Title of Invention

AN IMPROVED PROCESS FOR PREPARING A COMPOSITION CONTAINING CAROTENOIDS & NUTRIENTS RICH EXTRACT FROM CARROTS

Abstract

The invention describes an improved process for preparation of carotenoids and nutrients rich extract from carrots particularly to enhance stability of the carotenoids. As a result of this higher shelf life can be assigned to it and the formulations prepared therefrom. Combination of several principles gave synergistic protective effect for carotenoids, which are very sensitive to oxidation by air, light and heat.

Full Text

FORM 2 THE PATENTS ACT, 1970 As Amended by The Patent (Amendment) Act, 1999 COMPLETE SPECIFICATION SEE SECTION 10 "An Improved Process for preparing a Composition containing Carotenoids & Nutrients Rich Extract from carrots" APPLICANT Ajanta Pharma Limited an Indian Company, Registered under Companies Act, 1956 and having its Registered Office at: Ajanta House, 98 Govt. Industrial Area, Charkop, Kandivli (W), Mumbai - 400 067, Maharashtra, India GRANTED The following specification particularly describes the nature of the invention and the manner in which it is to be performed: 22-6-2004 FIELD OF THE INVENTION The present invention relates to an improved process for preparing a composition containing carotenoids and nutrients rich extract from carrots particularly to improve stability so that higher shelf life can be assigned to it and the formulations prepared therefrom. BACKGROUND OF THE INVENTION Carotenoids are a class of naturally - occurring yellow, orange or red tetra-terpenoids. The more commonly known carotenoids are alpha-carotene, beta-carotene , lutein, zeaxanthin, lycopene and cryptoxanthin. Carotenoids possess significant nutritional value. It is well known that carotene is the .precursor of vitamin A being considered as a provitamin A pre-cursors for the formation of retinal and vitamin A in humans. Of carotene isomers viz. alpha, beta and gamma carotene, beta-carotene isomer has the most vitamin A activity. Vitamin A, an essential vitamin, for life is not synthesized in the animal cell. In addition to the use of carotene as a precursor for vitamin A, there have been reports in the literature that suggest that carotene is itself useful in the prevention of certain types of cancer which are believed to be promoted by oxidising free radicals. It is postulated that carotene, which has an affinity for such free radicals may serve to reduce the free radical level in the body, thereby reducing the risks of development of several chronic disorders such as heart diseases, cancer, cataract and other ailments. Carotenoids are found in plant tissues, algae, bacteria and fungi. In particular they are found in vegetable sources such as carrots, spinach, tomatoes and fruits such as mango peach, pumpkin, papaya etc. It is well known that carrots are good sources of carotene. A little advertised fact about carrots is that only 20% of the total carotenes present are absorbed even when carrots are eaten in a finely grated form. The percentage of absorption from coarsely grated raw or cooked carrots is still less being around 5%. Besides this carotenoids and other vitamins are also destroyed to some extent during cooking. Therefore to get vitamin A and other beneficial activity of carotenoids a person needs to consume substantial amount of carrots. As the normal diet for the people can not accommodate such large quantities, there has been developed an extract having concentrated carotenoids. Oxidation of carotenoids is well known to degrade these substances, diminishing their colour stability creating undesirable flavours and aromas and also creating various oxidized forms thereof. Natural carotenoids are easily degraded when subjected to air, ultraviolet light or high temperatures. Presence of minerals increases degradation. Since nutrient rich products provide good media for microbes, they are also prone to microbial degradation. The stability of extract containing such substances must meet particularly stringent requirements when they are to be used for preparation of formulations. They are often exposed to influences such as elevated temperatures, moisture, mechanical friction or pressure, which are extremely damaging for the sensitive vitamins and carotenoids of the carotenoids and nutrients rich extract from carrot. Beta-carotene one of the most widely studied carotenoid is extremely sensitive to oxidation. It is usually stored in tightly sealed containers. However even with such a pre-cautionary measure there is sufficient oxygen available to cause a substantial decrease in activity in about eight days (US Patent 5,023,35). That is why there is a need for a process to provide improved stability to carotenoids. Inhibition of oxidation is normally accomplished commercially by the addition of synthetic antioxidants. Choice of an antioxidant depends on several factors including the desired functions, chemical and physical properties of the preservative, various constituents of the medicine, dispersibility, storage conditions and possible imparted flavour. Synthetic and natural food antioxidants are used routinely in some foods especially those containing oils and fats. Usage levels for natural oxidation inhibitors could be limited by other factors, though. In some applications, they may not be as effective as the synthetic antioxidants, and if they have to be used at higher levels, they might prove more expensive. Addition of natural antioxidants such as tocopherol usually provides little or no increase in oxidative stability of vegetable oils. Much of the literature states that tocopherols have a weak to moderate antioxidant effect. Added tocopherols used to stabilize vegetable oils are not very effective at preventing rancidity because of residual natural tocopherols in the oil. This "natural" antioxidant effect is often inadequate and synthetic antioxidants are added to fill stability needs. Since the introduction of phenolic antioxidants butylated hydroxyanisole and butylated hydroxytoluene into the food supply, these substances have been widely used. They are found in various prepared foods. Their wide use reflects both their effectiveness and their ability to increase the availability of low-cost convenient food. Phenolic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, tertiary butylhydroquinone are effective chain-breaking antioxidants and the most commonly used commercially. The antioxidants approved in the United States for use in foods include, among others, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, citric acid, ehtylenediaminetetra acetic acid, erythorbic acid, gum guaiac, lecithin, phosphates, propyl gallate, sulfites, tocopherols, glucose oxidase, . dilauryl thiodi-propionate, tertiary butylhydroquinone. The antioxidants approved in India include among others butylated hydroxyanisole, butylated hydroxytoluene, tertiary butylhydroquinone and tocopherols. However the incorporation of butylated hydroxytoluene in foods has regulatory problems in some parts of the world. During extraction, storage and processing in the formulation of dosage forms the carotenoids are exposed to air, temperature and light. Protective colloids in combination with the antioxidants offer added protection. Protective colloids have a stabilizing effect in combination with antioxidants that is the protective film is particularly impermeable to oxygen, which is important for the stability of the carotenoids. Also since the carotenoids and nutrients are sourced from natural raw material the natural bio-burden is high. The microorganisms are detrimental to the carotenoids since they are most likely to be destroyed by them. Therefore there is a need for preservation against microbial growth. Thus the present invention process provides a synergistic combination of different factors as described above for the improved stability of the carotenoids and nutrients rich extract from carrots. PRIOR ART Indian Patent No. 183575 dated 14 July 1995 assigned to Raptakos Brett & Co. Ltd. by Biyani who is also one of the inventors of the present invention, describes the use of carboxylic acid for the preparation of carotenoids and other micronutrients. However carotenoids were not stable and therefore not suitable to be used commercially. Indian Patent No 183599 dated 10th August 1998 to Biyani et al granted on a 'Process for making Pharmacologically and Biologically Active Compositions Extracted From . Carrots' assigned to Ajanta Pharma Ltd. describes the further addition of Saccharides to improve the stability of carotenoids and micronutrients. This has improved the stability of carotenoids upto six months for the formulations prepared therefrom which is inadequate as the desired stability of carotenoids and the shelf life of the formulations prepared therefrom preferably should be more than a year. US Patent No. 5,023,355 dated June 11, 1999 to Globus, et al. describes a process for the preparation of stabilized beta-carotene wherein Dolysorbate is heated under an inert atmosphere in the presence of B.HT followed by addition of beta-carotene in the formulations prepared therefrom which is inadequate as the desired stability of carotenoids and the shelf life of the formulations prepared therefrom preferably should be more than a year. US Patent No. 5,023,355 dated June 11, 1999 to Globus, et al. describes a process for the preparation of stabilized beta-carotene wherein polysorbate is heated under an inert atmosphere in the presence of butylated hydroxytoluene (BHT) followed by addition of beta-carotene in the presence of para-aminobenzoic acid and butylhydroxyquinone under inert atmosphere. The stability achieved in accordance to this prior art however is stated to be only 60 days. US patent No. 5,023,095 dated June 11, 1999 and assigned to BASF Corporation describes a colour stabilized food colouring composition. The composition contains beta-carotene dispersions, at least one edible oil and dl-alpha tocopherol. The maximum stability of 11 months is achieved using alpha - tocopherol as mentioned in this particular prior art. Moreover the composition containing beta-carotene as described in the prior art is for food colouring and not meant for pharmaceutical or other formulations like present invention. US patent No. 6,132,790 dated October 17, 2000 assigned to Betatene Limited describes a carotenoid composition comprising: a) a carotenoid in an oil solvent wherein the weight of carotenoid in the carotenoid is upto 12%, b) a dispersion of a water dispersible matrix and a stabilizer and optionally a non-oil solvent and c) an emulsifier wherein the carotenoid, non-oil solvent, water dispersible matrix, stabilizer Further, from the following detailed description it will be apparent that other various features, aspects and advantages of the present invention remains absent from the prior arts. OBJECTS OF THE INVENTION Therefore it is an object of the present invention to provide improved extract of carotenoids and nutrients from carrots. It is also an object of the present invention to increase stability of carotenoids and nutrients from carrots. Another object of the present invention is to enable increased stability of formulations comprising carotenoids and nutrients rich extract from carrots. It is still another object of the present invention to increase the shelf life of the formulations comprising carotenoids and nutrients rich extract from carrots. SUMMARY OF THE INVENTION Therefore process is disclosed for the preparation of carotenoids and nutrients rich extract comprising of using a synergistic combination of antioxidant, protective colloid, and antimicrobials to improve their stability and of the formulations prepared therefrom so that their shelf life can be increased from six months to twenty-four months. DETAILED DESCRIPTION OF THE INVENTION In accordance with presently preferred embodiments of the present invention an improved process is described for the preparation of carotenoids and nutrients of carrots with better stability. The term "carotenoid" embraces in the scope of the present invention all representatives of this class of substances including those from carrots (Daucus carota L) of all varieties. In accordance with the present invention process carotenoids and nutrients rich extract is prepared from carrots by such process that it contains most of the carotenoids and micronutrients of carrots. The term 'carotenoid and nutrients rich extract from carrots' embraces in the scope of the present invention the extract from carrots including 0.10 % - 30% of carotenoids mass as a percentage of total mass of extract. Typically the carotenoid fraction includes alpha-carotene, beta-carotene, lutein, zeaxanthin and lycopene. These carotenoids are present alongwith micro and macronutrients. Typically the micronutrients are vitamins, typically the B complex vitamins, Bl, B2, niacin and vitamin C and minerals and trace elements. Macronutrients, which are present, are lipids, proteins and carbohydrates. The process in accordance with the invention for the preparation of carotenoid preparations comprises : • antioxidants individually or in combination • Protective colloids individually or in combination that will effectively coat the granules for protection from air and increase ability to withstand the stresses of air and temperature exposure during processing • preservatives individually or in combination In a preferred form of the invention a process to prepare carotenoids and nutrients rich . carrot extract comprises the steps of: washing carrots with water and removal of tops and damaged portions; crushing the carrots; pressing the crush through coarse filter to separate the juice; centrifuging the said juice to get paste; addition of antioxidant in surfactant to the said paste; addition of the preservative dissolved in the water to the above paste; addition of protective colloid; cooling of the paste from 0 to -20° C; drying of the cooled paste in the fluid bed dryer to get the carotenoids and micronutrients rich extract in powder form. According to the present invention, typically the antioxidant is selected from the group of butylated hydroxyanisole, butylated hydroxytoluene, citric acid, ethylenediaminetetraacetic acid, erythorbic acid, gum guaiac, lecithin, phosphates, propyl gallate, sulfites, tocopherols, glucose oxidase, dilauryl thiodi-propionate, and tertiary butylhydroquinone. The oil soluble antioxidant comprises 0.01% to 3% and more preferably 0.2% to 1% by weight of the composition. The uniform incorporation of the antioxidant in the carotenoids and nutrients rich extract of carrots necessitates the use of a surfactant. Non-ionic surfactants individually or in combination are preferred. Typically the surfactant is selected from the group of Polysorbate 80, Polysorbate 60, sorbitan esters and the like, individually or in combination which comprises 0.01% to 3% by weight of the composition preferably 0.02% to 1% by weight of the composition. In the present invention process, typically the protective colloid is at least one, selected from a group consisting of gelatin, starch, gum acacia, dextrins or starch derivatives and/or combinations thereof. The protective colloids are used in the range of 1% to 40% preferably 5% to 20% by weight of the composition. The moisture content of the carotenoids and nutrient rich carrot extract was found to play an important role in the stability of the carotenoids. The carotenoids are stable at certain critical moisture content levels. At lower, levels' of moisture content the carotenoids degrade rapidly and at higher moisture content levels the material becomes sticky. This critical moisture content level was found to predispose the extract to microbiological spoilage. To provide protection from microbial growth during processing and storage in the present invention process preservative is added. Typically the preservative is at least one selected from a group consisting of methyl paraben, propyl paraben, sodium methyl paraben, sodium propyl paraben, sorbic acid, potassium sorbate, benzoic acid, sodium benzoate, and/or combinations thereof. The preservatives are used in the range of 0.01%) to 3% preferably 0.05% to 1% by weight of the composition. Drying procedures for extracts with carotenoid-based compositions have to take into consideration the sensitive nature of these constituents, especially to oxidation. Drying is normally carried out in a low oxygen atmosphere under vacuum. This however involves high drying times and increased costs. However in the present art with the improved process it was possible to carry out drying in the fluid bed dryer after cooling it, without affecting the stability of the carotenoids bringing down the drying times and costs considerably, making the product economical and viable. According to the present invention the paste was cooled to 0 to -20° C prior to the drying, this not only facilitates the drying in fluid bed dryer by improving densification of the extract and reducing the resultant volume. It also helps to minimize the oxidation of the carotenoids, which are sensitive to exposure to the higher temperature, due to lag time in drying. As per the present art the cooling was carried out in a Deepfreeze. In accordance with the present art process, fluid bed drying was carried out at an inlet temperature of between 30°C to 90°C, preferably between 40°C to 60°C and an outlet temperature between 30°C and 90°C, preferably between 30°C to 40°C, while controlling the moisture content between 5% and 15%, preferably between 5% to 10%. Care was taken during processing to ensure minimal exposure to air and light. Storage of material at all steps during processing and storage was in 40-micron aluminum foil. Foil was pressed to remove all residual air. Combination of antioxidant, protective colloid, and antimicrobials and other factors in accordance with the present invention process achieve stabilities, which none of the substances can achieve alone, and indeed the stabilities achievable, for all practical purposes, are greater than twenty-four months using present art. The heart of the invention lies in the totally unexpected effect, which a synergistic combination of different factors as described above had on the stability of the carotenoids and nutrients rich extract from carrots. Process as given in earlier patent No. 183575, gives procedure but not the stability or shelf life data of the carotenoids. Example 1 was prepared as per the process given in earlier patent No 183575, to check the stability of the product. However the product was not stable beyond one month, since the carotenoid content dropped to less than 90% of the indicated amount. Example 2 was prepared as per process given in earlier patent No. 183599. However the product was not stable beyond six. Example 3 was prepared using only butylated hydroxyanisole as antioxidant, however the product was not stable beyond twelve months. Example 4 was prepared as per the art described in the present invention and was found to be stable for twenty-four months. The stability data as mentioned above for example 1 to 4 are shown in Table 1. Carotenoids and nutrients rich extract from carrots of the present invention can be used in combination with other antioxidants, vitamins and minerals to prepare the formulations. The formulations are typical compositions for administration, which include specifically or more particularly oral and topical dosage forms. Oral formulations include granules, tablets, dispersible tablets, mouth dissolving tablets, chewable tablets and liquid dosage forms and the like. Topical dosage forms include creams, ointments, gels and the like. The compositions comprising carotenoids and nutrients rich extract from carrots will include typically about 0.1% to 30% of carotenoids by weight of the carrot extract preferably 0.25 to 10% of carotenoids by weight of the carotenoids and nutrients rich extract from carrots. These and other modifications and variations to the present invention may be practiced by those of the ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition it should be understood that aspects of various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in such appended claims. EXAMPLE 1 Prepared as per example 1 of patent No. 183575. 1.0 Kg of fresh, hard, good quality, dark coloured carrots with smooth surface were washed thoroughly with water using nylon brush. Tops and damaged portions were cut to get 960 gm good portion. This was crushed in a juicer. The extract was pressed through a coarse filter to" get 560 gm of liquid extract. 2.5 gm of fumaric acid was dissolved. The mixture/slurry was subjected to supercentrifuging to get 43 mg of concentrate containing 1-mg carotenoids per gm. 20 gm of this was filtered to get cake, which on drying under high vacuum (less than 10mm of Hg) gave 1.5 gm of carrot, extract powder containing 9 mg of carotenoids per gm of the product. It was stored in the refrigerator at a temperature of 2°C - 8 °C for studying stability. EXAMPLE 2 Prepared as per example 1 of patent No 183599. Fresh, hard, good quality carrots with a smooth surface, excluding those that were found defective, were selected and washed thoroughly with water. The sorted and washed carrots (1.0 Tone) were subjected to crushing in a fruit mill to provide a comminution which was subjected to pressing through a filter press for the purpose of separating the pulp from the juice to provide a juice (ca. 600 ml). To the juice, 3Kg of adipic acid was added with stirring. To the resulting mixture was added 60Kg of sorbitol and the mixture was subjected to centrifuging to provide paste (ca. 16.98Kg). The paste was dried under high vacuum, sieved through #16 s.s. sieve and stored in 40 micron aluminum foil, pressed to remove all air. Total carotenoid content was 1255 mg per gm of the product. It was stored in the refrigerator at a temperature of 2°C - 8 °C, for studying stability. from carrots Composition per 100 g product beta-Carotene alpha-Carotene Lycopene Lutein/Zeaxanthin Total Carotenoids Proteins Carbohydrates Phosphorus Lipids Vitamin C Range as per our Patent No. 183599 500 mg 100-4000 mg 25 mg 10-300mg 720 mg 10-2000 mg 10 mg 5 - 50 mg 1255 mg 250 - 5000 mg 30.8 g 10-50g 4.5 g l-25g 750 mg 0.1-lg 24.6 g 20 - 40 g 32 mg 10-500mg Vitamin B1 4.5 mg 1 -6 mg Vitamin B2 1.8 mg 0.5-4 mg Iron 89 mg 5- 100 mg Zinc 3 mg 1 -5mg Manganese 0.5 mg 0.1 - 1 mg Magnesium 192 mg 50 - 900 mg Calcium 1.681 g 0.5-3 g Potassium 3.20 g 1-4-g Sodium 1.07 g l-3g Total Minerals (Ash value) 4.08 g 3-10g EXAMPLE 3 The carrots were processed according to the procedure described in example 2.To the paste 17.4 Kg was added a dispersion of 13.76 gm of butylated hydroxyanisole in 275 ml of polysorbate 80. The paste was dried under high vacuum, to a moisture content of 7% to 8%, sieved through #16 S.S. sieve and stored in 40 micron aluminum foil, pressed to remove all air. Total carotenoid content was 8.6 mg per gm of the product. It was stored in the refrigerator at a temperature of 2°C - 8 °C, for studying stability. The other constituents of the product were in the range as given in example 2. EXAMPLE 4 The carrots were processed according to the procedure described in example 2.To the paste 17.4 Kg was added a dispersion of 13.76 gm of butylated hydroxyanisole in 275 ml of polysorbate 80. Sodium methyl paraben 23 gm and sodium propyl paraben 6 gm were dissolved in 70 ml of water and added to the paste, followed by gum acacia 688 gms. The paste was dried in the fluid bed dryer at an inlet temperature of 40°C to 60°C and an outlet temperature of 30°C to 60°C to moisture content of 7% to 8%. The dried granules were sieved through #16 S.S. sieve and stored in 40 micron aluminum foil, pressed to remove all air. Total carotenoid content was 9.2 mg per gm of the product. It was stored in the refrigerator at a temperature of 2°C - 8 °C for studying stability. The other constituents of the product were in the range as given in example 2. Table 1 STABILITY DATA (CAROTENOID CONTENT AS % OF INITIAL CONTENT) Storage - Refrigerator (2°C - 8°C) Initial mg/g 1 month 3 months 6 months 12 months 18 months 24 months Example 1 9.00 85.48 % Stability Discontd. Example 2 12.55 99.6 % 98.4 % 90.1 % Stability discontd. Example 3 8.6 98.4 % 97.8 % 95.1 % 90.12% Stability Discontd. Example 4 9.2 99.86 % 98.95 % 98.14% 97.56% 95.32 % 92.31% WE CLAIM: 1. A process for preparing a composition containing carotenoids and nutrients rich extract from carrots particularly to enhance stability of the carotenoids as a result of which higher shelf life can be assigned to it and the formulations prepared therefrom; comprising of steps of; i). Crushing of 1 Kg. of carrots, ii). Separation of juice; iii). Addition of 0.5 % of carboxylic acid (w/v of the juice) and 10 % (w/v of the juice) saccharide; iv). Separating the paste; v). Addition of at least one preservative in the range of 0.01% to 3% by weight of the extract, at least one antioxidant in the range of 0.01% to 3% by weight of the extract, at least one protective colloid in the range of 1.0% and 40% by weight of the extract; vi). Cooling the paste till it solidifies; vii). Drying in fluid bed dryer while controlling the moisture content. 2. A process as claimed in claim 1 in which at least one antioxidant is selected from a group consisting of butylated hydroxyanisole, butylated hydroxytoluene, citric acid, ethylenediaminetetraacetic acid, erythorbic acid, gum guaiac, lecithin, phosphates, propyl gallate, sulfites, tocopherols, glucose oxidase, dilauryl thiodi-propionate, and tertiary butylhydroquinone. 3. A process as claimed in claim 1 in which at least one protective colloid is selected from a group consisting of gelatin, starch, gum acacia, dextrins or starch derivatives. 4. A process as claimed in claim 1 in which at least one preservative is selected from a group consisting of methyl paraben, propyl paraben, sodium methyl paraben, sodium propyl paraben, sorbic acid, potassium sorbate, benzoic acid, sodium benzoate. 5. A process as claimed in claim 1 in which cooling of the paste is from 0°C to -20°C. 6. A process as claimed in claim 1 in which drying is carried out in the fluid bed dryer with an inlet temperature between 30°C to 90°C and outlet temperature between 30°C and 90°C. 7. A process as claimed in claim 1 and 8 in which inlet temperature is preferably between 40°C to 60°C and outlet temperature is preferably between 30°C to 60°C. 8. A process as claimed in claim 1 in which the moisture content of the extract is from 5% to 15%. Dated this 26th day of September 2003. For AJANTA PHARMA LIMITED MR.P. R. SONAWANE AJANTA PHAR MA LIMITED 98, Govt. Indl. Area, Charkop, Kandivali (W), Mumbai 67 To: Patent Office Mumbai Branch, Mumbai.

Documents:

133-mum-2001-abstract(22-06-2004).doc

133-mum-2001-abstract(22-06-2004).pdf

133-mum-2001-claim(granted)-(22-06-2004).pdf

133-mum-2001-claims(granted)-(22-06-2004).doc

133-mum-2001-correspondence(19-08-2004).pdf

133-mum-2001-correspondence(ipo)-(21-12-2004).pdf

133-mum-2001-form 1(07-02-2001).pdf

133-mum-2001-form 1(29-09-2003).pdf

133-mum-2001-form 2(granted)-(22-06-2004).pdf

133-mum-2001-form 3(02-05-2001).pdf

133-mum-2001-form 3(07-02-2001).pdf

133-mum-2001-form 5(02-05-2001).pdf

133-mum-2001-form 5(07-02-2001).pdf

133-mum-2001-form-2-(granted)-(22-06-2004).doc

133-mum-2001-form-pct-isa-210(02-05-2001).pdf