U.S. patent application number 10/705081 was filed with the patent office on 2005-05-12 for methods and kits for reducing cellular damage, inhibiting free radical production, and scavenging free radicals in mammals.
Invention is credited to Connor, Lynne M., Evans, Gregory S., Gellenbeck, Kevin W., Grochoski, Gregory, Hanausek-Walaszek, Margaret, Mayne, James R., Randolph, Keith, Roh-Schmidt, Haeri, Scimeca, John V., Slaga, Thomas J., Walaszek, Zbigniew.
Application Number | 20050100537 10/705081 |
Document ID | / |
Family ID | 34552278 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100537 |
Kind Code |
A1 |
Evans, Gregory S. ; et
al. |
May 12, 2005 |
Methods and kits for reducing cellular damage, inhibiting free
radical production, and scavenging free radicals in mammals
Abstract
Methods of reducing cellular damage are described that 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. Methods of inhibiting free radical
production, methods of scavenging free radicals, and kits for
reducing cellular damage are also described.
Inventors: |
Evans, Gregory S.; (Lowell,
MI) ; Grochoski, Gregory; (Ada, MI) ;
Randolph, Keith; (Anaheim, CA) ; Connor, Lynne
M.; (Grand Rapids, MI) ; Scimeca, John V.;
(Kentwood, MI) ; Gellenbeck, Kevin W.; (Poway,
CA) ; Mayne, James R.; (Kentwood, MI) ;
Roh-Schmidt, Haeri; (Ada, MI) ; Slaga, Thomas J.;
(Golden, CO) ; Hanausek-Walaszek, Margaret;
(Golden, CO) ; Walaszek, Zbigniew; (Golden,
CO) |
Correspondence
Address: |
ALTICOR INC.
7575 FULTON STREET EAST MAILCODE 78-2G
ADA
MI
49355
US
|
Family ID: |
34552278 |
Appl. No.: |
10/705081 |
Filed: |
November 10, 2003 |
Current U.S.
Class: |
424/94.1 ;
424/729; 424/766; 424/777; 514/458; 514/474; 514/763 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 36/736 20130101; A61K 31/22 20130101; A61K 36/35 20130101;
A61K 31/375 20130101; A61K 31/015 20130101; A61K 36/736 20130101;
A61K 31/015 20130101; A61K 31/355 20130101; A61K 36/82 20130101;
A61K 31/355 20130101; A61K 31/22 20130101; A61K 36/82 20130101;
A61K 36/87 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61P 17/18
20180101; A61K 2300/00 20130101; A61K 36/35 20130101; A61K 36/87
20130101; A61K 31/375 20130101 |
Class at
Publication: |
424/094.1 ;
424/729; 424/766; 424/777; 514/458; 514/474; 514/763 |
International
Class: |
A61K 038/43; A61K
031/355; A61K 035/78; A61K 031/375; A61K 031/015 |
Claims
1. A method of scavenging free radicals in a mammal comprising:
administering to the mammal an oral dosage form comprising a
therapeutically effective amount of a first antioxidant; and
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.
2. The method of claim 1 wherein both the first antioxidant and the
second antioxidant comprise acerola concentrate.
3. The method of claim 1 wherein only one of the first antioxidant
and the second antioxidant comprises acerola concentrate, and the
other comprises an antioxidant selected from the group consisting
of grape seed extract, pycnogenol, provatene, synthetic
.beta.-carotene, natural .beta.-carotene, vitamin E, L-ascorbic
acid, .quadrature.-tocopherol, green tea extract, elderberry
extract, lutein, coenzyme Q10, Complex 1, Complex 2, Complex 3, and
combinations thereof.
4. The method of claim 1 wherein only one of the first antioxidant
and the second antioxidant comprises acerola concentrate, and the
other comprises an antioxidant selected from the group consisting
of grape seed extract, pycnogenol, provatene, green tea extract,
elderberry extract, lutein, coenzyme Q10, Complex 1, Complex 2,
Complex 3, and combinations thereof.
5. The method of claim 1 wherein a first dosage is administered
comprised of either the oral dosage form or the topical dosage, a
second dosage is administered comprised of the remaining oral
dosage or topical dosage not used in the first dosage, and the
second dosage is administered at any time during the metabolism of
the first dosage.
6. The method of claim 1 wherein the administering of the oral
dosage form and the administering of the topical dosage form occur
within a time frame of about 30 minutes.
7. The method of claim 1 wherein the oral dosage form is selected
from the group consisting of a pill, capsule, gelcap, geltab,
beverage, chewing gum, chewable tablet, lozenge, viscous gel,
troche, toothpaste, gargling gel, mouth rinse, and combinations
thereof.
8. The method of claim 1 wherein the oral dosage form is selected
from the group consisting of a pill, capsule, gelcap, geltab,
chewable tablet, lozenge, troche, and combinations thereof.
9. The method of claim 1 wherein the topical dosage form is
selected from the group consisting of an emulsion, solution,
dispersion, gel, soap, transdermal patch, and combinations
thereof.
10. The method of claim 1 wherein the topical dosage form is a
lotion.
11. The method of claim 1 wherein each of the administering of the
oral dosage form and the administering of the topical dosage form
is repeated at least twice daily.
12. The method of claim 11 wherein both the first antioxidant and
the second antioxidant comprise acerola concentrate, and wherein
the therapeutically effective amount of acerola concentrate in the
oral dosage form is between about 50 mg and about 2000 mg.
13. The method of claim 11 wherein both the first antioxidant and
the second antioxidant comprise acerola concentrate, and wherein
therapeutically effective amount of acerola concentrate in the oral
dosage form is between about 350 mg and about 1500 mg.
14. The method of claim 11 wherein both the first antioxidant and
the second antioxidant comprise acerola concentrate, and wherein
the therapeutically effective amount of acerola concentrate in the
topical dosage form is between about 15 mg and about 200 mg.
15. The method of claim 11 wherein both the first antioxidant and
the second antioxidant comprise acerola concentrate, and wherein
the therapeutically effective amount of acerola concentrate in the
topical dosage form is between about 25 mg and about 100 mg.
16. A method of inhibiting free radical production in a mammal
comprising: administering to the mammal an oral dosage form
comprising acerola concentrate; and administering to the mammal a
topical dosage form comprising acerola concentrate.
17. The method of claim 16 wherein the acerola concentrate in the
oral dosage form is provided in an amount of between about 400 mg
and about 1200 mg, and the acerola concentrate in the topical
dosage form is provided in an amount of between about 50 mg and
about 75 mg.
18. A method of reducing cellular damage in a mammal comprising:
administering to the mammal an oral dosage form comprising a
therapeutically effective amount of a first antioxidant; and
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, and the administering of the oral
dosage form and the administering of the topical dosage form occur
within a time frame of about 24 hours.
19. A kit for reducing cellular damage in a mammal comprising: 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; wherein
at least one of the first antioxidant and the second antioxidant
comprises acerola concentrate.
20. The method of claim 19 wherein both the first antioxidant and
the second antioxidant comprise acerola concentrate.
Description
BACKGROUND
[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).
SUMMARY
[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.
BRIEF DESCRIPTION OF THE DRAWINGS
[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.
DETAILED DESCRIPTION
[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 .beta.-carotene, sodium bisulfite, vitamins
such as Vitamin E and Vitamin C (L-ascorbic acid),
.quadrature.-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.
1 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 (&)
Tetrahydrobisdemethoxydif- eruloylmethane 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.
Experimental Overview
[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.
In Vitro Research
[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 (&)
tetrahydrobisdemethoxydiferuloyl- methane (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.).
Tissue Culture
[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.times.10.sup.6 cells were grown to
confluence in a 25-mL flask in a 5-mL DMEM medium at 37.degree. 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.times.10.sup.6 cells were grown to confluence
in a 25-mL flask with a 5-mL RPMI 1640 medium at 37.degree. 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.degree. C. in a 5%
CO.sub.2 atmosphere. The cells (1.times.10.sup.6) 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.
Preparation Of Solutions
[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.
Linoleic Acid Auto-oxidation
[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.
Cytochrome C Reduction Assay
[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.times.10.sup.6 cell/mL) and 0.05 mM cytochrome
C. The reaction mixture was incubated for 15 min at 37.degree. C.
The reactions were terminated by placing the tubes on ice. The
mixtures were centrifuged at 1,500 g for 10 min at 4.degree. 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.times.10.sup.4 M/cm/15
min.
ATP-Bioluminescence Assay
[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.
MgATP.sup.2 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.
2TABLE 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.
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.
4TABLE 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
In Vivo Research Overview
[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.
Epidermal Hyperplasia Method
[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 .mu.g 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 .mu.m 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.
8-OH-dG Formation Method
[0059] DNA was isolated from freshly-frozen tissues of 5 mice per
group following non-phenol extraction and ethanol precipitation.
Approximately 100 .mu.g 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.times./wk for 4 wks) served as the positive control and skin from
solvent-treated and untreated animals served as negative
controls.
Ha-ras Mutation Method
[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.fwdarw.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.fwdarw.CTA transversion in codon 61
(located in exon 2) of the mouse Ha-ras gene.
In Vivo Results Summary
[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.
DMBA-induced Epidermal Hyperplasia
[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.
5TABLE 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.
6TABLE 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.
7TABLE 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.
* * * * *