KR100441927B1 - A Formulation for Providing Phytoceutical or Nutraceutical Benefits Comprising Cherry Isolates - Google Patents

Abstract

The present invention relates to a method for providing mammals, in particular humans, with the benefit of Neutralical or Phytoticall using cherry derivatives. It also relates to a method of inhibiting oxidation of biomass. Anthocyanins, bioflavonoids, phenols or mixtures thereof obtained from cherries are used in this method.

Description

Formulation for Providing Phytoceutical or Nutraceutical Benefits Comprising Cherry Isolates}

The present invention relates to a method of using cherry isolates with the benefits of beneficial Neutraceutical or Phytoceutical.

The present invention also relates to a method for inhibiting oxidation of a biomaterial in which oxidation inhibition is required using a composition obtained from a cherry. In particular, the present invention relates to compounds or compositions as dietary supplements or food additives. Most preferred are compositions containing mixtures of anthocyanins, bioflavonoids and phenols.

Many compounds derived from plants act as cellular antioxidants by keeping reactive oxygen intermediate levels low, as anti-inflammatory agents by inhibiting prostaglandin synthesis, or as inhibitors of enzymes involved in cell proliferation. Or the "neutratical" property can be imparted to food. Such activity may be important for the improvement of chronic diseases including cancer, arthritis and cardiovascular diseases (see Kinsella et al., Food Tech. 85-89 (1993)). Therefore, dietary supplements / food industry and Neutrostical companies have the opportunity to use compounds that use natural substances to improve food stability on an equally effective basis as synthetic antioxidants, as well as bring significant health benefits to consumers. Will have

Cherries are generally considered to have health benefits. Prunus cerasus el, the main mountain and cherry grown commercially in the United States. Prunus Cerasus L. (Rosacease), a cultivars Montmorency, is a preferred cherry. As a challenge to the single farming of Montmorency, new cultivars, BALATON® and cherry (Ujferbertoi furtos) were introduced to the United States in 1984, and Michigan, Utah And cultivation in Wisconsin. Balaton produces darker fruits than Montmorency.

Recent studies have shown that anthocyanins, such as cyanidin-3-glucoside, have strong antioxidant activity (Tsuda, T, et al., J. Agric. Food Chem. 42: 2407-2410 ( 1994). The addition of antioxidants is one of the representative ways to extend the shelf life of foods that are thought to be associated with lipid peroxidation reactions. Natural antioxidants can play an important role in the prevention of carcinogenesis. Dietary supplement antioxidants may be effective in damaging the peroxidation reactions of the biological system (Halliwell, B. and JMC Gutteridge, Free radicals in boilogy and medicine.Oxford University Press, New York 416-494 (1989); Osea, T., et al., Role of dietary antioxidants in protection against oxidative damage; Kuroda, Y., In antimutagenesis and anti carcinogenesis Mechanism, Shankel (DM), Waters, MD, Plenum Publishing.New York 139-153 (1990)).

Initial studies have shown that montmorency cherries contain anthocyanins, such as cyanidin-3-genthiobiosides and cyanidin-3-rutinosides (Li, KC, et al., J. Am. Chem). Soc. 78: 979-980 (1956)). Cyanidin-3-glucosilucitinoside was also found in six of seven acid and cherry varieties (see Harborne, J. B. et al. Phytochemistry 3: 453-463 (1964)). Dekazos is an anthocyanin pigment in montmorency cherries with cyanidin-3-sorboside, cyanidin-3-rutinoside and peonydine-3-rutinoside, peonidine and cyanidin. Cyanidin-3-glucoside (see Dekazos, ED, J. Food Sci. 35: 237-241 (1970)). However, it has been found that cyanidin-3-glucoside, cyanidin-3-phosphoside and cyanidin-3-rutinoside, as well as cyanidin-3-glucolulutinoside, are the main pigments of acids and cherries. Using HPLC retention values, Sandra et al. Stated that cyanidin-3-soporosides and cyanidin-3-glucosides are the main pigments and auxiliary pigments, respectively, in Montmorency Cherries grown in Michigan (Sandra See Chandra, A, et al., J. Agric. Food Chem. 40: 967-969 (1992). Similarly, it has been found that cyanidin-3-xylsilillotinoside is an auxiliary pigment in montmorency cherries (Shrikhande, AJ and FJ Francis, J. Food Sci. 38). : 649-651 (1973).

In the prior art, the production of pure anthocyanins (compounds 1 to 3 in Fig. 1) from balaton and montmorency cherry juice first gave this pigment to Amberlite XAD-2 (AMBERLITE XAD-2, Sigma Chemicals). ) By adsorption on a column (see Sandra A. et al., J. Agric. Food Chem. 41: 1062-1065 (1993)). This column was washed with water until the eluent showed a pH of about 7.0. The adsorbed pigments and other phenols were eluted with MeOH. The crude anthocyanins thus obtained were fractionated and purified by C-18 MPLC and HPLC, respectively, to obtain pure anthocyanins for spectral studies. 60 mg of pure anthocyanins 1-3 were obtained from 500 mg of crude montmorency anthocyanin obtained from Amberlite XAD-2, while 391.43 mg were obtained from balaton. The results indicate that the crude anthocyanins of montmorency cherries obtained from XAD-2 contain a high percentage of other organic compounds. Amberlite XAD-2 was unable to recycle the resin. No attempt has been made to use the flavonoids, isoflavonoids, crude mixtures of phenols and anthocyanins for any purpose. US Pat. No. 5,266,685 to Garbutt and US Pat. No. 5,665,783 to Katzakian et al. And US Pat. No. 5,817,354 to Mozaffar, disclose various adsorbent resins and related materials. The use for the separation of these free products is described. These patents only describe the general situation of the prior art for the use of adsorptive resins.

US Patent No. 5,503,867 to Pleva discloses the use of fully ground cherry and oat bran in ground meat. The amount of cherries used is 10-15% by weight and oat bran is believed to be added to compensate for cherry juice. At any rate, cherries certainly flavor meat, but the product is not universally palatable. The patent does not describe the benefits of Neutrastical or PhytoStical. Recent studies on stabilizing low-fat ground beef with cherry tissue have shown that such botanicals contain not only antioxidant lipid peroxidation but also potent antioxidants that inhibit the formation of heterocyclic aromatic amines and cholesterol oxidation products when fried. (Gomaa et al., ITF Abstracts No, 68E-7 (1996)). The hypothesis used to explain this observation was that polyphenols, such as flavonoids, anthocyanins and anthocyanidins, commonly found in vacuoles of higher plants such as cherries, exhibit such antioxidant effects.

In particular, there is a need for natural cherry-derived compositions for use as dietary supplements / nutraceuticals or food additives.

1 shows the structure of certain anthocyanins (pigments) isolated from balaton and montmorency cherries. The aglycone cyanidin has a hydroxyl group at position 3.

2 and 3 show major bioflavonoids isolated from cherries.

4 shows certain phenols separated from acid and cherry.

Figure 5 shows the steps of a method for separating anthocyanins, bioflavonoids and phenols from cherries.

6 is a schematic diagram showing an apparatus that may be used in the method shown in FIG. 5.

7 is a graph showing the antioxidant efficacy of anthocyanins and commercially available antioxidants in the liposome model system. Oxidation was initiated by adding ferrous ions. In the presence of the test compound, the decay rate of fluorescence decreased. The control standard sample did not contain any Fe ²⁺ and Fe ²⁺ did not contain any test compound. Another sample contained Fe ²⁺ and 2 μM test compound.

8 is a graph showing the antioxidant activity of isolated compounds 1 to 4, 7 and 8 and commercially available antioxidants TBHQ and BHT at concentrations of 10 μM. Data are averages of two 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'-trihydroxyflavone (FIG. 2).

FIG. 9 is a graph showing the antioxidant activity of isolated compounds 1, 3 and 4 (FIG. 4) and several commercially available antioxidants at 20 μM concentration. Antioxidant activity of Compound 2 was measured at 100 μM. The rate of peroxidation reaction is monitored by a decrease in fluorescence intensity as a function of time. Relative intensity is expressed as the fluorescence intensity at a given time divided by the initial intensity at the start of analysis. The value is the average of the values measured twice.

<Description of Preferred Embodiments>

A preferred method for preparing a mixture comprising anthocyanins, bioflavonoids and phenols from cherries into a composition comprises the steps of providing an aqueous solution containing anthocyanins, bioflavonoids and phenols obtained from cherries, anthocyanins, bioflavonoids and phenols from the aqueous solution. Moving to the resin surface, eluting the resin surface using an eluent to remove anthocyanins, bioflavonoids and phenols therefrom, and separating the eluent from the anthocyanins, bioflavonoids and phenols.

In particular, a preferred method for preparing anthocyanins, bioflavonoids and phenols from cherries in a composition comprises the steps of providing a first batch of cherries fresh or flash frozen to thaw, crushing the cherries and separating the juice and pulp, Extracting anthocyanins, bioflavonoids and phenols from the pulp into an aqueous solution, moving anthocyanins, bioflavonoids and phenols from the aqueous solution containing anthocyanins, bioflavonoids and phenols isolated from the pulp to adsorbent resin particles, and lowering the resin particles to lower alkanes Washing with ol to remove anthocyanins, bioflavonoids and phenols from resin particles, separating alkanols from anthocyanins, bioflavonoids and phenols, and resin particles from which anthocyanins, bioflavonoids and phenols have been removed And repeating the step using the separated alkanol and the second cherry batch.

In addition, preferred intake compositions for use in the method include a mixture of an edible carrier and a dry mixture of anthocyanins, bioflavonoids and phenols isolated from cherries, the weight ratio of the mixture to the carrier being about 0.1: 100 to 100: 0.1 to be.

Finally, a dry mixture of anthocyanins, bioflavonoids and phenols isolated from cherries, and a mixture of edible carriers, wherein the weight ratio of mixture to carrier is about 0.1: 100 to 100: 0.1 It provides a preferred method of inhibiting oxidation of a mammal comprising the. It is preferred that the composition contains at least partially dried cherry pulp.

The cherry used in the present invention may be persimmon or obstetric. Acids and cherries contain high levels of malic acid and other organic acids that contribute to the acidity of the acid and cherry. The process of the present invention isolates other organic acids containing malic acid and sugars that can be used in foods to provide acidity and flavor. Most preferred are balaton and montmorency cherries.

An isolated mixture of anthocyanins, bioflavonoids and phenols can be used as a natural nutraceutical / health supplement. In this regard the isolated mixture may be provided in powder, liquid or solid form. For example, the mixture may be a soluble powder composition that can be dissolved in water, milk or other similar liquid into a beverage. Alternatively, the mixture may be in solid form such as tablets, gel capsules, soft gels and the like. The mixture can also be incorporated into foods.

Generally, the mixture is in a form present in an amount of about 0.01% to about 50%, preferably about 0.1% to about 30%, more preferably about 0.5% to about 25% by weight of the total composition. It may be provided as.

For example, if the mixture is provided in the form of a tablet, the tablet may be in an daily dosage of about 0.1 mg to 300 mg, preferably 1 to 200 mg, preferably 60 to 100 mg of anthocyanin and bioflavonoids. Can be provided. 100 cherries provide 60-100 mg of anthocyanin. Phenols (FIG. 4) can be provided in daily dosages of 0.1 to 50 mg. 100 cherries provide 1-50 mg of phenols. The amounts of anthocyanins, bioflavonoids and phenols can be adjusted by isolating each compound and blending them together. In one embodiment a natural mixture of anthocyanins, bioflavonoids and phenols is used. The compositions may also be presented in liquid form using equivalent doses.

The resin has a surface on which anthocyanins, bioflavonoids and phenols are adsorbed. A preferred class of absorbent resins is crosslinked polymer resins consisting of styrene and divinylbenzene, such as amberlite-based resins, such as Rohm & Hass Co., Philadelphia, PA, USA. Commercially available Amberlite XAD-4 and Amberlite XAD-16. Another crosslinked polymeric styrene and divinylbenzene absorbent resin suitable for use in accordance with the present invention is XFS-4257, XFS-4022, manufactured by The Dow Chemical Company, Midland, Michigan, USA. , XUS-40323 and XUS-40322.

Preference is given to using a commercially available, nationally recognized (if necessary) styrene-divinyl-benzene (SDVB) crosslinked copolymer resin (eg Amberlite XAD-16). Thus, in a preferred embodiment, Amberlite XAD-16, commercially available from Rom and Haas Co., described in US Pat. No. 4,297,220, is used as the resin. This resin is a nonionic hydrophobic crosslinked polystyrene divinyl benzene absorbent resin. Amberlite XAD-16 has a macroreticular structure and has 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 of 100 to 200 microns.

It is also believed that other adsorbents, such as those belonging to the Amberlite XAD adsorbent family, containing hydrophobic macroreticular resin beads and having a particle diameter of 100 to 200 microns, may be effective in the process of the present invention. Moreover, different modified amberlites, such as the AMERCHROM CG adsorbent family, having a particle diameter of 100 to 200 microns, may also be suitable for use in the present invention. Amberlite XAD-16 is preferred because it can be reused many times (more than 100 times). However, in the case of food, it is considered important and / or preferred to use a nationally approved resin in the present invention.

Any solvent may be used to collect the adsorbed anthocyanins, bioflavonoids and phenols. Lower alkanols containing 1 to 4 carbon atoms are preferred, with ethanol (ethyl alcohol) licensed for use in food being most preferred. Typically ethanol is azeotropically distilled with water, but anhydrous ethanol may also be used. Water containing malic acid and sugars in the cherry passes through the column. It can be collected and used as a flavoring agent in foods.

Anthocyanins, bioflavonoids and phenols are preferably obtained from balaton and montmorency cherries. The composition of these cherries is disclosed in part in US Patent Application No. 08 / 799,788, filed February 12, 1997, and partially in US Patent Application No. 60 / 111,945, filed December 11, 1998. . As described in these patent applications, the Montmorency (Prunus cerasus) strain accounts for more than 95% of the mountains and cherries grown in Michigan, USA. However, new acid and cherry cultivars, Balaton Mountain and Cherry (Prunus cerasus), are cultivated in place of Montmorency in some Michigan orchards. This cherry has a higher anthocyanin content and is considered a better variety. The anthocyanin content of Montmorency and Balatonic acid and cherries is recorded (Wang et al., 1997 and Sandra et al., 1993). However, no detailed study of balatonic acid and other phenolic compounds in cherries has been performed before. According to an initial study, montmorency cherries contain cyanidin-3-genthiobioside and cyanidin-3-rutinoside (L. et al., J. Am. Chem. Soc. 78: 979-980 (1956). Cyanidin-3-glucosilutinoside was also found in six of the seven acid and cherry varieties (see Haborn et al. Phytochemistry 3: 453-463 (1964)). Decazos is an anthocyanin pigment in montmorency cherries with cyanidin-3-sorboside, cyanidin-3-rutinoside and cyanidin- together with peonidine-3-rutinoside, peonidine and cyanidin. Reported as 3-glucoside (see Decazos, J. Food Sci. 35: 237-241 (1970)). However, it has been found that cyanidin-3-glucoside, cyanidin-3-phosphoside and cyanidin-3-rutinoside, as well as cyanidin-3-glucolulutinoside, are the main pigments of acids and cherries. Using HPLC retention values, Sandra et al. Noted that cyanidin-3-soporosides and cyanidin-3-glucosides are the main pigments and auxiliary pigments, respectively, in Montmorency Cherries grown in Michigan (Sandra et al. See J. Agric. Food Chem. 40: 967-969 (1992). Similarly, it was found that cyanidin-3-xylsilillotinoside was an auxiliary pigment in montmorency cherries (see Shirikhand and Francis J. Food Sci. 38: 649-651 (1973)). ).

By "carrier" or "bulking agent" is meant a composition used to increase the volume of a composition purified from a cherry. Dry cherry pulp is preferred. Carriers included substances or proteins containing dietary starch, such as skim milk powder. Flour, sugar, soy flour, maltodextrin and various seasonings, such as salt, pepper, spices and herbs, belong to this group. Extenders are preferably used in amounts of about 10 ⁻⁶ to 10 ⁶ parts by weight based on the mixture.

The composition is placed in a food such that the food active ingredient is in an amount of about 0.1 to 10 mg / g. The feed amount is preferably selected to obtain the most beneficial result without affecting the taste of the food. The food may be in a high moisture (wet) or low moisture (dry) state as is well known to those skilled in the art. When used as a dietary supplement, tablets contain 0.1 to 1 g of active ingredient. Specific foods are cooked meat and other manufactured foods wherein the composition provides antioxidant properties and optionally color to the food. The compositions can be used as seasonings in the formulated foods to provide the benefits of Neutrastical or PhytoStical.

Methods for Extraction and Separation of Phytochemicals (Sandra et al., J. Agric. Food Chem. 41: 1062 (1992); Wang et al., J. Agric. Food Chem. 45: 2556-2560 (1997) ] And rapid screening methods of antioxidant activity (Arora, A. and Strasberg), J. Amer. Oil Chem. Soc. 74: 1031-1040 (1997). This method is used to identify, characterize, and test antioxidant compounds obtained from balaton and montmorency cherries. The juiced cherry tissue was extracted sequentially with hexane, ethyl acetate and methanol. Both the methanol fraction and the ethyl acetate fraction showed strong antioxidant activity in this screening assay. The ethyl acetate fraction was further purified by silica gel vacuum liquid chromatography to give four subfractions, which were further separated into seven fractions by preparative reverse phase HPLC. 2 and 3 show bioflavonoids isolated from balaton cherries. There are many analogous or homologous compounds in acids and cherries.

Two new phenolic compounds, (I) 1- (3'-4'-dihydroxy cinnamoyl) -2,3-dihydroxy cyclopentane and (II) 1- (3'-4'-dihydro Oxy cinnamoyl) -2,5-dihydroxy cyclopentane was identified.

Other compounds isolated from ethyl acetate extracts of cherry berries and analyzed by special methods include 1- (3'-methoxy, 4'-hydroxy cinnamoyl) quinic acid, 2-hydroxy-3- (2'-hydroxy). Hydroxyphenyl) propanoic acid, methyl 2-hydroxy-3- (2'-hydroxyphenyl) propanoate, D (+)-malic acid, β-sitosterol and β-sitosterol glycosides. 4 shows several isolated phenols. In addition, anthocyanin components obtained from the juice fractions have also been identified and analyzed completely (Sandra et al., J. Agric. Food Chem. 41: 1062 (1992); Wang et al. J. Agric. Food Chem. 45: 2556-). 2560 (1997)]), and the results indicate that such compounds contain potent antioxidant activity.

As shown in FIG. 4, the compound may be present in an isomeric or pure form wherein R ₁ and R ₂ are selected from the group consisting of hydroxyl and hydrogen and either of R ₁ and R ₂ is hydroxyl. The compound may be an isomer of the compound wherein R ₁ is hydroxyl and R ₂ is hydrogen. Particular isomers of these compounds are 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,5-diol, which can be isolated as pure compounds from acids and cherries. The compound may also be an isomer of the compound wherein R ₁ is hydrogen and R ₂ is hydroxyl. Specific isomers of these compounds are 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,3-diol, which is isolable as pure compounds from acids and cherries.

In addition, the present invention R ₁ and R ₂ is hydroxyl and selected from the group consisting of hydrogen and R ₁ and R ₂ of any of the hydroxyl jitsu, acid cherry isolated compounds or mixtures and pure form of the isomer of the formula from The present invention relates to a method for inhibiting oxidation of a substance requiring inhibition of oxidation, including providing a compound of the compound in an amount of antioxidant inhibition of the substance. In one embodiment the method uses an isomer of the compound wherein R ₁ is hydroxyl and R ₂ is hydrogen. Specific isomers of these compounds include 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,5-diol, which can be isolated as pure compounds from acids and cherries. In another embodiment the method comprises using an isomer of the compound wherein R ₁ is hydrogen and R ₂ is hydroxyl. Specific isomers of these compounds are 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,3-diol, which is isolable as pure compounds from acids and cherries. In another embodiment the method comprises using a mixture of isomers.

In one embodiment of the invention, the composition comprises 1- () as a pure compound isolable from acids and cherries or as a mixture with 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,5-diol. 3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,3-diol, and a nontoxic carrier or extender for the compound. In another embodiment of the invention, the composition is 1- in pure compound isolable from acid and cherry or in a mixture with 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,3-diol. (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,5-diol, and nontoxic carriers or extenders for the compounds. The composition may also contain other antioxidants obtained from cherries, such as anthocyanins, flavonoids or other phenols added to the pure compound. In addition, the carrier or extender used in the composition is a carrier suitable for use in animals or humans.

The composition of the present invention consists of the antioxidant of the present invention and about 0.1 to 30 denier particulate edible extender per part of the composition of the present invention, and the product inhibits oxidation of the oxidizing substance when it comes in. The composition containing the antioxidant of the present invention may be formed in any suitable solid or liquid dosage form and may be used as a nutraceutical / health supplement. Generally, the selected dosage form is 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,5- in neat compound or in a mixture containing two isomers or in admixture with other antioxidant compounds. Daily doses of diol or 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,3-diol are provided. The present invention may be useful as an anti-inflammatory agent in the form of a composition, mixture or in pure form or for the treatment of chronic diseases or for the prevention of diseases, for improving the effects on the diseases, or for stimulating the immune response to more effectively counter the diseases. In particular, the present invention may be useful for the prevention or treatment of various forms of diseases caused in part or in whole by free radicals in compositions, mixtures or pure forms.

As described above, the composition of the present invention may include one or more antioxidants selected from the group consisting of anthocyanins, bioflavonoids, phenols, and mixtures thereof. Anthocyanins, bioflavonoids, phenols are preferably isolated from cherries, in particular from balaton and montmorency cherries.

In a preferred embodiment, the anthocyanin is cyanidin-3-2 "-o-VII-D-glucopyranosyl-6" -o-∀-L-rhamnosyl-VII-D-glucopyranoside, cyanidin-3 -6 "-o-∀-L-lamnosyl-VII-D-glucopyranoside, cyanidin-3-VII-D-glucopyranoside and mixtures thereof. In a preferred embodiment , Bioflavonoids are 7-methoxy-5,8,4'-trihydroxyflavones.

The antioxidant of the present invention or a composition containing the compound of the present invention may be added to food as a conventional antioxidant food additive. In particular, food additives include 1- (3 ', 4'-dihydroxycinnamoyl) -cyclopenta-2,3-diol or 1- (3', 4'-, either as pure compounds or as mixtures containing two isomers. Dihydroxycinnamoyl) -cyclopenta-2,5-diol. In addition, the food additive may include other antioxidants selected from the group consisting of anthocyanins, bioflavonoids, phenols, and mixtures thereof. In a preferred embodiment, the anthocyanin is cyanidine-3-2 "-o-VII-D-glucopyranosyl-6" -o-∀-L-rhamnosyl-VII-D-glucopinoraside, cyanidin-3 -6 "-o-∀-L-lamnosyl-VII-D-glucopyranoside, cyanidin-3-VII-D-glucopyranoside and mixtures thereof. In a preferred embodiment , Bioflavonoids are 7-methoxy-5,8,4'-trihydroxyflavones Anthocyanins, bioflavonoids, phenols are preferably isolated from cherries, in particular from balatons and montmorency cherries. The inhibitor or the composition containing the compound of the present invention may be added to high moisture (wet) or low moisture (dry), fresh or uncooked, cured or cooked food. Compounds or compositions inhibit oxidation of lipids and thus inhibit the occurrence of odors . Occur during the cooking to the composition added to the meat prior to cooking and inhibits the formation of heterocyclic aromatic amines (HAA) indicating carcinogenic.

Summary of the Invention

The present invention relates to a method for providing a living mammal with the benefits of neutral or phytochemicals, including antioxidants, which method is selected from the group consisting of anthocyanins, bioflavonoids, phenols and mixtures thereof isolated from cherries. And feeding the mammal in an amount that provides the benefits of Neutrastical or PhytoStical.

The present invention provides a biomaterial of which oxidation inhibition is desired, comprising providing to the biomass a composition selected from the group consisting of anthocyanins, bioflavonoids, phenols and mixtures thereof isolated from cherries in an amount that inhibits oxidation of the biomass. It relates to a method of inhibiting oxidation.

The term "biomaterial" refers to living tissue in vivo or in vitro culture of an animal or human.

The term "anthocyanin" means a chromophoric compound contained in a cherry. For the purposes of the present invention the term includes aglycone cyanidin.

The term "bioflavonoid" refers to isoflavonoids and flavonoid compounds contained in cherries.

The term "phenols" means compounds having a phenyl group and one or more hydroxyl groups.

The term "neutrautical" refers to a substance that is beneficial to a mammal, such as vitamins, that affects the long-term health status of the mammal.

The term "phytochemical" means a product derived from a plant that provides the benefits of Neutrastical.

<Purpose>

Accordingly, it is an object of the present invention to provide a natural cherry composition that can be used in foods as an antioxidant to prevent oxidation of living tissue or as a dietary supplement or nutraceutical. This object will become more apparent with reference to the following detailed description and drawings.

<Examples 1 and 2>

As shown in FIG. 5, individual quick frozen (IQF) cherries (without seeds) were thawed and blended in an industrial WARING blender. The mixture was centrifuged at 10000 rpm and the juice decanted off. The residue pulp was further pressed with a cheese cloth to remove any excess juice.

The pulp was lyophilized at 15 ° C. The juice was treated on Amberlite XAD-16 HP resin to obtain anthocyanins, bioflavonoids and phenols, which are cherry acidity components. 1 kg of XAD-16 resin was washed with ethanol (1-2 L) and washed with water (6 L). The XAD-16 resin was left in water for 1 hour and then a glass column with a cotton plug. (10ID × 90 cm in length). The packed column was washed with water (2 L) before adding the juice for separation. 800 ml of juice were purified each time. The juice was added to the column surface and left to stand still without flow. It was then eluted with water and the first 1 liter was discarded. Then 2 liters of washings were collected because it contains acidic cherry juice because it contains malic acid and sugars from cherries. The column was then washed with an additional 4 L of water for Balaton Cherry Juice and 5 L of water for Montmorency Cherry Juice. Once the cherry juice was collected, the remaining washes washed with water were discarded. The column was then eluted with ethanol (1.3 L to 1.5 L) and a red solution (700-800 mL) containing anthocyanins, bioflavonoids and phenols was collected. The column was then dried and washed with 10 L of water, then this procedure was repeated several times (at least 100 times). The red alcoholic solution was then evaporated in vacuo (20 mTorr) to remove ethanol and aqueous solution, stabilized with 50 ppm of ascorbic acid and lyophilized at 10 ° C. Red powder was collected and stored.

Results of Example 1

Balaton Cherry

15.74 kg of IQF cherry

605 g of dry flesh

Juice volume 12.16 ℓ

Anthocyanins, Bioflavonoids

And 31.35 g in weight of phenols (red powder)

Volume of acidity by-products (malic acid and sugar) about 35 liters

Results of Example 2

Montmorency Cherries

IQF cherry weight 30.45 kg

895 g of dry flesh

Juice volume 24.03 ℓ

Anthocyanins, Bioflavonoids

And 47 g in weight of phenols (red powder)

Volume of cherry by-products (malic acid and sugar) about 75 l

The red powders of Examples 1 and 2 are preferably mixed with dry flesh as a carrier and tableted into tablets of 1 to 1000 mg, including the carrier (daily dose per adult).

To prevent degradation, various food grade acids can be added to the separated anthocyanins, bioflavonoids and phenols. Preferably they do not add flavor. Ascorbic acid (vitamin C) is preferred. The acid can be added before or after drying the cherry compound.

In small scale processes, lyophilization is used to remove the water. In larger scale processes, drying with an air circulation oven is preferred.

<Example 3>

As shown in Fig. 6, the open container 10 is equipped with an inlet pipe 11 and an outlet pipe 12 having valves 13 and 14, respectively. The resin beads 15 are filled in the open container 10. The water is introduced into the vessel 10 and then removed and disposed of through the discharge pipe 12. Cherry juice (not including flesh or seeds) as used in Example 1 is introduced into the container 10 and left for 25 minutes. The temperature of water and juice is about 20-30 ° C. The cherry juice residue containing malic acid and sugar is then removed through the outlet tube 12 and used as food flavourant. The resin 15 in the vessel is then washed again with water entering the inlet duct 11 and the water is removed through the outlet duct 12 and discarded. Subsequently, the anthocyanins, bioflavonoids and phenols on the resin particles are extracted by 95% ethanol introduced through the inlet pipe 11. Ethanol containing anthocyanins, bioflavonoids and phenols are collected from the vessel 10. Ethanol is removed from anthocyanins, bioflavonoids and phenols and the residue is flash dried in nitrogen. The powder thus obtained is preferably mixed with dried cherry pulp or other carrier as in Example 1. The resin particles are washed with water and the resin and ethanol are recycled several times.

<Example 4>

Crude ethyl acetate extracts from cherries (containing anthocyanins, bioflavonoids, phenols) were tested in aqueous solution using fluorescence analysis for antioxidant activity under various conditions. The fluorescence assay will be described first.

The need for testing antioxidant activity for many compounds or extracts requires the use of model systems (s) that adequately represent the structure and functional characteristics of the composition either alone or in foodstuffs. In addition, the test should be sensitive, fast and inexpensive. Fluorescence-based assays were used to evaluate antioxidant efficacy (J. Am. Chem. Soc. 1996, Aurora and Strasberg). A large monolayer vacuole consisting of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine was prepared, which is very similar to the properties of biological membranes, one of the main sites for peroxidation. The fluorescent probe 1,6-diphenylhexatriene propionic acid is incorporated into the membrane such that the polar head group holds the probe near the aqueous interface while the hydrophobic moieties are arranged parallel to the fatty acid chains. The probe reacts with the free radicals produced during the peroxidation reaction, reducing the intensity of fluorescence over time. Peroxidation reaction initiator (e.g., ferrous metal ion or free radical generator AAPH (azobis- [2-amidino propane hydrochloride])) to initiate the reaction and determine the rate of fluorescence reduction of the antioxidant composition to be tested. Measurements were made with or without. Analysis of the compound at a given concentration took only 21 minutes, only a few micrograms of lipid consumed, and could easily be performed with a simple fluorometer.

Large monolayer vacuoles (LUVs) were obtained from 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine by McDonald, R. C. et al. Biochim. Biophys. Acta 1061; 297-303 (1991). Briefly, lipids were dissolved in chloroform and dried using a rotary evaporator to form a thin film. The dried membrane is resuspended in a buffered aqueous solution and subjected to a liposofast piposome extruder (Avestin, Inc., Ottwa, Canada) via a polycarbonate filter having a pore size of 100 nm. Was extruded using. Homogeneity of size (80-100 nm) and monolayer properties of vacuoles were confirmed using freeze-broken scanning electron microscopy. Diphenylhexatriene-propionic acid (DPH-PA), a fluorescent probe, was incorporated into vacuoles in a molar ratio (probe: lipid) of 1: 350 during preparation. For fluorescence experiments, LUV containing DPH-PA was suspended at 100 μM final concentration in 50 mM Tris-HEPES buffer at 100 mM NaCl, pH 7. Fluorescent probes were excited at 384 nm and luminescence was monitored at 423 nm. Lipid oxidation in LUV is inhibited by the addition of ferrous ions or free radical generator AAPH. The progress was monitored through a decrease in fluorescence intensity of DPH-PA due to reaction with free radicals generated over 21 minutes. The rate of lipid oxidation reaction was determined by plotting the decrease in fluorescence intensity as a function of time. The results indicated that a mixture of crude anthocyanin extract and ethyl acetate was effective for inhibiting oxidation.

Solvent extraction of anthocyanins, bioflavonoids and phenols may be used but is undesirable when the product is used in food and due to cost concerns. If a preferred adsorptive resin is used, this step is unnecessary. It is also possible to separate and remix the components using chromatography, but for the purposes of the present invention this step is too expensive because it involves high pressure liquid chromatography.

Example 5

The antioxidant activity of cherry anthocyanin was analyzed using the method developed by Arora and Strasberg. As the reaction proceeds, the fluorescent probe will degrade and this will reduce the intensity of fluorescence. Thus, the rate of fluorescence attenuation is reduced in the presence of antioxidants. Experiments showed that the antioxidant activity of anthocyanins 1-3 and aglycone cyanidin is comparable to the commercially available antioxidants butylated hydroxyanisole (BRA) and butylated hydroxy toluene (BHT) (FIG. 7). . In addition, such cherry compounds showed better antioxidant activity than α-tocopherol. At 2 mM concentration, it was shown that the peroxidation of the sample containing tocopherol was indistinguishable from that of the Fe ²⁺ containing sample without the addition of any antioxidant (FIG. 7).

Cyanidins, the aglycones of anthocyanins, have higher efficacy than their glycosides, suggesting that the antioxidant activity of anthocyanins is due to their aglycone residues. Anthocyanins 1-3 contain 3, 2 and 1 saccharide residues, respectively, which explains why the lowest antioxidant activity in anthocyanin 1 is observed. The number of sugar residues at the C ₃ position appears to be very important for antioxidant activity. It has also been reported that the stability of aryloxy radicals can affect the antioxidant activity of the compounds and cause oxidation-promoting effects. Thus, the antioxidant activity of cyanidin may depend on the stability of its aryloxy radicals. Ortho-dihydroxy substitutions in the B-rings of anthocyanins and cyanidins are important for stabilizing the free radicals generated through the 3 'and 4'-OH moieties. In addition, the ortho-dihydroxy groups of anthocyanins are effective at complexing metal ions and thus prevent iron-induced lipid peroxidation reactions.

In the preparation of cyanidin, the anthocyanin mixture containing anthocyanins 1-3 (500 mg) was stirred with 3N HCl (20 mL) at 80 ° C. for 10 hours. The reaction mixture was purified on XAD-4 column as in anthocyanin preparation. The MeOH solution of cyanidin was evaporated to dryness to give a red amorphous powder (190 mg) which was stored at −30 ° C. until use.

In antioxidant assays, the buffer was stored in Selectex 100 to remove metal ions. 1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine 5 nM (available from Avanti Polar Lipids, Inc., Alabaster, AL) and fluorescent probe 3 Mixture containing-(p- (6-phenyl) -1,3,5-hexatrienyl) phenylpropionic acid 15 nM (Molecular Probes, Inc., Eugene, OR) Was dried under vacuum. The resulting membrane was suspended in 500 ml buffer (NaCl 0.15 M; EDTA 0.1 mM; MOPS 10 mM) and subjected to 10 freeze-thaw cycles in EtOH-dry ice bath. The suspension was then passed through a polycarbonate membrane of pore size 100 nm 29 times using a liposofast extruder (available from Avestin). The resulting liposomes (200 nM) were then suspended in 2 ml of buffer (100 mM NaCl, 50 mM HEPES, pH 7.0). Peroxidation reaction was initiated by the addition of Fe ²⁺ 4 nM. Anthocyanins, BHT, propyl gallate and ∀-tocopherol (vitamin E) were tested at 2 mM concentration. The control standard sample did not contain Fe ²⁺ or test compound added. The fluorescence intensity of the lipid suspension was monitored for 21 minutes using an SLM 4800 spectrofluorometer (SLM Instruments, Inc.) immediately after the addition of Fe ²⁺ in the presence or absence of the test compound. The relative fluorescence value was obtained by dividing the fluorescence value at a predetermined time by the fluorescence value at t = 0 minutes. The results are shown in FIG. Anthocyanins (1-3, FIG. 1) and cyanidins isolated from acids and cherries showed in vitro antioxidant activity comparable to commercially available products. Inhibition of lipid peroxidation of anthocyanins 1-3 and their aglycone cyanidin was 39, 70, 75 and 57% at 2 mM concentrations, respectively. Antioxidant activity of anthocyanins 1-3 and cyanidin was comparable to tert-butylhydroquinone and butylated hydroxytoluene at 2 mM concentration and superior to vitamin E.

<Examples 6 and 7>

As in Example 5, the antioxidant activity of the selected compounds as shown in Figures 2, 3 and 4 were tested. The results are shown in FIGS. 8 and 9. In Example 6, the antioxidant activity of the following compound is shown in FIG.

Compound 1 = naringenin

Compound 2 = Genistein

Compound 3 = chlorogenic acid

Compound 4 = Quercetin 3-Rhamnoside

Compound 5 = cafeferol 3-lutinoside

Compound 6 = 3'-methoxy caempferol 3-lutinoside

Compound 7 = geninistin 7-glucoside

Compound 8 = 5,8,4'-trihydroxy-6,7-dimethoxyflavone

In Example 7, the antioxidant activity of the following compound is shown in FIG.

Compound 1 = chlorogenic acid methyl ester

Compound 2 = 2-hydroxy-3- (o-hydroxyphenyl) -propanoic acid

Compound 3 = 1- (3'-4-dihydroxy cinnamoyl) -2,5-dihydroxy cyclopentane

Compound 4 = 1- (3'-4-dihydroxy cinnamoyl) -2,3-dihydroxy cyclopentane

In FIG. 9, antioxidant compounds 1, 2, 3 and 4 (shown in FIG. 4) were compared with various antioxidants. Antioxidants tested are caffeic acid, ferulic acid, chlorogenic acid, p-hydroxycinnamic acid and commercially available antioxidants, tert-butylhydroquinone (TBHQ) and butylated hydroxytoluene (BHT).

Data shown in FIGS. 8 and 9 were obtained using Fe ²⁺ -induced lipid peroxidation assay to measure antioxidant activity. 5 μmol of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine to prepare substrates used for Fe ²⁺ -induced lipid peroxidation analysis (Avanti Polar Lipiz, Inc.) ) And a mixture containing 0.015 μmol of the fluorescent probe 3- (p- (6-phenyl) -1,3,5-hexatrienyl) phenylpropionic acid (Molycula Probes, Inc.), a rotary evaporator Dried under vacuum. The resulting lipid membrane was suspended in 1000 μl of a solution containing 0.15 M NaCl, 0.1 mM EDTA, and 0.01 M 4-4-morpholine propanesulfonic acid (MOPS) buffer for Example 6, and suspended in 1000 μl of the solution for Example 7. The MOPS buffer was pretreated with a Selex 100 (Sigma Chemicals, St. Louis, Missouri) at a concentration of 5 g / 100 ml buffer to remove any residual metal ions. The suspension was then subjected to ten freeze-thaw cycles using dry ice / ethanol bath. The lipid-buffer suspension was then passed through a polycarbonate membrane with a pore size of 100 nm 29 times using a liposofast extruder (available from Avestin) to prepare a monolayer liposome.

20 μl aliquots of the liposome suspension were subjected to Fe ^{2 +} -induced lipid peroxidation assay, for example 6 N-2-hydroxyethylpiperazine-N'-2-ethane at 100 mM NaCl, pH 7.0. Dilute to 2 ml in a Selex 100 treated buffer containing 50 mM sulfonic acid (HEPES) buffer (200 mM NaCl for Example 7, 100 mM HEPES buffer at pH 7.0) and incubate at room temperature for 5 minutes, then 23 Incubation was continued for 5 minutes in a constant temperature cuvette holder of a spectrofluorometer fixed at &lt; RTI ID = 0.0 &gt; Then, peroxide reaction was initiated by adding 20 μl of 0.5 mM FeCl ₂ to bring the final concentration of Fe ²⁺ to 0.5 μM in the presence or absence of any test compound. The control standard sample did not contain Fe ²⁺ or any test compound. The fluorescence intensity of this liposome solution was measured every 3 minutes over 21 minutes at an excitation wavelength of 384 nm using a fluorescence spectrofluorometer (SLM4800, SLM Instruments, Inc.). The decrease in relative fluorescence intensity over time indicated the rate of peroxidation reaction. Percent inhibition of lipid oxidation (PI) was calculated by the following equation

PI = {[(F _rel ) _P1- (F _rel ) _Fe / [(F _ref ) _c- (F _rel ) _Fe ]} x 100

Wherein (F _rel ) _P1 is the relative fluorescence for Fe (II) and the test sample at the end of the 21 minute measurement time, and (F _ref ) _c is the relative fluorescence for the control standard sample at the end of the 21 minute measurement time. And (F _rel ) _Fe is the relative fluorescence for Fe (II) -containing samples at the end of the 21 minute measurement time (Arora et al. ((1997), J. Amer. Oil Chem. Soc. 74: 1031-1040].

In Example 6, the antioxidant activities of compounds 1, 2, 3, 4, 7 and 8 were analyzed at a concentration of 10 μM. The inhibitory effect of flavonoids on Fe ²⁺ lipid oxidation was due to its complexing ability to complex with Fe ²⁺ to form an inert complex that could not initiate a peroxidation reaction (Afanas et al., 1989). It is also contemplated that the complex of flavonoid Fe 2+ retains free radical capturing ability, thereby eliminating free radical intermediates in lipid peroxidation. Flavonoids can also act as free radical scavengers. As shown in FIG. 8, the antioxidant activity of Compound 8 (5,8,4-trihydroxyl-6,7-dimethoxyflavone) was higher than that of Compounds 1, 2, 4 and 7 at the 10 μM concentration studied. Excellent.

Conventional literature describes ortho-dihydroxy groups on the B ring (Bors et al., 1990), hydroxyl groups at position 3 on the C ring (Apana et al., 1989; Mora). Et al., 1990) and the presence of C ₂ -C ₃ double bonds (Bohr et al., 1990) together with 4-oxo functional groups are thought to be essential for effective radical removal by flavonoids. Although compound 8 has no 3-hydroxyl group and only one hydroxyl group on the B-ring, the antioxidant activity of compound 8 is higher than quercetin 3-rhamnoside. Quercetin 3-rhamnosides contain C ₂ -C ₃ double bonds with 3-hydroxyl and 4-oxo functional groups in addition to the ortho-dihydroxy group on the B ring. The enhanced antioxidant activity of compound 8 was presumably due to the hydroxyl and two methoxy groups of the A ring. Arora et al. (1997) reported that 7,8-dihydroxyflavones exhibited antioxidant activity similar to quercetin, although no substituents exist at the B ring and at position 3.

In FIG. 9, the inhibitory activity of compounds 3 and 4 on Fe ²⁺ induced lipid peroxidation reaction in large monolayer vacuoles was about 80% at 20 μM. Compound 1 showed about 50% inhibitory activity. However, Compound 2 did not show antioxidant activity even when tested at 100 μM concentration. The analysis also indicated that p-hydroxycinnamic acid was a weak antioxidant compared to ferulic acid. However, compounds 3 and 4, the caffeic acid homologues, showed the highest antioxidant activity in this assay. The percentage inhibition of TBHQ and BHT for lipid peroxidation reactions was greater than 90% at 20 μM concentration. The analysis indicated that caffeic acid is the best oxidant and is in order of compound 4, compound 3, chlorogenic acid and chlorogenic acid methyl ester.

<Example 8>

The antioxidant activity of the compositions of Examples 1 and 2 was tested and found to be comparable to Example 5.

Potential uses of antioxidant compounds include reducing or eliminating oxidative damage to cells, eliminating free radicals to reduce cell death, reducing atherosclerosis or atherosclerosis, reducing the incidence of heart attacks, reducing the incidence of arthritis, and gout. Pain and aging reduction.

The foregoing description is for illustrative purposes only and the invention is defined only by the appended claims.

Claims (45)

Anthocyanins, bioflavonoids isolated from cherries, and 1- (3'-4'-dihydroxy cinnamoyl) -2,3-dihydroxy cyclopentane, 1- (3'-4'-dihydric) Hydroxy cinnamoyl) -2,5-dihydroxy cyclopentane and a composition comprising a mixture of phenols selected from the group consisting of mixtures thereof in an amount to provide mammals with the benefits of nutraceuticals or phytochemicals, Formulations that provide the benefits of Neutrastical or PhytoStical. Wherein R ₁ and R ₂ are selected from the group consisting of hydroxyl and hydrogen, and one of R ₁ and R ₂ is hydroxyl The combination of claim 1, wherein the mammal is a human and the composition is provided in a formula. The combination of claim 1, wherein the composition is dried. The combination of claim 1, wherein said composition is a dry composition containing an edible carrier. The combination of claim 4, wherein the carrier is dried cherry pulp. The combination of claim 1, wherein the composition contains a carrier and the composition to carrier ratio is from 0.1: 100 to 100: 0.1. The combination of claim 1, wherein the composition is in liquid form. delete The combination of claim 1, wherein the composition is isolated from acid and cherry. The combination of claim 1, wherein the composition is isolated from persimmon and cherry. The combination of claim 1, wherein the composition is isolated from MONTMORENCY cherries. The combination of claim 1, wherein the composition is isolated from BALATON cherries. Anthocyanins, bioflavonoids isolated from cherry, 1- (3'-4'-dihydroxy cinnamoyl) -2,3-dihydroxy cyclopentane, 1- (3'-4'-dihydroxy Cinnamoyl) -2,5-dihydroxy cyclopentane and a mixture containing a mixture of phenols selected from the group consisting of a mixture of phenols, wherein Inhibitory formulation. Wherein R ₁ and R ₂ are selected from the group consisting of hydroxyl and hydrogen, and one of R ₁ and R ₂ is hydroxyl The combination of claim 13, wherein the biomass is present in the human body to which the composition is supplied. The combination of claim 13, wherein the biomass is present in the animal body to which the composition is supplied. The combination of claim 13, wherein the composition is dried. The combination of claim 13, wherein the composition is a dry composition containing a dry edible carrier. The combination of claim 13, wherein the carrier is dried cherry pulp. The combination of claim 13, wherein the composition contains a carrier and the ratio of composition to carrier is from 0.1: 100 to 100: 0.1. delete The combination of claim 13, wherein the composition is isolated from acid and cherry. The combination of claim 13, wherein the composition is isolated from persimmon and cherries. The combination of claim 13, wherein the composition is isolated from montmorency cherry. The combination of claim 13, wherein the composition is isolated from balaton cherries. delete delete delete delete a. Anthocyanins, bioflavonoids isolated from cherry, 1- (3'-4'-dihydroxy cinnamoyl) -2,3-dihydroxy cyclopentane, 1- (3'-4'-dihydroxy A composition containing a mixture of phenols selected from the group consisting of cinnamoyl) -2,5-dihydroxy cyclopentane and mixtures thereof is added to the food to improve the food quality and shelf life, b. Wherein the composition is included in food in an amount that provides the mammal with nutraceutical or phytochemical properties, A composition in which the composition is present in a food to provide the benefits of a nutraceutical or phytochemical, including antioxidant benefits and to preserve the quality of the food. Wherein R ₁ and R ₂ are selected from the group consisting of hydroxyl and hydrogen, and one of R ₁ and R ₂ is hydroxyl delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete