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WO2000033667A2 - Mode d'utilisation d'isolats de cerise aux proprietes phytoceutiques ou nutraceutiques antioxydantes - Google Patents

Mode d'utilisation d'isolats de cerise aux proprietes phytoceutiques ou nutraceutiques antioxydantes Download PDF

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Publication number
WO2000033667A2
WO2000033667A2 PCT/US1999/029440 US9929440W WO0033667A2 WO 2000033667 A2 WO2000033667 A2 WO 2000033667A2 US 9929440 W US9929440 W US 9929440W WO 0033667 A2 WO0033667 A2 WO 0033667A2
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WIPO (PCT)
Prior art keywords
composition
compound
cherries
anthocyanins
isolated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US1999/029440
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English (en)
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WO2000033667A3 (fr
Inventor
Muraleedharan G. Nair
Haibo Wang
Gale M. Strasburg
Alden M. Booren
James I. Gray
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Michigan State University MSU
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Michigan State University MSU
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Publication date
Priority claimed from US09/317,310 external-priority patent/US6423365B1/en
Priority claimed from US09/329,604 external-priority patent/US6150408A/en
Application filed by Michigan State University MSU filed Critical Michigan State University MSU
Priority to KR10-2001-7007239A priority Critical patent/KR100441927B1/ko
Priority to JP2000586180A priority patent/JP2002531473A/ja
Priority to AU21754/00A priority patent/AU2175400A/en
Publication of WO2000033667A2 publication Critical patent/WO2000033667A2/fr
Publication of WO2000033667A3 publication Critical patent/WO2000033667A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A23L33/11—Plant sterols or derivatives thereof, e.g. phytosterols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention provides a method for the use of cherry isolates having nutraceutical or phytoceutical properties to provide benefits.
  • the present invention also relates to a method for inhibiting oxidation of a living biological material in need thereof with a composition from cherries.
  • the present invention relates to a compound or composition as a dietary supplement or food additive. Most preferred is a composition containing a mixture of anthocyanins, bioflavonoids and phenolics. Many plant-derived compounds impart important positive pharmacological or
  • “nutraceutical” traits to foods by way of their abilities to serve as cellular 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.
  • antioxidants such as cyanidin-3-glucoside
  • Natural antioxidants may play an important role in the prevention of carcinogenesis. Dietary antioxidants may be effective against the peroxidative damage in living systems (Halliwell, B. and J. M. C. Gutteridge, Free radicals in biology and medicine. Oxford University Press, New York 416-494 (1989); Osawa, T.
  • 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.
  • cyanidin-3-glucosylrutinoside as well as cyanidin-3-glucoside, cyanidin-3-sophoroside and cyanidin-3-rutinoside were identified as main pigments in sour cherries.
  • U.S. Patent No. 5,503,867 to Pleva describes the use of whole ground cherries and oat bran in ground meat.
  • the amount of cherries used was 10 to 15% by weight and the oat bran is believed to be added to compensate for the juice in the cherries.
  • the cherries definitely contribute a flavor to the meat and the palatability of the product is not universally accepted.
  • This patent does not describe nutraceutical or phytoceutical benefits.
  • Recent studies on stabilization of low-fat ground beef with cherry tissue suggest that this plant source contains potent antioxidants, which not only suppress lipid peroxidation, but also inhibit formation of heterocyclic aromatic amines and cholesterol oxidation products during frying (Gomaa et al., IFT Abstracts No. 68E-7 (1996).
  • polyphenols such as flavonoids, anthocyanins and anthocyanidins, frequently found in the vacuoles of higher plants such as the cherries were responsible for this antioxidant effect.
  • the present invention relates to a method for providing nutraceutical or phytoceutical benefits, including antioxidant benefits, to a living mammal, which comprises: feeding the mammal a composition, isolated from cherries selected from the group consisting of anthocyanins, bioflavonoids, phenolics and mixtures thereof in an amount which provides the nutraceutical or phytoceutical properties to the mammal.
  • the present invention relates to a method for inhibiting oxidation in a living biological material in need thereof which comprises: providing a composition selected from the group consisting of isolated anthocyanins, bioflavonoids, phenolics and mixtures thereof from cherries with the living biological material in an amount which inhibits oxidation in the living biological material.
  • living biological material means living tissue in culture in an animal or in a human in vitro or in vivo.
  • anthocyanins means the compounds that impart color in cherries. This definition also includes the aglycone cyanidin for the purpose of the present application.
  • bioflavonoids means the isoflavonoids and flavonoid compounds contained in cherries.
  • phenolics refers to compounds with a phenyl group and having one or more hydroxyl groups from cherries.
  • the term “nutraceutical” means that there is a benefit to a living mammal, which affects the long term health of the mammal in the manner of vitamins.
  • the term “phytoceutical” means a plant derived product which provides the nutraceutical properties.
  • Figure 1 shows the structure of select anthocyanins (colorants) that have been isolated from BALATON and MONTMORENCY cherries.
  • the aglycone cyanidin has a hydroxyl group at position 3.
  • FIGS 2 and 3 are drawings showing the major bioflavonoids isolated from the cherries.
  • Figure 4 shows select phenolics isolated from tart cherries.
  • Figure 5 shows the steps in a method for isolating anthocyanins, bioflavonoids, and phenolics from cherries.
  • Figure 6 is a schematic drawing showing equipment that can be used in the method shown in Figure 5.
  • Figure 7 is a graph showing the antioxidant efficacy of anthocyanins and commercial antioxidants in a liposomal model system. Oxidation was initiated by the addition of ferrous ions. In the presence of test compounds, the rate of decay of fluorescence was decreased. Control samples contained no added Fe 2+ , and Fe 2+ contains no added test compounds. Other samples contained Fe 2+ plus 2 ⁇ M of test compound.
  • Figure 8 is a graph showing antioxidant activities of isolated compounds 1-4, 7, and 8 and commercial antioxidants TBHQ and BHT at 10 ⁇ M concentrations. Data represent the means of duplicate experiments.
  • Compound 1 is naringenin (R-i-OH,
  • Compound 3 is chiorogenic acid.
  • Compound 4 is quercetin 3-rhamnoside ( Figure 2).
  • Compound 7 is genistein 7-glucoside ( Figure 3).
  • Compound 8 is
  • Figure 9 is a graph showing antioxidant activities of isolated compounds 1 , 3, and 4 ( Figure 4) and some commercial antioxidants at 20 ⁇ M concentration.
  • the antioxidant activity of compound 2 was measured at 100 ⁇ M.
  • the rate of peroxidation was monitored by a decrease in fluorescence intensity as a function of time. Relative intensity represents the fluorescence intensity at a given time divided by the initial intensity at the start of the assay. Values represent the mean of duplicate measurements.
  • a preferred method for producing a mixture comprising anthocyanins, bioflavonoids and phenolics from cherries as a composition comprises providing an aqueous solution containing the anthocyanins, bioflavonoids and phenolics from the cherries; removing the anthocyanins, bioflavonoids and phenolics onto a resin surface from the aqueous solution; eluting the resin surface with a eluant to remove the anthocyanins, bioflavonoids and phenolics from the resin surface; and separating the eluant from the anthocyanins, bioflavonoids and phenolics.
  • a preferred method for producing anthocyanins, bioflavonoids and phenolics from cherries as a composition comprises providing a first batch of cherries, wherein the cherries are fresh or quick frozen and thawed; disrupting the cherries and separating pulp from the juice; extracting the anthocyanins, bioflavonoids and phenolics from the pulp into an aqueous solution; removing the anthocyanins, bioflavonoids and phenolics onto adsorbent resin particles from the aqueous solution containing the anthocyanins, bioflavonoids and phenolics separated from the pulp; washing the resin particles with a lower alkanol to remove the anthocyanins, bioflavonoids and phenolics from the resin particles; separating the alkanol from the anthocyanins, the bioflavonoids and phenolics; and repeating the above steps with the separated alkanol and the resin particles from which the anthocyanins, biofla
  • a preferred consumable composition for use in the method comprises in admixture: dried mixture of isolated anthocyanins, bioflavonoids and phenolics from cherries; and a food grade carrier, wherein the weight ratio of the mixture to the carrier is between about 0.1 to 100 and 100 to 0.1.
  • a preferred method for inhibiting oxidation in a mammal comprises feeding the mammal a consumable composition which comprises in admixture: dried mixture of isolated anthocyanins, bioflavonoids and phenolics removed from cherries; and a food grade carrier wherein the weight ratio of the mixture to the carrier is between about 0.1 to 100 and 100 to 0.1. It is preferred that the composition contain at least in part dried cherry pulp.
  • the cherries used in the present invention can be sweet or sour. Sour 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.
  • the isolated mixture of anthocyanins, bioflavonoids and phenolics can be used as a natural nutraceutical /dietary supplement.
  • the isolated mixture may be provided in a powdered, liquid, or solid form.
  • the mixture may be in a reconstitutable powder composition that, when reconstituted with, for example, water, milk or some other similar liquid will provide a drink.
  • the mixture may be in a solid form such as tablets, gel caps, soft gels, and the like.
  • the mixture may be incorporated into foodstuffs.
  • a mixture may be provided in a form such that the mixture is present in an amount in the range from about 0.01 % to about 50%, preferably from about 0.1% to about 30%, more preferably, from about 0.5% to about 25%, by weight of the total composition.
  • the tablet may provide a daily dose of the anthocyanins and bioflavonoids of about 0.1 mg to 300 mg, desirably from 1 to 200 mg, preferably a daily dose of 60-100 mg.
  • One hundred (100) cherries provide 60 to 100 mg of anthocyanins.
  • the phenolics ( Figure 4) may be provided in an amount of 0.1 to 50 mg as a daily dose.
  • One hundred cherries provide 1-50 mg of phenolics.
  • the amount of the anthocyanins, bioflavonoids and phenolics can be adjusted by isolating the individual compounds and blending them together. In one embodiment, a natural mixture of the anthocyanins, bioflavonoids and phenolics may be used.
  • the composition can also be provided in liquid form with equivalent dosages.
  • 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.
  • 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, Michigan, and the like. It is preferred to use commercially available, government-approved (where required), styrene-divinyl-benzene (SDVB) cross-linked copolymer resin, (e.g., AMBERLITE XAD-16).
  • AMBERLITE XAD-16 commercially available from Rohm and Haas Company, and described in U.S. Patent No. 4,297,220, 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.
  • 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.
  • 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.
  • 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 Serial No. 08/799,788 filed February 12, 1997 and in part U.S. application Serial No. 60/111 ,945, filed December 11 , 1998.
  • the Montmorency (Prunus cerasus) variety constitutes more than 95% of tart cherry cultivations in Michigan and USA.
  • Balaton tart cherry (P. cerasus) a new tart cherry cultivar, is being planted to replace Montmorency in several Michigan orchards. This cherry has higher anthocyanin contents and is regarded as a better variety.
  • 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-s-rutinoside and cyanidin-3- glucoside.
  • cyanidin-3-glucosylrutinoside as well as cyanidin-3-gluocoside, cyanidin-3-sophoroside and cyanidin-3-rutinoside were identified as main pigments in sour cherries.
  • 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 "6 and 10 6 parts by weight of the mixture.
  • 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.
  • the tablets When used as a dietary supplement the tablets contain between 0.1 to 1 gram of active ingredient.
  • a particular food is cooked meat and other prepared foods where the composition provides antioxidant properties to the food and optionally color.
  • the composition can be dispensed as a condiment on the prepared food to provide the nutraceutical or phytoceutical benefits.
  • Ri and R 2 are selected from the group consisting of a hydroxyl and a hydrogen and one of the R, and R2 is the hydroxyl.
  • the compound may be the isomer of the above compound wherein Ri is hydroxyl and R 2 is hydrogen. This particular isomer of the compound is 1-(3',
  • the compound may also be the isomer of the above compound wherein Ri is hydrogen and R 2 is hydroxyl.
  • This particular isomer of the compound is 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2,3-diol, which is isolatable as a pure compound from tart cherries.
  • the present invention also relates to a method of inhibiting oxidation in a material in need thereof which comprises providing a compound or a mixture of isomers isolatable from tart cherries and in pure form of the formula:
  • Ri and R 2 are selected from the group consisting of a hydroxyl or a hydrogen and one of the Ri and R 2 is the hydroxyl with the material in an amount which inhibits oxidation of the material.
  • the method uses the isomer of the above compound wherein Ri is hydroxyl and R 2 is hydrogen. This particular isomer of the compound is 1-(3 ⁇ 4'-dihydroxycinnamoyl)-cyclopenta-2, 5-diol, which is isolatable as a pure compound from tart cherries.
  • the method includes the use of the isomer of the above compound wherein Ri is hydrogen and R 2 is hydroxyl.
  • This particular isomer of the compound is 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 3-diol, which is isolatable as a pure compound from tart cherries.
  • the method includes the use of a mixture of the isomers.
  • the composition comprises 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 3-diol as a pure compound or as a mixture with 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 5-diol isolatable from tart cherries; and a non-toxic carrier or bulking agent for the compound.
  • the composition comprises 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 5-diol as a pure compound or as a mixture with 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 3-diol isolatable from tart cherries; and a non-toxic carrier or bulking agent for the compound.
  • the composition can further contain other antioxidants such as anthocyanins, flavonoids, or other phenolics from cherries added to the pure compound.
  • the carrier or bulking agent used in the compositions is a carrier suitable for animal or human use.
  • the composition consists of the antioxidant of the present invention and a particulate edible bulking agent in an amount between about 0. 1 to 30 parts per part of the invention, which product when introduced with an oxidizable material inhibits the oxidation of the material.
  • the composition containing the antioxidant of the present invention can be formed into any suitable solid or liquid dosage form and used as a nutraceutical/dietary supplement.
  • the selected dosage form provides a daily dose of the 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 5-diol or 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 3-diol as a pure compound, as an admixture containing both isomers, or as an admixture with other antioxidant compounds.
  • the present invention either as a composition, mixture, or in purified form could be useful as an anti- inflammatory agent, or for the treatment of chronic diseases, either in prevention of disease, ameliorating the effects of disease, or stimulating the immune response to more effectively combat disease.
  • the present invention either as a composition, mixture, or in purified form could be useful in preventing or treating various types of diseases, which can in part, or whole be caused by free radicals.
  • composition of the present invention can further include one or more antioxidants selected from the group consisting of anthocyanins, cyanidins, bioflavanoids, phenolics and mixtures thereof.
  • antioxidants selected from the group consisting of anthocyanins, cyanidins, bioflavanoids, phenolics and mixtures thereof.
  • the anthocyanins, bioflavanoids and phenolics are preferably isolated from cherries and in particular, BALATON and MONTMORENCY cherries.
  • the anthocyanin is selected from the group consisting of cyanidin-3-2"-o-3-D-glucopyranosyl-6"-o-V-L-rhamnosyl-3-D- glucopyranoside, cyanidin-3-6"-o-V-L-rhamnosyl-3-D-glucopyranoside, cyanidin-3-3- D-glucopyranoside, and mixtures thereof.
  • the bioflavonoid is 7-methoxy-5,8,4'-trihydroxyflavone.
  • composition containing the antioxidant of the present invention, or the compound of the present invention can be added to food as a general antioxidant food additive.
  • the food additive can include 1-(3', 4'-dihydroxycinnamoyl)-cyclopenta-2, 3-diol or 1-(3',
  • the food additive can further include other antioxidants selected from the group consisting of anthocyanins, cyanidins, bioflavanoids, phenolics and mixtures thereof.
  • the anthocyanin is selected from the group consisting of cyanidin-3-2"-o-3-D-glucopyranosyl-6"-o-V-L- rhamnosyl-3-D-glucopyranoside, cyanidin-3-6"-o-V-L-rhamnosyl-3-D-glucopyranoside, cyanidin-3-3-D-glucopyranoside, and mixtures thereof.
  • the bioflavonoid is 7-methoxy-5,8,4'-trihydroxyflavone.
  • the anthocyanins, bioflavanoids and phenolics are preferably isolated from cherries and, in particular from BALATON and MONTMORENCY cherries.
  • the composition containing the antioxidant of the present invention, or the compound of the present invention can be general antioxidant food additive that can be added to food which is either high (wet) or low moisture (dry), fresh or uncooked, cured or cooked.
  • the antioxidant compounds or compositions When added to food containing lipids, the antioxidant compounds or compositions inhibit oxidation of the lipids and thus inhibit the development of rancidity.
  • the addition of the composition to meat prior to cooking inhibits the formation of heterocyclic aromatic amines (HAA), which occurs upon cooking and which have shown to be carcinogenic.
  • HAA heterocyclic aromatic amines
  • 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 x 90 cm long) with a cotton plug.
  • the packed column was washed with water (2L) 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 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 the drying of the cherry compounds.
  • lyophilization is used to remove water.
  • drying in an air circulating oven is preferred.
  • an open vessel 10 is provided with an inlet line 11 and an outlet line 12, with valves 13 and 14, respectively.
  • the resin beads 15 are provided in the open vessel 10. Water is introduced into the vessel 10 and then removed through outlet line 12 and discarded.
  • the cherry juice (without the pulp or pits) as in Example 1 is introduced to the vessel 10 and allowed to stand for 25 minutes. The temperature of the water and juice is between about 20 and 30°C.
  • the cherry juice residue containing malic acid and sugars is then removed through the outlet line 12 and retained as a food flavoring.
  • the resin 15 in the vessel is then washed again with water from inlet line 11 and then removed and discarded through outlet line 12.
  • the anthocyanins, bioflavonoids and phenolics on the resin particles are then extracted using 95% ethanol introduced through inlet line 11.
  • the ethanol containing the anthocyanins, bioflavonoids and phenolics is removed from the vessel 10.
  • the ethanol is removed from the anthocyanins, bioflavonoids and phenolics and dried using flash drying under nitrogen.
  • the resulting powder is preferably then mixed with dried cherry pulp or other carrier as in Example 1.
  • the resin particles are washed with water and then the resins and ethanol are recycled many times.
  • a fluorescent probe 1 ,6-diphenylhexatriene propionic acid
  • This probe reacts with the free radicals generated during peroxidation, resulting in a decrease in fluorescence intensity with time.
  • a peroxidation initiator such as ferrous metal ions or the free radical generator AAPH (Azobis- [2-amidino propane hydrochloride]) is used to start the reaction, and the kinetics of fluorescence decrease are determined in the presence or absence of the antioxidant composition to be tested.
  • LUVs Large unilamellar vesicles
  • lipid was dissolved in chloroform, and was dried to a thin film using a rotary evaporator. The dried film was resuspended in an aqueous buffer, and was repeatedly extruded through a polycarbonate filter of 100 nm pore size using a
  • Liposofast piposome extruder (Avestin, Inc., Ottawa, Canada). The homogeneity of size (80-100 nm) and the unilamellar nature of the vesicles were confirmed using freeze-fracture scanning electron microscopy.
  • the fluorescent probe, diphenylhexatriene-propionic acid (DPH-PA) was incorporated into the vesicles during preparation at a mole ratio of 1 :350 (probe:lipid).
  • LUVs containing DPH-PA is suspended at a final concentration of 100 ⁇ M in 100 mM NaC1 , 50 mM tris-HEPES buffer at pH 7.0.
  • the fluorescent probe was excited at 384 nm and emission was monitored at 423 nm. Lipid oxidation is inhibited in the LUVs by addition of ferrous ions or the free radical generator AAPH; the progress was monitored by the decrease of the fluorescence intensity of DPH-PA resulting from reaction with free radicals generated over twenty-one minutes. A plot of the decrease of fluorescence intensity as a function of time was used to determine the kinetics of lipid oxidation. The results show that a mixture of the crude anthocyanin extract with ethylacetate was effective in inhibiting oxidation.
  • anthocyanins cyanidin
  • Anthocyanins 1-3 contain 3, 2, and 1 sugar residues, respectively, which explains the lowest antioxidant activity observed for anthocyanin 1.
  • the number of sugar residues at the C 3 position seems to be very important for antioxidant activity. The smaller the number of sugar units at C 3 the higher the antioxidant activity.
  • the stability of aryloxyl radical affected the antioxidant activities of compounds and may give rise to pro-oxidant effects. Therefore, the antioxidant activity of cyanidin may depend on the stability of its aryloxyl radical.
  • the o/ ⁇ t?o-dihydroxy substitution in the B ring of anthocyanins and cyanidin is important to stabilize the resulting free radical generated through the 3' and 4'-OH moieties.
  • orf 70-dihydroxy groups in anthocyanins have the potential to chelate metal ions and thus prevent iron-induced lipid peroxidation.
  • the anthocyanin mixture containing 1-3 500 mg was stirred with 3N HCI (20 mL) at 80°C for 10 h.
  • the reaction mixture was purified on a XAD-4 column as in the preparation of anthocyanins.
  • the MeOH solution of cyanidin was evaporated to dryness to yield a red amorphous powder (190 mg) and stored at -30° C until use.
  • the buffers were stored in Chelex 100 to remove metal ions.
  • the resulting film was suspended in 500 :L of buffer (NaCI, 0.15 M; EDTA 0.1 mM; MOPS 10 mM) and was then subjected to 10 freeze-thaw cycles in an EtOH-dry ice bath.
  • the suspension was then passed 29 times through a polycarbonate membrane with a pore size of 100 nm using a LiposoFast extruder (Avestin, Inc., Ottawa, Canada).
  • the resulting liposomes (200 nM) were then suspended in 2 mL of buffer (100 mM NaCI, 50 mM HEPES, pH 7.0). The peroxidation was initiated by the addition of 4 nM of Fe 2+ .
  • the antioxidant activities of 1-3 and cyanidin were comparable to the antioxidant activities of tert- butylhydroquinone and butylated hydroxytoluene and superior to vitamin E at 2- mM concentrations.
  • Compound 6 3' -methoxy kaempferol 3-rutinoside
  • Compound 7 Genistin 7-glucoside
  • Compound 8 5,8,4' - trihydroxyl-6,7-dimethoxyflavone.
  • Compound 3 1- 3'-4' -dihydroxy cinnamoyl) -2, 5-dihydroxy cyclopentane.
  • Compound 4 1-(3'-4'-dihydroxy cinnamoyl)-2, 3-dihydroxy cyclopentane,
  • antioxidant compounds 1 , 2, 3, and 4 were compared to various antioxidants.
  • the antioxidants tested were caffeic acid, ferulic acid, chlorogenic acid, p-hydroxycinnamic acid, and the commercially available antioxidants, tert butylhydroquinone (TBHQ) and butylated hydroxytoluene (BHT).
  • TBHQ tert butylhydroquinone
  • BHT butylated hydroxytoluene
  • Example 6 was dried under vacuum using a rotary evaporator.
  • the resulting lipid film was suspended in 1000 ⁇ L for Example 6 and 500 ⁇ L for Example 7 of a solution containing 0.15 M NaCI, 0.1 mM EDTA, and 0.01 M 4-morpholinepropanesulfonic acid (MOPS) buffer.
  • the MOPS buffer had been previously treated with CHELEX 100 (obtained from Sigma Chemicals, St. Louis, Missouri) at 5 g/100 mL buffer to remove any trace metal ions.
  • the suspension was then subjected to 10 freeze-thaw cycles using a dry ice/ethanol bath.
  • the lipid-buffer suspension was then passed 29 times through a polycarbonate membrane with a pore size of 100 nm using a LiposoFast extruder (Avestin, Inc., Ottawa, Canada) to produce unilamellar liposomes.
  • a LiposoFast extruder Avestin, Inc., Ottawa, Canada
  • a 20 ⁇ L aliquot of the above liposome suspension was diluted to 2 mL in CHELEX 100 treated buffer containing 100 mM NaCI , 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at pH 7.0 for Example 6 (200 mM NaCI , 100 mM HEPES buffer at pH 7.0 for Example 7), incubated for 5 minutes at room temperature, followed by incubation for another 5 minutes in the thermostatic cuvette holder of the spectrofluorometer which is held at 23°C.
  • CHELEX 100 treated buffer containing 100 mM NaCI , 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at pH 7.0 for Example 6 200 mM NaCI , 100 mM HEPES buffer at pH 7.0 for Example 7
  • Peroxidation was then initiated by addition of 20 ⁇ L of 0.5 mM FeCI 2 to achieve a final concentration of 0.5 ⁇ M of Fe 2+ in the absence or presence of any of the test compounds.
  • the control sample did not contain either Fe 2+ or any of the test compounds.
  • Fluorescence intensity of these liposome solutions were measured every 3 minutes over a period of 21 minutes at an excitation wavelength of 384 nm using a fluorescence spectrofluorometer (SLM4800 obtained from SLM Instruments, Inc., Urbana, Illinois). The decrease in relative fluorescence intensity over time indicated the rate of peroxidation.
  • the percent inhibition (PI) of the lipid oxidation was calculated using the equation:
  • Quercetin 3-rhamnoside contains an ortho-d ⁇ hydroxyl group in the B ring in addition to a 3-hydroxyl group and a double bond at C 2 -C 3 in conjunction with a 4-oxo functional group.
  • the enhanced antioxidant activity of compound 8 was probably due to the hydroxyl and two methoxy groups in ring A.
  • Arora et al (1997) reported that 7,8-dihydroxyflavone showed similar antioxidant activity to quercetin, though it lacked any substitution on the B-ring and at 3-position.
  • the inhibitory activities of Fe 2+ induced lipid peroxidation in the large unilamellar vesicles for compounds 3 and 4 were about 80% at 20 ⁇ M.
  • Compound 1 showed about 50% inhibitory activity.
  • compound 2 did not show antioxidant activity even when tested at a 100 ⁇ M concentration.
  • the assay results also showed that p-hydroxycinnamic acid was a weak antioxidant when compared to ferulic acid.
  • the caffeic acid analogues, compounds 3 and 4 showed the highest antioxidant activity in this assay.
  • the percent inhibition of lipid peroxidation for TBHQ and BHT were >90% at the 20 ⁇ M concentration.
  • the assay showed that caffeic acid is the best oxidant, followed by compound 4, compound 3, chlorogenic acid, and chlorogenic acid methyl ester.
  • EXAMPLE 8 The composition of Examples 1 and 2 were tested for antioxidant properties and they were found to be comparable to Example 5.
  • antioxidant compounds are to reduce or eliminate cellular oxidative damage, free radical scavenging to reduce cell death, reduce atherosclerosis or hardening of artery, reduce the incidence of heart attacks, reduce the incidence of arthritis and gout related pain and reduce aging. 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.

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Abstract

L'invention concerne une technique présentant des bienfaits nutraceutiques ou phytoceutiques pour un mammifère, en particulier pour l'homme, et qui consiste à utiliser des dérivés de cerise. L'invention porte également sur une technique permettant d'inhiber l'oxydation dans une matière vivante biologique. Selon ces techniques, on utilise une composition d'anthocyanines, de bioflavonoïdes, de phénoliques ou de mélanges en résultant à base de cerises.
PCT/US1999/029440 1998-12-11 1999-12-10 Mode d'utilisation d'isolats de cerise aux proprietes phytoceutiques ou nutraceutiques antioxydantes Ceased WO2000033667A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
KR10-2001-7007239A KR100441927B1 (ko) 1998-12-11 1999-12-10 체리 단리물을 포함하는 뉴트라슈티칼 또는 파이토슈티칼의 잇점을 제공하는 배합물
JP2000586180A JP2002531473A (ja) 1998-12-11 1999-12-10 酸化防止剤の植物的又は栄養的利益を与えるサクランボ単離物の使用方法
AU21754/00A AU2175400A (en) 1998-12-11 1999-12-10 Method for the use of cherry isolates providing antioxidant phytoceutical or nutraceutical benefits

Applications Claiming Priority (10)

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US11194598P 1998-12-11 1998-12-11
US60/111,945 1998-12-11
US12017899P 1999-02-16 1999-02-16
US60/120,178 1999-02-16
US09/317,310 1999-05-24
US09/317,310 US6423365B1 (en) 1998-12-11 1999-05-24 Method and compositions producing cherry derived products
US09/329,604 1999-06-10
US09/329,604 US6150408A (en) 1998-12-11 1999-06-10 Tart cherry compounds that have antioxidant activity and uses thereof
US34207699A 1999-06-28 1999-06-28
US09/342,076 1999-06-28

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1307212A4 (fr) * 2000-07-31 2004-12-08 World Resources Corp Antioxydants derives de plantes
WO2005009140A1 (fr) * 2003-07-17 2005-02-03 Biochem Laboratories As Utilisation d'anthocyanines dans un aliment pour poissons
US6967032B2 (en) 2001-11-09 2005-11-22 Medpalett Pharmaceuticals As Product
AU2005301315B2 (en) * 2004-07-29 2008-04-10 Michigan State University Insulin secretion by anthocyanins and anthocyanidins

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1269421C (zh) * 1998-12-11 2006-08-16 密执安州大学 生产源于浆果的产物的方法和组合物
US7306815B2 (en) * 2000-08-31 2007-12-11 Phenolics, Llc Compositions enriched in phenolic compounds and methods for producing the same
EP2108692A1 (fr) * 2008-04-11 2009-10-14 Brasseries Kronenbourg Procédé d'obtention d'extraits concentrés en polyphénols issus du procédé de brassage
JP2015051934A (ja) * 2013-09-05 2015-03-19 地方独立行政法人青森県産業技術センター 皮膚の酸化的ストレス抑制剤
CN112624928B (zh) * 2021-01-21 2023-03-17 黄河科技学院 一种抗凝血桃花有效成分及其应用
CN114317313B (zh) * 2021-08-24 2022-08-09 清枫链食苏打饮品(吉林)有限公司 酸樱桃提取物在制备降低尿酸或抑制痛风发作产品中的应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HUT63565A (en) * 1990-08-20 1993-09-28 Ohlenschlaeger Gerhard Process for producing curqtive mixture consisting of glutathione and anthocyan compounds
JPH06248267A (ja) * 1993-02-24 1994-09-06 S I I Techno Res:Yugen 新規抗酸化剤
JPH06271850A (ja) * 1993-03-23 1994-09-27 Nikken Food Kk 天然物を原料とした抗酸化剤およびその製造方法
US5762936A (en) * 1996-09-04 1998-06-09 Biotics Research Corporation Antioxidant derived from lentil and its preparation and uses
DE19720767A1 (de) * 1997-05-07 1998-11-12 Protekum Umweltinstitut Gmbh O Pharmazeutisches Produkt, insbesondere zur Therapie und Prophylaxe von Herz-Kreislauf-Erkrankungen

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1307212A4 (fr) * 2000-07-31 2004-12-08 World Resources Corp Antioxydants derives de plantes
US7109162B2 (en) 2000-07-31 2006-09-19 World Resourses Corporation Plant derived antioxidants
US6967032B2 (en) 2001-11-09 2005-11-22 Medpalett Pharmaceuticals As Product
WO2005009140A1 (fr) * 2003-07-17 2005-02-03 Biochem Laboratories As Utilisation d'anthocyanines dans un aliment pour poissons
AU2005301315B2 (en) * 2004-07-29 2008-04-10 Michigan State University Insulin secretion by anthocyanins and anthocyanidins

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