Treatment of diseases involving defective gap junctional communication
United States Patent: 7,514,107
Issued: April 7, 2009
Inventors: Sies; Helmut (Meerbusch,
Incorporated (McClean, VA)
Appl. No.: 10/392,641
Filed: March 20, 2003
George Washington University's Healthcare MBA
This invention relates to compositions
containing polyphenols, for example, cocoa polyphenols such as flavanols
and their related oligomers, and methods for treating abnormalities in gap
junctional communication of cells, such as cancer, heart arrhythmia, neuro-degenerative
diseases and cognitive dysfunction.
Description of the
SUMMARY OF THE INVENTION
The invention relates to compositions, products and methods for treating,
preventing, reducing the occurrences of, or reducing the risk of heart
arrhythmia in a mammal suffering from, or at risk of suffering from, heart
arrhythmia, a human or a veterinary animal such as a horse, a cat or a dog.
Moreover, the invention relates to methods and products for treating,
preventing, reducing the risk of, or slowing the progression of any
condition associated with abnormalities in the gap junctional communication
between cells such as cancer, heart arrhythmia, neuro-degenerative diseases
and cognitive dysfunction involving defective gap-junctional communication
In one aspect, the invention relates to a composition, such as a food, a
food additive, a dietary supplement, or a pharmaceutical comprising a cocoa
polyphenol. The composition may optionally contain L-arginine and/or a
cholesterol reducing agent.
Packaged products containing the above-mentioned compositions and a label
and/or instructions for use to treat, prevent, reduce the risk of, or slow
the progression of heart arrhythmia, a neuro-degenerative disease or any
condition associated with abnormalities in the gap junctional communication
of cells are also within the scope of the invention.
In another aspect, the invention relates to a method for treating,
preventing, reducing the risk of, or reducing the occurrences of heart
arrhythmia in a mammal, such as a human or a veterinary animal, by
administering a composition containing a cocoa polyphenol such as cocoa
flavanols and/or its related oligomers to the mammal in need thereof.
In yet another aspect, the invention relates to a method of treating,
preventing, reducing the progression of, or reducing the risk of a condition
associated with the abnormalities in the cellular gap junction communication
in a mammal, such as a human or a veterinary animal, by administering a
composition containing a cocoa polyphenol such as cocoa flavanols and/or its
related oligomers to the mammal in need thereof. For example, such condition
is a neuro-degenerative disease or cognitive dysfunction and the mammal
suffers or is at risk of suffering of the neuro-degenerative disease or
All patents, patent applications and references cited in this application
are hereby incorporated herein by reference. In case of any inconsistency,
the present disclosure governs.
The present invention relates to a composition containing a flavanol and/or
its related oligomers, such as at least one cocoa flavanol and/or its
related procyanidin oligomer, to a mammal in need thereof. The composition
may optionally contain a cholesterol lowering agent, for example, a sterol
and/or stanol based cholesterol lowering agent, L-arginine, calcium,
potassium, magnesium, an antioxidant, for example vitamin E and vitamin C,
any of the vitamins of the B complex, a carotenoid, guar gum, or a mono or
polyunsaturated fatty acid (e.g. omega-3 fatty acid), vitamin D3, retinal,
retinoids, soy protein and selenium. Compositions may contain polyphenols
from sources other than cocoa, e.g. peanut skins, which have properties
similar to those of cocoa polyphenols in addition to, or in place of, the
As used herein, the term "cocoa polyphenol" (CP) refers to a polyphenolic
substance such as flavanols and their related oligomers present in cocoa
beans or extracted from cocoa beans or cocoa ingredients. The term "flavanol"
includes the monomers catechin and epicatechin. Oligomers of catechin and
epicatechin are referred to as procyanidins. Any reference to cocoa
polyphenol herein should be understood to also apply to cocoa flavanols and
procyanidin, in combination and individually.
The term "cocoa ingredient" refers to a cocoa solids-containing material
derived from shell-free cocoa nibs such as chocolate liquor and partially or
fully-defatted cocoa solids (e.g. cake or powder).
The phrase "cholesterol lowering agent" means any compound, combination of
compounds, an extract or a plant component, naturally found or processed,
that has the property of lowering cholesterol levels in a mammal when
administered in an effective amount. When such cholesterol lowering agent is
a compound or a combination of compounds that is of a sterol or stanol type,
i.e., including derivatives and isomeric forms, the cholesterol lowering
agent is referred to as a "sterol and/or stanol based cholesterol lowering
agent." When the phrase is used in reference to a composition, for example,
"a cholesterol reducing dark chocolate," it means that the composition has
the property of lowering cholesterol.
The cocoa polyphenols may be of natural origin, i.e., derived from a cocoa
bean, or prepared synthetically. A person of skill in the art may select
natural or synthetic cocoa polyphenol based on availability or cost. Cocoa
polyphenols may be included in the composition in the form of a cocoa
ingredient containing cocoa polyphenols, for example, chocolate liquor
included in chocolate, or may be added independently of cocoa ingredients,
for example, as an extract, extract fraction, isolated and purified
individual compound, pooled extract fractions or a synthetically prepared
The cocoa polyphenols include cocoa flavanols and their oligomers. Flavanols
include (+)-catechin, (-)-epicatechin and their respective epimers (e.g.
(-)-catechin and (+)-epicatechin) and have the structure
-- see Original Patent.
The procyanidin oligomers may have from 2 to about 18, preferably from 2 to
about 12, and most preferably from 2 to about 10 monomeric units. For
example, oligomers may be dimers, trimers, tetramers, pentamers, hexamers,
heptamers, octamers, nonamers and decamers. In the oligomer, monomers are
connected via interflavan linkages of (4.fwdarw.6) and/or (4.fwdarw.8).
Oligomers with exclusively (4.fwdarw.8) linkages are linear; while the
presence of at least one (4.fwdarw.6) bond results in a branched oligomer.
In the case of peanut skins polyphenols, linkages, such as
C2.fwdarw.O.fwdarw.C7, are also present.
Thus, a polymeric compound of the formula A.sub.n, or a pharmaceutically
acceptable salt thereof, may be used in the compositions and methods of the
-- see Original Patent.
The cocoa polyphenol may be
prepared by extraction from cocoa beans, cocoa nibs, or cocoa ingredients
such as chocolate liquor, partially defatted cocoa solids, and/or fully
defatted cocoa solids. Preferably, the extract is prepared from a fully or
partially defatted cocoa powder. Beans from any species of Theobroma,
Herrania or inter- and intra-species crosses thereof may be used. The
extract may be prepared from fermented, underfermented or unfermented beans,
the fermented beans having the least amount of cocoa polyphenols and the
unfermented the most. The selection of beans may be made based on the
fermentation factor of the beans, for example, the extract may be made from
the beans having a fermentation factor of about 275 or less. Optimizing the
level of polyphenols in the cocoa ingredient and extract thereof by
manipulating the degree of fermentation may be done as described in the
International Appl. No. PCT/US97/15893 published as WO98/09533, the relevant
portions of which are hereby incorporated herein by reference.
Cocoa polyphenols may be extracted from cocoa ingredients that have been
processed using traditional methods of cocoa processing (described, for
example, in Industrial Chocolate Manufacture and Use, ed. Beckett, S. T.,
Blackie Acad. & Professional, New York, 1997, such as in Chapters 1, 5 and
6) or using an improved processing method described in U.S. Pat. No.
6,015,913 to Kealey et al. that preserves polyphenols (by preventing their
destruction) in cocoa ingredients in contrast to the traditional methods.
The improved cocoa processing method omits the traditional roasting step.
Thus, cocoa ingredients obtainable by (a) heating the cocoa bean for a time
and a temperature sufficient to loosen the cocoa shell without roasting the
cocoa nib; (b) winnowing the cocoa nib from the cocoa shell; (c) screw
pressing the cocoa nib and (d) recovering the cocoa butter and partially
defatted cocoa solids which contain preserved levels of cocoa polyphenols,
may be used. The method retains a much higher level of higher procyanidin
oligomers than traditional processing methods. Cocoa solids produced by this
method may contain greater than 20,000 .mu.g of total flavanol and/or
procyanidins per gram nonfat solids; preferably greater than 25,000 .mu.g/g,
more preferably greater than 28,000 .mu.g/g, and most preferably greater
than 30,000 .mu.g/g. For purposes of this invention, the total flavanol
and/or procyanidin amounts are determined as described in Example 2.
Cocoa polyphenols may be extracted from the sources indicated above using
solvents in which the polyphenols dissolve. Suitable solvents include water
or organic solvent such as methanol, ethanol, acetone, isopropyl alcohol and
ethyl acetate. Solvent mixtures may also be used. When water is used as the
solvent, it may be slightly acidified, for example with acetic acid.
Examples of some solvents are mixtures of water and organic solvent, for
example aqueous methanol, ethanol or acetone. Aqueous organic solvents may
contain, for example, from about 50% to about 95% of organic solvent. Thus,
about 50%, about 60%, about 70%, about 80% and about 90% organic solvent in
water may be used. The solvent may also contain a small amount of acid such
as acetic acid, for example, in the amount of about 0.5% to about 1.0%. The
composition of the extracts, i.e., the representation (i.e., oligomeric
profile) and the amount of procyanidin oligomers, will depend on the choice
of solvents. For example, the water extract contains primarily monomers, the
ethyl acetate extract contains monomers and lower oligomers, mainly dimers
and trimers, and the aqueous methanol, ethanol or acetone extract contains
monomers and a range of higher oligomers. One of the solvents for extraction
of monomer as well as higher procyanidin oligomers is about 70% acetone.
However, any extract containing polyphenols is useful in the invention. The
methods of cocoa polyphenol extraction are known in the art and are
described, for example, in the U.S. Pat. No. 5,554,645 to Romanczyk et al.
and the International Appl. No. PCT/US97/05693, published as WO97/36497.
Thus, in one embodiment, the cocoa extract is prepared by reducing cocoa
beans to cocoa powder, defatting the powder, extracting the cocoa
polyphenols, and purifying the extract. The cocoa powder can be prepared by
freeze-drying the cocoa beans and pulp, depulping and dehulling the
freeze-dried cocoa beans, and grinding the dehulled beans.
The cocoa polyphenol extract may be purified, for example, by removal of the
caffeine and/or theobromine, and further purified by gel permeation
chromatography and/or High Pressure Liquid Chromatography (HPLC). Gel
permeation chromatography (e.g. on Sephadex LH-20) may be used to enrich the
extract for higher procyanidin oligomers. For example, the eluate containing
monomers and lower oligomers may not be collected until the oligomer(s) of
choice begins eluting from the column. An example of such an extract is
known in the art and is described in Example 5 of the International Appl.
No. PCT/US97/05693, published as WO97/36497, the relevant portions of which
are hereby incorporated by reference herein. By using preparative HPLC, for
example, normal phase HPLC, the extract may be fractionated, for example,
into monomeric and oligomeric fractions containing at least 50% by weight of
the monomer or specific oligomer(s). When a particular fraction contains the
monomers or any of the lower oligomers (e.g. dimers, trimers or tetramers
fraction), the fraction contain about 90 to 95% by weight of the particular
oligomeric fraction. The desired fractions may be pooled after separation to
obtain a combination of oligomers of choice for example to contain oligomers
3-10 or 5-10. A person of skill in the art can manipulate the
chromatographic conditions to achieve the desired procyanidin profile in
view of the guidance in this specification, general knowledge in the art
and, for example, the teachings of U.S. Pat. No. 5,554,645 to Romanczyk et
al. and the International Appl. No. PCT/US97/05693, published as WO97/36497.
The monomeric fraction typically contains a mixture of monomers epicatechin
and catechin; and the oligomeric fraction typically contains a mixture of
dimers (in a dimer fraction), trimers (in a trimer fraction), tetramers (in
a tetramer fraction), etc. Mixtures of monomers and oligomers occur in
isolated fractions because cocoa contains more than one type of each of
monomer, dimer, etc. The oligomeric variability occurs as a result of two
monomers, epicatechin and catechin, that are building blocks of procyanidins,
as well as the chemical bond connecting monomers in the oligomer. Thus,
cocoa dimers are primarily B2 and B5, each of which contains two monomers of
epicatechin. Individual monomers and oligomers may be obtained using
reversed-phase HPLC, e.g. using a C18 column.
Cocoa polyphenol may be used in the compositions of the invention as a cocoa
extract, e.g. solvent-derived extract, cocoa fraction, isolated compounds or
in the form of a cocoa ingredient or a chocolate containing an effective
amount of cocoa flavanols and/or procyanidins. The cocoa ingredients may be
prepared using traditional cocoa processing procedures but is preferably
prepared using the method described in U.S. Pat. No. 6,015,913 to Kealey et
al. Alternatively, to enhance the level of cocoa polyphenols, chocolate
liquor and cocoa solids prepared from cocoa beans having a fermentation
factor of about 275 or less may be used. These ingredients have cocoa
polyphenol content that is higher than can be obtained using traditional
cocoa processing methods (e.g. with roasting) and fully fermented beans. The
chocolate may be prepared using conventional techniques from the ingredients
described above or using an improved process for preserving cocoa
polyphenols during chocolate manufacturing as described in the International
Appl. No. PCT/US99/05414 published as WO99/45788, the relevant portions of
which are hereby incorporated herein by reference. A chocolate prepared by
at least one of the following non-traditional processes is referred to
herein as a "chocolate having a conserved amount of cocoa polyphenols": (i)
preparing cocoa ingredients from underfermented or unfermented cocoa beans;
(ii) preserving cocoa polyphenol during cocoa ingredient manufacturing
process; and (iii) preserving cocoa polyphenol during chocolate
In some embodiments, the compositions contain at least one oligomer, such as
for example a dimer. Such compositions may additionally contain at least one
monomer or a combination of monomers, e.g. catechin and epicatechin. In
another embodiment, compositions containing a combination of monomers,
catechin and epicatechin, e.g. in a form of a monomer fraction isolated from
cocoa, are also prepared and used.
Synthetic procyanidins may also be used and are prepared by methods known in
the art and as described for example in the International Appl. No.
PCT/US98/21392 published as WO99/19319, the relevant portions of which are
hereby incorporated herein by reference. Cocoa polyphenol derivatives may
also be useful. These include gallated monomers and oligomers, glycosylated
monomers and oligomers, and mixtures thereof; metabolites of the procyanidin
monomers and oligomers, such as the sulphated, glucoronidated, and
methylated forms; and enzyme cleavage products of procyanidins generated by
colonic microflora metabolism or internal mammalian metabolism. The
derivatives may be from natural sources or prepared synthetically.
The composition may optionally comprise a cholesterol lowering agent. Any
cholesterol lowering agent irrespective of its mode of action may be used.
Suitable agents may act by reducing cholesterol absorption in the bile of a
mammal or by reducing cholesterol synthesis. Examples of suitable agents are
phytosterols, phytostanols and their derivatives and isomers; soy protein;
soluble fibers, e.g. beta-glucan from, for example, oat and psyllium, nuts,
rice bran oil, each of which is particularly suitable for use in food,
dietary supplements and food additive compositions. Known cholesterol
reducing drugs may also be used but are less preferred for use in the food
and food additive compositions, but may be used in a pharmaceutical. It will
be obvious to a person of skill in the art that the choice of the
cholesterol lowering agent depends on the intended delivery vehicle (e.g.
food, supplement, pharmaceutical) and the mode of administration. Thus, an
agent that reduces absorption of cholesterol in the bile will not be
preferred for intravenous administration. Similarly, if the delivery vehicle
is food, a cholesterol lowering agent having a strong medicinal taste or
smell may not be desirable.
Phytosterols are plant sterols that do not dissolve in water and have the
molecular weight and the structure similar to cholesterol. Phytosterol
reduce cholesterol absorption in the bile (of both endogenic and dietary
cholesterol) as well as serum cholesterol levels (total and LDL) without
being absorbed themselves. Over forty plant sterols have been identified but
beta-sitosterol, campesterol and stigmasterol are most abundant. Other
examples of useful sterols are brassicasterol, desmosterol, chalinosterol,
poriferasterol, an clionasterol. Individual sterols or a mixture of sterols,
isolated from natural sources or synthetic, and isomers and derivatives
thereof may be used. Particularly useful are saturated derivatives of
sterols, known as stanols, in which all carbon-carbon bonds in the rings are
saturated. Suitable stanols have 28 or 29 carbon atoms and include beta-sitostanol,
clionastanol, 22,23-dyhydrobrassicastanol and campestenol. Phytosterols may
be solid (e.g. powder, granules) or liquid (e.g. oil) form.
The sterols and stanols are found in several plant materials as described
for example in the International Appl. No. PCT/EP96/02344. Exemplary sources
of sterols/stanols are pine bark, soy oil, tall oil, bamboo shoot extract
(described in the International Appl. No. PCT/US98/12556, published as
WO98/57545), cocoa hulls and oil, and rice bran oil. Tall oil, a byproduct
of the pulp and paper industry, is a good source of stanol, i.e., beta-stanol.
Plant sterol may be obtained from natural sources such as vegetative oils,
vegetative oil sludge, vegetative oil distillates, and other plant oil
sources such as tall oil by relatively simple and inexpensive means. For
example, a preparation of sterols from vegetable oil sludge by using
solvents such as methanol is described in U.S. Pat. No. 4,420,427 to Hamunen.
Stanols are found in small amounts in nature but may be easily prepared from
sterols by hydrogenating sterols by any of the several methods known to
those of skill in the art. When a sterol starting material is prepared from
a plant material it will contain a mixture of several different sterols
thus, after hydrogenation, the resulting stanol will also be a mixture of
different stanols. The mixtures are suitable for use in the present
invention. However, pure specific sterol preparations may be hydrogenated as
well to produce pure stanols that can also be utilized.
Cocoa oil extracted from cocoa hulls is a good source of phytosterol. Cocoa
phytosterols are a mixture of free and bound sterols, with the free sterols
being up to about 90% of the phytosterols present. The phytosterols include
campesterol, .beta.-sitosterol, stigmasterol, cycloartenoyl, 24-methylene
cycloartenoyl, as well as minor amounts of other phytosterols. The bound
phytosterols include the fatty acid esters or ferulate derivatives of the
phytosterols. The cocoa oil also contains tocols, which include tocopherols
(which have antioxidant properties) and tocotrienols (which may have
cholesterol lowering activity. The cocoa oil is prepared by the process
comprising the steps of: (i) grinding the cocoa hulls; (ii) extracting the
ground cocoa hulls with a solvent for the phytosterols; (iii) removing the
solvent; and (iv) recovering the cocoa oil. The cocoa hulls, a byproduct of
the cocoa bean roasting, can be from dried fermented cocoa beans, micronized
cocoa beans, roasted cocoa beans, and preferably from dried unfermented
beans, which contain the highest total sterol content. The preferred cocoa
beans are from Theobroma cacao. The preferred solvents are petroleum ether,
hexane, pentane, and ethyl ether. The solvent may be recovered by vacuum
distillation. In one embodiment, freeze dried hulls are ground to a fine
powder with a Tekmar Mill (Cincinnati, Ohio) and the ground mass is
subjected to an overnight extraction with redistilled petroleum ether (b. p.
38-39.6.degree. C.) in a Soxtec apparatus (Fisher Scientific, Springfield,
N.J.). The following morning, the solvent is carefully removed by slow
evaporation under a stream of nitrogen, and the resultant extract is stored
at -40.degree. C. The phytosterols may then be purified by preparative HPLC
or column chromatography.
Esterified forms of both sterols and stanols may also be used.
Esterification renders the sterols/stanols more soluble in fats and oils,
which may, in some instances, help their incorporation into food products or
other delivery vehicles. For example, sterols may be esterified with fatty
acid esters. Examples of such esterified sterols include sitosterol acetate,
sitosterol oleate and stigmasterol oleate. Stanol esters may be prepared as
is known in the art and for example as described in U.S. Pat. No. 6,031,118
to van Amerongen et al., U.S. Pat. No. 5,892,068 to Higgins, U.S. Pat. No.
5,502,045 to Miettenen et al, and the International Appl. No. PCT/CA99/00655
(published as WO00/04887). In one embodiment, useful stanol esters are
prepared by esterifying at least one sterol with a C.sub.2 to C.sub.22 fatty
acid ester as described in U.S. Pat. No. 5,958,913 to Miettenen et al. Other
methods known in the art may be used to increase the solubility of sterols/stanols
upon administration to a mammal. One such method is described in U.S. Pat.
No. 5,932,562 to Ostlund, wherein the sterol/stanol is mixed with lecithin
to obtain a water soluble powder.
Sterols/stanols may be added to the composition in powder form by mixing
with other ingredients. In case of a food composition, stanol/sterols as
well as other cholesterol lowering agents are conveniently added at the step
of mixing. During the preparation of cholesterol lowering chocolate, for
example, sterols/stanols may be added to the dry mix containing sugar, the
melting butter; the nibs before milling; or the melted chocolate, which may
be less preferred. To facilitate mixing, sterols/stanols may be first
dissolved in a solubilizing agent such as fat, vegetable oil, monoglycerides,
diglycerides, triglycerides, tocopherols and mixtures thereof. Effective
carriers for making suspensions and emulsions of sterols/stanols are water,
alcohol, polyol, other edible compounds, for example chocolate liquor, in
which sterols/stanols are at least partially soluble and mixtures thereof.
Soy protein may be added to the composition in any known form, for example,
it may be soy protein isolate, soy protein concentrate, textured soy protein
or soy flour, flakes and grits. Whole grain or fragment thereof may also be
used as described, for example, in Example 5. Various forms of soy protein
are well known in the art and are commercially available. Its properties and
methods of obtaining are described, for example, in Soy Protein and Human
Nutrition, Wilcke et al., eds., Acad. Press, NY, 1979. Soy protein may be
used in combination with any sterol and/or stanol based cholesterol lowering
Soluble plant fibers, e.g. beta-glucan, are capable of reducing plasma
cholesterol. The fibers for use in the present invention may be obtained
from any source of beta-glucan, preferably oat grain and oat bran. The
fibers may be prepared and added to compositions according to methods known
in the art. They are particularly suitable for orally delivered compositions
such as foods and dietary supplements. Beta-glucan and other soluble plant
fibers may be used in combination with any sterol and/or stanol based
cholesterol lowering agent.
The composition of the invention may also contain L-arginine, which may be
provided in the composition of the invention in various forms, for example
as a purified compound, an extract from an L-arginine containing plant, or
in the form of a seed/nut ingredient, e.g. nut flour, or as an entire
seed/nut. Any L-arginine source may be used, synthetic or natural. Preferred
L-arginine sources are soy beans and nut meats such as peanuts, walnuts,
almonds, and hazelnuts. Defatted and partially defatted nut meats may be
used. These may be ground and are referred to as nut flour.
The composition may also contain calcium, potassium, magnesium, antioxidant
such as vitamin E and vitamin C, any of the vitamins of the B complex, a
carotenoid, guar gum, or a mono or polyunsaturated fatty acid (e.g. omega-3
fatty acid), which can be obtained according to the methods known in the
art. The mono or polyunsaturated fatty acids may be used in the form of an
olive oil, fish oil or a nut. Examples of nuts suitable for this use are:
peanuts, almonds and walnuts.
The composition of the invention is useful as a food, a food additive, a
dietary supplement, or a pharmaceutical. The compositions may contain a
carrier, a diluent, or an excipient. Depending on the intended use, the
carrier, diluent, or excipient may be chosen to be suitable for human or
veterinary use, food, additive, supplement or pharmaceutical use.
As used herein a "food" is a material consisting essentially of protein,
carbohydrate and/or fat, which is used in the body of an organism to sustain
growth, repair and vital processes and to furnish energy. Foods may also
contain supplementary substances such as minerals, vitamins and condiments.
See Merriam-Webster's Collegiate Dictionary, 10th Edition, 1993. The term
food includes a beverage adapted for human or animal consumption. As used
herein a "food additive" is as defined by the FDA in 21 C.F.R. 170.3(e)(1)
and includes direct and indirect additives. As used herein, a
"pharmaceutical" is a medicinal drug. See Merriam-Webster's Collegiate
Dictionary, 10th Edition, 1993. A pharmaceutical may also be referred to as
a medicament. As used herein, a "dietary supplement" is a product (other
than tobacco) that is intended to supplement the diet that bears or contains
the one or more of the following dietary ingredients: a vitamin, a mineral,
an herb or other botanical, an amino acid, a dietary substance for use by
man to supplement the diet by increasing the total daily intake, or a
concentrate, metabolite, constituent, extract or combination of these
Any conventional food including any beverage which has been improved by the
presence of a cocoa polyphenol or a derivative thereof, e.g. methylated
compounds or metabolic breakdown products, and optionally in combination
with a cholesterol lowering agent, L-arginine, calcium, potassium,
magnesium, an oxidant such as vitamin E and vitamin C, any of the vitamins
of the B complex, a carotenoid, guar gum, and/or a mono or polyunsaturated
fatty acid (e.g. omega-3), is within the scope of the invention.
The improvement is achieved either (i) by adding cocoa polyphenol or a
derivative thereof to a food that does not contain cocoa polyphenol or (ii)
when the food traditionally may contain cocoa polyphenols, such as for
example chocolate, by enhancing the polyphenol level over the one found in
the traditionally prepared food. The enhancement may be achieved by adding
additional cocoa polyphenols, for example, in a form of an extract, fraction
or isolated and purified compound there from; by adding cocoa polyphenol in
combination with another polyphenol containing ingredient (e.g. nut skins);
by manipulating the cocoa ingredients processing and cocoa bean selection,
as described above, to preserve cocoa polyphenol in the cocoa ingredient
used for the manufacture of the food product; or by manipulating the
chocolate manufacturing process as described above. Thus, these foods
(including beverages) contain an "elevated level of polyphenols" (including
cocoa procyanidins) in comparison to comparative conventional foods
(including beverages). An example of a chocolate having an elevated level of
polyphenol occurs when a chocolate manufacturer adds a cocoa extract
containing cocoa polyphenols to its previously commercially available
product. The foods may also be referred to as "high cocoa polyphenol foods,"
i.e., they contain higher levels of polyphenol than their traditional
The foods comprising cocoa polyphenols and optionally at least one
cholesterol lowering agent (e.g. a sterol and/or stanol based cholesterol
lowering agent), L-arginine, calcium, potassium, magnesium, an antioxidant
such as vitamin E and vitamin C, any of the vitamins of the B complex, a
carotenoid, guar gum, or a mono or polyunsaturated fatty acid (e.g. omega-3)
may be adapted for human or veterinary use, and include pet foods. The food
may be other than a confectionery, however, the preferred cholesterol
lowering food is a confectionery such as a standard of identity (SOI) and
non-SOI chocolate, such as milk, sweet and semi-sweet chocolate including
dark chocolate, low fat chocolate and a candy which may be a chocolate
covered candy. Other examples include a baked product (e.g. brownie, baked
snack, cookie, biscuit) a condiment, a granola bar, a toffee chew, a meal
replacement bar, a spread, a syrup, a powder beverage mix, a cocoa or a
chocolate flavored beverage, a pudding, a rice cake, a rice mix, a savory
sauce and the like. If desired, the foods may be chocolate or cocoa
flavored. Food products that contain L-arginine, in addition to the cocoa
polyphenol and the cholesterol lowering agent, are preferably chocolates and
candy bars, such as granola bars, containing nuts, for example, peanuts,
walnuts, almonds, and hazelnuts. It should be noted that the addition of
nuts with skins to the food described herein may also increase the total
polyphenol content since, for example, peanut skins contain about 17%
flavanols and procyanidins and almond skins contain about 30% flavanols and
procyanidins. In one embodiment, the nut skins are added to the nougat of a
chocolate candy containing a cholesterol lowering agent.
In certain embodiments, the non-chocolate food product contains from about
at least 5 micrograms/g to about 10 mg/g, and, for example, at least 5
micrograms/g food product, preferably at least 10 microgram/g, more
preferably at least 100 micrograms/g of cocoa flavanols and/or procyanidin
oligomers. If desired, the non-chocolate food products can contain much
higher levels of cocoa procyanidins than those found in the chocolate food
products described below.
In one embodiment, a chocolate confectionery contains an effective amount of
cocoa flavanol and/or procyanidin to treat, prevent, reduce the risk of, or
reduce the occurrences of heart arrhythmia, or generally any condition
associated with abnormalities in gap junctional communication, such as for
example, a neuro-degenerative disease. The chocolate confectionery may be
milk or dark chocolate. In certain embodiments, the chocolate comprises at
least 3,600 micrograms, preferably at least 4,000 micrograms, preferably at
least 4,500 micrograms, more preferably at least 5,000 micrograms, and most
preferably at least 5,500 micrograms cocoa flavanols and/or procyanidins
each per gram of chocolate, based on the total amount of nonfat cocoa solids
in the product. In other embodiments, the chocolate contains at least 6,000
micrograms, preferably at least 6,500 micrograms, more preferably at least
7,000 micrograms, and most preferably at least 8,000 micrograms of cocoa
procyanidins per gram, and even more preferably 10,000 micrograms/g based on
the nonfat cocoa solids in the product.
A milk chocolate confectionery may have at least 1,000 micrograms,
preferably at least 1,250 micrograms, more preferably at least 1,500
micrograms, and most preferably at least 2,000 micrograms cocoa flavanols
and/or procyanidins each per gram of milk chocolate, based on the total
amount of nonfat cocoa solids in the milk chocolate product. In the
preferred embodiment, the milk chocolate contains at least 2,500 micrograms,
preferably at least 3,000 micrograms, more preferably at least 4,000
micrograms, and most preferably at least 5,000 micrograms cocoa flavanols
and/or procyanidins each per gram of milk chocolate, based on the total
amount of nonfat cocoa solids in the milk chocolate product.
The amount of L-arginine in the food products can vary. Typically, cocoa
contains between 1 to 1.1 grams of L-arginine per 100 grams of partially
defatted cocoa solids. It can range from 0.8 to 1.5 per 100 grams of cocoa.
The chocolate food products of this invention contain L-arginine in an
amount greater than that which naturally occurs in the cocoa ingredients.
Knowing the amount of cocoa ingredients and L-arginine used in the food
product, one of ordinary skill in the art can readily determine the total
amount of L-arginine in the final product. The food product will generally
contain at least 5 micrograms/g, preferably at least 30 micrograms/g, or at
least 60 micrograms/g, even more preferably at least 200 micrograms/g food
When the cholesterol lowering agent is used in the food, its amount will
depend on the type of the agent used and can be determined by a person of
skill in the art based on the guidance in the specification, particularly
daily dosages provided below, and the knowledge in the art. A food
composition, for example, may contain from about 0.5 to about 10 g per 45 g
serving size, preferably about 1.5 to about 5 g per 45 g serving size, most
preferably about 2 to about 4.5 g per 45 g serving size of sterols/stanols.
With respect to soy protein and soluble fiber from oats, FDA has provided
minimum amounts per food serving to be able to make a health claim.
According to the FDA, a food serving containing beta-glucan must contain at
least 0.75 g, and the food serving containing soy protein must contain at
least 6.25 g soy protein. These values may also be used as a guide for
determining the amount of these cholesterol lowering agents in the food.
A daily effective amount of cocoa flavanols and/or procyanidins may be
provided in a single serving. Thus, a confectionery (e.g. chocolate) may
contain at least about 100 mg/serving (e.g. 150-200, 200-400 mg/serving)
cocoa procyanidins. When the cholesterol lowering agent is included in the
composition, at least 1.5 (e.g. 1.5-4.5 g) per serving sterol/stanol may be
Pharmaceuticals containing cocoa flavanols and/or procyanidins, optionally
in combination with a cholesterol lowering agent and/or L-arginine, may be
administered in a variety of ways such as orally, sublingually, bucally,
nasally, rectally, intravenously, parenterally and topically. A person of
skill in the art will be able to determine a suitable cholesterol lowering
agent depending on the mode of administration. Thus, dosage forms adapted
for each type of administration are within the scope of the invention and
include solid, liquid and semi-solid dosage forms, such as tablets,
capsules, gelatin capsules (gelcaps), bulk or unit dose powders or granules,
emulsions, suspensions, pastes, creams, gels, foams or jellies.
Sustained-release dosage forms are also within the scope of the invention
and may be prepared as described in U.S. Pat. Nos. 5,024,843; 5,091,190;
5,082,668; 4,612,008 and 4,327,725, relevant portions of which are hereby
incorporated herein by reference. Suitable pharmaceutically acceptable
carriers, diluents, or excipients are generally known in the art and can be
determined readily by a person skilled in the art. The tablet, for example,
may comprise an effective amount of the cocoa polyphenol-containing
composition and optionally a carrier, such as sorbitol, lactose, cellulose,
or dicalcium phosphate.
The dietary supplement containing cocoa flavanol and/or procyanidin, and
optionally at least one cholesterol lowering agent and/or L-arginine, may be
prepared using methods known in the art and may comprise, for example,
nutrient such as dicalcium phosphate, magnesium stearate, calcium nitrate,
vitamins, and minerals.
Further within the scope of the invention is a package comprising the
composition of the invention (e.g. a food, a dietary supplement, a
pharmaceutical) and a label indicating the presence of, or an enhanced
content of cocoa flavanol and/or procyanidin and/or a derivative thereof, or
directing use of the composition to treat, prevent, reduce the occurrences
of, or reduce the risk of heart arrhythmia or any condition associated with
the abnormality in the cellular gap junction communication, such as a neuro-degenerative
disease. Optionally, the label may indicate the cholesterol lowering agent
and/or L-arginine content, the beneficial properties of the combined
polyphenol(s), cholesterol lowering agent(s) and optionally L-arginine. The
package may contain the composition and the instructions for use to treat,
prevent, reduce the risk of, slow progression or reduce the occurrences of
heart arrhythmia or any condition associated with the abnormality/defect in
the cellular gap junction communication, such as a neuro-degenerative
disease or cancer.
As used herein, "treatment" means improving an existing medical condition,
for example, heart arrhythmia, or the symptoms associated therewith. The
term "preventing" means reducing the risks associated with developing a
disease, including reducing the onset of the disease. The prevention or
prophylaxis may be used in an individual known to be at high risk of
developing a disease or in a population at large for maintaining good
health, for example, reducing the risk of arrhythmia. The methods may be
used in a human or a veterinary animal, such as a dog, a cat, and a horse.
The methods comprise administering to a mammal, preferably a human or a
veterinary animal, for an effective period of time (for example, to induce
de novo connexin synthesis), an amount of the composition comprising a
flavanol and/or its related oligomer, such as a cocoa flavanol and/or its
related oligomer, optionally in combination with a cholesterol lowering
agent, L-arginine, an antioxidant (e.g. vitamin E, vitamin C), the flavanol
and/or its related oligomer being in the amount effective to treat, prevent
or reduce the occurrences of heart arrhythmia or any condition associated
with the gap junctional abnormalities/defects, e.g. neuro-degenerative
diseases (such as Parkinson's and Alzheimer's). Moreover, treatment of
cognitive dysfunctions which are connexin, for example, connexin 40 and/or
43, related is also within the scope of the invention. Thus, connexin
related functions may be treated or improved. Examples of cognitive
dysfunctions that may be treated according to the invention are short term
memory and depression. Other polyphenols such as those isolated from peanut
skins may also be used.
Thus, the following use is within the scope of the invention. Use of a cocoa
flavanol and/or its oligomer in the manufacture of a medicament, food,
nutraceutical or dietary supplement for use in treating, preventing,
reducing the risk of, or reducing the progression of a disease involving
defective gap junctional communication in a human or a veterinary animal.
The following uses are representative of some embodiments. Use of a cocoa
flavanol and/or its oligomer in the manufacture of a medicament, food,
nutraceutical or dietary supplement for use in treating, preventing,
reducing the risk of, or reducing the occurrence of heart arrhythmias in a
human or a veterinary animal. Use of a cocoa flavanol and/or its oligomer in
the manufacture of a medicament, food, nutraceutical or dietary supplement
for use in treating, preventing, reducing the risk of, or reducing the
progression of a cognitive dysfunction in a human or a veterinary animal.
A polymeric compound of the formula A.sub.n, or a pharmaceutically
acceptable salt thereof, may be used in the compositions and uses of the
-- see Original Patent.
The effective amount may be
determined by a person of skill in the art using the guidance provided
herein. For example, the effective amount may be such as to achieve a
physiologically relevant concentration in the body of a mammal. Such a
physiologically relevant concentration may be at least 20 nanomolar (nM),
preferably at least about 100 nM, and more preferably at least about 500 nM.
In one embodiment, at least about one micromole in the blood of the mammal
The methods may further comprise determining the effectiveness of the
treatment by, for example, determining the heart beat, such as using
electrocardiogram (ECG), ambulatory monitors, holter monitors,
transtelephonic monitors (optionally with memory loop), stress test,
echocardiogram, cardiac catheterization, electophysiology study and head
upright tilt test.
The composition may be administered to a healthy mammal for prophylactic
purposes or to a mammal in need of a treatment or having at least one of the
risk factors associated with heart arrhythmia. Any individual having at
least one of the risk factors associated with heart arrhythmia is a subject
for administration of the compositions described herein. The individuals
with a familial history of heart arrhythmia, hypertension, coronary artery
disease, heart attack, abnormal thyroid function as well as those smoking,
and having alcohol, caffeine, or illegal substance intake (e.g. cocaine), or
those recovering from heart surgery, are susceptible individuals in need of
the treatment described herein. Other populations of mammals that are
susceptible to developing vascular health problems will be apparent to a
person of skill in the art.
Veterinary animal, for example dogs, cats and horses, may be administered
the above described compositions to treat, prevent or reduce the occurrences
of heart arrhythmia.
The effective amount to treat, prevent, reduce the risk of and/or reduce the
occurrences of heart arrhythmia may be determined by a person skilled in the
art using the guidance provided herein and the general knowledge in the art.
Cocoa flavanols and/or related oligomers may be administered at from about
50 mg/day to about 1000 mg/day, preferably from about 100-150 mg/day to
about 900 mg/day, and most preferably from about 300 mg/day to about 500
mg/day. However, given that cocoa polyphenols are non-toxic, amounts higher
than stated above may be used.
To additionally obtain a cholesterol lowering effect, using sterols/stanols,
more than what is normally found in the average diet of a non-vegetarian
should be administered. A person on a typical North American diet consumes
about 200-400 mg/day. Thus, when phytosterol, such as sitosterol, is used to
reduce cholesterol levels, about at least 1 g/day, preferably at least about
3 g/day should be administered. Preferably, from at least about 1 g/day,
preferably at least about 4.5 g/d, to about 20 g/day is used. However, the
amount will vary depending on the cholesterol lowering potency of the
phytosterol so that, for example, if a more potent sitostanol is used, the
effective amount may be as low as about one to about three g/day. The
amounts may be determined using the analytical procedure described in Roger
et al., J. Amer. Oil Chem. Soc. 70(30) 1993 and Carpenter, et al., J. Amer.
Oil Chem. Soc. 71(8) 1994. Soy protein may be administered, for example, at
least about 25 g/day. Further guidance can be found in Recommended Daily
Allowances, 9.sup.th ed., National Res. Council/National Acad. Sci.,
Washington, D.C. Soluble fiber may be administered, for example, in the
amount of at least 3 g/day.
L-arginine, when used in the compositions and the treatment, may be
administered from about 2 g/day to about 50 g/day, preferably from about 3
g/day to about 10 g/day, and most preferably from about 6 g/day to about 8
g/day. Here as well, given that L-arginine naturally occurs in foods,
amounts higher than stated above may be used. Polyphenols generally may be
administered in the amounts known in the art. The enhanced absorption in the
presence of sterols/stanols may also be taken into consideration when
determining the effective amount.
The treatments/preventive administration may be continued as a regimen,
i.e., for an effective period of time, e.g., daily, monthly, bimonthly,
biannually, annually, or in some other regimen, as determined by the skilled
medical practitioner for such time as is necessary. The administration may
be continued for at least a period of time required to induce connexin
synthesis. Preferably, the composition is administered daily, most
preferably two or three times a day, for example, morning and evening to
maintain the levels of the effective compounds in the body of the mammal. To
obtain the most beneficial results, the composition may be administered for
at least about 30 to about 60 days. These regiments may be repeated
Claim 1 of 33 Claims
1. A method of treating a subject
suffering from cognitive dysfunction comprising administrating to the
subject a composition comprising an effective amount of a cocoa extract,
chocolate liquor, cocoa cake, cocoa powder, and/or cocoa nib, each
comprising at least the following cocoa polyphenol compounds: (a) a
flavanol having the formula -- see Original Patent, and (b) a dimer
composed of two units of the above flavanol connected via interflavan
linkages 4.fwdarw.6 and/or 4.fwdarw.8; wherein the subject is a human or a
veterinary animal, and wherein the composition comprises at least 100 mg
of the cocoa polyphenol compounds per unit of the composition.
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