BRPI0709723A2 - Nutritional compositions for the promotion of bone growth and maintenance of bone health and methods - Google Patents

Abstract

<B> NUTRITIONAL COMPOSITIONS FOR THE PROMOTION OF BONE GROWTH AND MAINTENANCE OF OSS0 HEALTH AND METHODS CONSIDERING THE SAME <D> The present invention relates to compositions and methods for maintaining bone health or preventing, relieving and / or treating bone disorders are presented. The present invention also provides for the manufacture of a nutritional product, a supplement or a medicine to promote bone growth or to maintain bone health and methods considering it. In one embodiment, the present invention provides a composition comprising an active ingredient having an effective amount of a plant or plant extract containing at least one phytochemical with the ability to induce bone morphogenetic protein expression.

Description

Report of the Invention Patent for "NUTRITIONAL COMPOSITIONS FOR PROMOTING BONE GROWTH AND BONE HEALTH MAINTENANCE AND CONSIDERING METHODS".

Background of the Invention

The present invention generally relates to nutritional compositions that provide health benefits. More specifically, the present invention relates to beneficial compositions which may be used, for example, to improve bone density and bone formation and methods thereof.

Bone mass develops throughout life and is regulated by genetic, mechanical and hormonal mechanisms. Mineral bone acquisition occurs during childhood and peak bone mass is reached in all 20 years of age. During this period, bone formation exceeds bone absorption. Later in life, and particularly around the time of death, or in the elderly population, bone mass and quality are impaired due to increased bone turnover with excessive bone resorption leading to a gradual loss of bone mass, microarchitecture. , structure and strength. To maintain bone, it is important to restore the balance between bone formation and bone resorption. This remolding process is regulated at the bone cell level involving a rigid interaction between bone formation cells (osteoblasts) and bone resorption cells (osteoclasts).

Phytonutrients, especially flavonoids, can positively influence the bone remodeling process. The most reported data are for soy isoflavones, which in some studies have been shown to prevent bone loss and improve bone mineral density (BMD) in postmenopausal women at doses of 50 - 90 mg / day. However, not all studies with isoflavones are positive and controversially still exist about their efficacy. In addition, some epidemiological evidence exists for the benefits of tea as an earlier study showed that tea drinks had a higher average BMD than non-tea drinks in an elderly population, however no intervention studies have been performed. to verify this discovery.

Currently, there is a strong interest in identifying agents that can stimulate bone formation. The discovery of recombinant BMP-2 has been shown to induce bone and cartilage formation. For example, bone morphogenetic protein 2 (BMP-2) is a member of the TGF3 family and is a key regulator of bone growth during embryonic development, and additional bone growth and repair. Statins (effective cholesterol-lowering drugs by inhibiting the HMG-CoA reductase enzyme) improve bone formation, partially mediated by BMP-2 induction (G. Mundy et al., Science 286: 1946-1949 (1999). ); CJ Edwards et al., Lancet, 355: 2218-2219 (2000)). Statins were also able to reduce the risk of hip fracture in namenopausal women (P. S. Wang, et al., JAMA 283: 3211 - 3216 (2000)).

Summary of the Invention

The present invention generally relates to nutritional compositions for maintaining bone health or preventing, alleviating and / or treating bone disorders. The present invention also provides for the manufacture of a nutritional product, supplement or medicament for promoting bone growth or for maintaining bone health and methods thereof. In particular, the present invention provides for the manufacture of a nutritional product, supplement or medicament to promote bone formation that is important for bone growth as well as for maintaining bone health through balanced bone remodeling and methods thereof.

In one embodiment, the present invention provides a composition comprising an active ingredient having an effective amount of a plant or plant extract containing at least one phytochemical having the ability to induce bone morphogenetic protein expression.

In one embodiment, the plant or plant extract further inhibits bone resorption.

In one embodiment, the plant is rosemary or cumin. In one embodiment, the phytochemical is selected from the group consisting of eupafoline, carnosol, scutellarin, gencuanin, caemp-ferol, acacetin, rosmarinic acid, rosmanol, cirsimaritin, luteolin. 7-epirosmanol, and compound C-0063-W-06 of Figure 7A and combinations thereof.

In one embodiment, the composition may be in a form selected from the group consisting of a nutritionally balanced diet, feed, a dietary supplement, a treat, a pharmaceutical composition and combinations thereof.

In one embodiment, the composition may be designed to assist bone regeneration during fracture healing, increased bone deformation and bone mineral density during growth, and optimize peak bone mass or decrease bone loss, in particular. particular, age-associated loss in humans or pets.

In one embodiment, the composition may be designed to build cartilage in humans or pets.

In one embodiment, the composition may be designed to prevent osteoarthritis in humans or pets.

In another embodiment, the present invention provides a composition comprising an active ingredient having an effective amount of a rosemary plant or rosemary plant extract containing at least one phytochemical having the ability to induce bone morphogenetic protein expression. For example, the phytochemical may be selected from the group consisting of eupafolin, carnosol, scutellarina, gentuanin, caempferol, acacetin and combinations thereof.

In an alternative embodiment, the present invention provides a method for making a food composition for the prevention, alleviation and / or treatment of bone disorders or maintenance of bone health in humans or pets, the method comprising providing a food composition; and adding to the food composition an active ingredient having a plant or plant extract containing at least one phytochemical having the ability to stimulate bone morphogenetic protein and / or inhibit bone resorption to prepare the composition. For example, the composition may include components chosen from the group consisting of chicory, tea, cocoa, bioactive substances, antioxidants, fatty acids, prebiotic fibers, glycosamine, chondroitin sulfate and combinations thereof.

In another embodiment, the present invention provides a method for treating, alleviating or preventing bone disorder or maintaining bone health. The method comprises administering a therapeutically effective amount of a composition comprising an activating ingredient having an effective amount of at least one plant. or plant extract containing at least one phytochemical having the ability to induce bone morphogenetic protein expression in an individual in need thereof.

In an alternative embodiment, the present invention provides a method of increasing bone formation, bone mineral density during growth and optimizing peak bone mass in humans or pets, the method comprising feeding an animal, a composition comprising an active ingredient. having an effective amount of at least one plant or plant extract containing at least one phytochemical having the ability to induce bone morphogenetic protein expression.

In another embodiment, the present invention provides a method for the treatment, relief and / or prophylaxis of osteoarthritis in pet animals and humans, the method comprises feeding an individual having arthritis or at risk for arthritis, a composition comprising an active ingredient having an effective amount of at least one plant or plant extract containing at least one phytochemical with the ability to induce bone morphogenetic protein expression in the subject.

In yet another embodiment, the present invention provides a method for treating or preventing osteoporosis, the method comprising administering to a subject having osteoporosis or at risk of having osteoporosis a therapeutically effective amount of a composition comprising an active ingredient having an effective amount of at least one. plant or plant extract containing at least one phytochemical with the ability to induce bone morphogenetic protein expression in the individual.

In still another alternative embodiment, the present invention provides a method for stimulating bone regeneration during fracture healing, the method comprises feeding an individual having a fracture, a therapeutically effective amount of a composition comprising an active ingredient with an amount It is effective for at least one plant or plant extract containing at least one phytochemical with the ability to induce bone morphogenetic protein expression in the subject.

In a further embodiment, the present invention provides a method for decreasing bone loss, the method comprises feeding an individual exhibiting bone loss, a composition comprising an active ingredient having an effective amount of at least one plant or plant extract containing at least one bone. least one phytochemical with the ability to induce bone morphogenetic protein expression in the individual.

Bone characteristics and advantages are described herein, and will be apparent from the following Detailed Description and the figures.

Brief Description of the Figures

Figure 1 illustrates an extraction protocol.

Figure 2 illustrates a summary of the extraction procedure and first fractionation.

Figure 3A illustrates BMP-2 results for cumin rosemary extracts.

Figure 3B illustrates alkaline phosphatase results for rosemary and cumin extracts.

Figure 3C illustrates a bone formation in an organ culture for rosemary and cumin extracts.

Figure 4 illustrates an extraction procedure from the MeOH / NRC initial water extract, and BMP-2 activity of the extracts (from 80g of dried leaves (hexane = 6g)).

Figure 5 illustrates bone formation in vivo with ale crim extract.

Figure 6A illustrates phenolics found positive in BMP-2 assays.

Figure 6B illustrates an alkaline phosphatase assay of BMP-2 positive phenolics.

Figure 6C illustrates an organ culture bone formation: examples with eupafolin and carnosol.

Figures 7A-B illustrate compounds isolated from ale crim extract 2188.

Figure 8A provides details of the effects of rosemary extract on the activity of human osteoclasts.

Figures 9A, B and C provide details on the effects of dealecrim extract and carnosol on articular cartilage metabolism.

Figure 10 shows induction of osteopontin mRNA (OPN) in human osteoblast cells (hPOBert) by rosemary or carnosol extract.

Figure 11 shows that Carnosol induces phase II enzyme expression NQ01, a protein / gene typically regulated by Nrf-1.

Detailed Description of the Invention

The present invention relates to beneficial compositions which may be used, for example, to improve bone density and bone formation and methods thereof. For example, in one embodiment, the present invention is directed to plants and plant extracts that stimulate bone formation and improve bone maintenance.

In plants, a number of isoprenoids (monoterpenes, sesqui-terpenes, etc.) are modulators of both HMG-CoA reductase and protein prenylation, mechanisms likely linked to either inhibition of bone resorption or enhancement of bone formation. . Therefore, certain plant compounds may be potential inhibitors of bone resorption and / or bone formation enhancers.

In embodiments of the present invention extracts have been prepared from medicinal and / or edible plant species, which have been proposed based on potential benefits to alleviate the symptoms of dopause or its ability to affect the synthesis pathway of cholesterole, thus having a potential to stimulate BMP-2 and bone formation. As discussed in more detail below, the extracts were generally prepared by a 4-step process (a) hexane, (b) methanol-water, (c) hydrolysed methanol-water extracts. glycosidases and cometylacetate re-extracted, and (d) removal of large polyphenols with a PVPP column. Methanol-water and ethylacetate extracts were used for in vitro tests. The extracts were hydrolyzed by oc- and β-glucosides rather than acid to ensure the release of flavonoid aglycones (biologically active form) from their glycosides.

The following bioassays were used for bone formation analysis:

- BMP-2 reporter gene assay (high speed scan)

- alkaline phosphatase in osteoblast cells

- in vitro calvary organ culture, bone formation

- In vivo calvary injection, bone formation.

For example, the extracts were examined for deosso formation by a high-speed reporter gene assay for BMP-2 followed by alkaline phosphatase assay and an organ culture model and finally rat calvary injection in vivo. Subtractions of positive extracts and / or pure compounds were further tested for activity. Analysis of the chemical composition of an active extract was performed to determine the active compounds.

Surprisingly it was found that compounds extracted from rosemary plants could be used as active compounds for bone development, growth and / or maintenance. For example, at least 2 phenolics (eg, eupafoline, carnosol, scutellarine) can contribute to the anabolic potential of rosemary extract. Additional rosemary phenolics include gentuanin, caempferol, acacetin. The extracts of rosemary and pure compounds were in an osteoblast / osteoclast co-culture system. Rosemary extract as well as the same 3 phenolics were shown to have an activity for cytokine regulation by controlling bone remodeling, ie OPG / RANKL. In addition, rosemary extract and carnosol have been found to stimulate the expression of osteopontin (OPN) in human osteoblast cells, possibly via AP-1 / Nrf-2 signaling pathways.

Three constituents of active rosemary extract are new, and have never been described in the literature. In addition, since several compounds belong to the same chemical class, a cross-linked biological evaluation of these constituents should lead to an interesting structure - activity correlation (eg carnosol / rosmanol / isorosmanol) that could provide additional strategies for the extracts of rosemary, but also for other plant species containing the most well-known active constituents.

In one embodiment of the present invention, the rosemary plant Me-OH / water extracts (25% of the initial dry leaf matter) obtained after a hexane fatty acid removal step contain the activity-responsible molecules and could be used in a food product. In vitro activity is only slightly observed in the MeOH / initial water extract due to the dilution of active compounds by other compounds and also by part due to the presence of bound forms. High activity is observed in BMP-2 assay after purification / concentration by ethyl acetate extraction and / or glycosidase hydrolysis then ethyl acetate extraction.

Plants or plant extracts according to the embodiments of the present invention may be used in the preparation of a food composition. The composition may be in the form of a nutritionally balanced food or feed, a dietary supplement, a candy or a pharmaceutical composition.

The plant or plant extract may be used alone or in association with other plants such as chicory, tea, cocoa, or other bioactive molecule such as antioxidants, fatty acids, prebiotic fibers, glycosamine, chondroitin sulfate, for example. In one embodiment of the present invention, a food composition or nutritional formula for human consumption is prepared. This composition can be a complete nutritional formula, a dairy product, a cold or non-refrigerated beverage, soup, a dietary supplement, a meal replacement, and a nutritional bar or candy.

Apart from the rosemary plant or rosemary plant extract according to the invention, the nutritional formula may comprise a source of protein. Dietary proteins are preferably used as a protein source. Dietary proteins may be any dietary protein; for example animal proteins (such as milk proteins, meat proteins, and egg proteins); vegetable proteins (such as soy protein, wheat protein, rice protein, and pea protein); free amino acid mixtures; or combinations of these. Milk proteins such as casein, whey proteins and soy proteins are particularly preferred. The composition may also contain a carbohydrate source and a fat source.

If the nutritional formula includes a fat source, it preferably provides from about 5% to about 55% of the nutritional formula e-energy; for example, approximately 20% to approximately 50% of energy. The lipids constituting the fat source may be any suitable fat or fat mixtures. Vegetable fats are particularly suitable; for example, soybean oil, palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canola oil, lecithins, and the like. Animal fats such as milk fats may also be added if desired.

A carbohydrate source can be added to the nutritional formula. It preferably provides approximately 40% to approximately 80% of the energy of the nutritional composition. Any suitable carbohydrates may be used, for example, sucrose, lactose, glucose, fructose, corn syrup solids, and maltodextrins, and mixtures thereof. Dietary fiber may also be added if desired. If used, it preferably comprises up to approximately 5% of the formula energy. Dietary fiber may be from any origin, including, for example, soybean, pea, oats, pectin, guar gum, arabic gum, and fructo-tooligosaccharides. Suitable vitamins and minerals may be included in the nutritional formula in an amount to meet the appropriate guidelines.

One or more food grade emulsifiers may be incorporated into the nutritional formula if desired; for example, tartaric diacetyl acid esters of mono- and diglycerides, Iecithin and mono- and diglycerides. Similarly suitable stabilizing salts may be included. Vitaminase minerals can also be combined with plant extract.

The nutritional composition is preferably administrable enterally; for example, in the form of a powder, tablet, capsule, liquid concentrate, solid product or ready-to-drink beverage. If a powdered nutritional formula is to be produced, the homogenized mixture is transferred to a suitable drying apparatus such as a spray dryer or freezer dryer and converted to powder.

In another embodiment, a nutritional composition comprises a milk-based cereal together with a prebiotic formula. Preferably, the milk-based cereal is an infant cereal that acts as a vehicle for the prebiotic formula.

In another embodiment, a usual food product may be enriched with at least one plant or plant extract according to the present invention. For example, a fermented milk, a yogurt, a fresh cheese, a quinosine-treated milk, a confectionery, for example, a sweet or sweet drink, a sweet bar, breakfast cereal flakes or bars, beverages, milk powders, soya-based products, non-milk fermented products, or clinical nutritional supplements.

The amount of the plant or plant extract in the composition may vary according to the plant source and its use. In a preferred embodiment, an effective daily dose amount is at least about 1 mg, and more preferably from 1 mg to 200 mg of the molecule per day.

In an alternative embodiment, a pharmaceutical composition containing at least one rosemary or phytochemical extract as described above in an amount sufficient to achieve the desired effect on an individual may be prepared. Such a composition may be a tablet, a liquid, capsules, soft capsules, pastes or pastilles, gums, or drinkable solutions or emulsions, a dry oral supplement, a wet oral supplement. The pharmaceutical composition may additionally contain carriers and excipients which are suitable for releasing the respective active molecule of a different nature to the target tissue. The type of vehicle / excipient and the amount thereof will depend on the nature of the substance and the mode of drug release and / or administration observed. It is appreciated that the natured versed person, based on his own knowledge, selects the appropriate components and galenic form.

The plant or plant extract according to the invention may be used in the preparation of a feed composition. Said composition may be administered to the pet as a supplement in its normal diet or as a component of a nutritionally complete diet, and more preferably in a hypocaloric diet. It may also be a pharmaceutical composition.

The plant or plant extract may be used alone or in association with other plants such as chicory, tea, cocoa, or other bioactive molecule such as antioxidants, fatty acids, prebiotic fibers, glycosamine, chondroitin sulfate, for example.

Preferably, the feed composition contains approximately 0.01 to 0.5 g of dried plants per gram of dry feed for a 15 kg feed; and 0.001 to 0.1 g of dry plants per gram of wet food for a 15 kg dog. The nutritionally complete feed composition according to the invention may be a powder, in dry form, a sweet or a chilled or uncooled feed product. It may be chilled or supplied as a product which does not need to be of refrigeration. These feeds can be produced in known natechnical ways.

The feed may optionally also contain a prebiotic, probiotic microorganism or another active agent, for example a long chain fatty acid. The amount of prebiotic in the feed is preferably less than 10% by weight. For example, the prebiotic may comprise from about 0.1% to about 5% of the feed weight. For feeds that use chicory as the prebiotic source, chicory may be included to comprise approximately 0.5% to approximately 10% dope of the feed mixture; more preferably, about 1% to about 5% by weight.

If a probiotic microorganism is used, the feed preferably contains approximately 104 to about 1010 cells per probiotic organism per gram of feed; more preferably, α-approximately 106 to approximately 106 cells of the probiotic microorganism per gram. The feed may contain approximately 0.5% to approximately 20% of the weight of the probiotic microorganism mixture; preferably about 1% to about 6% by weight; for example, about 3% to about 6% by weight.

If necessary, the feed may be supplemented with minerals and vitamins such that they are nutritionally complete. In addition, various other ingredients, for example sugar, salt, seasonings, seasonings, flavoring agents, and the like may also be incorporated into the feed as desired.

In another embodiment, dietary adjuncts may be prepared such as to improve feed quality. Like dietary aids, they may be encapsulated or may be supplied in powder form and packaged together or separately from a main meal, or dry or wet. By way of example, a powder containing extracts according to the invention may be packaged in powder sachets, or in a gel or lipid or other suitable carrier. These separately packaged units may be supplied together with a main meal or in multi-unit packs for use with a main or sweet meal according to user instructions.

The amount of feed to be consumed by the pet for a beneficial effect will depend on the size of the pet, the type, and the age of the pet. However, an amount of feed to provide a daily amount of approximately 0.5 to 5 g of plantlets per kg body weight would usually be suitable for dog puppies and cats.

Administering to a human or animal, the food or feed composition as described above, may result in improved bone regeneration during fracture healing. It can help to stimulate bone formation and bone mineral density during growth and to optimize bone mass peak. In particular, it can provide optimal bone growth during childhood. Such dietary composition may help to prevent bone loss, in particular, bone loss associated with age in mammals or bone loss associated with long-term hospitalization. It can reduce the risk of osteoporosis and improve recovery after fracture. In addition, it can help build cartilage in mammals and prevent osteoarthritis in pets and humans, which results in better activity or mobility of the individual (eg, pets and / or humans).

Examples

By way of example and not limitation, the following examples are illustrative of various embodiments of the present invention and further illustrate experimental testing conducted in accordance with embodiments of the present invention.

Example 1

Selected Plants and Pure Compounds

The plants were selected based on their phenolic knowledge:

(i) 25 plants in addition to soybean were selected from a Plant Database Containing Estrogen Substances (Table 1a) (ii) 9 plants + soybean containing substances affecting the cholesterol desynthesis pathway and thus having the potential to stimulate indire BMP-2 and bone formation (Table 1B)

(iii) Pure Compounds: estradiol-17β, genistein, daidzein, flavonoids, chicory compounds (sesquiterpene lactones).

Table 1A: List of plant species selected from the estrogen plant database

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Table 1B: List of plant species selected for potential effect on cholesterol and BMP-2

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Table 1C: List of Pure Compounds

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Plant Extraction Procedure

Referring to Figure 1, the extraction procedure generally included the following steps:

- Hexane, fatty acid withdrawal (extracts not examined)

- MeOH / H2 O (1a)

- Me0H / H2O, hydrolyzed with a, β-glycosidases, extracted with ethyl acetate (1b)

- 1a purified on PVPP column to remove high molecular weight polyphenols (2a)

- 1b purified on PVPP column to remove high molecular weight polyphenols (2b)

Extracts 2a and 2b provided similar results to extracts1a and 1b and therefore the polyphenol purification step was subsequently discontinued. The extraction procedure included treatment with glycosidase (rather than acid hydrolysis) to ensure the conversion of flavonoid glycosides to aglycones.

Subfractionation - Four subtractions were prepared by fractionation on solvent-eluting silica gel cartridge with variable polarity: ethyl acetate then ethyl acetate / methanol (95/5) followed by ethyl acetate / methanol (50/50) and finally methanol ( figure 2).

Scanning Steps and Bioassays

The scan for bone formation was performed at various stages:

(i) High speed BMP-2 reporter gene assay of unhydrolyzed MeOH / H2 O extracts (1a) and corresponding glycosidase hydrolyzed extract in ethyl acetate (1b). The extracts were tested twice at concentrations of 1 to 100 pg / ml, diluted in culture medium from plant extract stocks prepared as 50 pg / ml in DM-SO.

(ii) BMP-2 screen positive extracts were freshly prepared and re-examined with dose response to confirm as hits.

(iii) The BMP-2 test of subfractionation of pure candidate positive hits / hits

(iv) Extracts shown to be positive in the BMP-2 assay were further tested for osteoblast differentiation using alkaline phosphatase assay and in an organotypic model of bone formation using cranial bone culture and histomorphometry to demonstrate bone formation. as described by Traianedes et al. (1998).

(v) Final in vivo injection of "hit" extract into rat skull bones and monitoring of bone area and thickness. The extracts were evaluated in a neonatal cranial rat test in vitro. The bones were incubated with the extracts for the entire 4 days.

(vi) Monitored resorption activity by measuring the amount of type I collagen released into media such as osteoclast digestion bone.

(vii) The effects on cytokine-induced collagen type II degradation, MMP-mediated agrecana degradation, and agrecanase-mediated degradation in bovine articular cartilage explants.

Results: BMP-2 Reporter Gene Plant Extract Scan & Organ Culture

Tables 2 and 3 provide details of the scan results with BMP-2 as follows:

- 15 extracts were found positive in BMP-2. From these, 5 confirmed correct answers and active sub-fractions were identified (Rosmarinus officinalis; Taraxacum officinalis, Lindera benzoin, Cyperus ro-tundus, Iris pallida) and 5 positives of interest (Rosmarinus officinalis, Carumcarvi, Thymus vulgaris, Mentha spicata and Vitis). vinifera) from 2 BMP-2 scanning steps.

- Confirmed hits and active sub-fractions (Rosmarinus officina-lis; Taraxacum officinalis, Lindera benzoin, Cyperus rotundus, Iris pallida) e5 positives of interest (Rosmarinus officinalis, Carum carvi, Thymus vulga-ris, Mentha spicata and Vitis vinifera) Two-step BMP-2 scanning steps were also identified as active in the rat cranial organ culture model.

- The active extracts or sub-fractions were further confirmed to stimulate bone formation in the cranial organ culture model: Iris pallida, Cyperus rotundus, Rosmarinus officinalis (rosemary), Thymus vulgaris (thyme), Carum carvi (cumin). Table 2: Summary of BMP-2 scan positive extracts and confirmed in organ culture.

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Table 3: Total Summary of Plant Extract Results Until Identification of Hits

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Plant Extract Scan Conclusions

BMP-2 hits confirmed in bone formation assay in organ culture were extracted from soybean seeds, ale crim leaves, thyme leaves, cumin seeds.

Example of Rosemary & Cumin Hits for BMP-2 Activity, Confirmed in Alkaline Phosphatase Assay and Organ Culture

Figures 3A-C illustrate bone formation assay results for rosemary and cumin extract hits.

Effect of BMP-2 Active Extraction Procedure

After a first extraction of rosemary with methanol / water on leaves previously removed from fatty acid (ext. 2127), the induction of BMP-2 gene expression was 1.5X at 10 pg / ml (Figure 4). Specific extraction of this extract with ethyl acetate (2188) led to an increase in BMP-2 expression (8X induction). This suggests that the EtOAc extraction process resulted in the concentration of active compounds from the original MeOH / water extract. Following hydrolysis with glycosidases, the resulting ethyl acetate extract (2189) is also active, showing that additional molecules were extracted. The 2189 extract is even slightly more active than the unhydrolyzed one.

These results show unambiguously an activity in both extracts (unhydrolyzed: 2188 and hydrolyzed: 2189) suggesting the existence of active molecules in two forms: free and / or linked (glycosylated) in the original extract.

Cranial Bone Formation Following In Vivo Injection

Rosmarinus officinalis (rosemary extract) shows bone deforming activity in 3 independent in vitro bone formation assays (BMP-2, alkaline phosphatase, bone organ culture) as well as in the cranial bone inventive assay (see Figure 5).

Rosemary extract (here the leaves were first extracted with water, and water extract was hydrolyzed then extracted with ethyl acetate) is injected into the rat skull box, followed by ex vivo bone formation analysis.

Pure Compound Scan

Phenolics were tested at concentrations of 1 -10 μΜ. Active phenolics in the BMP-2 assay are listed in Table4. Figures 6A-C show certain positive phenolics in bone formation assays.

Table 4: Active phenolics in BMP-2, ALP and organ culture assays:

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Rosemary Compositional Analysis

Table 5 shows pure compounds found in extracts of crecrim.

Table 5: Rosemary Components

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Analysis of BMP-2 Active Rosemary Extracts

The extract for analysis was chosen following previous results showing that bone formation activity was concentrated in ethyl acetate extracts prepared from a methanol / water extract (2189) or without enzymatic hydrolysis (2188), ( see figure 4).

Ethyl acetate extract 2188 was selected by a phytochemical study of its main constituents including identification and purification of the compounds by HPLC / ELSD / UV / MS. This experience was done by Analyticon Discovery GmbH, Postdam. A depth phytochemical evaluation was then completed on the active Rosemary extract under BMP-2 assay. Preliminary results led to the isolation of 13 compounds. Nine compounds were identified. Four others required additional investigations. Further studies describe the structural elucidation of these 4 compounds performed by H-NMR and 2D-NMR (H, H-COZY, HMBC1 HMQC) by Analyticon Discovery GmbH (Postdam).

Analyticon provided thirteen pure identified molecules in order to perform the evaluation of their biological activity in bone health. These compounds are listed in Table 6 and their structures illustrated in Figures 7A-B. Among them, three are new compounds, never described in the literature (XI, XII, and XIII). A correlation: chemical structure - biological activity of these 13 constituents may provide an interesting and relevant tool for further development in bone health research.

Table 6: 14 compounds isolated from rosemary extract 2188

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Thirteen constituents of a rosemary extract (2188) for which structural elucidation has been achieved and validated are now available for evaluation of its biological activity in bone health trials.

Rosemary Extract and Its Anti-Bone Resorption Activity

Osteoporosis is a chronic disease characterized by slow bone loss. Bone is not dead tissue. On the contrary, it is constantly remodeled with old bone tissue being replaced by new bone. This remodeling is controlled by osteoblasts, the cells we deposit and osteoclasts, the cells that dissolve. Usually, there is a tight coupling between bone formation and bone resorption such that no liquid bone loss occurs. In osteoporosis, this coupling is not perfect as bone loss is more prominent than bone deformation. To treat osteoporosis, one may wish to increase bone deformation, decrease bone loss or both. In this example, it is shown that rosemary extracts may decrease bone loss.

Osteoclasts, differentiated from Peripheral Blood-Mononuclear Cells (PBMCs), were cultured in bovine bone slices. Their resorption activity was monitored by measuring the amount of type I collagen released into the media as bone differed.

Type I collagen is the main organic molecule of bone. As bone is digested, the mineral phase of bone is dissolved by exposing collagen fibers to the proteolytic activity of matrix metalloproteinases. Once digested, the collagen fibers become soluble and are released into the culture medium where their presence can occur. be quantified by trials

ELISA - CTX-I assay.

Figure 8A provides details of the effects of rosemary extract on human osteoclast activity as follows: rosemary extract 1 (P31 extract commercially available from Robertet) at a concentration of 10 pg / ml decreased the amount of collagen type I released from bone slices compared to culture media alone (control (CTL)) (Figure 8A). Rosemary Extract and Osteoarthritis

Osteoarthritis is a disease characterized by slow destruction of articular cartilage. This articular cartilage is due to an imbalance between the anabolic and catabolic activity of chondrocytes. Chondrocyte is the unique cell type present in cartilage and is responsible for maintaining the extracellular cartilage matrix. In osteoarthritis, catabolism is increased and is responsible for the loss of cartilage. The extracellular cartilage matrix is composed of 2 main molecules: collagen type II and aggregan. While collagen is mainly digested by matrix metalloproteins (MMPs) 1 agrecana can be degraded both by MMPs and another class of enzymes called agrecanases.

It was investigated here whether 2 different rosemary extracts and carnosol, one of the main components of this extract, could inhibit collagen and agrecana degradation in cultured articular cartilage explants. Former plants from healthy bovine articular cartilage were placed in culture. However, such explants naturally exhibit very low catabolic activity that is not ideal for testing potential anti-catabolic activity. To improve assay sensitivity, explants are cultured in the presence of 2 proinflammatory cytokines, TNF-α and on-costatin known to act together as potent MMP inducers and agrecanase activities.

Effects of rosemary and carnosol extracts and articular cartilage catabolism

Figures 9A, B and C provide details on the effects of rosemary extract and carnosol on articular cartilage metabolism as follows: rosemary extract 1 (commercially available P31 extract from Ro-bertet) at a concentration of 10 pg / ml completely nullified. collagen degradation induced by the proinflammatory cytokines TNF-α and on-costatin (Control (CTL)) (Figure 9A).

Rosemary extract 1 (commercially available P31 extract from Robertet) at a concentration of 10 μς / ιηΙ completely nullified the MMP-mediated degradation of TNF-α and oncostatin-induced cytokines (Control (CTL)) (Figure 9B) ).

Rosemary extract 1 (commercially available P31 extract from Robertet) at a concentration of 10 μg / ml also inhibited agrecanase-mediated degradation of agrecanase induced by TNF-α and oncostatin (Control (CTL)) cytokines. Similarly, another rosemary extract, rosemary extract 2 (from Nestec R&D Center in Tours obtained as described above) at a concentration of 1 or 5 μg / ml and carnosol at a concentration of 1 or 5 μΜ also inhibited collagen degradation. not cytokine-induced (Figure 9C).

OPN mRNA Induction:

Cell culture - HPOBTert osteoblasts were plated on collagen-coated plates and grown in MEM Eagle Modification medium supplemented with 10% fetal bovine serum, 1% L-glutamine and penicillin / streptomycin, 1 mM β-glycerolphosphate and 50 μg / ml ascorbic acid in a humidified atmosphere of 5% CO2, and 95% air at 37 ° C. When carbonocarbons and inhibitors were added, an equivalent amount of Me2SO was used as a control vehicle.

Real-time PCR mRNA analysis - Total cell RNA was extracted using the NucIeoSpin RNA Il kit (Macherey-Nagel, Switzerland). Equal amounts (1 pg) of RNA from different treatments were reverse transcribed using the First Filament cDNA Synthesis Kit for RT-PCR (Roche, Mannheim, Germany). For each sample, 2 μΙ 10x reaction buffer, 4 μΙ 25 mM MgCl2, 2 μΙ nucleotide mix, 2 μΙ random primers, 1 μΙ RNAse inhibitor 0.4 μΙ AMV reverse transcriptase from of the kit were added to the sample. Reverse transcription was performed under the following thermal cycling conditions (25 ° C for 10 min, 42 ° C for 60 min, and 75 ° C for 5 min using Concept PTC-100TM, Switzerland).

Real Time Quantitative PCR - Quantitative PCR was performed at 25 μΙ on three copies. This consisted of 12.5 μΙ Universal 2x Taqman MasterPCR Mixture, 1.25 μl on Demand Assay initiators and test leads (Applied Biosystems, USA) and 6.25 μl of RNA-free water itself. Amplification was conducted on an ABI 7000 (Applied Biosys-tems) machine with the following thermal profile: 50 ° C for 2 min, 10 min at 95 ° C, followed by 40 cycles of 95 ° C for 15s and 60 ° C for 1 min Degene expression levels were normalized to β-actin expression levels.

Figure 10 shows that rosemary and carnosol extract induces OPN expression in a dose-dependent manner by determining real-time PCR of OPN mRNA levels. HPOBtert cells were maintained for 48h in rosemary and carnosol extract at the indicated doses.

NQO1 induction

Preparation of Cytoplasmic Extracts - hPOBtert cells were washed twice with phosphate buffered saline and harvested with Iise buffer (1% Triton X-100, 20 mM tri / HCL pH 8, 137 mM NaCl 110% Glycerol, 2 mM EDTA pH 8, and newly added proteinase inhibitors: 1mM phenylmethylsulfonyl fluoride, 0.15 U / ml Aprotinin, 10 μg / ml Leupeptide and 10 μg / ml Pepstatin). The samples were centrifuged at 13,000 rpm, 4 ° C for 5 min and the supernatant transferred to a fresh tube. Protein concentration was determined using the BioRad protein assay. Approximately 50 μg of each sample was mixed with an appropriate volume of sample buffer, denatured for 5 min at 95 ° C along with 5 μl of ice-cooled standard protein and loaded onto a 10% ready-to-mix gel. and subjected to immunoblot analysis using anti-NQO1 antibodies.

Immunobloting - 50 μg of protein cell lysate was separated by SDS-PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane (Invitrogen) according to the manufacturer's protocol. The membranes examined by OPN and NOQ01 were blocked and examined in 5% milk in tri-buffered saline / Tween (20 mMBase Tri, pH 7.6, 137 mM, 0.1% Tween 20). Blots were visualized by chemiluminescence development, Wes-tern Blot detection system (Amersham Biosciences). Antibodies - NQ01-specific antibodies (SC-16464) were purchased from Santa Cruz Biotechnologies Inc (Santa Cruz, CA). The β-actin antibody (A-5441) was purchased from Sigma. Secondary antibodies were purchased from Sigma.

Figure 11 shows that Carnosol induces expression of the enzyme NQ01 phase II, a gene / protein typically regulated by Nrf-1. Safety Status Tolerance Test

The tolerance test was performed on young Sprague-Dawleymachos rats. The rats were orally fed "by fattening" for 5 days with daily administration of 1g (extract 2127, MeOH / water) per kg body weight of the animal. No abnormal behavior, mortality or signs of toxicity were observed during treatment or the subsequent 10 days observation period. Rosmarinus officinalis was therefore considered to be safe under these conditions.

Of 32 plant species, 120 extracts were examined in the BMP-2 assay, 15 were identified as positive hits. The most promising ones were from Rosmarinus officinalis (rosemary) along with those from Cyperus rotundus, Iris pallida, Thymus vulgaris (thyme and Carumcarvi (cumin)), which were also active in the rat organocranial culture model. BMP-2 assays have been confirmed in alkaline phosphatase and in functional organ culture assays for bone formation in vitro.

Rosmarinus officinalis (rosemary extract) was the most promising hit, showing bone formation activity in 3 independent in vitro bone formation assays (BMP-2, alkaline phosphatase, bone organ culture) as well as the in vitro test. alive from the skullcap. For example, rosemary extracts stimulated bone formation following injection into the rat skull in vivo.

Six phenolics that are components of rosemary (eupafolin, carosol, scutellarine, gentuanin, caempferol, acacetin) are active in the 3 bone formation assays. The most active are eupafolin and carnosol. Pure identified thirteen molecules were isolated from the active rosemary extract. Among them, 3 are new compounds (XI, X11 and XIII), never described previously in the literature.

The data presented further demonstrate that rosemary and carnosol extracts are capable of slowing down cartilage destruction. This property makes them interesting candidates for preventing arthritis or slowing their progression in humans and pets.

The data presented also show that ale-crim extract is capable of increasing bone formation but also decreasing bone re-absorption. It is not common to find a single compound / extract exhibiting both properties. This makes rosemary extract a highly interesting candidate for preventing osteoporosis or slowing its progression in humans or pets.

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is intended, therefore, that such changes in modifications are covered by the appended claims.

Claims (29)

A composition comprising an active ingredient having an effective amount of a plant or plant extract containing at least one phytochemical capable of inducing bone morphogenetic protein expression. The composition of claim 1, wherein the plant or plant extract further inhibits bone resorption. A composition according to claim 1, wherein the plant is rosemary. The composition according to claim 1, wherein the phytochemical is selected from the group consisting of eupafolin, carno sol, scutellarina, gentuanin, caempferol, acacetin and combinations thereof. Composition according to Claim 1, which is in a form selected from the group consisting of a nutritionally balanced diet, feed, a dietary supplement, a candy, a pharmaceutical composition. A composition according to claim 1 which is designed to assist in bone regeneration during fracture healing, increase bone formation and bone mineral density during growth and optimize bone mass peak or decrease bone mass. bone loss, in particular, age-associated bone loss in humans or pets. A composition according to claim 1 which is designed to construct cartilage and / or prevent cartilage loss in humans or pets. A composition according to claim 1 which is designed to prevent osteoarthritis in humans or pets. A composition comprising an active ingredient with an effective amount of a rosemary plant or rosemary plant extract containing at least one phytochemical with the ability to induce bone morphogenetic protein expression. A composition according to claim 9, wherein the phytochemical is selected from the group consisting of eupafolin, carno sol, scutellarina, gentuanin, caempferol, acacetin and combinations thereof. A method of making a food composition for the prevention, alleviation and / or treatment of bone disorders or maintenance of bone health in humans or pets, comprising: providing a food composition; Add to the food composition an active ingredient having a plant or plant extract containing at least one phytochemical capable of stimulating bone morphogenetic protein and / or inhibiting bone resorption to prepare the composition. A method according to claim 11, wherein the plant is rosemary. A method according to claim 11, wherein the phytochemical is selected from the group consisting of eupafolin, carnosol, esopelelin, gentuanin, caempferol, acacetin and combinations thereof. A method according to claim 11, wherein the composition includes components chosen from the group consisting of chicory, tea, cocoa, biatives, antioxidants, fatty acids, prebiotic fibers, glycosamine, chondroitin and combinations thereof. A method for treating, alleviating or preventing bone disorder or maintaining bone health, comprising: administering a therapeutically effective amount of a composition comprising an active ingredient having an effective amount of at least one plant or plant extract containing at least one phytochemical with the composition. ability to induce bone morphogenetic protein expression to an individual in need thereof. The method of claim 15, wherein the plant or plant extract further inhibits bone resorption. A method according to claim 15, wherein the plant is rosemary. The method according to claim 15, wherein the phytochemical is selected from the group consisting of eupafolin, carnosol, esopelelin, gentuanin, caempferol, acacetin and combinations thereof. A method of enhancing bone formation, bone mineral density during growth and optimizing peak bone mass in humans or pets, comprising: feeding an individual a composition comprising an active ingredient with an effective amount of at least one plant or extract of plant containing at least one phytochemical capable of inducing bone morphogenetic protein expression. The method of claim 19, wherein the plant or plant extract further inhibits bone resorption. The method of claim 19, wherein the plant is rosemary. The method according to claim 19, wherein the phytochemical is selected from the group consisting of eupafolin, carnosol, esopelelin, gentuanin, caempferol, acacetin and combinations thereof. A method for the treatment, relief and / or prophylaxis of osteoarthritis in pets and humans, comprising: feeding an or at risk osteoarthritis individual with a composition comprising an active ingredient having an effective amount of at least a plant or plant extract containing at least one phytochemical with the ability to induce bone morphogenetic protein expression in the subject. The method of claim 23, wherein the plant or plant extract further inhibits bone resorption. The method of claim 23, wherein the plant is rosemary. The method of claim 23, wherein the phytochemical is selected from the group consisting of eupafolin, carnosol, esopelelin, gentuanin, caempferol, acacetin and combinations thereof. A method for treating or preventing osteoporosis, comprising: administering to an individual having or at risk of osteoporosis a therapeutically effective amount of a composition comprising an active ingredient with an effective amount of at least one plant or plant extract containing at least a phytochemical with the ability to induce bone morphogenetic protein expression in the individual. A method of stimulating bone regeneration during fracture healing, comprising: feeding an individual having a fracture, a therapeutically effective amount of a composition comprising an active ingredient having an effective amount of at least one plant or plant extract containing at least one phytochemical with the ability to induce bone morphogenetic protein expression in the subject. 29. A method for decreasing bone loss, comprising: feeding an individual exhibiting bone loss with a composition comprising an active ingredient having an effective amount minus one plant or plant extract containing at least one phytochemical capable of inducing expression. of bone morphogenetic protein in the individual.