KR20060123663A - A composition containing natural cyclooxygenase inhibitors - Google Patents

Abstract

The present invention describes food supplements that contain one or more fruit extracts useful for pain relief and anti-inflammation. The food supplements may be used to inhibit inflammation mediated by cyclooxygenase and more particularly by cyclooxygenase-2.

Description

A composition containing a natural cyclooxygenase inhibitor {A COMPOSITION CONTAINING NATURAL CYCLOOXYGENASE INHIBITORS}

1 is a flowchart showing an embodiment of a process for obtaining and concentrating anthocyanins of requisite from anthocyanin-containing plants.

This application is directed to United States Division No. 60 / 151,280 ("PROCESS FOR CONCENTRATING FLAVONOIDS FROM PLANT", August 27, 1999). The present invention also discloses US divided application No. 60 / 151,278 (August 27, 1999).

The present invention relates to dietary supplements that are effective in alleviating pain or inflammation and inhibiting biological pathways associated with pain or inflammation propagation. These supplements contain flavonoids, in particular any anthocyanins.

Many consumers are now demanding natural substances to replace pharmaceutical synthetic products that support the various foods they experience in their daily lives. Therefore, dietary supplements containing natural substances, such as St. Johns wort, Gingko biloba, ginseng, etc., have recently been introduced to the market for various purposes. However, to date, no natural substance-containing products are available that provide relief of pain and / or inflammation at the same level as non-steroidal anti-inflammatory drugs (“NSAIDs”).

Currently, pain and inflammation are treated with aspirin, ibuprofen (Motrin, Advil) and other analogues known in the art (eg NSAIDs). Inflammation is propagated, in part, by a known group of compounds, such as prostaglandins, released in the host in response to mechanical, thermal, chemical injury and bacteria (Moncada et al., Handbook of Exp. Pharm. Vol 50-1, Springer Verlag, pp 588-616, 1978; Samuelsson, Science, 220: 568-575, 1983; Davies et al., Ann. Rev. Immunol. 2: 335-357, 1984). Prostaglandin synthesis is achieved stepwise by ubiquitous complex microsome enzymes. The first enzyme in this biosynthetic pathway is prostaglandin endopeperoxide synthase. This enzyme is referred to in the art as fatty acid cyclooxygenase. There are two allotropes in this enzyme, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) (Smith, Am. J. Physiol., 268: F181-F191, 1992).

Although substances such as aspirin inhibit prostaglandin production and thus pain or inflammation, they can cause gastrointestinal problems and / or gastric ulcers. To address this problem, drugs have been developed that target specific pain pathways for the purpose of reducing but not completely eliminating some of the problems associated with aspirin, ibuprofen and other similar substances. One such drug is Celebrex ^™ , which targets specific pain pathways and thus does not show some disadvantages associated with substances such as aspirin. In particular, NSAIDs inhibit enzymes of the human arachidonic acid / prostaglandin pathway to prevent the production of prostaglandins. However, drugs such as Celebrex ^™ distinguish COX-1 from COX-2 and are thought to have fewer side effects associated with conventional NSAIDs.

As mentioned above, many consumers prefer natural substances over synthetic drugs. In this regard, one method disclosed in PCT WO 00.33824 suggests a mixture of anthocyanins, bioflavonoids, phenolic compounds used as anti-inflammatory drugs as a result of inhibition of cyclooxygenase enzymes. Similarly, Wang et al., Antioxidant and anti-inflammatory compounds in tart cherries (anthocyanins, phenolics, flavonoids, Balaton, Montmorency), Dissertation Abstract, XP002137469 (1998) present compounds obtained from cherries and are hydrolyzed anthocyanins It is described that cyanidin exhibits inhibitory activity against COX-1 and COX-2 enzymes.

U.S. Patent No. 4,376,781 and 4,258,055 set forth the pharmacological properties of flavilium salts (organic salts of anthocyanidins) with anti-inflammatory, vascular-protective, hyperlipidemic, low cholesterol, hypoglycemic activity.

Loggia et al., Anti-inflammatory Activity of Benzopyrones that are inhibitors of Cyclo and Lipo-Oxygenase, Pharamcological Research Communications, Vol. 20, Supplement V, 91-94 (1998), describe the topical use of quercetin, camphorol, and esculletin. Lipoxygenase activity is shown.

Thus, there is a need for pharmacologically acceptable natural anti-inflammatory compositions that preferentially inhibit COX-2 enzymes. The present invention provides a dietary supplement comprising extracts derived from one or more plant materials of Vaccinium that have a naturally active aliquot that provides pain relief, anti-inflammatory activity, preferably COX-2 inhibition. To meet this need. The supplement contains aliquots in a ratio per dry weight which significantly exceeds the proportion of aliquots present per dry weight in the extract obtained from the plant material. In general, the active aliquot contains flavonoids and in particular anthocyanins.

Summary of the invention

The present invention relates to a dietary supplement containing one or more fruit extracts effective for pain relief and anti-inflammatory. Such supplements can be used to inhibit inflammation mediated by cyclooxygenase, in particular cyclooxygenase-2. More specifically, the present invention provides a supplement having anti-inflammatory properties, wherein the supplement is a flavonoid-rich fruit extract having a higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits; And pharmaceutically acceptable diluents or excipients.

In certain embodiments, the fruit is sweet cherry, tart cherry, acerola cherry, flop, bilberry, blackberry, cannant, chokeberry, blueberry, strawberry, cranberry, voyagerberry, grape, raspberry, elderberry, Selected from mixtures thereof. In certain embodiments, the anti-inflammatory activity of the extract is mediated by cyclooxygenase inhibition. In a suitable embodiment, the extract has a higher cyclooxygenase-2 inhibitory activity than cyclooxygenase-1 inhibitory activity. In a more suitable embodiment, the ratio of COX-2 inhibitory activity to COX-1 inhibitory activity is 1: 1 to 25: 1. Of course, this is a typical ratio range, and any ratio between these values is possible.

In certain embodiments, the anti-inflammatory activity is cyanidin-3-glucoside; Cyanidin-3-glucosilutinoside; Cyanidin-3-gentioside; Cyanidin-3-Lutinoside, Peonydin-3-Lutinoside, Peonidine, Cyanidin, Cyanidin-3-Soposide, Pelagonidine, Delphinidine, Petuninidine, Malbidine, Camperol Mediated by flavonoids selected from hesperidin, genthiodelpin, platinum, cinerarin, and mixtures thereof. In a suitable embodiment, the fruit extract is an elderberry extract, chokeberry extract, tart cherry extract or a mixture thereof. In another suitable embodiment, the supplement contains elderberry extract, bilberry extract, cherry extract, preferably tart cherry extract.

The present invention is also based on the observation that anthocyanidin, a hydrolyzed form of anthocyanin, exhibits higher COX inhibitory activity than anthocyanin. Therefore, the dietary supplement of the present invention is conceived to be mixed with the flavonoids (anthocyanin) and anthocyanin is hydrolyzed in vivo to provide COX inhibitory activity.

Supplements may be incorporated into gels, capsules, tablets, syrups, beverages or powders. Methods of making such formulations are known to those skilled in the art. In another aspect, the supplement may contain additional additives selected from vitamins, minerals, coenzymes, fibers, herbal extracts, or mixtures thereof. Particularly preferred herbal extracts include ginseng extract and boswellia extract. Vitamins can be selected from vitamin A, vitamin D, vitamin E, vitamin B ₁₂ , riboflavin, niacin, pantothenic acid, thiamine, choline, folic acid, biotin, vitamin K, vitamin C. The mineral may be selected from cobalt, copper, iron, manganese, zinc, selenium or mixtures thereof.

In certain embodiments, the anti-inflammatory activity is 2 to 100 times higher than the natural fruit anti-inflammatory activity. In another embodiment, the supplements exhibit higher pain relief properties than the pain relief properties of aspirin.

In addition, the present invention relates to a supplement having anti-inflammatory properties, wherein the supplement has a higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits, bilberry extract, cherry extract, elderberry extract. And fruit extracts selected from mixtures thereof. In certain embodiments, the supplement may further contain chokeberry extract or other fruit extracts described above.

In addition, the present invention relates to a method for inhibiting COX-2 activity in cells, which has a higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits, bilberry extract, cherry extract, elderberry extract. And contacting the cells with a fruit extract selected from a mixture thereof. In suitable embodiments, the cells are further contacted with chokeberry fruit extract or other fruit extracts described above. In a particularly suitable embodiment, the elderberry fruit extract and the chokeberry fruit extract are included in the same composition. In another embodiment, elderberry fruit extract and chokeberry fruit extract are included in other compositions. In certain embodiments of the invention, the cell is a mammalian cell. In another embodiment, the cell is a human cell. In certain embodiments, the cells are contacted with the fruit extract in vitro. In another embodiment, the cells are contacted with the fruit extract in vivo. In a suitable aspect, the method utilizes supplements made of gels, capsules, tablets, syrups, beverages or powders.

Another aspect of the invention provides a method of treating an inflammatory response in an animal, the method comprising: a fruit extract having a higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits; And administering to said animal a composition consisting of a pharmaceutically acceptable diluent or excipient.

In certain embodiments, the inflammatory response can be selected from arthritis, pain, allergic rash, inflammatory bowel symptoms, asthma. Of course, these are typical inflammatory diseases, and the supplements according to the present invention provide excellent herbal remedies for any disease caused by an inflammatory response. Supplements according to the invention are in the form of gels, capsules, tablets, syrups, beverages or powders.

As will be appreciated, the supplements disclosed herein can be used alone or in combination with other anti-inflammatory drugs. In these embodiments in combination with the supplements according to the invention and other drugs, selected from salicylates, glucocorticoids, para-aminophenol derivatives, opioids, indomethacin, sulindac, phenamate, propionic acid derivatives, oxycamps The anti-inflammatory drug is administered to the animal.

In another embodiment of the invention, the invention is directed to nutraceuticals comprising anthocyanin-containing fruit extracts, including elderberry (including tart cherry), bilberry, chokeberry fruit and other anthocyanin-containing fruits as described above. In this regard, the functional food is ingested or applied to the organism suffering from pain to relieve the pain. In particular, pain is arthritis, dysmenorrhea, headaches, insect bites, allergic rashes.

Another object of the present invention is to provide a supplement having an anti-inflammatory activity which is 2 to 100 times higher than the natural fruit anti-inflammatory activity. For example, supplements may contain elderberry fruit extracts that have higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits.

Still another object of the present invention is to provide a method of inhibiting COX-2 activity in cells by contacting a cell with elderberry fruit extract having a higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits. In addition, there is provided a method of treating an inflammatory response in an animal, the method comprising: a fruit extract having a higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits; And administering to the animal a composition consisting of a pharmaceutically acceptable diluent or excipient.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, the specific embodiments suggesting the detailed description and the appropriate embodiments of the present invention are not intended to limit the present invention to explain the present invention, because various modifications and variations of the present invention will be apparent to those skilled in the art from the detailed description. to be.

Unless otherwise stated, percentages used in the specification and claims are weight percentages.

Prostaglandins (including PGE ₂ , PGD ₂ , PGF ₂ , PGI ₂ and other related compounds) represent various autosecretory and side secretory hormones derived from the metabolism of fatty acids. They belong to a group of naturally occurring eicosanoids (prostaglandins, thromboxanes, rucotrienes) which are not stored in cells but are biosynthesized by the demand of arachidonic acid, a 20-carbon fatty acid derived from the degradation of cell-membrane phospholipids. In normal environments, eicosanoids are produced at low levels and function as important mediators of a wide variety of cellular functions in different types of cells. However, prostaglandins also play an important role in pathophysiology. In particular, inflammation occurs and is maintained, at least in part, in the overproduction of prostaglandins in damaged cells. The central role that prostaglandins play in inflammation is evident in the fact that aspirin-like non-steroidal anti-inflammatory drugs (NSAIDs), which are highly effective in the treatment of many pathological inflammatory conditions, act by inhibiting prostaglandin synthesis. Unfortunately, the usefulness of these drugs is limited by the side effects (gastrointestinal bleeding, gastric ulcers, kidney failure, etc.) due to the unwanted reduction of prostaglandins in normal cells that lack self-secreting and lateral secretory functions necessary to maintain normal physiology. The development of new drugs that more specifically achieve prostaglandin reduction in inflammatory cells without altering prostaglandin production in other cells is a goal for future pain and inflammation treatment.

The cyclooxygenase reaction is the first step in the prostaglandin synthesis pathway; An enzyme with prostaglandin G / H synthesis activity (PGHS) converts arachidonic acid to endoperoxide PGG ₂ , which is then degraded to PGH ₂ (two reactions are performed by a single enzyme). The PGH ₂ is then metabolized by one or a plurality of prostaglandin synthetases (PGE ₂ synthase, PGD ₂ synthase, etc.) and the final “2-series” of prostaglandins, PGE ₂ , PGD ₂ , PGF ₂ , PGI ₂ And other prostaglandins are made, including thromboxane (TAX ₂ ). The first step (PGHS) is the rate-limiting step of prostaglandin synthesis. Thus, PGHS-mediated responses are major targets of anti-inflammatory action; This action limits the production of prostaglandins (a target of therapy) in the inhibition and inflammation of PGHS activity that causes NSAIDs (aspirin, acetophene, ibuprofen, naproxen, indomethacin) activity and prostaglandins in non-inflammatory cells. Is to reduce normal production (which causes unwanted side effects).

In addition to abnormal changes associated with inflammation, various other factors are known to affect prostaglandin production under experimental conditions. These include growth factors, cAMP, tumor promoters, src activation and interleukin 1 and 2, all of which increase intracellular PGHS activity. Adrenergic glucocorticoid hormones and related anti-inflammatory synthetic steroids also inhibit prostaglandin synthesis, but their metabolic sites have not been identified.

The primary action of most non-steroidal anti-inflammatory drugs is to inhibit prostaglandin G / H synthase (cyclooxygenase), which acts as the first step in the biosynthesis of prostaglandins.

Cyclooxygenase exists in two allotropes, COX-1 and COX-2. COX-1, an essentially expressed form, has been studied extensively and has been reported to be involved in the maintenance of prostaglandin mediated physiological function. In contrast, the induced form, COX-2, is present in extremely small amounts under normal conditions but is significantly induced under inflammatory environments in vivo. Clearly, COX-1 and COX-2 perform different physiological and pathological functions.

The most frequently used NSAID is a non-selective cyclooxygenase inhibitor that indiscriminately inhibits both allotropes. After the discovery that the enzyme has two different allotropes, efforts are underway to find selective COX-2 inhibitors. Recently, although COX-2 specific inhibitors have been developed, they are thought to have side effects. There is a need for a safe and effective way to treat inflammation. The present invention fulfills this need.

The present invention provides natural substances that preferentially inhibit COX-2 activity, alleviate COX-2 mediated inflammation and replace NSAIDs. The present invention shows that certain fruit extracts possess higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits. These observations are used to provide supplements consisting of fruit extracts and pharmaceutically acceptable diluents or excipients with higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits. In other words, the present invention provides extracts obtained from anthocyanin-containing plants, particularly fruits, which selectively inhibit COX-2.

Alternatively, extracts of anthocyanin-containing plants can optionally inhibit COX-1 activity. Accordingly, the present invention contemplates one or more anthocyanin-containing supplements derived from extracts of anthocyanin-containing plants that selectively inhibit COX-1 or COX-2 activity.

More specifically, in one embodiment elderberry and chokeberry extracts have been found to possess high anti-inflammatory activity. This anti-inflammatory activity mediated by anthocyanin compounds (particularly hydrolyzed) in fruit extracts inhibits COX-2 more than COX-1. Methods and compositions utilizing these observations are described in detail below. However, extracts of this embodiment are selected from anthocyanin-containing plants and inhibit COX activity, preferably COX-2 activity. When such extracts are taken orally in mammals, the anthocyanins present therein are hydrolyzed in vivo to the corresponding anthocyanidins, which provides COX inhibition. In one embodiment, certain fruits have been found to have anti-inflammatory activity that overrides COX-2 over COX-1. In a suitable embodiment of the invention, it is preferred to obtain fruit extracts having anti-inflammatory activity in a COX-2 / COX-1 ratio of 2: 1 to 25: 1. In one embodiment, the cherries extract was found to have a COX-2 / COX-1 ratio of 4.6: 1, chokeberry 7.5: 1, and elderberry 10.1: 1. Indeed, this anti-inflammatory activity is greater than that of Celebrex ^™ , a widely recognized inhibitor of COX-2 synthesis.

Thus, extracts of chokeberry, elderberry, bilberry, tart cherry, or mixtures thereof are believed to possess excellent anti-inflammatory properties. In one embodiment, a dietary supplement containing one of these fruit extracts is contemplated. Alternatively, a dietary supplement containing two or more of these extracts is contemplated. Celebrex ^™ , a pharmaceutical anti-inflammatory drug believed to have specific COX-2 inhibitory activity, was found to possess a COX-2 / COX-1 inhibitory activity of 7.0.

In a suitable embodiment, the nutritional dietary supplement contains anthocyanin-containing 0.1% to 99%, preferably 5% to 95%, more preferably 10% to 90%, most preferably 30% to 90%. Contains extracts. The content of the anthocyanin-containing extract may be provided with any anthocyanin-containing fruit, the aforementioned extract, and any other anthocyanin-containing plant or extract.

In this regard, a single dosage form (ie a single tablet of liquid or solid, capsule) contains from 1 mg to 500 mg, preferably from 5 mg to 100 mg, more preferably from 20 mg to 70 mg of total anthocyanin. Tablets of the preferred formulation contain 50 mg total anthocyanin. "Total anthocyanin" herein means the total amount of anthocyanins present in a single dosage form.

The dietary supplement of the present invention is in a single dosage form, 0.1-99%, preferably 5% -95%, more preferably 10% -90%, most preferably 30% -90% anti- Provides inflammatory active ingredient content. Anti-inflammatory active ingredients can be obtained from anthocyanin-containing plants, extracts or plants or extracts that provide anti-inflammatory activity (eg ginseng, boswellia).

A. Anthocyanins, Natural Alternatives to NSAIDs

There is an increasing demand for dietary supplements containing superior phytochemicals found naturally in plants. These naturally occurring phytochemicals can be divided into several groups. One more important crowd is the flavonoids, which can be subdivided into several groups again. An important group in the flavonoid crowd is anthocyanins. Anthocyanins are red, green and neutral colors such as cherries (danyang, tart), acerola cherry, blue cherries, bilberry, blackberry, canned, chokeberry, strawberry, cranberry, boyzenberry, grape, raspberry and elderberry. Are most abundantly present in flowers and fruits. Anthocyanins have been found to be useful as antioxidants. The present invention provides the use of anthocyanins for providing anti-inflammatory activity in supplements.

According to the invention, anthocyanins are obtained from anthocyanin-containing plants. Methods for determining whether a plant contains anthocyanins are known in the art and are not mentioned herein. Suitable anthocyanin-containing plants include dandelions, tart cherries, acerola cherries, flops, bilberries, blackberries, cannes, chokeberries, blueberries, strawberries, cranberries, voyagerberries, grapes, raspberries, elderberries, these Fruits selected from a mixture of these are included.

Anthocyanins that are effective in providing anti-inflammatory activity include cyanidin-3-glucoside; Cyanidin-3-glucosilutinoside; Cyanidin-3-gentioside; Cyanidin-3-Lutinoside, Cyanidin-3-Samboonigreen, Cyanidin-3-Sam-5-Glucoside, Cyanidin-3-Galactoside, Peonidin-3-Lutinoside, Phe Onidine-3-glucoside, peonidine-3-galactosid, peonidine, cyanidin, cyanidin-3 soposide, pelargonidine, delphinidine, delphinidin-3-glucoside, delphinidin- 3-galactoside, phetunidine, petrounidine-3-glucoside, petrounidine-3-galactosid, malvidin, malvidin-3-arabinoside, malvidin-3-glucoside, malvidin 3-galactoside, camphorol, hesperidin, genthiodelpin, platinum, cinerarin and the like, but are not limited to these.

The chemical properties of these materials are based on 2-phenylbenzopyryllium (flavilium) having the formula:

When the 2,3,4,5,7,3 'or 5' position in this basic formula is substituted with a hydroxy or methoxy group, the resulting compound is known to be anthocyanidin, which is insoluble and photosensitive. It is rarely found in plants because it is unstable and rapidly destroyed by alkali. However, anthocyanins, glycosides of these compounds, are more stable and are found as natural substances in the leaves, flowers, and fruits of plants. Thus, anthocyanins are hydrolyzed to produce anthocyanidins (sugar removing type) and sugars.

The total number of anthocyanins found in nature is quite large because a large number of single, double, triple-saccharides are glycosylated at 3, 5 or 7 positions, and sugars are acetylated at 3 positions (using p-coumaric acid). Because). Thus, certain fruits include cyanidins, delphinidins, petunidines, pelargonidines, malvidin 3,5-diglucosides, 3-mono-glucosides, 3- (6-Op-coumaryl-glucose And 20 or more anthocyanins, including seed) -5-glucoside, 3- (6-O-coumaryl-glucoside). The color of anthocyanins is determined by their molecular structure and the biochemical properties of the medium in which they are present.

Extracts according to the present invention are peonydin, cyanidin, pelargonidine, delphinidin, phetonidine, malvidin, camphorol, hesperidin, genthiodelpin, platyconin, cinerarine, glucoside derivatives thereof, these At least one anthocyanin selected from mixtures of. In suitable embodiments, the anthocyanins are selected from cyanidins, peonidines, malbidines, petunidines, delphinidins, glycoside derivatives thereof, mixtures thereof.

Hydrolyzed anthocyanins, ie anthocyanidins, have been found to provide higher COX inhibitory activity compared to anthocyanins and their glycoside derivatives. As a result, it is believed that the present invention provides an advantage over currently available COX inhibitors such as NSAIDs. Anthocyanins are thought to provide very little COX inhibition, especially COX-1 inhibition. Therefore, intake of supplements containing anthocyanins can reduce side effects because there is little COX-1 inhibition in the gastrointestinal tract (“GI”).

B. How to Extract Anthocyanins

Various methods of extracting anthocyanins are known to those skilled in the art. Some of these methods are described, for example, in US Pat. 5,817,354; U.S. Patent No. 5,200,186; U.S. Patent No. 5,912,363; U.S. Patent No. 4,211,577; U.S. Patent No. 4,302,200.

U. S. Patent No. 5,817, 354 describes a method for extracting flavonoids from citrus products that cause bitter taste. This process involves contacting a fluid containing at least one bitter flavonoid with a polystyrene divinylbenzene resin to bind the flavonoid to the resin. Generally, centrifugation or ultrafiltration steps are used prior to contacting the polystyrene divinylbenzene resin. The flavonoids can then be recovered by eluting from the resin. This patent does not describe a method for eluting flavonoids from resins, but Chandra et al. (J. Agric. Food Chem., 1062-64, VOL. 41, No. 7 (1993)) describe the use of ethanol to elute anthocyanins. Describe the use.

U.S. Patent No. 5,912,363 describes a method for extracting proanthocyanidins from plant materials. The method optionally includes heating the aqueous mixture of solid plant material under increased pressure and reduced oxygen, followed by separation, filtration, absorption and eluting the absorbed proanthocyanidins with a polar solvent. The method also includes the reconstitution and recycling of the polar solvent in the elution step, reducing solvent consumption and resulting in a more economical process.

U.S. Patent No. 4,211,577 describes a method for extracting plant anthocyanin dyes, which process impurities to obtain individual anthocyanin monomer molecules in the solution, and pass the solution through the ultrafiltration membrane for further purification, soluble and / or blurred macromolecules, e.g. For example, it comprises a step of retaining upstream colloidal impurities that produce a hazy precipitate, and flowing an anthocyanin monomer downstream in liquid or powder to make a stable dye precipitate that can be used as a dye additive. In this way, the fruit solids are treated with sulfur dioxide solutions to ionize, decolorize and secure monomeric pigment molecules (change from anthocyanins to chromone 2- and 4-sulfonates). During ultrafiltration of the solution, upstream macromolecule components such as pectin, tannin, proteins, and complexes thereof remain, while anthocyanins flow downstream. Selective removal of sulfur dioxide from the ultrafiltered solution regenerates the original anthocyanin from the chromen sulfonate. Anthocyanins can then be evaporated to concentrate into a highly concentrated liquid, in which unstable pigments having acyl groups in the molecule can be selectively removed from the liquid by controlled precipitation at reduced temperatures.

U.S. Patent 4,302,200 describes a process for extracting anthocyanin from a natural product by contacting an anthocyanin containing natural product with a sulfite ion-containing aqueous solution at a temperature of 85 ° C. for a period of about 30 minutes, wherein the sulfite in the aqueous solution containing the natural product is The ion content is adjusted to at least 10,000 ppm in terms of SO ₂ .

US Pat. No. 3,963,700 describes a method for recovering anthocyanins from plant material, such as grape husks, by controlled precipitation of excess tartaric acid, such as tartaric acid-alkanol extraction and then potassium hydrogen tartrate. The patent also describes the use of these anthocyanins in the preparation of artificial grape drinks stained with anthocyanin extracts.

Although the method disclosed in this patent is effective in making anthocyanins with anti-inflammatory properties as disclosed herein, the inventors of the present application have developed other methods of extracting anthocyanins from natural sources.

The method relates to the process of concentrating flavonoids from plants without the use of unnecessary chemicals. The process includes passing a solution containing one or more flavonoids through an ultrafiltration membrane to provide a supernatant and a concentrate. The supernatant is then passed through a reverse osmosis membrane to provide a concentrate and a permeate, followed by recovery of the reverse osmosis concentrate.

The molecular weight cutoff of the ultrafiltration membrane is preferably 100,000 to 1,000,000. The molecular weight cutoff of the reverse osmosis membrane is from 1,000 to 10,000.

The recovered concentrate retains the anti-inflammatory COX-2 inhibitory properties identified herein. In a suitable embodiment, the concentrate is dried and mixed with one or more excipients to provide a dietary supplement.

In figure 1 a flowchart of an embodiment of a process according to the invention is shown. According to this embodiment, the plant source 1, in particular the fruit containing the flavonoids, preferably the fruit containing the anthocyanin compound, is processed by the extraction method 10 to obtain an extract or extract 2. For example, the plant may obtain lumps and extracts by an extraction process or pressurization process such as a screw or a back press. Alternatively, the plant material may be ground, crushed or processed to increase the surface area of the plant and to facilitate the extraction and separation of the required flavonoid compounds from the bulk.

To assist in this separation and to achieve a more efficient recovery of the required anthocyanin compound, the plant and extractant (3) are contacted to obtain an extract (extract) rich in flavonoids (especially anthocyanin compounds) and the mass residue (4) It is preferable to form. The extraction step can be carried out with conventional extraction devices of countercurrent, batch or multi-batch extraction.

In addition, the mass can be extracted to increase the recovery of the required anthocyanin compound. When carrying out this extraction step, the extract of this step is preferably mixed with the extract and / or extract of the previous step.

The extract can be separated from the mass in a known manner using a mass separation device such as centrifugation, screen, press or filter. Prior to ultrafiltration, the mass is preferably separated from the liquid by any known mass separator. For example, centrifugation, filters, screens, presses and the like are used.

The ultrafiltration process 20 is then used to remove suspended particles and colloidal high molecular weight components having a molecular weight of at least 200,000 daltons. The ultrafiltration membrane may be tubular, capillary, threaded, hollow fiber or other suitable form. The membrane consists of polysulfone, polyacrylonitrile, polyethersulfone, PVDF or other suitable material. Preferably, ultrafiltration is carried out in a cross flow. The molecular weight cutoff of the membrane is 20,000 to 300,000 daltons, preferably 200,000 daltons. In the absence of filtration prior to ultrafiltration, it is preferred to use higher molecular weight cutoff membranes in order to achieve acceptable filtration rates. Therefore, the scheme of integrating the microfiltration step prior to the ultrafiltration step.

For example, a microfilter may be used to remove suspended particles having a size in the range of 0.01 to 1 μm.

Ultrafiltration may be carried out at a pressure of 5 to 25 bar and a temperature of 20 ℃ to 80 ℃. This step primarily removes lipids, proteins and similar colloids, cell fragments, starches and the like, so that subsequent RO steps can be carried out without fouling the membrane, which lowers the filtration rate.

The ultrafiltration step results in a permeate (5) rich in anthocyanin compounds and a concentrate (7) containing unwanted compounds. In order to increase the final recovery of the flavonoids and the required anthocyanin compounds, a difiltrate 6 can be provided in the ultrafiltration membrane.

The ultrafiltration permeate (5) is treated with reverse osmosis (30) to provide a concentrate (8) rich in flavonoids, including anthocyanin compounds, and a permeate (10) substantially free of flavonoids, including anthocyanin compounds. In order to increase the final recovery of the flavonoids and the required anthocyanin compounds, the ultrafiltration membrane may be provided with difiltrate (9). Membranes that can be used in the RO of the present invention are polyethersulfones, polysulfones, cellulose acetates or polyamide films.

Reverse osmosis can be carried out at a pressure of 30 to 70 bar and a temperature of 30 ℃ to 80 ℃, preferably the temperature is maintained in the range of 30 ℃ to 45 ℃. In general, the reverse osmosis membrane has a molecular weight cutoff in the range of 1,000 to 10,000, preferably 2,000 to provide a concentrate.

The concentrate contains a higher concentration of the required anthocyanin compounds than is found in the first plant material. The concentrate may be left in solution, concentrated to dryness 40 to remove some of the moisture, or completely dried to make powder 11.

If a more concentrated solution is needed, some of the water can be removed by conventional methods, including the use of reverse osmosis membranes with a 90% NaCl retention.

Although spray drying is an appropriate drying method in this drying step, other drying methods may also be used, such as flash drying, freeze drying, fluid bed drying, ring drying, micron drying, tray drying, vacuum drying, high frequency drying or microwave drying. have.

Prior to drying, it is preferred to add one or more flow control agents, such as maltodextrin (eg, M100), magnesium hydroxide or other known flow control agents or carriers. In general, it is preferred to add the flow control agent in an amount of 20 to 60% per weight of solids in the concentrate.

When using spray drying, the total solids content of the concentrate should be at least 1% based on the total slurry weight, with a total solids content in the range of at least 20% to 35%. Higher levels of solids are preferred because of the reduced content of moisture that must be removed during the drying step. As a result, the solids content of the concentrate should be as high as possible while still being capable of effective treatment conditions. The upper limit of solids content in the concentrate is generally determined by the operating limits of the membrane used in the reverse osmosis / nanofiltration step, as well as the drying apparatus used.

The temperature of the concentrate is not important. In general, room temperature of 10-25 ° C. is preferred. High slurry temperatures may also be used, which are suitable for certain types of drying equipment.

Conventional spray drying apparatuses can be used and the operating procedures familiar to those skilled in the spray drying art can be applied to the spray-drying of the above processes. Dry gas effluent temperature is generally used to control the residual moisture level of the resulting powder. In the spray drying process, the dry gas outlet temperature is 40-100 ° C. Generally, in order to minimize the possibility of decomposition of the required anthocyanin compound, the outflow temperature is kept below 80 ° C. As will be appreciated, the corresponding dry gas inlet temperature is generally from 90 ° C. to 200 ° C., preferably less than 150 ° C.

The product recovered during the drying process is a fine, granular powder-free, particulate solid that is suitable for use as a dietary supplement.

The reverse osmosis permeate can be further processed with a concentrator 50 to provide a concentrate 12 that can be used to make fruit drinks.

However, as will be appreciated, any method for supplying fruit extracts with higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits other than the above methods may be utilized in the present invention.

While any of the foregoing methods are suitable for obtaining the required anthocyanins, commercially available extracts may be used which meet some or all of the requirements of the products according to the invention. For example, Artemis International (Fort Wayne, IN) provides extract concentrates and powders containing anthocyanins and other flavonoids. When using a commercial product, the anthocyanin content in the extract is preferably at least 10% per weight of the extract.

C. Identification of Anthocyanins and Novel Anti-Inflammatory Compounds

The present disclosure describes a method for extracting anti-inflammatory actives from fruit extracts, wherein the extracts possess higher anti-inflammatory activity than natural fruits. Based on these results, it is possible to obtain tablet compositions comprising novel compounds that contribute such anti-inflammatory activity to fruit extracts. This section provides general tips for the purification and identification of these compounds.

In general, fruit extracts are prepared as described above. These fruit extracts consist of a mixture of flavonoid compounds, some of which have selective COX-2 activity, some of which have selective COX-1 activity, and some of which have extensive cyclooxygenase inhibitory activity. Others retain no appreciable cyclooxygenase inhibitory activity.

After demonstrating that a particular fruit extract has anti-inflammatory activity, utilizing the assays described below to determine COX activity or any equivalent assay known to those skilled in the art, it is possible to isolate the individual components of the fruit extract. . Separation techniques are known to those skilled in the art. By way of example, those skilled in the art utilize chromatography such as thin layer chromatography, gas chromatography, high performance liquid chromatography, paper chromatography, affinity chromatography, ion exchange chromatography, supercritical fluid chromatography and the like to separate individual flavonoid components. Can be isolated (Freifelder, Physical Biochemistry and Co., New York, NY, 1982).

Partition chromatography is based on the theory that the solvent itself is partitioned into two phases when the two phases are in contact with each other and one or two phases consist of a solvent. Generally, partition chromatography uses columns packed with adsorbents and solvents. The solution containing the solvent is layered at the top of the column. The solvent then passes through the column continuously, which allows for the movement of the solvent through the column material. The solvent can then be recovered based on the speed of movement. The two most common types of distribution chromatography are paper chromatography and thin layer chromatography (TLC); All of these are absorption chromatography. In both cases, the matrix contains the trapped liquid. Another example of partition chromatography is gas-liquid and gel chromatography.

Paper chromatography is a variation of distribution chromatography, which is carried out in cellulose columns in the form of paper sheets. Cellulose contains a large amount of trapped moisture even after it is considerably dried. Distribution occurs between the captured water and the advanced solvent. The main solvent used is water. Generally, the small amount of solution mixture that is separated is placed on top of the paper and dried. The capillary attracts the solvent through the paper, decomposes the sample, and moves the component in the flow direction. Paper chromatograms can be developed for raising or lowering solvent flow. Two-dimensional separation is possible by varying the axis of movement by 90 ° after the first run.

Thin layer chromatography (TLC) is very routinely used to separate lipids and is therefore considered a suitable embodiment of the present invention. TLC can use any material that has the advantages of paper chromatography but can be finely divided and formed into a homogeneous layer. In TLC, the stationary phase is an adsorbent layer uniformly spread over the surface of a glass or plastic plate. These plates are generally made up of adsorbent slurry, which is placed on the surface of the gel after the wells are made by placing the tape at a selected height along the perimeter of the plate. The tape is removed after the adsorbent is dried and the plates are treated the same as in the paper chromatography paper. A sample is added and the plate is in contact with the adsorbent. By the time the adsorbent reaches the end of the plate, the plate is removed and dried. The spot can then be identified by fluorescence, immunological differentiation, radioactivity calculation, or color change after application of various reagents to the surface.

TLC of anthocyanins derived from bilberry extract is presented by Petri et al. (1994). This reference also presents additional spectroscopy and chromatographic techniques that can be used to identify and characterize anthocyanin reagents.

In gas liquid chromatography (GLC), the mobile phase is a gas and the stationary phase is a liquid absorbed on the inner surface of a tube or column or on a solid support. Generally, liquids are added as solids dissolved in volatile solvents such as ethers. Any sample that can be volatilized is introduced into the liquid together with an inert gas such as helium, argon or nitrogen and then heated. This gaseous mixture passes through the tube. The volatilized compound is continuously redistributed between the gaseous mobile phase and the liquid stationary phase depending on the partition coefficient.

The advantage of GLC lies in the separation of small molecules. Excellent sensitivity and speed, close to 1000 times standard liquid chromatography. By utilizing a non-destructive detector, GLC can be used preliminarily to purify the material in grams. Gel chromatography or molecular sieve chromatography is a specific form of distribution chromatography based on molecular size. The background theory of gel chromatography is that when molecules pass through pores, small molecules and larger molecules are separated by size in a column made of tiny particles of inert material with tiny pores. If the material that makes up the particles does not absorb the molecules, the only factor that determines the flow rate is size. Thus, when the form is relatively constant, molecules are eluted from the column in size order. Gel chromatography is most effective for separating molecules of different sizes because the separation is independent of other factors such as pH, ionic power, temperature and the like. In addition, there is substantially no absorption, less diffusion, and the elution volume is directly related to the molecular weight.

Gel materials for gel chromatography are arbitrary three-dimensional networks. Gels are generally composed of non-polar cross-linked polymers that are inert and do not bind or react with the analyte. The space in the gel is filled with liquid, which occupies most of the gel volume. Generally, the gel is dextran, agarose, polyacrylamide. These are used as aqueous solutions.

High performance liquid chromatography (HPLC) is characterized by very fast separations at full resolution. This is achieved by maintaining an appropriate flow rate with very fine particles and high pressure. Separation can be accomplished within minutes or at least 1 hour. In addition, very small amounts of sample are required because the particles are very small and dense so that the empty space is only a fraction of the fill volume. In addition, the concentration of the sample does not need to be very large because the band is so close that the sample is hardly diluted. HPLC with a photodiode array detection system has been used to study flavonoids such as rutin and other quercetin glycosides (pololidzin), including any anthocyanins (Paganga and Rice-Evans, FEBS Lett. 401 ( 1): 78-82, 1997). Reversed-HPLC gradients have been described in the isolation and quantitative evaluation of 12 anthocyanins (Petri et al., Acta Pharm. Hung. 64 (4) 117-122, 1994). Quercetin compounds can also be identified using HPLC techniques described by Laires et al. (Food Chem. Toxol., 31 (12) 989-994, 1993). This method is applicable to the present invention in characterizing and identifying novel flavonoids.

Affinity chromatography is a chromatography process that depends on the specific affinity between the substance to be separated and the molecules to which it can specifically bind. This is a receptor-ligand interaction. The column material is synthesized by co-bonding one of the binding partners with an insoluble matrix. The column material can then specifically absorb the material from the solution. Elution is caused by changing the bond to no state (by changing pH, ionic force, temperature, etc.).

The matrix should be a material with a wide range of chemical, physical and thermal stability without significantly absorbing the molecule itself. Ligands should be bound in a way that does not affect their binding properties. The ligand must also provide a relatively strong bond. In addition, it should be possible to elute the material without destroying the sample or ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography using antibodies to any detection agent.

The structures of the anthocyanins isolated by the aforementioned techniques are described in Saito et al. (Phytochemistry 41 (6) 1613-1620, 1996 and Phytochemistry 43 (6), 1365-1370, 1996) and Takeda et al. (Phytochemistry, 36 (3) 613-616, 1994) can be confirmed by mass spectra and NMR spectra. Additional NMR techniques are presented by Terahera et al. (BioSci. Biotech. Biochem., 58 (7) 1324-1325, 1994) and Nerdal et al. (Acta Chem. Scand. 46 (9) 872-876, 1992). Johansen et al. (Phytochemistry 30 (12) 4137-4141, 1991) describe various methods including ion-exchange resins for the separation of anthocyanins, droplet-counter chromatography, gel filtration, and subsequent chemical degradation for the characterization of separated anthocyanin compounds, We propose the use of techniques such as chromatography, spectroscopy, in particular homo- and heteronuclear two-dimensional NMR techniques. It will be apparent to those skilled in the art that any of the techniques described above can be used to isolate and purify the fruit extracts identified herein and to identify individual compounds that drive anti-inflammatory activity.

D. Assays for Testing Anti-inflammatory Activity

In the present invention, the anthocyanin-containing fruit extract retains anti-inflammatory activity. More specifically, these extracts have been shown to preferentially inhibit COX-2 activity over COX-1 activity. Such extracts provide an excellent alternative to traditional NSAIDs in that they are selective for COX-2. These inhibitory extracts are also superior to the recently developed COX-2 specific “super aspirin” because these extracts are natural extracts that are independent of increased predisposition to heart attack, seizures and other cardiovascular side effects. .

The concentration of any inhibitor that inhibits the enzyme by 50% of maximum activity is referred to as IC ₅₀ or I ₅₀ . The smaller the IC ₅₀ , the stronger the potency for the enzyme inhibition of the corresponding inhibitor. As a result, smaller amounts of inhibitors are needed for anti-inflammatory and pain-relieving supplements when the compound is absorbed, metabolized, and delivered to the site of dysfunction or disease.

Some substances, compounds or plant concentrates can selectively inhibit COX-1 or COX-2 enzymes. This may be referred to as the selectivity of the material. Selectivity can be expressed numerically in the ratio of I ₅₀ (COX-1) / I ₅₀ (COX-2). If the ratio is 1, the inhibitor is not selective for any of the isoenzymes; Inhibitors equally inhibit COX-1 and COX-2 enzymes. If the ratio is less than 1, the inhibitor is more selective for COX-1 inhibition. If the ratio is one or more, the inhibitor is more selective for COX-2 inhibition. For long-term anti-inflammatory and pain-relieving drugs or supplements, selectivity plays an important role in side effects. Side effects are gastrointestinal (GI) bleeding caused by inhibition of the COX-1 enzyme in the gastrointestinal tract where prostaglandins normally function in the GI tract.

Selectivity is an important issue in non-steroidal anti-inflammatory drugs (NSAIDs) because the NSAIDs inhibit COX-1 enzymes that are essentially expressed in the gastrointestinal tract, and besides the action of absorption and delivery to inflammation and pain sites, Because there is only one active form that can cause bleeding. Although not yet demonstrated, natural products, such as anthocyanin-containing plant extracts, may have the advantage of being non-active and active, thus not causing adverse effects on the GI tract. Different mechanisms of absorption, metabolism, and delivery may exist. Non-active forms (glycosidic forms with sugar) can be absorbed or passed through the gastrointestinal tract without inhibiting the COX-1 enzyme. As a result, the content of prostaglandins produced by the COX-1 enzyme in the gastrointestinal tract is normal or above enough to maintain the GI tract. After absorption, the sugar moiety is cleaved and the active form (glucose, anthocyanidin) is delivered to the site, where COX-2 enzymes are induced at high levels while COX-1 activity is inhibited (lower levels). . Inhibition of both enzymes at this site is very effective in anti-inflammatory and pain-relieving.

In one aspect of the invention, it is necessary to determine whether any fruit extract or component thereof has anti-inflammatory activity. Such activity can be measured by anti-inflammatory assays known to those skilled in the art. Using prostaglandin endoperoxide synthetase-1 and -2 isoenzyme, one can easily determine whether any extract possesses the appropriate activity. In these assays, the ability of these enzymes to convert arachidonic acid to prostaglandins is measured. Alternatively, the immunoassay described below can be used.

Reagents such as arachidonic acid and microsomal suspensions such as COX-1 and COX-2 enzymes are readily available to those skilled in the art (eg Oxford Biomedical Research, Oxford, MI, USA).

Thus, the COX-2 inhibitory activity of certain extracts can be obtained by (a) obtaining a COX-2 microsome composition; (b) mixing the candidate extract with the COX-2 microsome composition; (c) measuring the ability of the candidate extract to inhibit COX-2 activity.

COX-2 activity can be measured by obtaining a microsome membrane composition of COX-2 (5-10 mg protein / ml in appropriate buffer). COX-2 analysis is performed by monitoring the O ₂ uptake rate at 37 ° C. as described above (DeWitt et al., Am. J. med. 95 (2A) 40S-44S, 1993; Arch, Biochem. Biophys. 306 (1) 96-102; 1993). This assay basically measures the conversion of arachidonic acid to prostaglandin endoperoxide-2. Thus, one unit of cyclooxygenase activity shows oxygenation of 1 nmol arachidonic acid / min (DeWitt et al., 1993 supra). Alternatively, COX-2 activity can be determined by measuring the content of the COX-2 enzyme product using chromatography, such as thin layer chromatography, gas chromatography, high performance liquid chromatography, and the like. Another method of measuring COX-2 activity is to radio-label the substrate and monitor the radio-labeled end-product content of the COX-2 reaction. Regardless of the method used, one of ordinary skill in the art would appreciate cyclooxygenase activity, eg, O ₂ / cyclooxygenase mg / min used; Product mg / cyclooxygenase mg / min; The final measurement can be reported with the μCi radio-labeled arachidonic acid / cyclooxygenase mg / min used.

To determine whether fruit extracts can inhibit COX-2, the COX-2 activity of the microsome composition is measured in the absence of candidate extracts. The candidate extract is then added to the composition and the activity is re-measured in the presence of the candidate extract. Candidate extracts that reduce the content of oxidized arachidonic acid compared to arachidonic acid oxygenation in the absence are candidate extracts with COX-2 inhibitory capacity.

Control experiments can be conducted using known inhibitors of COX activity such as aspirin, ibuprofen, Celebrex ^™ , naproxen and the like. By comparing the results of the fruit extract with the results of COX-2 activity in the presence of these known inhibitors, the relative activity can also be determined.

When oxygen consumption is measured by O ₂ electrode chromatography techniques (quantification of end-products by densitometry or liquid scintillation spectroscopy), a significant reduction in arachidonic acid oxygenation is at least 20% to 40 at COX-2 inhibition levels. %, Preferably at least 50% reduction. Chromatographic assays to measure arachidonic acid metabolites and COX enzyme assays to measure prostaglandin formation are known in the art and can be performed in vitro or in vivo.

Quantitative in vitro testing of fruit extract inhibitory properties is not a requirement of the present invention, since the fruit extracts constituting the functional food of the present invention are generally the same compounds found naturally in whole fruits. Of course, as will be appreciated, the anthocyanin and flavonoid compounds that make up the COX-2 inhibitory component of the fruit extracts disclosed herein can be further modified to produce anti-inflammatory components upon ingestion in vivo.

Similarly, in vivo testing is not a requirement. However, those skilled in the art can utilize animal inflammation models to test the in vivo activity of these compounds. For example, rodents with inflammatory sites can be used to test the anti-inflammatory effects of COX-2 inhibitors identified by assays such as those described above. Such animal models can be utilized, for example, in assays using at least two animals with similar inflammation, one of which is in contact with a candidate anti-inflammatory composition and the other animal is deficient in anti-inflammatory components. Contact with a control or placebo composition comprising all components of the candidate composition. Inflammation reduction in animals in contact with the candidate composition as compared to animals in contact with the control or placebo composition suggests that the candidate composition retains anti-inflammatory activity.

E. Combination of Anthocyanins and Other Anti-inflammatory Drugs

In one aspect, the present invention describes the benefits of ingesting supplements with anti-inflammatory properties, wherein the supplements contain fruit extracts having higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits. As will be appreciated by those skilled in the art, such supplements may be mixed with other anti-inflammatory drugs. Such additional anti-inflammatory drugs aid the fruit extracts of the present invention, which are generally recognized anti-inflammatory drugs or any drug identified by the assays described above.

Regardless of whether the additional anti-inflammatory drug is a known anti-inflammatory drug or any drug identified herein, the present invention contemplates various combinations. Thus, when the fruit extract is “A” and the other anti-inflammatory drug is “B”, the combination is as follows:

A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B

B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A

B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

Fruit extracts and additional anti-inflammatory drugs inhibit the COX-2 activity of the cells by contacting or exposing the cells in vivo or in vitro. As used herein, “contact” and “exposure” refer to a process of delivering fruit extracts and secondary anti-inflammatory drugs to or in parallel with a target cell. In order to achieve a positive effect, it is contained in an amount effective to inhibit the production of prostaglandins that inhibit COX-2 activity and reduce inflammation and cause inflammation or other effects that reduce the inflammatory response in the individual in which the cells or cells are located. Both drugs can be delivered to the cell.

Anti-inflammatory drugs are known to those of skill in the art, including salicylic acid derivatives (e.g. aspirin), paraminophenol derivatives (e.g. acetamiphene), indole and indene acetic acid (indometacin, sulindac, etodalak) , Heteroaryl acetic acid (tolmethine dichlorophenac and ketorolac), aryl propionic acid derivatives (ibuprofen, naproxen, keoprene, phenoprene, oxaprozin), anthranilic acid (mephenamic acid, meclofenamic acid), enolic acid (pyoxy Drugs such as cam, tenoxycamp, phenylbutazone, oxypentatrazone). Other anti-inflammatory drugs are known to those skilled in the art and do not further describe these drugs.

F. Formulation

The present invention provides a natural supplement made from fruit extracts, wherein the supplement has a higher anti-inflammatory activity than the anti-inflammatory activity found in natural fruits. The present invention provides a fruit extract that can be provided in powder, liquid or solid form. Optional formulations are shown in the examples below, and this section describes the formulations and components of the formulations that may be preferred and readily produced in the present invention.

Fruit extracts are predominantly reconstitutable powder compositions, for example reconstituted with water, milk or other similar liquids to provide drinks, which can be used to provide anti-inflammatory activity to individuals in need of anti-inflammatory activity. Powder compositions and drinks made therefrom are enteric ingredients in pain or inflammation management programs that utilize a number of carefully designed products in various forms, such as shakes, soups, fruit drinks, snack bars, and other solid forms (eg, tablets, gel caps, etc.). In particular, they can be mixed or confused during pain management to provide more attractive and effective support for patients in prolonged protective situations.

In addition to drinks, the fruit extract of the present invention can be used in foodstuffs. Such fruit extracts may be mixed with any other foodstuff, for example oils containing extracts of the present invention may be used as edible oils, fried oils or salad oils, and may be used in any oil-based foods such as margarine, Can be used for mayonnaise or peanut butter. Starch fortified with the compounds of the present invention can be used in food products such as roast food, cereals, pasta, soups. Fruit extracts and oils containing the novel anthocyanins extracted therefrom can be emulsified and used in a variety of water-based foodstuffs, such as drinks, including the mixed drinks described above. Advantageously, such food products can be included in a low fat or low cholesterol diet regimen.

“Nutraceutical” is any functional food that provides additional benefits in addition to nutritional benefits, including sports vegetable drinks and other fortified drinks, as well as nutritional drinks and diet drinks (eg Slimfast ^™ , Boost ^™, etc.). The present invention provides a nutraceutical composition that can be used as an anti-inflammatory drug, as described above, including but not limited to headaches, allergic rashes, inflammatory bowel symptoms, joint pain, chronic fatigue, fibromyalgia. It can be used to alleviate diseases mediated by the action of COX-2.

Functional foods or foodstuffs may contain, in addition to purified fruit extracts, other beneficial ingredients, including but not limited to essential fatty acids, vitamins, minerals. These ingredients are known to those skilled in the art and are not limited to specific formulations and contents, but this section simply refers to ingredients that may form part of the supplements of the present invention. Additional information suggesting the content and production of dietary supplements can be found in US Pat. 5,902,797; U.S. Patent No. 5,834,048; U.S. Patent No. 5,817,350; U.S. Patent No. 5,792,461; U.S. Patent No. 5,707,657; U.S. Patent No. 5,656,312.

Essential fatty acids such as γ-linolenic acid (ω-3) and linoleic acid (ω-6) can be added to the supplement of the present invention. Studies show that the ratio of n-3 to n-6 fatty acids is much more important than the absolute amount of fatty acids in non-human animals (Boudreau, et al., "Lack of Dose Response by Dietary n-3 Fatty Acids at a Constant"). Ratioo of n-3 to n-6 Fatty Acids in Suppressing Eicosanoid Biosynthesis from Arachidonic Acid, "Am. J. Clin. Nutr. 54: 111-117 (1991)). Essential fatty acids are involved in cardiovascular health as well as in support of the immune system. Imbalances of these essential fatty acids can lead to cholesterol metabolism. In addition, immune system function may be impaired and cause inflammation.

Calcium and magnesium are, among other things, involved in bone health. One possible effect of the imbalance between calcium and magnesium is the imbalance of bone minerals, which can adversely affect bone growth and bone replacement (bone destruction and regeneration). Magnesium is as important as calcium in reducing the risk of bone health and osteoporosis in both men and women (Purvis, JR, "Effect of Oral Magnesium Supplementation Factors on Selected Cardiovascular Risk Factors in Non-Insulin-Dependent Diabetics," Archives of Family Medicine 3: 503 -50 (1994)).

  Zinc and copper are involved in cardiovascular health and are given in a 5: 1 zinc to copper ratio. The imbalance between these two minerals can lead to an antagonistic effect of zinc on copper. These effects can interfere with the body's ability to use copper to support cardiovascular health. Too much zinc compared to copper can also interfere with the body's ability to make SOD (superoxide dismutase), an important cardioprotective enzyme. In addition, an adequate zinc to copper ratio is required to achieve a proper balance of HDL (high density lipoprotein) to LDL (low density lipoprotein). In general, zinc intake in American male meals is only 33-75% of the RDA, thus contemplating dietary supplements containing zinc.

Selenium and iodine also have the most effective ratio, which is the 2: 1 selenium: iodine ratio. These minerals affect thyroid function and thus affect metabolism caused by changes in thyroid function. Unbalanced thyroid function leads to unreasonable stress on the body, which leads to disturbed absorption of nutrition from food. This can inhibit growth and development.

Pyridoxine, folate, cobalamin also have a ratio which works most effectively in the prevention of vascular diseases. The optimal ratio of pyridoxine (vitaman B6) to folate to cobalamin (vitamin B12) is 100: 4: 1, respectively. These vitamins affect cardiovascular function with the ability to reduce the potentially toxic amino acid homocysteine. This ratio reflects the imbalanced and inadequate levels of these vitamins consumed by food by individuals at risk for heart disease.

In addition to these, vitamin C, vitamin B1 (thiamine), vitamin E can be provided. Vitamin C demands increase in smokers, and smoking is a major cause of lung cancer. Vitamin B1 plays an important role in energy conversion. Thiamine diphosphate (TDP) is a coenzyme required to convert carbohydrates into energy. American men consume about 45% of their total calories from carbohydrates, so optimizing vitamin B1 in food is desirable.

Along with vitamin B6, vitamin B12 and folic acid supplementation help to regulate blood levels of homocysteine and are therefore an important ingredient in the dietary supplement formulations of the present invention. Vitamin D (calciferol) is essential for bone formation and mineral homeostasis. In the absence of vitamin D, the small intestine cannot absorb an appropriate amount of calcium, regardless of the amount of calcium available. Therefore, vitamin D is prescribed as a nutritional supplement to help build healthy bones.

The role of manganese in driving the metal-containing enzyme manganese-superoxide dismutase (Mn-SOD), along with similar roles in other metal-containing enzyme systems (glutamine synthase, arginase, pyruvate carboxylase) , Clearly identified. Many enzymatic systems other than manganese metal-containing enzymes have also been found to have manganese activation. The manganese-SOD relationship is of particular clinical importance because this type of metal-containing enzyme is the only active form in the cell's mitochondrial membrane, and thus plays a unique role in the protection of mitochondria and the security of the body's oxidative energy production system. Because. It is desirable to include manganese in dietary supplements.

Additional micronutrients that may be included in the supplement include vitamins A, vitamin C, vitamin E, riboflavin, niacin, niacinamide, pantothenic acid, pyridoxine, cobalamin, biotin, inositol, choline bitartrate, bitine, and vitamin K And minerals such as molybdenum, chromium, calcium, and the like.

Stress, exercise and other abnormalities generate free radicals in the body, which can cause damage to body components. In order to offset the free radicals, the present invention may contain the following antioxidants in addition to the vitamins C and E described above: citrus bioflavonoids, mixed carotenoids, green tea extracts, N-acetylcysteine.

In addition to these, spices and additives known to those skilled in the art can be added to the formulations of the present invention to enhance their taste. For example, the formulation may contain ginger, boswellia, fruit spices, colorants, preservatives, and the like.

The functional food of the present invention, which is ingested in a solid form, may further contain a solid carrier such as gelatin or adjuvant. When administered in liquid form, liquid carriers such as water, petroleum, animal or vegetable oils (eg peanut oil, mineral oil, soybean oil or sesame oil) or synthetic oils may be added. The nutraceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants or other additives known to those skilled in the art.

In a suitable embodiment, a dietary or nutritional functional food is provided, which contains from 0.1 to 99%, preferably from 30% to 90% of anthocyanin-containing fruit extract. In this regard, a single dosage form (ie a single tablet of liquid or solid, capsule) contains from 1 mg to 500 mg, preferably from 5 mg to 100 mg, more preferably from 20 mg to 70 mg of total anthocyanin. Tablets of the preferred formulation (single dosage form) contain 50 mg total anthocyanin. As used herein, “total anthocyanin” means the total amount of anthocyanins present in a single dosage form.

Extracts derived from anthocyanin-containing plants include peonidine, cyanidin, pelargonidine, delphinidin, petunidine, malvidin, camphorol, hesperidin, genthiodelpine, platyconin, cinerarin, glucosides thereof Derivatives, mixtures thereof. In suitable embodiments, the anthocyanins are selected from cyanidins, peonidines, malbidines, petunidines, delphinidins, glycoside derivatives thereof, mixtures thereof.

Preferably, the dietary supplement contains a stable anthocyanin, which is hydrolyzed to an anthocyanidin which is a sugar removing type in vivo to provide a COX inhibitory activity.

Suitable dietary supplements contain fruit extracts, wherein the fruit extracts are selected from elderberry, tart cherry, bilberry, and mixtures thereof. More specifically, the fruit extract is an elderberry extract of 2% to 98% per weight of fruit extract, 1% to 49% of tart cherry extract per weight of fruit extract and 1% to 49% content of weight of fruit extract Consists of bilberry extract. Preferably, the fruit extract is 90% to 98% (more preferably 96%) elderberry extract, 1% to 5% (more preferably 2%) tart cherry extract and 1% to 5 Consisting of bilberry extract in a% (more preferably 2%) content.

In suitable dietary supplements, cyanidin accounts for at least 90% of the total anthocyanin weight present in the elderberry extract. Preferably cyanidins comprise 95%, more preferably 96%, by weight of the total anthocyanins present in the elderberry extract. In this regard, cyanidin is present as a mixture of cyanidin-3-glucoside, cyanidin-3-sambunigrin, cyanidin-3,5-diglucoside, cyanidin-3-sambu-5-glucoside do.

Similarly, cyanidin accounts for at least 90% or more of the total anthocyanin weight present in the tart cherry extract. Preferably cyanidins comprise 95%, more preferably 96%, by weight of the total anthocyanins present in the tart cherry extract. In contrast to elderberry, the cyanidins are cyanidin-3-rutinoside-hakose, cyanidin-3-rutinoside-pentose, cyanidin-3-rutinoside, peonidin-3 Present in a mixture of lutinosides.

Finally, the bilberry contains a mixture of malvidin, peonidine, cyanidin, petunidine, delefinidine. Each of these anthocyanins accounts for 95%, more preferably 96%, by weight of the total anthocyanins present in the bilberry extract. More specifically, malvidin is present as malvidin-3-arabinoside, malvidin-3-glucoside, malvidin-3-galactosid. Peonydin is present as peonidine-3-rucoside, peonidine-3-galactoside. Cyanidin, petunidine, delphinidine are present as 3-glucoside and 3-galactoside.

The table below shows the content of each component in certain suitable embodiments.

fruit Anthocyanin Wt% of total anthocyanins Tart Cherry (Balaton) Cyanidin-3-Lutinoside-Hak Source 75% Cyanidin-3-Lutinoside-Pentose 3% Cyanidin-3-Lutinoside 18% Pheonidine-3-Lutinoside 4% synthesis 100% Elderberry Cyanidin-3-glucoside 42% Cyanidin-3-sambunigreen 43% Cyanidin-3.5-diglucoside 2% Cyanidin-3-sambu-5-glucoside 9% unknown 4% synthesis 100% Bilberry Malvidin-3-arabinoside 8% Malvidin-3-Glucoside 7% Malvidin-3-galactoside 3% Pheonidine-3-Lucoside 15% Pheonidine-3-galactoside 2% Cyanidin-3-glucoside 11% Cyanidin-3-galactoside 8% Petunidine-3-glucoside 5% Petunidine-3-galactoside 12% Delphinidin-3-glucoside 12% Delphinidin-3--galactoside 11% unknown 6%

Example

The following examples illustrate suitable embodiments of the present invention. However, as will be appreciated by those skilled in the art, many modifications may be made to achieve similar results without departing from the spirit and scope of the invention as disclosed in the detailed description.

Example 1

100 kg of cherries are pressed with a back press and the extract is recovered. The recovered extract is filtered through an ultrafiltration unit having a 200,000 molecular weight cutoff at a temperature of less than 38 ° C. Run the UF unit so that the concentrate contains less than 0.5% solids per weight.

The permeate of the UF unit is then subjected to reverse osmosis treatment using a membrane having a 4,000 molecular weight cutoff. The reverse osmosis step continues until the concentrate contains less than 1% solids by weight.

The concentrate is collected in a tank and concentrated to at least 20% solids per weight with a vacuum evaporator at a temperature below 52 ° C. to avoid degradation of the concentrated flavonoids.

The concentrate is mixed with maltodextrin and spray dried to the outlet temperature of the dry gas maintained at a temperature below 27 ° C.

The pulp remaining from the back press is recovered, dried and pulverized.

Example 2

A batch consisting of 38.8 kg tart cherries is pressurized in a back press to produce 19.3 kg of extract and 18.6 kg of lumps. The extract, pH 3.3, is pumped to the ultrafiltration membrane at a flow rate of 1770 to 1950 g / min, a pressure of 10 bar and a temperature of 29 ° C. at the start of filtration to 18 ° F. at the end of filtration. The difiltrate flow continues until the dissolved solids in the permeate are 0.2% by weight.

The ultrafiltration membrane is a PVDF polymer membrane of 100,000 molecular weight (Dalton) cutoff grade. Suitable membranes are available under the trade name PP from PCI Membrane Systems.

At the end of the ultrafiltration, a 53.7 kg permeate containing 5% solids per weight and a 3.49 kg concentrate containing 0.3% solids per weight are recovered. The permeate is then subjected to nanofiltration / reverse osmosis at a pressure of 40 bar, a flow rate of initial 1290 g / min to final 1380 g / min, 24 ° C. at the beginning of the process and 41 ° F. at the end of the process. 72.4 kg of difiltrate of water is used.

Polyethersulfone membranes with a 4,000 molecular weight (dalton) cutoff are used. Suitable membranes are available under the trade name ES404 from PCI Membrane Systems.

Immediately after the end of this step, 6.4 kg of concentrate is recovered, which contains 1% solids by weight. Recover 117 kg of permeate containing 2% solids by weight.

To make a powder, the concentrate is mixed with 79 g of maltodextrin M100 and the resulting product is introduced into a dry gas having an inlet temperature of approximately 140 ° C. and an outlet temperature of approximately 90 ° C. to obtain approximately 105 g of powder.

Example 3

Comparison of COX-1 and COX-2 Inhibitory Activities of Fruit Extracts

This example describes an enzyme assay for monitoring COX inhibitory activity of fruit extracts using an oxygen monitoring system. In this analysis, dissolved oxygen concentration changes are continuously monitored by the oxygen electrode in the Dissolved Oxygen Measuring System (Instech, Plymouth Meeting, PA). Results are recorded on a linear recorder (Fisher Scientific, Pittsburgh, PA).

Every day, fresh calcium chloride solution (15 g / 100 mL distilled water) is made and the electrode is installed according to the manufacturer's instructions. The chamber is kept at 37 ° C.

Using the prostaglandin assay kit, the assay is carried out in a manner similar to that found in the COX-1 assay. In brief, 50 μl phenol is added to 20 mL 100 mM Tris buffer and heated to 37 ° C. for 1 minute (work buffer). Add 0.9 ml working buffer to a tube of hematin. 50 μl of 0.1NaOH is added to the arachidonic acid vial and vortexed. 0.43 mL water is added and the solution is mixed once more. Extract samples are weighed and dissolved in working buffer at a final concentration of 0.1 g / ml. Buffers, samples or diluted samples are used for direct enzymatic analysis.

Enzyme assays are performed according to the manufacturer's instructions, which are known to those skilled in the art. In short, 600 μl of working buffer is drawn from the outlet outlet to the chamber with the injection valve closed, with the main outlet connected to the syringe in front. The stir bar is set at a speed of 3 k / min. At 1 minute intervals, 5 μl enzyme, 15 μl hematine solution, 6 μl buffer (or sample or diluted sample) and 8 μl arachidonic acid solution are injected.

Oxygen concentration changes are converted to mV and recorded. When arachidonic acid is added, the consumption of oxygen or a decrease in oxygen concentration becomes apparent.

As indicated in the table below, there is a clear trend in selectivity between experimental samples.

Table 1. COX-1 and COX-2 IC50 Values and Specific Activities

Sample COX-1 IC50 COX-2 IC50 Specific activity ^* aspirin 1 / 20,000 1 / 10,000 0.5 Montmorency Tart Cherry Primer 1 / 20,000 NA NA Balaton Tarts Cherry Prime 1 / 5,000 1 / 23,000 4.6 Milne Tart Cherry 1 / 2,500 NA NA Artemis Blueberries >> 1 / 1,000 1 / 10,000 NA Artemis Chokeberry 1 / 2,000 1 / 15,000 7.5 Artemis elderberry 1 / 3,000 1 / 23,000 10.1 Nutrilite Acerola Cherry 1 / 20,000 NA NA Celebrex 1 / 4,000 1 / 28,000 7 Quercetin Standard 1 / 3,500 1 / 22,000 6.3 Artemis bilberry 1 / 12,000 1 / 15,000 1.25

* The higher this value, the more selective the extract is for COX-2 than for COX-1.

In addition, COX-2 efficacy is monitored compared to Celebrex, a known COX-2 specific inhibitor.

Table 2. COX-2 Efficacy Relative to Celebrex

The method described above is used to measure COX-2 inhibition of various inhibitor candidates as compared to Celebrex, a known COX-2 inhibitor.

Sample COX-2 Efficacy compared to Celebrex Artemis Chokeberry 1 / 15,000 54% Artemis elderberry 1 / 23,000 82% Artemis Blueberries 1 / 10,000 36% Balaton Tarts Cherry Prime 1 / 23,000 82% Quercetin Standard 1 / 22,000 78% Celebrex 1 / 28,000 100%

The data clearly shows the selectivity of Artemis blueberry samples for COX-2.

Example 4

Appropriate methods for determining whether an inhibitor candidate inhibits COX-1 or COX-2 are described below. In general, six different concentrations are used for each inhibitor candidate for COX-1 and COX-2 enzyme reactions. The PG-f2α content from a standard or enzyme reactant is used to generate a standard curve (optical density vs. concentration), which standard curve is used to calculate (regress) the PG-f2α content in each enzyme reactant for the sample. Then, the PG-f2α content obtained from the aforementioned inhibitor candidates at six different reaction concentrations is used to prepare the sample curve. Finally, the concentration of inhibitor candidate, or IC ₅₀ , that inhibits the enzyme by 50% of its maximum activity (in the absence of inhibitors) is obtained from the same curve. To keep the results constant, one inhibitor candidate or drug is taken as a positive control for each set of experiments.

COX-1 and COX-2 enzymes are found in Dr. Kentucky University. Obtained from Daniel Tai. They are prepared as follows: The COX-1 enzyme is extracted from human platelet concentrate obtained from the Central Kentucky Blood Center. Platelet suspensions are centrifuged at 1,000 × g for 10 minutes. The pellet is washed with an equal volume of phosphate-buffered saline and the suspension is centrifuged once more. Platelets are suspended in 5-fold volume of 50 mM Tris-HCl buffer (pH 7.5) and sonicated at 4 ° C. for 3 × 20 seconds. The suspension is centrifuged at 5,000 × g for 10 minutes. The supernatant is further centrifuged at 100,000 × g for 60 minutes. Pellets (microsomes) are suspended in 5 ml of 50 mM Tris-HCl buffer (pH 7.5) and stored in 200 μl aliquots at −80 ° C. This aliquot is used as a source of COX-1 enzyme.

Recombinant human COX-2 enzyme is obtained from insect cells (Sf9) infected with recombinant baculovirus carrying COX-2 cDNA. In brief, Sf9 cells (1 × 10 ⁷ ) are plated in 20 ml complete TNF-FH medium in a 75 cm 2 tissue culture flask. Cells attach for 1 hour. Remove the medium; Add 4 ml Grace medium containing the recombinant virus with a multiplicity of approximately 10. Cells are grown continuously for 72 hours. Cells are harvested by centrifugation at 500 × g for 10 minutes. The cells are then suspended in 1 ml of 50 mM Tris-HCl buffer (pH 7.5) and sonicated at 0 ° C. for 3 × 10 seconds. The homogenate is spun at 5,000 × g for 5 seconds to remove cell debris. The supernatant is then stored in 200 μl aliquots at −80 ° C. This aliquot is used as a source of COX-2 enzyme.

Prepare the following buffers:

1.Coating Buffer: 0.1M NaHCO ₃ / Na ₂ CO ₃ , pH 9.5

2. Enzyme Immunoassay (“EIA”) buffer: 0.1 M KH ₂ PO ₄ / K ₂ HPO ₄ , pH 7.5 containing 0.9% NaCl and 0.1% bovine serum albumin (ELISA or RIA grade)

3. Antibody Stabilization Buffer: EIA Buffer + Sucrose (5 g / 100 mL)

4. Wash buffer: 0.01M KH ₂ PO ₄ / K ₂ HPO _{4 with} 0,05% Tween 20, pH 7.5

5. Enzyme and Sample Dilution Buffers: 50 mM Tris-HCl, pH 7.5

6. PBS (phosphate buffered saline): 10 mM KH ₂ PO ₄ / K ₂ HPO ₄ , pH 7.5 containing 0.9% NaCl.

7. Protein A solution: 1 mg / ml / PBS

Immunoassay wells were: (a) 100 μl Protein A solution was added to 19.9 mL coating buffer, mixed well and poured into a distribution tray; (b) pipette 200 μl into each well (wash several times prior to delivery to the well); (c) the plate was held at room temperature for 4-5 hours, at 37 ° C. for 2-3 hours or at 4 ° C. overnight; (d) Pipette 100 μl EIA buffer into each well to block unfilled sites. When water is supplied to the wells, the plates are kept at 4 ° C. for an unspecified time.

Prepare the following solution:

1.Arachidonic acid: 1 mg / ml / ethanol

2. Isoproterenol: 2.5 mg / ml, prepared immediately before use

3. Hemoglobin: 3.2 mg / ml, prepared immediately before use

4.SnCl ₂ 50mg / ml / ethanol

5.HCl 1N

6.K-Blue Substrate Buffer: Neogen, Lexington, KY

7. 30-fold dilution of COX-1 enzyme (as described above), 5 μl per assay

8. 5-fold dilution of COX-2 enzyme (as described above), 5 μl per assay

9. Dilute PGF-2α-antibody (Dr.Tai) 5,000 ×, 50 μl per well

10. Dilute PGF-2α-HRP (Dr.Tai) 2,000X, 100 μl per well

Prepare the following PGF-2α standard solutions:

A. 1 μg / ml

B. 20 μl A (1 μg / ml) + 980 μl EIA buffer ------ 1,000 pg / 50 μl

C. 200 μl B + 1.8 mL EA buffer -------------- 100 pg / 50 μl

D. 200 μl C + 1.8 mL EA buffer -------------- 10 pg / 50 μl

Standard (pg / 50 μl / well) B C D Buffer (ml) 0 1.0 5 0.5 0.5 10 1.0 20 0.2 0.8 50 0.5 0.5 100 1.0 200 0.2 0.8 500 0.5 0.5 1000 1.0

If the inhibitor candidate is an anthocyanin-containing plant, the anthocyanin is extracted, concentrated and hydrolyzed to provide a non-glycosaccharide type, such as anthocyanidin. Similarly, when the inhibitor candidate is a commercial extract, the extract is hydrolyzed to provide a nonglycoform. In each case, the nonglycotype of anthocyanin is examined. The hydrolyzed anthocyanins are then dissolved in 0.1% HCl dissolved in methanol for testing.

PGF-2α is prostaglandin, which undergoes an enzymatic reaction to determine whether an inhibitor candidate effectively inhibits COX-1 or COX-2 (depending on the enzyme used). PGF-2α is a stable and indirect prostaglandin reduced from prostaglandin products produced by enzymatic reactions.

The enzymatic reaction process is carried out as follows: (a) 385 μl of buffer (50 mM Tris-HCl, pH7.5) was prepared and 50 μl isoproterenol, 10 μl hemoglobin, 5 μl SnCl 2, 5 μl Mixing at room temperature with an enzyme (depending on the enzyme to be tested, for example COX-1 or COX-2 described above); (b) 455 μl of the mixture (a) is added to a tube containing 40 μl of inhibitor candidate and mixed; (c) 5 μl arachidonic acid solution was added to each tube, mixed and incubated at 37 ° C. for 5 minutes; (d) add 30 μl 1N HCl to stop the reaction and mix; (e) Neutralize by adding 30 μl of 1M Tris-base.

Enzyme immunoassay is a method of measuring the content of PGF-2α generated by the enzymatic reaction. The enzyme immunoassay is carried out in the following manner: (a) All liquids in the wells are stirred and blotted on paper towels; (b) wash twice with 200 μl wash buffer, stir and blot; (c) 50 μl of PGF-2α-antibody; (d) add 50 μl STD (PGF-2α) or the enzymatic product (50 × diluted with EIA buffer); (e) 100 μl PGF-2α-HRP (dissolved in EIA buffer); (f) the plate was kept at room temperature for 1 hour with stirring; (g) repeating step (b) to wash the well three times; (h) 100 μl substrate buffer was added to each well; (i) incubate at room temperature for 3 to 30 minutes depending on color development; (j) operating a computer and a 96-well bioanalytical reader (Molecular Devices, Vmax dynamic microplate reader) according to the operating instructions; (k) 30 μl of 1N HCl was added to terminate the reaction; (l) read at 420 nm; (m) Record the data.

The data is analyzed in the following steps: (a) copy the data into a spreadsheet, and (b) create a standard curve according to standard PGF-2α immunoassay results; (c) standard curve regression to find PGF-2α content in all sample enzyme reactions; (d) create a curve for each sample; (e) Confirm I ₅₀ . This process evaluated several inhibitors, the results of which are presented in Table 3 below.

Table 3.

Sample Sample weight (g) After purification, the final Vol. (Ml) Concentration of final anthocyanidins (mg / ml) Cyanidin concentration (mg / ml) Cyanidin (%) COX inhibition (hydrolyzed liquid sample) I ₅₀ (COX 1) (μg) I ₅₀ (COX 2) (μg) I ₅₀ (COX 1) / I ₅₀ (COX 2) A 1.33 2.1 1.53 0.93 61 13.2 10.7 1.2 B 0.15 2.0 1.68 0.74 44 19.2 21.4 0.9 C 0.15 2.0 2.92 1.36 47 8.7 8.7 1.0 D 0.2028 1.0 0.20 0.20 100 34.4 17.5 2.0 E 0.15213 1.0 0.17 0.17 100 31.5 15.8 2.0 F 0.30784 1.0 0.057 0.046 80 32.8 23.5 1.4 aspirin 100 100 One Ibuprofen 8 2 4 Celebrex 7 One 7 Vioxx 10 0.5 20

A is a tablet containing 40% bilberry with 7% anthocyanin.

B is a commercial extract (Nutritech) containing 25% bilberry.

C is a commercial extract (Nutritech) produced somewhat differently from Sample B and containing 25% bilberry.

D is a tablet containing 39.17% elderberry with 15% anthocyanin.

E is a tablet containing 39.17% elderberry with 20% anthocyanins.

F is a tablet containing 11.76% elderberry with 15% anthocyanin and 19% bilberry with 7.2% anthocyanin.

Elderberry has 98% cyanidin and its glycosides. Bilberry has 23% cyanidin and its glycosides.

In the results, it can be seen that the higher the cyanidin content, the higher the efficacy and selectivity.

According to the procedure disclosed in Example 4, a number of inhibitor candidates were evaluated. The results are shown in Table 4.

Table 4.

extract I ₅₀ COX-1 I ₅₀ COX-2 Selectivity Active substance content Artemis 29 22 1.3 10.0% Rubini (Artemis) 75 57 1.2 7.2% Elderberry Proto 18 14 1.3 21.0% Iprona * 41 34 1.2 2.3% Chokeberry (Iprona) * 117 97 1.2 13.6% Elderberry (Iprona) * 47 35 1.3 17.0% Nutmeg 14 10 1.4 10.6% Tart Cherry 01-01a * 250 200 1.3 3.48% Elderberry 004-03 * (23% CRR) 25 19 1.3 15.06% Elderberry 004-04 * (0% CRR) 18 14 1.3 20.15% Pomegranate Fruit Extract Powder 120 80 1.5 n / a Devilfish Extract 250 150 1.7 Boswellia 200 150 1.3 Panax Ginseng (Panax gotoginseng) 200 250 0.8 ginger 200 100 2 Green Tea Extract Powder 70 60 1.2 Green Tea Polyphenols 110 100 1.1

Note: 1. For all fruit samples total anthocyanin is quantified with cyanidin-3-glucoside.

      2. The content of active substance is calculated based on the experimental results and the percentage of active compound. For example, 100% anthocyanins in elderberry, chokeberry and tart cherry are active cyanidin glycosides.

      * Means testing COX inhibitory activity from the hydrolyzed and XAD column purified fruit.

According to the above results, the supplement for treating inflammation provides a COX-1 inhibition to COX-2 inhibition ratio of at least 1, preferably at least 1.3. As a result, the supplement provides for selective COX-2 inhibition.

Formulation

This example describes a formulation containing one or more fruit extracts derived from anthocyanin-containing plants for use as anti-inflammatory drugs. As will be appreciated by those skilled in the art, these formulations are merely exemplary and they may be changed to equivalents depending on the particular purpose.

Table 5. Anti-inflammatory Formulations 1:

ingredient 2 unit prescription 1 unit prescription system% Active Ingredients: Elderberry Extract (min. 7% Anthocyanin) 100mg 50 mg 11.277% Chokeberry Extract (min. 10% Anthocyanin) 100mg 50 mg 11.277% Tart Cherry Extract 5.00mg 2.50mg 0.564% Excipients: Rice grain powder 675.00mg 337.5mg 76.121% Magnesium stearate 4.50mg 2.25mg 0.507% Silicon dioxide 2.25mg 1.13mg 0.254%

Table 6. Anti-inflammatory Formulation 2:

ingredient 2 unit prescription 1 unit prescription Prescription% Active Ingredients: Elderberry Extract (min. 13% Anthocyanin) 100mg 50 mg 11.855% Chokeberry Extract (min. 10% Anthocyanin) 100mg 50 mg 11.855% Tart Cherry Extract 5.00mg 2.50mg 0.593% Other anti-inflammatory herbal extracts: boswellia (min. 65% boswellic acid) 600.00mg 300.00mg 71.132 Excipients: Rice grain powder 25.00mg 12.50mg 2.964% Magnesium stearate 9.00mg 4.50mg 1.067% Silicon dioxide 4.50mg 2.25mg 0.533%

Table 7. Anti-inflammatory Formulations 3:

ingredient 2 unit prescription 1 unit prescription Prescription% Active Ingredients: Elderberry Extract (min. 13% Anthocyanin) 100mg 50 mg 5.905% Chokeberry Extract (min. 10% Anthocyanin) 100mg 50 mg 5.905% Tart Cherry Extract 5.00mg 2.50mg 0.295% Other anti-inflammatory herbal extracts: Boswellia (min. 65% Boswellic acid) 600.00mg 300.00mg 35.430% Ginger extract (min. 5% 500.00mg 125.00mg 29.525% Excipients: Rice grain powder 375.00mg 93.75mg 22.143% Magnesium stearate 9.00mg 2.25mg 0.531% Silicon dioxide 4.50mg 1.13mg 0.266%

All compositions and / or methods disclosed and claimed herein can be practiced without undue experimentation in light of the specification herein. Although the compositions and methods of the present invention have been described in terms of suitable embodiments, it is to be understood that the compositions and / or methods disclosed herein and the order of any step or sequence of steps in the method may be changed without departing from the spirit and scope of the present invention. It is obvious to those skilled in the art. More specifically, the drugs disclosed herein may be replaced with any chemically, physiologically related drug that achieves the same or similar results. All such similar substitutions and alterations apparent to those skilled in the art are within the spirit and scope of the invention as defined by the appended claims.

Claims (10)

In a composition containing an anthocyanin-containing extract as an active ingredient in the manufacture of a drug that inhibits COX-2 activity, at least one of the anthocyanins is hydrolyzed to aglycone so that COX-1 inhibition is less than COX-2 inhibition. Less composition. In a composition containing a fruit extract as an active ingredient in the preparation of a drug that inhibits COX-2 activity, the composition inhibits COX-2 activity in contact cells, and the fruit extract is sweet cherry, tart cherry, acerola cherry, It is derived from two or more fruits selected from flop, bilberry, blackberry, cannula, chokeberry, blueberry, strawberry, cranberry, boyzenberry, grape, raspberry, elderberry, and mixtures thereof. A composition characterized by providing a higher anti-inflammatory activity than the anti-inflammatory activity found. The composition of claim 1 or 2, wherein the cell is a mammalian cell. The composition of claim 3, wherein the cell is a human cell. The composition of claim 1 or 2, wherein the composition is contacted with the cells in vitro. The composition of claim 1 or 2, wherein the composition is contacted with the cell in vivo. In the pharmaceutical composition used for the manufacture of a drug for treating an inflammatory response in an animal or human, a. Dry extract of fruit, wherein the extract has an anti-inflammatory activity that is 2 to 100 times higher than the anti-inflammatory activity found in natural fruits; b. A pharmaceutical composition, characterized in that it consists of a pharmaceutically acceptable carrier or excipient. 8. The pharmaceutical composition of claim 7, wherein the inflammatory response is selected from arthritis, pain, allergic rash, inflammatory bowel symptoms, asthma. 8. The fruit extract according to claim 7, wherein the fruit extract is peonidine, cyanidin, pelargonidine, delphinidin, phetunnidine, malvidin, camphorol, hesperidin, genthiodelpine, placoninine, cinerarine, glycoside derivatives thereof And an anthocyanin selected from mixtures thereof. 8. The pharmaceutical composition of claim 7, wherein the anthocyanin is hydrolyzed in vivo to selectively inhibit COX-2.

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