Title:
Anti-resorptive and bone building dietary supplements and methods of use
Kind Code:
A1


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, US)
Murray, Mary A. (Irvine, CA, US)
Lin, Yumei (Long Beach, CA, US)
Gellenbeck, Kevin W. (Poway, CA, US)
Da Costa, Silvia R. (Redondo Beach, CA, US)
Wilkins, Leon M. (North Andover, MA, US)
Roh-schmidt, Haeri (Stockton, CA, US)
Rana, Jatinder (Grand Rapids, MI, US)
Rebhun, John F. (Greenville, MI, US)
Fast, David J. (Grand Rapids, MI, US)
Krempin, Laurie (Temecula, CA, US)
Application Number:
11/977395
Publication Date:
08/21/2008
Filing Date:
10/24/2007
Primary Class:
Other Classes:
424/766, 435/375, 514/456, 424/725
International Classes:
A61K36/82; A23L33/00; A61K31/352; A61K36/00; A61K36/87; C12N5/02
View Patent Images:
Related US Applications:



Primary Examiner:
CHEN, CATHERYNE
Attorney, Agent or Firm:
BGL/Alticor (CHICAGO, IL, US)
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:

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

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.

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.

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.

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 κ 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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® is a synthetic ipriflavone compound available from Technical Sourcing International, Inc. (Missoula, Mont.).

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:

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.

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.

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.

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.

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.

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.

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.

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).

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.

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: dāngguī) 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.

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.

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.

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.

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.

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.

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.

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.

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° 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.

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 CO2-based fluids.” J. Agric Food Chem., 2004 May 5;52(9):2440-4.

Compositions of the Invention

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.

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.

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.

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.

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.

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 ½ 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Modes of Administration

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Parenteral preparations comprise particularly sterile or sterilized products.

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.

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

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 5×103 cells/100 μl/well. The cells are allowed to adhere and stabilize using a preincubation period of 24 hrs at 37° C. with 5% CO2. The media is removed and replaced with 50 μl of alpha-MEM 4% FCS. 50 μl of Serum Free (0.1% BSA) containing the compound or factor (2×) 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° C., 5% CO2 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 μl of cell culture lysing reagent (Promega # E153A) is added to each well and incubated for at least 10 min. 10 μl of the cell lysate is added to a 96 well white luminometric plate (Dynatech Labs # 0010107100) containing 100 μl 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/μg protein.

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 1
BMP2 Promoter assay (ratio to control)
Conc.R-1R-2R-3SFJSJSGQILCZB
μg/mlFOLD*
1001.000.900.802.000.801.105.204.501.400.90
500.900.900.901.600.700.904.804.401.400.90
250.901.100.951.000.841.303.603.901.001.10
12.50.901.300.931.000.771.202.903.600.801.90
6.31.001.100.880.900.871.302.302.900.801.40
3.21.101.100.930.900.871.401.802.100.901.10
1.61.001.200.951.400.881.201.301.500.701.30
0.81.101.100.980.900.851.201.151.200.801.20
0.40.901.000.990.900.851.101.000.900.801.20
0.21.000.900.860.900.791.201.101.000.801.10
0.10.900.900.870.800.821.101.100.901.201.10
0.050.800.901.081.200.841.001.001.000.701.10

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.

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° C. and 5% CO2. Twenty-four hours prior to experimentation, 3×105 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 μg/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 CT method established by Applied Biosystems (2001). Gene expression changes of 2-fold or greater are considered significant.

To measure BMP-2 protein expression, Hu09 cells are plated in 96-well culture plates at the density of 1×104 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.

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 2
BMP-2 Protein Expression Assay
Conc.R-1R-2R-3SFJSJSGQILCZB
μg/mlFOLD*
100<1.3<1.3<1.33.0<1.32.42.21.8<1.3<1.3
10<1.3<1.3<1.33.0<1.33.44.61.8<1.3<1.3
1<1.3<1.3<1.32.2<1.32.02.02.4<1.3<1.3
01.01.01.01.01.01.01.01.01.01.0

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.

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 3
BMP-2 Gene Expression Assay, 10 μg/ml
Test Ingredient:Fold Change in Gene Expression:
Rehmannia sp. extract (EUL)  28 × increase
Rehmannia sp. extract (Draco)11.7 × increase
Rehmannia sp. root (NuPharma) 6.3 × increase
Sophora fructus japonica  49 × increase
Sophora japonica (NuPharma) 8.3 × increase
Siberian ginseng  27 × increase
Quercetin 2.6 × increase
IpriflavoneNo effect
Licorice  45 × increase

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 4
BMP-2 Gene Expression Assay
BMP-2 Expression
Ingredient (dosage):N(fold change)
Q (100 μg/ml)114
Q (20 μg/ml)24.61 ± 0.07
Q (10 μg/ml)15
Q (10 μg/ml)23.48 ± 0.08
Q (10 μg/ml)23.54 ± 0.12
Q (10 μg/ml)23.52 ± 0.12
Q (5 μg/ml)22.51 ± 0.14
Q (1 μg/ml)12
Q (1 μg/ml)12.03
Q (0.1 μg/ml)1No change (−1.06)
SG (10 μg/ml)211.26 ± 0.14 
SG (10 μg/ml)212.38 ± 1.00 
SG (1 μg/ml)25.61 ± 1.45
SG (0.1 μg/ml)22.58 ± 0.38
SJ (10 μg/ml)229.10 ± 2.71 
SJ (10 μg/ml)232.45 ± 0.32 
SJ (10 μg/ml)232.80 ± 1.12 
SJ (5 μg/ml)223.43 ± 0.23 
SJ (2.5 μg/ml)214.89 ± 0.95 
SJ (1 μg/ml)211.64 ± 2.04 
SJ (0.1 μg/ml)23.72 ± 0.37
L (100 μg/ml)129
L (10 μg/ml)18
L (1 μg/ml)14
Q + SG (10 μg/ml each)2No change
(0.07 ± 1.82)
Q + SG (10 μg/ml each)1No change
(1.36)
Q + SJ (10 μg/ml each)212.01 ± 0.76 
Q (10 μg/ml) + SJ (1 μg/ml)27.09 ± 0.75
Q (10 μg/ml) + SJ (0.1 μg/ml)22.63 ± 0.15
Q (1 μg/ml) + SJ (10 μg/ml)2No change
(1.30 ± 0.17)
Q (5 μg/ml) + SJ (2.5 μg/ml)24.07 ± 0.02
Q (10 μg/ml) + SJ (5 μg/ml)213.86 ± 1.22 
Q (20 μg/ml) + SJ (10 μg/ml)216.86 ± 0.41 
SG (10 μg/ml) + SJ (10 μg/ml)21.97 ± 0.62
SG (10 μg/ml) + SJ (1 μg/ml)23.36 ± 0.04
SG (10 μg/ml) + SJ (0.1 μg/ml)24.57 ± 0.87
Q (100 μg/ml) + L (100 μg/ml)138
Q (100 μg/ml) + L (10 μg/ml)1252
Q (100 μg/ml) + L (1 μg/ml)135
Q (10 μg/ml) + L (100 μg/ml)192
Q (10 μg/ml) + L (10 μg/ml)171
Q (10 μg/ml) + L (1 μg/ml)115
Q (1 μg/ml) + L (100 μg/ml)131
Q (1 μg/ml) + L (10 μg/ml)137
Q (1 μg/ml) + L (1 μg/ml)16
Q + SG + SJ (10 μg/ml each)2No change
(1.39 ± 0.26)
Q (0 μg/ml)31.01 ± 0.05
Q (10 μg/ml)37.22 ± 0.51
L(0 μg/ml)31.01 ± 0.05
L(1 μg/ml)31.34 ± 0.12
L(5 μg/ml)30.81 ± 0.03
L(10 μg/ml)31.04 ± 0.04
Q (10 μg/ml) + L (1 μg/ml)38.54 ± 2.19
Q (10 μg/ml) + L (5 μg/ml)313.50 ± 4.49 
Q (10 μg/ml) + L (10 μg/ml)314.77 ± 3.04 

Example 20

Inhibition of RANK-L Study

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 μg/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.

Human osteosarcoma cell line, MG-63 (ATCC# CRL-1427), is maintained in phenol-red containing MEM (as recommended by ATCC) at 37° C. and 5% CO2. Twenty-four hours prior to experimentation, 3×105 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 μg/ml of each test ingredient or 30 ng/ml TGF-beta (+ control) for 4 hours at 37° C., 5% CO2. Stock solution of TGF-beta is 30 μg/ml (from R&D Systems, cat# 100-B-001).

After pretreatment incubation period, 10 μg/ml IL-1b (Calbiochem (catalog # 407615), and stock solution at 10 μg/ml, cat # 407615) were added to the cell cultures for 18 hours at 37° 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.

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 5
Production/Release of RANK-L Protein
(relative to untreated control)
% Change in Production/Release
Levels (compared to untreated
Ingredient (tested at 1 μg/ml):control)
Ginkgo biloba31% decrease
Green tea19% decrease
Sophora Fructus Japonica45% decrease
Rehmannia sp.74% decrease
Pomegranate20% decrease
Pomegranate (Naturex)14% decrease
Siberian ginseng50% decrease
IpriflavoneNo effect
Grape Seed Extract11% decrease
Dong Quai (20:1 extraction ratio)16% decrease
Sophora japonica (NuPharma)42% decrease

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 μg/ml of Pomegranate, 10 μg/ml Ipriflavone, 1 μg/ml Grape Seed extract, and 1 μg/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 6
RANK-L Inhibition
RANK-L
Ingredient (dosage):ninhibition (%)
Organic Olive Juice Powder (100 μg/ml)241.5 ± 4.0
Organic Olive Juice Powder (10 μg/ml)619.7 ± 2.2
Organic Olive Juice Powder (1 μg/ml)6No inhibition
Organic Olive Juice Powder (0.1 μg/ml)3No inhibition
Pomegranate extract (30 μg/ml)258.6 ± 1.5
Pomegranate extract (20 μg/ml)256.2 ± 2.6
Pomegranate extract (10 μg/ml)348.3 ± 3.1
Pomegranate extract (10 μg/ml)2 46.9 ± 10.7
Pomegranate extract (10 μg/ml)248.4 ± 1.1
Pomegranate extract (10 μg/ml)349.4 ± 3.3
Pomegranate extract (1 μg/ml)319.3 ± 2.1
Pomegranate extract (0.1 μg/ml)3 4.7 ± 4.2
Ipriflavone (Ostivone) (10 μg/ml)3  0 ± 4.3
Ipriflavone (Ostivone) (10 μg/ml)3 2.8 ± 3.9
Ipriflavone (Ostivone) (10 μg/ml)3  0 ± 2.5
Ipriflavone (Ostivone) (1 μg/ml)2 0.8 ± 0.3
Grape Seed Extract (40% OPC) (10 μg/ml)318.7 ± 6.9
Grape Seed Extract (40% OPC) (10 μg/ml)221.9 ± 0.6
Grape Seed Extract (40% OPC) (5 μg/ml)313.1 ± 3.4
Grape Seed Extract (40% OPC) (2 μg/ml)210.3 ± 1.6
Grape Seed Extract (40% OPC) (1 μg/ml)3 8.0 ± 2.2
Grape Seed Extract (40% OPC) (1 μg/ml)210.7 ± 1.0
Grape Seed Extract (40% OPC) (0.1 μg/ml)2  0 ± 4.3
Green Tea Extract (40% EGCG) (10 μg/ml)323.9 ± 1.4
Green Tea Extract (40% EGCG) (10 μg/ml)322.6 ± 7.5
Green Tea Extract (40% EGCG) (1 μg/ml)310.2 ± 1.6
Green Tea Extract (40% EGCG) (0.1 μg/ml)3  0 ± 2.6
Pomegranate + Ipriflavone (Ostivone) (10 μg/ml each)245.7 ± 2.4
Pomegranate + Ipriflavone (Ostivone) (10 μg/ml each)248.0 ± 2.8
Pomegranate + Ipriflavone (Ostivone) (10 μg/ml each)346.9 ± 5.3
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (1 μg/ml)248.3 ± 3.5
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (0.1 μg/ml)245.6 ± 4.0
Pomegranate (1 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml)321.6 ± 5.2
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +263.3 ± 0.7
Grape Seed Extract (10 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +264.6 ± 4.9
Grape Seed Extract (1 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +249.9 ± 2.5
Grape Seed Extract (0.1 μg/ml)
Pomegranate + Ipriflavone (Ostivone) + Grape Seed Extract242.8 ± 8.9
(10 μg/ml each)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +339.0 ± 2.9
Green Tea Extract (10 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +254.6 ± 1.1
Green Tea Extract (1 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +237.5 ± 8.1
Green Tea Extract (0.1 μg/ml)
Pomegranate + Ipriflavone (Ostivone) + Green Tea Extract (10323.5 ± 2.3
μg/ml each)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +342.9 ± 1.1
Grape Seed Extract (10 μg/ml) + Green Tea Extract (10 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +260.8 ± 0.9
Grape Seed Extract (10 μg/ml) + Green Tea Extract (0.1 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +355.1 ± 1.8
Grape Seed Extract (1 μg/ml) + Green Tea Extract (0.1 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +368.6 ± 2.6
Grape Seed Extract (1 μg/ml ) + Green Tea Extract (1 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +365.2 ± 1.1
Grape Seed Extract (1 μg/ml) + Green Tea Extract (10 μg/ml)
Pomegranate (10 μg/ml) + Ipriflavone (Ostivone) (10 μg/ml) +352.3 ± 0.7
Grape Seed Extract (0.1) + Green Tea Extract (1 μg/ml)
Pomegranate + Ipriflavone (Ostivone) + Grape Seed Extract +338.0 ± 3.5
Green Tea Extract (10 μg/ml each)
Ipriflavone (Ostivone) + Grape Seed Extract + Green Tea3 9.7 ± 2.8
Extract (10 μg/ml each)
Pomegranate + Grape Seed Extract + Green Tea Extract (10326.9 ± 4.8
μg/ml each)
Pomegranate (10 μg/ml) + Grape Seed Extract (1 μg/ml)252.7 ± 1.0
Pomegranate (10 μg/ml) + Grape Seed Extract (1 μg/ml)251.1 ± 1.8
Pomegranate (20 μg/ml) + Grape Seed Extract (2 μg/ml)258.2 ± 0.5
Pomegranate (30 μg/ml) + Grape Seed Extract (1 μg/ml)264.3 ± 2.1
Pomegranate (30 μg/ml) + Grape Seed Extract (2 μg/ml)259.9 ± 1.2
Pomegranate (30 μg/ml) + Grape Seed Extract (5 μg/ml)257.2 ± 3.1

Example 3

Isolation of Punicalagins from Pomegranate

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

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.

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 μg/ml.

After four hours of treatment, IL-1b (Calbiochem (catalog # 407615), stock solution at 10 μg/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° C.

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 μL of purified RNA. Annealing temperature is 57° C. in the Stratagene Mx4000 with 1 μL of RANK-L gene specific primers in a 50 μL reaction (10 μM initial concentration; HLUX3013920, Invitrogen Inc.).

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 7
Effect of Punicalagins on RANK-L Expression
IngredientNRANK-L Expression (fold increase)
Untreated21.0 ± 0.1
IL1B (3 ng/ml)23.3 ± 0  
Punicalagins 1 μg/ml22.6 ± 0.5
Punicalagins 10 μg/ml21.9 ± 0.4
Punicalagins 100 μg/ml20.7 ± .02

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 μg/mL.

Example 5

Inhibition of Type IV Collagenase (MMP9) Protein Expression by Punicalagins from Pomegranate

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 μg/ml, 10 μg/ml, and 100 μg/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 8
Effect of Pomegranate Extracts on MMP9 (type
IV collagenase) protein Expression
Tested Pomegranate ExtractNInhibition of MMP9
Untreated21.0 ± 0.1
IL1B (10 ng/ml)21.65
Punicalagins 1 μg/ml41.5 + .06
Punicalagins 10 μg/ml41.2 ± .04
Punicalagins 100 μg/ml41.0 ± 0.1

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

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.

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 μg/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.

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).

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 9
Inhibition of C. histolyticum collagenase activity
by Grape Seed and Pomegranate Extracts
% Collagenase
SampleConcentration (μg/ml)Activity
Pomegranate extract1 96.7 ± 10.0%
1086.3 ± 5.9%
10029.2 ± 5.7%
Grape seed extract198.9 ± 3.0%
1048.8 ± 2.3%
100−7.2 ± 4.3%
Collagenase only0100.0 ± 0.8% 

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.