Methods and kits for reducing cellular damage, inhibiting free radical production, and scavenging free radicals in mammals

[0001] The present invention relates to methods, materials and kits for reducing cellular damage in mammals and, more particularly, to methods and materials and kits for protecting against injurious environmental stresses and their damaging effects on DNA and cellular structure, function, and growth. Kits discussed herein comprise combinations of oral and topical dosage forms.

[0002] Free radicals, or reactive oxygen species (“ROS”) and other oxidizing species (“OOS”) are thought to contribute to the development and progression of a variety of diseases or other abnormal conditions of the human body, ranging from skin conditions to cancer and cardiovascular disease. Increasingly, free radicals and their metabolites are being implicated in tissue injuries that lead to the initiation and/or promotion of multistage carcinogenesis.

[0003] The ROS species include superoxide (O2-), hydrogen peroxide (H2O2), peroxy radicals (HO2 and RO2) alkyl peroxide (R2O2), hydroxyl radical (—OH), alkoxy radical (—OR), and singlet oxygen. The OOS species include hypohalous acids (HOX) (where X is chloride, bromide, iodide), Z-amines (where Z is either chlorinated or ammoniated amine containing compounds, nitric oxide (NO), ammonia, cyclooxygenase, phospholipase A2, phospholipase C and transition metals.

[0004] Each of the ROS, directly or acting as an intermediate, are thought to act on various parts of cells through the cell membrane to adversely impact the human body. In view of the suspected causative or contributory role played by free radicals and their metabolites in the development and growth of cancerous cells, antioxidants and free radical scavengers have emerged as potential prophylactics for the prevention of cancer.

[0005] For reasons including reduced cost, increased bioavailability, and potentially reduced toxicity, it would be generally preferable to employ antioxidants and free radical scavengers or inhibitors obtained from natural sources, as opposed to specialty chemicals prepared synthetically, as prophylactics in therapies aimed at preventing or inhibiting the growth of cancerous cells. In addition, it would be desirable to identify naturally occurring antioxidants and free radical scavengers or inhibitors that exhibit high efficacy and potency in inhibiting the growth of free radical species both topically (e.g., at a region of skin, such as a portion of the hands or face, routinely subjected to potentially carcinogenic environmental stimuli) as well as systemically (e.g., inside the body, such as in an internal organ).

[0006] The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary. By way of introduction, a first method of reducing cellular damage in a mammal includes (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first antioxidant, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant comprises acerola concentrate. Desirably, either the oral dosage form or the topical dosage form is administered first to the mammal, with the second of the two dosage forms being administered at any time during the metabolism of the first dosage form.

[0007] A first method of inhibiting free radical production in a mammal includes (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first antioxidant, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant comprises Acerola concentrate.

[0008] A first method of scavenging free radicals in a mammal includes (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first antioxidant, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant comprises Acerola concentrate.

[0009] A first kit for reducing cellular damage in a mammal includes (a) an oral dosage form comprising a therapeutically effective amount of a first antioxidant, and (b) a topical dosage form comprising a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant comprises Acerola concentrate.

[0010] A second method of reducing cellular damage in a mammal includes (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first phytochemical, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second phytochemical, wherein at least one of the first phytochemical and the second phytochemical comprises Acerola concentrate. Desirably, either the oral dosage form or the topical dosage form is administered first to the mammal, with the second of the two dosage forms being administered at any time during the metabolism of the first dosage form.

[0011] A second method of inhibiting free radical production in a mammal includes (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first phytochemical, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second phytochemical, wherein at least one of the first phytochemical and the second phytochemical comprises Acerola concentrate.

[0012] A second method of scavenging free radicals in a mammal includes (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first phytochemical, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second phytochemical, wherein at least one of the first phytochemical and the second phytochemical comprises Acerola concentrate.

[0013] A second kit for reducing cellular damage in a mammal includes (a) an oral dosage form comprising a therapeutically effective amount of a first phytochemical, and (b) a topical dosage form comprising a therapeutically effective amount of a second phytochemical, wherein at least one of the first phytochemical and the second phytochemical comprises Acerola concentrate.

[0014] FIG. 1 shows a graph of dose response of 3PC cells to treatment with pycnogenol, grape seed extract, ascorbic acid, and Acerola concentrate.

[0015] FIG. 2 shows a graph of dose response of 3PC cells to treatment with elderberry, lutein, and green tea extract.

[0016] It has been discovered that powerful antioxidant protection against injurious environmental stresses and their damaging effects on DNA and cellular structure, function and growth is provided by the administration of an oral dosage form containing a therapeutically effective amount of a first antioxidant and a topical dosage form containing a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant contains acerola concentrate. Moreover, it has been discovered that powerful antioxidant protection against injurious environmental stresses and their damaging effects on DNA and cellular structure, function and growth is likewise provided by the administration of an oral dosage form containing a therapeutically effective amount of a first phytochemical and a topical dosage form containing a therapeutically effective amount of a second phytochemical, wherein at least one of the first phytochemical and the second phytochemical contains acerola concentrate. Desirably, one or the other of the oral dosage form and the topical dosage form is administered first, with the second of the two dosage forms being administered at any time during the metabolism of the first dosage form.

[0017] Acerola, the ripe fruit of Malpighia punicifolia known as the Barbados cherry or the West-Indian cherry, is one of the very richest natural sources of ascorbic acid (i.e., Vitamin C). While the antioxidant activity of ascorbic acid to protect cells against damage from environmental stresses is documented, it has now been discovered in accordance with the present invention that the protective activity of acerola concentrate is, surprisingly and unexpectedly, more than four times higher than the activity one would expect based solely on ascorbic acid content. Methods and kits embodying features of the present invention are described hereinbelow.

[0018] Throughout this description and in the appended claims, the following definitions are to be understood. Terms that are not defined have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs:

[0019] The phrase “reducing cellular damage” refers to one or more of (i) preventing damage to normal DNA or healthy cell structure, function or growth, (ii) partially or completely inhibiting further damage to DNA or unhealthy cell structure, function or growth, and (iii) reversing damage inflicted on previously healthy cells.

[0020] The phrases “inhibiting free radical production” and “scavenging free radicals” have the same meaning as commonly understood by one of ordinary skill in the art, namely where “inhibiting” generally refers to the inhibition of initiating events that occur in the free radical production pathway while “scavenging” refers to the inhibition of free radicals once they are produced. However, since there are situations where the production of one free radical creates another via a sequence of self-perpetuating chain reactions called “propagation”, the phrase “inhibiting free radical production” applies to both inhibiting the initiating events and inhibiting free radicals that occur during propagation.

[0021] The phrases “acerola cherry concentrate” and “acerola concentrate” refer to either a liquid or solid concentrate of acerola fruit obtained by subjecting the naturally occurring fruit to a concentrative process, including but not limited to counter current extraction, ultrafiltration (UF), and the like.

[0022] The phrase “therapeutically effective amount” refers to an amount of an antioxidant or phytochemical, such as acerola concentrate that, when used in accordance with methods embodying features of the present invention, enables a target effect (e.g., preventing or reducing damage to DNA, cell structure, function, or growth, inhibiting free radical production, scavenging free radicals, etc.) to be achieved in a particular subject.

[0023] The term “metabolism” refers to every stage in the metabolic transformation of a dosage form, including the initial introduction of a dosage form into a subject (e.g., either by topical application, peroral consumption, or the like), the subsequent migration of one or more ingredients of the dosage form to a treatment site in or on the body, the consumption and/or degradation of one or more ingredients of the dosage form by the subject, and the eventual excretion of one or more ingredients of the dosage form and/or metabolites thereof.

[0024] The term “phytochemical” refers to any species produced by and/or obtained from a plant, including species that possess antioxidant properties.

[0025] A first series of representative methods of preventing or reducing cellular damage and/or inhibiting free radical production and/or scavenging free radicals in a mammal that embody features of the present invention, include (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first antioxidant, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant comprises acerola concentrate. Desirably, both the first antioxidant and the second antioxidant comprise acerola concentrate.

[0026] A second series of representative methods of reducing cellular damage and/or inhibiting free radical production and/or scavenging free radicals in a mammal that embody features of the present invention, include (a) administering to the mammal an oral dosage form comprising a therapeutically effective amount of a first phytochemical, and (b) administering to the mammal a topical dosage form comprising a therapeutically effective amount of a second phytochemical, wherein at least one of the first phytochemical and the second phytochemical comprises acerola concentrate. Desirably, both the first antioxidant and the second antioxidant comprise acerola concentrate.

[0027] Antioxidants suitable for use in accordance with the present invention may be derived from natural sources or prepared synthetically. Phytochemicals obtained from fruits and vegetables (e.g. polyphenols), particularly those with antioxidant properties, are especially desirable for use in accordance with the present invention. Suitable representative antioxidants and phytochemicals, in addition to acerola concentrate, include but are not limited to bioflavonoids, catechin-based preparations such as proanthanol and proanthocyanidin, grape seed extract, pycnogenol, provatene, carotenoids such as β-carotene, sodium bisulfite, vitamins such as Vitamin E and Vitamin C (L-ascorbic acid), □-tocopherol, green tea extract, elderberry extract, lutein, coenzyme Q10, complexes such as Complex 1, Complex 2, and Complex 3 shown in Table 1 below, and the like, and combinations thereof. Grape seed extract, pycnogenol, provatene, green tea extract, elderberry extract, lutein, coenzyme Q10, Complex 1, Complex 2, and Complex 3 are particularly desirable at present.

	TABLE 1
	Complex 1	Complex 2	Complex 3
Tocopheryl Acetate	25-75%	25-75%	25-75%
Tocopherol	5-25%	5-25%	5-25%
Bioflavonoids	1-10%	1-10%
Tetrahydrodiferuloylmethane (&)	1-10%	1-10%
Tetrahydrodemethoxydiferuloylmethane (&)
Tetrahydrobisdemethoxydiferuloylmethane
Grape Seed Extract (&) Phospholipids	5-25%	1-10%
Glutathione	5-25%	5-25%	5-25%
Palmitoyl Hydroxypropyltrimonium	5-25%	5-25%	5-25%
Amylopectin/Glycerin Crosspolymer (&)
Lecithin (&) Camellia Sinensis Extract
Superoxide Dismutase	0.01-2%	0.01-2%	0.01-2%
Tetrahexyldecyl Ascorbate		10-30%
Ubiquinone		1-10%
Retinyl Acetate		1-10%
Magnesium Ascorbyl Phosphate			10-30%
Bitter Orange Peel Extract in Butylene Glycol			0.01-2%
Cyclodextrin & Soybean (Glycine Soja) Germ			1-10%
Extract
Retinyl Palmitate			1-10%
Licorice Extract			0.01-2%
TOTAL	100%	100%	100%

[0028] The amounts shown in Table 1 represent percentage ranges of ingredients used to prepare each of Complexes 1, 2, and 3. The complexes shown in Table 1 are further defined in U.S. patent application Ser. No. 10/155305, herein incorporated by reference.

[0029] Generally, the oral dosage form and the topical dosage form are administered to the mammal any time during the metabolism of the other dosage form. In other embodiments, the two administrations occur within a time frame of about 24 hours, 12 hours, 8 hours, 4 hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, and 2 minutes. A substantially concomitant administration of the two dosage forms (i.e., the second dosage form is administered shortly after administration of the first dosage form, or in about 1 hour or less) is desirable at present.

[0030] All manner of oral dosage forms suitable for peroral administration of a pharmaceutical are contemplated for use in accordance with the present invention. Representative oral dosage forms for use in accordance with the present invention include but are not limited to pills, capsules, gelcaps, geltabs, beverages, chewing gums, chewable tablets, lozenges, viscous gels, troches, toothpastes, dental implants, gargling gels, mouth rinses, and the like, and combinations thereof. Presently preferred oral dosage forms include pills, capsules, gelcaps, geltabs, chewable tablets, lozenges, and troches.

[0031] In alternative embodiments in accordance with the present invention, the oral dosage form containing the first antioxidant is provided in the form of a controlled release delivery system of a type known in the art (e.g., see U.S. Pat. No. 6,004,582 to Faour et al.). The programmed release of the first antioxidant (e.g., acerola concentrate) into an individual's system may be desirable in order to minimize the number of oral dosage forms consumed by the individual in the course of a day (i.e., one controlled release dosage form may be ingested as opposed to multiple conventional dosage forms). Moreover, a controlled release delivery system used as an oral dosage form in accordance with the present invention, which contains acerola concentrate in its interior, may optionally be coated with an outer layer that likewise contains acerola concentrate, thus providing the rapid release of a bolus dose of acerola concentrate upon consumption.

[0032] All manner of topical dosage forms suitable for external application of a pharmaceutical are contemplated for use in accordance with the present invention. Representative topical dosage forms for use in accordance with the present invention include but are not limited to emulsions (e.g., creams, lotions, and the like), solutions, dispersions, gels, soaps, transdermal patches, and the like, and combinations thereof. Presently preferred topical dosage forms include emulsions, solutions, and gels.

[0033] The specific amounts of the first antioxidant and the second antioxidant in the oral dosage form and the topical dosage form, respectively, may vary with the subject, type of cells to be treated, format of dosage form, etc. For example, the weight, age, and overall health of a subject may be factors in determining what constitutes a therapeutically effective amount for the particular subject. Similarly, the physical properties of a dosage form (e.g., solid, liquid, concentrated, dilute, etc.) may be additional factors in determining a therapeutically effective amount. The therapeutically effective amounts of first and second antioxidants may be the same or different, and are preferably selected to provide optimum efficacy.

[0034] In presently preferred therapies embodying features of the present invention, both the first and second antioxidants comprise acerola concentrate. In presently preferred representative formulations of oral dosage forms for use in accordance with such therapies, the therapeutically effective amount of acerola concentrate is between about 50 mg and about 2000 mg, more preferably between about 350 mg and about 1500 mg, and still more preferably between about 400 mg and about 1200 mg. A particularly preferred oral dosage formulation at present is a tablet containing about 950 mg of acerola concentrate, which has an ascorbic acid content of about 120 mg or about 12.5% by weight. It is to be understood that the amount of acerola concentrate contained in an oral dosage form used in accordance with the present invention is dependent on the frequency of administration of the oral dosage form during the course of day. The presently preferred ranges described above correspond to a twice-daily peroral administration.

[0035] In presently preferred representative formulations of topical dosage forms in accordance with the above-described presently preferred therapies, the therapeutically effective amount of acerola concentrate is between about 15 mg and about 200 mg, more preferably between about 25 mg and about 100 mg, and still more preferably between about 50 mg and about 75 mg. A particularly preferred topical dosage formulation at present is a cream containing acerola concentrate in a concentration of about 5% by weight of the composition, such that an application of about 1.2 grams of cream provides about 65 mg of acerola concentrate. It is to be understood that the amount of acerola concentrate contained in a topical dosage form used in accordance with the present invention is dependent on the frequency of administration of the topical dosage form during the course of day and on the surface area of exposed skin that is to be covered. The presently preferred ranges described above correspond to a twice-daily topical administration to the hands and face of a subject.

[0036] The frequency of repetition of methods embodying features of the present invention is not restricted, and corresponds to a therapeutically effective frequency. Presently preferred dosing frequencies include once-daily and twice-daily administrations of an oral dosage form and a topical dosage form.

[0037] The type of acerola concentrate used in accordance with the present invention is not limited. Concentrates of acerola fruit obtained via counter current extraction or an ultrafiltration (UF) method are preferred, with UF-prepared acerola concentrate being especially preferred at present. The acerola concentrate manufactured and sold by Nutrilite (Buena Park, Calif.) is a particularly preferred material for use in accordance with the present invention. Analysis of a representative sample of Nutrilite acerola concentrate indicates the presence of multiple flavonoids and the presence of ascorbic acid in a concentration of between about 14 and about 17 percent by weight.

[0038] Representative kits for reducing cellular damage and/or inhibiting free radical production and/or scavenging free radicals in a mammal that embody features of the present invention, include (a) an oral dosage form comprising a therapeutically effective amount of a first antioxidant, and (b) a topical dosage form comprising a therapeutically effective amount of a second antioxidant, wherein at least one of the first antioxidant and the second antioxidant comprises acerola concentrate.

[0039] Dosage forms embodying features of the present invention can be included in a kit, container, pack, or dispenser together with instructions for their use. The oral dosage form and topical dosage form may be provided in packaged combination in forms suitable for immediate application or in forms requiring modification prior to use. For example, a cream for use as a topical dosage form may be provided as a ready-to-use dermopharmaceutical containing a cream base in combination with a second antioxidant (e.g., acerola concentrate) or in two separate packages (e.g., cream base and acerola concentrate) which are to be combined and mixed prior to application. Packaging the ingredients of a dosage form in separate containers may permit long-term storage without substantially diminishing the functioning of the active components. Furthermore, ingredients can be packaged under inert environments (e.g., under a positive pressure of nitrogen gas, argon gas, or the like), which is especially preferred for ingredients that are sensitive to air and/or moisture.

[0040] Oral and topical dosage forms embodying features of the present invention can be supplied in all manner of containers such that the activities of the different components are substantially preserved, while the components themselves are not substantially adsorbed or altered by the materials of the container. Suitable containers include but are not limited to ampules, bottles, test tubes, vials, flasks, syringes, envelopes (e.g., foil-lined), and the like. The containers may be comprised of any suitable material including but not limited to glass, organic polymers (e.g., polycarbonate, polystyrene, polyethylene, etc.), ceramic, metal (e.g., aluminum), metal alloys (e.g., steel), cork, and the like. In addition, the containers may comprise one or more sterile access ports (e.g., for access via a needle), such as may be provided by a septum. Preferred materials for septa include rubber and polytetrafluoroethylene of the type sold under the trade name TEFLON by DuPont (Wilmington, Del.). In addition, the containers may comprise two or more compartments separated by partitions or membranes that can be removed to allow mixing of the components.

[0041] Kits embodying features of the present invention may also be supplied with instructional materials. Instructions may be printed (e.g., on paper) and/or supplied in an electronic-readable medium (e.g., floppy disc, CD-ROM, DVD-ROM, zip disc, videotape, audio tape, etc.). Alternatively, instructions may be provided by directing a user to an Internet web site (e.g., specified by the manufacturer or distributor of the kit) and/or via electronic mail.

[0042] In another embodiment of this invention, various methods are provided directed to reducing damage cause by reactive oxidative species. In one embodiment, the method involves administering to a mammal an oral dosage form comprising a therapeutically effective amount of a first antioxidant and a topical dosage form comprising a therapeutically effective amount of a second antioxidant. In this method, at least one of the first antioxidant or the second antioxidant comprises acerola concentrate.

[0043] The following examples and comparative study of antioxidant activities illustrate features in accordance with the present invention, and are provided solely by way of illustration. They are not intended to limit the scope of the appended claims or their equivalents. As will be seen from the following examples, the use of both an oral and topical dosage form, one of which contains an effective amount of acerola concentrate is more effective at preventing experimental carcinogenesis than either the topical or oral form alone. In addition, the examples provided below will show that acerola concentrate is more effective at preventing experimental carcinogenesis than an equivalent amount of synthetic Vitamin C.

[0044] Research has increasingly established antioxidants as protective or preventative agents against reactive oxygen species' potential to damage to DNA, cell structure, function and growth ( see Recent Results in Cancer Res. 1999, 151, 29; Adv. Exp. Med. Biol. 1995, 369, 167). The research undertaken in the present invention evaluated the ability of the above described antioxidants to protect DNA, cellular structure and function using in vitro and in vivo experimental models. Antioxidants were initially evaluated in vitro for their antioxidant properties in a variety of reactive oxygen species assay systems. Subsequent to identifying top performing antioxidants in vitro, antioxidants were evaluated in vivo for their effectiveness when administered orally alone, topically alone, and in combined oral and topical applications.

[0045] Antioxidants were subjected to various in vitro biochemical assays to assess their antioxidant capabilities against different reactive oxygen species. Inhibition of linoleic acid auto-oxidation was assayed to address protection against peroxyl radical (J. Org. Chem., 1993, 58, 3532); inhibition of cytochrome C reduction was assayed to address protection against superoxide anion (J. Clin. Invest., 1973, 52, 741). In addition, the properties of antioxidants to inhibit reactive oxygen species-stimulated growth of cells was evaluated using an ATP bioluminescence assay (J. Immunol. Meth., 1993, 160, 81).

[0046] The concentration-dependent reactive oxygen scavenging abilities of several natural source and synthetic antioxidants were assessed. The natural source antioxidants and other chemicals used in this study were obtained from the following sources (product names and/or identifying characteristics are included in parentheses): tocopheryl acetate (VITAMIN E, SYNTHETIC N.F.—1000 lU/g) from Hoffman-LaRoche, Inc. (Nutley, N.J.); tocopherol (COVI-OX T-50 NATURAL) from Cognis Corp, USA (Cincinnati, Ohio); bioflavonoids (LEMON BIOLFLAVONOIDS) from Access Business Group LLC (Lakeview, Calif.); tetrahydrodiferuloylmethane (&) tetrahydrodemethoxydiferuloylmethane (&) tetrahydrobisdemethoxydiferuloylmethane (TETRAHYDROCURCUMINOIDS) from Sabinsa Corporation (Piscataway, N.J.); grape seed extract (&) phospholipids (LEUCOSELECT PHYTOSOME) from Indena S.p.A. (Milan, Italy); glutathione (GLUTHAM) from Silab (Saint Viance, France); palmitoyl hydroxypropyltrimonium amylopectin/glycerin crosspolymer (&) lecithin (&) camellia sinensis extract; green tea (GLYCOSPHERE—GREEN TEA DECAFFEINATED) from Kobo Products (East Brunswick, N.J.); superoxide dismutase (S.O.D.C.am) from Silab; tetrahexyldecyl ascorbate (BV-OSC) from Barnet (Englewood Cliffs, N.J.); ubiquinone (UBIDECARENONE) from Seltzer Chemicals, Inc. (Carlsbad, Calif.); retinyl acetate (VITAMIN A ACETATE 2.8 M lU/g USP) from BASF (Mount Olive, N.J.); magnesium ascorbyl phosphate (VC-PMG-U5) from Nikko Chemicals, Co. (Tokyo, Japan); bitter orange peel extract in butylene glycol (BITTER ORANGE EXTRACT) from Centerchem (Norwalk, Conn.); cyclodextrin & soybean (glycine soja) germ extract (ISOFLAVONE SG-10) from Barnet; retinyl palmitate (VITAMIN A PALMITATE, USP, FCC, TYPE P1) from Hoffman-LaRoche, Inc.; licorice extract (LICHALCONE LR-15) from Barnet; elderberry extract (13% anthocyanosides) from Access Business Group, LLC; pyctiogenol from Natural Health Sciences (Hillside, N.J.); provatene from Provatene Partners; green tea extract (95% polyphenols) from TSI; lutein (10% esters, 5% lutein) from Cognis Corp, USA; acerola concentrate (15% ascorbic acid) from Access Business Group, LLC; and CoQ10 from Kyowa Hakko U.S.A., Inc. (Aliso Viejo, Calif.).

[0047] The macrophage cell lines J774A.1 and P388D1 were obtained from the American Type Culture Collection (ATCC, Rockville, Md.). The 3PC-cell line was obtained from the University of Texas M. D Anderson Cancer Center, Science Park-Research Division (Smithville, Tex.). The macrophage J774A.1 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (Cyclone, Logan, Utah). The 1×10⁶ cells were grown to confluence in a 25-mL flask in a 5-mL DMEM medium at 37° C. The medium was supplemented with L-glutamine and penicillin/streptomycin purchased from Gibco Laboratories (Grand Island, N.Y.). Another line of macrophage cells, P338D1, was maintained in RPMI 1640 containing 10% fetal bovine serum (Cyclone, Logan, Utah). The 1×10⁶ cells were grown to confluence in a 25-mL flask with a 5-mL RPMI 1640 medium at 37° C., containing L-glutamine, penicillin/streptomycin, sodium bicarbonate, HEPES, D-glucose, and sodium pyruvate, all of which were purchased from Gibco Laboratories (Grand Island, N.Y.). The basal 3PC keratinocytes were maintained in Eagle Minimum Essential Minimum (EMEM) containing no calcium and 8% fetal bovine serum (Cyclone, Logan, Utah), and were grown at 37° C. in a 5% CO₂ atmosphere. The cells (1×10⁶) were grown to confluence in a 12-mL flask in a 5-mL EMEM medium containing L-glutamine and penicillin/streptomycin (Gibco Laboratories Grand Island, N.Y.), ethanolamine, phosphoethanolamine, insulin, epidermal growth factor, and transferrin (Sigma Chemical Co, St Louis, Mo.). The cells were more than 90% viable as estimated by trypan blue exclusion.

[0048] A stock solution of 0.3 phosphate buffer (pH 7.4) was treated overnight with Chelex-100 at room temperature and stored in a plastic bottle for no more than 2 weeks. Stock solutions of SDS and HDTBr (both 0.12 M) were prepared and used within 2 weeks. A solution of 0.5 M ABAP was freshly made in 0.05 phosphate buffer (pH 7.4). Stock solutions of antioxidants, except for L-ascorbic acid, acerola concentrate, and the mixed carotenoids, were freshly prepared in 0.05 phosphate buffer (pH 7.4) as 5 mg/mL stock solutions. The two water-soluble antioxidants were freshly prepared 30 min before use.

[0049] The ability of peroxides to form free radicals and, consequently, to initiate lipid peroxidation and DNA damage may be measured by a simple, rapid, and convenient spectrophotometric technique, as described by W. A. Pryor et al. (J. Org. Chem., 1993, 58, 3532). The method utilizes linoleic acid as an auto-oxidizable substrate, and monitors the appearance of linoleic acid hydroperoxide, which has an absorption at 233 nm. The method of forming radicals from peroxides in water employs a solution of 2.6 mM linoleic acid in 0.12 M SDS micelles in a 0.05 M phosphate buffer at pH 7.4. This solution is prepared and thermostated in a spectrophotometric cuvette. The initiator ABAP is then added in the presence or absence of various phytochemicals, and the rate of development of absorbance at 233 nm is followed.

[0050] Superoxide anion production by macrophages was measured by the cytochrome C reduction assay described by B. M. Babior et al. (J. Clin. Invest., 1973, 52, 741). The reaction mixture contained 1 mL of macrophages (3×10⁶ cell/mL) and 0.05 mM cytochrome C. The reaction mixture was incubated for 15 min at 37° C. The reactions were terminated by placing the tubes on ice. The mixtures were centrifuged at 1,500 g for 10 min at 4° C., and the supernatant fractions were transferred to clean tubes for subsequent spectrophotometric measurements at 550 nm. Absorbance values were converted into nanomoles of cytochrome C reduced by using the extinction coefficient of 2.1×10⁴ M/cm/15 min.

[0051] Many methods have been used for ATP determination, but the most widely used at present, in large part due to its sensitivity, is the luciferin-luciferase bioluminescent assay (see J. Immunol. Meth., 1993, 160, 81). ATP bioluminescence has been used for determining levels of ATP in a number of different cell types. MgATP² converts the luciferin into a form capable of being catalytically oxidized by the luciferase in a high quantum yield chemiluminescent reaction. Under optimum conditions and at low ATP concentration, light intensity is linearly related to ATP concentration. Most ATP is found within living cells and links catabolic and anabolic processes. Cell injury or oxygen/substrate depletion results in a rapid decrease of the cytoplasmic ATP. Cellular ATP can be measured by direct lysis of the cells with a suitable detergent. The released ATP is then free to react with the luciferin-luciferase leading to light emission. The ATPLite-M system (Packard Instrument Co., Meriden, Conn.) is an adenosine triphosphate (ATP) monitoring system based on firefly luciferase. The ATPLite-M assay system is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin.

[0052] The abilities of the natural source antioxidants to scavenge oxygen free radicals was assessed using one or more in vitro assays, including but not limited to: linoleic acid autoxidation, cytochrome c reduction, and ATP-bioluminescence. Concentration-dependent responses for each antioxidant in each of the assays were utilized to estimate 50% effective inhibitory concentrations for each antioxidant (IC50; mg/L).

[0053] Table 2 below summarizes estimated IC50 values for selected top performing antioxidants as evaluated in the linoleic acid auto-oxidation and cytochrome c reduction assays. Note that the antioxidants shown in Table 2 are rank ordered in terms of increasing IC50, they were not statistically different from each other, i.e., they performed similarly well.

TABLE 2
Estimated IC50 Values of Selected Top Performing
Natural Antioxidants (mg/L)
	Linoleic Acid Auto-oxidation	Cytochrome C Reduction
	(peroxyl radical scavenging)	(superoxide anion scavenging)
	Grape Seed Extract	Elderberry Extract
	0.8	6
	Elderberry Extract	Complex 1
	0.8	12
	Pycnogenol	Grape Seed Extract
	2.5	14
	Green Tea Extract	Green Tea Extract
	2.7	25
	Complex 1	Acerola Concentrate
	3.0	38

[0054] The data shown in FIGS. 1 and 2 depict dose-response relationships for selected top performing antioxidants in the ATP-bioluminescence assay. The estimated IC50 values obtained from dose response relationships for these antioxidants are summarized in Table 3.

	TABLE 3
		Estimated IC50 (mg/L) in ATP
	Antioxidant	Bioluminescence Assay
	Lutein	70.8
	Green Tea Extract	43.0
	Pycnogenol	35.8
	Elderberry Extract	24.5
	Grape Seed Extract	21.5
	Acerola Concentrate	8.2
	Ascorbic Acid	4.6

[0055] It is clear from the data shown in FIGS. 1 and 2 and Table 3 above that the natural source antioxidants examined in this study—particularly those containing a variety of different antioxidants, such as acerola concentrate—are effective inhibitors of oxygen free radical formation.

[0056] A comparison of the performance of acerola concentrate to that of ascorbic acid on a weight basis reveals that these two antioxidant materials possess similar antioxidant capacity. This result is surprising and unexpected as acerola concentrate contains about only 15% by weight as ascorbic acid. This strongly suggests that one or more compositional ingredients of acerola concentrate other than vitamin C (e.g., flavonoids) significantly enhances the effectiveness of endogenous vitamin C antioxidant potency. Calculation of IC50 values based upon ascorbic acid content reveals that acerola concentrate is approximately four times more potent than can be explained based solely on vitamin C content, as shown in Table 4 below.

TABLE 4
	Estimated IC50	Estimated IC50
	based on weight	Based on vitamin C
Antioxidant	(mg/L)	content (mg/L)	Percent
Acerola Concentrate	8.16	1.22	15%
(AC)
Ascorbic Acid (AA)	4.60	4.60	100%
Ratio of Effectiveness AC:AA = 3.76

[0057] One of the best studied in vivo models for evaluation of environmental stress response effects on DNA and cell damage is the mouse skin system in which the skin is treated with a chemical compound known to damage DNA and cell structure and function. Multiple topical applications of low dose dimethylbenzanthracene (DMBA) results in predictable irritation/inflammation in the skin which is accompanied by damage to DNA and normal cell structure, function and growth (Adv. Exp. Med. Biol. 1995, 369, 167). The aforementioned antioxidant compounds were evaluated for their ability to reduce or prevent this damage via oral or topical or combined oral plus topical application prior to or during DMBA treatment. Following dosing and treatment, skin DNA and cell structure and function were evaluated using three endpoints, epidermal hyperplasia, 8-OH-dG formation, and Ha-ras mutation. Each method is described briefly below.

[0058] Seven week old, pathogen free, female SENCAR mice were purchased from the National Cancer Institute (NCI, Frederick, Md.). Mice were randomized by weight and separated into groups (n=5 mice per group). Experimental groups of mice were treated on shaved dorsal skin with antioxidants, 15 min prior to treatment with DMBA (25 μg per treatment). In topical experiments, different doses, i.e., 0.5, 1.0, 2.0, and/or 4.0 mg, of test or reference antioxidants per mouse were applied topically twice weekly for a total of 8 treatments (4 weeks). In each experiment, a positive control group was treated with DMBA only, no antioxidants were administered. Negative control groups were treated with vehicle (acetone) only. The DMBA solution was prepared in acetone immediately before use, under yellow light. Most antioxidants were administered in acetone. Antioxidants that were not soluble in acetone to the desired concentration, were dissolved in the necessary volume: mixture of acetone and water or ethanol. All topical treatments were administered in a final volume of 0.2 mL. Dietary antioxidants were administered in at least two different doses, i.e., 0.5%, 1.0% and/or 5.0%. Test and reference antioxidants were administered in AIN-93G based diets beginning 2 weeks prior to the first topical application of DMBA. DMBA was again administered a total of 8 times over 4 weeks with no other topical treatments. The same control groups were maintained. Both positive and negative control groups were fed a standard AIN-93G diet (i.e., not supplemented with antioxidants). Animals were sacrificed 48 hours after the final DMBA treatment. At sacrifice the shaved dorsal skin section was removed. A one square centimeter section was removed from the center of the skin, preserved in 10% buffered formalin, and embedded for histological preparation. Epidermal thickness was determined in each animal from at least 20 randomly selected sites per animal using formalin-fixed, paraffin-embedded 5 μm sections stained with hematoxylin and eosin. The remaining skin was frozen in liquid nitrogen. All frozen sections were stored at −70C. until analysis for isolation of DNA.

[0059] DNA was isolated from freshly-frozen tissues of 5 mice per group following non-phenol extraction and ethanol precipitation. Approximately 100 μg of isolated DNA was digested to nucleosides with nuclease P1 and alkaline phosphatase. Quantification of modified DNA bases was accomplished by high performance liquid chromatography (Shimadzu, Japan) with electrochemical detection unit (ECD) using an ESA system (ESA, Inc. Chelmsford, Mass.); normal bases (dG) were quantified by HPLC (78) using an UV detection system. Data were expressed as pmol 8-OH-dG/105 pmol dG. All analyses were performed in duplicate or triplicate, with appropriate standard curves to correlate area units or peak height with concentration. Skin from mice treated with DMBA (100 nmols, 2×/wk for 4 wks) served as the positive control and skin from solvent-treated and untreated animals served as negative controls.

[0060] DNA isolated from freshly-frozen tissues of 5 mice per group was analyzed for mutations in codon 61 of c-Ha-ras by PCR analysis. The procedure used for Ha-ras codon 61 was derived from Nelson et al (Proc. Natl. Acad. Sci. USA 89, 6398). The 3MSP61 mutant reverse primer was designed so that its 3′ end nucleotide (A) pairs with the middle nucleotide (underlined) of a CAA→CTA transversion in codon 61, and selectively amplifies mutated DNA under the conditions described below. The assay was based on the fact that Taq polymerase lacks 3′ exonuclease activity and thus cannot repair a mismatch at the 3′ end of the annealed primer. The conditions of the assay depend on the reverse primer failing to anneal sufficiently to the wild type sequence so that extension does not occur. Using the same forward primer, one reaction was run with the reverse mismatch primer (3MSP61mut) and another reaction was run with a reverse wild type primer (3MSP61wt). This protocol detects only CAA->CTA transversion, mutations that are the most prevalent in codon 61 point mutations. The ratio of the amount of wild type DNA to mutated DNA was determined by quantifying intensity of 32P labeling on autoradiograms. The DNA from the plasmid pHras61mut was used as a positive control sample. The plasmid pHras61 contains cloned exon 2 Ha-ras DNA from a Sencar mouse tumor. The cloned mutation was verified by DNA sequencing. The mutation is the CAA→CTA transversion in codon 61 (located in exon 2) of the mouse Ha-ras gene.

[0061] Acerola concentrate and selected other antioxidants exhibited potent antioxidant activity in the in vivo assays, whether administered topically, orally, or in combination. Significantly, Acerola concentrate was a component among the five top performing combination therapies, including a topical/oral combination consisting only of Acerola concentrate.

[0062] As shown below in Table 5, and similar to the results of the ATP Bioluminescence assay discussed above, acerola concentrate containing approximately 15% vitamin C tended to performed on par with pure ascorbic acid when applied topically or when consumed orally, both inhibiting DMBA-induced epidermal hyperplasia 66% to 70%. These results for either dosage route are a manifestation of the synergism between the ascorbic acid and, presumably, other flavonoids contained in acerola concentrate.

[0063] Surprisingly, combination oral and topical acerola concentrate delivered more antioxidant protection than either dosage form alone (see combination treatment in Table 5). When equivalent doses are administered as a combination of oral and topical dosing, DMBA-induced epidermal hyperplasia was essentially 100% inhibited, i.e., epidermal cells were apparently completely protected from the damaging insult of DMBA treatment.

TABLE 5
Inhibition of DMBA-Induced Epidermal Hyperplasia
	Inhibition (%)
	Topical application
	Ascorbic Acid (0.5 mg)	66%
	Ascorbic Acid (2.0 mg)	66%
	Acerola Concentrate (0.5 mg)	68%
	Acerola Concentrate (1.0 mg)	80%
	Acerola Concentrate (2.0 mg)	70%
	Dietary
	Ascorbic Acid (1.0%)	83%
	Ascorbic Acid (5.0%)	86%
	Acerola Concentrate (1.0%)	70%
	Acerola Concentrate (5.0%)	82%
	Combination topical and oral
	Acerola Concentrate diet alone (0.5%)	73%
	Acerola Concentrate (0.5%) + Acerola	98%
	Concentrate (1.0 mg)
	Acerola Concentrate(0.5%) + Acerola	105%
	Concentrate(2.0 mg)
	Acerola Concentrate diet alone (1.0%)	81%
	Acerola Concentrate (1.0%) + Acerola	96%
	Concentrate (1.0 mg)
	Acerola Concentrate (1.0%) + Acerola	96%
	Concentrate (2.0 mg)

[0064] Thus, an enhancement in epidermal hyperplasia inhibiting activity of acerola concentrate is observed when a first therapeutically effective amount is administered in an oral dosage form and a second therapeutically effective amount is administered in a topical dosage form, in accordance with the present invention. Moreover, there is additional enhancement in the antioxidant activity of acerola concentrate when it is administered in combination with other antioxidants orally and/or topically. Representative synergistic combinations of antioxidants in accordance with the present invention include but are not limited to a mixture of acerola concentrate, vitamin E, and Complex 2, and a mixture of acerola concentrate, vitamin E, and Complex 1.

[0065] As shown in Table 6, combination application of oral and topical acerola concentrate surprisingly exhibits synergistic antioxidant protection against DMBA-induced formation of 8-OH-dG, a marker of genetic damage. When acerola concentrate is administered either orally or topically alone, there is 15% to 30% inhibition of 8-OH-dG formation in vivo in response to DMBA. When the same doses of acerola concentrate is administered both orally and topically, there is 81% inhibition of of 8-OH-dG formation, more than twice the inhibition observed with each dosage form alone, a clear demonstration of synergistic protection against 8-OH-dG formation by oral and topical acerola concentrate.

TABLE 6
Inhibition of DMBA-induced 8-OH-dG formation
	8-OH-dG formation	% Inhibition
	Acerola Concentrate 2.0 mg (topical alone)	−30.8
	Acerola Concentrate 1% (dietary alone)	−15.1
	Acerola Concentrate (1% dietary) + Acerola	−81.5
	Concentrate (2 mg topical)

[0066] As shown in Table 7, topical acerola concentrate surprisingly exhibits almost complete antioxidant protection against DMBA-induced formation Ha-ras formation, another marker of genetic damage.

TABLE 7
Inhibition of DMBA-induced Ha-ras mutation
	Topical	Ha-ras Formation
	DMBA (pos. control)	18.39
	Acetone (neg. control)	3.05
	Acerola Concentrate (4 mg) + DMBA	3.73
	Ascorbic Acid (4 mg) + DMBA	6.19

[0067] The foregoing detailed description and examples have been provided by way of explanation and illustration, and are not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be obvious to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.