U.S. patent application number 11/977395 was filed with the patent office on 2008-08-21 for anti-resorptive and bone building dietary supplements and methods of use.
Invention is credited to Silvia R. da Costa, David J. Fast, Kevin W. Gellenbeck, David W. Krempin, Laurie Krempin, Yumei Lin, Mary A. Murray, Jatinder Rana, John F. Rebhun, Haeri Roh-Schmidt, Leon M. Wilkins.
Application Number | 20080199546 11/977395 |
Document ID | / |
Family ID | 39186019 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080199546 |
Kind Code |
A1 |
Krempin; David W. ; et
al. |
August 21, 2008 |
Anti-resorptive and bone building dietary supplements and methods
of use
Abstract
Disclosed herein are dietary supplement compositions and methods
for increasing or stimulating bone growth and, decreasing or
preventing bone resorption comprising a first composition
comprising a combination of at least two of: quercetin, Rehmannia
sp., Rehmannia sp. root, Siberian ginseng, Sophora japonica,
licorice, and ipriflavone, wherein the combination of the first
composition increases BMP-2 gene expression, promoter activity, or
protein expression; and a second composition comprising an extract
of pomegranate in combination with at least one of Siberian
ginseng, Ginkgo biloba, green tea, Sophora japonica, Rehmannia sp.,
grape seed, Dong Quai, and ipriflavone, wherein the combination of
the second composition inhibits the expression of RANK-L.
Inventors: |
Krempin; David W.;
(Temecula, CA) ; Murray; Mary A.; (Irvine, CA)
; Lin; Yumei; (Long Beach, CA) ; Gellenbeck; Kevin
W.; (Poway, CA) ; da Costa; Silvia R.;
(Redondo Beach, CA) ; Wilkins; Leon M.; (North
Andover, MA) ; Roh-Schmidt; Haeri; (Stockton, CA)
; Rana; Jatinder; (Grand Rapids, MI) ; Rebhun;
John F.; (Greenville, MI) ; Fast; David J.;
(Grand Rapids, MI) ; Krempin; Laurie; (Temecula,
CA) |
Correspondence
Address: |
IN RE: 28533;BRINKS, HOFER, GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Family ID: |
39186019 |
Appl. No.: |
11/977395 |
Filed: |
October 24, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60854312 |
Oct 24, 2006 |
|
|
|
60925914 |
Apr 23, 2007 |
|
|
|
Current U.S.
Class: |
424/729 ;
424/725; 424/766; 435/375; 514/456 |
Current CPC
Class: |
A61K 31/352 20130101;
A61K 36/185 20130101; A61K 36/804 20130101; A61K 36/258 20130101;
A61P 19/08 20180101; A23L 33/105 20160801; A61K 36/484 20130101;
A61P 19/10 20180101; A61K 36/484 20130101; A61K 36/489 20130101;
A61K 36/185 20130101; A61K 36/489 20130101; A61K 2300/00 20130101;
A61K 36/87 20130101; A61K 31/352 20130101; A61P 43/00 20180101;
A61K 36/87 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61P 19/00 20180101; A23V 2002/00 20130101; A61K 36/258
20130101; A61K 36/254 20130101 |
Class at
Publication: |
424/729 ;
435/375; 514/456; 424/766; 424/725 |
International
Class: |
A61K 36/82 20060101
A61K036/82; C12N 5/02 20060101 C12N005/02; A61K 36/87 20060101
A61K036/87; A61K 36/00 20060101 A61K036/00; A61K 31/352 20060101
A61K031/352 |
Claims
1. A method of increasing BMP-2 expression in a cell comprising
administering to the cell a composition comprising quercetin,
wherein the quercetin increases BMP-2 expression in the cell.
2. The method of claim 1, wherein administering a composition
comprising quercetin increases BMP-2 gene expression.
3. The method of claim 1, wherein administering a composition
comprising quercetin increases BMP-2 protein expression.
4. The method of claim 1, wherein administering a composition
comprising quercetin increases BMP-2 promoter activity.
5. The method of claim 1, wherein the composition further comprises
an extract of licorice.
6. The method of claim 5, wherein the extract of licorice is an
ethanol extract.
7. The method of claim 5, wherein the composition comprises
approximately 10-1000 mg quercetin and approximately 10-500 mg
extract of licorice.
8. The method of claim 7, wherein the composition comprises
approximately 250 mg quecetin and approximately 125 mg extract of
licorice.
9. A method of increasing bone growth comprising administering a
composition comprising a composition comprising quercetin, wherein
the quercetin increases BMP-2 expression.
10. A method of inhibiting RANK-L synthesis in a cell comprising
administering to the cell a composition comprising one or more of
pomegranate extract, grape seed extract, olive juice powder, and
green tea extract, wherein the composition inhibits RANK-L
synthesis.
11. The method of claim 10, wherein the composition comprises a
pomegranate extract and a grape seed extract.
12. The method of claim 11, wherein the pomegranate extract
comprises punicalagins.
13. The method of claim 10, wherein the composition comprises
approximately 10-2000 mg pomegranate extract and approximately
35-250 mg grape seed extract.
14. The method of claim 13, wherein the composition comprises
approximately 1250 mg of pomegranate extract and approximately 125
mg grape seed extract.
15. A method of inhibiting bone resorption comprising administering
a composition comprising one or more of pomegranate extract, grape
seed extract, olive juice powder, and green tea extract, wherein
the composition inhibits RANK-L synthesis.
16. The method of claim 15, wherein the composition comprises a
pomegranate extract and a grape seed extract.
17. The method of claim 16, wherein the pomegranate extract
comprises punicalagins.
18. The method of claim 16, wherein the composition comprises
approximately 10-200 mg pomegranate extract and approximately
35-250 mg grape seed extract.
19. The method of claim 18, wherein the composition comprises
approximately 1250 mg of pomegranate extract and approximately 125
mg grape seed extract.
Description
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/854,312, filed Oct. 24, 2006 and U.S.
Provisional Application Ser. No. 60/925,914, filed Apr. 23, 2007,
the entire contents of which are hereby incorporated by
reference.
BACKGROUND
[0002] Healthy bones continuously undergo a remodeling process,
where an equilibrium is reached between bone resorption and bone
formation through the concerted action of active bone cells, i.e.
bone forming osteoblasts and bone resorbing osteoclasts. The bone
remodeling process begins with activation of cells covering
unmineralized bone, i.e. lining cells. The lining cells resorb the
unmineralized bone, then retract and leave room for the osteoclasts
which resorb the old, mineralized bone and create an environment
which attracts the osteoblast to the same site. The osteoblasts
thereafter lay down an organic matrix, which subsequently becomes
mineralized to form new bone. Thus bone mass is determined by the
balance between bone resorption by osteoclasts and bone formation
by osteoblasts.
[0003] The amount of mineral in bone is largely responsible for its
hardness, while substances like the structural protein collagen
also contribute to bone's mechanical strength. The dense outermost
bone is known as cortical bone while the more spongy internal form
is known as cancellous or trabecular bone.
[0004] Most bone diseases are due to a disruption in the
equilibrium of the bone remodeling process. Generally, the
disruption is an increase in bone resorption. For example,
osteoporosis, one of the most common bone diseases, is
characterized by a decrease in bone mass along with a
microstructural change in bone, but there is no effect on the
chemical composition of bone itself which results in increased
susceptibility to bone fractures. Osteoporosis may be considered
the result of a negative balance in the bone remodeling cycle, i.e.
less bone is formed than is being resorbed.
[0005] Thus, therapeutic agents for treating bone disorders are
directed at inhibiting bone resorption and increasing bone
formation. There are many different molecules and pathways involved
in the bone remodeling process and the various therapeutic agents
presently available target different molecules and pathways. For
example, bisphosphonates (such as aledronate and risedronate)
inhibit bone resorption by blocking osteoclast activity. Other
therapeutic agents seek to inhibit bone resorption by blocking
binding to members of the TNF receptor/ligand family, such as
Receptor Activator for Nuclear Factor .kappa. B Ligand (RANK-L), a
cytokine that activates osteoclasts, the cells that are involved in
bone resorption. Inhibition of release of RANK-L prevents bone
mineral loss.
[0006] Still other therapeutic agents target increasing bone
formation. For example, activated bone morphogenic protein gene is
known to have direct effects on triggering osteoblast cell
differentiation and promoting bone formation. Delivery of
recombinant bone morphogenic protein-2 (BMP-2) has been shown to
induce bone or cartilage formation. However, systemic
administration of pharmaceutical and biological agents, such as
recombinant BMP-2, can have deleterious effects on the intestine
and other tissues. Therefore, there is a need in the art for
natural and plant-derived extracts, that can be used in dietary
supplement interventions for preventing and/or treating bone
disorders by inhibiting bone resorption and/or increasing bone
formation.
BRIEF SUMMARY
[0007] The present invention is based on the discovery that novel
combinations of various natural and plant-derived extracts can (1)
inhibit bone resorption by inhibiting, decreasing, or preventing
the expression and/or release of RANK-L, and/or by preventing
calcium release from bones; (2) increase bone growth by increasing
or stimulating gene and/or protein expression of BMP-2 and (3)
improve or maintain bone strength.
[0008] In one example, the invention is a composition for
increasing or stimulating bone growth comprising natural,
plant-derived extracts, including a combination of at least two of
the following: quercetin dihydrate, quercetin anhydrate, extract of
Rehmannia sp., extract of Rehmannia sp. root, extract of Siberian
ginseng, extract of Sophora fructus japonica, extract of Sophora
japonica, extract of licorice, and ipriflavone, wherein the
combination increases BMP-2 gene or protein expression.
[0009] In another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
quercetin anhydrate or dihydrate, an extract of Siberian ginseng,
an extract of Sophora japonica, and an extract of licorice, wherein
the combination increases BMP-2 gene or protein expression.
[0010] In another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
approximately 10-1000 mg of quercetin anhydrate or dihydrate,
approximately 100-800 mg of an extract of Siberian ginseng, and
approximately 10-500 mg of an extract of licorice, wherein the
combination increases BMP-2 gene or protein expression.
[0011] In a further example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
approximately 10-1000 mg of quercetin anhydrate or dihydrate, and
approximately 10-500 mg of an extract of licorice, wherein the
combination increases BMP-2 gene or protein expression.
[0012] In another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
approximately 10-1000 mg of quercetin anhydrate or dihydrate, and
approximately 100-800 mg of an extract of Siberian ginseng, wherein
the combination increases BMP-2 gene or protein expression.
[0013] In a further example, the invention is a method of
increasing or stimulating bone growth using a formulation
comprising a combination of at least two of the following:
quercetin anhydrate, quercetin dihydrate, extract of Rehmannia sp.,
extract of Rehmannia sp. root, extract of Siberian ginseng, extract
of Sophora japonica, extract of licorice, and ipriflavone, wherein
the combination increases BMP-2 gene or protein expression in the
cell.
[0014] A further example of the invention is a method of increasing
or stimulating bone growth using a formulation comprising a
composition comprising a combination of approximately 10-1000 mg of
quercetin anhydrate or dihydrate, approximately 100-800 mg of an
extract of Siberian ginseng, and approximately 10-500 mg of an
extract of licorice, wherein the combination increases BMP-2 gene
or protein expression in a cell.
[0015] Another example of the invention is a method of increasing
or stimulating bone growth using a formulation comprising a
combination of approximately 10-1000 mg quercetin anhydrate or
dihydrate, and approximately 10-500 mg of an extract of licorice,
wherein the combination increases BMP-2 gene or protein expression
in a cell.
[0016] A further example of the invention is a method of increasing
or stimulating bone growth in a cell, comprising administering to
the cell a composition comprising a combination of approximately
10-1000 mg quercetin anhydrate or dihydrate, and approximately
100-800 mg of an extract of Siberian ginseng, wherein the
combination increases BMP-2 gene or protein in the cell.
[0017] In another example, the present invention may be a
composition for inhibiting, decreasing, or preventing bone
resorption comprising a pomegranate extract, punicalagins, or both,
in combination with one or more of the following: quercetin
dihydrate, quercetin anhydrate, extract of Rehmannia sp., extract
of Rehmannia sp. root, extract of Siberian ginseng, extract of
Sophora fructus japonica, extract of Sophora japonica, extract of
licorice, and ipriflavone, wherein the pomegranate extract, the
punicalagins, or both inhibit expression, production, and/or
release of RANK-L.
[0018] In one example, compositions and methods of the present
invention utilize extracts of pomegranate (Punica granatum) fruit
and peel which contain compounds known as punicalagins, to inhibit
or decrease bone resorption. The extracts of pomegranate useful in
the present invention, for example a pomegranate extract comprised
of punicalagins, can be used in compositions and methods for
inhibiting, decreasing, or preventing bone resorption, wherein the
pomegranate extract or punicalagins inhibit, decrease or prevent
the expression, production, and/or release of RANK-L. Accordingly,
in one example the present invention is a composition for
inhibiting, decreasing, or preventing bone resorption that
comprises a pomegranate extract, at least one punicalagin, or both.
Alternatively, the present invention contemplates a method of
inhibiting, decreasing, or preventing bone resorption comprising
administering a composition comprising a pomegranate extract, at
least one punicalagin, or both, wherein the composition inhibits
one of expression, production, and/or release of RANK-L.
[0019] In yet another example, the present invention is a
composition for inhibiting, decreasing, or preventing bone
resorption comprising natural, plant-derived extracts, including a
combination of at least two of the following: an extract of
pomegranate, an olive extract, an extract of Siberian ginseng, an
extract of Ginkgo biloba, an extract of green tea, an extract of
Sophora japonica, an extract of Rehmannia sp., an extract of grape
seed, an extract of Dong Quai, and ipriflavone, wherein the
combination inhibits expression, production, and/or release of
RANK-L.
[0020] In a further example, the present invention is a composition
for inhibiting, decreasing, or preventing bone resorption
comprising a combination of an extract of pomegranate preferably
containing punicalagins, an extract of grape seed, ipriflavone, and
an extract of green tea, wherein the combination inhibits
expression, production, and/or release of RANK-L.
[0021] In another example, the present invention is a composition
for inhibiting, decreasing, or preventing bone resorption
comprising a combination of approximately 10-2000 mg of an extract
of pomegranate, which may contain punicalagins, approximately
35-250 mg of an extract of grape seed, and approximately 400-700 mg
of ipriflavone, wherein the combination inhibits expression,
production, and/or release of RANK-L.
[0022] In yet another example, the present invention is a
composition for inhibiting, decreasing, or preventing bone
resorption comprising approximately 10-2000 mg of an extract of
pomegranate, approximately 35-250 mg of an extract grape seed, and
approximately 400-700 mg of ipriflavone, wherein the composition
inhibits release of calcium from bones.
[0023] Another example of the invention is a method of inhibiting,
decreasing, or preventing bone resorption comprising administering
a formula comprising a composition of natural, plant-derived
extracts, including a combination of at least two of the following
extracts: an extract of pomegranate, an olive extract, an extract
of Siberian ginseng, an extract of Ginkgo biloba, an extract of
green tea, an extract of Sophora japonica, an extract of Rehmannia
sp., an extract of grape seed, an extract of Dong Quai, and
ipriflavone, wherein the combination inhibits expression,
production, and/or release of RANK-L in a cell.
[0024] In a further example, the invention is a method of
inhibiting, decreasing, or preventing bone resorption by
administering a formula comprising a combination of an extract of
pomegranate, an extract of grape seed, ipriflavone, and an extract
of green tea, wherein the combination inhibit expression,
production, and/or release of RANK-L in a cell.
[0025] In a further example, the invention is a method of
inhibiting, decreasing, or preventing bone resorption by
administering a formula comprising a combination of approximately
10-2000 mg of an extract of pomegranate, approximately 35-250 mg of
an extract of grape seed, and approximately 400-700 mg of
ipriflavone, wherein the combination inhibits expression,
production, and/or release of RANK-L in a cell.
[0026] In yet another example, the invention is a method of
inhibiting, decreasing, or preventing bone resorption by
administering a formula comprising a combination of approximately
10-2000 mg of an extract of pomegranate, approximately 35-250 mg of
an extract of grape seed, and approximately 400-700 mg of
ipriflavone, wherein the combination inhibits release of calcium
from bones.
[0027] In a further example, the invention is a dietary supplement
regimen for increasing or stimulating bone growth and inhibiting,
decreasing, or preventing bone resorption comprising a first
composition comprising a combination of at least two of quercetin
dihydrate, quercetin anhydrate, an extract of Rehmannia sp., an
extract of Rehmannia sp. root, an extract of Siberian ginseng, an
extract of Sophora japonica, an extract of licorice, and
ipriflavone, wherein the combination of the first composition
increases the expression and/or activity of BMP-2; and a second
composition comprising a combination of at least two of an extract
of pomegranate, an olive extract, an extract of Siberian ginseng,
an extract of Ginkgo biloba, an extract of green tea, an extract of
Sophora japonica, an extract of Rehmannia sp., an extract of grape
seed, an extract of Dong Quai, and ipriflavone, wherein the
combination of the second composition inhibits the expression of
RANK-L.
DETAILED DESCRIPTION
[0028] It is to be understood that this invention is not limited to
the particular compositions, methodology, or protocols described
herein. Further, unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
invention belongs. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention, which will be limited only by the claims.
[0029] The present invention is based on the surprising discovery
that unique combinations of ingredients including two or more of
the following: quercetin anhydrate, quercetin dihydrate, extract of
Rehmannia sp., extract of Rehmannia sp. root, extract of Siberian
ginseng, extract of Sophora japonica, extract of licorice, extract
of ipriflavone, extract of pomegranate, extract of olive, extract
of Ginkgo biloba, extract of green tea, extract of grape seed, and
extract of Dong Quai, which are described more fully below,
increase bone growth by increasing or stimulating expression and/or
activity of the BMP-2 promoter, gene, and or/protein, or inhibit
bone resorption by either inhibiting, decreasing, or preventing
RANK-L expression, production, or release or by inhibiting,
decreasing, or preventing release of calcium from bones.
[0030] BMP-2 is a member of a family of bone morphogenic proteins,
which are novel factors in the extended transforming growth factor
B superfamily. Recombinant BMP-2 and BMP-4 can induce new bone
formation when injected locally into the subcutaneous tissues of
rats (Wozney J. Molec (1992) 32: 160-67). BMP-2 and BMP-4 are
expressed by normal osteoblasts as they differentiate, and have
been shown to stimulate osteoblast differentiation and bone nodule
formation in vitro as well as bone formation in vivo. Thus, by
increasing or stimulating BMP-2 promoter activity, gene expression,
and/or protein expression, the unique compositions of the present
invention are useful for increasing or stimulating bone growth and
treating or preventing a variety of bone disorders.
[0031] RANK-L, receptor activator of nuclear factor (NF)-kB ligand
(also: Osteoprotegerin ligand, OPGL) a member of the Tumor Necrosis
Factor (TNF) family, is the main stimulatory factor for the
formation of mature osteoclasts, bone cells that aid in bone
resorption, and is essential for their survival. RANK-L is produced
by osteoblastic lineage cells and activated T lymphocytes. It
activates the specific receptor RANK that is located on osteoclasts
and dendritic cells. One strategy for a formulation of the present
invention is to directly inhibit RANK-L expression when stimulated
by IL-1. Activation of RANK-L when stimulated with IL-1 can
directly trigger osteoclastogenesis (and thus bone resorption).
Thus, by inhibiting, decreasing, or preventing RANK-L expression,
production, or release, the unique compositions of the present
invention are useful for inhibiting, decreasing, or preventing bone
resorption and treating or preventing a variety of bone disorders
including bone loss, osteoporosis, osteolytic bones, etc.
[0032] Quercetin, which refers to quercetin extract(s), and the
various forms of quercetin, such as quercetin dihydrate, quercetin
anhydrate, etc., is one compound that is useful in unique
compositions of the present invention. Quercetin is a flavonoid
that forms the "backbone" for many other flavonoids, including the
citrus flavonoids rutin, hesperidin, naringin and tangeritin.
Quercetin is found to be the most active of the flavonoids in
studies, and many medicinal plants owe much of their activity to
their high quercetin content. Quercetin has demonstrated
significant anti-inflammatory activity because of direct inhibition
of several initial processes of inflammation. For example, it
inhibits both the manufacture and release of histamine and other
allergic/inflammatory mediators. In addition, it exerts potent
antioxidant activity and vitamin C-sparing action.
[0033] Quercetin also may have positive effects in combating or
helping to prevent cancer, prostatitis, heart disease, cataracts,
allergies, inflammations, and respiratory diseases such as
bronchitis and asthma. In addition, according to U.S. Pat. No.
5,478,579, when used in amounts ranging from 50-1500 mg/day,
quercetin anhydrate and/or quercetin dihydrate can enhance
absorption of calcium into bone tissues.
[0034] Foods rich in quercetin include apples, black & green
tea, onions, raspberries, red wine, red grapes, citrus fruits,
broccoli, fava beans, other leafy green vegetables, and
cherries.
[0035] As discussed more fully below, the present invention is
based in part on the discovery that quercetin is a potent activator
of BMP-2 promoter activity and protein expression. In addition, the
assay results discussed below demonstrate that when quercetin
dihydrate is administered in combination with Siberian ginseng,
licorice, or both, the combination achieves a surprising synergy
resulting in BMP-2 promoter activity and protein expression beyond
that achieved with any ingredient alone.
[0036] A quercetin ingredient used in the present invention may be
obtained commercially from various sources including, for example,
Twinlab (American Fork, Utah), Jarrow Formulas (Los Angeles,
Calif.), Natural Factors (Coquitlam, British Columbia, Canada), and
NOW Foods (Bloomingdale, Ill.). In addition, quercetin may be
obtained by any of the extraction methods discussed more fully
below, or described or known in the art.
[0037] Rehmannia, another plant useful in unique compositions of
the present invention, is a genus of six species of flowering
plants in the order Lamiales, endemic to China. Known as dihuang ()
in Chinese, this medicinal herb is used for a variety of ailments
such as anemia, dizziness and constipation. Rehmannia contains the
vitamins A, B, C, and D, as well as other useful compounds.
[0038] Rehmannia sp. extracts or extracts of Rehmannia sp.,
including extracts of Rehmannia sp. roots or Rehmannia sp. root
extracts may be obtained commercially from various sources
including EUL Herb Manufacturing (La Verne, Calif.) and NuPharma
Neutraceuticals (Miami Beach, Fla.). In addition, extracts of
Rehmannia sp. may be obtained by any of the extraction methods
discussed more fully below or known or described in the art.
[0039] Siberian ginseng, one of the compounds useful in unique
compositions of the present invention, which is also known as
Eleutherococcus senticosus, is a species of small, woody shrub in
the family Araliaceae native to Northeastern Asia. Siberian ginseng
is a powerful tonic herb with a wide range of health benefits. For
example, Siberian ginseng has immunoprotective effects against
breast (mammary gland) carcinoma, stomach carcinoma, oral cavity
carcinoma, skin melanoma and ovarian carcinoma. It was found to
have a pronounced effect on T lymphocytes, predominantly of the
helper/inducer type, but also on cytotoxic and natural killer
cells. In addition, Siberian ginseng is known to have a protective
effect against osteoporosis. See, e.g., Kropotov et al., "Effects
of Siberian ginseng extract and ipriflavone on the development of
glucocorticoid-induced osteoporosis." Bull Exp Biol Med., 2002.
133(3):252-4.
[0040] As discussed more fully below, the present invention is
based in part on the discovery that extracts of Siberian ginseng
(or Siberian ginseng extracts) are potent activators of BMP-2
promoter activity, BMP-2 gene expression and BMP-2 protein
expression. In addition, the assay results discussed below
demonstrate that when extracts of Siberian ginseng are administered
in combination with quercetin dihydrate, the combination achieves a
surprising synergy resulting in BMP-2 promoter activity, gene
expression and protein expression beyond that achieved with either
ingredient alone.
[0041] Siberian ginseng extracts may be commercially obtained from
various suppliers such as Xi'an Tianxingjian Natural Bio-products
Group (Xi'an, Shaanxi, China). In addition, Siberian ginseng
extract may be obtained using any of the extraction techniques
discussed more fully below or known in the art. In one example, a
Siberian ginseng extract may be obtained as an alcoholic fluid
extraction of the root and/or rhizome of Siberian ginseng.
[0042] Sophora japonica, or Sophora fructus japonica, another of
the compounds useful in unique compositions of the present
invention, is also referred to as the Pagoda Tree and is native to
eastern Asia. Rutin, an active compound that may be found in a
Sophora japonica extract, may be used to increase the permeability
(e.g. the resolution and porousness of the dilation) of
capillaries. In addition to rutin, quercetin anhydrate and
quercetin dihydrate may be extracted from Sophora japonica plants,
including from the leaves, stem, flower, seeds, root, etc.
[0043] As discussed more fully below, the present invention is
based in part on the discovery that extracts of Sophora japonica
(or Sophora japonica extracts) are potent activators of BMP-2
promoter activity, gene expression and protein expression.
[0044] Sophora japonica extracts may be commercially obtained from
various suppliers such as NuPharma Nutraceuticals (Miami Beach,
Fla.). In addition, Sophora japonica extract may be obtained using
any of the extraction techniques discussed more fully below or
known in the art. In one example, the ripe seeds of Sophora
japonica may be used to obtain a Sophora japonica extract useful in
the present invention.
[0045] Licorice extracts, useful in unique compositions of the
present invention, are widely used in treating bronchial problems
such as catarrh, bronchitis and coughs in general. It also forms an
important ingredient in controlling peptic ulcerations, gastritis
and ulcers. According to JP 2002 179585 and US 20020009506, when
administered in an amount ranging from 50-100 mg per day, licorice
can be used to treat osteoporosis, improve bone metabolism, and
promote calcification.
[0046] As discussed more fully below, the present invention is
based in part on the discovery that licorice extracts (or extracts
of licorice), for example ethanol and ethanol-water extracts of
licorice, are potent activators of BMP-2 promoter activity, gene
expression and protein expression. In addition, the assay results
discussed below demonstrate that when licorice extract is
administered in combination with quercetin dihydrate, the
combination achieves a surprising synergy resulting in BMP-2
promoter activity, gene expression and protein expression beyond
that achieved with either ingredient alone.
[0047] Licorice extracts may be commercially obtained from various
suppliers such as Herbs Forever, Inc. (Los Angeles, Calif.). In
addition, a licorice extract may be obtained using any of the
extraction techniques discussed more fully below or known in the
art. In one example, a licorice extract can be an ethanol extract
of licorice obtained from the root, runner, and/or rhizome of
Glycyrrhiza glabra.
[0048] Ipriflavone, another compound useful in compositions of the
invention, is an isoflavone. Ipriflavone may be found in small
amounts in legume plants, such as alfalfa, but generally is
synthetically manufactured as 7-isopropoxy isoflavone, using
polyphenols as a starting material.
[0049] As discussed more fully below, the present invention is
based in part on the discovery that ipriflavone is a potent
inhibitor of RANK-L expression. Ipriflavone may be commercially
obtained from various sources. For example, Ostivone.RTM. is a
synthetic ipriflavone compound available from Technical Sourcing
International, Inc. (Missoula, Mont.).
[0050] Pomegranates can be extracted to yield an extract of
pomegranate (pomegranate extract) that is useful in compositions of
the present invention. When extracted, pomegranate, known as Punica
granatum, is generally standardized to ellagic acid or punicalagin
content. Punicalagins exist as isomers of
2,3,hexahydroxydiphenoyl-gallagyl-D-glucose. An exemplary structure
is shown below:
##STR00001##
[0051] Pomegranate extracts also may be high in polyphenols, such
as hydrolysable tannins, and particularly punicalagins, which may
be responsible for the free-radical scavenging ability of
pomegranate juice.
[0052] In Japan, pomegranate has been used to inhibit bone quantity
reduction. See JP 1999 0049884. The present invention is based in
part on the discovery that an extract of pomegranate is a potent
inhibitor of RANK-L expression, production, or release. In one
example, punicalagins present in extracts of pomegranate inhibit or
decrease RANK-L expression. Therefore, pomegranate extracts
comprising punicalagins, ellagic acid or both are useful in
compositions and methods for inhibiting, decreasing, or preventing
the production, release, and/or expression of RANK-L.
[0053] Pomegranate extracts may be commercially obtained from
various sources including Nature's Way (Springville, Utah),
Nature's Herbs (American Fork, Utah), Swansen's Health Products
(Fargo, N. Dak.) and Doctor's Trust Vitamins (Orlando, Fla.). In
addition, a pomegranate extract may be obtained using any of the
extraction techniques discussed more fully below or known in the
art.
[0054] Extracts of Ginkgo biloba (or Ginkgo biloba extracts),
another of the extracts useful in unique compositions of the
present invention, are known to have three effects: (1) improve
blood flow (including microcirculation in small capillaries) to
most tissues and organs; (2) protect against oxidative cell damage
from free radicals (antioxidant); and (3) block the effects of
platelet aggregation and blood clotting.
[0055] Ginkgo biloba extracts may be commercially obtained from
various sources including Puritan's Pride (Long Island, N.Y.). In
addition, Ginkgo biloba extracts may be obtained using any of the
extraction process disclosed herein or known in the art. For
example, Ginkgo biloba extracts may be obtained using any of the
extraction processes disclosed herein or known in the art to
extract dried or fresh leaves, or seeds of Gingko biloba.
[0056] Green Tea, which can be extracted to yield one of the
compounds useful in unique compositions of the present invention,
has long been used by the Chinese as medicine to treat headaches,
body aches, poor digestion, and improve well-being and life
expectancy. Green tea extract is rich in bioflavonoids, including
the anti-oxidant epigallocatechin gallate (EGCG). The EGCG in green
tea extract protects against digestive and respiratory infections,
blocks the actions of carcinogens, can function as an
anti-bacterial, and can also help lower cholesterol levels.
[0057] As discussed more fully below, the present invention is
based in part on the discovery that green tea extract (or an
extract of green tea) is a potent inhibitor of RANK-L expression.
Green tea extracts may be commercially obtained from various
sources including Life Extension (Fort Lauderdale, Fla.). In
addition, a green tea extract may be obtained using any of the
extraction techniques discussed more fully below or known in the
art.
[0058] Grape Seed extracts (or extracts of grape seeds), another of
the compounds useful in unique compositions of the present
invention, contain a class of flavonoid complexes known as
oligomeric proanthocyanidins or OPCs that act as antioxidants (free
radical scavengers) in the human body. OPCs may help protect
against the effects of internal and environmental stresses (that
is, cigarette smoking, pollution, and supporting normal body
metabolic processes).
[0059] The extracts of grape seed used in the present invention may
be obtained from commercially available sources. For example, the
grape seed extract may be obtained from Kikkoman Corporation
(Tokyo, Japan), Polyphenolics, Inc. (Madera, Calif.), Bio Serae
Laboratories SA (Bram, France), OptiPure (Los Angeles, Calif.), Dry
Creek Nutrition, Inc. (Modesto, Calif.), or other suitable sources.
In addition, the extraction techniques discussed more fully below,
or those known or described in the art may be used to produce a
grape seed extract to be used in the present invention.
[0060] Dong Quai extracts, useful in unique compositions of the
present invention, also are known as extracts of Angelica sinensis
or "female ginseng." Dong Quai is an herb from the family Apiaceae,
indigenous to China. Its root is commonly known in Chinese as dong
quai or danggui (Chinese: ; pinyin: d nggu ) and is widely used in
Chinese traditional medicine to treat gynecological ailments,
fatigue, mild anemia and high blood pressure. Dong Quai has
analgesic, anti-inflammatory, antispasmodic and sedative effects.
The plant's phytochemicals consist of coumarins, phytosterols,
polysaccharides, ferulate, and flavonoids.
[0061] Dong Quai extract (or extract of Dong Quai) may be
commercially obtained from a variety of different sources,
including Capricorns Lair (Ogden, Utah). In addition, Dong Quai can
be extracted using any of the extraction techniques described more
fully below, or any extraction techniques known in the art.
[0062] Although each of the extracts used in the present invention
is commercially available, there are numerous extraction methods
that can be used to produce an extract to be used in the present
invention without commercially purchasing the extract. Some
examples of extraction methods that can be used to produce an
extract to be used in the present invention are described below.
Other examples are known and described in the art, including in
various publications and patents. The extraction methods described
more fully below are exemplary and one of ordinary skill in the art
will appreciate that other extraction techniques and methods may be
used to obtain an extract useful in the present invention.
[0063] Extracts used in the present invention may be from a variety
of sources, including different varieties and species. For example,
grape seeds from grapes of any color or variety may be used to
obtain a grape seed extract. In addition, any of the parts of a
plant may be extracted, including the fruit, peel, seeds, stem,
leaves, roots, bark, rhizome, runner, etc.
[0064] In one example, an extract useful in the unique compositions
of the present invention might be obtained using an organic solvent
extraction technique. More specifically, an extract useful in the
present invention, such as a licorice extract or a licorice root
extract, can be produced by extracting licorice or licorice root
with an organic solvent, for example, hexane, ethyl acetate,
ethanol, or hydro-ethanol.
[0065] In another example, solvent sequential fractionation may be
used to obtain an extract useful in the unique compositions of the
present invention. For example, using this technique, a grape seed
extract could be obtained by sequentially extracting grape seeds
with hexane, ethyl acetate, ethanol, and hydro-ethanol. The
extracts obtained after each step (fractions) of the sequence will
contain chemical compounds in increasing order of polarity similar
to the solvents used for extracting them. The fractions are dried
to evaporate the solvents, resulting in an extract of grape seed.
Those of skill in the art will appreciate that many other solvents
can be used in practicing solvent sequential fractionation
extraction.
[0066] Total hydro-ethanolic extraction techniques might also be
used to obtain an extract useful in the unique compositions of the
present invention. Generally, this is referred to as a lump-sum
extraction. The extract generated in this process will contain a
broad variety of phytochemicals present in the extracted material
including fat and water solubles. Following collection of the
extract solution, the solvent will be evaporated, resulting in the
extract. In one example, pomegranates might be extracted using this
technique.
[0067] Total ethanol extraction may also be used in the present
invention. This technique uses ethanol, rather than hydro-ethanol,
as the solvent. This extraction technique generates an extract that
may include fat soluble and/or lipophilic compounds in addition to
water soluble compounds. An extract of green tea might be obtained
using this technique.
[0068] Another example of an extraction technique that might be
used to obtain an extract useful in the present invention is
supercritical fluid carbon dioxide extraction (SFE). In this
extraction procedure the material to be extracted is not exposed to
any organic solvents. Rather, the extraction solvent is carbon
dioxide, with or without a modifier, in supercritical conditions
(>31.3.degree. C. and >73.8 bar). Those of skill in the art
will appreciate that temperature and pressure conditions can be
varied to obtain the best yield of extract. This technique
generates an extract of fat soluble and/or lipophilic compounds,
similar to the total hexane and ethyl acetate extraction technique
described above.
[0069] Those of skill in the art will appreciate that there are
many other extraction processes, both known in the art and
described in various patents and publications that can be used to
obtain the extracts to be used in practicing the present invention.
For example, the extraction procedures described in the following
references, which are incorporated herein by reference, could be
used in practicing the present invention: Murga et al., "Extraction
of natural complex phenols and tannins from grape seeds by using
supercritical mixtures of carbon dioxide and alcohol." J. Agric
Food Chem. 2000 August:48(8):3408-12; Hong et al.,
"Microwave-assisted extraction of phenolic compounds from grape
seed." Nat Prod Lett. 2001; 15(3): 197-204; Ashraf-Khorassani et
al., "Sequential fractionation of grape seeds into oils,
polyphenols, and procyanidins via a single system employing
CO.sub.2-based fluids." J. Agric Food Chem., 2004 May
5;52(9):2440-4.
[0070] Compositions of the Invention
[0071] Compositions of the present invention may be formulated in
an acceptable carrier and may be prepared, packaged, and labeled
for increasing or stimulating bone growth, inhibiting, decreasing,
or preventing bone resorption, increasing bone strength, improving
bone architecture, or treatment, prevention, or management of
various bone disorders including, but not limited to, fractures,
osteoporosis, periodontal disease, metastatic bone disease, and
osteolytic bone disease.
[0072] In one example, the invention is a composition for
increasing or stimulating bone growth, comprising a combination of
at least two of the following: quercetin anhydrate, quercetin
dihydrate, Rehmannia sp. extract, Siberian ginseng extract, Sophora
japonica extract, licorice extract, and ipriflavone, wherein the
combination increases BMP-2 gene or protein expression.
[0073] In another example, the invention is a composition for
increasing bone growth, comprising a combination of at least two of
the following: quercetin, anhydrate, quercetin dihydrate, Rehmannia
sp. extract, Siberian ginseng extract, Sophora japonica extract,
licorice extract, and ipriflavone, wherein the combination
increases BMP-2 gene or protein expression, and further wherein, if
present: quercetin anhydrate or quercetin dihydrate, is present in
an amount ranging from approximately 10-1000 mg, more preferably in
an amount ranging from approximately 200-750 mg, more preferably in
an amount ranging from approximately 300-700 mg, more preferably in
an amount ranging from approximately 400-600 mg, more preferably in
an amount of approximately 500 mg; Rehmannia sp. extract is present
in an amount ranging from approximately 10-1000 mg, more preferably
in an amount ranging from approximately 100-900 mg, more preferably
in an amount ranging from approximately 200-800 mg, more preferably
in an amount ranging from approximately 300-700 mg, more preferably
in an amount ranging from approximately 400-600 mg, more preferably
in an amount of approximately 500 mg; Siberian ginseng extract is
present in an amount ranging from 100-800 mg, more preferably in an
amount ranging from approximately 200-750 mg, more preferably in an
amount ranging from approximately 300-700 mg, more preferably in an
amount ranging from approximately 400-600 mg, more preferably in an
amount of approximately 500 mg; Sophora japonica extract is present
in an amount ranging from 10-1000 mg, more preferably in an amount
ranging from approximately 100-900 mg, more preferably in an amount
ranging from approximately 200-800 mg, more preferably in an amount
ranging from approximately 300-700 mg, more preferably in an amount
ranging from approximately 400-600 mg, more preferably in an amount
of approximately 500 mg; licorice extract is present in an amount
ranging from approximately 10-500 mg, more preferably in an amount
ranging from approximately 25-450 mg, more preferably in an amount
ranging from approximately 50-400 mg, more preferably in an amount
ranging from approximately 75-350 mg, more preferably in an amount
ranging from approximately more preferably in an amount ranging
from approximately 100-300 mg, more preferably in an amount ranging
from approximately 125-250 mg, more preferably in an amount ranging
from approximately 25-175 mg; and ipriflavone is present in an
amount ranging from 200-700 mg, more preferably in an amount
ranging from approximately 250-650 mg, more preferably in an amount
ranging from approximately 300-600 mg, more preferably in an amount
ranging from approximately 400-500 mg, more preferably in an amount
of approximately 600 mg.
[0074] In another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
quercetin anhydrate, quercetin dihydrate, Siberian ginseng extract,
Sophora japonica extract, and licorice extract, wherein the
combination increases BMP-2 gene or protein expression, and further
wherein the quercetin anhydrate or quercetin dihydrate is present
in an amount ranging from approximately 10-1000 mg, more preferably
in an amount ranging from approximately 200-750 mg, more preferably
in an amount ranging from approximately 300-700 mg, more preferably
in an amount ranging from approximately 400-600 mg, more preferably
in an amount of approximately 500 mg; Siberian ginseng extract is
present in an amount ranging from approximately 100-800 mg, more
preferably in an amount ranging from approximately 200-750 mg, more
preferably in an amount ranging from approximately 300-700 mg, more
preferably in an amount ranging from approximately 400-600 mg, more
preferably in an amount of approximately 500 mg; Sophora japonica
extract is present in an amount ranging from approximately 10-1000
mg, more preferably in an amount ranging from approximately 100-900
mg, more preferably in an amount ranging from approximately 200-800
mg, more preferably in an amount ranging from approximately 300-700
mg, more preferably in an amount ranging from approximately 400-600
mg, more preferably in an amount of approximately 500 mg; and
licorice extract is present in an amount ranging from 10-500 mg,
more preferably in an amount ranging from approximately 25-450 mg,
more preferably in an amount ranging from approximately 50-400 mg,
more preferably in an amount ranging from approximately 75-350 mg,
more preferably in an amount ranging from approximately 100-300 mg,
more preferably in an amount ranging from approximately 125-250 mg,
more preferably in an amount ranging from approximately 25-175
mg.
[0075] In another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
approximately 10-1000 mg of quercetin anhydrate, quercetin
dihydrate, more preferably approximately 250-750 mg, more
preferably approximately 300-700 mg, more preferably approximately
400-600 mg, more preferably approximately 500 mg; approximately
100-800 mg of Siberian ginseng, more preferably approximately
200-750 mg, more preferably approximately 300-700 mg, more
preferably approximately 400-600 mg, more preferably approximately
500 mg; and approximately 10-500 mg of licorice extract, more
preferably approximately 25-450 mg, more preferably approximately
50-400 mg, more preferably approximately 75-350 mg, more preferably
approximately 100-300 mg, more preferably approximately 125-250 mg,
more preferably approximately 25-175 mg, wherein the combination
increases BMP-2 gene or protein expression.
[0076] In a further example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
quercetin anhydrate or quercetin dihydrate, Siberian ginseng
extract, and licorice extract, wherein the combination increases
BMP-2 gene or protein expression, and further wherein the quercetin
anhydrate or quercetin dihydrate, Siberian ginseng extract, and
licorice extract are present in equal amounts, more preferably
wherein the Siberian ginseng extract is present in an amount that
is 1/2 that of quercetin anhydrate or quercetin dihydrate and the
licorice extract is present in an amount that is 1/10 that of
quercetin anhydrate or quercetin dihydrate.
[0077] In another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
quercetin anhydrate or quercetin dihydrate, and Siberian ginseng
extract, wherein quercetin anhydrate or quercetin dihydrate forms
approximately 10-75% w/w of the composition, more preferably
approximately 50% w/w of the composition and Siberian ginseng
extract forms approximately 10-75% w/w of the composition, more
preferably approximately 50% w/w of the composition, wherein the
combination of quercetin anhydrate or quercetin dihydrate and
Siberian ginseng extract increases BMP-2 gene or protein
expression.
[0078] In yet another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
at least two of: quercetin anhydrate, quercetin dihydrate, Siberian
ginseng extract, Sophora japonica extract, and licorice extract,
wherein if present, quercetin anhydrate or quercetin dihydrate
forms approximately 10-75% w/w of the composition, more preferably
approximately 50% w/w of the composition; Siberian ginseng extract
forms approximately 10-75% w/w of the composition, more preferably
approximately 50% w/w of the composition; Sophora japonica extract
forms approximately 1-50% w/w of the composition, more preferably
approximately 2-15% w/w of the composition, more preferably
approximately 5-10% w/w of the composition; and licorice extract
forms approximately 1-50% w/w of the composition, more preferably
approximately 2-15% w/w of the composition, more preferably
approximately 5-10% w/w of the composition, further wherein the
combination increases BMP-2 gene or protein expression.
[0079] In a further example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
approximately 10-1000 mg of quercetin anhydrate or quercetin
dihydrate, more preferably approximately 250-750 mg, more
preferably approximately 300-700 mg, more preferably approximately
400-600 mg, more preferably approximately 500 mg; and approximately
10-500 mg of licorice extract, more preferably approximately 20-125
mg, more preferably approximately 20-100 mg, more preferably
approximately 25-75 mg, more preferably approximately 50 mg,
wherein the combination increases BMP-2 gene or protein
expression.
[0080] In another example, the invention is a composition for
increasing or stimulating bone growth comprising a combination of
approximately 10-1000 mg of quercetin anhydrate or quercetin
dihydrate, more preferably approximately 250-750 mg, more
preferably approximately 300-700 mg, more preferably approximately
400-600 mg, more preferably approximately 500 mg; and approximately
100-800 mg of Siberian ginseng extract, more preferably
approximately 200-750 mg, more preferably approximately 300-700 mg,
more preferably approximately 400-600 mg, more preferably
approximately 500 mg, wherein the combination of quercetin
anhydrate or quercetin dihydrate, and Siberian ginseng extract
increases BMP-2 gene or protein expression.
[0081] In a further example, the invention is a method of
increasing or stimulating bone growth by administering a formula
comprising a combination of at least two of the following:
quercetin anhydrate, quercetin dihydrate, Rehmannia sp. extract,
Rehmannia sp. root extract, Siberian ginseng extract, Sophora
japonica extract, licorice extract, and ipriflavone, wherein the
combination increases BMP-2 gene or protein expression in a
cell.
[0082] A further example of the invention is a method of increasing
or stimulating bone growth by administering a formula comprising a
combination of approximately 10-1000 mg of quercetin anhydrate or
quercetin dihydrate, more preferably approximately 250-750 mg, more
preferably approximately 300-700 mg, more preferably approximately
400-600 mg, more preferably approximately 500 mg; approximately
100-800 mg of Siberian ginseng extract, more preferably
approximately 200-750 mg, more preferably approximately 300-700 mg,
more preferably approximately 400-600 mg, more preferably
approximately 500 mg; and approximately 10-500 mg of licorice
extract, more preferably approximately 25-450 mg, more preferably
approximately 50-400 mg, more preferably approximately 75-350 mg,
more preferably approximately 100-300 mg, more preferably
approximately 125-250 mg, more preferably approximately 25-75 mg,
wherein the combination increases BMP-2 gene or protein expression
in a cell.
[0083] Another example of the invention is a method of increasing
or stimulating bone growth by administering a formulation
comprising a combination of approximately 10-1000 mg quercetin
anhydrate or quercetin dihydrate, more preferably approximately
250-750 mg, more preferably approximately 300-700 mg, more
preferably approximately 400-600 mg, more preferably approximately
500 mg; and approximately 10-500 mg of licorice extract, more
preferably approximately 20-125 mg, more preferably approximately
20-100 mg, more preferably approximately 25-75 mg, more preferably
approximately 50 mg, wherein the combination increases BMP-2 gene
or protein expression in a cell.
[0084] A further example of the invention is a method of increasing
or stimulating bone growth by administering a formula comprising a
combination of approximately 10-1000 mg quercetin anhydrate or
quercetin dihydrate, more preferably approximately 250-750 mg, more
preferably approximately 300-700 mg, more preferably approximately
400-600 mg, more preferably approximately 500 mg; and approximately
100-800 mg of Siberian ginseng extract, more preferably
approximately 200-750 mg, more preferably approximately 300-700 mg,
more preferably approximately 400-600 mg, more preferably
approximately 500 mg, wherein the combination of quercetin
anhydrate or quercetin dihydrate, and Siberian ginseng extract
increases the expression and/or activity of the BMP-2 gene or
protein in a cell.
[0085] In another example, the present invention is a composition
for inhibiting, decreasing, or preventing bone resorption
comprising a pomegranate extract and at least one of the following
natural, plant-derived extracts: Siberian ginseng extract, Ginkgo
biloba extract, green tea extract, Sophora japonica extract,
Rehmannia sp. extract, grape seed extract, Dong Quai extract, and
ipriflavone, wherein the combination inhibits expression,
production, and/or release of RANK-L, and further wherein, the
pomegranate extract is present in an amount ranging from
approximately 10-2000 mg, more preferably in an amount ranging from
approximately 300-1700 mg, more preferably in an amount ranging
from approximately 400-1500 mg, more preferably in an amount
ranging from approximately 500-1250 mg, more preferably in an
amount ranging from approximately 600-1000 mg, more preferably in
an amount ranging from approximately 700-900 mg; more preferably in
an amount of approximately 500 mg; and if present, Siberian ginseng
extract is present in an amount ranging from approximately 100-2000
mg, more preferably in an amount ranging from approximately
300-1700 mg, more preferably in an amount ranging from
approximately 400-1500 mg, more preferably in an amount ranging
from approximately 500-1250 mg, more preferably in an amount
ranging from approximately 600-1000 mg, more preferably in an
amount ranging from approximately 700-900 mg; more preferably in an
amount of approximately 500 mg; Ginkgo biloba extract is present in
an amount ranging from approximately 10-1000 mg, more preferably in
an amount ranging from approximately 100-900 mg, more preferably in
an amount ranging from approximately 200-800 mg, more preferably in
an amount ranging from approximately 300-700 mg, more preferably in
an amount ranging from approximately 400-600 mg, more preferably in
an amount of approximately 500 mg; green tea extract is present in
an amount ranging from approximately 300-700, more preferably in an
amount ranging from approximately 350-650 mg, more preferably in an
amount ranging from approximately 400-600 mg, more preferably in an
amount of approximately 500 mg; Sophora japonica extract is present
in an amount ranging from 10-1000 mg, more preferably in an amount
ranging from approximately 100-900 mg, more preferably in an amount
ranging from approximately 200-800 mg, more preferably in an amount
ranging from approximately 300-700 mg, more preferably in an amount
ranging from approximately 400-600 mg, more preferably in an amount
of approximately 500 mg; Rehmannia sp. extract is present in an
amount ranging from approximately 10-1000 mg, more preferably in an
amount ranging from approximately 100-900 mg, more preferably in an
amount ranging from approximately 200-800 mg, more preferably in an
amount ranging from approximately 300-700 mg, more preferably in an
amount ranging from approximately 400-600 mg, more preferably in an
amount of approximately 500 mg; grape seed extract is present in an
amount ranging from 35-250 mg, more preferably in an amount ranging
from approximately 50-150 mg, more preferably in an amount ranging
from approximately 75-125 mg; Dong quai extract is present in an
amount ranging from approximately 10-1000 mg, more preferably in an
amount ranging from approximately 100-900 mg, more preferably in an
amount ranging from approximately 200-800 mg, more preferably in an
amount ranging from approximately 300-700 mg, more preferably in an
amount ranging from approximately 400-600 mg, more preferably in an
amount of approximately 500 mg; and ipriflavone is present in an
amount ranging from approximately 400-700 mg, more preferably in an
amount ranging from approximately 450-650 mg, more preferably in an
amount ranging from approximately 500-600 mg, more preferably in an
amount of approximately 600 mg; wherein the combination inhibits
expression, production, and/or release of RANK-L or inhibits
release of calcium from bone.
[0086] In a further example, the present invention is a composition
for inhibiting, decreasing, or preventing bone resorption
comprising a combination of approximately 10-2000 mg of pomegranate
extract, more preferably approximately 400-1700 mg, more preferably
approximately 500-1500 mg, more preferably approximately 600-1250
mg, more preferably approximately 700-1000 mg, more preferably
approximately 800-900 mg; approximately 35-250 mg of grape seed
extract, more preferably approximately 50-150 mg, more preferably
approximately 75-125 mg; approximately 400-700 mg of ipriflavone,
more preferably approximately 450-650 mg, more preferably
approximately 500-600 mg, more preferably approximately 600 mg; and
approximately 300-700 mg of a green tea extract, more preferably
approximately 350-650 mg, more preferably approximately 400-600 mg,
more preferably approximately 500 mg, wherein the combination
inhibits expression, production, and/or release of RANK-L or
inhibits release of calcium from bone.
[0087] In another example, the present invention is a composition
for inhibiting, decreasing, or preventing bone resorption
comprising a combination of approximately 10-2000 mg of pomegranate
extract, more preferably approximately 400-1700 mg, more preferably
approximately 500-1500 mg, more preferably approximately 600-1250
mg, more preferably approximately 700-1000 mg, more preferably
approximately 800-900 mg; approximately 35-250 mg of grape seed
extract, more preferably approximately 50-150 mg, more preferably
approximately 75-125 mg; and approximately 400-700 mg of
ipriflavone, more preferably approximately 450-650 mg, more
preferably approximately 500-600 mg, more preferably approximately
600 mg, wherein the combination inhibits expression, production,
and/or release of RANK-L or inhibits release of calcium from
bone.
[0088] In another example, the invention is a composition for
inhibiting, decreasing, or preventing bone resorption comprising a
combination of pomegranate extract, grape seed extract, and
ipriflavone, wherein the grape seed extract is present in an amount
of approximately 1/10 the pomegranate extract and the ipriflavone
is present in an amount of approximately 400-700 mg, more
preferably approximately 450-650 mg, more preferably approximately
500-600 mg, more preferably approximately 600 mg, wherein the
combination inhibits expression, production, and/or release of
RANK-L or inhibits release of calcium from bone.
[0089] In a further example, the invention is a composition for
inhibiting, decreasing, or preventing bone resorption comprising a
combination of at least two of pomegranate extract, grape seed
extract, ipriflavone, and green tea extract, wherein, if present,
the pomegranate extract is present in an amount ranging from
approximately 25-100% w/w of the composition, more preferably from
approximately 30-90% w/w of the composition, more preferably from
approximately 40-80% w/w of the composition, more preferably from
approximately 45-60% w/w of the composition, more preferably
approximately 50% w/w of the composition; the grape seed extract is
present in an amount ranging from approximately 1-25% w/w of the
composition, more preferably approximately 2-15% w/w of the
composition, more preferably approximately 5-10% w/w of the
composition; the Ipriflavone is present in an amount ranging from
approximately 25-100% w/w of the composition, more preferably from
approximately 30-90% w/w of the composition, more preferably from
approximately 40-80% w/w of the composition, more preferably from
approximately 45-60% w/w of the composition, more preferably
approximately 50% w/w of the composition; and the green tea extract
is present in an amount ranging from approximately 1-25% w/w of the
composition, more preferably approximately 2-15% w/w of the
composition, more preferably approximately 5-10% w/w of the
composition, further wherein the combination inhibits expression,
production, and/or release of RANK-L.
[0090] Another example of the invention is a method of inhibiting,
decreasing, or preventing bone resorption by administering a
formula comprising natural, plant-derived extracts, including at
least one of the following extracts: pomegranate, Siberian ginseng,
Ginkgo biloba, green tea, Sophora japonica, Rehmannia sp., grape
seed, Dong Quai, and ipriflavone, wherein the composition inhibits
expression, production, and/or release of RANK-L in a cell.
[0091] In a further example, the invention is a method of
inhibiting, decreasing, or preventing bone resorption by
administering a formula comprising a combination of approximately
10-2000 mg of pomegranate extract, more preferably approximately
400-1700 mg, more preferably approximately 500-1500 mg, more
preferably approximately 600-1250 mg, more preferably approximately
700-1000 mg, more preferably approximately 800-900 mg;
approximately 35-250 mg of grape seed extract, more preferably
approximately 50-150 mg, more preferably approximately 75-125 mg;
approximately 400-700 mg of ipriflavone, more preferably
approximately 450-650 mg, more preferably approximately 500-600 mg,
more preferably approximately 600 mg; and approximately 300-700 mg
of a green tea extract, more preferably approximately 350-650 mg,
more preferably approximately 400-600 mg, more preferably
approximately 500 mg, wherein the combination inhibits expression,
production, and/or release of RANK-L in a cell.
[0092] In a further example, the invention is a method of
inhibiting, decreasing, or preventing bone resorption by
administering a formula comprising a combination of approximately
10-2000 mg of pomegranate extract, more preferably approximately
400-1700 mg, more preferably approximately 500-1500 mg, more
preferably approximately 600-1250 mg, more preferably approximately
700-1000 mg, more preferably approximately 800-900 mg;
approximately 35-250 mg of grape seed extract, more preferably
approximately 50-125 mg, more preferably approximately 75-100 mg;
and approximately 400-700 mg of ipriflavone, more preferably
approximately 450-650 mg, more preferably approximately 500-600 mg,
more preferably approximately 600 mg, wherein the combination
inhibits expression, production, and/or release of RANK-L in a
cell.
[0093] In a further example, the invention is a dietary supplement
regimen for increasing or stimulating bone growth and inhibiting,
decreasing, or preventing bone resorption comprising a first
composition comprising a combination of at least two of: quercetin
dihydrate, quercetin anhydrate, Siberian ginseng extract, licorice
extract, and Sophora japonica extract, wherein the combination of
the first composition increases the expression and/or activity of
BMP-2; and a second composition comprising a combination of at
least two of: pomegranate extract, grape seed extract, ipriflavone,
and green tea extract, wherein the combination of the second
composition inhibits the expression of RANK-L.
[0094] In a further example, the invention is a dietary supplement
regimen for increasing or stimulating bone growth and inhibiting,
decreasing, or preventing bone resorption comprising a first
composition comprising a combination of at least two of: quercetin
anhydrate, quercetin dihydrate, Siberian ginseng extract, licorice
extract, and Sophora japonica extract, wherein, if present,
quercetin anhydrate or quercetin dihydrate is present in an amount
ranging from approximately 10-1000 mg, more preferably in an amount
ranging from approximately 200-750 mg, more preferably in an amount
ranging from approximately 300-700 mg, more preferably in an amount
ranging from approximately 400-600 mg, more preferably in an amount
of approximately 500 mg; wherein, if present, Siberian ginseng
extract is present in an amount ranging from approximately 100-800
mg, more preferably in an amount ranging from approximately 200-750
mg, more preferably in an amount ranging from approximately 300-700
mg, more preferably in an amount ranging from approximately 400-600
mg, more preferably in an amount of approximately 500 mg; wherein,
if present, licorice extract is present in an amount ranging from
10-500 mg, more preferably in an amount ranging from approximately
25-450 mg, more preferably in an amount ranging from approximately
50-400 mg, more preferably in an amount ranging from approximately
75-350 mg, more preferably in an amount ranging from approximately
100-300 mg, more preferably in an amount ranging from approximately
125-250 mg, more preferably in an amount ranging from approximately
25-175 mg; and wherein if present, Sophora japonica extract is
present in an amount ranging from approximately 10-1000 mg, more
preferably in an amount ranging from approximately 100-900 mg, more
preferably in an amount ranging from approximately 200-800 mg, more
preferably in an amount ranging from approximately 300-700 mg, more
preferably in an amount ranging from approximately 400-600 mg, more
preferably in an amount of approximately 500 mg, and further
wherein the combination of the first composition increases the
expression and/or activity of BMP-2; and a second composition
comprising a combination of at least two of: pomegranate extract,
grape seed extract, ipriflavone, and green tea extract, wherein, if
present, pomegranate extract is present in an amount ranging from
approximately 10-2000 mg, more preferably approximately 400-1700
mg, more preferably approximately 500-1500 mg, more preferably
approximately 600-1250 mg, more preferably approximately 700-1000
mg, more preferably approximately 800-900 mg; wherein, if present,
grape seed extract is present in an amount ranging from
approximately 35-250 mg, more preferably approximately 50-150 mg,
more preferably approximately 75-125 mg; wherein, if present,
ipriflavone is present in an amount ranging from approximately
400-700 mg, more preferably approximately 450-650 mg, more
preferably approximately 500-600 mg, more preferably approximately
600 mg; and wherein, if present, green tea extract is present in an
amount ranging from approximately 300-700 mg, more preferably
approximately 350-650 mg, more preferably approximately 400-600 mg,
more preferably approximately 500 mg, and further wherein the
combination of the second composition inhibits the expression of
RANK-L.
[0095] Modes of Administration
[0096] The compositions of the invention may be administered
systemically or locally. For systemic use, the compositions of the
invention are formulated for parenteral (e. g., intravenous,
subcutaneous, intramuscular, intraperitoneal, intranasal or
transdermal) or enteral (e. g., oral or rectal) delivery according
to conventional methods. Intravenous administration can be by a
series of injections or by continuous infusion over an extended
period. Administration by injection or other routes of discretely
spaced administration can be performed at intervals ranging from
weekly to once to three times daily. Alternatively, the
compositions disclosed herein may be administered in a cyclical
manner (administration of disclosed composition; followed by no
administration; followed by administration of disclosed
composition; and the like). Treatment can continue until the
desired outcome is achieved. Alternatively, administration of the
compositions of the present invention may be continual, and thereby
be a preventative administration, rather than an administration for
treatment.
[0097] In general, compositions of the present invention can
include a cosmetically or pharmaceutically acceptable vehicle, such
as saline, buffered saline, 5% dextrose in water, borate-buffered
saline containing trace metals or the like. Compositions of the
present invention may further include one or more excipients, for
example, vitamin A, vitamin D, or calcium; preservatives;
solubilizers; buffering agents; albumin to prevent protein loss on
vial surfaces; lubricants; fillers; stabilizers; etc. Methods of
formulation are well known in the art and are disclosed, for
example, in Remington's Pharmaceutical Sciences, Gennaro, Mack
Publishing Co., Easton Pa., 1990, which is incorporated herein by
reference.
[0098] Compositions for use within the present invention can be in
the form of sterile, non-pyrogenic liquid solutions or suspensions,
coated capsules, suppositories, lyophilized powders, transdermal
patches or other forms known in the art. Local administration may
be by injection at the site of injury or defect, or by insertion or
attachment of a solid carrier at the site, or by direct, topical
application of a viscous liquid, or the like. For local
administration, the delivery vehicle preferably provides a matrix
for the growing bone or cartilage, and more preferably is a vehicle
that can be absorbed by the cell without adverse effects.
[0099] Aqueous suspensions may contain the extract ingredients of
the present invention in admixture with pharmacologically
acceptable excipients such as vitamin A, vitamin D, and calcium,
suspending agents, such as methyl cellulose; and wetting agents,
such as lecithin, lysolecithin or long-chain fatty alcohols. The
said aqueous suspensions may also contain preservatives, coloring
agents, flavoring agents, sweetening agents and the like in
accordance with industry standards.
[0100] Preferably, compositions of the present invention are orally
administered in the form of a pill, tablet, powder, bar, food,
beverage, lozenge, etc. Additionally, compositions of the present
invention may be presented as a dried or powdered product for
reconstitution with water or other suitable vehicle before use.
Liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, or fractionated vegetable
oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates
or sorbic acid).
[0101] When administered in the form of a beverage, compositions of
the present invention may be water-based, milk-based, tea-based,
fruit juice-based, or some combination thereof.
[0102] Compositions of the present invention may also be orally
administered in the form of a solid prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinized maize starch, polyvinyl pyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The solids may be
coated by methods well-known in the art. In a preferred embodiment,
the composition of the present invention may take the form of a
two-piece hard shell capsule, soft gelatin capsule, or powder to be
dissolved in a liquid for oral consumption. Preparations for oral
administration may be suitably formulated to give controlled
release of the active compound.
[0103] Compositions of the present invention that are orally
administered can further comprise thickeners, including xanthum
gum, carboxymethyl-cellulose, carboxyethyl-cellulose,
hydroxypropyl-cellulose, methyl-cellulose, microcrystalline
cellulose, starches, dextrins, fermented whey, tofu, maltodextrins,
polyols, including sugar alcohols (e.g., sorbitol and mannitol),
carbohydrates (e.g. lactose), propylene glycol alginate, gellan
gum, guar, pectin, tragacanth gum, gum acacia, locust bean gum, gum
arabic, gelatin, as well as mixtures of these thickeners. These
thickeners are typically included in the formulations of the
present invention at levels up to about 0.1%, depending on the
particular thickener involved and the viscosity effects
desired.
[0104] Orally administered compositions of the present invention
can, and typically will, contain an effective amount of one or more
sweeteners, including carbohydrate sweeteners and natural and/or
artificial no/low calorie sweeteners. The amount of the sweetener
used in the formulations of the present invention will vary, but
typically depends on the type of sweetener used and the sweetness
intensity desired.
[0105] In addition to the formulations described previously, the
compounds may also be a formulated as a sustained and/or timed
release formulation. Common timed and/or controlled release
delivery systems include, but are not be restricted to, starches,
osmotic pumps, or gelatin micro capsules.
[0106] The compositions may, if desired, be presented in a pack or
dispenser device which may comprise one or more unit dosage forms
comprising a composition of the present invention. The pack may for
example comprise metal or plastic foil, such as a blister pack. The
pack or dispenser device may be accompanied by instructions for
administration.
[0107] Preparations of compositions of the present invention for
topical and local application comprise aerosol sprays, lotions,
gels and ointments in cosmetically or pharmaceutically appropriate
vehicles which may comprise lower aliphatic alcohols, polyglycols
such as glycerol, polyethylene glycol, esters of fatty acids, oils
and fats, and silicones. The preparations may further comprise
antioxidants, such as ascorbic acid or tocopherol, and
preservatives, such as p-hydroxybenzoic acid esters.
[0108] Parenteral preparations comprise particularly sterile or
sterilized products.
[0109] Injectable compositions may be provided containing a
combination of the extracts of the present invention and any of the
well known injectable carriers. These may contain salts for
regulating the osmotic pressure.
[0110] Other useful dosage forms can be prepared by methods and
techniques that will be well understood by those of skill in the
art and may include the use of additional ingredients in producing
tablets, capsules, or liquid dosage forms. Although exemplary
dosages, dose frequencies, and methods of administration are
discussed herein, these are merely exemplary and it is appreciated
that the dose, dose frequency, and mode of administration may vary
according to the age, body weight, condition and response of the
individual consumer or patient, and the particular composition of
the present invention that is used.
EXAMPLES
Example 1
Expression/Activity of BMP-2 Promoter, Gene, and Protein
[0111] 2T3-BMP2-Luciferase cells, which are murine fibroblast cells
transfected with BMP-2 promoters linked to the reporter gene
luciferase, are cultured using alpha-MEM 10% FCS with 1%
penicillin/streptomycin and 1% glutamine and are split 1/5 once per
week. The cells are plated in microtiter plates at a cell density
of 5.times.10.sup.3 cells/100 .mu.l/well. The cells are allowed to
adhere and stabilize using a preincubation period of 24 hrs at
37.degree. C. with 5% CO.sub.2. The media is removed and replaced
with 50 .mu.l of alpha-MEM 4% FCS. 50 .mu.l of Serum Free (0.1%
BSA) containing the compound or factor (2.times.) to be tested is
added to each well. The final volume is 100 ul and the final serum
concentration is 2% FCS. A routine positive control used is
recombinant human BMP2 ("rhBMP2") or Chinese Hamster Ovary-BMP2
("CHO-BMP2") conditioned media. The treated cells are then
incubated at 37.degree. C., 5% CO.sub.2 for 48 hrs. Media is then
removed and the cells are rinsed 3 times with PBS. Excess PBS is
removed from the wells and 100 .mu.l of cell culture lysing reagent
(Promega # E153A) is added to each well and incubated for at least
10 min. 10 .mu.l of the cell lysate is added to a 96 well white
luminometric plate (Dynatech Labs # 0010107100) containing 100
.mu.l of luciferase assay buffer with substrate (Promega # E152A).
The luciferase activity is read using a Dynatech ML2250 automated
96 well luminometer. The data is then expressed as either
percentage of luciferase activity/well or percentage of luciferase
activity/.mu.g protein.
[0112] The following compounds were tested for ability to activate
the BMP-2 promoter: quercetin, Rehmannia sp. extract, Rehmannia sp.
root extract, Siberian ginseng extract, Sophora japonica extract,
licorice extract, ipriflavone, and cal-z-bone. The results are
reported below, Table 1:
TABLE-US-00001 TABLE 1 BMP2 Promoter assay (ratio to control) Conc.
R-1 R-2 R-3 SFJ SJ SG Q I L CZB .mu.g/ml FOLD* 100 1.00 0.90 0.80
2.00 0.80 1.10 5.20 4.50 1.40 0.90 50 0.90 0.90 0.90 1.60 0.70 0.90
4.80 4.40 1.40 0.90 25 0.90 1.10 0.95 1.00 0.84 1.30 3.60 3.90 1.00
1.10 12.5 0.90 1.30 0.93 1.00 0.77 1.20 2.90 3.60 0.80 1.90 6.3
1.00 1.10 0.88 0.90 0.87 1.30 2.30 2.90 0.80 1.40 3.2 1.10 1.10
0.93 0.90 0.87 1.40 1.80 2.10 0.90 1.10 1.6 1.00 1.20 0.95 1.40
0.88 1.20 1.30 1.50 0.70 1.30 0.8 1.10 1.10 0.98 0.90 0.85 1.20
1.15 1.20 0.80 1.20 0.4 0.90 1.00 0.99 0.90 0.85 1.10 1.00 0.90
0.80 1.20 0.2 1.00 0.90 0.86 0.90 0.79 1.20 1.10 1.00 0.80 1.10 0.1
0.90 0.90 0.87 0.80 0.82 1.10 1.10 0.90 1.20 1.10 0.05 0.80 0.90
1.08 1.20 0.84 1.00 1.00 1.00 0.70 1.10
[0113] In Table 1, R-1=Rehmannia sp. extract (EUL), R-2=Rehmannia
sp. extract (Draco), R-3=Rehmannia sp. root (NuPharma), SFJ=Sophora
fructus japonica, SJ=Sophora japonica (NuPharma), SG=Siberian
ginseng, Q=Quercetin, I=Ipriflavone, L=Licorice, CZB=Cal-Z-bone.
*Note: If the fold value is 2 or greater, the treatment
significantly activated BMP-2 promoter. If the fold value is less
than 2, the treatment had no effect on BMP-2 promoter. Thus,
according to this assay, quercetin dihydrate and ipriflavone were
the most potent activators of the BMP-2 promoter.
[0114] It is also possible to test for BMP-2 gene expression and
protein expression using assays similar to those described above
for testing BMP-2 promoter activity. In particular, for gene
expression, the human osteosarcoma cell line, MG-63 (ATCC#
CRL-1427), is maintained in phenol-red containing MEM (as
recommended by ATCC) at 37.degree. C. and 5% CO.sub.2. Twenty-four
hours prior to experimentation, 3.times.10.sup.5 cells are seeded
in 12-well plates in phenol-red free MEM. For each experiment,
cells are treated with test extracts at treatment concentrations of
10, 1, and 0.1 .mu.g/ml. After an overnight incubation, RNA is
extracted using conventional trizol/guanidine isothiocyanate based
lysis. The isolated RNA was digested with RNase-free DNase I to
remove any DNA contamination and then reverse transcribed to cDNA
using random hexamer as well as oligo(dT) primers according to the
manufacturer's instructions (Stratagene). Quantitative real time
PCR is performed using FAM-labeled specific primers for BMP-2 and
HEX-labeled specific primers for 18S rRNA (Invitrogen). All
reactions are carried out in triplicate and the relative amount of
mRNAs in treated versus untreated samples is calculated using the
comparative C.sub.T method established by Applied Biosystems
(2001). Gene expression changes of 2-fold or greater are considered
significant.
[0115] To measure BMP-2 protein expression, Hu09 cells are plated
in 96-well culture plates at the density of 1.times.10.sup.4
cells/well and cultured with alpha-MEM supplemented with 10% FCS
for 24 hours. The cells are treated with different proteasome
inhibitors for 24 hours. After incubation, the conditioned media is
transferred into microcentrifuge tubes and centrifuged at 14,000
rpm for 2-3 minutes to remove cellular debris. The concentration of
BMP-2 protein in the supernatant is then determined using the
standard BMP-2 Elisa Kit (R&D ELISA KIT DBP200). Recombinant
hBMP-2 (R&D) is used as a standard.
[0116] The following compounds were tested for their ability to
increase the expression of BMP-2 gene expression and protein
expression: quercetin, Rehmannia sp. extract, Rehmannia sp. root
extract, Siberian ginseng extract, Sophora japonica extract,
licorice extract, ipriflavone, and cal-z-bone. The results and
concentrations tested in the protein expression assay are reported
below in Table 2.
TABLE-US-00002 TABLE 2 BMP-2 Protein Expression Assay Conc. R-1 R-2
R-3 SFJ SJ SG Q I L CZB .mu.g/ml FOLD* 100 <1.3 <1.3 <1.3
3.0 <1.3 2.4 2.2 1.8 <1.3 <1.3 10 <1.3 <1.3 <1.3
3.0 <1.3 3.4 4.6 1.8 <1.3 <1.3 1 <1.3 <1.3 <1.3
2.2 <1.3 2.0 2.0 2.4 <1.3 <1.3 0 1.0 1.0 1.0 1.0 1.0 1.0
1.0 1.0 1.0 1.0
[0117] In Table 2, R-1=Rehmannia sp. extract (EUL), R-2=Rehmannia
sp. extract (Draco), R-3=Rehmannia sp. root (NuPharma), SFJ=Sophora
fructus japonica, SJ=Sophora japonica (NuPharma), SG=Siberian
ginseng, Q=Quercetin, I=Ipriflavone, L=Licorice, and
CZB=Cal-Z-bone. *Note: If the fold value is 2 or greater, the
treatment significantly activated BMP-2 protein expression. If the
fold value is less than 2, the treatment had no effect on BMP-2
protein expression.
[0118] The results and concentrations tested in the gene expression
assay are listed below in Table 3. An increase in gene expression
that is a change of 2-fold or greater is considered
significant.
TABLE-US-00003 TABLE 3 BMP-2 Gene Expression Assay, 10 .mu.g/ml
Test Ingredient: Fold Change in Gene Expression: Rehmannia sp.
extract (EUL) 28 .times. increase Rehmannia sp. extract (Draco)
11.7 .times. increase Rehmannia sp. root (NuPharma) 6.3 .times.
increase Sophora fructus japonica 49 .times. increase Sophora
japonica (NuPharma) 8.3 .times. increase Siberian ginseng 27
.times. increase Quercetin 2.6 .times. increase Ipriflavone No
effect Licorice 45 .times. increase
[0119] Additional results of the gene expression assay are listed
below in Table 4. In Table 4, Q=quercetin, SG=Siberian ginseng,
SJ=Sophora japonica, and L=licorice. An increase in gene expression
that is 2-fold or greater is considered significant. Synergy was
found at a 10:5 or 2:1 and 10:10 or 1:1 ratio of quercetin to
licorice over quercetin alone.
TABLE-US-00004 TABLE 4 BMP-2 Gene Expression Assay BMP-2 Expression
Ingredient (dosage): N (fold change) Q (100 .mu.g/ml) 1 14 Q (20
.mu.g/ml) 2 4.61 .+-. 0.07 Q (10 .mu.g/ml) 1 5 Q (10 .mu.g/ml) 2
3.48 .+-. 0.08 Q (10 .mu.g/ml) 2 3.54 .+-. 0.12 Q (10 .mu.g/ml) 2
3.52 .+-. 0.12 Q (5 .mu.g/ml) 2 2.51 .+-. 0.14 Q (1 .mu.g/ml) 1 2 Q
(1 .mu.g/ml) 1 2.03 Q (0.1 .mu.g/ml) 1 No change (-1.06) SG (10
.mu.g/ml) 2 11.26 .+-. 0.14 SG (10 .mu.g/ml) 2 12.38 .+-. 1.00 SG
(1 .mu.g/ml) 2 5.61 .+-. 1.45 SG (0.1 .mu.g/ml) 2 2.58 .+-. 0.38 SJ
(10 .mu.g/ml) 2 29.10 .+-. 2.71 SJ (10 .mu.g/ml) 2 32.45 .+-. 0.32
SJ (10 .mu.g/ml) 2 32.80 .+-. 1.12 SJ (5 .mu.g/ml) 2 23.43 .+-.
0.23 SJ (2.5 .mu.g/ml) 2 14.89 .+-. 0.95 SJ (1 .mu.g/ml) 2 11.64
.+-. 2.04 SJ (0.1 .mu.g/ml) 2 3.72 .+-. 0.37 L (100 .mu.g/ml) 1 29
L (10 .mu.g/ml) 1 8 L (1 .mu.g/ml) 1 4 Q + SG (10 .mu.g/ml each) 2
No change (0.07 .+-. 1.82) Q + SG (10 .mu.g/ml each) 1 No change
(1.36) Q + SJ (10 .mu.g/ml each) 2 12.01 .+-. 0.76 Q (10 .mu.g/ml)
+ SJ (1 .mu.g/ml) 2 7.09 .+-. 0.75 Q (10 .mu.g/ml) + SJ (0.1
.mu.g/ml) 2 2.63 .+-. 0.15 Q (1 .mu.g/ml) + SJ (10 .mu.g/ml) 2 No
change (1.30 .+-. 0.17) Q (5 .mu.g/ml) + SJ (2.5 .mu.g/ml) 2 4.07
.+-. 0.02 Q (10 .mu.g/ml) + SJ (5 .mu.g/ml) 2 13.86 .+-. 1.22 Q (20
.mu.g/ml) + SJ (10 .mu.g/ml) 2 16.86 .+-. 0.41 SG (10 .mu.g/ml) +
SJ (10 .mu.g/ml) 2 1.97 .+-. 0.62 SG (10 .mu.g/ml) + SJ (1
.mu.g/ml) 2 3.36 .+-. 0.04 SG (10 .mu.g/ml) + SJ (0.1 .mu.g/ml) 2
4.57 .+-. 0.87 Q (100 .mu.g/ml) + L (100 .mu.g/ml) 1 38 Q (100
.mu.g/ml) + L (10 .mu.g/ml) 1 252 Q (100 .mu.g/ml) + L (1 .mu.g/ml)
1 35 Q (10 .mu.g/ml) + L (100 .mu.g/ml) 1 92 Q (10 .mu.g/ml) + L
(10 .mu.g/ml) 1 71 Q (10 .mu.g/ml) + L (1 .mu.g/ml) 1 15 Q (1
.mu.g/ml) + L (100 .mu.g/ml) 1 31 Q (1 .mu.g/ml) + L (10 .mu.g/ml)
1 37 Q (1 .mu.g/ml) + L (1 .mu.g/ml) 1 6 Q + SG + SJ (10 .mu.g/ml
each) 2 No change (1.39 .+-. 0.26) Q (0 .mu.g/ml) 3 1.01 .+-. 0.05
Q (10 .mu.g/ml) 3 7.22 .+-. 0.51 L(0 .mu.g/ml) 3 1.01 .+-. 0.05 L(1
.mu.g/ml) 3 1.34 .+-. 0.12 L(5 .mu.g/ml) 3 0.81 .+-. 0.03 L(10
.mu.g/ml) 3 1.04 .+-. 0.04 Q (10 .mu.g/ml) + L (1 .mu.g/ml) 3 8.54
.+-. 2.19 Q (10 .mu.g/ml) + L (5 .mu.g/ml) 3 13.50 .+-. 4.49 Q (10
.mu.g/ml) + L (10 .mu.g/ml) 3 14.77 .+-. 3.04
Example 20
Inhibition of RANK-L Study
[0120] Weigh between 150 to 250 mg of each ingredient into a 15 ml
conial bottomed tube. Dissolve in a solution of 50% DMSO:30%
Ethanol:20% water, such as to end with a final stock solution of 50
.mu.g/ml. For solvent control, mix 1.5 ml DMSO, 0.9 ml ethanol, and
0.6 ml water. Vortex thoroughly. Sonicate for 10 minute in water
bath, room temperature. Vortex thoroughly again. Dilute ingredient
stocks in fresh phenol red-free media.
[0121] Human osteosarcoma cell line, MG-63 (ATCC# CRL-1427), is
maintained in phenol-red containing MEM (as recommended by ATCC) at
37.degree. C. and 5% CO.sub.2. Twenty-four hours prior to
experimentation, 3.times.10.sup.5 cells are seeded in 12-well
plates in phenol-red free MEM. Spent medium is removed from wells
and MG-63 cells (ATCC # CRL-1427) are pretreated with 1, 0.1, 0.01
.mu.g/ml of each test ingredient or 30 ng/ml TGF-beta (+ control)
for 4 hours at 37.degree. C., 5% CO.sub.2. Stock solution of
TGF-beta is 30 .mu.g/ml (from R&D Systems, cat# 100-B-001).
[0122] After pretreatment incubation period, 10 .mu.g/ml IL-1b
(Calbiochem (catalog # 407615), and stock solution at 10 .mu.g/ml,
cat # 407615) were added to the cell cultures for 18 hours at
37.degree. C. Supernatant media from the treated and stimulated
cells were removed and total RANK-L proteins measured using RANK-L
ELISA (in triplicate) as described by manufacturer (from APOTECH,
catalog # APO-54N-016-k101). The resulting protein quantity was
compared to that of media (null) treated stimulated cells to
determine percent decrease in total RANK-L protein.
[0123] The above-described assay procedure was used to determine
the ability of extracts of: Ginkgo biloba, green tea, Sophora
fructus japonica, Rehmannia sp., pomegranate, Siberian ginseng,
ipriflavone, grape seed, Dong quai, and Sophora japonica, to
inhibit RANK-L expression, production, or release. The results of
this assay are reported below in Table 5. Decreases of 10% or
greater are considered significant.
TABLE-US-00005 TABLE 5 Production/Release of RANK-L Protein
(relative to untreated control) % Change in Production/Release
Levels (compared to untreated Ingredient (tested at 1 .mu.g/ml):
control) Ginkgo biloba 31% decrease Green tea 19% decrease Sophora
Fructus Japonica 45% decrease Rehmannia sp. 74% decrease
Pomegranate 20% decrease Pomegranate (Naturex) 14% decrease
Siberian ginseng 50% decrease Ipriflavone No effect Grape Seed
Extract 11% decrease Dong Quai (20:1 extraction ratio) 16% decrease
Sophora japonica (NuPharma) 42% decrease
[0124] Based on the results reported at Table 5, it was determined
that pomegranate extract, ipriflavone (Ostivone), grape seed
extract (40% proanthocyanidins) and green tea extract (40% EGCG)
showed the positive effect on inhibition of RANK-L
production/release. Therefore, various combinations of these
ingredients were tested, using the RANK-L inhibition assay
described above, to determine what level of inhibition of RANK-L
production/release could be achieved. The results are reported
below in Table 6. As these reports demonstrate, the combination of
10 .mu.g/ml of Pomegranate, 10 .mu.g/ml Ipriflavone, 1 .mu.g/ml
Grape Seed extract, and 1 .mu.g/ml Green tea was found to be the
formula that maximized the inhibition of RANK-L synthesis in
response to IL-1B protein stimulus of osteoblast cells. The results
also show that anything with pomegranate generally performed well.
Further, no major interference was found among the ingredients.
TABLE-US-00006 TABLE 6 RANK-L Inhibition RANK-L Ingredient
(dosage): n inhibition (%) Organic Olive Juice Powder (100
.mu.g/ml) 2 41.5 .+-. 4.0 Organic Olive Juice Powder (10 .mu.g/ml)
6 19.7 .+-. 2.2 Organic Olive Juice Powder (1 .mu.g/ml) 6 No
inhibition Organic Olive Juice Powder (0.1 .mu.g/ml) 3 No
inhibition Pomegranate extract (30 .mu.g/ml) 2 58.6 .+-. 1.5
Pomegranate extract (20 .mu.g/ml) 2 56.2 .+-. 2.6 Pomegranate
extract (10 .mu.g/ml) 3 48.3 .+-. 3.1 Pomegranate extract (10
.mu.g/ml) 2 46.9 .+-. 10.7 Pomegranate extract (10 .mu.g/ml) 2 48.4
.+-. 1.1 Pomegranate extract (10 .mu.g/ml) 3 49.4 .+-. 3.3
Pomegranate extract (1 .mu.g/ml) 3 19.3 .+-. 2.1 Pomegranate
extract (0.1 .mu.g/ml) 3 4.7 .+-. 4.2 Ipriflavone (Ostivone) (10
.mu.g/ml) 3 0 .+-. 4.3 Ipriflavone (Ostivone) (10 .mu.g/ml) 3 2.8
.+-. 3.9 Ipriflavone (Ostivone) (10 .mu.g/ml) 3 0 .+-. 2.5
Ipriflavone (Ostivone) (1 .mu.g/ml) 2 0.8 .+-. 0.3 Grape Seed
Extract (40% OPC) (10 .mu.g/ml) 3 18.7 .+-. 6.9 Grape Seed Extract
(40% OPC) (10 .mu.g/ml) 2 21.9 .+-. 0.6 Grape Seed Extract (40%
OPC) (5 .mu.g/ml) 3 13.1 .+-. 3.4 Grape Seed Extract (40% OPC) (2
.mu.g/ml) 2 10.3 .+-. 1.6 Grape Seed Extract (40% OPC) (1 .mu.g/ml)
3 8.0 .+-. 2.2 Grape Seed Extract (40% OPC) (1 .mu.g/ml) 2 10.7
.+-. 1.0 Grape Seed Extract (40% OPC) (0.1 .mu.g/ml) 2 0 .+-. 4.3
Green Tea Extract (40% EGCG) (10 .mu.g/ml) 3 23.9 .+-. 1.4 Green
Tea Extract (40% EGCG) (10 .mu.g/ml) 3 22.6 .+-. 7.5 Green Tea
Extract (40% EGCG) (1 .mu.g/ml) 3 10.2 .+-. 1.6 Green Tea Extract
(40% EGCG) (0.1 .mu.g/ml) 3 0 .+-. 2.6 Pomegranate + Ipriflavone
(Ostivone) (10 .mu.g/ml each) 2 45.7 .+-. 2.4 Pomegranate +
Ipriflavone (Ostivone) (10 .mu.g/ml each) 2 48.0 .+-. 2.8
Pomegranate + Ipriflavone (Ostivone) (10 .mu.g/ml each) 3 46.9 .+-.
5.3 Pomegranate (10 .mu.g/ml) + Ipriflavone (Ostivone) (1 .mu.g/ml)
2 48.3 .+-. 3.5 Pomegranate (10 .mu.g/ml) + Ipriflavone (Ostivone)
(0.1 .mu.g/ml) 2 45.6 .+-. 4.0 Pomegranate (1 .mu.g/ml) +
Ipriflavone (Ostivone) (10 .mu.g/ml) 3 21.6 .+-. 5.2 Pomegranate
(10 .mu.g/ml) + Ipriflavone (Ostivone) (10 .mu.g/ml) + 2 63.3 .+-.
0.7 Grape Seed Extract (10 .mu.g/ml) Pomegranate (10 .mu.g/ml) +
Ipriflavone (Ostivone) (10 .mu.g/ml) + 2 64.6 .+-. 4.9 Grape Seed
Extract (1 .mu.g/ml) Pomegranate (10 .mu.g/ml) + Ipriflavone
(Ostivone) (10 .mu.g/ml) + 2 49.9 .+-. 2.5 Grape Seed Extract (0.1
.mu.g/ml) Pomegranate + Ipriflavone (Ostivone) + Grape Seed Extract
2 42.8 .+-. 8.9 (10 .mu.g/ml each) Pomegranate (10 .mu.g/ml) +
Ipriflavone (Ostivone) (10 .mu.g/ml) + 3 39.0 .+-. 2.9 Green Tea
Extract (10 .mu.g/ml) Pomegranate (10 .mu.g/ml) + Ipriflavone
(Ostivone) (10 .mu.g/ml) + 2 54.6 .+-. 1.1 Green Tea Extract (1
.mu.g/ml) Pomegranate (10 .mu.g/ml) + Ipriflavone (Ostivone) (10
.mu.g/ml) + 2 37.5 .+-. 8.1 Green Tea Extract (0.1 .mu.g/ml)
Pomegranate + Ipriflavone (Ostivone) + Green Tea Extract (10 3 23.5
.+-. 2.3 .mu.g/ml each) Pomegranate (10 .mu.g/ml) + Ipriflavone
(Ostivone) (10 .mu.g/ml) + 3 42.9 .+-. 1.1 Grape Seed Extract (10
.mu.g/ml) + Green Tea Extract (10 .mu.g/ml) Pomegranate (10
.mu.g/ml) + Ipriflavone (Ostivone) (10 .mu.g/ml) + 2 60.8 .+-. 0.9
Grape Seed Extract (10 .mu.g/ml) + Green Tea Extract (0.1 .mu.g/ml)
Pomegranate (10 .mu.g/ml) + Ipriflavone (Ostivone) (10 .mu.g/ml) +
3 55.1 .+-. 1.8 Grape Seed Extract (1 .mu.g/ml) + Green Tea Extract
(0.1 .mu.g/ml) Pomegranate (10 .mu.g/ml) + Ipriflavone (Ostivone)
(10 .mu.g/ml) + 3 68.6 .+-. 2.6 Grape Seed Extract (1 .mu.g/ml ) +
Green Tea Extract (1 .mu.g/ml) Pomegranate (10 .mu.g/ml) +
Ipriflavone (Ostivone) (10 .mu.g/ml) + 3 65.2 .+-. 1.1 Grape Seed
Extract (1 .mu.g/ml) + Green Tea Extract (10 .mu.g/ml) Pomegranate
(10 .mu.g/ml) + Ipriflavone (Ostivone) (10 .mu.g/ml) + 3 52.3 .+-.
0.7 Grape Seed Extract (0.1) + Green Tea Extract (1 .mu.g/ml)
Pomegranate + Ipriflavone (Ostivone) + Grape Seed Extract + 3 38.0
.+-. 3.5 Green Tea Extract (10 .mu.g/ml each) Ipriflavone
(Ostivone) + Grape Seed Extract + Green Tea 3 9.7 .+-. 2.8 Extract
(10 .mu.g/ml each) Pomegranate + Grape Seed Extract + Green Tea
Extract (10 3 26.9 .+-. 4.8 .mu.g/ml each) Pomegranate (10
.mu.g/ml) + Grape Seed Extract (1 .mu.g/ml) 2 52.7 .+-. 1.0
Pomegranate (10 .mu.g/ml) + Grape Seed Extract (1 .mu.g/ml) 2 51.1
.+-. 1.8 Pomegranate (20 .mu.g/ml) + Grape Seed Extract (2
.mu.g/ml) 2 58.2 .+-. 0.5 Pomegranate (30 .mu.g/ml) + Grape Seed
Extract (1 .mu.g/ml) 2 64.3 .+-. 2.1 Pomegranate (30 .mu.g/ml) +
Grape Seed Extract (2 .mu.g/ml) 2 59.9 .+-. 1.2 Pomegranate (30
.mu.g/ml) + Grape Seed Extract (5 .mu.g/ml) 2 57.2 .+-. 3.1
Example 3
Isolation of Punicalagins from Pomegranate
[0125] Fresh pomegranates were peeled to separate the seeds from
the peels. The seeds, peels, and fruit flesh were separately
extracted using a water and alcohol combination (80:20). Each of
the peels, seeds, and flesh yielded extracts with punicalagins but
the pomegranate peel extract yielded the highest levels of
punicalagins. In addition, water was shown to be the best
extraction solvent for extracting punicalagins.
Example 4
Inhibition of RANK-L by Punicalagins
[0126] Punicalagin test samples are extracted from pomegranates,
including from pomegranate peels, skins, fleshy fruit, and seeds
using 100% DMSO at 100 mg/ml. Particulate matter in the extraction
is not removed. The punicalagin test samples are diluted in MEM
without phenol red (estrogenic) and 0.1% FBS to lower background
signaling to ten times the final concentrations. Final DMSO
concentrations are kept below 0.2% and are kept constant during
treatments. DMSO solvent control is used in the untreated
group.
[0127] MG63 cells (ATCC # CRL-1472), a human-derived osteosarcoma
cell line, are plated at 200,000 cells per well in a 12 well plate
with phenol red free MEM at 10% FBS. The next day the media is
changed to phenol red free MEM at 0.1% FBS. The cells are incubated
for four hours and then treated with punicalagin test samples at
concentrations of 1, 10, and 100 .mu.g/ml.
[0128] After four hours of treatment, IL-1b (Calbiochem (catalog #
407615), stock solution at 10 .mu.g/ml, cat # 407615) are added to
a final concentration of 3 ng/mL. The treated cells are then
allowed to incubate for 16 hours at 37.degree. C.
[0129] After 16 hours the cells are lysed and the RNA is extracted
from the cells with the RNasy purification kit (Qiagen). RNA is
reverse transcribed to cDNA and quantified by qPCR using 2-step
qRT-PCR reagents (Invitrogen), and 4 .mu.L of purified RNA.
Annealing temperature is 57.degree. C. in the Stratagene Mx4000
with 1 .mu.L of RANK-L gene specific primers in a 50 .mu.L reaction
(10 .mu.M initial concentration; HLUX3013920, Invitrogen Inc.).
[0130] RANK-L and GAPDH gene expression Ct data are obtained. RNA
is quantified with adjustments for GAPDH expression. The untreated
control values for RANK-L expression are used to evaluate treatment
affects. The results are reported below in Table 7.
TABLE-US-00007 TABLE 7 Effect of Punicalagins on RANK-L Expression
Ingredient N RANK-L Expression (fold increase) Untreated 2 1.0 .+-.
0.1 IL1B (3 ng/ml) 2 3.3 .+-. 0 Punicalagins 1 .mu.g/ml 2 2.6 .+-.
0.5 Punicalagins 10 .mu.g/ml 2 1.9 .+-. 0.4 Punicalagins 100
.mu.g/ml 2 0.7 .+-. .02
[0131] As shown in Table 7 punicalagins purified from pomegranates
inhibit IL-1b stimulated RANK-L gene expression in a dose dependent
manner and completely inhibit RANK-L expression at 100
.mu.g/mL.
Example 5
Inhibition of Type IV Collagenase (MMP9) Protein Expression by
Punicalagins from Pomegranate
[0132] Keratinocytes and fibroblasts were co-cultured in DMEM
containing 0.5% BSA. Co-cultures were exposed to various
concentrations of punicalagins (ranging from 0.1%-10%) extracted
from pomegranates. Specifically, the ability of the pomegranate
extracts to inhibit type IV collagenase protein (matrix
metalloproteinase-9/MMP9) expression at concentrations of 1.0
.mu.g/ml, 10 .mu.g/ml, and 100 .mu.g/ml were tested. Following
exposure to the pomegranate extracts, the co-culture cells were
stimulated with 10 ng/ml of IL-1B for 18 hours. Following
stimulation with IL-1B for 18 hours, and MMP9 concentration was
determined in the media as shown below in Table 8.
TABLE-US-00008 TABLE 8 Effect of Pomegranate Extracts on MMP9 (type
IV collagenase) protein Expression Tested Pomegranate Extract N
Inhibition of MMP9 Untreated 2 1.0 .+-. 0.1 IL1B (10 ng/ml) 2 1.65
Punicalagins 1 .mu.g/ml 4 1.5 + .06 Punicalagins 10 .mu.g/ml 4 1.2
.+-. .04 Punicalagins 100 .mu.g/ml 4 1.0 .+-. 0.1
[0133] The results reported at Table 8 demonstrate that
punicalagins from pomegranate inhibit IL-1B stimulated collagenase
release (MMP9) from keratinocytes in vitro. These results
demonstrate that punicalagins inhibit inflammation-stimulated
breakdown of the extracellular matrix. Activated osteoclasts reduce
bone strength and increase bone loss by secreting matrix digesting
enzymes (MMPs) to break down the bone's collagen/calcium phosphate
framework. Blocking the destruction of bone's collagen/calcium
framework will be expected to maintain/improve bone strength and
bone structure. Increased bone strength and bone structure are
characterized by increased bone mineral density, increased bone
volume, increased trabecular cell number, decreased trabecular
separation, improved bone architecture, increase in maximum force
needed to fracture bone, and increase in stiffness of bone.
Example 6
Inhibition of C. histolyticum Collagenase Activity by Grape Seed
and Pomegranate Extracts
[0134] Samples are prepared by weighing out 100 mg of powder. A 50
mg/ml total extract of the sample is then prepared by sequential
addition of DMSO:Ethanol:water in a ratio of 5:3:2. Therefore for
100 mg of powder, 1 ml DMSO, 0.6 ml ethanol, and 0.4 ml water would
be used. The solutions are extensively mixed by vortexing and are
then incubated for 10 min in a sonic water bath. The samples are
diluted from the stock concentration of 50 mg/ml to test
concentrations.
[0135] Inhibition of collagenase activity is assayed using a
commercially available kit (Molecular Probes, Eugene, Oreg.). The
kit is based on an ability to digest a collagen substrate labeled
with a fluorescent tag. Prior to digestion, the fluorescence of the
substrate is quenched. After exposure to collagenase, the substrate
is cleaved abolishing the quenching effect so that the fluorescence
increases. The samples (prepared according to above procedure) are
first added to the collagenase (0.2 Units/ml) provided with the
kits. The fluorescent substrate (50 .mu.g/ml) is then added and the
reaction is incubated for an hour at ambient temperature.
Fluorescence is read on a plate reader at excitation/emission of
495/515 nm. Data are expressed as % control compared to MMP without
any inhibitor added. A decrease from 100% total enzyme activity is
considered a positive response.
[0136] A dose dependent response towards reduction of C.
histolyticum collagenase activity was observed for both the
pomegranate extract and the grape seed extract (Table 9).
[0137] Activated osteoclasts reduce bone strength and increase bone
loss by secreting matrix digesting enzymes (MMP's) to break down
the bone's collagen/calcium phosphate framework. By the mechanism
suggested here, the grape seed and pomegranate extracts in
particular are found to be potent inhibitors of C. histolyticum
collagenase activity. By this activity a net positive balance of
collagen production may be achieved resulting in the maintenance or
improvement of bone strength and bone structure.
TABLE-US-00009 TABLE 9 Inhibition of C. histolyticum collagenase
activity by Grape Seed and Pomegranate Extracts % Collagenase
Sample Concentration (.mu.g/ml) Activity Pomegranate extract 1 96.7
.+-. 10.0% 10 86.3 .+-. 5.9% 100 29.2 .+-. 5.7% Grape seed extract
1 98.9 .+-. 3.0% 10 48.8 .+-. 2.3% 100 -7.2 .+-. 4.3% Collagenase
only 0 100.0 .+-. 0.8%
[0138] It is therefore intended that the foregoing detailed
description be regarded as illustrative rather than limiting, and
that it be understood that it is the following claims, including
all equivalents, that are intended to define the spirit and scope
of this invention.
* * * * *