Title of Invention

"A PROCESS FOR THE EXTRACTION OF THE ANTIOXIDANTS FROM POMEGRANATE PEELS (PUNICA GRANATUM)"

Abstract

The present invention provides a process for the extraction of antioxidants from pomegranate peels {Punica granatum). The process is comprised of powdering the dried peel followed by organic solvent extraction , concentration and vacuum distillation. The extract obtained from the present process has 78 % of phenolic content and shows the 70 % radical scavenging activity at 10 ppm concentration. Phenolic content of the extract has 78 % of phenolic content.

Full Text

The present invention relates to a process for the extraction of the antioxidants from pomegranate peels (Punka granatum). Antioxidants have become an indispensable group of food additives mainly become of their unique properties of enhancing the shelf life of a host of food products without any damage to sensory or nutritional qualities. Both synthetic and natural antioxidant activities are used in food products to retard lipid oxidation. Synthetic antioxidants are mainly phenolics e.g. BHA, BHT, TBHQ and the gallates. Of the natural antioxidant activity two important groups, tocopherols and ascorbic acid are highly effective in many food products. Many natural products such as plant phenolic compounds, spice extracts have significant antioxidant activity. Antioxidants are compounds, having the capacity to prevent, retard or delay the oxidation process. Antioxidants are affecting in vitro scavengers of reactive oxygen species and free radicals. Antioxidants protect LDL oxidation. The shelf life of processed foods is expected to improve with the use of the above food additives (Adegoke GO, Vijay Kumar M, Gopala Krishna AG, Varadaraj, MC, Sambaiah K and Lokesh BR, J. Food Sci. Technol., 35(4), 283-298,1998). Reference may be made to Shin et al. (Shin, Z., Chang, Y.S., Kaung, W.S. and Jung, S.U. US Patent US 175012, 1992) wherein the isolation of antioxidant from defatted rice bran is reported. The rice bran was extracted with water and ethanol (1:0.8) to get an extract. Reference may be made to Nguyen et al. (Nguyen, Uy., Frakman, G. and Evans, D.A. US Patent 5017397, 1991), wherein the isolation of antioxidant from Labiatae herbs by application of a supercritical fluid extraction using carbon dioxide under specific conditions is reported. Reference may be made to Shin et al. (Shin, Z., Chang, Y.S., Kaung, W.S. and Jung, S.U. US Patent US 175012, 1992) wherein the isolation of antioxidant from defatted ricebran is reported. The ricebran was extracted with water and ethanol (1:0.8) to get an extract. Reference may be made to Todd and Paul (US Patent 5209870,1993), wherein the isolation of Labiatae antioxidants using aqueous alkaline lower aliphatic alcoholic or polyol solution is reported. Reference may be made to Gill et al., (Gill, M.I., Francisco, A., Barberan, T, Hess-Pierce, B, Holcroft, D.M. and Kader, A.A., J. Agric. Food Chemistry, 48(10), 4581-4589, 2000), wherein the juice of pomegranate was used for antioxidant activity. The drawback of this method is that polyphenolic content is However, the exhaustive literature survey reveals that, there is no existing process reported for the extraction of antioxidants from pomegranate peels (Punica granatum). Invention concerns a process for the production of natural antioxidants from pomegranate peels. Recently, the demand for natural antioxidants for the stabilization of animal and vegetable fats and oils has increased considerably in the food and cosmetics industry after understanding that synthetic antioxidants, such as BHA (butylhydroxyanisole), BHT (butylhydroxytoluene) as well as TBHQ (tertiary butylhydroquinone) cannot be regarded as toxicologically harmless and safe. The main object of the present invention is to provide a process for the extraction of antioxidants from pomegranate peels (Punica granatum). Another object of the present invention is to provide a concentrated preparation of phenoHcs from pomegranate peels. It provides maximum utilization of the antioxidant properties of the phenoHcs, which are non-ionic, non-toxic and water-soluble antioxidants. Another object of this invention is to utilize the pomegranate peels for the production of value added preparation, which are the byproducts from food processing industries. Therefore, the object of the present invention is to provide a process for the production of natural antioxidants from pomegranate peels by extraction using acetone. The pomegranate peel extract is obtained according to the present invention, which is essentially neutral in flavour, odor and Hght brown in colour and which is more effective as compared to synthetic antioxidants. The novelty of the process is: 1. To utilize the waste material from agro-food industries. 2. There is no report on the antioxidant activity of pomegranate peels. 3. The present invention exploits the use of a solvent, which gives highest yield of polyphenols and antioxidants. Accordingly, the present invention provides the process for the extraction of antioxidants from pomegranate peels (Punica granatum), which comprises, i). separation of pomegranate peels from pomegranate fruit manually, ii). drying of the peels either mechanically for 3 hours at 80°C or sun drying or shadow drying for 8-12 hours to moisture content of less than 7%, iii). particle size reduction of peels using mechanical grinder to obtain 40 - 60 mesh size, iv). antioxidants extraction using known methods with solvents such as hexane, ethyl acetate and acetone in a successive manner at temperature 40-70 °C for 4-8 h, v). concentrating antioxidant extract using distillation under vacuum to obtain maximum 40% yield. In another embodiment of the present invention, the peels may be dried by conventional method to moisture content of less than 7%. In another embodiment of the present invention, the peel powder may be subjected to successive extraction preferably at 60-70°C for 6-8 h. In another embodiment of the present invention, the extract recovery may be upto maximum 40% with acetone. The extraction of antioxidants from pomegranate peels was done according to following flow diagram (Diagram Removed) The radical scavenging activity was measured as following (Blios, M.S. Nature, 181,1199,1958). Aliquots of 0.025, 0.05, 0.1 ml of pomegranate peel extracts and standard were taken in different test tubes. To this, 5.0 ml of methanolic solution of l,l-Diphenyl-2-picryl hydrazyl (DPPH) was added, shaken well and the mixture was allowed to stand at room temperature for 20 min. The blank was prepared as above without extract. The changes in the Optical density (OD) of the samples were measured at 517 run. Radical scavenging activity was expressed as the inhibition percentage calculated using the following formula, % Radical scavenging activity = Blank OD - Sample OD/ Blank OD x 100. The radical scavenging activity was found to be 68-74% at 10 ppm concentration. The concentration of phenolic compounds in the extracts was determined by the method of Ock-Sook Yi (Ock-Sook Yi., Anne S. Meyer and Edvin N. Frankel, /. Am. Oil. Chem. Soc. 1997, 74, 1301) and results were expressed as (+) catechin equivalents. The pomegranate peel extracts were dissolved in a mixture of methanol and water (6:4 v/v). Samples (0.2 ml) were mixed with 1.0 ml of ten fold diluted Folin-Ciocalteu reagent and 0.8 ml of 7.5% Sodium Carbonate solution. After standing for 30 min at room temperature, the absorbance was measured at 765 nm using Genesys-5 UV-visible spectrophotometer (Milton Roy, USA). The estimation of phenolic compounds in the fractions was carried out in triplicate and the results were averaged. The phenolics were found to be in the range of 70-80%. The following examples are given by way of illustration of the present invention and therefore should not be constructed to limit the scope of the present invention. Example 1 Pomegranate peels were manually removed from the fruits and were cleaned of adhering tissues. The peels were sun dried to moisture content of 7%. The dried peels were powdered in a kitchen blender and sieved to get 40 mesh size. 50 g of the peel powder was extracted with hexane (250 ml) for 4 h using soxhlet extractor. The left over pomegranate residue was re-extracted with ethyl acetate (250 ml) in a soxhlet extractor for 4 h. The filtrates were discarded. The remaining residue was dried and it was subjected to extraction with acetone (250 ml) using soxhlet extractor for 8 h. The extract was filtered and concentrated under vacuum to get powder (34.4% yield). The radical scavenging activity was measured as following (Blios, M.S. Nature, 181,1199,1958). Aliquots of 0.025, 0.05, 0.1 ml of pomegranate peel extracts and standard were taken in different test tubes. To this, 5.0 ml of methanolic solution of l,l-Diphenyl-2-picryl hydrazyl (DPPH) was added, shaken well and the mixture was allowed to stand at room temperature for 20 min. The blank was prepared as above without extract. The changes in the Optical density (OD) of the samples were measured at 517 nm. Radical scavenging activity was expressed as the inhibition percentage calculated using the following formula, % Radical scavenging activity = Blank OD - Sample OD/ Blank OD x 100. The radical scavenging activity was found to be 68-74% at 10 ppm concentration. The concentration of phenolic compounds in the extracts was determined by the method of Ock-Sook Yi (Ock-Sook Yi., Anne S. Meyer and Edvin N. Frankel, /. Am. Oil. Chem. Soc. 1997, 74, 1301) and results were expressed as (+) catechin equivalents. The pomegranate peel extracts were dissolved in a mixture of methanol and water (6:4 v/v). Samples (0.2 ml) were mixed with 1.0 ml of ten fold diluted Folin-Ciocalteu reagent and 0.8 ml of 7.5% Sodium Carbonate solution. After standing for 30 min at room temperature, the absorbance was measured at 765 ran using Genesys-5 UV-visible spectrophotometer (Milton Roy, USA). The estimation of phenolic compounds in the fractions was carried out in triplicate and the results were averaged. This fraction has 84% phenolic content and it showed 74% radical scavenging activity at 10 ppm concentration. Example 2 Pomegranate peels were manually removed from the fruits and were cleaned of adhering tissues. The peels were oven dried to moisture content of 5.3% for 3 hours at 80°C. The dried peels were powdered in a kitchen blender and sieved to get 60 mesh size. 100 g of the peel powder was extracted with hexane (500 ml) for 4 h using soxhlet extractor. The left over pomegranate residue was re-extracted with ethyl acetate (500 ml) in a soxhlet extractor for 4 h. The filtrates were discarded. The remaining residue was dried and it was subjected to extraction with acetone (500 ml) using soxhlet extractor for 8 h. The extract was filtered and concentrated under vacuum to get powder (32.42% yield). The phenolic content and radical scavenging activity were assayed as mentioned in the earlier example (1). This fraction has 78% phenolic content and it showed 70% radical scavenging activity at 10 ppm concentration Example 3 Pomegranate peels were manually removed from the fruits and were cleaned of adhering tissues. The peels were sun dried to moisture content of 4.8%. The dried peels were powdered in a kitchen blender and sieved to get 40 mesh size. 25 g of the peel powder was extracted with hexane (125 ml) for 4 h using soxhlet extractor. The left over pomegranate residue was re-extracted with ethyl acetate (125 ml) in a soxhlet extractor for 4 h. The filtrates were discarded. The remaining residue was dried and it was subjected to extraction with acetone (125 ml) using soxhlet extractor for 8 h. The extract was filtered and concentrated under vacuum to get powder (41.0 % yield). The phenolic content and radical scavenging activity were assayed as mentioned in the earlier example (1). This fraction has 68% phenolic content and it showed 65% radical scavenging activity at 10 ppm concentration We Claim: 1. A process for the extraction of antioxidants from pomegranate peels (Punica granatum) which comprises, i). separating the pomegranate peels from pomegranate fruit, ii). drying the peels for 2-3 hours at 80-90°C for 8-12 hours to obtain the desired moisture content less than 7%, iii). reducing the particle size of the peels using mechanical grinder to obtain 40 - 60 mesh size, iv). extracting the antioxidants using known methods with an organic solvents, in a successive manner at temperature ranging between 40-70 °C for 4-8 h, v). concentrating the above extract using distillation under vacuum to obtain the desired product, 2. A process as claimed in claims 1 wherein the peels is dried by conventional method to moisture content of less than 7%. 3. A process as claimed in claim 1&2, wherein the peel powder used is subjected to successive extraction preferably at 60-70°C for 6-8 hours. 4. A process as claimed in claim 1-3 wherein, the extract recovery is upto maximum 40% with acetone. 5. A process for the extraction of antioxidants from pomegranate peels substantially as herein described with reference to the examples.

Documents:

392-del-2001-abstract.pdf

392-del-2001-claims.pdf

392-del-2001-complete specification (granted).pdf

392-del-2001-correspondence-others.pdf

392-del-2001-correspondence-po.pdf

392-del-2001-description (complete).pdf

392-del-2001-form-1.pdf

392-del-2001-form-18.pdf

392-del-2001-form-2.pdf

392-del-2001-form-3.pdf