USOO6569446B1 (12) United States Patent (10) Patent No.: US 6,569,446 B1 Howard (45) Date of Patent: May 27, 2003 (54) SOLUBILIZATION OF FLAVONOLS 5,780,060 A 7/1998 Levy .......................... 424/489 5,908,650 A * 6/1999 Lenoble et al. ............. 426/262 (75) Inventor: Alan N. Howard, Cambridge (GB) FOREIGN PATENT DOCUMENTS (73) Assignee: The Howard Foundation, Cambridge EP O O24731 3/1981 (GB) EP O 169 347 1/1986 EP O 267 630 5/1988 (*) Notice: Subject to any disclaimer, the term of this EP O 384 796 8/1990 patent is extended or adjusted under 35 EP O 692 480 1/1996 U.S.C. 154(b) by 0 days. EP O 713 TO6 5/1996 EP 1 O72265 A1 1/2001 FR 2 577 437 2/1985 (21) Appl. No.: 09/504,747 GB 1092 269 11/1967 1-1. GB 1, 195 OSO 6/1970 (22) Filed: Feb. 16, 2000 GB 1 235 379 6/1971 O O GB 1349 483 4/1974 Related U.S. Application Data GB 2317561 A 4/1998 WO WO 94/22321 10/1994 63) Continuation-in-part of application No. 08/978,158, filed on Nov. 25, 1997 p Pat RE 6,086910, which i ti WO WO95/13360 5/1995 ation-in-partOV. Z, of, applicationnow Pat. No. No. OUSO,91U, 08/934,055, which filed is on a continuSep. 19, W W SR SEC 1997, now Pat. No. 6,099,854. (30) Foreign Application Priority Data OTHER PUBLICATIONS Sep. 20, 1996 (GB) ............................................. 96.177OO Scheffeldt et al, Journal of Food Science, 43:517-520, 1978. May 31, 1997 (GB) ...... ... 9711171 Derwent Publications, AN 1980–19056C (XP-002167810). May 31, 1997 (GB) ...... ... 9711172 Derwent Publications, AN 1990–1591.25 (XP-002167811). May 31, 1997 (GB) ............................................. 971. 1173 Ziemelis, G., et al., Precipitation of flavonols in a dry red table wine, Chemistry and Industry, Dec. 6, 1969, pp. (51) Int. Cl." ................................................ A23K 1/165 1781-1782. (52) U.S. Cl. ....................... 424/442; 464/441; 464/443; Database WPI, AN95-280915, “New Catechin Glycoside 464/456; 464/464; 514/456 Derivative Phenol Oxidase Inhibit Antioxidant Hypochol (58) Field of Search .......................... 424/442; 464/441, lesterolaemic Agent Health Food Addivitive', Derwent Pub 464/443, 456, 464; 514/456 lications Ltd., Jul. 18, 1995. Frankel, E.N., et al., “Principal Phenolic Phytochemicals in (56) References Cited Selected California Wines and Their Antioxidant Activity in U.S. PATENT DOCUMENTS Inhibiting Oxidation of Human Low-Density Lipoproteins”, J. Agric. Food Chem., 1995, 43,890-894. 4,698.360 A 10/1987 Masquelier ................. 514/456 4,808,574 A 2/1989 Brekhman ................... 514/23 * cited by examiner 4,857,327 A 8/1989 Virdalm ..... 424/195.1 4,913.909 A 4/1990 Hara .......................... 424/688 Primary Examiner. Theodore J. Criares 5,141,611 A 8/1992 Ford ....................... 204/182.4 ASSistant Examiner Jennifer Kim 5,352,384 A 10/1994 Shen .......................... 252/398 (74) Attorney, Agent, or Firm-Piper Rudnick LLP, Perry 5,464,619 A 11/1995 Kuznicki ... 424/195.1 E. Van Over 5,470,589 A 11/1995 Shi ............................ 424/698 5,474,774 A 12/1995 Walker .................... 424/195.1 (57) ABSTRACT 5,525,341. A 6/1996 Walker .................... 424/195.1 5,554,645 A 9/1996 Romanczyk .. ... 514/453 A method of increasing the solubility in water of a flavonol 5,587,176 A 12/1996 Warren ......... ... 424/443 component of a flavonol-containing composition, the 5,591,771 A 1/1997 Markonius . ... 514/456 method including the Steps of providing a flavonol 5,607.965 A 3/1997 Kondo ...... ... 514/456 containing composition, providing an anthocyanin 5,637,561. A 6/1997 Shen ............................. 514/2 containing composition and mixing the two compositions. 5,650,432 A 7/1997 Walker ...... ... 514/456 5,686,082 A 11/1997 N’Guyen ... ... 424/401 5,762.936 A 6/1998 Ronzio .................... 424/195.1 28 Claims, 4 Drawing Sheets U.S. Patent May 27, 2003 Sheet 1 of 4 US 6,569,446 B1 U.S. Patent May 27, 2003 Sheet 2 of 4 US 6,569,446 B1 mg/L Quercetin 75 Anthocyanin 60 45 30 15 - - 1 Ethonol O O 5 10 15 20 25 mg/ml Anthocyanin Powder or % Ethanol mg/L Quercetin 200 100 50 O O 10 20 30 40 mg/ml Anthocyanin Powder (Grape) FIG.5 U.S. Patent May 27, 2003 Sheet 3 of 4 US 6,569,446 B1 mg/L Quercetin 20 15 O 10 O O 5 O O 10 20 30 40 50 mg/ml Anthocyanin Powder (Black Carrot) F G 6 mg/L Quercetin 300 200 100 O O 25 50 100 200 mg/ml Anthocyanin Powder (Red Cabbage) FIG.7 U.S. Patent May 27, 2003 Sheet 4 of 4 US 6,569,446 B1 mg/L Quercetin 1000 O 800 500 O 200 O O 125 200 500 800 1000 mg/ml Anthocyanin Powder (Malvin Chloride) FIG.8 US 6,569,446 B1 1 2 SOLUBILIZATION OF FLAVONOLS type of grape, the climate in which it is grown, and the manufacturing process used in making the juice. Some grape This Application is a continuation-in-part of U.S. appli juice, especially that made from the Concord grape, may cation Ser. No. 08/978,158, filed Nov. 25, 1997, now U.S. contain as much as 2.5g polyphenol per litre of juice. Grape Pat. No. 6,086,910, which is a continuation-in-part of U.S. skins and Seeds are commercially extracted with water and application Ser. No. 08/934,055 filed Sep. 19, 1997, now other Solvents to obtain polyphenols. In addition, polyphe U.S. Pat. No. 6,099,854. nols from grape skins and Seeds become incorporated into wine during the Vinification process. Red wine is made by FIELD OF THE INVENTION maintaining contact between the fermenting liquor and the crushed grape residue (pomace) for prolonged periods, This invention relates to a method of increasing the whilst in the manufacture of white wine the grape Skins are solubility of flavonols and their related compounds in water removed relatively quickly. Accordingly, wine in general, and aqueous Solutions, and to a method of providing Soluble and red wine in particular, contains reasonable amounts of flavonol-containing compositions. polyphenols, amounting to about 1-3 g/L and is thus a BACKGROUND TO THE INVENTION 15 potential commercial Source of polyphenolic compounds. Polyphenols are known to have antioxidant properties and Polyphenols are those compounds which comprise more have potential use in the food, cosmetic and pharmaceutical than one phenolic group. Among the polyphenols are the industries. Among the polyphenols, the flavonols have been following classes: flavonoids (a term often used to denote shown to have many useful properties as antioxidants, and polyphenols in general, but more commonly in Europe to to decrease platelet Stickiness. denote only the flavones), the flavanols, proanthocyanidins Epidemiological Studies have shown that countries and (also called procyanidols, procyanins, procyanidins and people with a high flavonol intake have leSS coronary heart tannins) and anthocyanins. disease (Hertog et al., 1995 Arch. Int. Med. 155, 381-6). The flavones are compounds with a basic Structure shown In unprocessed fruit and vegetables the flavonols occur as in FIG. 1 in which two benzene rings (A and B) are linked 25 glycosides and the aglycone is absent. The most abundant with a heterocyclic Six member ring C containing a carbonyl flavonol is quercetin. Among vegetables the highest concen group. Ring B can be joined in position 2 (as illustrated) to tration of quercetinglycosides is in onions (3 to 500 mg/kg), give a flavone or to position 3 to give an iso flavone. kale (100 mg/kg) French beans (30 to 45 mg/kg), and Hydroxylation can occur at one or more of positions 3, 5, 7, broccoli (30 mg/kg). Among fruit examined quercetin con and 3', 4', 5' to give compounds called flavonols. Typical centration averageS 15 mg/kg, with apples having the high examples of flavonols are: quercetin (hydroxylated at posi est concentration of 21 to 72 mg/kg (Hertogetal, 1992 J. Ag. tions 3, 5, 7,3', 4'), kaempferol (hydroxylated at positions 3, Food Chem. 40, 2379–83). 5, 7, 4), and myricetin (hydroxylated at positions 3, 5, 7,3', Flavonol glycosides are present in grapes and values 4, 5). Flavonols can exist naturally as the aglycone or as ranging from 8 to 97 mg/Kg fresh weight have been reported O-glycosides (e.g. with D-glucose, galactose, arabinose, 35 (Macheix et al., 1990 Fruit Phenolics pp 378 CRC Press rhamnose etc). Other forms of substitution such as Boca Raton). During the fermentation process, Some of the methylation, Sulphation and malonylation are also found. Sugar is split off and the aglycone formed. On average about The flavanols have a basic structure shown in FIG. 2. The 50% of the flavonol exists in wine as the aglycone. The two most common flavanols are catechin (hydroxyl groups flavonol content of grape skins and wine is very variable and at positions 5, 7, 3', 4') and its Stereo-isomer epi-catechin. 40 depends on the variety of grape and more especially the The hydroxyl groups can be esterified with gallic acid. The amount of Sunshine in which the grapes are grown. The proanthocyanidins are polymers of catechin and/or epicat flavonols have a yellow color and act as filters to blue and echin and can contain up to 8 or more repeat units. These ultraViolet light which is very injurious to the grape.