Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF STABILIZED ANTHOCYANIN CONCENTRATE

Abstract

The present invention provides an improved process for the preparation of natural colour cough syrup mixture comprising peeling of black java plum fruits followed by blending the peels in acidified water containing ascorbic acid. Blended broth was filtered through nylon filters followed by centrifugation and passing through ion exchange column. Anthocyanin concentrate was obtained by recovering the liquid phase followed by concentrating the solution. This improved cough syrup with salbutamol and red natural pigment anthocyanin provides the two fold improvement in the syrup formulation induced due to the incorporation of natural colourant. Further, the levels of incorporation act as nutraceutical owing to their antioxidant effect in human systems

Full Text

The present invention relates to an improved process for the preparation of natural colour stabilized cough syrup mixture. 'The present invention particularly relates to an improved formulation with natural pigment - anthocyanin and betalaines for cough syrup. This formulation has use as an anti - asthmatic and anti-spasmodic for human use. Commercial syrup samples are pale-pink in color imparted due to synthetic dyes (carmine and sun set yellow FCF); The market for salbutamol in India is estimated to be Rs. 2.7 billion, and is growing at 7-8% each year. Around 44% of the market has been for oral preparations, wherein sales have been increasing 2-3% per annum, (http.www.patentindia.org). In recent years the use of natural colorants is preferred in food and pharmaceutical products because of the consumer preference towards more natural products, due to the health-promoting effects of natural colours and safety in comparison with synthetic dyes (http://www.africangreys.com/articles/nutrition/chemicals.htm). Certain natural colorants/pigments from plant sources are known to possess functional properties such as anti-oxidants, hence having health promoting effects. Among the several natural red colourants from plant sources, red pigments such as anthocyanin from fruits and vegetables (Psczczola DE, 1998, Food Technology 52:70-82) and betalaines from red beet (Wyler and Dreiding, 1961, Helv. Chem. Acta. 44,249) have been suggested for food applications (Psczczola DE, 1998, Food Technology 52:70-82). Reference may be made to Piatelli and Minale (Phytochemistry 3: 307-310, 1964) wherein they reported that the colour of red beet is due to water-soluble nitrogenous pigments, chemically known as betalaines. Betalaines contain two major pigments betacyanins - a red colored pigment and the betaxanthins - a yellow colored one, in addition to a few other minor similar pigments. Betalaines are present in restricted number of plants belonging to order Centrospermae (Piatelli, 1976, in The Biochemistry of Plants, Vol.7, Conn EE., Academic Press, New York, pp 557-575). The hue value of betalaines is very high, often higher than the synthetic colourant FD & C Red No. 2 (Von Elbe JH, 1975, Food Technology, 29: 42-46). The sensitivity of betalaines to heat, oxygen and gamma and UV radiation suggests that the specific application of betalaines as colorants in certain food / pharmaceutical formulations. The other natural red colour, anthocyanin is chemically grouped as "flavonoid" (Harborne JB et al, 1975, The Flavonoids, Chapman & Hall, London) is synthesized in a vast number of higher plants. Several edible fruits are good sources of anthocyanin (Shrikhande AJ, 1986, Anthocyanins in foods, CRC Grit. Rev. Food Sci & Nutrition, 7: 193-213). Apart from imparting red attractive color, anthocyanins are excellent anti-oxidants (Stahl W & Sies H, 1993, Annals of the New York Academy of Sciences 691: 10-19, (Narayan et al., 1999, Prostagland. Leuk. Essent. Fatty Acid 60:1-4) and thus impart health-promoting effect to higher animals and human beings. Some anthocyanins were tested in a standard cough syrup formulation. Black Java plum fruit serve as an excellent source of anthocyanin (Timberlake CF & Henry BS, 1986, Plant pigments as natural food colours, Endeavor NS 10: 31-36) and anthocyanins impart attractive red color. Anthocyanin extracts prepared from vegetables and edible fruits are used as edible colours throughout the world (Diplock AT et al, 1998, British Jour. Nutrition, Suppl. 1, S77-S112). Reference may be made to von Elbe (1986) who observed that the anthocyanins underwent chemical changes when incorporated in food products (von Elbe JH, 1986, Chemical changes in plant and animal pigments during food processing. In. Role of Chemistry in the Quality of Processed Food. Ed. Owen R. Fennerna et al, Food and Nutrition Press 41-64, Westport, Connecticut). The stability of natural red pigments, anthocyanins, from plant sources is strongly affected by light, pH, temperature, oxygen and water activity. For these reasons, the suitability of the pigment in any food or pharmaceutical formulations needs to be tested where the food / pharmaceutical formulation may need to be appropriately altered to stabilize the colour as well as the active ingredient, Keeping the levels of other ingredients within permissible limits. The present invention relates to a syrup formulation where the anthocyanins obtained from purple edible fruit, the "Black Java Plum" also known as Jambolan or Jamun fruit, from the tree botanically identified as Syzygium cumini, are used to colour the standard cough syrup formulation with salbutamol as the active ingredient where the ingredients of the syrup are appropriately chosen and their levels altered to allow the pigment to be stable under desired light and temperature conditions for over a period of 120 days. In the present invention, the a cough syrup formulation with Salbutamol was developed where the synthetic dye is replaced with several levels of anthocyanin extracted from the peel of edible Black Java plum to match the hue of synthetic dye in syrup. The levels of components such as propylene glycol, ascorbic acid and glycerol in the syrup were varied to obtain good stability of anthocyanin for shelf life of over 120 days under varied light and temperature conditions. Reference may be made to the Food and Drug Act, which permits anthocyanin from grape skin as a food additive. The World Health Organization (1982), recommended an average daily intake of 2.5-mg anthocyanin/Kg-body weight/day from grape skin extract (Ames and Hoffmann, 2001, Chemistry and Physiol. of Selected Food Colorants, American Chemical Society Series 775, Washington, DC). Production of anthocyanin concentrates from various plant parts and their use in food products has been studied for many years and there are many research articles and patents concerning it. The advantages of incorporating natural anthocyanin in food formulations are many. Influence of different factors on anthocyanin concentration and color in several food products such as jams, pomegranate juice during storage are studied by Gorcia-Viguera et al., (1999 J. Food Sci. 64: 243-247, and Food Science and Technology International 1999, 4: 99-105). The use of anthocyanins in foods and beverages is well known. Reference may be made to the patent filed (NF105/02/11348/DEL/02) where a syrup containing paracetamol as the active ingredient was formulated with grape skin anthocyanin with a colour stability of about 90 days. However, the use of Black Java plum fruit as a source of anthocyanin for cough syrup formulation with an extended colour stability of over 100 days has not been documented so far. Salbutamol is an extensively used active ingredient in cough syrups and other formulations targeted to asthmatic patients. The draw-backs associated with commercial formulation of cough syrups containing the active ingredient, salbutamol, are pale pink or yellow in colour caused due to the addition of synthetic dye (carmine or sunset yellow). Such synthetic dyes are known to cause harmful effects in human beings. Since there is a worldwide preference for natural colourants in food and Pharmaceuticals, the replacement of synthetic colours with natural ones such as anthocyanin will be advantageous due to health reasons. The improvement in syrup formulation induced due to the incorporation of natural colourant, anthocyanin, is two-fold. Firstly it will replace the undesirable synthetic dye while imparting a similar color effect to the formulation. Secondly, the levels of incorporation act as nutraceutical owing to their anti-oxidant effect in human systems. Novelties of the present invention are: a) The new formulation developed in the present study replaces undesirable synthetic dye in cough syrup targeted for treatment of human beings as an anti-asthmatic and spasmodic. b) Anthocyanin being a potent antioxidant, the new syrup developed here is expected to impart health benefit effect on human beings. c) The formulation is expected to impart better sensory properties to the product. d) The natural color does not involve any significant changes in the standard procedure of syrup preparation. e) The color is stable at different light and temperature conditions, for which the syrup is expected get exposed to. f) The active component i.e., salbutamol does not get affected by change in the formulation and the use of anthocyanin as a coloring agent. g) The process described here can be easily scaled up to industrial level. The main object of the present invention is to provide an improved combination of ingredients for preparing a cough syrup formulation with natural colour extract from edible vegetable/fruit sources which obviates the draw-backs of synthetic colour as referred above. Another object of the present invention is to explore the possibility of developing cough syrup formulation with natural red color with a hue value comparable to the commercial formulation. Yet another object of the present invention is to experiment and find out the level of pigment that may be needed to obtain a stable hue of the natural colour under expected fluctuations in the light and temperature conditions during a know time period Still yet another object of the present invention is to explore the stability of the natural color, in the final formulation, under various light and temperature conditions Still yet another object of the present invention is to explore the effect of the natural color on the active ingredient i.e., salbutamol. Accordingly, the present invention provides an improved process for the preparation of stabilized anthocyanin concentrate, wherein the steps comprising of: a) peeling of Black Java plum fruits, b) blending the said peels in acidified water containing 0.001 toO.002% ascorbic acid to obtain a broth, c) filtering the broth as obtained in step (b) through nylon filters and collecting the filtrate, d) centrifuging the filtrate as obtained in step (c) at 8000 to lOOOOx g at 4-5 C for 10-15 minutes to collect clear solution, e) passing the clear solution as obtained in step (d) through ion exchange column and recovering the liquid phase, f) concentrating the liquid phase obtained in step (e) under vacuum to get anthocyanin concentrate In an embodiment of the process wherein the above obtained anthocyanin concentrate at a range of l-10mg may be added to a 500ml syrup made up of salbutamol (0.200g),propylene glycol (100-500g),Podium benzoate (533g), Methyl paraban sodiate (533mg), Propyl paraban sodiate (53.3 mg), Sugar (200- 350g), Glycerol (50-200g), Ascorbic acid (100-300mg), Disodium ethylene diaminotetra acetic acid (65mg). The following examples are given by way of illustration of the present invention and therefore, should not be construed to limit the scope of the invention. Example 1 1000 Grams of healthy and uniformly ripened Black Java plum fruits are collected washed with tap water followed by washing with distilled water to remove surface dust, insect debris and pesticides if any. Surface water is gently removed by blotting with clean blotters. Fruit skins are separated, homogenized in a blender with a known volume of acidified water (with 0.0001% ascorbic acid) of pH 5.0, passed through nylon filter, the filtrate is centrifuged at 10000 x gravity at 5°C, supernatant is collected, passed through ion-exchange column (Dowex 1 and Dowex 50), concentrated the liquid phase under vacuum at 30°C. The concentrated pigment is collected and estimated the anthocyanin content by spectrophotometry following the method described by Garcia-Viguera et al (1999. J. Food Sci: 64: 243-247). Example 2 500 ml of syrup containing ingredients as in Annexure 1 is prepared (based on the formulation of Howerd C.Ansel, Introduction to pharmaceutical dosage forms, 2nd edition:107-141 ), 1.25 milligram of anthocyanin extract (prepared as described in Example 1) is added, pH adjusted to 5.5, uniformly mixed by vortexing the content, dispensed to 50 tubes each holding 10 ml of syrup. The coloured syrup formulation is divided into batches and tested for colour stability as well as salbutamol stability at temperatures of 25, 30, 35, 40 and 45°C; light regimes of 1000 lux, 2000 lux and dark for 120 days. Colour stability is tested by using spectrophotometer and by using colour-measuring system. The colour of anthocyanin is stable till the end of 120th day with a partial loss of red hue in samples stored in light resulting in about 5% loss of red hue in the syrup. There is no change in the Salbutamol content checked using standard method (1997 Quantitative analysis of drugs in pharmaceutical formulations, third edition, P.D.Sethi; New Delhi pp-342-344). Example 3 500 ml of syrup containing ingredients as in Annexure 1 is prepared to which, 2.5 milligram of anthocyanin extract (prepared as described in Example 1) is added, pH adjusted to 5.5 using 0.1N hydrochloric acid, uniformly mixed by vortexing the content, dispensed to 50 tubes each holding 10 ml of syrup. The coloured syrup formulation is divided into batches and tested for colour stability as well as salbutamol stability at temperatures of 25, 30, 35 40 and 45°C light regimes of 1000 lux, 2000 lux and dark for 120 days. Colour stability is tested by using spectrophotometer as well as by using colour-measuring system. The colour of anthocyanin is stable till the end of 120th day with a negligible (about 2%) loss of red hue in samples stored in light resulting in slight loss of red hue in the syrup. There is no change in the salbutamol content. Example 4 500 ml of syrup containing ingredients as in Annexure 1 where 100 grams of propylene glycol and 75 milli litre of glycerol is added, 3.75 milligram of anthocyanin extract (prepared as described in Example 1) is added, pH adjusted to 5.5, uniformly mixed by vortexing the content, dispensed to 50 tubes each holding 10 ml of syrup. The coloured syrup formulation is divided into batches and tested for colour stability as well as salbutamol stability at temperatures of 25, 30, 35 40 and 45°C light regimes of 1000 lux, 2000 lux and dark for 120 days. Colour stability is tested by using spectrophotometer and by using colour-measuring system. The colour of anthocyanin was stable for 120 days with a partial loss of red hue in samples stored in light however, the final hue after storage at all conditions may be similar to that required for commercial sample. There is no change in the salbutamol content. Example 5 500 ml of syrup containing ingredients as in Annexure 1 where 125 grams of propylene glycol and 75 millilitre of glycerol is added, 3.75 milligram of anthocyanin extract (prepared as described in Example 1) is added, pH adjusted to 5.5, uniformly mixed by vortexing the content, dispensed to 50 tubes each holding 10 ml of syrup. The coloured syrup formulation is divided into batches and tested for colour stability as well as salbutamol stability at temperatures of 25, 30, 35 40 and 45°C light regimes of 1000 lux, 2000 lux and dark for 90 days. Colour stability is tested by using spectrophotometer and by using colour-measuring system. The colour of anthocyanin remain stable till the end of 90th day with a negligible loss of red hue in samples stored in light however, the final hue after storage at all conditions may appear similar to that required for commercial sample. There is no change in the salbutamol content. Example 6 500 ml of syrup containing ingredients as in Annexure 1 was prepared to which 135 grams of propylene glycol and 75 millilitre of glycerol is added, 3.75 milligram of anthocyanin extract (prepared as described in Example 1) is added, pH adjusted to 5.5, uniformly mixed by vortexing the content, dispensed to 50 tubes each holding 10 ml of syrup. The coloured syrup formulation is divided into batches and tested for colour stability as well as salbutamol stability at temperatures of 25, 30, 35 40 and 45°C light regimes of 1000 lux, 2000 lux and dark for 120 days. Colour stability tested by measuring optical density using spectrophotometer as well as by using colour-measuring system confirmed the stability of colour of anthocyanin till the end of 120th day with a no loss of red hue in samples stored in light however, the final hue after storage at all conditions appeared similar to that required for commercial sample. However, there is no change in the salbutamol content. • The cough syrup formulation proposed in the present claim retains most of the incorporated anthocyanin under the maximum and minimum fluctuations of light and temperature conditions. • This formulation is new, not claimed by others. • Incorporation of anthocyanin does not alter the activity and stability of the active ingredient - salbutamol compound. We claim: 1. An improved process for the preparation of stabilized anthocyanin concentrate, wherein the steps comprising of: a) peeling of Black Java plum fruits, b) blending the said peels in acidified water containing 0.001 to0.002% ascorbic acid to obtain a broth, c) filtering the broth as obtained in step (b) through nylon filters and collecting the filtrate, d) centrifuging the filtrate as obtained in step (c) at 8000 to 1 0000xg at 4-5° C for 10-15 minutes to collect clear solution, e) passing the clear solution as obtained in step (d) through ion exchange column and recovering the liquid phase, f) concentrating the liquid phase obtained in step (e) under vacuum to get anthocyanin concentrate 2. Use of the anthocyanin concentrate as prepared by the process of claim 1, for the preparation of an improved cough formulation.

Documents:

395-DEL-2003-Abstract-(03-09-2008).pdf

395-del-2003-abstract.pdf

395-DEL-2003-Claims-(03-09-2008).pdf

395-DEL-2003-Claims-(14-11-2008).pdf

395-del-2003-claims.pdf

395-DEL-2003-Correspondence-Others-(03-09-2008).pdf

395-DEL-2003-Correspondence-Others-(14-11-2008).pdf

395-del-2003-correspondence-others.pdf

395-del-2003-correspondence-po.pdf

395-del-2003-description (complete)-03-09-2008.pdf

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

395-DEL-2003-Form-1-(03-09-2008).pdf

395-del-2003-form-1.pdf

395-del-2003-form-18.pdf

395-DEL-2003-Form-2-(03-09-2008).pdf

395-del-2003-form-2.pdf

395-DEL-2003-Form-3-(03-09-2008).pdf

395-del-2003-form-3.pdf