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 This application is a continuation-in-part of U.S. Ser. No. 09/317,310, filed May 24, 1999. This application is based upon Provisional application Ser. No. 60/111,945, filed Dec. 11, 1998. This application is also based upon U.S. Provisional Application Serial No. 60/120,178, filed Feb. 16, 1999.
 (1) Summary of the Invention
 The present invention relates to a method of use of at least one compound isolated from cherries as cyclooxygenase (COX-1 and COX-2) inhibitors. In particular, the present invention provides a natural cherry composition containing a mixture of anthocyanins, bioflavonoids and phenolics for use as anti-inflammatory agents as a result of inhibition of the cyclooxygenase enzymes.
 (2) Description of Related Art
 Many plant-derived compounds may also impart important positive pharmacological or “nutraceutical/phytoceutical” traits to foods by way of their abilities to serve as antioxidants by maintaining low levels of reactive oxygen intermediates, as anti-inflammatory agents by inhibiting prostaglandin synthesis, or as inhibitors of enzymes involved in cell proliferation. These activities may be important in ameliorating chronic diseases including cancer, arthritis, and cardiovascular disease (Kinsella et al., Food Tech. 85-89 (1993). Thus, with natural products, the dietary supplement/food industry and neutraceutical/phytoceutical companies have the opportunity to employ compounds which can not only enhance food stability as effectively as synthetic antioxidants, but can also offer significant health benefits to the consumer.
 Cherries are thought to have beneficial health properties in general. Consumption of cherries was reported to alleviate arthritic pain and gout (Hamel, P. B., et al. Cherokee Plants 28: Herald: Raleigh, N.C. (1975)) although there is no evidence for its active components or mode of action. These beneficial effects may be partially associated with the abundance of anthocyanins, the glycosides of cyanidin.
 Colorants like anthocyanins have been regarded as the index of quality in tart cherries. Most importantly, recent results showed that anthocyanins such as cyanidin-3-glucoside have strong antioxidant activities (Tsuda, T., et al, J. Agric. Food Chem. 42:2407-2410 (1994)).
 Early studies have showed that MONTMORENCY cherry contains the anthocyanins cyanidin-3-gentiobioside and cyanidin-3-rutinoside (Li, K. C., et al., J. Am. Chem. Soc. 78:979-980 (1956)). Cyanidin-3-glucosylrutinoside was also found in six out of the seven sour cherry varieties (Harborne, J. B., et al., Phytochemistry 3:453-463 (1964)). Dekazos (Dekazos, E. D., J. Food Sci. 35:237-241 (1970)) reported anthocyanin pigments in MONTMORENCY cherry as peonidin-3-rutinoside, peonidin and cyanidin along with cyanidin-3-sophoroside, cyanidin-3-rutinoside and cyanidin-3-glucoside. However, cyanidin-3-glucosylrutinoside as well as cyanidin-3-glucoside, cyanidin-3-sophoroside and cyanidin-3-rutinoside were identified as main pigments in sour cherries. Using HPLC retention values, Chandra et al (Chandra, A., et al., J. Agric. Food Chem. 40:967-969 (1992)) reported that cyanidin-3-sophoroside and cyanidin-3-glucoside were the major and minor anthocyanins, respectively, in Michigan grown MONTMORENCY cherry. Similarly, cyanidin-3-xylosylrutinoside was detected as a minor pigment in MONTMORENCY cherry (Shrikhande, A. J. and F. J. Francis, J. Food Sci. 38:649-651 (1973)).
 In the prior art, production of pure anthocyanins (compounds 1-3 of
 Cyclooxygenase (COX) or prostaglandin endoperoxide H synthase (PGHS-1, PGHS-2 or COX-1/COX-2) enzymes are widely used to measure the anti-inflammatory effects of plant products (Bayer, T., et al., Phytochemistry 28 2373-2378 (1989); and Goda, Y., et al., Chem. Pharm. Bull. 40 2452-2457 (1992)). COX enzyme is the pharmacological target site for the nonsteroidal anti-inflammatory drug discovery (Humes, J. L., et al., Proc. Natl. Acad. Sci. U.S.A. 78 2053-2056 (1981); and Rome, L. H., et al., Proc. Natl. Acad. Sci. U.S.A. 72 4863-4865 (1975)). Two isozymes of cyclooxygenase involved in prostaglandin synthesis are cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), respectively (Hemler, M., et al., J. Biol. Chem. 25 251, 5575-5579 (1976)). It is hypothesized that selective COX-2 inhibitors are mainly responsible for anti-inflammatory activity (Masferrer, J. L., et al., Proc. Natl. Acad. Sci. U.S.A. 91 3228-3232 (1994)). Flavonoids are now being investigated as anti-inflammatory substances as well as their structural features for cyclooxygenase (COX) activity. The 5,7-dihydroxyflavone, galangin with an IC
 There is a need for natural product derived compositions for use as cyclooxygenase inhibitors and as anti-inflammatory agents.
 The present invention relates to a method for inhibiting cyclooxygenase or prostaglandin H synthase enzymes which comprises: providing at least one compound isolatable from a cherry with at least one of the enzymes to inhibit the enzymes.
 Further, the present invention relates to a method for inhibiting cyclooxygenase or prostaglandin H synthase enzymes which comprises: providing at least one bioflavonoid compound isolatable from a cherry with at least one of the enzymes to inhibit the enzymes.
 Further, the present invention relates to a method for inhibiting inflammation in a mammal which comprises: administering at least one compound isolatable from a cherry to the mammal to inhibit inflammation.
 Further, the present invention relates to a method for inhibiting inflammation in a mammal which comprises: administering at least one bioflavonoid, anthocyanin or phenolic compound isolated from a cherry to the mammal to inhibit the inflammation.
 Finally, the present invention relates to a method for inhibiting inflammation in a mammal which comprises administering cyanidin to the mammal to inhibit inflammation.
 The term “anthocyanins” includes the color producing compounds contained in cherries. For the purpose of this application this incudes the aglycone cyanidin.
 The term “bioflavonoids” means the isoflavonoid and flavonoid compounds contained in cherries.
 The term “phenolics” refers to compounds with a phenyl group and having one or more hydroxyl groups.
 The compounds isolated from cherries are most useful with living material. The living material can be in an animal or human. It can also be in tissue culture.
 It is therefore an object of the present invention to provide a cherry compound which can be used as cyclooxygenase inhibitors and anti-inflammatory agents. Further, it is an object of the present invention to provide a method for isolating the composition on a commercial scale. Finally, it is an object of the present invention to provide a natural source compound which is economical to prepare and easy to use. These and other objects will become increasingly apparent by reference to the following description and the drawings.
 The isolates are preferably prepared as a mixture of anthocyanins, bioflavonoids and phenolics by a method for producing a mixture comprising anthocyanins, bioflavonoids and phenolics from cherries as a composition which comprises:
 (a) providing an aqueous solution containing the anthocyanins, biof lavonoids and phenolics from the cherries;
 (b) removing the anthocyanins, bioflavonoids and phenolics onto a resin surface from the aqueous solution;
 (c) eluting the resin surface with an eluant to remove the anthocyanins, bioflavonoids and phenolics from the resin surface; and
 (d) separating the eluant from the anthocyanins, bioflavonoids and phenolics.
 The cherries used to produce the isolates can be sweet or sour. Tart cherries contain high levels of malic acid in addition to other organic acids which contributes to the sour taste of tart cherries. The method isolates malic acid and other organic acids containing sugars which can be used in foods to provide tartness and flavor. Most preferred are the BALATON and MONTMORENCY cherries.
 The isolated mixture of anthocyanins, bioflavonoids and phenolics can be tableted and used as a natural nutraceutical, phytoceutical or dietary supplement. In general, the tablets provide a daily dose of the anthocyanins and bioflavonoids of about 1 to 200 mg, preferably a daily dose of 10-100 mg. One hundred (100) cherries provide 10 to 100 mg of anthocyanins and bioflavonoids. The phenolics (
 The resin has a surface to which the anthocyanins, bioflavonoids and the phenolics are adsorbed. A preferred class of adsorptive resins are polymeric crosslinked resins composed of styrene and divinylbenzene such as, for example, the AMBERLITE series of resins, e.g., AMBERLITE XAD-4 and AMBERLITE XAD-16, which are available commercially from Rohm & Haas Co., Philadelphia, Pa. Other polymeric crosslinked styrene and divinylbenzene adsorptive resins suitable for use according to the invention are XFS-4257, XFS-4022, XUS-40323 and XUS-40322 manufactured by The Dow Chemical Company, Midland, Mich., and the like.
 It is preferred to use commercially available, FDA-approved, styrene-divinyl-benzene (SDVB) cross-linked copolymer resin, (e.g., AMBERLITE XAD-16). Thus, in the preferred embodiment, AMBERLITE XAD-16, commercially available from Rohm and Haas Company, and described in U.S. Pat. No. 4,297,220, herein incorporated by reference, is used as the resin. This resin is a non-ionic hydrophobic, cross-linked polystyrene divinyl benzene adsorbent resin. AMBERLITE XAD-16 has a macroreticular structure, with both a continuous polymer phase and a continuous pore phase. In a particularly preferred embodiment, the resin used in the present invention has a particle size ranging from 100-200 microns.
 It is contemplated that other adsorbents such as those in the AMBERLITE XAD adsorbent series which contain hydrophobic macroreticular resin beads, with particle sizes in the range of 100-200 microns, will also be effective in the methods of the present invention. Moreover, different variations of the AMBERLITES, such as the AMERCHROM CG series of adsorbents, used with particle sizes in the range of 100-200 microns, may also be suitable for use in the present invention. The AMBERLITE XAD-16 is preferred since it can be re-used many times (over 100 times). However, it is contemplated that for food, the use of governmentally-approved resins in the present invention will be considered important and/or desirable.
 Any solvent can be used to remove the adsorbed anthocyanins, bioflavonoids and phenolics. Preferred are lower alkanols containing 1 to 4 carbon atoms and most preferred is ethanol (ethyl alcohol) since it is approved for food use. Typically the ethanol is azeotroped with water; however, absolute ethanol can be used. Water containing malic acid and sugars in the cherries pass through the column. These are collected and can be used in foods as flavors.
 The anthocyanins, bioflavonoids and phenolics are preferably isolated from the BALATON and the MONTMORENCY cherries. The composition of the cherries is in part shown in part by U.S. application Ser. No. 08/799,788 filed Feb. 12, 1997 and in part U.S. application Ser. No. 60/111,945, filed Dec. 11, 1998 and 60/120,178, filed Feb. 16, 1999, which are incorporated by reference herein.
 The term “carrier” or “bulking agent” is used to mean a composition which is added to increase the volume of the composition of the purified composition from the cherry. Preferred is dried cherry pulp. These include any edible starch containing material, protein, such as non-fat dry milk. Within this group are flour, sugar, soybean meal, maltodextrin and various condiments, such as salt, pepper, spices and herbs, for instance. The bulking agent is used in an amount between about 10
 The ratio of anthocyanins, bioflavonoids and phenolics to the carrier is between 0.1 to 100 and 100 to 0.1.
 The composition is introduced into the food in an amount between about 0.1 and 10 mg/gm of the active ingredients of the food. The amount is preferably selected so as to not affect the taste of the food and to produce the most beneficial result. The food can be high (wet) or low moisture (dry) as is well known to those skilled in the art. When used as a dietary supplement the tablets contain between 0.1 to 1 gram of active ingredient.
 Methods have been developed for extraction and isolation of phytochemicals (Chandra, A., et al., J. Agric. Food Chem. 41:1062 (1992); Wang, H., et al., J. Agric. Food Chem. 45:2556-2560 (1997)) and for rapid screening of antioxidant activity (Arora, A. and G. M. Strasburg, J. Amer. Oil Chem. Soc. 74:1031-1040 (1997)). These methods are being utilized to identify, characterize and test the compounds from BALATON and MONTMORENCY cherries. Juiced cherry tissue was sequentially extracted with hexane, ethyl acetate and methanol. Both methanol and ethyl acetate fractions showed strong antioxidant activity in the screening assay. The ethyl acetate fraction was further purified by silica gel vacuum liquid chromatography to yield four subfractions; the subfraction was further separated into seven fractions by preparative reverse phase HPLC.
 Two novel phenolic compounds were identified:
 I) 1-(3′-4′-dihydroxy cinnamoyl)-2,3-dihydroxy cyclopentane, and II) 1-(3′-4′-dihydroxy cinnamoyl) -2,5-dihydroxy cyclopentane. Other compounds isolated from the ethyl acetate extract of cherry fruits and characterized by spectral methods include: 1-(3′-methoxy, 4′-hydroxy cinnamoyl) quinic acid, 2-hydroxy-3-(2′-hydroxyphenyl) propanoic acid, methyl 2-hydroxy-3-(2′-hydroxyphenyl) propanoate, D(+)-malic acid, β-sitosterol ad β-sitosterol glucoside.
 As shown in
 The pulp was lyophilized at 15° C. The juice was processed on AMBERLITE XAD-16 HP resin to produce cherry sour, anthocyanins, bioflavonoids and phenolics. The XAD-16 resin, 1 kg, was washed with ethanol (1-2 L) and then washed with water (6 L). The XAD-16 resin was allowed to stand in water for 1 hour before loading into a glass column (10 ID×90 cm long) with a cotton plug. The packed column was washed with water (2 L) before loading the juice for separation. 800 mL juice was purified each time. The juice was added onto the surface of the column and allowed to settle with no flow. It was then eluted with water and the first 1 L was discarded. The next 2 L of washing was collected, since it contained the cherry juice which was sour since it contained malic acid and sugars from the cherries. The column was then washed with an additional 4 L of water in the case of BALATON and 5 L for MONTMORENCY cherry juice. Once the cherry juice was collected, the remainder of the washing with water were discarded. The column was then eluted with ethanol (1.3-1.5 L) and collected the red solution containing anthocyanins, bioflavonoids and phenolics (700-800 ml). The column was then run dry and washed with 10 L of water before repeating the process many of times (over 100).
 The red alcoholic solution was then evaporated under vacuum a (20 millitorr) to remove ethanol and the aqueous solution, stabilized with 50 ppm ascorbic acid, was lyophilized at 10° C. The red powder was collected and stored.
 Example 1 results:
BALATON cherry Weight of IQF cherries 15.74 kg Weight of dried pulp 605 g Volume of juice 12.16 L Weight of anthocyanins, bioflavonoids 31.35 g and phenolics (red powder) Volume of sour byproduct (malic acid and sugars) @ 35 L
 Example 2 results:
MONTMORENCY cherry Weight of TQF cherries 30.45 kg Weight of dried pulp 895 g Volume of juice 24.03 L Weight of anthocyanins, bioflavonoids and 47 g phenolics (red powder) Volume of cherry by-product 75 L (malic acid and sugars) @
 The red powders of Examples 1 and 2 were preferably mixed with dried pulp as a carrier and tableted into 1 to 1000 mg tablets including the carrier (1 adult daily dose).
 Various food grade acids can be added to the isolated anthocyanins, bioflavonoids and phenolics to prevent decomposition. Preferably they do not add flavor. Ascorbic acid (vitamin C) is preferred. The acid can be added before or after, preferably before drying of the cherry compounds.
 For small scale processing, lyophilization is used to remove water. For larger scale production, drying in an air circulating oven is preferred.
 As shown in
 The antiinflammatory assays on the anthocyanins and cyanidin were conducted using prostaglandin endoperoxide H synthase-1 and -2 isozymes (PGHS-1, and -2) and were based on their ability to convert arachidonic acid to prostaglandins (PGs). The positive controls used in this experiment were aspirin, naproxen, and ibuprofen. Aspirin gave an IC
 For measurements of time-dependent inhibition of PGHS-2 enzyme activity by cyanidin, the enzyme was preincubated at 37° C. with 15 nM of cyanidin (one-fourth of the concentration of IC
 The specific inhibition of the PGHS-2 enzyme is a major advance in antiinflammatory therapy because it significantly reduces the adverse effects of nonsteroidal antiinflammatory drugs (NSAIDs). It is generally believed that ulcerogenic and other adverse properties of NSAIDs result from the inhibition of PGHS-1, whereas the therapeutically desirable effects come from the inhibition of PGHS-2 enzyme.
 Similarly, cyanidin showed better antiinflammatory activity than aspirin in the inflammatory assays. The antioxidant and antiinflammatory properties of anthocyanins and cyanidin suggest that consumption of cherries may have the potential to reduce cardiovascular or chronic diseases in humans.
 In particular, arachidonic acid and a microsomal fraction of ram seminal vesicles containing PGHS-1 enzyme suspended in 100 mM Tris pH 7.8 and 300 μM diethyldithiocarbamic acid (DDC) as a preservative were purchased from Oxford Biomedical Research (Oxford, Mich.). Recombinant human PGHS-2 enzyme was initially obtained from Dr. David Dewitt (Department of Biochemistry, Michigan State University, East Lansing, Mich.) and then purchased from Oxford Biomedical Research (Oxford, Mich.). Naproxen, ibuprofen, and hemoglobin were purchased from Sigma Chemical Co. (St. Louis, Mo.). Anthocyanins 1-3 were purified from Balaton tart cherry by HPLC and were identified by
 To prepare cyanidin, the anthocyanin mixture containing 1-3 (
 In the antiinflammatory assay, cyclooxygenase activities were measured by using PGHS-1 enzyme (ca. 5 mg protein/mL in 0.1 M TrisHCl, pH 7.8), a homogeneous protein purified from ram seminal vesicles. Microsomal preparations from recombinant human prostaglandin synthase-2 (COX-2) were obtained from insect cell lysate. Assays were performed at 37° C. by monitoring the initial rate of O
 This is an antiinflammatory assay for cyclooxygenase inhibition activity of flavonoids and isoflavonoids. Arachidonic acid and microsomal suspensions of PGHS-1 (COX-1) and COX-2 (PGHS-2) were purchased from Oxford Biomedical Research (Oxford, Mich., USA). Genistein, genistin, naringenin, quercetin, 5,8,4′-trihydroxy-6,7-dimethoxyflavone, kaempferol-3-rutinoside and 3′-methoxy kaempferol 3-rutinoside were purified from BALATON tart cherry by HPLC and were identified by
 For measuring the COX activity, flavonoids or isoflavonoids were dissolved in DMSO to yield 40 mM stock solution and was further diluted to the desired concentration according to the COX-1/COX-2 inhibitory activity of each compound assayed.
 Anti-inflammatory assay: COX activities were measured using microsomal suspensions of PGHS-1 and PGHS-2. Microsomal membranes (5 mg protein/mL in 0.1M Tris HCl, pH 7.4) were prepared and assayed on the same day. COX-1 and COX-2 assay was performed at 37° C. controlled by a circulation bath (Model-1166, VWR Scientific Products, Chicago, Ill.) by monitoring the rate of O
 Each assay mixture contained 600 μL of 0.1 M Tris-HCl, pH 8.0, 1 mM phenol, 17 μg hemoglobin and 10 μM arachidonate and were mixed in a microchamber (INSTECH Laboratory, Plymouth Meeting, Pa., USA). For anthocyanins and cyanidin pH 7 is preferred to prevent decomposition in absence of additives. Reactions were initiated by adding 5 μg of microsomal protein (5 μL). Instantaneous inhibition was determined by measuring the cyclooxygenase activity initiated by adding microsomal suspensions of PGHS-1 or PGHS-2 in the assay mixtures containing 10 μM arachidonate and various concentrations of test compounds. The IC
 The COX-1/COX-2 activity of BALATON cherry biof lavonoids was determined by monitoring the O
 COX-1/COX-2 inhibitory activities of each compound at different concentrations was calculated by comparing the tangent of O
 Among the flavonoids tested, kaempferol showed the highest COX-1 inhibition, followed by luteolin, quercetin, naringenin and quercetin 3-rhamnoside (
 Among the isoflavonoids (
 Thus several flavonoids and isoflavonoids isolated from BALATON tart cherry were assayed for prostaglandin H endoperoxide synthase (PGHS-1 or PGHS-2) enzyme activity. Genistein showed the highest COX-1 inhibitory activity among the isoflavonoids studied with an IC
 The composition of Examples 1 and 2 were tested for anti-inflammatory activity using cyclooxygenase 1 and 2 (COX-1 and COX-2) in an assay as described in Wang et al., J. Nat. Products 62:294-296 (1999); Wang et al., J. of Ag. and Food Chemistry, 47: 840-844 (1999) and Wang et al., J. of Nat. Products, 62:86-88 (1999) and Examples 4 and 5. The results were that the compositions exhibited anti-inflammatory activities, specifically strong inhibition of COX-1 and COX-2.
 It is intended that the foregoing description be only illustrative of the present invention and that the present invention be limited only by the hereinafter appended claims.