HK1190328B - Method of preparing a muscadine pomace extract - Google Patents

Description

Preparation method of muscadine pomace extract

The application is a divisional application of international application PCT/US2009/052346, with the application number of 200980137635.3 and the invention name of 'a preparation method of muscadine pomace extract', entering the Chinese national stage at 24/3/2011.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit and priority to U.S. provisional application No.61/085,371, filed on 31/7/2008, which is hereby incorporated by reference in its entirety.

Technical Field

The present disclosure relates to muscadine pomace extracts, e.g., for use in anti-aging supplements, and methods of making muscadine pomace extracts.

Background

Reactive Oxygen Species (ROS) are inevitable by-products of aerobic metabolism and are therefore continuously produced in humans and other organisms. Humans are also exposed to ROS from external/environmental sources such as pollution, sunlight and diet. Although ROS have different chemical forms, they all have deleterious effects on the structure and function of cellular components and macromolecules. The intensity of ROS generation/exposure is referred to as oxidative stress.

Oxidative stress is associated with the pathogenesis of chronic inflammatory diseases such as diabetes, cancer, atherosclerosis and other cardiovascular diseases as well as degenerative diseases such as alzheimer's disease and parkinson's disease. In addition, multiple lines of evidence support the notion that oxidative stress is the central mechanism responsible for normal aging. Therefore, there is a need to develop therapeutic methods to inhibit oxidative stress.

Disclosure of Invention

Minimizing oxidative stress is believed to be critical for health and longevity. Epidemiological data show that diets rich in antioxidant nutrients provide great benefits. In addition to the classical antioxidant vitamins (vitamins C and E), dietary plants contain many polyphenols which, after consumption, exhibit a rapid and long-term reduction in oxidative stress. Several in vitro assays for measuring antioxidant capacity of different foods have been established and there is a strong correlation between polyphenol content and antioxidant capacity.

Although plants, including grapes, provide a promising source of antioxidants, there are significant obstacles to the widespread use of phytochemicals in the human population. For example, bioavailability of active agents in food is often not optimal for use as an antioxidant. Active agents may also be concentrated in certain parts of the plant that are in lower ratios or are discarded in typical preparations of food products for consumption. Different varieties of related foods may also have biochemical differences, making them less suitable for therapeutic applications.

The inventors determined the antioxidant capacity of the muscadine pomace extract and the polygonum cuspidatum extract, alone and in combination (in a mixture or dietary supplement), as measured by an Oxygen Radical Absorbance Capacity (ORAC) assay. Both the hydrophilic antioxidant capacity and the lipophilic antioxidant capacity of the samples were measured. These studies demonstrate a strong synergistic effect of muscadine pomace extract and polygonum cuspidatum root extract in a mixture or dietary supplement in producing lipophilic antioxidant capacity. The selective synergy shown under lipophilic conditions is unexpected. These findings demonstrate that mixtures and dietary supplements containing at least as a substantially pure source of resveratrol, both an extract of giant knotweed, and an extract of muscadine pomace, have increased antioxidant capacity as compared to either of the extracts alone.

Disclosed herein are compositions having increased antioxidant activity. In certain disclosed embodiments, the compositions comprise a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% and resveratrol from a source other than muscadine (e.g., a polygonum cuspidatum root extract) having a minimum purity of at least 5%, wherein the ratio of muscadine polyphenols to trans-resveratrol is in the range of 0.1/1 to 10/1 (weight/weight), thereby providing a composition having antioxidant activity. In a particular example, a composition includes a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% and resveratrol having a minimum purity of at least 5% from a source other than muscadine (e.g., a polygonum cuspidatum root extract), wherein the ratio of muscadine pomace extract to resveratrol is in the range of 0.2/1 to 50/1 (weight/weight), such as 5/1 to 50/1 (weight/weight), or 20/1 to 50/1 (weight/weight), thereby providing a composition having antioxidant activity. In certain examples, the composition has a total ORAC of at least 24 μmolTrolox equivalents per mg polyphenol (μmolTE/mg polyphenol).

In certain examples, the muscadine (vitis rotundifolia) pomace extract has a total polyphenol content of about 4% and the composition has a muscadine pomace extract to trans-resveratrol ratio of about 18 to 1 (weight to weight). In an example, the composition has a muscadine polyphenol to resveratrol ratio of about 0.75 to 1 (weight/weight). In certain examples, the muscadine pomace extract may include 20% to 50% solids in a liquid. For example, muscadine pomace extracts contain about 40% solids in a liquid. In certain examples, the resveratrol comprises at least 98% trans-resveratrol extracted from the root of polygonum cuspidatum (Polygonum cuspidatum).

In certain examples, the disclosed compositions can also include elderberry extract, purple carrot extract, excipients (such as glycerin, sorbitol, colloidal silicon dioxide, or natural flavoring additives), or combinations thereof. In a specific example, the composition includes a muscadine pomace extract at 23% to 32% of the total composition, resveratrol at 1.2% to 3.0% of the total composition, a purple carrot extract at 0.12% to 0.20% of the total composition, an elderberry extract at 0.4% to 0.6% of the total composition, sorbitol at 50% to 61% of the total composition, a 99.7% glycerin composition at 8% to 12% of the total composition, and colloidal silicon dioxide at 1% to 2% of the total composition.

In a more specific example, the composition includes a muscadine pomace extract (containing about 4.1% polyphenols) at about 28.9% of the total amount, resveratrol at about 1.65% of the total amount, a purple carrot extract at about 0.16% of the total amount, an elderberry extract at about 0.48% of the total amount, sorbitol at about 54.8% of the total amount, a 99.7% glycerin composition at about 10% of the total amount, colloidal silicon dioxide at about 2% of the total amount, a Concord (concod) grape extract at about 1.27%, a Cabernet (Cabernet) grape extract at about 0.16% and red grape powder at about 0.48%.

In another specific example, the composition includes a muscadine pomace extract (containing about 5% polyphenols) at about 23.77% of the total amount, resveratrol at about 1.65% of the total amount, a purple carrot extract at about 0.16% of the total amount, an elderberry extract at about 0.48% of the total amount, sorbitol at about 60% of the total amount, a 99.7% glycerin composition at about 10% of the total amount, colloidal silicon dioxide at about 2% of the total amount, a concord grape extract at about 1.27%, a cabernet grape extract at about 0.16% and red grape powder at about 0.48%.

Any of the disclosed compositions can be provided in a non-beverage food, beverage, or dietary supplement in liquid or solid form. In addition, any of the disclosed compositions can be used as antioxidants, such as anti-aging supplements, including skin improving agents. For example, methods of preventing or inhibiting cellular aging are disclosed, the methods comprising administering to a subject in need thereof a composition of the present disclosure in an amount sufficient to prevent or inhibit one or more processes associated with cellular aging, such as preventing or inhibiting the formation or activity of free radicals, in a subject ingesting the composition.

Also provided herein are methods of producing the disclosed antioxidant compositions. These methods may include combining a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% with resveratrol from a source other than muscadine having a trans-resveratrol minimum purity of at least 5%, wherein the ratio of muscadine polyphenols to trans-resveratrol is in the range of 0.1/1 to 10/1 (weight/weight), thereby producing a muscadine pomace extract and trans-resveratrol mixture having antioxidant activity. In certain examples, the methods may include combining a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% with resveratrol from a source other than muscadine (e.g., a polygonum cuspidatum root extract) having a trans-resveratrol minimum purity of at least 5%, wherein the ratio of muscadine pomace extract to resveratrol is in the range of 0.2/1 to 50/1 (weight/weight), such as 5/1 to 50/1 (weight/weight), or 20/1 to 50/1 (weight/weight), including 18 to 1 (weight/weight), thereby providing a composition having antioxidant activity.

The disclosed methods may also include preparing a muscadine pomace extract and then combining the muscadine pomace extract with trans-resveratrol. In one particular example, preparing the muscadine pomace extract includes combining a bronze muscadine pomace extract with a purple muscadine pomace extract. In other examples, preparing the muscadine pomace extract includes preparing the extract from a mixture of bronze muscadine pomace and purple muscadine pomace.

In a particular example, the method includes preparing the muscadine pomace extract by combining a bronze muscadine pomace extract with a purple muscadine pomace extract, wherein the ratio of bronze muscadine pomace extract to purple muscadine pomace extract ranges from 0.1 to 10 (weight to weight), such as 0.3 to 3 (weight to weight). In a more specific example, the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is about 2.25 to 1 (weight to weight).

The above and other features of the present disclosure will become more apparent from the following detailed description.

Detailed Description

I. Abbreviations and terms

(a) Abbreviations

FRAP: plasma ferric iron reduction capacity

mg: milligrams of

ml: milliliter (ml)

ORAC: oxygen radical absorption capacity

ROS: active oxygen

TE: trolox equivalent

TEAC: trolox equivalent antioxidant capacity

wt: weight (D)

(b) Term(s) for

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein and in the claims, the noun form without a specific numerical designation includes plural referents thereof unless the context clearly dictates otherwise. The term "or" refers to a single element or a combination of two or more elements of the recited optional elements, unless the context clearly dictates otherwise. As used herein, "comprising" means "including". Thus, "comprising a or B" means "including A, B or a and B," without excluding other elements.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. Moreover, it should be understood that any numerical value is approximate, whether or not the word "about" or "approximately" is stated, etc. All percentages and ratios are by weight unless otherwise indicated.

Application: providing or administering to the subject an agent, such as a composition comprising muscadine pomace extract and trans-resveratrol, by any effective means. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, transdermal, intranasal, topical, and inhalation routes.

Antioxidant activity: including activity in scavenging and neutralizing oxidative free radicals. Antioxidant activity can be measured using the methods disclosed herein as well as methods known in the art, including an Oxygen Radical Absorbance Capacity (ORAC) assay, a ferric plasma reduction capacity (FRAP) assay, and a Trolox Equivalent Antioxidant Capacity (TEAC) assay. For example, if the composition has a total ORAC of at least 24 μmolTrolox equivalents/mg polyphenol (μmolTE/mg polyphenol), it has antioxidant activity and can be used as an antioxidant.

Elderberry (sambucus nigra): a plant belonging to the family of the five-Formica Fusca (Adoxaceae) found in Europe and North America has several regional varieties or subspecies. The flowers are flat umbrella-shaped inflorescences. Berries are black to blue-green and contain anthocyanins and other polyphenolic substances, the amount and type of which vary from species to species.

The "elderberry extract" may be a material obtained by extracting elderberry according to any extraction method known to those skilled in the art, as long as it has the desired activity (e.g., color-stabilizing activity, antioxidant activity, or a combination thereof). For example, the elderberry extract may include a fruit juice obtained by pressing elderberry fruit, or an extract obtained by extracting the whole fruit of elderberry or a suitable portion of the pericarp or seed of the fruit by an optional extraction method generally known to those skilled in the art, and the like. In addition, elderberry fruit crushed products or dried elderberry fruit concentrates, may be used as "elderberry extract".

Excipient: inactive materials which act as carriers for the active ingredients of the compositions. Excipients may include substances used to add bulk to a formulation with a very strong active ingredient, to allow convenient and precise dosing, to stabilize the active ingredient and to make the delivery system optically and/or organoleptically acceptable. Examples of pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. In particular examples, the disclosed anti-aging supplement includes the following excipients: glycerin, sorbitol, colloidal silicon dioxide and natural flavoring additives.

Inhibition (including prevention) of cellular senescence: inhibiting (e.g., arresting) one or more processes associated with cellular aging, such as inhibiting the formation or activity of free radicals, in a subject ingesting the composition. Preventing cellular aging refers to an intervention that ameliorates the signs or symptoms of cellular aging. Preventing includes preventing to delay the occurrence of one or more processes associated with cellular aging. The prevention or inhibition of cellular senescence need not be completely absent. In particular examples, the disclosed compositions reduce or delay processes associated with cellular aging by at least 10%, at least 20%, at least 50%, or even at least 90%. Such reduction can be measured using the methods disclosed herein as well as methods known in the art.

Muscadine (vitis rotundifolia): grapes, native to the southeast united states, are found in the field from the terra hua (Delamare) to the gulf of mexico and westward to missouri, kansas, oklahoma and texas. Muscadines are well suited to the warm and humid conditions of the southeast united states. The fruit that comes out is a loose small cluster of 3-40 grapes, which is significantly different from the large, tightly bunched characteristics of european and american grapes. The fruit is round, 1 to 1-1/2 inches, has a thick, tough peel, and contains up to 5 hard oval seeds. The color of the fruit ranges from greenish bronze to bronze, pink, purple and almost black.

Muscadine is available in many different varieties, including female (pistil-only) varieties such as BlackBeauty, BlackFry, Darlene, Fry, Higgins, Jumbo, Scuppernong, Surgarate, Summit, Supreme, and SweetJenny, and self-pollinated varieties such as Carlos, Cowart, Dixieland, DixieRed, FrySeedless, Magnolia, Nesbit, Noble, Redgate, Regale, and Sterling.

For example, bronze muscadine varieties identified by those skilled in the art include Carlos, Chowan, Doreen, Higgins, Magnolia, Nevermiss, Pamlico, Roanoke, Scupperong, Sterling, and Summit cultivars. Purple to black varieties include Albermarle, Bountiful, Cowart, GA-1, Hunt, NC-1, Noble, Regale, Tareel, and Jumbo.

The phytochemical composition of whole muscadine is different from that of Vitis vinifera (Vitisvinifera). Muscadine has a high total phenolic content and is characterized by high concentrations of ellagic, galloic and flavonoid glycosides. The presence of ellagic acid in muscadines is unique and is found in the form of free ellagic acid, ellagic acid glycosides, methoxylated derivatives, and ellagitannins. Another unique feature is the anthocyanin chemistry observed in muscadine. These compounds are present in non-acylated form as 3, 5-diglucosides of delphinidin, anthocyanidins, petunidin, methylcyanidin and malvidin (as opposed to 3-glucoside), which, together with the natural colour influence of the other anthocyanins present in the grapes, impart a deep purple colour to the juice and pomace. The purple pomace extract contains anthocyanin, while the bronze pomace extract does not.

The red and purple anthocyanins are polyphenolic compounds with antioxidant properties. Flavonols are the second most abundant flavonoid found in whole muscadines. The major phenolic substances reported in the pericarp fraction of muscadine (in descending order) are ellagic acid, myricetin, quercetin and kaempferol, while epicatechin, catechin and gallic acid are reported in the seeds (Patrana-Bonilla et al, J.Agric.FoodChem.51: 5497-.

A pharmaceutically acceptable medium: the pharmaceutically acceptable media (carriers) useful in the present disclosure are conventional. Compositions and formulations suitable for drug delivery of one or more compositions, such as one or more muscadine compositions, with other agents are described in Remington's pharmaceutical sciences, mack publishing co, Easton, PA,19 th edition (1995), master ed by martin, w.

In general, the nature of the medium will depend on the particular mode of administration used. For example, parenteral formulations typically comprise injectable fluids, which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as vehicles. For solid compositions (e.g., in the form of powders, pills, tablets or capsules), conventional non-toxic solid media may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral medium, the pharmaceutical composition to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Purification of: the term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified substance is one in which the substance is more enriched than the substance in its natural environment, for example in a fruit (e.g. grape). In one embodiment, the preparation is purified such that the substance comprises at least about 5% (e.g., without limitation, at least 10%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, 95%, 98%, or 99%) of the total content of the preparation. In examples, the disclosed compositions having antioxidant activity include trans-resveratrol with a minimum purity of at least 50%, 70%, 80%, 90%, 95%, 98%, or 99% (by weight) of the total resveratrol preparation.

Fruit residues: the pericarp, seed and pulp remaining after juice extraction. In one example, the pomace extract is a bronze muscadine pomace extract, a purple muscadine pomace extract, or a combination thereof. Many different varieties of muscadine pomace are available as starting materials, including female (pistachio-only) varieties such as BlackBeauty, BlackFry, Darlene, Fry, Higgins, Jumbo, scupperenong, sugarrate, Summit, Supreme, and SweetJenny, and self-pollinated varieties such as Carlos, Cowart, Dixieland, DixieRed, frysedleless, magnola, neubitt, Noble, Redgate, Regale, and Sterling.

Muscadine pomace contains phenolic compounds with antioxidant properties, including gallic acid and ellagic acid.

Purple carrot (Daucuscarota): carrot cultivars containing anthocyanin pigments. The "purple carrot extract" may be a substance obtained by extracting purple carrot according to any extraction method known to those skilled in the art, as long as it has the desired activity (e.g., color-stabilizing activity, antioxidant activity, or a combination thereof). In one example, the purple carrot extract has the property of stabilizing muscadine color pigments and can therefore be subsequently used as a color stabilizing additive.

Resveratrol: a phytoalexin, belonging to the stilbene class of compounds, is a derivative of stilbene and is produced in plants with the aid of stilbene synthases. Resveratrol exists as two structural isomers: cis-and trans-resveratrol. Trans-resveratrol can undergo isomerization to the cis form upon heating or exposure to ultraviolet radiation.

Resveratrol is present in various amounts in the roots and stems of grapes, raspberries, morous alba, plums, peanuts, bilberry species including blueberries, bilberry and cranberry, certain pine trees such as Pinus sylvestris and eastern white pine, and giant knotweed. In grapes, resveratrol is found mainly in the pericarp and seeds. The amount of resveratrol found in grape pericarp varies with the grape cultivar, its geographical origin and exposure to fungal infection.

The term resveratrol as used herein may include natural trans-resveratrol extracted from a plant such as grape or synthetic trans-resveratrol. As used herein, the term resveratrol may include modified formulations of trans-resveratrol, such as microencapsulated or water dispersible forms.

Object: living multicellular is a vertebrate organism, a category of which includes both human and veterinary subjects.

A therapeutically effective amount of: the amount of the composition that can induce a desired response (e.g., prevent or inhibit cellular aging) alone or in combination with other agents (e.g., other antioxidants). The preparations disclosed herein can be administered in a therapeutically effective amount.

In one example, the desired response is to inhibit or reduce the production or activity of free radicals associated with one or more cellular aging processes in a subject to which the composition is administered. The generation or activity of free radicals need not be completely eliminated for the effectiveness of the composition. For example, the composition can reduce the production or activity by a desired amount, e.g., by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, as compared to the production or activity of free radicals in the absence of the composition.

A therapeutically effective amount of the disclosed muscadine pomace extract compositions can be administered, for example, in a single dose or in several doses daily during the course of treatment. However, a therapeutically effective amount may depend on the subject to be treated, the severity and type of the condition being treated, and the mode of administration. For example, if administered orally, the therapeutically effective amount of such agents may vary from about 1 to about 500 milligrams of trans-resveratrol, muscadine polyphenols, or combinations thereof per day.

Unit dose: physically separate units containing a predetermined amount of active material calculated to produce, individually or collectively, a desired effect, e.g., a therapeutic effect. A single unit dose or multiple unit doses may be used to provide a desired effect or activity, such as antioxidant activity. In one example, a unit dose includes an amount of an agent required to prevent or inhibit one or more processes associated with cellular aging.

Description of several embodiments

Plant agents have been extensively studied as potential sources of nutrients that can be used to reduce oxidative stress. Red grapes are among the many foods that have been the subject of intensive research. The antioxidant effect of red grapes is generally attributed to polyphenolic compounds such as resveratrol and procyanidins, the latter being present in significant amounts in the grapes.

The present inventors have conducted a series of studies to develop a muscadine pomace extract sufficient to increase the maximum solubility of ellagic acid. It has now been found that the unconcentrated 65% to 35% ratio of bronze muscadine pomace extract to purple muscadine pomace extract (volume/volume) increases the maximum solubility of ellagic acid between the two pomace extracts.

Determining the maximum solubility of ellagic acid (which is unique to muscadine) in muscadine pomace extracts allows for the preparation of extracts with increased ellagic acid solubility (and thus obtaining polyphenol profiles of the pomace), which further allows for the preparation of extracts with improved antioxidant activity. Accordingly, muscadine pomace extracts having improved ellagic acid solubility and methods of using these extracts are disclosed herein. For example, the disclosed muscadine extracts can be administered alone or in combination with other compounds in non-beverage foods, beverages, dietary supplements, or topical ointments. Also provided are methods of producing a muscadine pomace extract comprising combining a bronze muscadine pomace extract with a purple muscadine pomace extract to produce a muscadine pomace extract, wherein the ratio of bronze muscadine pomace extract to purple muscadine pomace extract ranges from 0.3 to 3 (weight to weight).

In addition, the inventors determined the antioxidant capacity of the disclosed muscadine pomace extract and polygonum cuspidatum extract normalized to 98% trans-resveratrol, separately or in combination (in a mixture or dietary supplement), as measured by ORAC analysis. Both the hydrophilic antioxidant capacity and the lipophilic antioxidant capacity of the samples were measured. These studies demonstrate a strong synergistic effect of muscadine pomace extract and polygonum cuspidatum root extract normalized to 98% trans-resveratrol in producing lipophilic antioxidant capacity in a mixture or dietary supplement. The selective synergy shown under lipophilic conditions is unexpected. Furthermore, while the muscadine extract used in the composition may be a natural extract that may vary by species, extraction process, etc., the synergistic lipophilic antioxidant activity may be retained so long as the disclosed ratio of muscadine polyphenols to resveratrol is maintained.

These findings demonstrate that mixtures and dietary supplements containing both giant knotweed extract, normalized to 98% trans-resveratrol, and muscadine pomace extract have increased lipophilic antioxidant capacity compared to either extract alone. Importantly, oxidative processes occurring in lipophilic environments are believed to cause and/or initiate the pathological development of a variety of disease states, such as Low Density Lipoprotein (LDL) oxidation in atherosclerosis and obesity-induced insulin resistance in type II diabetes. In addition, oxidation of dietary lipids in the gastrointestinal tract results in the absorption of cytotoxic and genotoxic lipid peroxidation products such as Malondialdehyde (MDA). Thus, the disclosed compositions with high lipophilic antioxidant capacity may provide beneficial effects, counteracting the deleterious consequences of lipid oxidation mentioned above.

This discovery has enabled the provision of muscadine pomace extract compositions having improved antioxidant activity and methods of producing these compositions. For example, the muscadine pomace extracts disclosed herein having antioxidant activity can be administered in a non-beverage food, beverage, liquid or solid dietary supplement, or topical ointment. A method of producing the disclosed compositions may include combining a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% with trans-resveratrol from a source other than muscadine having a minimum purity of at least 5%, wherein the ratio of muscadine polyphenols to trans-resveratrol is in the range of 0.1/1 to 10/1 (weight/weight), thereby producing a muscadine pomace extract and trans-resveratrol mixture having antioxidant activity. The method of producing the disclosed compositions may further comprise combining a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% with trans-resveratrol from a source other than muscadine having a minimum purity of at least 5%, wherein the ratio of muscadine pomace extract to trans-resveratrol is in the range of 0.2/1 to 50/1 (weight/weight), such as 5/1 to 50/1 (weight/weight), including 20/1 to 50/1 (weight/weight), such as 18 to 1 (weight/weight), thereby producing a muscadine pomace extract and trans-resveratrol mixture having antioxidant activity.

Lipophilic antioxidants have been found to be effective in preventing various types of skin damage, such as, but not limited to, inhibition of lipid peroxidation and products resulting from lipid peroxidation, such as cross-linking agents. Oxidative stress is known to be the central mechanism underlying normal aging. It is shown herein that the disclosed compositions have improved lipophilic antioxidant activity. Based on these observations, methods of inhibiting cellular aging, e.g., preventing or inhibiting the production or activity of free radicals, are disclosed. The methods include using the disclosed compositions (e.g., dietary supplements) with improved antioxidant capacity as anti-aging supplements. For example, methods can be provided for preventing or reducing one or more processes associated with cellular aging, such as reducing or inhibiting the production or activity of free radicals, in a subject ingesting the mixture or dietary supplement composition.

A. Musk grape pomace extract

Disclosed herein are muscadine pomace extracts derived from bronze muscadine pomace and purple muscadine pomace. In certain embodiments, the ratio of bronze muscadine pomace extract to purple muscadine pomace extract in the muscadine pomace extract ranges from 0.3 to 3 (weight to weight). For example, the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is about 2.75 to about 1 (weight to weight), 2.5 to about 1 (weight to weight), about 2.25 to about 1 (weight to weight), about 2 to about 1 (weight to weight), about 1.5 to about 1 (weight to weight), or about 1 to about 1 (weight to weight). The term "about" as used herein is defined as ± 0.5. In a specific example, the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is about 2.25 to about 1 (weight to weight).

In certain embodiments, the muscadine (vitis rotundifolia) pomace extract disclosed has a polyphenol content of at least 2%. For example, the polyphenol content is at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 12%, or at least 14%. In a specific example, a muscadine (vitis rotundifolia) pomace extract has a polyphenol content of about 4%.

In certain embodiments, the disclosed muscadine pomace extracts include 20% to 50% solids in a liquid, such as at least 25%, at least 30%, at least 35%, at least 37%, at least 40%, at least 42%, at least 44%, at least 46%, or at least 48%. In a specific example, the extract comprises about 40% solids in the liquid.

B. Preparation method of muscadine pomace extract

Also disclosed are methods of preparing muscadine pomace extracts wherein the ratio of bronze muscadine pomace extract to purple muscadine pomace extract ranges from 0.3 to 3 (weight to weight). In one disclosed embodiment, the muscadine pomace extract is prepared by combining bronze muscadine pomace extract with purple muscadine pomace extract. In a particular embodiment, the muscadine pomace extract is prepared by combining a bronze muscadine pomace extract with a purple muscadine pomace extract, wherein the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is in the range of 0.1 to 10 (weight to weight), including from 0.3 to 3 (weight to weight), such as about 2.75 to about 1 (weight to weight), 2.5 to about 1 (weight to weight), about 2.25 to about 1 (weight to weight), about 2 to about 1 (weight to weight), about 1.5 to about 1 (weight to weight), or about 1 to about 1 (weight to weight). In a more specific example, the muscadine pomace extract is prepared by combining a bronze muscadine pomace extract with a purple muscadine pomace extract, wherein the ratio of bronze muscadine extract to purple muscadine pomace extract is about 2.25 to 1 (weight to weight).

In certain examples, a method of preparing a muscadine pomace extract can include preparing a bronze muscadine pomace extract and a purple muscadine pomace extract, respectively. For example, bronze muscadine pomace and purple muscadine pomace are extracted separately using water, preferably heated water. Preparing the bronze muscadine pomace extract and the purple muscadine pomace extract may also include fermenting the bronze muscadine pomace extract and the purple muscadine pomace extract to remove extracted sugars. In one example, fermentation is performed after extraction of the bronze muscadine pomace and the purple muscadine pomace, but before the bronze muscadine pomace extract is combined with the purple muscadine pomace extract to produce the disclosed muscadine pomace extract. In other examples, fermentation is performed after combining the bronze muscadine pomace extract and the purple muscadine pomace extract in a desired post-extraction ratio (e.g., a bronze to purple ratio of about 2.25 to 1).

Fermentation can be carried out by any method known to those skilled in the art, including the methods described herein. For example, fermentation may include adding yeast and yeast nutrients to the pomace and fermenting the extract until the residual sugar content is converted to ethanol. Typically, one uses 2 pounds of yeast per 1000 gallons of 1 × (unconcentrated) extract. In such instances, fermentation is typically complete after 3 days. In other examples, the amount and/or strain of yeast and the duration and temperature of fermentation may vary depending on the particular method known to those skilled in the art. In certain examples, enzymes may be used to clarify and/or settle residues in pomace extracts, or to improve extraction yields. For example, these enzymes may include pectinase or a mixture of enzymes from Aspergillus niger (Aspergillus niger) and are commercially available from sources such as Scott laboratories. These enzymes may be added to the pomace extract before or during fermentation.

In certain embodiments, preparing the bronze muscadine pomace extract and the purple muscadine pomace extract comprises filtering the bronze muscadine pomace extract and the purple muscadine pomace extract. For example, the bronze muscadine pomace extract and the purple muscadine pomace extract can be filtered before and/or after fermentation. Filtration can be performed according to general methods known to those skilled in the art, including the filtration methods disclosed herein. In a specific example, the extract is filtered through a screen having a suitable mesh size, such as a USP screen (typically 120 mesh) or similar filter cloth (e.g. filters commercially available from millipore corporation).

In other embodiments, preparing the muscadine pomace extract can include combining bronze muscadine pomace with purple muscadine pomace in a desired ratio and simultaneously extracting the bronze muscadine pomace and the purple muscadine pomace. For example, bronze muscadine pomace and purple muscadine pomace are simultaneously extracted using water, preferably heated water, to produce the disclosed muscadine pomace extracts. In certain embodiments, the method may further comprise fermenting the muscadine pomace extract to remove extracted sugars. As previously mentioned, any fermentation process known to those skilled in the art may be used, including the processes described herein. For example, fermentation may include adding yeast and yeast nutrients to the pomace and fermenting the extract until the residual sugar content is converted to ethanol. In one example, fermentation involves adding 2 pounds of yeast per 1000 gallons of 1X extract and allowing the extract to remain at room temperature for about 3 days or until the residual sugar content is converted to ethanol. The disclosed method may further comprise filtering the muscadine pomace extract. For example, the muscadine pomace extract can be filtered before and/or after fermentation. Filtration can be performed according to general techniques known to those skilled in the art, including the filtration methods disclosed herein.

In certain embodiments, the methods of making muscadine pomace extracts further comprise concentrating the bronze muscadine pomace extract and the purple muscadine pomace extract such that each extract comprises 20% to 50% solids in the liquid, e.g., at least 25%, at least 30%, at least 35%, at least 37%, at least 40%, at least 42%, at least 44%, at least 46%, or at least 48%. In a specific example, the extracts are concentrated so that each extract comprises about 40% solids in the liquid. The bronze and violet extracts may be concentrated using well known methods for concentrating samples, including the methods disclosed herein for concentrating samples.

In a specific example, to prepare a muscadine pomace extract containing 40% solids, the muscadine pomace extract is dried to a powder form and reconstituted in water to a 40% solids level. Alternatively, and more commercially acceptable, the concentration is carried out by removing the extraction solvent by evaporation under vacuum. This can be achieved by either a batch or continuous process. The batch process involves placing the extract in a container, pulling a vacuum of 20-29 "Hg while heating the container jacket to provide energy to increase the vapor pressure of the solvent. The solvent vapor is condensed outside the vessel, and the rate of condensation controls the temperature of the condensate. The same principle applies to a continuous evaporation process, but with the advantage of reducing the length of time the condensate is exposed to high temperatures. Both processes are suitable for the concentration of the muscadine pomace extract described herein.

In particular embodiments, the methods of making muscadine pomace extracts include separately extracting bronze and purple pomace, then filtering each extract, and then combining the bronze and purple pomace extracts in the desired ratio. In some examples, the method may further comprise fermenting the combined muscadine pomace extract to remove extracted sugars. As previously mentioned, any fermentation process known to those skilled in the art may be used, including the processes described herein. In one example, the method comprises more than one filtration step, such as filtration of the extract before and after fermentation. In certain examples, the method may further comprise concentrating the extract as described herein. For example, the method may comprise concentrating the extract by evaporation under vacuum to remove the extraction solvent.

C. Composition with antioxidant activity

Disclosed herein are compositions having improved antioxidant activity. In certain disclosed embodiments, the compositions include a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% and trans-resveratrol from a source other than muscadine (e.g., a polygonum cuspidatum root extract) having a minimum purity of at least 5%, wherein the ratio of muscadine polyphenols to trans-resveratrol is in the range of 0.1/1 to 10/1 (weight/weight), thereby providing a composition having antioxidant activity.

In certain examples, the composition comprises a muscadine (vitis rotundifolia) pomace extract having a total polyphenol content of at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 12%, or at least 14%, and trans-resveratrol having a minimum purity of at least 5%, at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98% from a source other than muscadine. In a particular example, the composition includes a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of about 4% and trans-resveratrol from a source other than muscadine having a minimum purity of at least 98%.

In particular embodiments, the compositions include a muscadine pomace extract having 20% to 50% solids in a liquid, e.g., at least 23%, at least 25%, at least 30%, at least 35%, at least 37%, at least 40%, at least 42%, at least 44%, at least 46%, or at least 48% solids. In a specific example, the extract comprises about 40% solids in the liquid.

In certain embodiments, the resveratrol comprises at least 5% trans-resveratrol, such as at least 10% trans-resveratrol, at least 20% trans-resveratrol, at least 30% trans-resveratrol, at least 40% trans-resveratrol, at least 50% trans-resveratrol, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98% trans-resveratrol. In specific embodiments, the resveratrol comprises at least 50% trans-resveratrol, such as at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98% trans-resveratrol extracted from the root of polygonum cuspidatum (polygonumcuspitatum). It is contemplated that other sources of trans-resveratrol may be used in the compositions of the present disclosure, including synthetic trans-resveratrol.

In certain embodiments, the disclosed antioxidant compositions have a muscadine polyphenol to trans-resveratrol ratio of at least 0.1 to 1 (weight/weight), such as 0.25 to 1, 0.5 to 1, 0.6 to 1, 0.65 to 1, 0.7 to 1, 0.75 to 1, 0.8 to 1, 0.9 to 1, or 1 to 1 (weight/weight). In other embodiments, the ratio of muscadine polyphenol to trans-resveratrol may be as high as 10 to 1 (weight to weight), such as 2 to 1,3 to 1, 4 to 1, 5 to 1, 7.5 to 1, or 9 to 1 (weight to weight). In a specific example, the disclosed antioxidant composition has a muscadine polyphenol to trans-resveratrol ratio of 0.75 to 1 (weight/weight).

In certain embodiments, the disclosed antioxidant compositions have a muscadine pomace extract to trans-resveratrol ratio ranging from 0.2/1 to 50/1 (weight/weight), for example, 0.5 to 1, 1 to 1, 5 to 1, 10 to 1, 15 to 1, 20 to 1, 22 to 1, 25 to 1, 30 to 1, 35 to 1, 40 to 1, or 45 to 1 (weight/weight).

In certain embodiments, the disclosed compositions having antioxidant activity have a total ORAC of at least 21 μmol trolox equivalents per mg polyphenol (μmol te/mg polyphenol), such as at least 22 μmol te/mg polyphenol, at least 24 μmol te/mg polyphenol, at least 26 μmol te/mg polyphenol, at least 28 μmol te/mg polyphenol, or at least 30 μmol te/mg polyphenol. In one example, the disclosed composition has a total ORAC of 24 μmolTE/mg polyphenol.

In certain examples, the disclosed compositions can also include elderberry extract, purple carrot extract, excipients (such as glycerin, sorbitol, colloidal silicon dioxide, or natural flavoring additives), or combinations thereof. For example, elderberry extract and purple carrot extract may be included to provide color or additional antioxidant activity to the composition.

In particular examples, the composition comprises from 23% to 32% (e.g., 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, or 32%) of a muscadine pomace extract (containing from about 4% to 5% polyphenol content) of a total composition, from 1.2% to 3.0% (e.g., 1.3%, 1.5%, 1.7%, 1.9%, 2.1%, 2.3%, 2.5%, 2.7%, or 2.9%) of trans-resveratrol of a total composition, from 0.12% to 0.20% (e.g., 0.12%, 0.14%, 0.16%, 0.18%, or 0.20%) of a purple carrot extract of a total composition, from 0.4% to 0.6% (e.g., 0.4%, 0.45%, 0.5%, 0.55%, or 0.6%) of an elderberry extract of a total composition, from 50% to 61% (e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%) of sorbitol of a total composition, from 8% to 12% (e.8% of a total composition, 9%, 10%, 11%, or 12%) of a 99.7% glycerin composition, and 1% to 2% of the total (e.g., 1.2%, 1.5%, 1.7%, or 2%) of colloidal silica. In other specific examples, the composition comprises from 8% to 60% of the total composition of a muscadine pomace extract (containing from 2% to 14% muscadine polyphenols by weight of the liquid) and from 1.2% to 3.0% of the total composition of trans-resveratrol.

In a more specific example, the composition comprises from 28% to 30% of the total muscadine pomace extract, from 1% to 2% of the total resveratrol, from 0.1% to 0.2% of the total purple carrot extract, from 0.4% to 0.5% of the total elderberry extract, from 53% to 56% of the total sorbitol, from 9% to 11% of the total 99.7% glycerin composition, from 1.5% to 2% of the total colloidal silica, from about 1% to 2% of the concord grape extract, from about 0.1% to 0.2% of the cabernet grape extract, and from 0.4% to 0.5% of the red grape powder.

In a more specific example, the composition comprises muscadine pomace extract (containing 4.1% polyphenol content) at about 28.9% of total, trans-resveratrol at about 1.65% of total, purple carrot extract at about 0.16% of total, elderberry extract at about 0.48% of total, sorbitol at about 54.8% of total, a 99.7% glycerin composition at about 10% of total, colloidal silicon dioxide at about 2% of total, concord grape extract at about 1.27%, cabernet grape extract at about 0.16% and red grape powder at about 0.48%.

In another embodiment, the composition comprises from 23% to 25% of the total muscadine pomace extract, from 1% to 2% of the total resveratrol, from 0.1% to 0.2% of the total purple carrot extract, from 0.4% to 0.5% of the total elderberry extract, from 58% to 61% of the total sorbitol, from 9% to 11% of the total 99.7% glycerin composition, from 1.5% to 2% of the total colloidal silica, from about 1% to 2% of the concord grape extract, from 0.1% to 0.2% of the cabernet grape extract, and from 0.4% to 0.5% of the red grape powder.

In another more specific example, the composition comprises a muscadine pomace liquid extract (containing 5% polyphenol content) at about 23.77% of total, trans-resveratrol at about 1.65% of total, a purple carrot extract at about 0.16% of total, an elderberry extract at about 0.48% of total, sorbitol at about 60.05% of total, a 99.7% glycerin composition at about 10% of total, colloidal silicon dioxide at about 2% of total, a concord grape extract at about 1.27%, a cabernet grape extract at about 0.16% and a red grape powder at about 0.48%.

Any of the disclosed compositions can be provided in a non-beverage food, beverage, or liquid or solid dietary supplement. In certain examples, the disclosed compositions are provided as beverages. The compositions herein (particularly food, beverage and dietary supplement compositions) may be fortified with one or more nutrients, particularly one or more vitamins and/or minerals. The U.S. recommended daily intakes of vitamins and minerals (USRDI) are specified and described in the recommended daily dietary allowance of the food and nutrition board (food and nutrition board, national academy of sciences-national research and study) under the national academy of sciences — national research council (recommended daily dietary allowance). Unless otherwise specified herein, when a given mineral is present in a product, the product comprises at least about 1%, e.g., at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150% of the USRDI of such mineral. Unless otherwise specified herein, when a given vitamin is present in a product, the product comprises at least about 1%, e.g., at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 75%, at least about 100%, at least about 150% of the USRDI of such vitamin. The amount of vitamins and minerals added may depend on the processing method and the concentration of other ingredients in the formulation.

Non-limiting examples of such vitamins and minerals include iron, zinc, copper, calcium, phosphorus, niacin, thiamin, folic acid, pantothenic acid, iodine, vitamin a, vitamin C, vitamin B2, vitamin B3, vitamin B6, vitamin B12, vitamin D, vitamin E, and vitamin K. Commercially available sources of vitamins and minerals may also be included in the compositions of the present invention.

For example, any soluble salt of these minerals suitable for inclusion in an edible product may be used, such as magnesium citrate, magnesium gluconate, magnesium sulfate, zinc chloride, zinc sulfate, potassium iodide, copper sulfate, copper gluconate, and copper citrate. Sources of calcium may include, for example, amino acid chelated calcium, calcium carbonate, calcium oxide, calcium hydroxide, calcium sulfate, calcium chloride, calcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium citrate, calcium malate, calcium titanate, calcium gluconate, calcium realate, calcium tantrate, and calcium lactate, particularly calcium citrate-malate. As used herein, "vitamin a" includes, but is not limited to, retinol, beta-carotene, retinol palmitate, and retinol acetate. Vitamin a may be in the form of an oil, beads (beads) or encapsulated. Encapsulated ascorbic acid and edible salts of ascorbic acid may also be used. Sources of iodine include iodine-containing salts such as sodium iodide, potassium iodate, sodium iodate, or mixtures thereof. These salts may be encapsulated. Acceptable forms of iron are well known in the art. The amount of iron compound incorporated into the composition will vary widely depending on the level of supplementation desired in the final product and the consumer targeted. The iron fortifying compositions of the present disclosure typically comprise from about 5% to about 100%, e.g., about 15%, about 25%, about 40%, about 50%, about 70%, about 80%, or about 90% of the USRDI of iron.

Ferrous iron is typically better utilized by the body than ferric iron. Highly bioavailable ferrous salts that can be used in the ingestible compositions of the present disclosure are ferrous sulfate, ferrous fumarate, ferrous succinate, ferrous gluconate, ferrous lactate, ferrous tartrate, ferrous citrate, ferrous amino acid chelates, and mixtures of these ferrous salts. Although ferrous iron is typically more bioavailable, certain ferric salts can also provide a highly bioavailable source of iron. Highly bioavailable ferric salts that can be used in the food or beverage compositions of the present disclosure are ferric saccharate, ferric ammonium citrate, ferric sulfate, and mixtures of these ferric salts. Combinations or mixtures of highly bioavailable ferrous and ferric salts can be used in these edible mixtures and ready-to-eat beverages. In addition to these highly bioavailable ferrous and ferric salts, other sources of bioavailable iron can be included in the food and beverage compositions of the present disclosure. Other sources of iron particularly suitable for fortifying products include certain iron-sugar-carboxylic acid complexes.

The zinc compounds useful in the compositions of the present disclosure may take any of the commonly used forms, such as zinc sulfate, zinc chloride, zinc acetate, zinc gluconate, zinc ascorbate, zinc citrate, zinc aspartate, zinc picolinate, zinc amino acid chelates, and zinc oxide.

In certain examples, the food and beverage compositions may further comprise one or more dietary fibers. "dietary fiber" refers to complex carbohydrates that are resistant to digestion by mammalian enzymes, such as the carbohydrates found in plant cell walls and seaweeds, as well as carbohydrates produced by microbial fermentation. Examples of these complex carbohydrates are bran, cellulose, hemicellulose, pectin, gums and mucilages, seaweed extracts, and biosynthetic gums. Sources of cellulosic fibers include vegetables, fruits, seeds, cereals, and man-made fibers (e.g., synthesized by bacteria). Commercially available fibers such as purified plant cellulose or cellulose powder may also be used. Naturally occurring fibers include fibers from whole citrus peel, citrus endothelium, sugar beet, citrus pulp and capsule solids, apple, apricot and watermelon peel.

Beverage acidity can be adjusted to and maintained within the requisite range by known and conventional methods, e.g., using food grade acid buffers. Typically, beverage acidity within the above-mentioned range is a balance between the highest acidity for microbial inhibition and the optimum acidity for the desired beverage taste. In certain examples, the beverage composition has a pH of about 2 to about 8, such as about 2 to about 4.5 or about 2.7 to about 4.2.

Organic as well as inorganic edible acids can be used to adjust the pH of the beverage composition. The acid may be present in its undissociated form or, alternatively, as its corresponding salt, for example potassium or sodium hydrogen phosphate, potassium or sodium dihydrogen phosphate. In certain examples, the acid comprises citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, pyruvic acid, or a mixture thereof. Acidulants may also act as antioxidants to stabilize beverage components. Examples of commonly used antioxidants include, but are not limited to, ascorbic acid, EDTA (ethylenediaminetetraacetic acid) and salts thereof.

D. Muscadine compositions having antioxidant activity

Also discloses a preparation method of the muscadine pomace extract composition with antioxidant activity. The compositions of the present invention may be manufactured according to methods well known to the ordinary skilled artisan. In general, the compositions may be prepared by bringing together all the ingredients separately or suitably, dissolving, dispersing or otherwise mixing in water, where appropriate with a mechanical stirrer until all the ingredients have been dissolved or sufficiently dispersed. All of the separated solutions may be combined as appropriate. When a storage stable composition is desired, the final mixture may optionally be pasteurized or aseptically filled under suitable process conditions.

These methods can include combining a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% with resveratrol from a source other than muscadine having a minimum purity of at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%) wherein the ratio of muscadine polyphenols to trans-resveratrol is in the range of 0.1/1 to 10/1 (weight/weight), thereby producing a muscadine pomace extract and trans-resveratrol mixture having antioxidant activity. The disclosed methods may also include preparing a muscadine pomace extract and then combining the muscadine pomace extract with trans-resveratrol.

In one particular example, preparing the muscadine pomace extract includes combining a bronze muscadine pomace extract with a purple muscadine pomace extract. In other examples, preparing the muscadine pomace extract includes simultaneously extracting a mixture of bronze muscadine pomace and purple muscadine pomace. In certain examples, the muscadine pomace extract included in the compositions of the present disclosure is prepared according to the methods disclosed herein. However, it is contemplated that the muscadine pomace extract can be prepared according to methods known in the art, including concentrating the extract by crushing, pressing, extracting, filtering (several times), and evaporating under vacuum followed by freezing. In one example, only water is used in the extraction process, and no other ingredients such as solvents, carriers, or preservatives are added to the extract itself. The process is carried out under conditions that preserve the polyphenolic compounds while reducing the presence of other molecules such as most sugars. In other examples, the extraction process utilizes ethanol or a mixture of ethanol and water. In particular examples, the extraction process may further include the use of enzymes to clarify or facilitate extraction. For example, mixtures of enzymes from Aspergillus niger (Aspergillus niger) or pectinases may be used for these purposes. Examples of commercialization include ScottzymeKS and scottzymeec 5L from scott laboratories.

In other examples, the disclosed compositions are prepared by utilizing a muscadine pomace extract having a total phenolic concentration of at least 2%. For example, commercially available muscadine extracts having a total phenolic concentration of at least 2% can be used to prepare the disclosed compositions having antioxidant activity.

In particular examples, the method of preparing a composition having antioxidant activity can further comprise combining the muscadine pomace extract and resveratrol with an elderberry extract, a purple carrot extract, an excipient (e.g., glycerin, sorbitol, colloidal silicon dioxide, or a natural flavoring additive), or a combination thereof. In a specific example, the method includes combining a muscadine pomace extract (containing about 4% to about 5% polyphenol content) at 23% to 32% of the total composition with trans-resveratrol at 1.2% to 3.0% of the total, a purple carrot extract at 0.12% to 0.20% of the total, an elderberry extract at 0.4% to 0.6% of the total, sorbitol at 50% to 61% of the total, a 99.7% glycerin composition at 8% to 12% of the total, and colloidal silicon dioxide at 1% to 2% of the total.

In a more specific example, the composition includes a muscadine pomace extract (containing 4.1% polyphenol content) combined with resveratrol in a total amount of about 1.65%, a purple carrot extract in a total amount of about 0.16%, an elderberry extract in a total amount of about 0.48%, sorbitol in a total amount of about 54.8%, a 99.7% glycerin composition in a total amount of about 10%, colloidal silicon dioxide in a total amount, a concord grape extract in a total amount of about 1.27%, a cabernet grape extract in a total amount of about 0.16%, and red grape powder in a total amount of about 0.48%.

In another more specific example, the composition includes combining a muscadine pomace liquid extract (containing 5% polyphenol content) at about 23.77% of total amount with trans-resveratrol at about 1.65% of total amount, a purple carrot extract at about 0.16% of total amount, an elderberry extract at about 0.48% of total amount, sorbitol at about 60.05% of total amount, a 99.7% glycerin composition at about 10% of total amount, colloidal silicon dioxide at about 2% of total amount, a concord grape extract at about 1.27%, a cabernet grape extract at about 0.16% and a red grape powder at about 0.48%.

In the preparation of a ready-to-drink composition, a beverage concentrate may optionally be formed first. One method of preparing a beverage composition in a concentrated form begins with a volume of water that is less than the volume of water required in the preparation of the beverage composition. Another approach is to partially dehydrate the finally prepared beverage composition to remove only a portion of the water and any other volatile liquids present. Dewatering can be accomplished according to well known procedures such as vacuum evaporation. The concentrate may take the form of a relatively concentrated liquid. Thick syrup is typically formed by adding suitable ingredients, such as electrolytes or emulsions, to the beverage concentrate. The thick syrup is then mixed with water to form a finished beverage or finished beverage concentrate.

Substantially dry mixtures of the disclosed compositions can be prepared by mixing together all of the desired dry ingredients in appropriate amounts and ratios. Optionally, the finally prepared beverage composition may be dehydrated to give a substantially dry mixture of the beverage composition. The substantially dry mixture, as for example a powder, granules or tablets, can be later dissolved in an appropriate amount of carbonated or non-carbonated water to make the final drinkable beverage, or taken with water.

As a form of food, the disclosed composition may be formulated in any optional form, such as a granular state, a grain state, a paste state, a gel state, a solid state, or a liquid state. In these forms, various other substances may be included, such as binders, disintegrants, thickeners, dispersants, reabsorption promoters, flavoring agents, buffers, surfactants, dissolution aids, preservatives, emulsifiers, isotonicity agents, stabilizers, or pH control agents. In particular examples, when the disclosed composition is used as a health-preserving (including but not limited to skin-improving) food, functional food, or the like, it preferably comprises the active ingredients of the compositions and extracts of the present invention (e.g., muscadine pomace extract, resveratrol, purple carrot extract, and elderberry extract) in amounts such that the predetermined effects of the present disclosure are exhibited sufficient to provide antioxidant activity.

E. Methods and kits for inhibiting (e.g., preventing) cellular aging

Disclosed compositions having improved lipophilic antioxidant activity are shown herein. Oxidative stress is known to be the central mechanism responsible for normal aging. Lipophilic antioxidants are also known to inhibit various types of skin damage. Based on these observations, methods of inhibiting cellular senescence, e.g., inhibiting or reducing the production and activity of free radicals, are disclosed. In one example, a method comprises administering to a subject in need thereof a disclosed composition having antioxidant activity in an amount sufficient to inhibit or reduce one or more processes associated with cellular aging. For example, inhibiting one or more processes associated with cellular aging includes reducing or inhibiting the formation or activity of free radicals in a subject ingesting the composition.

In certain disclosed embodiments, the methods comprise administering an ingestible composition, such as a composition described herein, comprising a muscadine (vitis rotundifolia) pomace extract derived from bronze muscadine pomace and purple muscadine pomace. In certain embodiments, the ratio of bronze muscadine pomace extract to purple muscadine pomace extract in the muscadine pomace extract ranges from 0.1 to 10 (weight to weight), such as 0.3 to 3 (weight to weight). For example, the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is about 2.75 to about 1 (weight to weight), 2.5 to about 1 (weight to weight), about 2.25 to about 1 (weight to weight), about 2 to about 1 (weight to weight), about 1.5 to about 1 (weight to weight), or about 1 to about 1 (weight to weight). The term "about" as used herein is defined as ± 0.5. In a specific example, the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is about 2.25 to about 1 (weight to weight).

In certain embodiments, the muscadine (vitis rotundifolia) pomace extract administered orally has a polyphenol content of at least 2%. For example, the polyphenol content is at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 12%, or at least 14%. In a specific example, a muscadine (vitis rotundifolia) pomace extract has a polyphenol content of about 4%.

In certain embodiments, the methods comprise administering an ingestible composition comprising a muscadine pomace extract along with other additional ingredients, including, but not limited to, resveratrol. In certain examples, the methods comprise administering an oral composition having a ratio of muscadine polyphenol to resveratrol of about 0.75 to 1 (weight/weight). In other examples, the method comprises administering a composition comprising at least from 8% to 60% of a total composition of a muscadine pomace extract (comprising from 2% to 14%, e.g., 4%, 6%, 8%, 10%, or 12% muscadine polyphenols by weight of the liquid) and from 1.2% to 3.0% of total trans-resveratrol.

In other embodiments, the methods comprise administering an oral composition comprising a muscadine pomace extract having a polyphenol content of at least 2% and resveratrol from a source other than muscadine (e.g., a polygonum cuspidatum root extract) having a minimum purity of at least 5%, wherein the ratio of muscadine polyphenols to trans-resveratrol is in the range of 0.1/1 to 10/1 (weight/weight), thereby providing a composition having antioxidant activity sufficient to inhibit or reduce one or more processes associated with cellular aging, including but not limited to improving skin texture. In a particular example, the method comprises administering a composition comprising a muscadine (vitis rotundifolia) pomace extract having a polyphenol content of at least 2% and resveratrol from a source other than muscadine (e.g., a polygonum cuspidatum root extract) having a minimum purity of at least 5%, wherein the ratio of muscadine pomace extract to resveratrol is in the range of 0.2/1 to 50/1 (weight/weight), such as 5/1 to 50/1 (weight/weight), or 20/1 to 50/1 (weight/weight), thereby providing a composition having antioxidant activity. In certain examples, the composition has a total ORAC of at least 24 μmolTrolox equivalents per mg polyphenol (μmolTE/mg polyphenol).

In other embodiments, the application of a solution or topical ointment containing muscadine pomace extract alone or in combination with other active ingredients, including but not limited to resveratrol, provides a method of improving skin quality by inhibiting or reducing free radical formation or activity in skin cells. In one example, a solution or topical ointment comprises the disclosed muscadine pomace extract without resveratrol. In another example, a solution or topical ointment includes both the muscadine pomace extract and resveratrol disclosed, e.g., the muscadine pomace extract includes muscadine polyphenol concentrations such that the ratio of muscadine polyphenol to resveratrol is about 0.75 to 1 (weight to weight). The method may be performed by a clinician or other health care provider. The method is also designed for home use. The method can reduce aging-related changes in skin appearance, visibly reduce wrinkles in human skin, and improve the texture quality of the skin. Also provided are compositions and kits for improving skin condition that may comprise the disclosed compositions (including oral and topical) having antioxidant activity or muscadine extract with one or more other anti-aging compositions, such as one or more other antioxidants.

Any skin surface (e.g., the epidermis of the skin) can be treated using the methods provided herein. Skin surfaces that may be treated include, but are not limited to, the eye orbit, lips, cheeks, nasolabial folds, forehead, neck, upper labial folds (rhytides), stomach, neck, back, chest, hands, legs, feet, or any combination thereof. In an example, the skin of any facial surface can be treated using the methods provided herein. The method may be applied to any facial area and/or any body surface area, other areas that may be directly applied are the chest and neck. More than one skin surface may be treated during the same treatment. In a specific example, the composition or extract of the present disclosure in liquid or cream form is applied substantially uniformly over the skin surface, forming a layer of the composition or extract on the skin.

The disclosed compositions or extracts may contain other materials conventionally used in cosmetics, such as perfumes; an antimicrobial agent; an antibacterial agent; adding a fat liquor; complexing and chelating agents; a pearlescent agent; a plant extract; vitamins such as retinol or vitamin C; an active agent; a preservative; a bactericide; a surfactant; dyes, pigments or any substance having a coloring effect; an emulsifier; a thickener; softening, moisturizing and/or hydrating substances; or other common constituents of cosmetic or dermatological preparations, such as alcohols, polyols, polymers, foam stabilizers, electrolytes, organic solvents or silicone derivatives. The composition may also contain functional additives such as keratolytic agents, oxidizing agents, sunscreens and skin smoothing agents. The composition may also contain components believed to be beneficial in the injection therapy of mesoderm in the skin and underlying subcutaneous tissue, including antioxidants such as dimethylaminoethanol, alpha-lipoic acid and ascorbic acid. The composition may also contain agents that enhance the efficacy of the composition or extract, such as lipophilic and lipophobic permeation enhancers (e.g., triethyl citrate, propylene glycol, fatty acid esters, and the like). Agents that enhance vascular perfusion of the skin, such as aminophylline or pentoxifylline, may also be included in the composition. The compositions may also contain agents that increase the filling and tone of the skin and allow the underlying subcutaneous tissue structures to contract or contract, such as phosphatidylcholine and deoxycholic acid sulfate. Physiological substances that can provide hormonal benefits, such as substances that cause estrogen or androgen stimulation of the skin, including estriol and testosterone, can also be included in the composition.

The disclosed muscadine compositions may include one or more preservatives. Such preservatives include, for example, Opthiphen^TM(from International specialty products, Inc.)）、(from Lonza corporation), preservatives listed in the european union cosmetic directive, and the like, for example, formaldehyde donors (e.g., DMDM hydantoin, which may be available under the trade name DMDM hydantoin)Hereinafter from Lonza corporation), iodopropyl butylcarbamate (e.g., as available under the trade name glycoacil-STM from Lonza corporation and/or dekabenkker corporation under the trade name dekabenmbtm), parabens (e.g., alkyl esters of parabens, such as methyl, ethyl, propyl, and/or butyl parabens), phenoxyethanol, ethanol, benzoic acid, and salicylic acid. The preservative system may also comprise a preservative aid such as octoxyglycerol or soybean oil (glycerinesoya). Other preservatives or preservative aids include dibromocyanobutane (2-bromo-2-bromomethylglutaronitrile), 3-iodo-2-propynylbutylcarbamate, 2-bromo-2-nitropropane-1, 3-diol, imidazolidinyl urea, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-chloroacetamide, benzalkonium chloride, and benzyl alcohol.

The composition may also comprise one or more conditioning agents, such as water soluble conditioning agents. Other conditioning agents include compounds known as polyquaternary ammonium salts, for example according to International Nomenclature for Cosmetic Ingredients (INCI), particularly polyquaternary ammonium salt-1 through polyquaternary ammonium salt-56.

The composition may be dispensed from a hose, a jar, a bottle, a pump, a can, a spray can or a spray bottle, or from some other known container.

Also disclosed herein are muscadine pomace extract compositions and kits for use at home or by a clinician for improving skin quality. In one example, the kit is suitable for use by a clinician or cosmetologist. In such an example, the kit may further comprise an applicator for assisting in the application of the composition. Typically, the kit further comprises instructions for use. These instructions may be written or in a digital format (e.g., videotape, DVD, or CD) for an electronic device such as a computer, CD player, mp3 player, or DVD player, etc. In another example, the kit is suitable for use at home. The kit may include an applicator, such as a sponge or cloth, for applying the composition. Alternatively, the composition may be applied using one or more fingers. Generally, the kit will also include instructions for use. These instructions may be written or in a digital format (e.g., videotape, DVD, or CD) for an electronic device such as a computer, CD player, mp3 player, or DVD player.

In certain examples, a kit comprises one composition or extract of the present disclosure and one or more additional anti-aging compounds, such as another antioxidant (e.g., vitamin C, vitamin E, selenium, and/or beta-carotene), in two separate containers or as a single composition in a single container. In one example, the composition or extract is applied to a target skin area. In a specific, non-limiting example, the muscadine pomace extract anti-aging kit can be used by an individual at home as follows. First, an appropriate amount of the composition or extract is applied to the skin surface by using an applicator, such as a sponge or cloth, or by using one or more fingers, to provide a uniform layer of the composition or extract on the skin surface. The composition or extract is allowed to passively absorb into the skin surface over a length of time, or absorption of the composition/extract by the skin surface may be facilitated by gentle rubbing of the composition into the skin surface with the fingertips. Generally, after application of the muscadine pomace composition/extract, normal skin care procedures, such as applying makeup and applying other moisturizers, can be continued.

This process may be performed as described twice daily, once every two days, twice weekly, once every two weeks, or once monthly, or using some other time interval, such as once every 3 to 5 days. Improving skin condition includes reversing, slowing down the progression of, or preventing skin changes associated with natural or intrinsic aging. As used herein, deterring and variations thereof refers to any degree of delay in the occurrence of skin changes. For example, improving skin quality includes reversing, slowing the progression of, or preventing skin changes associated with free radical formation and activity. In one example, improving skin quality includes reversing, slowing the progression of, or preventing sun damage or photoaging skin changes associated with exposure to sunlight or other forms of actinic radiation (e.g., UV radiation and tanning machines). As another example, improving skin quality may also include reversing, slowing the progression of, or preventing skin changes caused by external factors including, but not limited to, radiation, air pollution, wind, cold, moisture, heat, chemicals, smoke, smoking, and combinations thereof. Improving skin quality may also include reversing, arresting or reducing scarring that may be caused by certain skin conditions (e.g., acne), infections (e.g., leishmaniasis), or injuries (e.g., abrasions, punctures, lacerations or surgical wounds). The improvement in skin may also include at least one of: making facial lines appear less prominent, filling facial lines and/or wrinkles, improving the appearance of infraorbital lines and/or periorbital lines, improving the appearance of fishtail lines, reducing and/or eliminating the appearance of wrinkles, particularly facial wrinkles on the cheeks, forehead (e.g., vertical wrinkles between the eyes, lateral wrinkles above the eyes) and/or around the mouth, and particularly the appearance of deep wrinkles, folds, or creases, improving skin softness, reducing and/or eliminating fine and/or deep lines, folds, and creases, and smoothing the skin. Methods for measuring skin improvement are known in the art. See, for example, U.S. patent nos. 6,866,856 and 6,682,763.

Skin changes that can be treated by practicing the methods disclosed herein and using the kits disclosed herein include, for example, wrinkles (including but not limited to human facial wrinkles), folds, deep lines, folds and fine lines, deepening of skin lines, thinning of skin, preventing or reducing the appearance of scarring, yellowing of skin, formation of color spots, hyperpigmentation, pigmentation and/or non-pigmented age spots, dermatification, loss of elasticity, loss of resilience, loss of collagen fibers, abnormal changes in elastic fibers, degeneration of dermal small blood vessels, formation of visible blood vessels on the skin surface with increased sun exposure, and combinations thereof.

Improving skin condition includes reducing, alleviating, and/or minimizing one or more of the skin changes discussed above. Improving skin quality can result in skin having a younger appearance. Improving skin texture can result in the skin having a smoother, more watery (less dry) or less flaky appearance. For example, in certain embodiments, improving skin quality may include reducing roughness, dryness, or flaking. Improving skin quality includes removing and improving lines and wrinkles, improving filling and tone, with the desired lifting and tightening effects observed.

The texture properties of the skin, including softness, suppleness and smoothness, can be improved, resulting in an increase in shine, purity and brightness. Other and important characteristics of the skin that can be measured objectively and subjectively include, but are not limited to, skin laxity or, conversely, skin firmness, and the presence and degree of textural fine lines and coarser lines in the skin.

These properties are the same as those judging apparent external conditions (e.g. skin aging). Improving these properties through the treatment methods and kits disclosed herein yields benefits based on visual judgment of appearance. The skin is altered by the methods disclosed herein, reducing the appearance of skin aging.

The desired benefits may include not only physiological benefits to the skin, but also therapeutic and pharmacological benefits, such as possible malignancy prevention and treatment, whether by chemoprevention or modified photodynamic therapy. Benefits may also include acne treatment and inhibition by including compositions that inhibit sebaceous gland activity.

F. Pharmaceutical composition

The disclosed muscadine pomace extracts and compositions are useful at least for inhibiting (e.g., preventing) one or more oxidative processes, such as free radical formation associated with cellular events such as cellular aging. Accordingly, also described herein are pharmaceutical compositions comprising at least one disclosed muscadine pomace extract alone or in combination with resveratrol.

The formulation of pharmaceutical compositions is well known in the art. For example, Remington pharmacology, e.w. martin (Remington's pharmaceutical sciences), mack publishing co, Easton, PA,19 th edition, 1995, describes exemplary formulations (and components thereof) suitable for drug delivery of at least one of the disclosed muscadine pomace extracts, alone or in combination with resveratrol. In certain examples, the composition further comprises other agents such as purple carrot root extract or elderberry extract. Pharmaceutical compositions comprising at least one of these compounds may be formulated for use in human or veterinary medicine. The specific formulation of the disclosed pharmaceutical compositions may depend, for example, on the mode of administration (e.g., oral, topical, or parenteral) and/or the condition to be treated (e.g., production or activity of free radicals). In certain embodiments, the formulation includes a pharmaceutically acceptable carrier and at least one active ingredient, such as a muscadine pomace extract.

Pharmaceutically acceptable carriers useful in the methods and compositions of the present disclosure are conventional in the art. The nature of the pharmaceutically acceptable carrier will depend on the particular mode of administration used. For example, parenteral formulations typically comprise an injectable fluid as a medium, which includes a drug and a physiologically acceptable fluid such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. For solid compositions such as powder, pill, tablet or capsule forms, conventional non-toxic solid carriers may be included such as pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to the biologically neutral carrier, the pharmaceutical composition to be administered may optionally contain minor amounts of non-toxic auxiliary substances or excipients such as wetting or emulsifying agents, preservatives, pH buffering agents and the like; such as sodium acetate or sorbitan monostearate. Other non-limiting excipients include non-ionic solubilizers such as cremophor (cremophor), or proteins such as human serum albumin.

The disclosed pharmaceutical compositions may be formulated as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are non-toxic salts of the free base form of the compounds which possess the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydroiodic acid, and phosphoric acid. Non-limiting examples of suitable organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, aspartic acid (aspartic acid), benzenesulfonic acid, methylbenzenesulfonic acid, naphthalenesulfonic acid, and the like. A further list of suitable pharmaceutically acceptable salts can be found in Remington pharmacy (Remington's pharmaceutical sciences), 19 th edition, Mack publishing company, Easton, PA, 1995. Pharmaceutically acceptable salts may also be used to adjust the osmotic pressure of the composition.

The dosage form of the disclosed pharmaceutical compositions is determined by the mode of administration selected. For example, oral dosage forms may be used. Oral formulations may be liquids such as syrups, solutions or suspensions, or solids such as powders, pills, tablets or capsules. Methods of preparing these dosage forms are disclosed herein or will be apparent to those skilled in the art.

Certain embodiments of pharmaceutical compositions comprising a compound of the present disclosure may be formulated in unit dosage forms suitable for single administration of precise dosages. The amount of active ingredient administered depends on the subject to be treated, the severity of the disease and the mode of administration and is well known to those skilled in the art. Within these limits, the formulation to be administered comprises an amount of the extract or compound disclosed herein that is effective to achieve the desired effect in the subject. In a specific example, for oral administration, the composition is provided in a liquid form containing about 75mg of muscadine pomace extract polyphenol and 100mg of trans-resveratrol. In other examples, for oral administration, the compositions are provided in the form of tablets containing from about 1.0 to about 500mg of the active ingredient, particularly about 10.0mg, about 50mg, 100mg, about 150mg, about 175mg, about 200mg, about 210mg, about 225mg, about 250mg, about 300mg, about 400mg, or about 500mg of the active ingredient, for symptomatic adjustment of the dosage to the subject. In an exemplary oral dosage regimen, tablets containing from about 1mg to about 500mg (e.g., from about 150mg to about 225 mg) of the active ingredient are administered at least once daily. In other examples, tablets containing from about 1mg to about 500mg (e.g., from about 150mg to about 225 mg) of active ingredient are administered at least once a week, such as at least twice, three times, at least four times, at least five times, or at least six times a week, or daily.

G. Administration of the disclosed extracts and compositions

The methods of administration of any of the disclosed compositions and extracts are conventional. For example, the disclosed methods (e.g., methods comprising compositions having antioxidant activity for preventing or reducing one or more processes associated with aging) can be administered by injection, intratumorally, orally, topically, transdermally, parenterally, or by inhalation or spray. In particular examples, the compositions are orally administered to a mammalian subject, such as a human, in the form of a non-beverage food, beverage, or dietary supplement. In another example, the composition is topically applied to the skin surface of a mammalian subject, such as a human.

The therapeutically effective amount of the agent administered may vary depending on the desired effect and the subject to be treated. In one example, the method comprises administering at least 1mg of the composition to a subject (e.g., a human subject) daily. For example, a human may administer at least 1g or at least 10g of the composition per day, e.g., 1g to 5g per day, 5g to 10g per day, e.g., 7g per day. In one example, the subject is administered at least 5g of a composition comprising a muscadine pomace extract and resveratrol. In other examples, the subject is orally administered at least 6.3g of such a composition. The dose may be administered in divided doses (e.g. 2, 3 or 4 divided doses per day) or in a single dose per day.

In particular examples, a therapeutic composition (e.g., the disclosed anti-aging supplement containing resveratrol and muscadine extract) is administered to a subject in a multi-dose daily dosing schedule, e.g., for a period of at least two consecutive days, 10 consecutive days, etc., e.g., weeks, months, or years. In one example, a therapeutic composition (e.g., the disclosed anti-aging supplement containing resveratrol and muscadine extract) is administered to a subject comprising daily administration for a period of at least 30 days, e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

Therapeutic compositions, such as the disclosed anti-aging supplements comprising trans-resveratrol and muscadine pomace extract or muscadine pomace extract alone, may also include one or more bioactive or inactive compounds (or both), such as other antioxidants and conventional non-toxic pharmaceutically acceptable carriers, respectively. In particular examples, the therapeutic composition further comprises one or more biologically inactive compounds. Examples of such biologically inactive compounds include, but are not limited to: carriers, thickeners, diluents, buffers, preservatives and carriers. Pharmaceutically acceptable carriers which can be used in these formulations are conventional (see e.w. martin "Remington pharmacology" (Remington's pharmaceutical sciences), mack publishing co., Easton, PA,19 th edition (1995)). In general, the nature of the carrier will depend on the particular mode of administration to be used. For example, parenteral formulations may include injectable fluids as the medium, including drugs and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the pharmaceutical composition to be administered may include minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monostearate.

Although the present disclosure provides a detailed description of the use of any of the disclosed compositions and extracts to prevent or inhibit one or more processes associated with cellular aging, it is contemplated that the disclosed compositions and extracts may be used not only as antioxidants to prevent or inhibit one or more processes associated with cellular aging, but also to treat any diseases associated with oxidative stress. For example, it is contemplated that the extracts and compositions of the present invention may be used to reduce, prevent or treat oxidative stress associated with the pathogenesis of chronic inflammatory diseases such as diabetes, cancer, atherosclerosis and other cardiovascular diseases, as well as with degenerative diseases such as Alzheimer's disease and Parkinson's disease.

The presently disclosed subject matter is further illustrated by the following non-limiting examples.

Detailed Description

Example 1

Determination of ellagic acid solubility in muscadine pomace extract

This example illustrates the effect of various ratios of bronze to purple pomace extracts on ellagic acid solubility.

The separately fermented bronze and purple pomace extracts were heated and then mixed in the ratios provided in table 1 to a total volume of 100 ml.

TABLE 1.1 various ratios of bronze/purple pomace extracts at X concentrations

Bronze (ml)	Purple (ml)
		75	25
65	35
		50	50
25	75

The resulting extract mixture was 1X concentrated because the bronze and purple pomace extracts were not concentrated separately. A 1X concentration typically contains about 2% solids solution (100 grams of solution equals 2 grams of dry extract). Approximately 20 ml of each ratio was transferred to a corresponding glass test tube and sealed with a foil. The sample was heated at 85 ℃ for 30 minutes and then removed from the heat source and allowed to return to room temperature. The samples were then filtered through a 0.45 μm TFE filter w/GMP and analyzed by HPLC/MS to determine their ellagic acid content. Bronze and purple muscadine pomace extracts were also analyzed separately by the same method to determine the baseline value of ellagic acid in both bronze and purple pomace extracts and to determine the anthocyanin content in purple pomace extracts. Then passed through a reversed phase HPLC tandem ion trap mass detector with UV-VIS detection at 254nm, using at [ M-H ]]^-Extracted Ion Chromatogram (EIC) at 301amu, determining the total area of ellagic acid in the sample.

To prepare bronze/purple pomace extracts at various ratios containing 40% solids, 1X bronze and purple muscadine pomace extracts were each dried to powder form and reconstituted in water to produce a liquid containing 40% solids. The two separate solutions are then mixed in various ratios by weight (wt) rather than volume. The various ratios evaluated are provided in tables 2A and 2B.

TABLE 2 determination of ellagic acid solubility in various bronze/purple extract ratios at A.1X concentrations

TABLE 2B determination of ellagic acid solubility in various bronze/purple extract ratios at about 20X concentration or 40% solids level

Approximately 10 ml of each ratio was transferred to a corresponding glass test tube and sealed with a foil. The sample was heated at 85 ℃ for 30 minutes and then removed from the heat source and allowed to return to room temperature. The samples were then filtered through a 0.45 μm TFE filter w/GMP and analyzed by HPLC/MS to determine their ellagic acid content. Bronze and purple muscadine pomace extracts were also analyzed separately by the same procedure to determine baseline values of ellagic acid in both bronze and purple muscadine pomace extracts, and to determine the anthocyanin content in purple muscadine pomace extracts. Using validated test method C205, tandem ion trap mass Detector, [ M-H ]]^-Extracted Ion Chromatogram (EIC) at 301amu, determining the total area of ellagic acid in the sample.

These studies found that at 1X concentration levels, a ratio (volume/volume) of 65% to 35% bronze to purple pomace extract was sufficient to promote maximum solubility of ellagic acid in the mixture of the two pomace extracts. Furthermore, at the 40% solids level, the solubility of ellagic acid continues to increase as the content of purple muscadine pomace extract in the mixture increases. These findings indicate that at 40% solids level, a greater amount of ellagic acid is present in the solid form, thus requiring a higher content of purple pomace extract to completely dissolve all ellagic acid present in the mixture. These studies may also indicate that anthocyanin contained in purple muscadine pomace extract affects the solubility of ellagic acid contained in bronze muscadine pomace extract.

Example 2

Anti-aging dietary supplement and preparation thereof

The present embodiments provide dietary supplements that can be consumed to prevent or inhibit one or more processes associated with cellular aging.

The disclosed anti-aging dietary supplement is prepared according to the formulations provided in table 3a or table 3b. TABLE 3A. anti-aging dietary supplement

TABLE 3B anti-aging dietary supplements

The dietary supplement includes instructions for dosing. The instructions indicate that 5 ml of supplement (about 1 teaspoon) can be taken daily prior to a meal and used as an anti-aging supplement, for example, to inhibit one or more processes associated with cellular aging.

Example 3

Antioxidant capacity of muscadine pomace extract and polygonum cuspidatum root extract mixture and anti-aging supplement

This example demonstrates the improved antioxidant capacity of mixtures and supplements containing giant knotweed extract and muscadine pomace extract as measured by Oxygen Radical Absorbance Capacity (ORAC) analysis.

The antioxidant capacity of the two plant extracts, alone or in combination in a mixture or supplement, was evaluated using the ORAC assay. This assay has been used to measure the antioxidant capacity of a wide variety of foods and beverages and is the basis for the data contained in the USDAORAC database. Both hydrophilic antioxidant capacity and lipophilic antioxidant capacity can be measured by this test.

The following samples were analyzed: (1) dried/powdered polygonum cuspidatum root extract normalized to a minimum of 98% trans-resveratrol (actual content is 100% trans-resveratrol); (2) a dry/powdered muscadine pomace extract (bronze to purple pomace in a 2:1 ratio) containing 14.4% total polyphenol content; (3) mixing the above dried/powdered rhizoma Polygoni Cuspidati and dried/powdered muscadine pomace extract at a ratio of 1:5.36 (wt: wt) (total polyphenol content is 27.5% of the mixture, ratio of muscadine polyphenol to trans-resveratrol is 0.75 to 1); and (4) the anti-aging supplements provided in table 3a of example 2. The hydrophilicity, lipophilicity and total ORAC values were measured (total ORAC value is the sum of the hydrophilicity and lipophilicity values) and the results are expressed as μmolTrolox equivalents per mg polyphenol (μmolTE/mg polyphenol). The results are shown in table 4.

TABLE 4 hydrophilic, lipophilic and Total ORAC values expressed as μmolTE/mg polyphenol for samples 1-4.

As shown in table 4, the measured value of the hydrophilicity ORAC of the mixture was close to the value predicted from the additive effect of the two extracts. However, the measured lipophilicity ORAC value of the mixture was 5-fold higher than the predicted accumulation, resulting in a 20% increase in the total ORAC value. Similar synergy was observed for the compositions, approaching 3-fold the predicted ORAC score based on additivity. The results shown in table 5 are expressed as ORAC values per gram of material (relative to the values shown in table 4 for each mg of polyphenol). As shown in table 5, the synergistic effect of the muscadine pomace extract and polygonum cuspidatum root extract mixture and the anti-aging supplement in producing lipophilic antioxidant capacity was maintained when the values were expressed as ORAC per gram of material:

TABLE 5 hydrophilic, lipophilic and total ORAC values expressed as ORAC values per gram of material for samples 1-4.

These studies demonstrate a strong synergistic effect of muscadine pomace extract and polygonum cuspidatum root extract mixture and anti-aging supplement in producing lipophilic antioxidant capacity. The selectivity synergy shown under lipophilic conditions is unexpected. Thus, the present findings demonstrate the increased antioxidant capacity of a mixture containing a polygonum cuspidatum extract and a muscadine pomace extract when compared to either extract alone, as measured by the ORAC assay.

Example 4

Antioxidant capsule preparation and preparation

The present example provides antioxidant capsule formulations and their preparation that can be used to inhibit one or more oxidative processes, including preventing or inhibiting free radical formation.

An aqueous extract of bronze to purple pomace having a solids content of 40% and a polyphenol content measured by Gallic Acid Equivalent (GAE) of 10% in solids in a ratio of 2:1 was treated as follows: to 10Kg of the above-mentioned pomace concentrate, 1.33Kg of maltodextrin MD100 was added. The mixture was mixed to dissolve the maltodextrin. Subsequently, the liquid concentrate was freeze-dried to yield a dry material. The chemical values of the dried muscadine/maltodextrin material were as follows: (1) total polyphenols (as% GAE), NLT7.0% (by dry weight); gallic acid (HPLC), NLT0.3% (by dry weight), and free ellagic acid (HPLC), NLT0.15% (by dry weight). The characteristics of the dried muscadine/maltodextrin are provided in table 6.

TABLE 6 characteristics of dried muscadine/maltodextrin materials

Appearance:	magenta powder
		And (3) taste:	characteristics of grape extract
Odor:	characteristics of grape extract

Capsules were prepared by weighing batches of each raw material. Passing the raw materials throughA grinder, which finally sends the maltodextrin to the grinder. The ground material was mixed until homogeneous and then filled into two "0" size capsules on an encapsulation machine. Examples of the dosage forms manufactured are provided in table 7 and the results of the analysis of muscadine/resveratrol capsules are provided in table 8.

TABLE 7 dosage forms manufactured-3 capsules/dose

Raw materials	Statement/dosage	Excess amount	RM assay	mg/dose	mg/capsule
						muscadine/MD 100	75mg	10%	75mg/g	1100	366.67
Trans-resveratrol	100mg	10%	98%	112.245	37.415
						Microcrystalline cellulose				195	65.0
Cabosil				25.0	8.333
						Magnesium stearate				14.0	4.667
Maltodextrin				203.755	67.918
									Total up to	1650	550

TABLE 8 muscadine/resveratrol Capsule analysis results

Test parameters	Specifications (each capsule)	Measurement/results
			Appearance:	two-piece '0' dark purple capsule	Conform to
Odor:	characteristics of grape extract	Conform to
			Capsule size:	0.864”-0.876”	conform to
Total weight (10 capsules):	6.36-6.75g	6.48gm
			filling weight	0.539-0.578g	0.556gm
Disintegration time:	not more than 45min	28min
			Polyphenol (as GAE)	Minimum 58.3mg	61.34
Trans-resveratrol	Minimum 33.3mg	36.8

Example 5

Reducing oxidative stress in a human subject

This example demonstrates the in vivo efficacy of the antioxidant capsules described in example 4, as evaluated in a placebo-controlled clinical trial.

The capsules contained a mixture of muscadine pomace extract and polygonum cuspidatum extract providing 75mg muscadine polyphenols and 100mg trans-resveratrol per 3 capsules (tables 7 and 8). It is known that eating a meal rapidly increases oxidative stress and produces an inflammatory response. For example, it has been shown that in healthy normal weight humans, consumption of 900kcal snack foods causes a significant increase in the production of Reactive Oxygen Species (ROS) in circulating Monocytes (MNC) and polymorphonuclear leukocytes (PMN) (Aljada et al, am.J.Clin.Nutr.79: 682-. These changes are accompanied by elevated expression of the p47phox protein in MNCs, a subunit of NADPH oxidase, a mediator of ROS production.

To test the effect of antioxidant capsules in this postprandial oxidative stress model, a group of 10 healthy subjects (age 37 ± 4 years, BMI22.6 ± 0.5kg/m 2) was provided 910kcal snack food on two separate days and a single dose (3 capsules) of antioxidant formulation or placebo (3 capsules) was taken 10 minutes prior to a meal. Blood samples were collected at baseline and at 1,3 and 5 hours post-meal. Circulating concentrations of glucose, insulin and lipids were similar after each treatment, indicating that the disclosed antioxidant capsules did not alter digestion/absorption of the diet. However, the disclosed antioxidant capsule treatment greatly reduced the increase in ROS production in both MNC (15% versus 62% placebo) and PMN (8% versus 64% placebo) 1 hour after the meal. Furthermore, ingestion of diet plus placebo resulted in a significant increase in P47phox protein levels in MNCs of 148% above baseline (P < 0.05), while ingestion of diet plus antioxidant capsules completely prevented any significant change in P47phox levels in MNCs (P <0.05 using two-way RMANOVA). Furthermore, at 3 hours after ingestion of the diet plus antioxidant capsules, the DNA binding activity of the antioxidant stress transcription factor Nrf-2 was significantly increased, 150% above baseline (P <0.05 with RMANOVA and two-way RMANOVA), compared to the placebo treated group, where the DNA binding activity of the protective transcription factor was actually decreased. Nrf-2 binds to promoter sequences of Antioxidant Response Elements (ARE) of multiple genes encoding endogenous antioxidant enzymes (e.g., glutathione-S-transferase), and thus an increase in DNA binding activity of Nrf-2 upon ingestion of the disclosed antioxidant preparations suggests the potential for upregulation of various cellular defense mechanisms.

Finally, plasma endotoxin levels increased significantly after ingestion of diet and placebo, exceeding 60% at baseline (P < 0.05) at 3 hours, while endotoxin concentrations decreased significantly when the diet was consumed with the disclosed antioxidant capsules, being below 28% at 1 hour (P <0.05 using rmanovoa and two-way RMANOVA). Endotoxin is a powerful inflammatory signal that contributes to the activation of various cytokines that increase oxidative stress.

These results demonstrate the novel in vivo antioxidant effect of a mixture of muscadine pomace extract and polygonum cuspidatum (muscadine polyphenol to trans-resveratrol ratio of 0.75 to 1), thus extending the in vitro discovery of the synergistic antioxidant capacity of this mixture (example 3).

Based on these findings, a method for suppressing oxidative stress can be provided. For example, a composition of the present disclosure is administered to a subject to inhibit oxidative stress, such as that associated with a meal. In one example, the disclosed compositions are administered 30 minutes before or after a meal, e.g., 10 minutes before or after a meal. In a specific example, the composition is administered about 10 minutes before eating.

Since the principles of the present disclosure may be applied to many possible embodiments, it should be recognized that the described embodiments are only examples of the disclosed subject matter and should not be taken as limiting the scope of the present disclosure. Rather, the scope of the disclosure is defined by the claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims (34)

1. A method of producing a muscadine pomace extract, the method comprising:

combining a bronze muscadine pomace extract with a purple muscadine pomace extract, wherein the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is in the range of 0.1 to 10 on a weight/weight basis to produce a muscadine pomace extract with increased ellagic acid solubility, and the muscadine pomace extract has a polyphenol content of at least 2%.

2. A muscadine pomace extract composition comprising bronze muscadine pomace extract and purple muscadine extract, wherein the ratio of bronze muscadine pomace extract to purple muscadine pomace extract is in the range of 0.1 to 10 on a weight/weight basis, the mixture of bronze muscadine pomace extract and purple muscadine extract increases the solubility of ellagic acid in the extract, and the muscadine pomace extract has a polyphenol content of at least 2%.

3. The method of claim 1, wherein the muscadine pomace extract has a total polyphenol content of 4%.

4. The composition of claim 2, wherein the muscadine pomace extract has a total polyphenol content of 4%.

5. The method of claim 1 or 3, further comprising preparing a bronze muscadine pomace extract and a purple muscadine pomace extract prior to combining the bronze muscadine pomace extract with the purple muscadine pomace extract to produce the muscadine pomace extract having a polyphenol content of at least 2%.

6. The method of claim 5, wherein preparing the bronze muscadine pomace extract and the purple muscadine pomace extract comprises extracting the bronze muscadine pomace and the purple muscadine pomace with water to produce the bronze muscadine pomace extract and the purple muscadine pomace extract, respectively, and then combining the bronze muscadine pomace extract with the purple muscadine pomace extract to produce the muscadine pomace extract having a polyphenol content of at least 2%.

7. The method of claim 6, wherein preparing the bronze muscadine pomace extract and the purple muscadine pomace extract further comprises filtering the bronze muscadine pomace extract and the purple muscadine pomace extract separately and then combining the bronze muscadine pomace extract with the purple muscadine pomace extract to produce the muscadine pomace extract having a polyphenol content of at least 2%.

8. The method of claim 7, wherein preparing the bronze muscadine pomace extract and the purple muscadine pomace extract further comprises fermenting the bronze muscadine pomace extract and the purple muscadine pomace extract to remove extracted sugars after filtering the bronze muscadine pomace and the purple muscadine pomace separately but before combining the bronze muscadine pomace extract with the purple muscadine pomace extract to produce the muscadine pomace extract having a polyphenol content of at least 2%.

9. The method of claim 8, wherein preparing the bronze muscadine pomace extract and the purple muscadine pomace extract further comprises filtering the bronze muscadine pomace extract and the purple muscadine pomace extract after fermenting the bronze muscadine pomace extract and the purple muscadine pomace extract but before combining the bronze muscadine pomace extract with the purple muscadine pomace extract to produce the muscadine pomace extract having a polyphenol content of at least 2%.

10. The method of claim 9, further comprising concentrating the bronze muscadine pomace extract and the purple muscadine pomace extract such that each extract comprises 20% to 50% solids in a liquid prior to combining the bronze pomace extract with the purple pomace extract.

11. The method of claim 10, wherein the bronze muscadine pomace extract and the purple muscadine pomace extract each comprise 40% solids in a liquid.

12. The method of claim 6, further comprising filtering the muscadine pomace extract after combining the bronze muscadine pomace extract with the purple muscadine pomace extract to produce a muscadine pomace extract having a polyphenol content of at least 2%.

13. The method of claim 12, further comprising fermenting the muscadine pomace extract after filtering the muscadine pomace extract.

14. The method of claim 13, further comprising filtering the muscadine pomace extract after fermenting the muscadine pomace extract.

15. The method of claim 14, further comprising concentrating the muscadine pomace extract after filtering the muscadine pomace extract such that the muscadine pomace extract comprises 20% to 50% solids in a liquid.

16. The method of claim 15, wherein the muscadine pomace extract comprises 40% solids in a liquid.

17. The method of claim 1 or 3, further comprising extracting the bronze muscadine pomace and purple muscadine pomace simultaneously with water after combining the bronze muscadine pomace and purple muscadine pomace.

18. The method of claim 17, further comprising filtering the muscadine pomace extract after simultaneously extracting the bronze muscadine pomace and the purple muscadine pomace.

19. The method of claim 18, further comprising fermenting the muscadine pomace extract after filtering the muscadine pomace extract to remove extracted sugars.

20. The method of claim 19, further comprising filtering the muscadine pomace extract after fermenting the muscadine pomace extract to produce the muscadine pomace extract having a polyphenol content of at least 2%.

21. The method of claim 20, further comprising concentrating the muscadine pomace extract such that the extract contains 20% to 50% solids in a liquid.

22. The method of claim 21, wherein the muscadine pomace extract comprises 40% solids in a liquid.

23. The method of claim 1 or 3, further comprising adding excipients or other ingredients to the muscadine pomace extract.

24. The method of claim 23, wherein the excipient comprises glycerin, sorbitol, colloidal silicon dioxide, a natural flavoring additive, or a combination thereof.

25. The composition of claim 2 or 4, wherein the composition further comprises an excipient or other ingredient.

26. The method of claim 1 or 3, wherein the composition is provided in a non-beverage food, beverage, or dietary supplement or cosmetic composition.

27. The composition of claim 2 or 4, wherein the composition is provided in a non-beverage food, beverage, or dietary supplement or cosmetic composition.

28. The composition of claim 2 or 4 for use as an antioxidant.

29. The composition of claim 2 or 4 for use as an anti-aging supplement.

30. Use of a composition of any one of claims 2, 4, 25, and 27-29 in a dose in the manufacture of a medicament for preventing or inhibiting cellular senescence, wherein the dose is sufficient to prevent or inhibit one or more processes associated with cellular senescence.

31. The use of claim 30, wherein preventing or inhibiting one or more processes associated with cellular aging comprises preventing or inhibiting the formation or activity of free radicals in a subject ingesting the composition.

32. Use of a dose of the composition of any one of claims 2, 4, 25, and 27-29 in the manufacture of a medicament for inhibiting oxidative stress, wherein the dose is sufficient to reduce or inhibit oxidative stress.

33. Use according to claim 32, wherein the composition is administered 10 to 30 minutes before or after a meal.

34. The use of claim 33, wherein the composition is administered 10 minutes prior to a meal.