U.S. patent application number 10/689197 was filed with the patent office on 2004-04-15 for method of reducing low density lipoprotein cholesterol concentration.
This patent application is currently assigned to The Solae Company, LLC.. Invention is credited to Henley, E. C., Potter, Susan M., Waggle, Doyle H..
Application Number | 20040071800 10/689197 |
Document ID | / |
Family ID | 32074289 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040071800 |
Kind Code |
A1 |
Waggle, Doyle H. ; et
al. |
April 15, 2004 |
Method of reducing low density lipoprotein cholesterol
concentration
Abstract
The present invention is a method for decreasing the blood
concentration of total and LDL cholesterol in a human in which a
plant sterol and a soy hypocotyl material are co-administered to
the human, where the plant sterol comprises at least 0.49%, by
weight, of the combined weight of the plant sterol and the soy
hypocotyl material
Inventors: |
Waggle, Doyle H.; (St.
Louis, MO) ; Potter, Susan M.; (St. Louis, MO)
; Henley, E. C.; (St. Louis, MO) |
Correspondence
Address: |
Richard B. Taylor
Patent Dept. 4C
Protein Technologies International, Inc.
P.O. Box 88940
St. Louis
MO
63188
US
|
Assignee: |
The Solae Company, LLC.
|
Family ID: |
32074289 |
Appl. No.: |
10/689197 |
Filed: |
October 20, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10689197 |
Oct 20, 2003 |
|
|
|
10461751 |
Jun 13, 2003 |
|
|
|
10461751 |
Jun 13, 2003 |
|
|
|
09826346 |
Apr 4, 2001 |
|
|
|
6579534 |
|
|
|
|
09826346 |
Apr 4, 2001 |
|
|
|
09298528 |
Apr 23, 1999 |
|
|
|
6544566 |
|
|
|
|
Current U.S.
Class: |
424/757 ;
514/169; 514/27; 514/456 |
Current CPC
Class: |
A23V 2002/00 20130101;
A23V 2002/00 20130101; A61K 36/48 20130101; A61K 36/48 20130101;
A61K 31/78 20130101; A23V 2002/00 20130101; A23L 33/185 20160801;
A23L 33/11 20160801; A61K 31/78 20130101; Y10T 436/104165 20150115;
A23V 2002/00 20130101; A23V 2250/2116 20130101; A23V 2250/2116
20130101; A23V 2250/5488 20130101; A23V 2250/21372 20130101; A61K
2300/00 20130101; A23V 2250/2116 20130101; A23V 2250/5488 20130101;
A23V 2250/156 20130101; A23V 2250/156 20130101; A23V 2250/2136
20130101; A61K 2300/00 20130101; A23V 2250/5488 20130101; A23V
2250/70 20130101; A23V 2250/156 20130101; A23V 2250/70 20130101;
A23V 2250/70 20130101; A23V 2250/21368 20130101 |
Class at
Publication: |
424/757 ;
514/169; 514/456; 514/027 |
International
Class: |
A61K 035/78; A61K
031/7048; A61K 031/56; A61K 031/353 |
Claims
What is claimed is:
1. A method for lowering blood concentration of total and low
density lipoprotein cholesterol in a human comprising:
co-administering a plant sterol and a soy hypocotyl material to a
human to decrease the blood concentration of total and LDL
cholesterol in said human, where said plant sterol comprises at
least 0.49%, by weight, of the combined weight of the
co-administered plant sterol and soy hypocotyl material.
2. The method of claim 1, further comprising administering an
isoflavone selected from the group consisting of genistein,
daidzein, glycitein, biochanin A, formononetin, and their naturally
occuring glycosides and glycoside conjugates.
3. The method of claim 2 wherein said plant sterol, said soy
hypocotyl material, and said isoflavone are further administered to
prevent or minimize the development of atheroscelerosis.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions for and
methods of reducing low density lipoprotein cholesterol and total
cholesterol concentrations in the blood. In particular, the present
invention relates to compositions containing plant sterols, soy
protein, and isoflavones, and combinations thereof, which are
useful for lowering LDL-cholesterol and total cholesterol blood
concentrations and for preventing or minimizing development of
atherosclerosis.
BACKGROUND OF THE INVENTION
[0002] Cardiovascular disease is a leading cause of morbidity and
mortality, particularly in the United States and in Western
European countries. Several causative factors are implicated in the
development of cardiovascular disease including hereditary
predisposition to the disease, gender, lifestyle factors such as
smoking and diet, age, hypertension, and hyperlipidemia, including
hypercholesteremia. Several of these factors, particularly
hyperlipidemia and hypercholesteremia, contribute to the
development of atherosclerosis, a primary cause of vascular and
heart disease.
[0003] High blood cholesterol concentration is one of the major
risk factors for vascular disease and coronary heart disease in
humans. Elevated low density lipoprotein cholesterol (hereafter
"LDL-cholesterol") and elevated total cholesterol are directly
related to an increased risk of coronary heart disease. Cholesterol
and Mortality: 30 Years of Follow-Up from the Framingham Study,
Anderson, Castelli, & Levy, JAMA, Vol 257, pp. 2176-80
(1987).
[0004] Ingestion of soy protein materials in the diet is associated
with a lower risk of coronary heart disease, which may reflect
decreases in serum cholesterol levels. Soy protein materials are
known to reduce total cholesterol and LDL-cholesterol levels in the
blood of animals. A recent meta-analysis of the effects of soy
protein intake on serum lipids in humans has shown that dietary soy
protein is significantly related to lowering serum concentrations
of total cholesterol and LDL-cholesterol in humans. Meta-Analysis
of the Effects of Soy Protein Intake on Serum Lipids, Anderson,
Johnstone, and Cook-Newell, N. Engl. J Med., Vol. 333, No. 5, pp.
276-82 (1995).
[0005] Ingestion of phytosterols, compounds which are found in
whole soybeans, has also been shown to reduce the circulating total
and LDL cholesterol levels in the blood. Dietary Phytosterols: A
Review of Metabolism, Benefits, and Side Effects, Ling & Jones,
Life Sci., Vol. 57:3, pp. 195-206 (1995). Phytosterols are sterol
compounds produced by plants which are structurally very similar to
cholesterol except that they always contain some substitutions at
the C.sub.24 position on the sterol side chain. Common plant
sterols include the unsaturated sterols .beta.-sitosterol,
campesterol, and stigmasterol, which are shown in FIG. 1, and their
saturated counterparts sitostanol and campestanol, shown in FIG. 2.
Dietary sources of phytosterols are corn oil, soybean oil, and
other plant oils which contain the relatively hydrophobic
compounds.
[0006] The phytoestrogenic isoflavones, shown in FIGS. 3 and 4, are
another set of compounds that are found in whole soybeans which
have recently been recognized as a significant factor in reducing
LDL and total cholesterol concentrations in the blood. Purified
concentrated isoflavones, particularly genistein, suppress the
biosynthesis of cholesterol as well as prevent the oxidation of LDL
cholesterol, an important step in the development of
atherosclerosis. See e.g., Korean Patent No. 97-20103; Oxidized Low
Density Lipoprotein-Mediated Activation of Phospholipase D in
Smooth Muscle Cells: a Possible Role in Cell Proliferation and
Atherogenesis, Natarajan et al., J. Lipid Res. 36:9 pp. 2005-16
(September 1995).
[0007] In the conventional processing of whole soybeans to produce
soy oils, soy protein, and pharmaceutical or dietary supplement
compositions, the phytosterols, isoflavones, and soy protein are
separated. The plant sterols are separated from soy protein and the
isoflavones into a soy oil fraction which contains fats and fat
solubles by mechanical or chemical extraction of dehulled soybeans,
The soy oil fraction is separated from the remaining soy
materials--the "defatted soy material"--for use in soy and
vegetable oils, which may be further processed to form shortenings,
margarines, and lecithin.
[0008] The defatted soy materials, usually defatted soy flakes, are
used as a starting material to produce products containing
substantial amounts of soy protein such as soy protein concentrates
and soy protein isolates. The defatted soy materials used to
produce soy protein products also contain the isoflavones. However,
in conventional processes for the production of soy protein
materials such as soy protein concentrates and soy protein
isolates, substantial amounts of the isoflavones are separated from
the soy protein material by washing the soy protein material with
an alcohol extract or an aqueous extract. See, e.g., Soy Protein
Products, Characteristics, Nutritional Aspects, and Utilization,
pp. 3-6 (Pub. Soy Protein Council, 1987).
[0009] In the commercial production of soy protein materials the
focus has been to separate the isoflavones from the soy protein
material since isoflavones have been associated with poor taste,
odor, and/or color when present in a soy protein material. For
example, U.S. Pat. No. 5,804,234 teaches a process for removing
isoflavones from a soybean material by contacting the protein
material with an adsorbent resin to produce a better tasting
soybean protein. Alcohol extraction of soy protein materials is
particularly preferred since alcohol extraction is purported to
produce a better tasting vegetable protein material than aqueous
extraction, in part because alcohol is quite effective in removing
isoflavones from the soy protein material. See, e.g., Japanese
Patent No. 63-245,648A.
[0010] Recent efforts have also been focused on separating the
isoflavones from plant materials in which they occur, particularly
soy beans and clover, to provide a purified isoflavone material
which can be used to provide health benefits. For example, the
following patents disclose various methods of separating
isoflavones from plant materials: U.S. Pat. Nos. 4,428,876;
5,702,752; 5,679,806; 4,390,559; 4,366,248; 4,366,082; 4,264,509;
4,232,122; 4,157,984; Japanese Patent Nos. 1-258669A; 5-170756A;
41-90720A; 62-126186A; 62-126185A; and PCT Patent Application No.
WO 93/23069. The separated isoflavone compounds may be used in
pharmaceutical compositions or dietary supplement compositions to
prevent or treat a variety of deleterious health conditions
including lowering elevated cholesterol levels. For example, the
separated isoflavone materials may be utilized in pharmaceutical or
dietary supplement compositions as described in U.S. Pat. Nos.
5,516,528; 5,424,331; 5,569,459; 5,654,011 and PCT Patent
Application No. WO 93/23069.
[0011] New methods and compositions utilizing soy components and
which are capable of providing significant lowering of LDL and
total cholesterol levels in the blood to significantly reduce the
risk of atherosclerosis and coronary heart disease remain
desirable.
SUMMARY OF THE INVENTION
[0012] In one aspect, the present invention is a composition
containing a soy protein material containing at least 49% soy
protein by dry weight of the soy protein material, at least one
isoflavone selected from the group consisting of genistein,
daidzein, glycitein, biochanin A, formononetin, and their
respective naturally occurring glycosides and glycoside conjugates,
and a plant sterol, where the plant sterol forms at least 0.49% of
the composition by weight.
[0013] In another aspect, the present invention is a composition
containing a soy protein material which contains at least 49% soy
protein by dry weight of the soy protein material, and a plant
sterol, where the plant sterol forms at least 0.49% of the
composition by weight.
[0014] In a further aspect, the present invention is a composition
containing at least one isoflavone selected from the group
consisting of genistein, daidzein, glycitein, biochanin A,
formononetin, and their respective naturally occurring glycosides
and glycoside conjugates, and a plant sterol, where the plant
sterol forms at least 0.49% of the composition by weight.
[0015] In yet another aspect, the present invention is a method for
lowering the concentration of LDL and total cholesterol in the
blood of a human in which a soy protein material containing at
least 49% soy protein by dry weight of the soy protein material, an
isoflavone selected from the group consisting of genistein,
daidzein, glycitein, biochanin A, formononetin, and their
respective naturally occurring glycosides and glycoside conjugates,
and a plant sterol are co-administered to the human to decrease the
concentration of LDL and total cholesterol in the blood of the
human. The plant sterol is co-administered in an amount such that
the plant sterol comprises at least 0.49% of the combined weight of
the co-administered soy protein material, isoflavone, and plant
sterol. In a preferred aspect, the plant sterol, soy protein
material, and isoflavone are co-administered to the human to
prevent or minimize the development of atherosclerosis in the
human.
[0016] In still another aspect, the present invention is a method
for lowering the concentration of low density lipoprotein
cholesterol and total cholesterol in the blood of a human in which
a soy protein material containing at least 49% soy protein by dry
weight of the soy protein material and a plant sterol are
co-administered to a human to decrease the concentration of LDL and
total cholesterol in the blood of the human. The plant sterol is
co-administered in an amount such that the plant sterol comprises
at least 0.49% of the combined weight of the co-administered soy
protein material and plant sterol. In a preferred aspect, the plant
sterol and the soy protein material are co-administered to the
human to prevent or minimize the development of atherosclerosis in
the human.
[0017] In yet another aspect, the present invention is a method for
lowering the concentration of low density lipoprotein cholesterol
and total cholesterol in the blood of a human in which a plant
sterol and an isoflavone selected from the group consisting of
genistein, daidzein, glycitein, biochanin A, formononetin, and
their respective naturally occurring glycosides and glycoside
conjugates are co-administered to the human to decrease the
concentration of LDL and total cholesterol in the blood of the
human. The plant sterol is co-administered in an amount such that
the plant sterol comprises at least 0.49% of the combined weight of
the co-administered isoflavone and the plant sterol. In a preferred
aspect, the plant sterol and the isoflavone are co-administered to
the human to prevent or minimize the development of atherosclerosis
in the human.
[0018] In a further aspect, the present invention is a composition
containing a soy hypocotyl material and a plant sterol, where the
plant sterol forms at least 0.49% of the composition by weight.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a depiction of the molecular structure of the
unsaturated plant sterols .beta.-sitosterol, campesterol, and
stigmasterol.
[0020] FIG. 2 is a depiction of the molecular structure of the
saturated plant sterols sitostanol and campestanol.
[0021] FIG. 3 is a depiction of the molecular structure of the
aglucone isoflavones utilized in the invention.
[0022] FIG. 4 is a depiction of the molecular structure of
representative isoflavone glycosides and isoflavone glycoside
conjugates.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] As used herein "Mal" represents "malonyl" and "Ac"
represents "acetyl". The term "minimize", or a derivative thereof,
includes a complete or partial inhibition of a specified biological
effect (which is apparent from the context in which the term
minimize is used). Also, as used herein, the term "isoflavone" or
"phytoestrogen" may mean both a single isoflavone or phytoestrogen,
or plural isoflavones or phytoestrogens when the "isoflavone" or
"phytoestrogen" is defined as at least one of a selected group of
isoflavones. The term "isoflavone glycoside" refers to an
isoflavone moiety having a carbohydrate monomer moiety covalently
bonded thereto. The term "isoflavone glycoside conjugate" refers to
an isoflavone glycoside having another molecular moiety, such as an
ester, bonded to the carbohydrate portion of the isoflavone
glycoside. Also, as used herein, a "pharmaceutical preparation" is
a compound or a mixture of compounds combined with an excipient
which is effective to deliver the compound or compounds to a human
as prescribed by a physician. An "over-the-counter preparation" is
a compound or mixture of compounds combined with an excipient which
is effective to deliver the compound or compounds to a human which
does not require a prescription from a physician in order to be
administered to the human.
[0024] The present invention resides in the discovery that plant
sterols in combination with soy protein materials and/or
isoflavones synergistically lower low density lipoprotein
cholesterol and total cholesterol levels in the blood of a human
when co-administered in amounts effective to induce the cholesterol
lowering activity of each of the co-administered components. The
present invention, therefore, is directed to compositions for and
methods of decreasing LDL and total cholesterol concentrations in
the blood with combinations of plant sterols (also known as
"phytosterols") soy protein materials and/or selected isoflavones.
Furthermore, plant sterols in combination with soy protein
materials and particularly with isoflavones are effective to
prevent or minimize the development of atherosclerosis by reducing
the LDL and total cholesterol concentrations in the blood, as well
as by antioxidant effects and tyrosine kinase inhibition.
Therefore, the present invention is also directed to compositions
for and methods of preventing or minimizing the development of
atherosclerosis.
[0025] Compositions
[0026] In one embodiment, the compositions of the present invention
include a plant sterol in combination with either a soy protein
material containing at least 49% soy protein by dry weight of the
soy protein material or with at least one isoflavone selected from
genistein, daidzein, glycitein, biochanin A, formononetin, and
their naturally occurring glycosides and glycoside conjugates, or a
plant sterol in combination with both an isoflavone and a soy
protein material. Most preferably the composition contains all
three components, a plant sterol, a soy protein material, and an
isoflavone, so that the LDL and total cholesterol decreasing
effects of each component are cooperatively utilized.
[0027] In another embodiment, the compositions of the present
invention include a plant sterol in combination with a soy
hypocotyl material, which typically contains from about 37% to
about 44% soy protein by weight. The soy hypocotyl/plant sterol
composition may additionally include at least one isoflavone
selected from genistein, daidzein, glycitein, biochanin A,
formononetin, and their naturally occuring glycosides and glycoside
conjugates.
[0028] Plant Sterols
[0029] The plant sterols which may be used in the composition
include both saturated and unsaturated plant sterols. Preferred
unsaturated plant sterols which may be utilized in the composition
of the invention are .beta.-sitosterol, campesterol, and
stigmasterol, shown in FIG. 1, and preferred saturated plant
sterols which may be used in the composition are campestanol and
sitostanol, shown in FIG. 2. The unsaturated sterols
.beta.-sitosterol and campesterol are particularly preferred.
[0030] The amount of plant sterols in the present composition is at
least 0.49% of the total weight of the composition, more preferably
from about 0.5% to about 99.5% of the total weight of the
composition, and most preferably from about 1% to about 60% of the
total weight of the composition. The relative amount of plant
sterols in the composition will depend on whether the composition
contains a soy protein material. If the composition contains a soy
protein material, generally the amount of plant sterols in the
composition, as a percent of the total weight of the composition,
will be significantly less than if the composition does not contain
a soy protein material.
[0031] Soy Protein Materials
[0032] In one embodiment, the soy protein materials which may be
used in a composition of the present invention are soy protein
materials which contain at least 49% soy protein by dry weight of
the soy protein containing material. Preferred soy protein
materials which contain at least 49% soy protein by dry weight are
defatted soy flours and soy flakes, which contain from 49% soy
protein to about 65% soy protein by weight; soy protein
concentrates, which contain from at least 65% soy protein to about
90% soy protein by weight; and soy protein isolates, which contain
at least 90% soy protein by weight. Soy protein concentrates and
soy protein isolates are particularly preferred. In another
embodiment, the soy protein materials that may be used in a
composition of the present invention are soy hypocotyl
materials.
[0033] The amount of soy protein materials in the present
composition ranges from 0% to 99.55% by total weight of the
composition. When a soy protein material is included in the
composition, it preferably comprises at least a majority of the
composition, more preferably being present from about 55% to about
99.55%, even more preferably from about 75% to about 99.55%, and
most preferably from about 80% to about 99.5% of the total weight
of the composition. A soy protein material will be always be
included in the composition if the composition contains no
isoflavones.
[0034] Isoflavones
[0035] The isoflavones which may be used in a composition of the
present invention are the aglucone isoflavones genistein, daidzein,
glycitein, biochanin A, formononetin, shown in FIG. 3, and the
naturally occurring glycosides and glycoside conjugates of these
aglucone isoflavones including genistin, 6"-O-Mal genistin, 6"-O-Ac
genistin, daidzin, 6"-O-Mal daidzin, 6"-O-Ac daidzin, glycitin, and
6"-O-Mal glycitin, shown in FIG. 4. One or more of the isoflavones
may be used in the composition. The aglucone isoflavones are
particularly preferred since they are the most biologically active
of the isoflavones. The most preferred isoflavones for use in the
composition are genistein, daidzein, biochanin A and/or
formononetin.
[0036] The amount of isoflavone in the composition ranges from 0%
to about 66%, by total weight of the composition When the soy
protein material is included in the composition the amount of
isoflavone in the composition ranges from about 0% to about 15% by
total weight of the composition. Most preferably the isoflavone is
present in an amount from about 0.0066% to about 10% of the
composition by total weight. The isoflavone will always be included
in the composition if the composition contains no soy protein
material.
[0037] The composition of the present invention may be prepared by
obtaining and combining the above described components using
processes and procedures well known in the art.
[0038] The plant sterol may be obtained by extracting and isolating
a plant sterol from a plant or by chemically synthesizing the
sterol. Alternatively, an oil containing a plant sterol which is
extracted from a plant containing plant sterols may be utilized in
the composition in sufficient amounts to provide the desired levels
of plant sterol in the composition, where the oil is extracted from
the plant in accordance with conventional methods for recovering
plant oils.
[0039] In a preferred embodiment the plant sterol is obtained from
soy oil as a nonsaponifiable fraction of the soy oil according to
the method provided in U.S. Pat. No. 3,993,756 (particularly
Example 10), which is incorporated herein by reference. Briefly, a
nonsaponifiable fraction of soybean oil containing about 40-50% by
weight plant sterols is obtained from whole soybeans. Soy oil is
obtained from the whole soybeans by conventional cracking,
dehulling, flaking, and extraction procedures. The soy oil is
purified by conventional soy oil purification procedures until the
final deodorization step. The deodorization of the soy oil is
conducted at 225.degree. C.-235.degree. C. at reduced pressures of
2-3 mm Hg to distill and separate an extract containing the plant
sterols from the soy oil. The free fatty acids contained in the
extract are converted to methyl esters by refluxing the extract
with methanol and concentrated sulfuric acid. (by weight Ig
extract: 1.72 g methanol: 0.019 g sulfuric acid) at 65-70.degree.
C. for 3-4 hours. After stopping the reaction, the methanol is
removed under reduced pressure and the residual oily material is
washed with water at 80'-100.degree. C. to remove the sulfuric
acid. The methyl esters of the fatty acids are then removed by
distillation at 170-190.degree. C. at 20-30 mm Hg. The resulting
residue is purified to provide a nonsaponifiable fraction of
soybean oil containing 40-50% plant sterols. The plant sterols in
the nonsaponifiable fraction are .beta.-sitosterol, campesterol,
and stigmasterol. The non-saponifiable fraction may be further
purified by HPLC to obtain each individual plant sterol, or more
preferably, may be used directly as the plant sterol source in the
present composition.
[0040] .beta.-sitosterol may also be extracted from several other
plant oils according to known procedures. .beta.-sitosterol can be
isolated from wheat germ oil and from corn oil according to the
procedures set forth by Anderson et al in J. Am. Chem. Soc., 48,
2897 (1926); from rye germ oil by the method of Gloyer et al in J.
Am. Chem. Soc., 61, 1901 (1939); from cottonseed oil by the method
of Wallis in J. Org. Chem., 2, 335 (1937); and from tall oil by the
method of Sandqvist et al in Ber. 64, 2167 (1931), all incorporated
herein by reference.
[0041] Campesterol may be extracted from other plant oils as well
according to known procedures provided by Fernholz et al in J. Am.
Chem. Soc. 63, 1155 (1941), which is incorporated herein by
reference. Campesterol may also be synthesized by the procedure of
Tarzia et al, Gazz. Chim. Ital., 97, 102 (1967) and Chem Abstr.,
67, 32883q (1967), which are incorporated herein by reference.
Stigmasterol may be isolated from soy or calabar beans.
[0042] The saturated plant sterols may be obtained from their
unsaturated counterparts by hydrogenation. Utilizing conventional
hydrogenation procedures .beta.-sitosterol and stigmasterol may be
converted to sitostanol and campesterol may be converted to
campestanol.
[0043] Certain of the plant sterols are commercially available, and
may purchased directly for incorporation into the present
composition. For example, campesterol and stigmasterol may be
purchased from Aldrich Chemical Company, Inc., 940 West Saint Paul
Avenue, Milwaukee, Wis. .beta.-sitosterol and sitostanol may be
purchased from Sigma Chemical Company, 3050 Spruce Street, St.
Louis, Mo.
[0044] In one embodiment, the soy protein material is a defatted
soy protein flour or grit, a soy protein concentrate, or a soy
protein isolate, each of which contains at least 49% soy protein by
weight. To obtain a defatted soy flour or soy grit, whole soybeans
are cleaned, cracked, flaked, and defatted according to
conventional soybean processing methods to produce defatted soy
flakes, chips, meal, or cake, from which soy flour and soy grit may
be produced. Defatted soy flakes and soy meal are also commercially
available. Defatted soy flour and defatted soy grit are produced
from the defatted soy flakes, chips, meal, or cake by comminuting
the material into a finely divided form according to conventional
processes. The defatted soy flour is comminuted into particles
sized 150.mu. or less (sufficient to permit the particles to pass
through a No. 100 U.S. screen mesh), and the defatted soy grit is
comminuted into particles sized from about 150.mu. to about 2000%
(sufficient to permit the particles to pass through No. 10-No. 80
U.S. screen meshes). The defatted soy flour and the defatted soy
grit contain from 49% soy protein to about 65% soy protein by
weight, where the remainder of the soy flour and soy grit is formed
of primarily of carbohydrates, soy fiber, and ash. Defatted soy
flour and soy grit are commercially available and may be acquired
for direct incorporation into the present composition.
[0045] Preferably the soy protein material used in the composition
is a soy protein concentrate or a soy protein isolate, which
contain significantly more soy protein than a soy flour or grit. A
soy protein concentrate contains from about 65% soy protein to
about 90% soy protein by weight, where the remainder of the soy
protein concentrate is primarily soy fiber and other
polysaccharides. A soy protein concentrate for use in the
composition of the present invention may be formed by conventional
processes for producing soy protein concentrates which include (i)
washing defatted soy flakes or soy flour with an aqueous alcohol,
preferably aqueous ethanol or methanol; (ii) washing defatted soy
flakes or soy flour with a dilute acid having a pH at about the
isoelectric point of soy protein (pH 4.4-4.6); or (iii) heating
defatted soy flakes or soy flour with moisture to denature the
protein, then washing the flakes or flour with water. Soy protein
concentrates are commercially available and may be acquired for
direct incorporation into the present compositions.
[0046] Most preferably the soy protein material used in the
composition of the invention is a soy protein isolate (also known
as an isolated soy protein), which has the highest soy protein
content of processed soy protein materials. A soy protein isolate
contains at least about 90% soy protein by weight, where the
remainder of the soy protein isolate is primarily formed of ash and
moisture. A soy protein isolate for use in the composition of the
present invention may be formed by a conventional process for
forming soy protein isolates in which defatted soy flakes are
extracted with an aqueous solution having a pH of from about 6 to
about 11, preferably a dilute aqueous sodium, calcium, or ammonium
hydroxide solution, and an extract containing soluble protein is
separated from insoluble materials such soy fiber and other
insoluble polysaccharides. The pH of the separated extract is
adjusted to about the isoelectric point of soy protein, preferably
about pH 4.4-4.6, to precipitate the protein. The precipitated
protein is separated from the liquid fraction of the extract to
separate the protein from soluble carbohydrates. The separated
protein is then washed, and may be neutralized, to provide the soy
protein isolate. Soy protein isolates are commercially available
and may be acquired for direct incorporation into the composition
of the present invention.
[0047] In another embodiment, the soy protein material is a soy
hypocotyl material, which contains from about 37% to about 44% soy
protein by weight. To obtain a soy hypocotyl material, whole
soybeans are dehulled, and the soy cotyledons are split in
accordance with conventional soy processing techniques. The
hypocotyls become separated following the splitting of the
cotyledons, and may be separated and isolated by passing the
disturbed soybeans over a sieve of sufficient pore size to
selectively remove the small hypocotyl. The raw hypocotyl may be
ground or milled to produce a powder or flour.
[0048] The isoflavone compounds utilized in the present
compositions are naturally occurring substances which may be found
in plants such as legumes, clover, and the root of the kudzu vine
(pueraria root). Common legume sources of these isoflavone
compounds include soy beans, chick peas, and various other types of
beans and peas. Clover sources of these isoflavone compounds
include red clover and subterranean clover. Soy beans are a
particularly preferred source of the isoflavone compounds (except
biochanin A and its glycosides which are not present in soy).
[0049] The isoflavone compounds may be obtained from the plant
sources in which they naturally occur, and several of the
isoflavone compounds may be synthetically prepared by processes
known in the art. For example, daidzein may be isolated from red
clover as disclosed by Wong (J. Sci. Food Agr., Vol. 13, p. 304
(1962)) or may be isolated from the mold Micromonospora halophytica
as provided by Ganguly and Sarre (Chem. & Ind. (London), p. 201
(1970)), both references of which are incorporated by reference
herein. Daidzein may be synthetically prepared by the methods
provided by Baker et al (J. Chem. Soc., p. 274 (1933)), Wesley et
al. (Ber. Vol. 66, p. 685 (1933)), Mahal et al. (J. Chem. Soc., p.
1769 (1934)), Baker et al. (J. Chem. Soc., p. 1852 (1953)), or
Farkas (Ber. Vol. 90, p. 2940 (1957)), each reference of which is
incorporated herein by reference. The isoflavone glycoside daidzin
may be synthetically prepared by the method of Farkas et al. (Ber.,
Vol. 92, p. 819 (1959)), incorporated herein by reference. The
daidzein isoflavone glycoside conjugates 6'-O-Mal daidzin and
6'-O-Ac daidzin can be prepared by a conventional saponification of
daidzin with a malonyl or an acetyl anhydride, respectively.
[0050] Genistein may be synthetically prepared by the methods
provided by Baker et al (J. Chem. Soc., p. 3115 (1928));
Narasimhachari et al. (J. Sci. Ind. Res., Vol. 12, p. 287 (1953));
Yoder et al., (Proc. Iowa Acad. Sci., Vol. 61, p. 271 (1954); and
Zemplen et al. (Acta. Chim. Acad. Sci. Hung., Vol. 19, p. 277
(1959)), each reference of which is incorporated herein by
reference. The isoflavone glycoside genistin may be synthetically
prepared by the method of Zemplen et al. (Ber., Vol 76B, p. 1110
(1943)), incorporated herein by reference. The isoflavone
conjugates of genistin, 6'-O-Mal genistin and 6'-O-Ac genistin, can
be prepared by a conventional saponification of genistin with a
malonyl or an acetyl anhydride, respectively.
[0051] Biochanin A can be synthetically prepared by the method
provided by Baker et al. (Nature 169:706 (1952)), incorporated
herein by reference. Biochanin A can also be separated from red
clover by the method provided by Pope et al. (Chem. & Ind.
(London) p. 1092 (1953)), incorporated herein by reference.
Formononetin can be synthetically prepared by the methods disclosed
by Wessely et al. (Ber. 66:685 (1933)) and Kagel et al.
(Tetrahedron Letters, p. 593 (1962)), both references of which are
incorporated herein by reference. Formononetin can be isolated from
soybean meal by the method of Walz (Ann. 489:118 (1931)) or can be
isolated from clover species by the method of Bradbury et al. (J.
Chem. Soc. p. 3447 (1951)), both references of which are
incorporated herein by reference.
[0052] In a most preferred embodiment, the isoflavone component of
the present composition is recovered in a soy protein material
which is also utilized in the composition. As noted above,
isoflavones are naturally occurring compounds in soy. In the
production of soy protein flours, grits, concentrates, and isolates
some of the isoflavones may be retained in the soy protein
materials. Utilization of appropriate processing techniques while
processing the soy protein material as described above to produce
soy protein flours, grits, concentrates, or isolates will increase
the amount of isoflavones retained in the soy protein materials
significantly over conventionally processed soy protein materials.
Particularly, alcohol washing or extraction of the soy protein
material should be avoided since isoflavones are highly soluble in
aqueous alcohol, and soy protein concentrates should not be
produced by alcohol washing if isoflavones are to be retained in
the soy protein material. Furthermore, aqueous washing of the soy
protein material should be minimized since washing removes the
isoflavones from the soy protein. Washing, if conducted, should be
conducted at cool to cold temperatures (from 0.degree. C. to
45.degree. C.) to maximize the insolubility of the isoflavones in
the water wash and minimize the loss of isoflavones in the wash.
The isoflavones in the soy protein material should be converted to
their aglucone form, as described below, to minimize solubility in
water prior to washing or extracting the soy protein material with
water. Use of the above measures aids in the retention of the
isoflavones in the soy protein material soy that the resulting soy
protein material contains significant amounts of isoflavones.
[0053] In another embodiment, the isoflavone or isoflavones are
separated from plant materials in which they naturally occur for
use in the present compositions. A preferred method of isolating
the isoflavone compounds from plant materials in which they
naturally occur is to extract the plant materials with an alcohol,
preferably methanol or ethanol, or an aqueous solution, preferably
an aqueous alkaline solution, to remove the isoflavones from the
plant material. It is preferred to comminute the plant material
before extracting the isoflavone compounds to maximize recovery of
the isoflavone compounds from the plant material. The isoflavone
compounds can be isolated from the extract by conventional
separation procedures such as reverse phase high performance liquid
chromatography ("HPLC").
[0054] In a preferred embodiment, the isoflavone compounds
genistein, genistin, 6'-O-Mal genistin, 6'-O-Ac genistin, daidzein,
daidzin, 6'-O-Mal daidzin, 6'-O-Ac daidzin, glycitein, glycitin,
and 6'-O-Mal glycitin are isolated from a soy material, preferably
a commercially available soy material. Soy materials from which the
isoflavone compounds can be isolated include: soy beans, dehulled
soy beans, soy meal, soy flour, soy grits, soy flakes (full fat and
defatted), soy cotyledons, soy hypocotyls, soy molasses, soy
protein concentrate, soy whey, soy whey protein, and soy protein
isolate. In one embodiment, the isoflavones are extracted from soy
beans, dehulled soy beans, soy meal, soy flour, soy grits, soy
flakes, soy protein concentrate, soy whey protein, or soy protein
isolate, preferably soy meal, soy flour, soy grits, or soy flakes,
with a low molecular weight organic extractant, preferably an
alcohol, ethyl acetate, acetone, or ether, and most preferably
aqueous ethyl alcohol or methyl alcohol. Most preferably the
extractant has a pH of about the isoelectric point of soy protein
(about pH 4 to pH 5) to minimize the amount of soy protein
extracted by the extractant.
[0055] The extractant containing the isoflavones is separated from
the insoluble soy materials to form an isoflavone enriched extract.
If desired, an isoflavone enriched material may be recovered by
concentrating the extract to remove the solvent, thereby producing
a solid isoflavone enriched material which may be utilized in the
compositions and methods of the present invention.
[0056] In a more preferred embodiment the isoflavone compounds are
further purified from other soy materials soluble in the extract.
The isoflavone containing extract is contacted with a material
which adsorbs the isoflavones in the extract, and the adsorbed
isoflavones are eluted out of the adsorbent material with a solvent
which causes the isoflavones to be differentially eluted from the
adsorbent material.
[0057] In a preferred embodiment, the isoflavones are separated
from impurities in the extract by a conventional reverse phase HPLC
separation. After extraction of the isoflavones from the soy
material and separation of the extract from the insoluble soy
materials, the extract is filtered to remove insoluble materials
that could plug an HPLC column. An HPLC column is prepared by
packing a conventional commercially available HPLC column with a
particulate adsorbent material which will releasably bind the
isoflavones and impurities in the extract in a compound specific
manner. The adsorbent material may be any reverse phase HPLC
packing material, however, a preferred packing material may be
chosen by the criteria of load capacity, separation effectiveness,
and cost. One such preferred packing material is Kromasil C18 16
.mu.m 100 .ANG. beads available from Eka Nobel, Nobel Industries,
Sweden.
[0058] The filtered extract is passed through the packed HPLC
column until all the binding sites of the column are fully
saturated with isoflavones, which is detected by the appearance of
isoflavones in the effluent from the column. The HPLC column may
then be eluted with a solvent to effect the separation. In a
preferred embodiment, the eluent is a polar solvent such as
ethanol, methanol, ethyl acetate, or acetonitrile, and preferably
is an aqueous alcohol having an alcohol content of between about
30% and about 90%, most preferably about 50%, and most preferably
the alcohol is ethanol.
[0059] The isoflavone compounds and impurities are separately
collected from the column effluent. The isoflavone fractions of the
eluent may be identified from other eluent fractions in accordance
with conventional HPLC and analytical chemistry techniques. The
isoflavone fractions of the eluent may be collected from the
column, and the volatile content of the solvent (e.g. alcohol) can
be removed by evaporation. The isoflavone compounds can be
recovered directly if the all of the solvent is removed by
evaporation, or may be recovered by chilling the remaining solvent
(e.g. water) and centrifuging or filtering the isoflavones from the
remaining solvent.
[0060] In a particularly preferred embodiment isoflavone glycoside
conjugates and isoflavone glycosides recovered from a plant
material are converted to their respective aglucone isoflavone
forms. The conversion of the isoflavone glycoside conjugates and
isoflavone glycosides to the aglucone isoflavones can be effected
in the substrate from which the phytoestrogenic isoflavones are to
be extracted prior to the extraction, or may be effected in the
isoflavone enriched extract after separation of the extract from
the insoluble plant materials. The aglucone isoflavone compounds
are believed to be particularly active in decreasing LDL and total
blood cholesterol concentrations and in preventing or minimizing
the development of atherosclerosis. The aglucone isoflavones are
also more easily separated from an extract containing water than
their respective glycoside conjugate and glycoside forms since the
aglucones are less water soluble.
[0061] The isoflavone glycoside conjugates (e.g. 6"-O-Mal genistin,
6"-O-Ac genistin, 6"-O-Mal daidzin, 6"-O-Ac daidzin, and 6"-O-Mal
glycitin) can be converted to their respective glycosides (e.g.
genistin, daidzin, and glycitin) by forming an aqueous alkaline
solution of an extract or substrate containing the isoflavones
having a pH of about 6 to about 13, preferably about pH 9 to about
pH 11, and treating the aqueous alkaline solution at a temperature
of about 2.degree. C. to about 121.degree. C., preferably about
25.degree. C. to about 75.degree. C., for a period of time
sufficient to effect the conversion, preferably about 30 minutes to
about 5 hours, more preferably about 30 minutes to about 1.5 hours.
The isoflavone glycosides (e.g. genistin, daidzin, and glycitin)
can be converted to their respective aglucone forms (e.g.
genistein, daidzein, and glycitein) by contacting the isoflavone
glycosides with an enzyme capable of cleaving a
1,4-.beta.-glycoside bond--preferably a commercially available
beta-glucosidase enzyme, an alpha- or beta-galactosidase enzyme, a
pectinase enzyme, a lactase enzyme, or a gluco-amylase enzyme--at a
pH at which the enzyme is active, typically from about pH 3 to
about pH 9, and at a temperature of about 25.degree. C. to about
75.degree. C., more preferably about 45.degree. C. to about
65.degree. C., for a period of time sufficient to effect the
conversion, typically about 1 hour to about 24 hours, preferably
about 1 hour to about 3 hours.
[0062] The aglucone isoflavones can be separated from a plant
substrate using conventional separation procedures. For example,
the aglucone isoflavones may be extracted from a soy or clover
substrate with a low molecular weight alcohol. The aglucone
isoflavones may be separated from the extract by conventional
recrystallization processes, or by HPLC. In a particularly
preferred embodiment, an isoflavone composition isolated from a soy
substrate and utilized in the present compositions or methods
includes at least 40% genistein, at least 15% daidzein, and at
least 1% glycitein. In another particularly preferred embodiment of
the invention, an isoflavone composition isolated from a soy
substrate and utilized in the present compositions or methods
contains at least 85% genistein, at least 5% daidzein, and at least
0.5% glycitein.
[0063] Several of the isoflavone compounds utilized in the present
composition are commercially available, and may be purchased for
formulation into the present compositions or used in the present
methods of the invention. For example, genistein, daidzein, and
glycitein are commercially available and may be purchased from
Indofine Chemical Company Inc., P.O. Box 473, Somerville, N.J.
08876. Biochanin A is available from Aldrich Chemical Company,
Inc., 940 West Saint Paul Avenue, Milwaukee, Wis. 53233.
[0064] The plant sterol and the soy protein material and/or the
isoflavone may be combined into a composition of the present
invention by adding the components together, and preferably
thoroughly mixing them. For example, the plant sterol and the soy
protein material and/or the isoflavone may be combined by placing
the materials in a chopping or a mixing bowl and mixing the
materials for a sufficient time to thoroughly blend them.
[0065] Preferably the components are mixed together so that the
resulting composition contains a biologically active amount of each
of the components, although the composition is not limited to
having biologically active amounts of each component therein since
the composition may be administered more than once in order to
achieve a desired decrease in LDL and total cholesterol blood
concentrations. Preferably the composition contains from about 500
mg to about 50 g of plant sterols therein, and more preferably from
about 1 g to about 10 g of the plant sterols. When the composition
contains a soy protein material preferably from about 5 g to about
100 g of the soy protein material is present in the composition,
and more preferably from about 10 g to about 40 g of the soy
protein material is present in the composition. When the
composition contains isoflavones preferably from about 10 mg to
about 1000 mg of the isoflavones are present in the composition,
and more preferably from about 25 mg to about 200 mg of the
isoflavones are present in the composition.
[0066] In a preferred embodiment the plant sterol and the soy
protein material and/or the isoflavone are formulated into a
pharmaceutical or over-the-counter delivery system with suitable
excipients. Pharmaceutical or over-the-counter formulations
incorporating the plant sterol and the soy protein material and/or
the isoflavone can be prepared by procedures known in the art. For
example, the components of the present invention can be formulated
into tablets, capsules, powders, suspensions, solutions for
parenteral administration including intravenous, intramuscular, and
subcutaneous administration, and into solutions for application
onto patches for transdermal application with common and
conventional carriers, binders, diluents, and excipients.
[0067] Inert pharmaceutically acceptable excipients useful to form
pharmaceutical or over-the-counter formulations in accordance with
the present invention include starch, mannitol, calcium sulfate,
dicalcium phosphate, magnesium stearate, silicic derivatives,
and/or sugars such as sucrose, lactose, and glucose. Binding agents
include carboxymethyl cellulose and other cellulose derivatives,
gelatin, natural and synthetic gums including alginates such as
sodium alginate, polyethylene glycol, waxes and the like. Diluents
useful in the invention include a suitable oil, saline, sugar
solutions such as aqueous dextrose or aqueous glucose, and glycols
such as polyethylene or polypropylene glycol. Other excipients
include lubricants such as sodium oleate, sodium acetate, sodium
stearate, sodium chloride, sodium benzoate, talc, and magnesium
stearate, and the like; disintegrating agents including agar,
calcium carbonate, sodium bicarbonate, starch, xanthan gum, and the
like; and adsorptive carriers such as bentonite and kaolin.
Coloring and flavoring agents may also be added to the
pharmaceutical or over-the counter formulations.
[0068] In another preferred embodiment dietary supplements
incorporating the plant sterol and the soy protein material and/or
the isoflavone can be prepared by adding each of the components to
a food as a food ingredient, or by adding a mixture of the
components to a food as a food ingredient. The foods to which these
components may be added include almost all foods, but most
preferably are foods in which soy protein materials or vegetable
oils containing plant sterols are used as functional ingredients.
For example, the plant sterol and the soy protein material and/or
the isoflavone can be added to foods including, but not limited to,
meats such as ground meats, emulsified meats, and marinated meats;
beverages such as nutritional beverages, sports beverages, protein
fortified beverages, juices, milk, milk alternatives, and weight
loss beverages; cheeses such as hard and soft cheeses, cream
cheese, and cottage cheese; frozen desserts such as ice cream, ice
milk, low fat frozen desserts, and non-dairy frozen desserts;
yogurts; soups; puddings; bakery products; salad dressings; and
dips and spreads such as mayonnaise and chip dips. Most preferably
the plant sterol and the soy protein material, with or without the
isoflavones, are mixed in a aqueous slurry to form an emulsion
which is particularly useful in emulsive foods and beverages such
as salad dressings, yogurts, soy cheeses, emulsified meats, and
powdered beverages.
[0069] The following non-limiting formulations illustrate
pharmaceutical and dietary formulations including the plant sterols
and the soy protein and/or the isoflavone compounds in accordance
with the compositions of the present invention and which may be
used in accordance with the methods of the present invention.
Formulations
[0070] The following Formulations 1-4 illustrate pharmaceutical or
over-the-counter formulations including a plant sterol and a soy
protein material and/or an isoflavone useful in the compositions of
the present invention.
[0071] Formulation 1
[0072] Gelatin Capsules
[0073] Hard gelatin capsules are prepared using the following
ingredients: Isoflavone 25-100 mg/capsule; Plant sterol 500-2000
mg/capsule; Starch, NF 0-1000 mg/capsule; Starch flowable powder
0-1000 mg/capsule; Silicone fluid 350 centistokes 0-20 mg/capsule.
The ingredients are mixed, passed through a sieve, and filled into
capsules.
[0074] Formulation 2
[0075] Tablets
[0076] Tablets are prepared using the following ingredients: Plant
sterol 250-500 mg/tablet; Soy protein material 1000-2000 mg/tablet;
Microcrystalline cellulose 20-300 mg/tablet; Starch 0-50 mg/tablet;
Magnesium stearate or stearate acid 0-15 mg/tablet; Silicon
dioxide, fumed 0-400 mg/tablet; silicon dioxide, colloidal 0-1
mg/tablet, and lactose 0-100 mg/tablet. The ingredients are blended
and compressed to form tablets.
[0077] Formulation 3
[0078] Suspensions
[0079] Suspensions are prepared using the following ingredients:
Isoflavone 0.1-200 mg/5 ml; Plant sterol 250-2000 mg/5 ml; Soy
protein material 500-2000 mg/5 ml; Sodium carboxymethyl cellulose
50-700 mg/5 ml; Sodium benzoate 0-10 mg/5 ml; Purified water 5 ml;
and flavor and color agents as needed.
[0080] Formulation 4
[0081] Parenteral Solutions
[0082] A parenteral composition is prepared by stirring 1.5% by
weight of active ingredients (active ingredients being soy protein
material, plant sterol, and isoflavone in a weight ratio of 5:4:1)
in 10% by volume propylene glycol and water. The solution is made
isotonic with sodium chloride and sterilized.
[0083] The following Formulations 5-8 illustrate dietary
supplements that may be formed using a plant sterol and a soy
protein isolate rich in the isoflavone compounds utilized in the
present compositions. The isoflavone rich soy protein isolate in
the following examples typically contains between about 1 to about
7 milligrams of the isoflavone compounds per gram of soy
protein.
[0084] Formulation 5
[0085] Ready to Drink Beverage
[0086] A ready to drink beverage is formed of the following
components:
1 Ingredient Percent of composition, by weight Water 75-82
Isoflavone rich soy protein isolate 10-15 Plant sterol 3-5 Sucrose
5-8 Cocoa 0.1-1 Vitamins/Minerals 0.1-1 Flavor 0.1-1 Cellulose gel
0.1-0.5
[0087] The ready to drink beverage may be served in 8 ounce
servings containing about 20 grams of isolated soy protein
including about 20 to about 140 milligrams of the isoflavone
compounds and about 6 grams of plant sterol.
[0088] Formulation 6
[0089] Powdered Beverage
[0090] A powdered beverage is formed of the following
components:
2 Ingredient Percent of composition, by weight Isoflavone rich soy
protein isolate 75-85 Plant sterol 5-10 Sucrose 8-15 Maltodextrin
1-5 Vitamins/Minerals 0.5-2 Aspartame 0-0.5 Flavor 0-0.5
[0091] 30 grams of the powdered beverage formulation may be added
to water to form a serving containing about 23 grams of soy protein
isolate including about 23 to about 161 milligrams of the
isoflavone compounds and about 2.5 grams of plant sterol.
[0092] Formulation 7
[0093] Food Bar
[0094] A food bar is formed of the following components:
3 Ingredients Percent of composition, by weight Isoflavone rich soy
protein isolate 20-30 Plant sterol 1-5 Corn syrup 30-45 Rice syrup
solids 7-14 Glycerin 1-5 Cocoa 2-7 Compound coating 15-25
[0095] The food bar may be served in 70 gram portions containing
about 15 grams of soy protein isolate having about 15 to about 105
milligrams of the isoflavone compounds and about 2 grams of plant
sterol.
[0096] Formulation 8
[0097] Soy Yogurt
[0098] A soy yogurt is formed of the following components:
4 Ingredients Percent of composition, by weight Water 50-70
Isoflavone rich soy protein isolate 5-15 Plant sterol 5-15 Sucrose
3-8 Corn starch 1-5 Dextrin 0.3-1 Cellulose gel 1-3 Culture
(yogurt) 0.01-0.1 Fruit 10-20 Vitamins/Minerals 0.05-0.3
[0099] The soy yogurt may be served in a 170 gram serving
containing about 17 grams of soy protein isolate having about 17 to
about 170 milligrams of isoflavone compounds and containing about
17 grams of plant sterol.
[0100] Methods
[0101] The present invention is also directed to methods of
decreasing the blood concentration of low density lipoprotein
cholesterol or total cholesterol in a human with a plant sterol and
a soy protein material and/or an isoflavone compound. To decrease
the blood concentration of LDL cholesterol and total cholesterol
the plant sterol is co-administered to the human with the soy
protein material and/or the isoflavone compound on an ongoing
regular basis, preferably daily. The plant sterol is preferably
.beta.-sitosterol, campesterol, stigmasterol, sitostanol or
campestanol, as described above. The soy protein material is either
a soy hypocotyl material or a soy protein material containing at
least 49% soy protein by dry weight of the soy protein material
such as a soy flour, grit, concentrate, or isolate. The isoflavone
compound is selected from one or more of genistein, daidzein,
glycitein, biochanin A, formononetin, and their naturally occurring
glycosides or glycoside conjugates, as described above. The plant
sterol and the soy protein material and/or the isoflavone are
preferably administered in a pharmaceutical or over-the-counter
composition or as a dietary supplement, as described above,
depending on which route is more effective and/or acceptable.
[0102] The plant sterol and the soy protein material and/or the
isoflavone compound may be co-administered either concurrently or
sequentially within a specified period of time, preferably daily,
on a periodic basis. Most preferably the plant sterol and the soy
protein material and/or the isoflavone compound are co-administered
concurrently in a composition of the present invention, described
above. Alternatively, the plant sterol and the soy protein and/or
the isoflavone compound are administered sequentially as separate
components. "Sequentially" as used herein is intended to mean
administration of each component individually within a specified
periodic period of time, for example one day, and is not intended
to be limited to immediate consecutive administration of each
component.
[0103] The plant sterol and the soy protein material and/or the
isoflavone compound are administered to a human in an amount which
is effective to deliver a blood cholesterol concentration
decreasing amount of each component to the human. The particular
dosage of each component effective to deliver a blood cholesterol
concentration decreasing dosage of each component will depend on
several factors including the size of the human to which the
component is administered, the blood cholesterol concentration of
the human to which the component is administered, and the route of
administration of the component. Preferably, the plant sterol is
administered in a dose of from 500 mg/day to 50 g/day, and more
preferably from 1 g/day to 5 g/day, on an ongoing basis, to deliver
a blood cholesterol decreasing dose of the plant sterol.
Preferably, if administered, the soy protein material is
administered in a dose of from 5 g/day to 100 g/day, and more
preferably from 10 g/day to 30 g/day, to deliver a blood
cholesterol decreasing dose of the soy protein material.
Preferably, if administered, the isoflavone is administered in a
dose of 10 mg/day to 1000 mg/day, more preferably from 25 mg/day to
200 mg/day, to deliver a blood cholesterol decreasing dose of the
isoflavone compound.
[0104] When co-administered in amounts effective to individually
decrease LDL and total blood cholesterol concentrations, the plant
sterol and the soy protein material and/or the isoflavone
cooperatively decrease these blood cholesterol concentrations in a
human. Plant sterols, soy protein materials, and the isoflavones
utilized in the present method decrease LDL and total blood
cholesterol concentrations in humans by distinct mechanisms, or
distinct combinations of mechanisms which may overlap to some
extent. The cholesterol lowering mechanism(s) of each of these
materials is believed to interact with such mechanisms of the other
materials to accelerate the activity of the cholesterol lowering
mechanisms of the other materials. The accelerated activity of the
cholesterol lowering mechanisms of the combined cholesterol
reducing components causes a collective decrease in LDL and total
cholesterol concentrations in the blood which is greater than the
decrease in LDL and total cholesterol blood concentrations caused
by each of the components separately.
[0105] For example, one mechanism by which co-administration of a
plant sterol, a soy protein material, and an isoflavone may
synergistically decrease LDL and total cholesterol concentrations
in the blood combines certain aspects of proposed cholesterol
lowering mechanisms of each material. It is known that plant
sterols appear to affect the enterohepatic circulation balance
between cholesterol and bile acids by preventing absorption of
dietary cholesterol in the intestine. This reduces cholesterol
feedback in enterohepatic cholesterol circulation regulation,
creating a need for cholesterol in the liver to synthesize bile
acids. The liver can obtain the necessary cholesterol from the
blood through LDL cholesterol receptors in liver cells, which are
upregulated by administration of plant sterols, or can synthesize
the required cholesterol. Blood cholesterol concentrations drop if
the cholesterol required by the liver is removed from the
blood.
[0106] One known mechanism by which soy protein appears to decrease
LDL and total cholesterol concentrations in the blood is by
increasing fecal bile acid excretion. Increased fecal bile acid
excretion induces the liver to produce more bile acids, utilizing
cholesterol as a substrate in the production of the bile acids. As
noted above, the liver can obtain cholesterol for the synthesis of
bile acids from the blood, lowering blood cholesterol
concentrations.
[0107] Combined administration of a plant sterol and soy protein in
accordance with the present invention may cause a cooperative
adjustment of enterohepatic cholesterol homeostasis which results
in significantly lower LDL and total cholesterol concentrations in
the blood. Inhibition of intestinal absorption of dietary
cholesterol by a plant sterol may produce a hepatic deficiency of
cholesterol which is relieved in part by pulling cholesterol from
the blood. Increased fecal bile acid excretion induced by soy
protein may exacerbate the hepatic cholesterol deficiency caused by
the plant sterol by increasing the removal of cholesterol from the
liver as a bile acid substrate, which is relieved in part by
drawing further cholesterol from the blood.
[0108] The isoflavones utilized in the present invention are
effective to cooperatively lower LDL and total cholesterol
concentrations in combination with a plant sterol, or with a plant
sterol and a soy protein material, in part because the isoflavones
may upregulate or increase the number of hepatic LDL
cholesterol-receptors. The isoflavones utilized in the present
invention are genistein, daidzein, glycitein, formononetin,
biochanin A, and their respective glycosides and glycoside
conjugates, which are phytoestrogens that are known to have
estrogenic effects in vivo. Estrogen increases the number of
hepatic LDL cholesterol receptors, which increases the clearance of
LDL cholesterol particles from blood plasma into the liver. In
accordance with the present invention, increased upregulation or
numbers of hepatic LDL cholesterol receptors induced by the
estrogenic effects of the isoflavone(s), in coordination with an
increased demand for cholesterol in the liver induced by the plant
sterol and/or the soy protein material, may significantly promote
the clearance of LDL cholesterol from the blood into the liver.
[0109] The method of the present invention, however, is not to be
limited to a specific mechanism of action, particularly since there
may be several mechanisms by which the co-administered plant
sterol, isoflavone(s), and/or soy protein material reduce LDL and
total cholesterol concentrations in the blood. For example, in
addition to altering cholesterol homoestasis by increasing
excretion of bile acids, soy protein may decrease the concentration
of LDL and total cholesterol in the blood as result of its high
arginine amino acid content, by specific action of the 7S storage
unit of the protein, by altering the ratio of serum glucagon to
serum insulin which affects cholesterol metabolism in the liver, or
by increasing serum free thyroxine concentrations. Plant sterols
also function to reduce cholesterol by mechanisms other than
inhibition of intestinal absorption of cholesterol since plant
sterols have been shown to reduce cholesterol levels by direct
injection into the bloodstream.
[0110] The present invention is also directed to methods of
preventing or minimizing the development of atherosclerosis by
co-administering to a human a plant sterol and a soy protein
material which is either a soy hypocotyl material or a soy protein
material containing at least 49% soy protein by weight and/or at
least one isoflavone selected from genistein, daidzein, glycitein,
biochanin A, formononetin, and their respective naturally occurring
glycosides. The plant sterol is preferably at least one of
P-sitosterol, campesterol, stigmasterol, sitostanol, and
campestanol. The soy protein material is preferably soy flour, soy
grit, soy protein concentrate, or soy protein isolate. The
preferred methods of administration and dosages are the same as
described above with respect to administering a plant sterol and a
soy protein material and/or an isoflavone to lower LDL and total
blood cholesterol concentrations.
[0111] As described above, the plant sterol and the soy protein
material and/or the isoflavones are effective to cooperatively
decrease LDL and total blood cholesterol concentrations when
administered to a human in amounts sufficient to induce the
cholesterol lowering activity of each compound. Elevated levels of
LDL cholesterol in the blood have been linked to the development of
atherosclerosis, since the LDL cholesterol is the key component in
the formation of atherosclerotic plaque. Therefore, the combination
of LDL cholesterol lowering plant sterols, soy protein materials,
and/or isoflavones are effective to prevent or minimize the
development of atherosclerosis.
[0112] In a preferred embodiment, the isoflavone is always
co-administered in combination with the plant sterol to prevent or
minimize the development of atherosclerosis, regardless whether the
soy protein material is administered, since the isoflavone provides
anti-atherosclerotic activity in addition to lowering the blood
concentration of LDL cholesterol. Specifically, these isoflavones
provide significant protection against atherosclerosis at the
vascular level. The isoflavones--particularly the aglucone
isoflavones genistein, daidzein, biochanin A, and
formononetin--inhibit oxidation of LDL cholesterol. Oxidation of
LDL cholesterol is a key step in the formation of atherosclerotic
plaques since oxidation of LDL cholesterol increases its
atherogenicity. Some of the isoflavones, particularly genistein,
daidzein, and biochanin A, are also tyrosine kinase inhibitors
which inhibit the formation of atherosclerotic lesions and thrombin
by inhibiting enzymatic tyrosine kinase activity. Therefore,
co-administration of plant sterols and at least one of the
isoflavones in accordance with the present invention, with or
without a soy protein material, prevents or minimizes the
development of atherosclerosis, both by cooperatively lowering LDL
and total cholesterol levels in the blood and by inhibiting the
mechanisms of formation and growth of atherosclerotic plaques.
EXAMPLE 1
[0113] Five to fifty men are selected for clinical study. The men
are diagnosed with moderate to high hypercholesteremia, e.g. having
total blood cholesterol levels of 250 mg/dl or higher. The men are
divided into five groups, the first of which receives 30 g of an
isoflavone-free isolated soy protein dietary supplement (from which
the isoflavones have been removed by alcohol extraction) (the "soy"
group), the second of which receives 5 g of a plant sterol dietary
supplement which contains the plant sterol .beta.-sitosterol (the
"plant sterol" group), the third of which receives 75 mg of an
isoflavone dietary supplement which contains the isoflavones
genistein and daidzein (the "isoflavone" group), the fourth of
which receives a dietary supplement containing 30 g isolated soy
protein, 5 g of the plant sterol .beta.-sitosterol, and 75 mg of
the isoflavones genistein and daidzein (the "combination" group),
and the fifth of which receives no dietary supplement (the
"control" group). The diets of the groups are selected to include
no further source of isoflavones, plant sterols and soy protein,
and no source of estrogen is administered to either of the two
groups. The diets are continued for 6 months.
[0114] Prior to beginning the diets, the patients are benchmarked
as to total and low density lipoprotein blood cholesterol levels.
The benchmarked symptoms are measured again for each group after
the groups have been on the respective diets for 6 months. The
results are compared between members of each group and between the
beginning of the study and end of the study for each member.
Enhanced activity in reducing LDL and total blood cholesterol
levels by the combination of a plant sterol, a soy protein
material, and an isoflavone is shown by a greater percent decrease
of the LDL or total blood cholesterol levels in the combination
group relative the soy group, the plant sterol group, the
isoflavone group, and the control group.
[0115] Utility of the combination of the plant sterols and soy
protein material and/or isoflavones for lowering LDL blood
cholesterol concentrations in a human is evidenced by activity in
the above example.
EXAMPLE 2
[0116] Five to fifty women are selected for clinical study. The
women are postmenopausal and have been diagnosed with
atherosclerosis, i.e., have been diagnosed with atherosclerotic
plaque including stenotic plaque and occlusive plaque. The women
are divided into five groups, the first of which receives 30 g of
an isoflavone-free isolated soy protein dietary supplement (from
which the isoflavones have been removed by alcohol extraction) (the
"soy" group), the second of which receives 5 g of a plant sterol
dietary supplement which contains the plant sterol
.beta.-sitosterol (the "plant sterol" group), the third of which
receives 75 mg of an isoflavone dietary supplement which contains
the isoflavones genistein and daidzein (the "isoflavone" group),
the fourth of which receives a dietary supplement containing 30 g
isolated soy protein, 5 g of the plant sterol .beta.-sitosterol,
and 75 mg of the isoflavones genistein and daidzein (the
"combination" group), and the fifth of which receives no dietary
supplement (the "control" group). The diets of the groups are
selected to include no further source of isoflavones, plant sterols
and soy protein, and no source of estrogen is administered to
either of the two groups. The diets are continued for 6 months.
[0117] Prior to beginning the diets, the patients are benchmarked
as to total blood cholesterol levels; high density and low density
lipoprotein blood cholesterol levels; estrogen levels; and degree
of stenosis and atherosclerotic plaque in a selected blood vessel
to be studied for vascular reactivity non-invasively (e.g., using
high resolution ultrasound), such as the superficial femoral artery
or the brachial artery. The benchmarked symptoms are measured again
for each group after the groups have been on the respective diets
for 6 months. The results are compared between members of each
group and between the beginning of the study and end of the study
for each member. Enhanced anti-atherosclerotic activity of the
combination of compounds relative to .beta.-sitosterol, isolated
soy protein, the isoflavones genistein and daidzein, and no
treatment is shown by either a greater reduction of atherosclerotic
plaque and stenosis or the least amount of incremental increase of
atherosclerotic plaque or stenosis in the combination group
relative to the soy group, the plant sterol group, the isoflavone
group, and the control group.
[0118] Utility of the combination of the plant sterols and soy
protein material and/or isoflavones for preventing or minimizing
the development of atherosclerotic plaque in a human is evidenced
by activity in the above example.
* * * * *