JP7745247B2 - Bone strengthening agent - Google Patents

Description

The present invention relates to a bone strengthening agent, and more specifically, to a bone strengthening agent containing isoflavones and certain other components as active ingredients.

In recent years, osteoporosis has been on the rise, especially among elderly women. It is believed that osteoporosis is caused by poor calcium absorption from the intestines and a decrease in the secretion of female hormones that occurs with age.

On the other hand, soy isoflavones, also known as phytoestrogens, have effects similar to the female hormone estrogen and are known to promote bone formation and inhibit bone resorption (bone destruction).

Such preparations utilizing soy isoflavones have been proposed, for example, a preparation for preventing periodontal disease characterized by containing soy isoflavone aglycone (see Patent Document 1), a plant fermented extract blend characterized by containing a plant fermented extract and isoflavone (see Patent Document 2), and a bone strengthening agent containing a milk-derived basic protein fraction and/or a milk-derived basic protein fraction hydrolysate and soy isoflavone as active ingredients (see Patent Document 3).

Japanese Patent Application Laid-Open No. 2002-121146 JP 2012-240946 A JP 2013-184959 A

The objective of the present invention is to provide a bone strengthening agent that has a high bone formation promoting effect.

The inventors conducted extensive research and studies into the bone-strengthening ability of isoflavones, and discovered that a high bone formation promoting effect can be achieved by combining isoflavones with specific other components, leading to the completion of this invention. In other words, they discovered that by combining isoflavones with specific other components that have little or no bone formation promoting ability, it is possible to synergistically improve bone formation promoting ability.

That is, the present invention relates to a bone-strengthening agent characterized by containing, as active ingredients, isoflavone and at least one component selected from the group consisting of the following (a) to (e):
(a) at least one plant material selected from barley, sweet potato, longevity grass, pine, terminalia, coconut, maqui berry, and kale; (b) at least one additive selected from reduced maltose and calcium stearate; (c) at least one amino acid selected from asparagine, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, phenylalanine, serine, and valine; (d) a polyphenol consisting of chlorogenic acid; and (e) at least one vitamin or mineral selected from vitamin B1, vitamin B2, vitamin B6, vitamin B12, sodium pantothenate, niacin, folic acid, vitamin K, magnesium, iron, selenium, and calcium.

The bone strengthening agent of the present invention may be obtained by adding an active ingredient consisting of isoflavone and at least one component selected from the group consisting of (a) to (e).

In the bone strengthening agent of the present invention, the blending mass ratio of isoflavones to other active ingredients is preferably in the range of 0.5:1 to 70:1.
The bone strengthening agent of the present invention is preferably in the form of a tablet, capsule, powder, granule, or liquid.

The present invention also relates to a bone strengthening method (excluding medical procedures) that involves ingesting the bone strengthening agent of the present invention.

The bone strengthening agent of the present invention can achieve a high bone formation promoting effect and strengthen bones.

This figure shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + plant material) was applied to an osteoblast cell line (MC3T3-E1), and shows, from left to right, cases when barley, sweet potato, chomeisou grass, and pine were used as plant materials. This figure shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + plant material) was applied to an osteoblast cell line (MC3T3-E1), and from left to right, shows the cases when Terminalia, coconut palm, and maqui berry were used as plant materials. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones plus additives) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when reduced maltose was used as the additive, and the graph on the right shows the results when calcium stearate was used as the additive. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + amino acids) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when asparagine was used as the amino acid, and the graph on the right shows the results when cysteine was used as the amino acid. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + amino acids) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when glutamine was used as the amino acid, and the graph on the right shows the results when glutamic acid was used as the amino acid. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + amino acids) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when glycine was used as the amino acid, and the graph on the right shows the results when leucine was used as the amino acid. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + amino acids) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when lysine hydrochloride was used as the amino acid, and the graph on the right shows the results when phenylalanine was used as the amino acid. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + amino acids) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when serine was used as the amino acid, and the graph on the right shows the results when valine was used as the amino acid. FIG. 1 shows the results of Runx2 gene expression when the bone strengthening agent of the present invention (isoflavone + polyphenol) was applied to an osteoblast cell line (MC3T3-E1), and chlorogenic acid was used as the polyphenol. 1 shows the results of Runx2 gene expression when the bone strengthening agent of the present invention (isoflavones plus vitamins and minerals) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when vitamin B1 was used as the vitamin, and the graph on the right shows the results when vitamin B2 was used as the vitamin. 1 shows the results of Runx2 gene expression when the bone strengthening agent of the present invention (isoflavones plus vitamins and minerals) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when vitamin B6 was used as the vitamin, and the graph on the right shows the results when vitamin B12 was used as the vitamin. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones plus vitamins and minerals) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when sodium pantothenate was used as the vitamin, and the graph on the right shows the results when niacin was used as the vitamin. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones plus vitamins and minerals) was applied to an osteoblast cell line (MC3T3-E1). The graph on the left shows the results when folic acid was used as the vitamin, and the graph on the right shows the results when vitamin K was used as the vitamin. This figure shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + vitamins and minerals) was applied to an osteoblast cell line (MC3T3-E1), and shows, from left to right, the cases when magnesium, iron, and serine were used as minerals. This figure shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + vitamins and minerals) was applied to an osteoblast cell line (MC3T3-E1), and shows, from left to right, the results when eggshell calcium, seaweed calcium, and fish calcium were used as minerals. FIG. 1 shows the results of Runx2 gene expression when the bone-strengthening agent of the present invention (isoflavones + kale) was applied to an osteoblast cell line (MC3T3-E1).

The bone strengthening agent of the present invention is characterized by containing, as active ingredients, isoflavones and at least one component selected from the group consisting of (a) to (e) below (hereinafter sometimes referred to as "other component"). The bone strengthening agent of the present invention has a high ability to promote bone formation and strengthens bones by increasing bone mass, thereby preventing and treating osteoporosis.

[Isoflavone]
The isoflavones that are the active ingredients of the bone strengthening agent of the present invention are flavonoid compounds that are abundant in legumes, particularly soybeans, and the isoflavones used in the present invention are preferably soybean isoflavones derived from soybeans. Isoflavones may be aglycones or glycosides. Furthermore, isoflavones may be purified, or may be a mixture with other components of the raw material (e.g., soybeans). Soybean isoflavones are commercially available, and commercially available products can be used.

[Other ingredients]

(a) Plant Material In the bone strengthening agent of the present invention, it is preferable to use at least one plant material selected from barley, sweet potato, chou-mei-sou, pine, terminalia, coconut, maqui berry, and kale as an active ingredient together with isoflavones.

These plant materials may be any part of the plant, such as leaves, stems, roots, flowers, fruits, trunks, or branches. In addition to the plant material itself (including dried products), processed plant material products such as crushed products, juice, and extracts can be used. Crushed products include powder and granules. Juice and extracts may be in liquid form, but can also be used as a paste or dry powder. Extracts can be obtained by extraction using an appropriate solvent, such as water (hot water), ethanol, or aqueous ethanol. These plant materials can be commercially available.

Barley that can be used includes two-row barley and six-row barley. Stems and leaves are preferred as plant materials for the present invention, with young leaves being particularly preferred.

Sweet potato refers to a plant belonging to the Convolvulaceae family, commonly known as Ipomoea batatas. There are no particular limitations on the variety of sweet potato, and examples include Suio, Joy White, Koganesengan, Shiroyutaka, Satsuma Starch, and Ayamurasaki. Stems and leaves are preferred as plant materials for the present invention, with young leaves being particularly preferred.

Chou-mei-sou is a perennial plant also known as Peucedanum japonicum, belonging to the Apiaceae family and the genus Peucedanum. The leaves and roots are preferred plant parts to be used as plant materials in the present invention.

Examples of pines include French maritime pine (Pinus Martima), larch, Japanese black pine, and Japanese red pine. Of these, French maritime pine (Pinus Martima), which is rich in proanthocyanidins, is preferred. The bark is preferred as the plant material for the present invention, and an extract obtained from this pine bark is particularly preferred. This extract contains high concentrations of oligomeric proanthocyanidins in addition to proanthocyanidins; for example, an extract containing 20% or more by mass of oligomeric proanthocyanidins based on the dry mass of the extract is preferred.

Examples of Terminalia include Terminalia bellirica (belerica), Terminalia catappa, Terminalia tomentosa, Terminalia citrina, Terminalia phellocarpa, Terminalia copelandii, Terminalia brassi, Terminalia ivorensis, Terminalia superba, Terminalia arjuna, and Terminalia chebula. Of these, Terminalia bellirica (belerica) and Terminalia chebula are preferred. The fruit is the preferred part of the plant to be used as the plant material of the present invention.

Coconut is a plant of the palm family, and the part of the coconut that is preferably used as the plant material for the present invention is the fruit (endosperm).

Maqui berry is a plant of the Portunaceae family native to Patagonia, Chile, and its scientific name is Aristotellia chilensis. The fruit is preferably used as the plant material for the present invention.

Kale is a plant of the Brassicaceae family, and the leaves and stems are preferably used as the plant material of the present invention. Specifically, dried and crushed kale, dried and powdered kale slurry or squeezed juice, and extracts obtained from kale or its dried products are preferred. Kale that has been dried, extracted, or otherwise processed immediately after harvest is particularly preferred. If time is required before processing, it is preferable to store the kale using storage methods commonly used by those skilled in the art, such as low-temperature storage, to prevent deterioration. There are no particular limitations on the variety of kale, and various types of kale can be used, such as kitchen kale, tree kale, bush kale, marrow kale, collard greens, and green-leaf kale.

(b) Additives In the bone strengthening agent of the present invention, it is preferable to use at least one additive selected from reduced maltose and calcium stearate as an active ingredient together with isoflavone. Reduced maltose and calcium stearate are additives used in the production of tablets, etc., with reduced maltose usually used as an excipient and calcium stearate usually used as a lubricant.

(c) Amino Acids In the bone strengthening agent of the present invention, it is preferable to use, together with isoflavones, at least one amino acid (including a salt) selected from asparagine, cysteine, glutamine, glutamic acid, glycine, leucine, lysine, phenylalanine, serine, and valine as an active ingredient. Examples of the salt include sodium salts and hydrochlorides.

(d) Polyphenols In the bone strengthening agent of the present invention, it is preferable to use polyphenols consisting of chlorogenic acid as an active ingredient together with isoflavones.

(e) Vitamins and Minerals In the bone strengthening agent of the present invention, it is preferable to use, together with isoflavones, at least one vitamin and mineral selected from vitamin B1, vitamin B2, vitamin B6, vitamin B12, sodium pantothenate, niacin, folic acid, vitamin K, magnesium, iron, selenium, and calcium as an active ingredient. Note that iron, selenium, magnesium, and calcium as minerals in the present invention include the form of compounds containing these metals.

The bone strengthening agent of the present invention is not particularly limited as long as it contains isoflavones and other specified ingredients and can be distinguished from other products in that it is used to strengthen bones. For example, the scope of the present invention includes products that display a bone-strengthening effect (bone formation promoting effect) on the product itself, packaging, instructions, or promotional materials. Examples of such products include so-called health foods such as pharmaceuticals (including quasi-drugs), functional foods such as specified health foods, nutrient-functional foods, and functional food products whose efficacy has been approved by a designated organization, as well as feed. Examples of so-called health foods include those that display claims such as "for maintaining bone health," "for those concerned about bone health," and "preventing bone brittleness."

The bone strengthening agent of the present invention may be in the form of, for example, a tablet, capsule, powder, granule, liquid, granule, bar, plate, block, solid, pill, paste, cream, caplet, gel, chewable, or stick. Among these, tablets, capsules, powder, granule, and liquid are particularly preferred. Specific examples include packaged beverages filled in PET bottles, cans, jars, etc., and instant powdered or granular beverages that can be dissolved in water (hot water), milk, fruit juice, green juice, or the like. These are preferred because they are easy to drink during meals and can be palatable.

The amount of isoflavones and other ingredients (active ingredients) contained in the bone strengthening agent of the present invention may be appropriately determined within the range that achieves the desired effect.

In general, when the bone strengthening agent of the present invention is in the form of a pharmaceutical or supplement (tablet, capsule), the active ingredient preferably accounts for 0.01 to 100% by mass, more preferably 0.1 to 85% by mass, and even more preferably 0.5 to 70% by mass, of the total dry mass.

When the bone strengthening agent of the present invention is a packaged beverage (liquid), the active ingredient is preferably contained in an amount of 0.01 to 15% by mass, more preferably 0.03 to 12.5% by mass, and even more preferably 0.05 to 10% by mass, calculated on a dry mass basis.

Furthermore, when the bone strengthening agent of the present invention is an instant powdered drink (powder) or an instant granular drink (granules), the active ingredient is preferably contained in an amount of 0.1 to 80% by mass, more preferably 0.5 to 70% by mass, and even more preferably 1 to 60% by mass, of the total amount converted to dry mass.

To more effectively achieve the effects of the present invention, the active ingredient preferably accounts for 80% or more of the total dry mass of the bone strengthening agent of the present invention (excluding water), more preferably 90% or more, even more preferably 95% or more, and particularly preferably 100%. Furthermore, when the bone strengthening agent of the present invention contains any of components (a) to (e), it is preferable that the components contained therein consist solely of the active ingredient of the present invention. In other words, when the bone strengthening agent of the present invention contains, for example, component (a) (plant material), it is preferable that it does not contain any plant material other than barley, sweet potato, chou-mei-sou, pine, terminalia, coconut, maqui berry, and kale.

There are no particular restrictions on the intake amount of the bone strengthening agent of the present invention, but from the perspective of more significantly exerting the effects of the present invention, it is preferable to ingest the active ingredient so that the daily intake is 0.1 mg/day or more, more preferably 1 mg/day or more, and even more preferably 10 mg/day or more. There is no particular upper limit, but it is, for example, 8 g/day, preferably 4 g/day. It is also preferable to ingest the bone strengthening agent of the present invention so that the intake of isoflavones is 30 mg/day or less (aglycone equivalent).

The bone strengthening agent of the present invention can be stored as a daily dose in a single container or divided into, for example, two or three containers so that the daily intake amount is the above-mentioned amount.

The blending mass ratio of isoflavones to other ingredients, calculated on a dry mass basis, is preferably in the range of 0.5:1 to 70:1, more preferably in the range of 0.75:1 to 60:1, even more preferably in the range of 1:1 to 60:1, and particularly preferably in the range of 1:1 to 50:1. By ensuring that the blending ratio of isoflavones to other ingredients is within the above range, the effects of the present invention can be more effectively achieved.

The bone strengthening agent of the present invention can be manufactured by known formulation methods, adding ingredients other than the orally acceptable active ingredient, as needed.

In addition to bone-strengthening foods containing the active ingredient, the bone-strengthening agent of the present invention can also include bone-strengthening foods obtained by adding the active ingredient to a food, such as a food that does not normally contain the active ingredient of the present invention to which the active ingredient has been added. The active ingredients may be added separately or simultaneously, or together with other ingredients other than the active ingredient.

The bone-strengthening foods of the present invention include, for example, beverages such as carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, smoothies, and green juice; frozen desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; sweets such as candy, candy, gum, chocolate, candy tablets, snacks, biscuits, jelly, jam, cream, baked goods, and bread; processed seafood and livestock foods such as kamaboko, ham, and sausage; processed milk, fermented milk, Examples include dairy products such as yogurt; oils and fats such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, and processed foods thereof; condiments such as sauces and soy sauce; and retort pouch foods such as curry, stew, oyakodon (chicken and egg rice bowl), porridge, rice porridge, Chinese rice bowl, katsudon (pork cutlet bowl), tempura bowl, beef bowl, hayashi rice, omelet rice, oden, mapo dolphin, gyoza (dumplings), shumai (steamed dumplings), hamburger steak, meatballs, various sauces, and various soups.

The bone strengthening method of the present invention is characterized by ingesting the bone strengthening agent of the present invention described above, but does not include medical procedures. For example, the method of the present invention can be a method of strengthening bones by providing the bone strengthening food of the present invention at a restaurant or other eating and drinking establishment.

The present invention will be described below based on examples.
[Example 1]
Osteoblast cell line (MC3T3-E1) cultured in culture medium (MEMa; Alpha modification of Eagle's Minimum Essential Media, 10% FBS, 1% PS) was seeded into a 96-well plate at a density of 5 x 10 ³ cells/well.

Subsequently, 0.1 mL of 0.5 μg/mL isoflavone or a solution containing other components dissolved at specified concentrations as shown in Tables 1 to 5 was added, and the cells were cultured for 48 hours. Runx2 gene expression was measured by real-time PCR (preliminary WST confirmation confirmed that the concentration was such that more than 90% of cells survived compared to the control). The Runx2 gene is a transcription factor essential for osteoblast differentiation, and is known to induce osteoblast differentiation and promote bone formation.

The results are shown in Tables 1 to 5 and Figures 1 to 15. Each graph in the figure represents, from left to right, the results of "Control- (no addition)," "Control+ (0.5 μg/mL isoflavones added alone)," "Other ingredients added alone," and "0.5 μg/mL isoflavones + other ingredients added." The numbers in the legend below the graphs indicate the sample concentration; for example, "250-" for barley in the Figure 1 graph indicates 250 μg/mL barley. The concentration unit for added ingredients is μg/mL in all graphs. The graphs are expressed as relative values, with the value for Control+ set to 1.

For barley, dried and ground powder of young leaves was used.
For sweet potatoes, dried and crushed young leaves were used.
For Chomeisou, dried and crushed powder of leaves was used.
For pine, a hot water extract (dried powder) of the bark of French maritime pine was used.
For Terminalia, a hot water extract (dried powder) of the fruit was used.
For the coconut, commercially available coconut milk was used after drying and removing the oil.
For maqui berry, commercially available dried powder of the fruit was used.
For kale, dried and crushed powder of leaves was used.
For mugwort, finely ground fresh leaves were used.

As shown in Tables 1 to 5 and Figures 1 to 15, the combination of isoflavones with specific other components of the present invention synergistically increased the expression level of the Runx2 gene in osteoblast cell lines. Therefore, the bone-strengthening agent of the present invention promotes bone formation and effectively strengthens bones.

[Example 2] (Production of tablets)
A mixture consisting of 25 mg of soy isoflavones, 10 mg of aspartame, 50 mg of reduced maltose, 200 mg of calcium stearate, 10 mg of cysteine, 5 mg of lysine hydrochloride, 5 mg of phenylalanine, 0.1 mg of serine, 5 mg of niacin, 0.05 mg of folic acid, 0.1 mg of vitamin K, 0.01 mg of iron, 0.02 mg of magnesium, 50 mg of maqui berry, 0.2 mg of pine bark extract, and 50 mg of fish calcium was compressed into tablets.

[Example 3] (Production of capsules)
A mixture consisting of 50 mg of soy isoflavone, 180 mg of aspartame, 50 mg of reduced maltose, 350 mg of calcium stearate, 10 mg of cysteine, 5 mg of lysine hydrochloride, 5 mg of phenylalanine, 0.1 mg of serine, 5 mg of niacin, 0.05 mg of folic acid, 0.1 mg of vitamin K, 0.01 mg of iron, 0.02 mg of magnesium, 50 mg of coconut, and 0.2 mg of seaweed calcium was encapsulated in a soft capsule to produce a capsule preparation.

Example 4 (Production of Instant Powder 1)
A powder formulation was prepared by mixing 50 mg of soy isoflavones, 180 mg of aspartame, 200 mg of reduced maltose, 200 mg of maltose, 200 mg of calcium stearate, 250 mg of asparagine, 25 mg of glutamine, 25 mg of glutamic acid, 20 mg of glycine, 20 mg of leucine, 10 mg of chlorogenic acid, 0.1 mg of vitamin B1, 0.1 mg of vitamin B2, 0.1 mg of vitamin B6, 0.05 mg of vitamin B12, 0.05 mg of sodium pantothenate, 0.2 mg of folic acid, 0.1 mg of vitamin K, 2000 mg of indigestible dextrin, 2000 mg of barley leaf powder, 2000 mg of kale powder, 500 mg of finely ground green tea powder, and 250 mg of shellfish calcium. This powder formulation showed good solubility when dissolved in 100 ml of water.

Example 5 (Production of Instant Powder 2)
A powder formulation was produced by mixing 100 mg of soy isoflavones, 1000 mg of reduced maltose, 1200 mg of maltose, 300 mg of sugar, 2000 mg of indigestible dextrin, 250 mg of asparagine, 25 mg of glutamine, 25 mg of glutamic acid, 20 mg of glycine, 20 mg of leucine, 10 mg of chlorogenic acid, 0.1 mg of vitamin B1, 0.1 mg of vitamin B2, 0.1 mg of vitamin B6, 0.05 mg of vitamin B12, 0.05 mg of sodium pantothenate, 0.2 mg of folic acid, 0.1 mg of vitamin K, 2000 mg of young sweet potato leaf powder, 2000 mg of kale powder, 500 mg of finely ground green tea powder, and 10 mg of uncalcined coral calcium.
This powder had good solubility when dissolved in 200 ml of water.

[Example 6] (Production of a liquid preparation in a PET bottle)
A liquid formulation was prepared by filling a plastic bottle with 75 mg of soy isoflavones, 2500 mg of reduced maltose, 350 mg of asparagine, 50 mg of glutamine, 50 mg of glutamic acid, 50 mg of glycine, 20 mg of leucine, 20 mg of valine, 20 mg of chlorogenic acid, 0.2 mg of vitamin B1, 0.2 mg of vitamin B2, 0.2 mg of vitamin B6, 0.2 mg of vitamin B12, 0.1 mg of sodium pantothenate, 0.2 mg of folic acid, 0.1 mg of vitamin K, 2500 mg of young barley leaf powder, 2500 mg of young sweet potato leaf powder, 1000 mg of Chou-mei-so, 50 mg of pine bark extract, 250 mg of Terminalia bellirica, 500 mg of uncalcined coral calcium, and 500 ml of water, and sealing the bottle.

The bone strengthening agent of the present invention has the effect of strengthening bones and can be used as an oral agent, making the present invention highly industrially useful.

Claims (1)

A tablet, capsule, powder or granule (excluding foods containing natto, miso or miso-like foods) characterized in that it contains isoflavone as an active ingredient for maintaining bone health and further contains at least one amino acid selected from the group consisting of valine, phenylalanine, cysteine or serine (excluding those derived from soybean or whey protein hydrolysate ) .