Method for the use of cherry isolates providing antioxidant phytoceutical or nutraceutical benefits

Abstract

A method for providing nutraceutical or phytoceutical benefits to a mammal, particularly a human, using cherry derivatives is described. A method for inhibiting oxidation in a living biological material is also described. A composition of anthocyanins, bioflavonoids, phenolics or mixtures thereof from cherries is used in the methods.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

None

BACKGROUND OF THE INVENTION

(1) Summary of the Invention

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. In particular 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.

(2) Description of Related Art

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. 85-89 (1993). Thus, with natural products, the dietary supplement/food industry and nutraceutical 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. A preferred cherry is Prunus Cerasus L. (Rosacease), cv. MONTMORENCY which is the major tart cherry commercially grown in the United States. In order to challenge the MONTMORENCY monoculture, a new cultivar, BALATON tart cherry ( Ujferbertoi furtos ), was introduced into the United States in 1984, and has been tested in Michigan, Utah, and Wisconsin. BALATON produces fruits darker than MONTMORENCY.

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)). The addition of antioxidants is one of the popular methods to increase the shelf life of food products which is thought to be associated with lipid peroxidation. 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., et al, Role of dietary antioxidants in protection against oxidative damage. In antimutagenesis and anticarcinogenesis Mechanisms; Kuroda, Y.; Shankel, D. M., Waters, M. D., Eds.; Plenum Publishing. New York 139-153 (1990)). 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 FIG. 1 ) from BALATON and MONTMORENCY cherry juices was carried out first by adsorbing the pigment on an AMBERLITE XAD-2 (Sigma Chemicals) column (Chandra, A., et al., J. Agric. Food Chem. 41:1062-1065 (1993)). The column was washed with water until the eluant gave a pH of approximately 7.0. The adsorbed pigments along with other phenolics were eluted with MeOH. The resulting crude anthocyanins were fractionated and purified by C-18 MPLC and HPLC, respectively, to afford pure anthocyanins for spectral studies. Purification of 500 mg crude MONTMORENCY anthocyanins from AMBERLITE XAD-2 yielded 60 mg of pure anthocyanins 1-3 compared to 391.43 mg from BALATON. This research indicated that crude anthocyanins from MONTMORENCY obtained from the XAD-2 contained a high percentage of other organic compounds. The AMBERLITE XAD-2 did not allow recycling of the resin. There was no attempt to use the crude mixture of flavonoids, isoflavonoids, phenolics and anthocyanins for any purpose. U.S. Pat. No. 5,266,685 to Garbutt, U.S. Pat. No. 5,665,783 to Katzakian et al and U.S. Pat. No. 5,817,354 to Mozaffar describe various adsorbent resins and their use for unrelated products. These patents are only illustrative of the general state of the art in the use of adsorbent resins.

U.S. Pat. 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. In any event, 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). The hypothesis used to explain these observations was that 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.

There is a need for natural cherry derived compositions for use, particularly as dietary supplements/nutraceutical or food additives.

SUMMARY OF THE INVENTION

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.

The term “living biological material means living tissue in culture in an animal or in a human in vitro or in vivo.

The term “anthocyanins” means the compounds that impart color in cherries. This definition also includes the aglycone cyanidin for the purpose of the present application.

The term “bioflavonoids” means the isoflavonoids and flavonoid compounds contained in cherries.

The term “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.

OBJECTS

It is therefore an object of the present invention to provide a natural source cherry composition which can be used in foods or as dietary supplements or nutraceuticals as antioxidants which prevent oxidation in living tissue. This and other objects will become increasingly apparent by reference to the following description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the isolated anthocyanins (colorants) 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, as described in provisional application Serial No. 60/111,945, filed Dec. 11, 1998.

FIG. 4 shows the phenolics isolated from tart cherries.

FIG. 5 shows the steps in the method of the present invention as described in Examples 1 and 2.

FIG. 6 is a schematic drawing showing the use of an open vessel 10 for holding resin beads, which remove anthocyanins and phenolics from the cherry juice.

FIG. 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 μM of test compound.

FIG. 8 is a graph showing antioxidant activities of isolated compounds 1-4, 7, and 8 and commercial antioxidants TBHQ and BHT at 10 μM concentrations. Data represent the means of duplicate experiments. Compound 1 is naringenin (R 1 —OH, R 4 —OH and R 6 —OH; R 2 , R 3 , R 5 and R 7 —H). Compound 2 is genistein (FIG. 3 ). Compound 3 is chlorogenic acid. Compound 4 is quercetin 3-rhamnoside (FIG. 2 ). Compound 7 is genistein 7-glucoside (FIG. 3 ). Compound 8 is 6,7-dimethoxy-5,8,4 1 -trihydroxyflavone (FIG. 2 ).

FIG. 9 is a graph showing antioxidant activities of isolated compounds 1, 3, and 4 ( FIG. 4 ) and some commercial antioxidants at 20 μM concentration. The antioxidant activity of compound 2 was measured at 100 μM. 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 means of duplicate measurements.

DESCRIPTION OF PREFERRED EMBODIMENTS

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.

In particular 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, bioflavonoids and phenolics have been removed with a second batch of the cherries.

Further, 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.

Finally, a preferred method is provided for inhibiting oxidation in a mammal which 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. 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/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 60-100 mg. One hundred (100) cherries provide 60 to 100 mg of anthocyanins. The phenolics ( FIG. 4 ) are 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. It is preferred to use the natural mixture of the anthocyanins, bioflavonoids and phenolics which is isolated by the resin. 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. 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 Serial No. 60/111,945, filed December 11, 1998 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 −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. 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.

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 and characterize the antioxidant 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 which showed the strongest antioxidant activity was further separated into seven fractions by preparative reverse phase HPLC. FIGS. 2 and 3 show the bioflavonoids isolated from the BALATON cherries. There are thus numerous analogous or homologous compounds in the tart cherries.

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. FIG. 4 shows some of the phenolics which were isolated. The anthocyanin components obtained from the juice fraction also have been identified and fully characterized (Chandra, A., et al., J. Agric. Food Chem. 41:1062 (1992); Wang, H., et al., J. Agric. Food Chem. 45:2556-2560 (1997)); the results indicate that these compounds contain potent antioxidant activity.

EXAMPLES 1 and 2

As shown in FIG. 5 , individual quick frozen (IQF) cherries (which had been pitted) were defrosted and blended in an industrial WARING blender. The mixture was centrifuged at 10,000 rpm and the juice was decanted. The residue, pulp, was further pressed with cheese cloth to remove any additional juice.

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	@35	L
	(malic acid and sugars)

Example 2 results:

	MONTMORENCY cherry
	Weight of IQF cherries	30.45	kg
	Weight of dried pulp	895	g
	Volume of juice	24.03	L
	Weight of anthocyanins, bioflavonoids	47	g
	and 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 the 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.

EXAMPLE 3

As shown in FIG. 6 , 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.

EXAMPLE 4

Crude ethyl acetate extracts from cherries (containing anthocyanins, bioflavonoids and phenolics) were tested in aqueous solution under various conditions using a fluorescent assay for antioxidant activity. The fluorescent assay is described first.

The need to screen large numbers of compounds or extracts for antioxidant activity requires that a model system (or systems) be employed which reasonably well represents the structural and functional characteristics of the composition alone or in the food product. The test must also be sensitive, rapid, and inexpensive. A fluorescence-based assay for evaluating antioxidant efficacy was used (Arora, A., and G. M. Strasburg, J. Am. Chem. Soc. 1996)). Large unilamellar vesicles consisting of 1-stearoyl-2-linoleoly-sn-glycero-3-phosphocholine were prepared, which closely resemble the properties of biological membranes, one of the primary sites of peroxidation. A fluorescent probe, 1,6-diphenylhexatriene propionic acid, is incorporated into the membranes such that the polar head group anchors the probe near the aqueous interface, while the hydrophobic portion lies parallel to the fatty acid chains. 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. An assay for a compound at a given concentration presently takes only twenty-one minutes, consumes only a few micrograms of lipid, and can be readily conducted with a simple fluorometer.

Large unilamellar vesicles (LUVs) were prepared from 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine according to the procedure outlined by MacDonald et al (MacDonald, R. C., et al., Biochim. Biophys. Acta 1061:297-303 (1991)). Briefly, the 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). For the fluorescence experiments, LUVs containing DPH-PA is suspended at a final concentration of 100 μM in 100 mM NaCl, 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 which are set forth in Ser. No. 08/799,788 filed Feb. 12, 1997, now U.S. Pat. No. 5,985,636 show that -a mixture of the crude anthocyanin extract with ethylacetate was effective in inhibiting oxidation.

Solvent extraction of the anthocyanins, bioflavonoids and phenolics can be used; however this is not preferred where the product is to be used as a food and for expense reasons. Where the preferred adsorbent resins are used, this step is unnecessary. It is also possible to separate and recombine the components using chromatography; however, for the purpose of the present invention, this is far too expensive since it involves high pressure liquid chromatography.

EXAMPLE 5

The cherry anthocyanins were assayed for antioxidant activity using the method developed by Arora and Strasburg. As the reaction proceeds, the fluorescent probe will be degraded, resulting in the decline of fluorescent intensity. Therefore, in the presence of an antioxidant, the rate of fluorescence decay will be reduced. Experiments indicated that antioxidant activities of anthocyanins 1-3 and of the aglycon, cyanidin, compared favorably with the commercial antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (FIG. 7 ). Also, these cherry compounds showed better antioxidant activity than α-tocopherol. At 2-mM concentrations, the extent of peroxidation of the sample containing α-tocopherol was indistinguishable from that of the Fe 2+ containing sample with no added antioxidant (FIG. 7 ).

The aglycon of anthocyanins, cyanidin, has higher efficacy than its glycosides, suggesting that the antioxidant activity of anthocyanins is due to their aglycon moiety. 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. Also, it is reported that 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 ortho-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. Also, ortho-dihydroxy groups in anthocyanins have the potential to chelate metal ions and thus prevent iron-induced lipid peroxidation.

In the preparation of cyanidin, the anthocyanin mixture containing 1-3 (500 mg) was stirred with 3N HCl (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.

In the antioxidant assay, the buffers were stored in Chelex 100 to remove metal ions. A mixture containing 5 μM of 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine (Avanti Polar Lipids, Inc., Alabaster, Ala.) and 15 nM of the fluorescence probe 3-(p-(6-phenyl)-1,3,5-hexatrienyl) phenylpropionic acid (Molecular Probes, Inc., Eugene, Oreg.) was dried under vacuum. The resulting film was suspended in 500 μL of buffer (NaCl, 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 NaCl, 50 mM HEPES, pH 7.0). The peroxidation was initiated by the addition of 4 nM of Fe 2+ . Anthocyanins, BHT, propyl gallate, and α-tocopherol (vitamin E) were tested at 2-μM concentration. Control samples contained no added Fe 2+ or test compound. Fluorescent intensity of lipid suspension was monitored 21 min with or without test compounds, immediately followed by the addition of Fe 2+ , using a SLM 4800 spectrofluorometer (SLM Instruments, Urbana, Ill.). The values of relative fluorescence were determined by dividing the fluorescence value at a given time point by that at t=0 min. The results are shown in FIG. 7 . The anthocyanins (1-3— FIG. 1 ) and cyanidin isolated from tart cherries exhibited in vitro antioxidant activity comparable to commercial products. The inhibition of lipid peroxidation of anthocyanins 1-3 and their aglycon, cyanidin, were 39, 70, 75 and 57%, respectively, at 2-mM concentrations. 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.

EXAMPLES 6 AND 7

In the same manner as in Example 5, the compounds of FIGS. 2 and 3 were tested for antioxidant activity. The results are shown in FIGS. 8 and 9 . For FIG. 8 , compound 8 was superior to the other compounds. For FIG. 9 , compounds 3 and 4 were effective as antioxidants.

EXAMPLE 8

The composition of Examples 1 and 2 were tested for antioxidant properties and they were found to be comparable to Example 5.

Potential uses of 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.

Claims

1. A method for providing nutraceutical or phytoceutical benefits to a living mammal which comprises:feeding the mammal a composition isolated from cherries which contains a mixture of antriocyanins, bioflavonoids and phenolics, wherein the phenolics are selected from the group consisting of 1-(3′,4′-dihydroxy cinnamoyl)-2,3-dihydroxy cyclopentane, 1-(3′-4′-dihydroxy cinnamoyl)-2,5-dihydroxy cyclopentane, and mixtures thereof, in an amount which provides the nutraceutical or phytoceutical properties to the mammal.

2. The method of claim 1 wherein the mammal is a human and the composition is provided in a prepared food.

3. The method of claim 1 wherein the composition is dried.

4. The method of claim 1 wherein the composition is dried and contains a food grade carrier.

5. The method of claim 4 wherein the carrier is dried cherry pulp.

6. The method of claim 1 wherein the composition contains a carrier and contains a ratio of composition to carrier between 0.1 to 100 and 100 to 0.1.

7. The method of claim 1 wherein the composition is in liquid form.

8. The method of claim 1 wherein the composition is isolated from tart cherries.

9. The method of claim 1 wherein the composition is isolated from sweet cherries.

10. The method of claim 1 wherein the composition is isolated from MONTMORENCY cherries.

11. The method of claim 1 wherein the composition is isolated from BALATON cherries.

12. A method for inhibiting oxidation in a living biological material in need thereof which comprises:providing a composition containing a mixture of anthocyanins, bioflavonoids and phenolics, wherein the phenolics are selected from the group consisting of 1-(3′,4′-dihydroxy cinnamoyl)-2,3-dihydroxy cyclopentane, 1-(3′-4′-dihydroxy cinnamoyl)-2,5-dihydroxy cyclopentane, and mixtures thereof, from cherries with the living biological material in an amount which inhibits oxidation in the living biological material.

13. The method of claim 12 wherein the living biological material is in a human which is fed the composition.

14. The method of claim 12 wherein the living biological material is in an animal which is fed the composition.

15. The method of claim 12 wherein the composition is dried.

16. The method of claim 12 wherein the composition is dried and contains a dried food grade carrier.

17. The method of claim 16 wherein the carrier is dried cherry pulp.

18. The method of claim 12 wherein the composition contains a carrier and contains a ratio of composition to carrier between 0.1 to 100 and 100 to 0.1.

19. The method of claim 12 wherein the composition is isolated from tart cherries.

20. The method of claim 12 wherein the composition is isolated from sweet cherries.

21. The method of claim 12 wherein the composition is isolated from MONTMORENCY cherries.

22. The method of claim 12 wherein the composition is isolated from BALATON cherries.

23. A method for providing nutraceutical or phytoceutical benefits, including antioxidant benefits, to a living mammal, wherein a composition is present in a food and preserving food quality which comprises:(a) adding a composition, which contains a mixture of anthocyanins, bioflavonoids and phenolics, wherein the phenolics are selected from the group consisting of 1-(3′,4′-dihydroxy cinnamoyl)-2,3-dihydroxy cyclopentane 1-(3′-4′-dihydroxy cinnamoyl)-2,5-dihydroxy cyclopentane, and mixtures thereof, isolated from cherries to the food to enhance quality of the food and shelf life; and (b) feeding the mammal the composition in the food, wherein the composition is provided in the food in an amount which provides the antioxidant benefits to the mammal.