role in the physiological and metabolical functions of the human body. Inorganic nutrients like zinc, iron, manganese, magnesium, copper and iodine are found to be necessary for the health and well being of the human body. Many enzymes required for human metabolic activity are metalloenzymes having one or more of the above inorganic elements as their constituents. For instance, zinc is a constituent of many netallo enzymes involved in the metabolic pathways of synthesising carbohydrates, lipids and proteins. They are also found to be beneficial in cell mediated immunity in convalescing patients. Iron is an essential component of many important metalloprotelns such as haemoglobin and myoglobin. Oxidation reduction enzymes and cytochrome which are of great importance in cell oxidation acting as intermediate oxygen carriers also contain iron. Many metabolic processes such as nucleic acid and protein synthesis in the human body depend on the presence of enzymes and proteins containing iron. Similarly, it is essential that daily intake of iodine and manganese as dietary supplements is essential for growth, sexual development and in building resistance to certain disorders. Magnesium is an essential constituent of all soft tissues and bones and is required for the activity of a large number of enzymes particularly in phosphorylation, which is a process for converting sugars to phosphates and splitting the later to provide energy for vital biological processes. Magnesium deficiency symptoms in man include emotional liability, irritability, hyperflexia and the like. Copper forms a part of cytochrome oxidase, tyrrosinase, catalase and ascorbic acid oxidase. Copper also plays an important role in the biochemical and metabolic activities such as introduction of iron into haemoglobin and cytochrome molecules. As copper facilitates absorption of iron, a diet deficient in copper leads to severe retardation of growth, anaemia and anorexia. Another essential component of enzyme glutathione peroxidase which catalysis the oxidation of glutothione, is selenium. Gluthothione protects haemoglobin from oxidative damage. Selenium also plays an important role in tissue respiration. The object of this invention is to provide a synergistic composition of essential amino acids, vitamins and trace elements. Normal diet in many developing and underdeveloped countries do not provide adequate requirements of vitamins, minerals and amino acids leading to serious health problems. It is found that inclusion or addition of minerals (inorganic trace elements) disclosed hereinabove enhances protein synthesis and assimilation of essential trace elements during metabolic activity. It is also found that such a composition exhibits properties fundamentally different from that of the constituting components and shows unforeseen properties. Different components of this composition compliment each other in rejuvenating and revitalising the human body by enhancing protein synthesis. providing extra energy, supplementing growth requirements, metabolic maintenance and immunity development. The synergestic, rejuvenating and revitalising pharmaceutical composition according to this invention, comprises a blend of of essential amino acids, vitamins and inorganic trace elements, listed hereunder, in the range specified thereagainst with known pharmaceutically acceptable adjuvants and/or exoepients: L Leucine - 13 to 23 mg L Iso leucine - 4.5 to 7.5mg L Lysine hydrochloride - 19 to 31 mg L Phenyl alanine - 3.5 to 8.5 mg L Threonine - 3 to 5 mg L Valine - 5 to 8 mg L Tryptophane - 3.5 to 8.5 mg DL Methionine - 13 to 23 mg 5 - Hydroxy anthro^ilic acid - 0.15 to 0.25 mg Vitamin A acetate - 2000 to 3000 lU Vitamin Dg - 150 to 250 lU Vitamin Bl Mononitrate - 3.5 to 6.5 mg Vitamin B2 - 2 to 4 mg Niacinamide - 19 to 31 mg Vitamin B6 hydrochloride - 1.2 to 2.6 mg Folic acid - 0.2 to 0.9 mg Calcium Pantothenate - 3.6 to 6.5 mg Vitamin B12 - 1.0 to 3.1 meg Vitamin C - 20 to 60 mg Vitamin E - 1 to 8 lU Potassium iodide - 0.1 to 0.3 mg Ferrous sulphate - 14.3 to 26.6 mg Copper Sulphate - 3.2 to 4.8 mg. Manganese Sulphate - 1.1 to 1.7 mg Magnesium Sulphate - 5.9 to 8.9 mg Zinc Sulphate - 3 to 4.3 mg Sodium Selenite - 10 to 30 meg Conventional colourants and excipients and additives are added depending on the nature of the composition desired. If a liquid composition is preferred, adjuvants like distilled water and pharmaoeutically acceptable co-solvents, sweeteners, preservatives, flavours, etc. are added and the active components specified above either solubilized or dissolved therein. Tablets or capsules may be prepared by blending the components with suitable solid adjuvants, known in the art and tabletting the blend by known methods. Active components are spheronised either together or in preferred groups and then encapsulated in shells or capsules made from pharmaoeutically acceptable and biodegradable materials. Comonly used excipients or adjuvants and colourants are stated herein below: Col - Tartrazine Col - Erythrooine Col - Indigo-carmine Col - Sunset yellow Titanium dioxide. Lactose, Potato starch, Microcrystalline Cellulose, Calcium, Carboxy Methyl Cellulose, Methyl cellulose. Hydroxy propyl cellulose, polyetylene glycol,Synthetic aluminium, Silicate, Stearic acid, Industrial Methylated spirit. Poly vinyl pyrolidone, Iso-propylalcohol, and distilled water. In a preferred embodiment, compatible components of the composition are grouped together, spheronised, dried and then encapsulated. For instance,a capsule may contain a plurality of granules, the total composition of which falls within the range specified hereinabove, but each group of granules may contain a group of components different from the other. For example, a capsule may contain white, yellow, red, chocolate and orange granules having a composition specified herein below. The values indicated include the averages of vitamins. 1. White granules I: Vitamin B6 HCl - 1.725 mg Niacinamide - 30 mg Calcium pantothenate - 6.5 mg Lactose - 8.4 mg Potato starch - 3.2 mg Micro crystalline cellulose- 6.0 mg Calcium carboxymethyl cellulose- 5.5 mg Titanium dioxide - 0.1 mg Distilled water - 0.0.1 ml These components are mixed in a planetary mixer for one hour to obtain a soft mass which is extruded and spheronised using a murmeriser. The spheronised granules are dried at 45° for about an hour till the moisture content is reduced to less than IX. 2. White Granules II: Vitamin Bl (Thiamine mononitrate) - 6 mg Microcrystalline Cellulose - 2 mg Potato starch - 1 mg Methyl cellulose - 0.4 mg Calcium Carboxymethyl cellulose - 0.9 mg Titanium dioxide - 0.1 mg Water purified - 0.01 mg The above ingredients are mixed and spheronised as stated in in the preparation of White granules No.I. 3. Yellow Granules: DL Methonine - 18.4 mg L Lysine Hydrochloride - 25.0 mg L Leucine - 18.3 ng L Isoleucine - 5.9 ng L Valine - 6.7 ng L Phenyl Alanine - 5.0 mg L Threonine - 4.2 ng Microcrystalline Cellulose - 11.6 ng Potato starch - 13.0 ng Methyl Cellulose - 3.5 ng Hydroxy Propyl Cellulose - 0.7 mg Tartarazine (Colour) - 0.27 ng Distilled water - 0.01 nl The above ingredients are blended and spheronised as given under White Granules Ho.I. 4. Orange Granules: Riboflavin (vitanin B2) - 3.3 ng Folic acid - 0.975 ng Vitanin B12 - 3 nog 5 Hydroxy Anthranilic acid - 0.22 ng Lactose - 12 ng Potato starch - 9 ng Microcrystalline cellulose - 6 ng Polyethylene glycol - 0.3 ng Calciun Carboxynethyl cellulose - 0.25 ng Hydroxy Propyl cellulose - 0.8 Bg Distilled Water - 0.01 ng These compounds are mixed and spheronized as stated hereinabove. 5. Red Granules: DL-L~ToGo pherol Acetate (vitamin E acetate) - 16.5 mg L Tryptophan - 5 mg Synthetic Aluminium Silicate - 3.3 mg Lactose - 13.7 mg Micro Crystalline Cellulose - 2 mg Polyethylene Glycol 8000 - 0.1 mg Calcium Carboxy methyl cellulose - 2 mg Hydroxy Propyl Cellulose - 2 mg Erythrocine (Colour) - 0.116 mg Tartrazine (Colour) - 0.012 mg Distilled Water - 0.01 ml All the above items are spheronised in a manner stated hereinabove. 6. Chocolate Granules: Ascorbic acid - 52.0 mg Stearic acid - 1.56 mg Industrial Methylated Spirit - 0.008 ng Micro Crystalline cellulose - 2.6 ng Polyethylene Glycol 8000 - 1.6 mg Calcium Carboxy Methyl cellulose - 2.0 ng Methyl Cellulose - 0.6 ng Colour Tartrazine - 0.252 ng Colour Erythrocine - 0;15 ng Colour Indigo Carmine - 0.078 ng Potato starch - 1.8 ng Distilled water - 0.01 nl Spheronised granules are nade fron the above ingredients in a known manner. 7. A and D Granules: Vitanin A Acetate - 8 ng Vitamin D3 - 0.008 ng Lactose - 12.8 ng Micro Crystalline Cellulose - 3.392 ng Polyvinyl Pyrolidone - 2 ng Isopropyl alcohol - 0.01 nl The first four compounds are blended and then granulated and spheronised after adding a solution of polyvinyl pyrolidone in isopropyl alcohol. 8. Mineral Granules: Potassium Iodide — 0.1 mg Ferrous Sulphate - 20.5 mg Copper Sulphate - 4 mg Manganese Sulphate - 1.4 mg Magnesium Sulphate - 7.425 mg Zinc Sulphate - 3.3 mg Sodium Selenite - 10 mg Maize starch - 3.75 mg Lactose - 20.5 mg Microcrystalline cellulose - 5 mg Sodum carboxy methyl cellulose - 1.5 mg Polyvinyl Pyrolidone - 2 mg Distilled water - Q.S. The first 11 compounds are mixed in a planatory mixer for 30 minutes and 2 mg of polyvinyl pyrolidine in distilled water is added to the mixture to obtain a soft mass which is extruded through an extruder and then spheronised in a murmeriser. Capsules are prepared by mixing the required amount of granules referred under items 1 to 8 and then encapsulating them in a conventional manner. Obvious variations and equivalents are well within the scope of this invention and the appended claims. Example 1: Effect of present invention (Composition 1) in reducing the oxidative stress in diabetic rats. Seven formulations of the following compositions were prepared in a suitable form, along with appropriate excipients, by standard method known to those of ordinary skill in the art. An increase in oxidative stress has been proposed to play a crucial role in the development of the diabetic vascular complications. The purpose of this study is to evaluate the effectiveness of the above said compositions in reducing the oxidative stress in diabetic mellitus. Method The study involves male Sprague-Dawley (SD) rats, (age 10 weeks and 210-240 g body weight). They were housed in an air-conditioned room at 24 ± 2°C with a lighting schedule of 12 hours light and 12 hours dark for 14 days for acclimatization. An isocaloric diet and purified water were supplied ad libitium. Rats were fasted for 16 hours before the induction of diabetes with streptozotocin. Animals were injected intraperitoneally (Dose: 60 mg/Kg of body weight) with 0.23 ml of a freshly prepared streptozotocin solution (60 mg/ml in 0.01 M citrate buffer, pH 4.5). Then the diabetic state was assessed by measuring serum glucose concentration 48 hours post Streptozotocin injection. Only rats with serum glucose levels greater than 300 mg/dl were selected and used for the experiment. They were randomly divided into eight groups (n= 14). On Day 0, blood samples were collected from the tail veins of the 16-hour fasted rats just prior to the initiation of the various treatment schedules. Then the rats were the treated as follows. Group 1, served as control and received placebo. Group 2, given composition 1 (Animal Dose: 243.79 mg/kg of body weight) Group 3, given composition 2 (Animal Dose: 194.95 mg/kg of body weight) Group 4, given composition 3 (Animal Dose: 154.13 mg/kg of body weight) Group 5, given composition 4 (Animal Dose: 138.10 mg/kg of body weight) Group 6, given composition 5 (Animal Dose: 105.49 mg/kg of body weight) Group 7, given composition 6 (Animal Dose: 48.64 mg/kg of body weight) Group 8, given composition 7 (Animal Dose: 89.46 mg/kg of body weight). The rats were given above compositions for a period of 10 days. Daily measurements of the body weight, food and fluid intakes of the rats were also recorded. On the Day 10, all the rats were fasted for 16 hours and the following morning they were killed by decapitation. Kidneys and liver samples were frozen in liquid nitrogen and immediately stored at -70°C. Thiobarbituric acid reactive substances (TBARS) measurement The TBARS level was measured as an index of malondialdehyde (MDA) production. Malondialdehyde is an end product of lipid peroxidation. The measurement of MDA levels by thiobarbituric acid reactivity is the most widely used method for assessing lipid peroxidation. When it reacts with thiobarbituric acid a red coloured complex is formed. About 1 g of the liver and kidney samples were homogenised in 4 ml of 1.15% ice cold potassium chloride using a homogeniser. To this 0.1 ml of the homogenate, 0.2 ml of 8.1 % dodecyl sodium sulphate salt, 1.5 ml of 1% phosphoric acid, 0.2 ml of distilled water and 1.0 ml of 0.6 % 2-thiobarbituric acid were added. The mixture was heated in a boiling water bath for 45 minutes. Subsequently, the heated mixture was cooled in an ice bath, followed by an addition of 4.0 ml of n-butanol to extract the cold thiobarbituric acid reactants. This is then centrifuged at 1000 g for 5 minutes and the n-butanol layer was separated. The optical density of the n-butanol layer was determined at 353 imi and expressed as nmol MDA/25 mg wet weight. The results are tabulated in table 1 Superoxide dismutase (SOD) measurement SOD activity was measured based on the ability of the enzyme to inhibit the autoxidation process of pyrogallol. The liver tissue was homogenized in 50 mmol/L phosphate buffer (pH 7.8) using a homogeniser. The homogenate was centrifuged at 1,600 g for 15 minutes. Then the 20 |xl of 10 mmol/L of pyrogallol solution was added to various concentrations of the tissue supematants and the rate of autoxidation was measured spectrophotometrically at 420 nm. SOD activity is expressed as units of SOD/mg protein (1.0 U is defined as the amount of the enzyme, which causes 50% inhibition of pyrogallol autoxidation).The results are shown in table 2. Catalase (CAT) measurement CAT activity was measured based on the ability of the enzyme to break down the hydrogen peroxide. The liver tissue was homogenised in isotonic buffer (pH 7.4). The homogenate was centrifuged at 1000 g for 10 minutes. 20 ^1 of 100-fold diluted tissue supernatant was added to 980 \xl of the assay mixture containing 900 jil of 10 mmol/L of hydrogen peroxide, 50 |j,l of Tris HCI buffer (pH 8.0) and 30 jxl of distilled water. The rate of decomposition of H2O2 was monitored spectrophotometrically at 240 run. CAT activity is expressed as k/mg protein, where k is the first order rate constant. The results are given in table 2. Reduced glutathione (GSH) measurement Reduced glutathione levels were estimated based on the ability of the SH group to reduce 5, 5'-dithiobis- (2-nitrobenzoic acid) to form 1 mole of 2-nitro-5-mercaptobenzoic acid per mole of SH. The liver tissue was homogenized in 50 mmol/L Tris HCI buffer (pH 7.4). The homogenate was centrifuged sequentially at 10,000 g for 20 minutes, then at 100,000 g for 60 minutes. To 0.5 ml of tissue supernatant, 1.5 ml of 0.2 mol/L Tris HCI buffer (20 mmol/L EDTA, pH 8.2), 0.1 ml of 0.01 mol/L of 5, 5'-dithiobis-(2- nitrobenzoic acid) and 7.9 ml of methanol were added. The mixture was incubated at 37°C with occasional shaking for 30 minutes. It was then centrifuged at 3,000 g for 15 minutes. The absorbance of the supernatant was determined at 412 mn. The GSH concentrations of the samples were derived from the standard curve prepared using known amounts of GSH. GSH levels are expressed as |a,mol/mg protein. The results obtained were given in table 2. The hepatic SOD, CAT activities and GSH levels were significantly higher in the group 2 than the other groups. This shows that the composition 1 according to the present invention reduces the oxygen free radicals and improves the activities of the hepatic anti oxidant en2ymes. Conclusion: From the above study it is evident that only the present invention stated in (Composition 1) surprisingly has significant anti oxidant property and thus reduces the oxidative stress in diabetic mellitus. Example 2: The effect of the present invention in reducing glycosylated hemoglobin in diabetes. This study was performed to evaluate the effect of the present invention in combination with metformin in reducing the glycosylated hemoglobin. Method The study was performed in women with type II diabetes, experiencing poor blood sugar control while taking metformin 500 mg b.i.d. Along with continued metformin therapy, the patients were given an oral daily supplementation of the compositions stated in the present invention. About 96 women of age in between 45 and 55 who were under metformin therapy (500 mg b.i.d) were selected. A baseline level of glycosylated hemoglobin HbAlc was measured. They were randomly assigned into eight groups (n=12) and were treated as follows. Group 1, served as control and received placebo. Group 2, received composition 1. Group 3, received composition 2. Group 4, received composition 3. Group 5, received composition 4. Group 6, received composition 5. Group 7, received composition 6. Group 8, received composition 7. All the patients received isocaloric diet and followed similar work pattern. The compositions were given twice daily for a period of 3 months. After 3 months the level of HbAlc was measured from venous blood sample through usual laboratory procedure known to prior art. The results are tabulated in table 3 Conclusion; The above result clearly shows that only the Composition 1 as per the present invention has unexpected synergistic effects in lowering the glycosylated hemoglobin level and improves blood sugar control in diabetes. Example 3: Evaluation of the plasma levels of Amino acids in oral alimentation of the present invention. A study was undertaken to evaluate the effectiveness of the compositions in improving amino acids level in the blood. The objective of the study is to determine whether oral intake of the compositions results in an improvement of the nutritional status with respect to amino acids present in the administered compositions. Method: A double-blind, placebo controlled study was conducted over a one month period. A total of 96 subjects, aged 30-45 years, were chosen for the study. An initial assessment of the nutritional status for amino acid of each subject was conducted. The subjects were randomly separated into 8 groups of 12 subjects each and they were treated as follows. Group 1, served as control and received placebo. Group 2, received composition 1. Group 3, received composition 2. Group 4, received composition 3. Group 5, received composition 4. Group 6, received composition 5. Group 7, received composition 6. Group 8, received composition 7. All the subjects were given isocaloric diet and received no other nutritional supplements during the one month assessment period. Blood samples were drawn from the antecubital vein into a syringe having heparin sodium in the dead space. It was centrifuged immediately and the plasma removed. The amino acid levels were determined using High-pressure liquid chromatography (HPLC) separation with post column Ninhydrin quantitation. WE CLAIM: 1. A synergistic, rejuvenating and revitalising pharmaceutical composition comprising a blend of essential amino acids, vitamins and inorganic trace elements listed hereinunder in the range specified thereagainst with known pharmaceutically acceptable adjuvants and/or excipients. 2. The pharmaceutical composition as claimed in claim 1, wherein the blend is in the form of an emulsion or suspension. 3. The pharmaceutical composition as claimed in claim 1, wherein the blend is in the form of tablets. 4. The pharmaceutical composition as claimed in claim 1, wherein the blend is in the form of granules/pellets encapsulated in a pharmaceutically acceptable casing. 5. The pharmaceutical composition as claimed in claims 1 to 4 wherein blends of compatible compounds are encapsulated. 6. The pharmaceutical composition as claimed in any of the above claims, having colourants added thereto. 7. The pharmaceutical composition as claimed in claims 1 to 5, and 6 wherein different granules have different colours. 8. A synergistic, rejuvenating and revitalising pharmaceutical composition, substantially as herein described.

Documents:

1370-mas-1996 abstract duplicate.pdf

1370-mas-1996 abstract.pdf

1370-mas-1996 claims duplicate.pdf

1370-mas-1996 claims.pdf

1370-mas-1996 correspondence others.pdf

1370-mas-1996 correspondence po.pdf

1370-mas-1996 description (complete) duplicate.pdf

1370-mas-1996 description (complete).pdf

1370-mas-1996 form-1.pdf

1370-mas-1996 form-19.pdf

1370-mas-1996 form-26.pdf

1370-mas-1996 others.pdf