Title of Invention	POLYPHENOL-ENRICHED COMPOSITION FROM COCOA SHELL EXTRACTION
Abstract	The invention relates to a process for the extraction of cocoa shells to provide a theobromine-enriched fraction of composition and a polyphenol-enriched fraction or composition

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FORM 2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION (See Section 10; rule 13) TITLE POLYPHENOL-ENRICHED COMPOSITION FROM COCOA SHELL EXTRACTION APPLICANT KRAFT FOODS R & D, INC. Bayerwaldstrasse 8 D-81737 Munich Germany Nationality : a US corporation The following specification particularly describes the nature of this invention and the manner in which it is to be performed POLYPHENOL-ENRICHED COMPOSITION FROM COCOA SHELL EXTRACTION FIELD OF THE INVENTION The present invention relates to a process for the extraction of cocoa shells to provide a theobromine-enrlched fraction or composition and a polyphenol-enriched fraction or composition. BACKGROUND OF THE INVENTION Naturally-occurring polyphenois derived from plants or plant materials (e.g, tea, cocoa beans, and the like) are known to have antioxidant properties as well as providing other potential health benefits. Thus, considerable research has been carried out in recent years with regard to methods for obtaining such po'lyphenols, including cocoa polyphenois, as well as methods for using them. U.S. Patent 5,554,645 (September 10,1996) provides a method for extraction of polyphenols from dehulled, defatted, freeze-dried cocoa bean powder using a 70:30 acetone;water solvent. The extract was then purified using.gel permeation chromatography or high performance liquid chromatography to obtain polyphenols consisting essentially of oligomers 3 through 12. International Patent Publication WO 00/45769. (August 10, 2000) and corresponding U.S. Patent 6,576,275 (June 10, 2003) provide a method for extraction of polyphenols from cocoa and other plant materials to provide a reduced-purine-containing polyphenol material. Starting cocoa materials including, for example, fresh cocoa beans, defatted cocoa solids, cocoa powder, low-fat cocoa powder, cocoa shells, cocoa waste materials, or other cocoa-containing raw materials are preferably ground to a particle size of less than 250:m. The ground cocoa material is then extracted with a hydroxylic solvent (e.g., water, lower alcohols, and mixtures thereof). The resulting extract Is then applied to an adsorption material having a high polyphenol/purine adsorption ratio of st least about 5/1. Suitable absorbents 2 Include polyvlnylpolypyrrolidone and chitosan, as well as derivatives, modifications, and Wends thereof. The polyphenols are then removed from the adsorption material using a desorption solvent such as water, a water- miscible alcohol, mixtures of water and a weter-miscible alcohol, and mixtures of water and a water-miscible Ketone. The polyphenol-containing material can then be concentrated by evaporation of the desorptlon solvent. Although this method reduces the relative amounts of purines (i e,, theobromine and caffeine), significant amounts of purines remain in the collected polyphefrol-contafnfng materials. O.S. Patent 8r159,451 (December 12,2000) provides a method of producing a fraction of cacao bean husk having activity against glucosyltranferase in the prevention of tooth decay. In this method, 4 to 10 parts of a 50 percent acetone aqueous solution was added to 1 part dried cacoa bean husk, stirred under reflux at 40 to 80°C for 4 to 6 hours, to form an extract. This procedure was repeated twice The extract was then concentrated and dried using vacuum techniques. The resulting extract was then added to a styrene-based adsorption resin, washed with 1 to 2 pails of 20 percent ethanol, followed by the addition of 1 to 2 parts of 50 percent ethanol: The fractions from the 50 percent ethanol elution were collected and concentrated under reduced pressure at 40 to 50°C to provide the desired fraction having activity against glucosyltranferase. International Patent Publication WO 00/62631 (October 28,2000) provides a method to produce a cacao extract containing dietary fiber (especially insoluble dietary fiber) which Is reported to be useful In treating diabetes. Heat-treated cacao husks are extracted with ethanol and then centrifuged and then fractionated into a supernatant and a residue. The residue faction is reportedly useful In the treatment of diabetes. The supernatant is reported to contain polyphenols and can be used In beverages, European Patent Application EP 1304047 (published April 23, 2003) provides a method for obtaining an extract of cacao bean or cacoa bean husks which reportedly Inhibits the suppression of gap functional Intercellular 3 communication and DNA synthesis of cancer cells. After removing cocoa butter, the cacao bean husks were extracted with 50 percent acetone for 5 hours at 60°C under reflux. The extract was centrifuged and the supernatant was collected, filtered, concentrated, and freeze dried. The resulting cacao bean husk fraction was adsorbed on a polystyrene hydrophobic adsorption resin and fractionated with water/acetone or water/methanol mixtures to obtain the active material. European Patent Application EP1346640 (published September 24, 2003) provides a method for producing a low fat cocoa extract having a high level of cocoa flavors and/or cocoa flavor precursors and a high level of antioxidants (e.g., polyphenols). This method involves placing cocoa seeds In an acetic acid solution, heating the mixture, removing the seeds, and concentrating the dissolved solids from the solution. U.S. Patent 6,627,232 (September 30,2003) provides a method for selectively extracting tetrarners, pentamers, and higher molecular weight cocoa procyanidin oligomers from partially defatted or fully defatted cocoa solids prepared from cocoa beans that have not been roasted. In one embodiment, this method includes the steps of (a) extracting the cocoa solids with ethyl acetate; (b) recovering the extracted cocoa solids; (c) extracting the recovered, extracted cocoa solids with a solvent selected from the group consisting of acetone, ethanol and mixtures thereof with up to 50% water by volume and; (d) separating the cocoa Solids from the cocoa extract of step (c) to obtain the procyanidin extract U.S. Patent Publication US 2004/0096566 (May 20,2004) provides a method for obtaining polyphenols from cocoa beans. The fresh beans (which have not been pro-treated or defatted) are treated to remove the shells and pulp, ground In the presence of solvents, infused with the solvent for a few hours to a few days, filtered and rinsed with the same solvent, and the solvent removed by distillation under vacuum to obtain the desired residue. Although these methods are generally able to provide polyphenols from various portions of the cocoa bean, it would still be desirable to provide a methods in which a polyphenol-enriched fraction and a theobromine-enrlched 4 fraction can be obtained so the more of the components can be used. Moreover, it would be desirable to provide such fractions using what is normally considered a waste product (namely, the cocoa shells), The present Invention provides such methods. SUMMARY OP THE The present Invention relates to a process for the manufacture of a theobromine-enriched composition and a polyphenol-enriched composition from cocoa shells, said process comprising (1) providing defatted cocoa shells; (2) extracting the defatted cocoa shells with an aqueous acetone solution containing about 30 to about 80 percent acetone to provide a cocoa shell extract material; (3) treating the cocoa shell extract material to remove suspended solids to provide a treated cocoa shell extract material; (4) removing acetone from the treated cocoa shell extract material to provide an aqueous extract material; (5) concentrating (he aqueous extract material to about 1.5 to about 5 percent solids to provide a concentrated extract material; (6) applying the concentrated extract material to a gel filtration column containing a gel filtration medium suitable for separating theobromine from polyphenols; (7) rinsing the gel filtration column with water to collect a thaobromine enriched fraction; (8) eluting the water-rinsed gel filtration column with a low molecular weight polar organic solvent to collect a polyphenol-enrlched fraction, wherein the polyphenol-enriched fraction is essentially free of phytosterols and theobromine; (9) concentrating the theobromine-enrlched fraction to obtain the theobromine-enriched c6mposltlon; and (10) concentrating the polyphenol-enriched fraction to obtain the polyphenol-enriched composition. Preferred gel filtration media for use in this 5 invention include Sephadex™-type gel filtration media from Amersham Biosciences (a part of GE Healthcare); Sephadex™ LH-20 is especially preferred. The low molecular weight polar organic solvent is preferably methanol. The cocoa shells used in the present invention can be derived from roasted or unroasted cocoa beans and/or from fermented or unfermented cocoa beans. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a general flow diagram illustrating the present invention. Figure 2 is a detailed flow diagram illustrating a preferred embodiment of the present invention. DETAILED DESCRIPTION As indicated in Figure 1, defatted cocoa shells are obtained. The cocoa shells may be derived from fermented or unfermented beans, either of which may be unroasted or roasted; the shells used In the extraction process of this invention may be whole, broken, ground, or combinations thereof. Such defatted cocoa shells can be prepared using conventional techniques; for example, the cocoa shells can be defatted by extraction using hexane or other low molecular weight, non-polar or weakly-polar solvent (e.g., penfene, ethyl ether, petroleum ether, and the like); the non-polar solvent hexane Is generally preferred. Although not wishing to be limited by theory, it is thought that defatted the cocoa shells assists In removing phytosterols prior to the next extraction step as well as providing for improved separation In the later stages of the process. The defatted cocoa shells are extracted with an aqueous acetone solution. Generally, this aqueous acetone solution contains about 30 to about 80 percent acetone,- preferably about 40 to about 60 percent acetone, and more preferably about 50 percent acetone. Suspended solids are then removed from the aqueous acetone extracted material using conventional techniques (e.g., filtration, decantation, centrifugation, and the like), At least a portion of the acetone (generally about 80 percent or more and preferably about 95 to essentially 100 percent) is removed using 6 conventional techniques (e.g., distillation, vacuum distillation, and the like) to obtain an aqueous extract material. The resulting aqueous extract material is then subjected to gel filtration using a gel filtration medium suitable for separating both theobromine and polyphenols. Preferred get filtration media for use In this Invention Include SephadexTM-type gel filtration media (generally derived from three-dimensional crosslinked polysaccharfde dextran) from Amersham Blosclences (a part of GE Healthcare); Sephadex™ LH-20 Is especially preferred. The gel filtration column containing the aqueous extract material Is first rinsed with water to remove a theobromine-enriched fraction; the theobromine-enrlched fraction is collected. The gel filtration column Is then eluted with a low molecular weight polar organic solvent to remove the polyphenol-enriched fraction; the polyphenol-enriched fraction Is then collected. Suitable low molecular weight polar organic solvents Include, for example, methanol, ethanol, ethyl acetate, acetone, and the like; generally, methanol Is preferred, The polyphenol-enriched fraction is essentially free of phytosterols and theobromine. For purposes of this Invention, “essentially free of phytosterols” is Intended to include polyphenol-enriched compositions wherein at least about 90 percent, preferably about 95, and most preferably essentially 100 percent (I.e., only a trace remaining), of the phytosterols present in the original cocoa shells have been removed. Likewise, for purposes of this Invention, “essentially free of theobromine” is Intended to Include polyphenol-enriched compositions wherein at least about 90 percent, preferably about 95, and most preferably essentially 100 percent (le., only a trace remaining), of the theobromine present In the original cocoa shells have been removed. As noted above, the cocoa shells used in the present invention can be derived from roasted or unroasted cocoa beans and/or from fermented or unferrnented cocoa beans. Generally, higher yields of polyphenols are expected from unfermented shells as compared to fermented shells as well as from roasted shells as compared to unroasted shells. Figure 2 illustrates a preferred embodiment of the present Invention. In this embodiment, the defatted cocoa shells are extracted multiple times 7 with an aqueous acetone solution. Generally, this aqueous acetone solution contains 30 to about 80 percent acetone, preferably about 40 to about 60 percent acetone, and more preferably about 50 percent acetone, For purposes of this Invention, “multiple times” Is Intended to mean at least two extractions, preferably about 2 to 5 extractions, and more preferably about 2 to 3 extractions. Preferably, each extraction Is carried out with a fresh (I.e., one which has not been previously used for extraction) aqueous acetone solution. The liquid extracts are preferably combined and then treated to remove suspended solids using conventional techniques (e.g., filtration. decantation with or without centrifugation, and the like). At least a portion of the acetone (generally about 80 percent or more and preferably about 95 to essentially 100 percent) is removed using conventional techniques (e.g., distillation, vacuum distillation, and the like) to obtain an aqueous extract material. The resulting aqueous extract material is then subjected to gel filtration using a gel filtration medium suitable for separating both theobromlne and polyphenols. Preferred gel filtration media for use in this Invention include Sephadex™-type gel filtration media (generally derived from three-dimensional crosslinked polysaccharide dextran) from Amersham Biosciences (a part of GE Healthcare); Sephadex™ LH-20 is especially preferred. The gel filtration column containing the aqueous extract material is first rinsed with water to remove a theobromine-enriched fraction; the theobromine-enrtehed fraction is collected. The gel filtration column is then eluted with a low molecular weight polar organic solvent, preferably methanol, to remove the polyphenol-enriched fraction; the polyphenol enriched fraction Is then collected. The collected theobromine-enriehed fraction can be used as the theobromine-enriched composition or, if desired, concentrated to obtain the theobromlne-enrfched composition. Such concentration can be carried out using conventional techniques such as, for example, freeze drying, thermal drying, crystallization, liquid partition techniques using chloroform or methylene chloride, and the like; generally freeze drying is preferred, 8 The collected polyphenol-enriched fraction can be used ae the polyphenol-enriched composition or, If desired, concentrated to obtain the polyphenol-enriched composition. Such concentration can be carried out by first removing at least a portion of the methanol using conventional techniques (e.g., evaporation, vacuum distillation, and the like) followed by removing at least a portion of the water by conventional techniques (e.g., freeze drying, thermal drying, crystallization, liquid partition, and the like) with freeze drying being preferred, The invention will now be illustrated by specific examples which describe preferred embodiments of the present invention. They are not intended to limit the scope of the invention. Unless otherwise indicated, all ratios and percentages throughout this specification are by weight AH patents and other publications discussed In this specification are hereby Incorporated by reference. Example 1. This example illustrates the isolation of a polyphenol-enriched fraction and a theobromine-enriched fraction from cocoa shells. Cocoa shells were obtained from unroasted fermented cocoa beans in the usual way. Roasted or unroasted cocoa shells from fermented or unfermented cocoa beans can be used in the same manner if desired. The cocoa shells (BOg) were extracted with hexane (200 ml) for 6 hours in a soxhlet extractor. After drying In vacuum, the defatted shells were slurrled with 50 percent aqueous acetone (350 ml) at 70°C for 45 minutes in a flask fitted with a condenser. After centrifuging and decanting the extract, the residue was further extracted with 50% acetone solution (200mi); the extraction/separation procedure was repeated up to 4 times. The extracts were combined and concentrated to about 2X by rotary evaporation. The resulting concentrated aqueous solution was taken to dryness by freeze drying, Polyphenol contents were measured by the Folln-Clocalteu method (see, e.g.,Singleton et at.,Am.J. Bra/. W..37 144-158(1965)). The . following results were: obtained: 9 The freeze dried concentrated extract obtained from combining all five extractions was added to a Sephadex LH-20 gat permeation column (diameter 4.5 cm, length 30 cm). The column was rinsed with water to yield a thaobromine-enriched fraction which was obtained in the dry state by freeze drying. Methanol was then used to elute a polyphenol-enriched fraction which was then dried by evaporation of the methanol and freeze drying to obtain a solid polyphenol-enriched composition or residua. The following results were obtained: This example clearly demonstrates that a theobromine-enriched fraction 0.e, the water eluate) and a polyphenol-enriched faction (Le, the methanol eluate) can be obtained from cocoa shells using the present Invention, Moreover, this example also demonstrates that the polyphenol-enriched fraction Is essentially free of theobromine (as well as caffeine), After the methanol elution, polyphenols remain on the column (about 1,90 - 0.75 - 0.77 = 0,38 g/100g shells); further recovery of polyphenols could be obtained, if desired, by additional methanol elutate of the column gnd treatment of the eluate as above. 10 The antloxldant capacity of the polyphenol-enriched fraction methenol was measured using a TEAC (Trotox Equivalent Antloxldant Capacity) method (Re et at, Free Radical Bblogy & Medicine, 26,1231-1237 (1989)). An antfoxldant capacity of 1.8 μmol Trototfmg was found This antloxldant capacity Is significantly higher than that typically found for commercial rosemary, grape, and elderberry extract samples (0,83,0.78 and 0,42 μmol Trolox/mgr respectively). Example 2. This example illustrates a modification of the procedure used In Example 1. Essentially the same procedure was used as In Example 1 except that, subsequent to water rinsing, the column was eluted with 50% aqueous methanol followed by pure methanol. The eluants wane dried as described In Example 1. The following results were obtained: E^mpla3T This example Illustrates the isolation of a high theobromine content solid preparation from the theobromine-enriched faction* The theobromine-enriched faction was obtained as In Example 1, The water rinse was collected and reduced to a low volume (to the point where cloudiness became apparent). A small amount of active carbon was added and the mixture filtered. Hot water was passed through the residue and the washings collected and added to the filtrate. The solution was reduced to a volume of 100 ml and poured into a separating funnel. Beaker washings and methytene chloride (50 ml) were added, the mixture shaken (3 to 4 times), allowed to settle, and the lower phase (chlorinated solvent) ran out. Methylene chloride addition, the shaking, settling, and separation steps were repeated three times, The methylene chloride was then removed by drying 11 over sodium sulfate, the latter removed by filtration, and the theobromine enriched product obtained by evaporation on a rotary evaporator. The theobromine content of the solid preparation, as determined by HPLC, was about 84 percent. The preparation of a similar solid composition prepared using chloroform rather than methylene chloride as solvent contained about 64 percent theobromine.

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