WO1997039632A1

WO1997039632A1 - Natural antioxidant composition

Info

Publication number: WO1997039632A1
Application number: PCT/US1997/006821
Authority: WO
Inventors: Harald P. Wulff
Original assignee: Henkel Corp
Current assignee: Henkel Corp
Priority date: 1996-04-22
Filing date: 1997-04-22
Publication date: 1997-10-30
Anticipated expiration: 1998-10-22
Also published as: EP0897264A1; AU2741197A; EP0897264A4

Abstract

The present invention provides a composition comprising pulverized, lyophilized grape seeds. This natural antioxidant composition may readily be formulated into oral and topical pharmaceutical products for administration to humans or animals to provide protection from the harmful effects of free radicals. The natural antioxidant composition of the present invention may also be used as a natural additive in food, cosmetic, or pharmaceutical products to prevent or retard their oxidation and spoilage. The present invention also provides a stable, natural source of proanthocyanodin antioxidant compounds which may be extracted by conventional means to provide a stable, more highly purified antioxidant composition.

Description

NATURAL ANTIOXIDANT COMPOSITION

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

This invention provides a composition comprising pulverized, lyophilized grape seeds, having beneficial antioxidant properties. This composition may be

used in pharmaceutical compositions which may be administered orally or topically to humans and animals, to provide protection from the harmful effects of free radicals in the human or animal body. This composition also may be used as a natural food additive to prevent the oxidation of food products during

processing. The present invention also provides a stable, natural source of proanthocyanodin antioxidant compounds which may be extracted by conventional means to provide a stable, highly purified, high potency antioxidant composition.

BACKGROUND Recent scientific studies have provided evidence that antioxidants are capable of protecting healthy cells from free radical damage. Free radicals are unstable chemicals formed in the body during metabolism and from exposure to environmental sources, such as air pollution, cigarette smoke, and dietary fats. When there is an excessive number of free radicals in the body, they can attack healthy cells and can contribute to a number of degenerative diseases, such as cancer. The following is a list of diseases and conditions to which free radicals have been linked: Conditions Involving Multiple Organs

Inflammatory-immune injury Nutritional deficiencies Glomerulonephritis Kwashiorkor

(idiopathic, membranous) Vitamin E deficiency Vasculitis (hepatitis B virus, drugs) Autoimmune diseases Alcohol damage

Ischemia-reflow states Radiation injury

Drug and toxin-induced reactions Aging

Disorders of "premature aging"

Iron overload Immune deficiency of age

Idiopathic hemochromatosis Dietary iron overload Cancer Thalassemia and other chronic anemias Amyloid diseases

Conditions Involving Single Organs

Erythrocytes Joint abnormalities

Phenylhydrazine Rheumatoid arthritis

Primaquine

Lead poisoning Heart and cardiovascular

Protoporphyrin photo-oxidation Alcohol cardiomyopathy Malaria Keshan diseases (selenium

Sickle-cell anemia deficiency)

Favism Atherosclerosis

Fanconi anemia Doxorubicin toxicity

Lung Kidney Cigarette-smoke effects Nephrotic antiglomerular basement

Emphysema membrane disease

Hyperoxia Aminoglycoside nephrotoxicity

Bronchopulmonary dysplasia Heavy metal nephrotoxicity

Oxidant pollutants Renal graft rejection Acute respiratory distress syndrome

Mineral dust pneumoconiosis

Bleomycin toxicity

Paraquat toxicity Gastrointestinal tract Eye Endotoxin liver injury Cataractogenesis Carbon tetrachloride liver injury Ocular hemorrhage Diabetogenic action of alloxan Degenerative retinal damage Free fatty acid-induced pancreatitis Retinopathy of prematurity Nonsteroidal anti- inflammatory drug- Photic retinopathy induced lesions

Skin

Brain Solar radiation

Hyperbaric oxygen Thermal injury Neurotoxins Porphyria

Senile dementia Contact dermatitis Parkinson disease - MPTP Photosensitive dyes Hypertensive cerebrovascular injury; Bloom syndrome cerebral trauma Allergic encephalomyelitis and other demyelinating diseases Ataxia-telangiectasia syndrome Potentiation of traumatic injury Aluminum overload Abetalipoproteinemia

Antioxidants quench free radicals before they have a chance to attack healthy cells. Antioxidants have been shown in the majority of studies to lower the risk for certain cancers, such as lung, stomach, cervix, breast, bladder, and

oral cancers. Other health benefits include protection against heart diseases, cataracts, and age-related macular degeneration. While free radicals can cause or complicate many diseases, including cancer, arthritis, cataracts, and heart disease, antioxidants can help protect the body from these chronic disorders. They also enhance the body's immune system.

Typical antioxidant compounds used in pharmaceutical compositions include vitamin A and other carotenoid compounds, vitamin E and other tocopherols, and proanthocyanodins and their derivatives. These compounds may be produced synthetically (e.g. , by the method of U.S. Patent 2,917,539 (beta-

carotene) and U.S. Patent 5,208,381 (lycopene)). Alternatively, these compounds may be extracted from plant matter using solvent-based extraction systems, such as the method of Karrer et al. (Helv. Chim. Acta 16:641 (1933) (extraction of alpha carotene or lutein); the method of U.S. Patent 4,698,360 (extraction of proanthocyanodins from pine bark); the method of U.S. Patent 2,848,508 (extraction of beta-carotene); and the method of German Patent 1 , 168,890 (extraction of lycopene). Proanthocyanodins are antioxidant pycnogenols. They are characterized among the plant polyphenols by their low toxicity (LD₅₀ = 3g/kg), and corresponding low risk of acute or chronic intoxication upon therapeutic administration. Proanthocyanodins are also nonteratogenic, nonmutagenic, and nonantigenic (thus providing for a very low risk of allergic reaction upon therapeutic administration), and have high

bioavailability following oral administration. Antioxidants are also useful in the preservation of food, cosmetic, and pharmaceutical products. Common antioxidants used in food, cosmetic, and pharmaceutical processing include sulfites, such as sodium bisulfite (a mixture of monosodium sulfite, NaHSO₃, and sodium metabisulfite, Na₂S₂O₅), sodium metabisulfite, potassium metabisulfite (K₂S_:O₅), sulfur dioxide, and acids such as ascorbic and benzoic acid. However, acids are unsuitable for use in many food, cosmetic, and pharmaceutical formulations. Furthermore, exposure to sulfites, which are commonly used as antioxidants in the processing of many food products, including wine, can produce acute airway obstruction and respiratory distress in sensitive individuals, particularly those suffering from asthma.

Concern for avoiding such reactions has prompted a decrease in the use of sulfiting agents as sanitizing and preservative agents in food products; however,

in many instances, no reliable substitute exists. In view of the aforementioned deficiencies attendant with prior art antioxidant compositions, it should be apparent that there still exists in the art a need for an improved, natural antioxidant composition. Such a composition

would be useful for its therapeutic benefits, and to prevent oxidation of pharmaceutical, cosmetic, and food compositions, without provoking the undesirable and sometimes dangerous side effects associated with prior art antioxidant compositions.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a natural antioxidant composition comprising pulverized, lyophilized grape seeds. Grape seeds provide a natural source of antioxidant proanthocyanodins. The composition of the present invention is thus a completely namral antioxidant which may readily be formulated into oral and topical pharmaceutical products for administration to humans or animals to provide protection from the harmful effects of free

radicals. The antioxidant composition of the present invention may also be used as a natural additive in food, cosmetic, or pharmaceutical products to prevent or retard their oxidation and spoilage. The present invention also provides a stable, natural source of antioxidant

proanthocyanodins which may be extracted by conventional means to provide a stable, highly purified, high potency antioxidant proanthocyanodin composition for use in any application for which antioxidants are suitable. With the foregoing and other objects, advantages and features of the

invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Accordingly, the present invention provides a composition with beneficial antioxidant properties, comprising pulverized, lyophilized grape seeds. Such a composition may be formulated as a stable, completely natural product. The pulverized, lyophilized grape seeds which comprise the antioxidant composition of the present invention provide a natural source of antioxidant proanthocyanodins. Proanthocyanodins form a class of natural polyphenols, defined by an exclusive property, namely the production of a red pigment (anthocyanodin) by the Bate-Smith reaction (Bate-Smith, E.C , and Swain, T. (1953) Chemistry & Industry pp 377-378). Natural proanthocyanodins are typically an oligomeric mixture of proanthocyanodins having the general formula:

where rings A and/or B are substituted with one or more hydroxy groups. In the practice of the present invention, grape seeds may be ground or pulverized into a fine powder using any suitable method. The whole or pulverized grape seeds are then lyophilized (freeze-dried) in order to remove at least 99%, and preferably more than 99% , of the water naturally contained in the grape seeds.

Lyophilization is a process of drying in which water is sublimed from the product after it is frozen. This serves to enhance their stability and shelf life, while maintaining the status of the grape seeds as a completely natural product. Lyophilization may be carried out using any means known in the art. Lyophilization essentially comprises freezing the product (in this case, pulverized grape seeds), evacuating the air from the freezer such that the air pressure falls usually below 0.1 torr (ice sublimes at pressures below 3 torr), subliming ice in the freezer on a cold condensing surface at a temperature below that of the product, and introducing heat to the product under controlled conditions, thus providing energy for sublimation of the ice in the product while maintaining the product below its eutectic temperature.

Once pulverized and lyophilized, the grape seeds of the present invention may be used directly in any application in which a completely natural antioxidant is desired. In another embodiment of the present invention, the pulverized, lyophilized grape seeds of the present invention are subject to conventional chemical extraction procedures in order to extract the antioxidant proanthocyanodins contained therein. This provides a stable, highly purified, high potency antioxidant proanthocyanodin composition for use in any application for which antioxidants are suitable. Proanthocyanodins may be extracted from

grapeseeds by any method known in the art, for example, by the method of U.S.

Patents 3,436,407 and 4,698,360.

In an exemplary method, proanthocyanodins are extracted from grape seeds by the following steps: grape seeds are first pulverized and extracted with boiling water (the extracted grape seeds can then be compressed or pressed to physically remove all liquid possible). The liquid is cooled to 20 ^°C and filtered. Sodium chloride is added to saturation, and the precipitate formed is separated. The filtrate is extracted 3X with ethyl acetate at the rate of 1/10 the volume of

the aqueous phase. The ethyl acetate collected is dried on anhydrous Na₂SO₄ and brought back to 1/5 of its volume by distillation under reduced pressure. It is then poured into three volumes of chloroform, while stirring mechanically. The proanthocyanodins precipitate out, and may be collected by filtration. They may be further purified by redissolution in ethyl acetate and reprecipitation in chloroform. They may be finally washed with chloroform and dried at reduced pressure at a temperature no higher than 50 ^°C.

In another embodiment of the present invention, the grape seeds are lyophilized first; in this form they may be stored for long periods prior to pulverizing and/or extraction of proanthocyanodins. The lyophilized grape seeds may be pulverized while in the lyophilized state; alternatively, the grape seeds may be reconstituted prior to pulverizing.

Oral antioxidant pharmaceutical compositions

The present invention also provides an oral antioxidant pharmaceutical composition. Such a composition may comprise the pulverized, lyophilized grapeseeds according to the present invention, where a completely natural

product is desired. Alternatively, the composition may comprise purified proanthocyanodins extracted from pulverized, lyophilized grapeseeds, where a more purified antioxidant composition is desired. In either case, the oral antioxidant pharmaceutical composition according to the present invention will

further comprise a pharmaceutically acceptable carrier suitable for oral administration.

The oral compositions of the present invention can be made by conventional compounding procedures known in the pharmaceutical art, that is, by mixing the active substances with edible pharmaceutically acceptable non-toxic inert, solid or liquid carriers and/or excipients suitable for systemic administration and conventionally used in oral dosage forms. Preferably, carriers for use in the present invention will be natural carriers, such as edible oils. The

edible oil may be a vegetable oil such as avocado, coconut, corn, grape seed, palm, peanut, olive, sesame, soya, rapeseed, or walnut oil or any other edible vegetable oil or mixture of one or more of these oils. Additionally, edible, non- toxic pharmaceutically acceptable stabilizers usually used as stabilizers in oral dosage forms or edible, non-toxic salts thereof can be included in the

compositions. Preferably, the stabilizer will be beeswax or silica. All the above carriers, excipients and stabilizers are intended to include only those suitable for oral administration and all are conventional and known to the pharmaceutical art. The pharmaceutical compositions for oral administration may be in the

form of tablets, including sustained release forms, lozenges, chewing gum, and capsules. The soft gelatin capsule dosage form is most preferred. These dosage forms may be prepared by those skilled in the art in accordance with known

techniques in the art, for example, as described in Remington 's Pharmaceutical Sciences, 17th Edition (1985), Mack Publishing Co. , Easton, PA.

According to the present invention, the patient ingests the composition preferably daily to obtain the benefit of the administration of the proanthocyanodins. The dosage of the antioxidant composition of the present invention administered to a patient will vary depending upon several factors, including, but not limited to, the age and weight of the patient, the general health of the patient, the severity of the symptoms, and the like.

Topical antioxidant pharmaceutical compositions, and cosmetic compositions containing antioxidants

The present invention also provides topical antioxidant pharmaceutical compositions, and cosmetic compositions containing antioxidants. Such a

composition may comprise the pulverized, lyophilized grapeseeds according to the present invention, where a completely natural product is desired. Alternatively, the composition may comprise purified proanthocyanodins extracted from pulverized, lyophilized grapeseeds, where a more purified antioxidant composition is desired. In either case, the topical antioxidant

pharmaceutical composition or cosmetic composition according to the present invention will further comprise a carrier suitable for topical administration.

For use in topical pharmaceutical and cosmetic compositions, the pulverized lyophilized grape seeds which comprise the natural antioxidant

composition of the present invention may be used in all galenical forms normally used for topical application, in particular lotion or serum-type solutions or dispersions, liquid or semi-liquid milk emulsions produced by dispersions of a fatty phase in an aqueous phase (O/W) or the reverse (W/O), or cream- or gel- type emulsions having a soft consistency, or microgranulates or vesicular ionic and/or non- ionic dispersions.

The natural antioxidant composition of the present invention may be added to a wide variety of products for topical application, including makeup, creams for cleansing, protecting, treating, or caring for the skin, in particular, the face, hands, and feet (e.g. , day and night creams, makeup removal creams, foundation creams and sunscreens), liquid foundations, makeup removal lotions, protective or skin-care body lotions, sunscreen lotions, skin care lotions, gels, or foams, such as cleansing, sunscreen, and artificial tanning lotions, bath preparations, deodorant compositions, after-shave gels or lotions, depilatory creams, and compositions used for insect stings and against pain. The topical compositions according to the invention may also consist of solid preparations used for soaps and cleansing bars.

In conventional fashion, the topical compositions according to the present invention may also contain additives customarily used in cosmetics, such as

water-absorbent or lipophilic gelling agents, water-absorbent or lipophilic active ingredients, preservatives, solvents, perfumes, fillers, screens, and coloring substances. The quantities of these various additives are those conventionally used in cosmetics; for example, from 0.1 % to 10% of the total weight of the

composition. These additives may, depending on the nature thereof, be added to the fatty phase, the aqueous phase, and/or in lipidic spherules. Preferably, such additives are natural substances.

Oils suitable for use in the topical compositions of the present invention include mineral oils (e.g. , vaseline oil), vegetable oils (e.g., sunflower oil), animal oils (e.g., perhydrosqualene), synthetic oils (e.g., Purcellin oil), silicone containing oils, (e.g. , cyclomethicone) and fluorinated oils (e.g. , perfluoropolyethers). Fatty alcohols and fatty acids (e.g. , stearic acid) can be

added to these oils. Preferably, the oil is a natural vegetable oil.

Emulsifiers suitable for use in the topical compositions of the present invention include, for example, glycerol stearate, polysorbate 60, and a PEG-6/PEG-32/glycol stearate mixture (Tefose^®).

Solvents suitable for use in the topical compositions of the present invention include, for example, the lower alcohols, in particular ethanol and

isopropanol. Water-absorbent gelling agents suitable for use in the topical compositions

of the present invention include carboxyvinyl polymers, acrylic copolymers (e.g. , acrylate/alkylacrylate copolymers), poly aery lamides, polysaccharides (e.g. , hydroxypropylcellulose), and, preferably, natural gums and clays. Lipophilic gelling agents suitable for use in the topical compositions of the present invention

include modified clays (e.g., bentonites), the metallic salts of fatty acids (e.g. , aluminum stearates), and hydrophobic silica.

Water-absorbing active ingredients include proteins and protein hydrolysates, amino acids, polyalcohols, urea, allantoin, sugars and sugar

derivatives, vitamins, and hydroxy acids.

Lipophilic active ingredients include retinol (vitamin A) and derivatives thereof, tocopherol (vitamin E) and derivatives thereof, essential fatty acids,

ceramides, essential oils, and salicylic acid and derivatives thereof.

The pulverized, lyophilized grape seeds of the present invention may also be combined with active ingredients intended, in particular, for the prevention and/or treatment of skin disorders. Such active ingredients may include, for example:

- agents modulating the differentiation, proliferation, and/or skin pigmentation, such as retinoic acid and isomers thereof, retinol and esters thereof, vitamin D and derivatives thereof, estrogens such as estradiol, kojik acid and hydroquinone;

- antibacterial agents such as clindamycin phosphate, erythromycin, tetracycline antibiotics, and the like; - antifungal agents, such as imidazoles (e.g. , econazole, ketoconazole,

miconazole) and salts thereof, polyenes (e.g. , amphotericin B), allylamines (e.g. , terbinafine, octopirox), and the like;

- steroidal anti-inflammatory agents, such as hydrocortisone, betamethasone valerate, clobetasol propionate, and the like, or nonsteroidal anti- inflammatory agents, such as ibuprofen, ketoprofen, and salts thereof, diclofenac

and salts thereof, acetylsalicylic acid, acetaminophen, glycyrrhetinic acid, and the like;

- anesthetic agents, such as lidocaine chlorhydrate and derivatives thereof, and the like;

- anti-pruriginous agents, such as thenaldine, trimeprazine, and cyproheptadine, and the like;

- antiviral agents, such as acyclovir, and the like;

- keratolytic agents, such as a- and ^-hydroxycarboxylic and β- ketocarboxylic acids, and salts, amides, and esters thereof, hydroxy acids, such as glycolic, lactic, salicylic, and citric acid, and, in general, fruit acids, and the like;

- other antioxidants, such as α-tocopherol and esters thereof, superoxide dismutases, metal chelating agents, ascorbic acid and esters thereof, and the like; - anti-seborrhea agents, such as progesterone, and the like; and

- acne-fighting products, such as retinoic acid or benzoyl peroxide, and

the like. Method of treatment

The present invention also includes a method for quenching free radicals comprising administering to a mammal, including a human, in need of such treatment an antioxidant effective amount of an antioxidant composition according to the present invention. Such a method may comprise administering

an effective amount of pulverized, lyophilized grape seeds to quench free radicals. Alternatively, the method may comprise administering an antioxidant effective amount of purified proanthocyanodins extracted from pulverized, lyophilized grapeseeds. This method may be practiced by administration of an antioxidant composition according to the present invention by itself, or in combination with other active ingredients, including other antioxidants, and/or therapeutic agents in

a pharmaceutical composition. Other therapeutic agents suitable for use herein are any compatible drugs that are effective by the same or other mechanisms for the intended purpose, or drugs that are complementary to those of the present agents. Examples include carotenoids (e.g. , beta carotene derived from Dunaliella salina) and vitamins (e.g. , natural tocopherols derived from soybean oil, such as d-α-tocopherol), among others. In the case where a totally natural product is desired, the compatible compounds will preferably be natural antioxidants.

The compounds utilized in combination therapy may be administered simultaneously, in either separate or combined formulations, or at different times than the compositions according to the present invention, e.g. , sequentially, such that a combined effect is achieved. The amounts and regime of administration will be adjusted by the practitioner, by preferably initially lowering their standard doses and then titrating the results obtained.

Antioxidant food additive The present invention also provides an antioxidant composition which may be added to food products to prevent oxidation. Such a composition may comprise the pulverized, lyophilized grapeseeds according to the present invention, where a completely natural product is desired. Alternatively, the composition may comprise purified proanthocyanodins extracted from pulverized, lyophilized grapeseeds, where a more purified antioxidant composition is desired. In particular, the antioxidant composition of the present invention is suitable for addition to wine, in order to prevent oxidation of the wine. In the typical fermentation process, wines may be dosed with the antioxidant composition of the present invention at any time during fermentation. Preferably, wines are dosed with the antioxidant composition of the present invention at the time of bottling, as a substitute for all or part of the sulfur dioxide typically used to prevent oxidation in winemaking. The present invention also provides a process for preventin the oxidation of food products comprising adding to a food product an antioxidant effective amount of an antioxidant composition according to the present invention during the preparation or processing of the food product. While the invention has been described and illustrated herein by

references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material, combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied and will be appreciated by those skilled in the art.

Claims

What is claimed is:

1. A composition comprising lyophilized grape seeds.

2. The composition of claim 1 wherein the grape seeds are pulverized prior to lyophilization.

3. The composition of claim 1 wherein the grape seeds are pulverized subsequent to lyophilization.

4. The composition of claim 1 , wherein the grape seeds are lyophilized by a process comprising the steps of: freezing the grape seeds in a freezer; reducing the air pressure in the freezer; subliming ice in the freezer on a cold condensing surface at a temperature below that of the grape seeds; and introducing heat to the grape seeds while maintaining them below their eutectic temperature.

5. The composition of claim 4, wherein the pressure in the freezer is reduced below 0.1 torr.

6. The composition of claim 5, wherein more than 99% of the water naturally occurring in the grape seeds is removed.

7. A process for producing lyophilized grape seeds comprising the steps of: freezing grape seeds in a freezer;

reducing the air pressure in the freezer; subliming ice in the freezer on a cold condensing surface at a temperature below that of the grape seeds; and introducing heat to the grape seeds while maintaining them below their

eutectic temperature.

8. The process of claim 7, wherein the pressure in the freezer is reduced below 0.1 torr.

9. The process of claim 7, wherein more than 99% of the water naturally occurring in the grape seeds is removed.

10. The process of claim 7, further comprising the step of pulverizing the grape seeds prior to lyophilization.

11. The process of claim 7, further comprising the step of pulverizing the grape seeds subsequent to lyophilization.

12. An antioxidant pharmaceutical composition comprising an effective

antioxidant amount of pulverized, lyophilized grape seeds and a pharmaceutically acceptable carrier therefor.

13. The antioxidant composition of claim 12 suitable for oral administration.

14. The antioxidant composition of claim 12 suitable for topical administration.

15. A cosmetic composition comprising an effective amount of the composition of pulverized, lyophilized grape seeds and a cosmetically acceptable carrier therefor.

16. A method of preventing or inhibiting the damaging effects of free radicals comprising administering to a mammal in need of such treatment an effective antioxidant amount of the composition of claim 1.

17. A composition for preventing or inhibiting the oxidation of food products comprising the composition of claim 1.

18. A process for preventing the oxidation of food products comprising adding to a food product an antioxidant effective amount of a natural antioxidant composition comprising the composition of claim 1 during the preparation or processing of the food product.

19. The process of claim 18, wherein the food product is wine.

20. The process of claim 18, wherein the wine is dosed with the antioxidant

composition at the time of bottling, as a substitute for all or part of the sulfur dioxide typically used to prevent oxidation in winemaking.

21. An antioxidant composition comprising an antioxidant effective amount of

proanthocyanodins extracted from pulverized, lyophilized grape seeds and a carrier therefor.

22. A method for producing purified proanthocyanodins comprising:

freezing grape seeds in a freezer; reducing the air pressure in the freezer; subliming ice in the freezer on a cold condensing surface at a temperature below that of the grape seeds; introducing heat to the grape seeds while maintaining them below their eutectic temperature such that water sublimes from the grape seeds; and extracting proanthocyanodins from the lyophilized grape seeds.

23. The method of claim 22, further comprising the step of pulverizing the grape seeds prior to freezing.

24. The method of claim 22, further comprising the step of pulverizing the grape seeds following drying and prior to extraction of the proanthocyanodins.