Bone metabolism improver

Abstract

To provide a bone metabolism improver composed of a novel ingredient.SOLUTION: A feature of the present invention for solving the problem is as follows: 1) an osteoclast differentiation inhibitor containing at least one of ferulic acid, astaxanthin, α-lipoic acid, Coprinus comatus extract, Cistanche plant extract, Subgen. Cerasus extract, γ-oryzanol, plant- and / or processed plant-derived polyamine composition, and Petasites extract as an active ingredient; 2) an osteoblast differentiation promotor containing at least one of α-lipoic acid, Cistanche plant extract, and ferulic acid as an active ingredient; 3) a promoter for expression of BSPII in an osteoblast precursor containing Cistanche plant extract as an active ingredient; 4) a promoter for expression of type 1 collagen in an osteoblast precursor containing Cistanche plant extract as an active ingredient; and 5) a bone metabolism improver containing at least one agent of 1) to 4) as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to health foods and pharmaceuticals for improving bone metabolism. [Background technology]

[0002] Osteoporosis is a serious social problem affecting over 10 million people in Japan. Furthermore, the rapid aging of the population means an increase in the number of people aged 65 and over, and it is predicted that the number of osteoporosis patients will continue to increase. Osteoporosis is a condition in which bone strength is reduced, increasing the risk of fractures. Therefore, its treatment and prevention are necessary to realize a healthy, long-lived society.

[0003] In the human body, bones are constantly undergoing repeated cycles of bone resorption and bone formation. The cells involved in bone resorption are osteoclasts, and the cells involved in bone formation are osteoblasts. Bone formation, maintenance, and repair depend on the balance between formation and resorption of these cells, and it is known that disruption of this balance can cause bone resorption to exceed bone formation, resulting in bone metabolic disorders such as osteoporosis. Therefore, it is thought that ensuring that bone formation does not exceed bone resorption is important for maintaining and increasing bone mass.

[0004] Because bone resorption is carried out by osteoclasts, the greater the differentiation and activation of osteoclasts, the higher the bone resorption rate. On the other hand, because bone formation is carried out by osteoblasts, the greater the differentiation and activation of osteoblasts, the higher the bone formation rate. Therefore, if a composition could be obtained that has a bone resorption inhibitory effect (reducing the bone resorption rate) and an osteogenic effect (enhancing the bone formation rate), it would be effective in treating osteoporosis. Examples of compositions that have been reported to have such effects include a bone metabolism-improving composition containing, as active ingredients, a ginger extract containing 6-gingerol and 6-shogaol, the pungent components of ginger (see Patent Document 1), and a bone metabolism-improving composition containing, as an active ingredient, toddaculin derived from Toddalia asiatica (scientific name: Toddalia asiatica) (see Patent Document 2) (Patent Document 3).

[0005] In addition, various bone metabolism markers are being developed to complement bone mineral density measurements. The non-collagenous bone matrix protein BSPII has also been suggested to be related to bone metabolism, as its blood concentration increases in postmenopausal women (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2014-043416 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-069355 [Patent Document 3] Japanese Patent Application Publication No. 2018-123111 [Non-patent literature]

[0007] [Non-Patent Document 1] W. Withold et al., Clinical Chemistry, Volume 43, Number 1, pages 85-91 (1997). Summary of the Invention [Problem to be solved by the invention]

[0008] Under these circumstances, the present inventors have discovered that ferulic acid, astaxanthin, α-lipoic acid, Coprinus comatus extract, plant extracts of the Orobanchaceae family, cherry blossom extract, γ-oryzanol, polyamine compositions derived from plants and / or processed plant products, and butterbur extract have the effect of inhibiting osteoclast differentiation; that α-lipoic acid, plant extracts of the Orobanchaceae family, and ferulic acid promote osteoblast differentiation; and that plant extracts of the Orobanchaceae family promote the expression of BSPII and type 1 collagen in preosteoblasts, thereby completing the present invention. That is, an object of the present invention is to provide a bone metabolism improving agent containing a novel component as an active ingredient. [Means for solving the problem]

[0009] The features of the present invention for solving the above problems are as follows. 1. An osteoclast differentiation inhibitor containing as an active ingredient at least one of ferulic acid, astaxanthin, alpha-lipoic acid, Coprinus comatus extract, Orobanchaceae plant extract, cherry blossom extract, gamma-oryzanol, a polyamine composition derived from plants and / or processed plant products, and butterbur extract. 2. An osteoblast differentiation promoter containing at least one of the following active ingredients: alpha-lipoic acid, plant extract from the Orobanchaceae family, and ferulic acid. 3. A BSPII expression promoter in osteoblast precursor cells, containing an extract of a plant from the Orobanchaceae family as an active ingredient. 4. A type 1 collagen expression promoter in osteoblast precursors, containing an extract of a plant from the Orobanchaceae family as its active ingredient. 5. A bone metabolism improving agent containing at least one of the agents selected from 1. to 4. above as an active ingredient. [Effects of the Invention]

[0010] According to the present invention, ferulic acid, astaxanthin, α-lipoic acid, Coprinus comatus extract, Orobanchaceae plant extract, cherry blossom extract, γ-oryzanol, a polyamine composition derived from plants and / or processed plant products, and butterbur extract have an inhibitory effect on osteoclast differentiation, making these components useful as inhibitors of osteoclast differentiation. Furthermore, according to the present invention, α-lipoic acid, the plant extract of the Orobanchaceae family, and ferulic acid have the effect of promoting osteoblast differentiation, making these components useful as agents for promoting osteoblast differentiation. Furthermore, according to the present invention, an extract of a plant belonging to the Orobanchaceae family has an effect of promoting BSPII expression in osteoblast precursors, and is therefore useful as a BSPII expression promoter. Plant extracts from the Orobanchaceae family have the effect of promoting type 1 collagen expression in osteoblast precursors, making them useful as agents for promoting type 1 collagen expression in osteoblast precursors. For these reasons, ferulic acid, astaxanthin, α-lipoic acid, Coprinus comatus extract, Orobanchaceae plant extract, cherry blossom extract, γ-oryzanol, polyamine compositions derived from plants and / or processed plant products, and butterbur extract, particularly Orobanchaceae plant extract, are useful as bone metabolism improvers. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing the results of an investigation into the effects of various food extracts on osteoclast differentiation. [Figure 2] 1 is a graph showing the results of an investigation into the effects of various food extracts on osteoblast differentiation. [Figure 3] FIG. 1 shows the effect of Cistanche Tubulosa on osteoblast differentiation markers. [Figure 4] 1 shows photographs showing the effect of Cistanche Tubulosa on the localization and expression of type I collagen during osteoblast differentiation. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. The bone metabolism improving agent of the present invention contains at least one active ingredient selected from the group consisting of ferulic acid, astaxanthin, α-lipoic acid, Coprinus comatus extract, Orobanchaceae plant extract, cherry blossom extract, γ-oryzanol, a polyamine composition derived from processed plants, and butterbur extract.

[0013] The ferulic acid is a compound represented by the following chemical formula (1).

[0014] [ka] Chemical formula (1)

[0015] The method for obtaining ferulic acid is not particularly limited, and a commercially available product may be used. For example, "Ferulic Acid" manufactured by Oryza Oil & Fat Chemical Co., Ltd. may be used.

[0016] The astaxanthin is a compound represented by the following chemical formula (2).

[0017] [ka]

[0018] As the astaxanthin used in the present invention, it is preferable to use an ester thereof. Examples of astaxanthin esters include, but are not limited to, monoesters or diesters of saturated fatty acids such as palmitic acid and stearic acid, or unsaturated fatty acids such as oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, bishomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. These can be used alone or in appropriate combinations. Astaxanthin has a structure in which an extra oxo group and a hydroxyl group are present at both ends of the β-carotene backbone, and therefore, unlike β-carotene, the molecule is less stable. In contrast, esters in which the hydroxyl groups at both ends are esterified with unsaturated fatty acids (e.g., krill extract) are more stable.

[0019] The astaxanthin and / or its esters used in the present invention may be chemically synthesized or derived from natural products. Examples of the latter natural products include red yeast, Tigliopus (red water flea), crustacean shells such as krill, and algae such as Haematococcus, which contain astaxanthin and / or its esters. In the present invention, extracts containing astaxanthin and / or its esters produced by any method can be used, as long as they utilize the properties of astaxanthin and / or its esters. Generally, extracts from these natural products are used, and they may be in the form of an extracted extract or, if necessary, purified. In the present invention, crude extracts or crushed powders containing astaxanthin and / or its esters, or those that are optionally purified or chemically synthesized, can be used alone or in combination. Considering stability in the body, esters are preferably used. As the astaxanthin, a commercially available product may be used, for example, "Astaxanthin" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0020] The α-lipoic acid is also called thioctic acid and is a compound represented by the following chemical formula (3).

[0021] [ka]

[0022] The method for producing the α-lipoic acid is not particularly limited, and it can be produced by a known method. In addition, the α-lipoic acid can be a commercially available product, such as "α-lipoic acid-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0023] Coprinus comatus, the raw material for the above-mentioned Coprinus comatus extract, is an edible mushroom with a cylindrical, white, silky cap covered in white, furry scales. However, because its beauty only lasts for a few days in the wild, it is called a phantom mushroom, and due to its rarity, it is known as a luxury food ingredient in Italy. The extraction solvent is not particularly limited, but polar solvents such as water, methanol, ethanol, isopropyl alcohol, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, etc. Two or more of these solvents may be mixed. Preferably, water or ethanol is used as the extraction solvent to efficiently extract the active ingredient. In particular, water is a preferred extraction solvent because it is less likely to reduce the activity of the active ingredient during extraction and is also safe for use in food. The type of water used for extraction is not particularly limited, and tap water, distilled water, mineral water, alkaline ionized water, deep sea water, etc. can be used.

[0024] As the extraction method, any method such as continuous extraction, immersion extraction, countercurrent extraction, or supercritical extraction can be employed, and any apparatus can be used at room temperature or under reflux heating.

[0025] Specifically, the extraction method involves placing the raw material (Coprinus comatus) in a treatment tank filled with an extraction solvent and stirring to extract the active ingredients. For example, when using water as the extraction solvent, approximately 5 to 100 times the amount (by weight) of the extraction solvent as the raw material is used, and extraction is carried out for approximately 30 minutes to 2 hours. After the active ingredients have been extracted into the solvent, the extract is obtained by filtering to remove the extraction residue.

[0026] Thereafter, the extract is subjected to treatments such as dilution, concentration, drying and purification according to conventional methods to obtain the bone metabolism improving agent of the present invention. The active ingredient may be purified by passing the extract through a synthetic adsorption resin, gel filtration resin, or the like to adsorb the active ingredient, eluting it with methanol, ethanol, or the like, and concentrating it under reduced pressure.

[0027] Furthermore, the extract of Coprinus comatus is characterized by containing ergothioneine. Ergothioneine is a type of amino acid represented by the following formula (4), and has been found to be widely distributed in the red blood cells and liver of animals, including humans. It has been reported to have antioxidant activities such as scavenging hydroxyl radicals, inhibiting the generation of hydroxyl radicals from hydrogen peroxide in an Fe- and Cu-dependent manner, inhibiting the oxidation of copper-dependent oxyhemoglobin, and inhibiting the oxidation of arachidonic acid by myoglobin and hydrogen peroxide.

[0028] [ka]

[0029] The content of ergothioneine contained in the above-mentioned extract of Coprinus comatus is not particularly limited, but can be 0.01 wt% or more, or even 0.1 to 20 wt%, when the total mass of the extract of Coprinus comatus is 100 wt%.

[0030] The above-mentioned Coprinus comatus extract can be a commercially available product, for example, "Coprino (registered trademark) Extract-P0.5" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0031] It is known that fresh stems of herbs belonging to the Cistanche family are effective in treating infertility, impotence, constipation, etc. (See Patent Document 1). In addition, preparations obtained from the fresh stems of such perennial herbs nourish the blood and kidneys. These parasitic and perennial herbs are widely cultivated in northwestern China and are known locally as "desert ginseng." The most widely cultivated Cistanche species is the holoparasitic plant of the Cistanche family, Cistanche tubulosa.

[0032] The plant of the Orobanchaceae family used as a raw material is not particularly limited, and examples thereof include, but are not limited to, the holoparasitic plant Cistanche tubulosa (Cistanche tubulosa), Cistanche salsa (Cistanche salsa), Aeginetia indica (Aeginetia indica), and Boschniakia rossica (Boschniakia rossica). These may be used alone or in combination. Of these, the holoparasitic plant Cistanche tubulosa (Cistanche tubulosa) is particularly preferred.

[0033] The method for obtaining an extract from the raw material plant of the Orobanchaceae family is not particularly limited, and examples thereof include solvent extraction and supercritical extraction.

[0034] In addition, when extraction is performed by solvent extraction, the solvent to be used is not particularly limited, but it is preferable to use a polar solvent. In addition, the polar solvent is not particularly limited, but examples thereof include water, methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, acetic acid, ethyl acetate, ether, etc. These may be used alone or in combination of two or more. It is preferable to then further subject the extract to treatment such as dilution, concentration, drying, purification, etc., since this allows an extract containing phenylethanoid glycosides at a high concentration to be obtained. Examples of purification methods include activated carbon treatment, resin adsorption treatment, ion exchange resin treatment, and liquid-liquid countercurrent distribution. Commercially available products can also be used as the plant extract of the Orobanchaceae family, such as "Cistanche Tubulosa Extract-P25" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0035] Sakura (cherry blossom, cherry tree) used as a raw material for cherry blossom extract is a general term for plants of the genus Prunus in the family Rosaceae, excluding plum, peach, apricot, etc., and generally refers to those belonging to the subgenus Cerasus.

[0036] The cherry species used in the present invention is not particularly limited, and for example, cherry trees belonging to groups such as the Yamazakura group, Edohigan group, Mamezakura group, Chojizakura group, Miyamazakura group, and Shinamizakura group can be used, but are not limited to these groups.

[0037] There are no particular limitations on the parts of the cherry tree that can be used as raw materials in the present invention, and examples include leaves, stems, trunks, flowers, roots, and fruits, with the use of leaves or flowers being preferred.

[0038] When extracting by polar solvent extraction, the polar solvent to be used is not particularly limited, but examples include water, methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, acetic acid, ethyl acetate, ether, hexane, etc. Among these, water, methanol, and ethanol are preferred because they allow for efficient extraction of active ingredients. These solvents may be used alone or in combination of two or more.

[0039] When using water as the extraction solvent, the extraction temperature should be 20-100°C, preferably 40-70°C. Too low an extraction temperature makes it difficult to extract the active ingredients, while too high an extraction temperature is undesirable because it can lead to residual cyanide compounds in the cherry blossoms and the active ingredients being easily decomposed. The type of water used for extraction is not particularly limited; tap water, distilled water, mineral water, alkaline ionized water, etc. can be used.

[0040] When using aqueous alcohol as the extraction solvent, the alcohol concentration is preferably 20 wt%, more preferably 25 wt% to 50 wt%, and even more preferably 25 wt% to 30 wt%. If the alcohol concentration is less than 20 wt%, it becomes difficult to extract a high amount of active ingredients, and if the alcohol concentration exceeds 50 wt%, the yield decreases due to the influence of impurities, etc. When the alcohol concentration is 30 wt % or more, the extraction temperature should be 0 to 95° C., preferably about 0 to 60° C. The aqueous ethanol extraction should be repeated at various concentrations to increase the content of the active ingredient.

[0041] Furthermore, when extraction is performed using a polar solvent, the extraction method is not particularly limited, and any method such as continuous extraction, immersion extraction, or countercurrent extraction can be used, and any device can be used at room temperature or under reflux heating. When extraction is performed using the above-mentioned methods, only one of these methods may be performed, or these methods may be combined. Furthermore, these extractions may be performed only once or two or more times.

[0042] The cherry blossom extract may be a commercially available product, for example, "Sakura Flower Extract-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0043] γ-oryzanol is a mixture of esters of ferulic acid and plant sterols or triterpene alcohols. Plant sterols include campesterol, β-sitosterol, and stigmasterol. Triterpene alcohols include cycloartenol, 24-methylenecycloartanol, cyclobulanol, and cyclosadol.

[0044] The γ-oryzanol is not particularly limited as long as it contains the above-mentioned compounds, and commercially available products can also be used. For example, "γ-oryzanol" manufactured by Oryza Oil & Fat Chemical Co., Ltd. can be used.

[0045] Polyamines, a general term for aliphatic hydrocarbons with two or more primary amino groups, are natural products that are ubiquitously present in living organisms, with over 20 types having been discovered. Representative polyamines include putrescine, spermidine, and spermine. The main physiological effects of polyamines are known to be (1) stabilization and structural changes of nucleic acids through interactions with nucleic acids, (2) promotion of various nucleic acid synthesis pathways, (3) activation of protein synthesis pathways, (4) stabilization of cell membranes and enhancement of membrane permeability of substances, (5) scavenging of reactive oxygen species, (6) promotion of cell proliferation, and (7) anti-allergic effects.

[0046] In the present invention, "plants and / or processed plant products" refers to various plants or processed plant products, and includes, but is not limited to, dicotyledonous plants, monocotyledonous plants, herbaceous plants, woody plants, Cucurbitaceae plants, Solanaceae plants, Gramineae plants, Cruciferae plants, Leguminosae plants, Malvaceae plants, Asteraceae plants, Chenopodiaceae plants, Leguminosae plants, processed plant products, plant extracts, etc. For example, sweet potato, tomato, cucumber, pumpkin, melon, watermelon, tobacco, Arabidopsis, bell pepper, eggplant, bean, taro, spinach, carrot, strawberry, potato, rice, corn, alfalfa, wheat, barley, soybean, rapeseed, sorghum, eucalyptus, poplar, kenaf, eucommia, sugarcane, sugar beet, cassava, sago palm, pigweed, lily, orchid, carnation, rose, chrysanthemum, petunia, torenia, goldfish Examples of ingredients that may be used include grass, cyclamen, baby's breath, geranium, sunflower, lawn grass, cotton, enoki mushroom, shimeji mushroom, matsutake mushroom, shiitake mushroom, mushrooms, ginseng, agaricus, turmeric, ginseng, citrus fruits, green tea, black tea, oolong tea, banana, kiwi, natto, soy milk, soybean extract, wheat extract, germ extract, germ extract, fruit juice, okara, rice germ, wheat germ, barley germ, soybean germ, corn germ, milo germ, and sunflower germ. Preferably, monocotyledonous or dicotyledonous plants are used, more preferably grasses or legumes, and particularly preferably corn, mushrooms, soybeans, wheat, rice, natto, soy milk, okara, wheat germ, soybean germ, corn germ, soybean extract, wheat extract, germ extract, and germ extract. Polyamines may be recovered from plants that provide a large amount of net food per capita per year, such as soybeans, rice, and wheat.

[0047] The plant tissue from which the polyamine composition is recovered is not particularly limited. It is preferably in the form of a seed or in the process of growth. Plants in the process of growth can be recovered from the whole or partial tissue. Parts from which the polyamine composition can be recovered include, but are not particularly limited to, the whole plant, flowers, buds, ovaries, fruits, leaves, cotyledons, stems, buds, roots, seeds, dried seeds, embryos, germs, and roots. Fruits, leaves, stems, buds, seeds, dried seeds, germs, and embryos are preferred, and seeds, dried seeds, germs, and embryos are particularly preferred. Among these, it is particularly preferred to use at least one of soybean germ, rice germ, and wheat germ.

[0048] The polyamine composition derived from plants and / or processed plant products can be produced by the following method. That is, a plant-derived polyamine composition can be obtained by subjecting a plant and / or a processed plant product to acidic conditions and then separating the liquid fraction.

[0049] Here, "under acidic conditions" refers to conditions where the pH is 6 or less. By maintaining the pH under acidic conditions during purification, the effect of efficient and stable recovery of polyamine compositions from plant tissues can be obtained. This effect is uniformly achieved at a pH of 6 or less, but a pH of 6 to 3 is preferred, and a pH of 4.5 to 3.5 is particularly preferred.

[0050] In the present invention, "processed plant products" refer to products obtained from plants or processed products thereof. The preparation method involves extracting plants using water, organic solvents, or mixtures of water and organic solvents under low, room, or heated conditions, using methods such as impregnation, distillation, compression, ultrasonication, supercritical fluid processing, and subcritical fluid processing. Furthermore, processed products obtained by fermenting plants or extracts collected from plants are also included. Examples include plant extracts, soy milk, okara, wheat flour, fermented extracts, and natto.

[0051] Examples of the acid solution to be added to create an acidic condition include mineral acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; organic acids such as acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, lactic acid, tartaric acid, citric acid, and benzoic acid; and acidic water. Examples of the acid solution to be added to create an acidic condition include inorganic acids and organic acids such as 0.01N to 6N hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, trichloroacetic acid, sulfosalicylic acid, formic acid, citric acid, and lactic acid; and 0.1 to 10% perchloric acid.

[0052] In addition, polyamines contained in plants and / or processed plant products are thoroughly extracted into an acid solution (liquid fraction), and then the liquid fraction is separated from residues and precipitates by centrifugation or filtration. The recovered liquid fraction contains a large amount of polyamines and is obtained as a "polyamine composition."

[0053] In the present invention, "polyamine" refers to a general term for aliphatic hydrocarbons with two or more primary amino groups, and is a natural product ubiquitously present in living organisms. More than 20 types of polyamines have been discovered. Examples include 1,3-diaminopropane, putrescine, cadaverine, caldine, spermidine, homospermidine, aminopropylcadaverine, thermine, spermine, thermospermine, canavalmine, aminopentylnorspermidine, N,N-bis(aminopropyl)cadaverine, homospermine, caldopentamine, homocaldopentamine, caldohexamine, and homocaldohexamine. Representative polyamines include putrescine, spermidine, and spermine.

[0054] "Putrescine" as referred to in this invention is a typical polyamine, a common natural product ubiquitously present in living organisms, and an aliphatic hydrocarbon compound with two primary amino groups. "Spermidine" is a typical polyamine, a common natural product ubiquitously present in living organisms, and an aliphatic hydrocarbon compound with three primary amino groups. "Spermine" is a typical polyamine, a common natural product ubiquitously present in living organisms, and an aliphatic hydrocarbon compound with four primary amino groups. Cadaverine is a typical polyamine, a common natural product that is ubiquitously present in living organisms, and is an aliphatic hydrocarbon compound with two primary amino groups.

[0055] If necessary, the polyamine composition may be desalted or purified by ion exchange, membrane fractionation, gel filtration, or electrodialysis. By performing at least one of these methods, a more highly purified polyamine composition can be obtained. For example, in the ion exchange method, a polyamine solution is passed through a column packed with an ion exchange resin to separate the polyamine from impurities such as amino acids, peptides, proteins, and sugars. The ion exchange resin used may have ion exchange groups such as sulfonic acid groups, sulfopropyl groups, phosphate groups, carboxymethyl groups, aminoethyl groups, diethylamino groups, quaternary aminoethyl groups, or quaternary ammonium groups. Either a cation exchange resin or an anion exchange resin can be used. When a cation exchange resin is used, the polyamine is adsorbed onto the cation exchange resin. After sufficient separation of non-adsorbed substances, the polyamine is eluted with an acidic solution such as sulfuric acid or hydrochloric acid, or a salt solution such as sodium chloride. When an anion exchange resin is used, the polyamine is not adsorbed onto the anion exchange resin, and the non-adsorbed fraction containing the polyamine is recovered. For example, in membrane fractionation, a polyamine composition is subjected to ultrafiltration (UF) using a cellulose-based, cellulose acetate-based, polysulfone-based, polyamide-based, polyacrylonitrile-based, polytetrafluoroethylene-based, polyester-based, polypropylene-based, or other ultrafiltration membrane with a molecular weight cutoff of 1,000 to 100,000, and a permeate containing polyamines is recovered. Alternatively, a polyamine solution is subjected to nanofiltration (NF) using a nanofiltration membrane with a salt rejection of 30 to 80% to desalt it. For example, in gel filtration, a polyamine composition is neutralized and passed through a column packed with a gel filtration carrier to recover polyamines by molecular weight fractionation. The gel filtration carrier used is a dextran-based, acrylamide-based, agarose-based, cellulose-based, polyvinyl-based, glass-based, polystyrene-based, or other gel filtration carrier with a molecular weight cutoff of 100 to 100,000. For example, in electrodialysis, a polyamine composition and saline are alternately supplied between cation-exchange and anion-exchange membranes to perform electrodialysis.

[0056] Commercially available polyamine compositions derived from plants and / or processed plant products can be used, and examples thereof include "Polyamine-P" and "Oryza Polyamine-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0057] Butterbur is a perennial plant of the Petasites genus in the Asteraceae family, and is known as "Houtosai" in Chinese herbal medicine. It is dioecious and has a rhizome that runs horizontally underground, with broad-brimmed leaf blades growing on petioles that emerge from this rhizome. The flowers appear in early spring before the leaves unfold, and are formed by corymbs of tubular flower heads (flower stalks: butterbur shoots) at the top of short, upright stems. It has been cultivated as a vegetable native to Japan since ancient times, and its inflorescences, flower stalks, and petioles are edible. Wild butterbur is also harvested and used. Cultivated varieties include Aichi Wase-buki, Mizu-buki (Kyoto-buki), Akita-buki, and Rawan-buki.

[0058] In the present invention, the part of the butterbur used as the extraction material is not limited, and any of the petiole, leaf blade, inflorescence, rhizome, etc. may be used. However, it is preferable to use the above-ground part of the butterbur (petiole and leaf blade). This is because the petiole and leaf blade of the butterbur contain a large amount of components that have the effect of improving bone metabolism. The above-ground part of the butterbur may also be the inflorescence (butterbur stalk), fruit, etc. Regarding the plant species, either male or female plants may be used. There are no limitations on the cultivated variety or cultivation period of butterbur.

[0059] The solvents that can be used to extract the butterbur extract from the raw material include water, methanol, ethanol, isopropyl alcohol, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, acetone, etc. Two or more of these solvents may be mixed. It is preferable to use aqueous ethanol as the extraction solvent. In particular, aqueous ethanol is less likely to reduce the activity of the active ingredients during extraction, making it a preferred extraction solvent in terms of safety when using the extract in food. The type of water used for extraction is not particularly limited, and tap water, distilled water, mineral water, alkaline ionized water, deep sea water, etc. can be used.

[0060] Regarding the extraction temperature, for example, when aqueous ethanol is used, the extraction temperature should be 20 to 80° C., preferably about 60 to 70° C. If the extraction temperature is too low, it becomes difficult to extract the active ingredient, and if the extraction temperature is too high, the activity of the active ingredient is likely to decrease.

[0061] The aqueous ethanol used as the extraction solvent should have an ethanol concentration of 40 to 90% (wt / wt). The ethanol concentration should be 40% (wt / wt) or higher because too little ethanol reduces the amount of extraction, while too high an ethanol concentration makes it difficult to extract the bone metabolism-improving components. An ethanol concentration of 60 to 80% (wt / wt), and even more preferably about 70% (wt / wt), is desirable. Furthermore, ethanol extraction can be repeatedly performed while gradually changing the ethanol concentration to increase the content of the active ingredient.

[0062] As a method for extracting the butterbur extract, any method such as continuous extraction, immersion extraction, countercurrent extraction, or supercritical extraction can be used, and any device can be used at room temperature or under reflux heating.

[0063] The extraction solvent may be a polar solvent such as water, methanol, ethanol, isopropyl alcohol, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, ethyl acetate, etc. Two or more of these solvents may be mixed. Preferably, when aqueous ethanol or a mixture of aqueous ethanol is used as the extraction solvent, the active ingredients can be extracted efficiently.

[0064] When water is used as the extraction solvent, the type of water is not particularly limited, and tap water, distilled water, mineral water, alkaline ionized water, deep sea water, etc. can be used.

[0065] When using aqueous ethanol as the extraction solvent, the ethanol concentration is not particularly limited, but it is particularly preferred that the ethanol concentration be 10 to 90% (wt / wt), preferably 20 to 80% (wt / wt).The reason for limiting the ethanol concentration to 90% (wt / wt) or less is that if the ethanol concentration is too high, the active ingredient cannot be extracted.

[0066] The extraction temperature should be 20 to 80° C., preferably about 40 to 50° C. If the extraction temperature is too low, it becomes difficult to extract the active ingredients, and if the extraction temperature is too high, the active ingredients will decompose, resulting in a decrease in physiological activity (health functionality).

[0067] As the extraction method, any method such as stirring extraction, continuous extraction, immersion extraction, countercurrent extraction, and supercritical extraction can be employed, and any apparatus can be used at room temperature or under reflux heating.

[0068] In addition, a commercially available product can be used as the butterbur extract, for example, "Butterbur Extract-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0069] The bone metabolism improving agent of the present invention can be used as an ingredient in various foods and beverages. Examples of foods and beverages include general foods such as confectioneries (gum, candy, caramel, chocolate, cookies, snacks, jelly, gummy candy, tablet candy, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, yogurt, etc.), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, black tea, green tea, carbonated drinks, sports drinks, etc.), as well as health foods (tablets, capsules, etc.), and nutritional supplements (nutrition drinks, etc.). The bone metabolism improving agent of the present invention can be appropriately blended into these foods and beverages.

[0070] These foods and beverages can contain various ingredients depending on their type, such as glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, colorants, flavorings, and preservatives.

[0071] Specifically, the bone metabolism improving agent can be spray-dried or freeze-dried together with powdered cellulose, and then formed into a powder, granules, tablets, or solution, which can be easily incorporated into foods and beverages (e.g., instant foods). Alternatively, the bone metabolism improving agent can be dissolved in, for example, fats and oils, ethanol, glycerin, or a mixture thereof to form a liquid, which can then be added to beverages or solid foods. If necessary, the agent can also be mixed with a binder such as gum arabic or dextrin to form a powder or granules, which can then be added to beverages or solid foods.

[0072] When the bone metabolism improving agent of the present invention is applied to food and drink, the amount of the active ingredient added is preferably 1 to 20 wt % in total relative to the food and drink, since the main purpose is disease prevention and health maintenance.

[0073] The bone metabolism improving agent of the present invention may be used as a material for medicines (including pharmaceuticals and quasi-drugs). Pharmaceutical preparations can be produced by appropriately blending the bone metabolism improving agent of the present invention with raw materials for pharmaceutical preparations. Examples of raw materials that can be blended into the bone metabolism improving agent of the present invention include excipients (glucose, lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, cacao butter, hardened vegetable oil, kaolin, talc, etc.), binders (distilled water, physiological saline, ethanol water, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrants (sodium alginate, agar, carbonated water, etc.), and the like. Examples of suitable additives include: sodium carbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, starch, lactose, powdered acacia, gelatin, ethanol, etc.), disintegration inhibitors (white sugar, stearin, cocoa butter, hydrogenated oil, etc.), absorption enhancers (quaternary ammonium base, sodium lauryl sulfate, etc.), adsorbents (glycerin, starch, lactose, kaolin, bentonite, silicic acid, etc.), lubricants (purified talc, stearates, polyethylene glycol, etc.).

[0074] The bone metabolism improving agent of the present invention can generally be administered orally in the form of tablets, pills, soft or hard capsules, fine granules, powders, granules, liquids, etc., but may also be administered parenterally. When administered parenterally, it can be administered in the form of a solution or with the addition of a dispersing agent, suspending agent, stabilizer, etc., by local tissue administration, intradermal, subcutaneous, intramuscular, or intravenous injection. It may also be in the form of a suppository, etc. Furthermore, it can be administered as an eye drop.

[0075] The dosage may vary depending on the administration method, the condition, the age of the patient, etc., but typically, adults can be given 0.5 to 5000 mg of the active ingredient per day, and children can be given 0.5 to 3000 mg. The compounding ratio of the bone metabolism improving agent can be changed appropriately depending on the dosage form, but is usually about 0.3 to 15.0 wt% when administered orally or via mucosal absorption, and about 0.01 to 10 wt% when administered parenterally. Note that the dosage varies depending on various conditions, so in some cases a smaller dosage than the above-mentioned dosage is sufficient, and in other cases it may be necessary to administer an amount exceeding the range. [Example]

[0076] Examples of the present invention will be described below. Note that the examples shown below are provided to confirm the various actions and effects of the bone metabolism improving agent obtained by the present invention, and the scope of the present invention is not limited to these products and manufacturing methods.

[0077] Test materials The test material samples used in this example were: ferulic acid: "Ferulic Acid" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; astaxanthin: "Astaxanthin-20" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; α-lipoic acid: "α-Lipoic Acid-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; Coprino mushroom extract: "Coprino Extract-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; Orobanchaceae plant extract: "Cistanche Tubulosa Extract-P25" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; cherry blossom extract: "Cherry Blossom Extract-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; γ-oryzanol: "γ-oryzanol" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; polyamine derived from plant products: "Polyamine-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; butterbur extract: "Butterbur Extract-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.; and bird's nest extract (comparative example): "Butterfly's Nest Extract-P" manufactured by Oryza Oil & Fat Chemical Co., Ltd.

[0078] Test Example 1: Test method for examining the effect on osteoclast differentiation Ferulic acid, astaxanthin, α-lipoic acid, Coprino extract, Cistanche Tubulosa extract, cherry blossom extract, γ-oryzanol, polyamine, butterbur extract, and bird's nest extract were prepared as stock solutions at 10 mg / ml (in 10% DMSO). Mouse-derived RAW264.7 cells were used as osteoclast precursors. Cell growth medium was α-MEM supplemented with penicillin-streptomycin, L-glutamine solution, and 10% fetal bovine serum. Osteoclast differentiation was induced by adding sRANKL (ORIENTAL YEAST) to the growth medium at 100 ng / ml (final concentration). RAW264.7 cells were treated with RANKL and each sample extract, or with 0.1% DMSO (final concentration) as a control. Four days after osteoclast differentiation induction, the cells were subjected to TRAP staining and osteoclast counts. The results are shown in Figure 1.

[0079] Results and Effects of Examples in Test Example 1 As shown in Figure 1, all compounds except bird's nest reduced the number of TRAP-positive osteoclasts. Cistanche Tubulosa, astaxanthin, and α-lipoic acid reduced the number of TRAP-positive osteoclasts at a concentration of 20 μg / ml. Cistanche Tubulosa, astaxanthin, and α-lipoic acid are thought to effectively suppress osteoclast differentiation, confirming their usefulness as bone resorption inhibitors.

[0080] Test Example 2: Examination of the effect on osteoblast differentiation Test Method MC3T3-E1 cells were used as osteoblast precursors. Osteoblast differentiation was induced in α-MEM supplemented with penicillin-streptomycin and fetal bovine serum (10%), supplemented with 10 mM glycerophosphate, 50 μg / ml ascorbic acid, and 10 nM dexamethasone, with the medium replaced every three days. Differentiation was induced in the presence of each sample or vehicle (0.1% DMSO) as a control. After two weeks, the cells were fixed in 10% formalin and stained with Alizarin Red S and alkaline phosphatase. The results are shown in Figure 2.

[0081] Results and Effects of Examples in Test Example 2 As shown in Figure 2, α-lipoic acid strongly promoted alkaline phosphatase expression locally. Cistanche Tubulosa and ferulic acid showed calcium deposition around the wells. It was confirmed that α-lipoic acid, Cistanche Tubulosa, and ferulic acid promote osteoblast differentiation and bone mineralization.

[0082] Test Example 3: Effect of Cistanche Tubulosa on osteoblast differentiation markers Test Method Osteoblast precursor cells (MC3T3-E1) were cultured at 37°C for 3 days, then replaced with differentiation medium (described above) and cultured in the presence of Cistanche Tubulosa (10 μg / ml) or control 0.1% DMSO. Proteins were extracted using RIPA buffer over time (0, 2, 9, 16, and 21 days). Western blot analysis was performed using anti-BSPII, anti-CDH11, and anti-RUNX2 antibodies. The results are shown in Figure 3.

[0083] Results and Effects of Examples in Test Example 3 As shown in Figure 3, Cistanche Tubulosa had no effect on CDH and RUNX2 expression, but increased BSPII expression was observed 16 to 21 days after induction of osteoblast differentiation, confirming that Cistanche Tubulosa promotes the secretion of BSPII, an extracellular matrix protein important for bone mineralization.

[0084] Test Example 4: Effect of Cistanche Tubulosa on type I collagen localization and expression during osteoblast differentiation Test Method Osteoblast precursor cells (MC3T3-E1) were cultured at 37°C for 3 days, then the medium was replaced with differentiation-inducing medium (described above) and cultured in the presence of Cistanche Tubulosa (10 μg / ml) or control 0.1% DMSO. After 3 days, immunostaining was performed. Nuclei were stained with DAPI, actin was stained with phalloidin-FITC, and type I collagen was stained with an anti-type I collagen antibody (secondary antibody: anti-mouse Alexa-594). The results are shown in Figure 4.

[0085] Results and Effects of Examples in Test Example 4 As shown in FIG. 4, in the presence of Cistanche Tubulosa, increased density of actin fibers and significant accumulation of type I collagen in the cytoplasm were observed. This confirmed that Cistanche Tubulosa exerted pseudo-mechanical stress on cells, resulting in the densification of actin fibers and the associated accumulation of type I collagen.

[0086] Formulation examples of the bone metabolism improving agent according to the present invention are shown below, but the following formulation examples do not limit the present invention. Example 1: Chewing gum Sugar 53.0wt% Gum base 20.0 Glucose 10.0 Starch syrup 16.0 Fragrance 0.5 Bone metabolism improver 0.5 100.0wt%

[0087] Mixing example 2: Gummies Reduced starch syrup 40.0wt% Granulated sugar 20.0 Glucose 20.0 Gelatin 4.7 Wednesday 9.68 Yuzu juice 4.0 Yuzu flavor 0.6 Dye 0.02 Bone metabolism improver 1.0 100.0wt%

[0088] Mixing example 3: Candy Sugar 50.0wt% Starch syrup 33.0 Wednesday 14.4 organic acid 2.0 Fragrance 0.2 Bone metabolism improver 0.4 100.0wt%

[0089] Blend Example 4: Yogurt (hard / soft) Milk 41.5wt% Skim milk powder 5.8 Sugar 8.0 Agar 0.15 Gelatin 0.1 Lactic acid bacteria 0.005 Bone metabolism improver 0.4 fragrance trace amount water residue 100.0wt%

[0090] Formulation example 5: Soft drink High fructose corn syrup 30.0wt% Emulsifier 0.5 Bone metabolism improver 0.3 Fragrance (appropriate amount) Purified water remainder 100.0wt%

[0091] Formulation example 6: Tablet confectionery Sugar 76.4wt% Glucose 19.0 Sucrose fatty acid ester 0.2 Bone metabolism improver 0.5 Purified water 3.9 100.0wt%

[0092] Formulation example 7: Soft capsule Brown rice germ oil 47.0wt% Yuzu seed oil 40.0 Emulsifier 12.0 Bone metabolism improver 1.0 100.0wt%

[0093] Formulation example 8: Tablets Lactose 54.0wt% Microcrystalline cellulose 30.0 Starch decomposition product 10.0 Glycerin fatty acid ester 5.0 Bone metabolism improver 1.0 100.0wt%

[0094] Formulation example 9: Eye drops Ketotifen fumarate 0.7wt% Sodium azulene sulfonate 0.2 Sodium cromoglycate 9.8 Potassium L-aspartate 8.5 Allantoin 3.0 Tetrahydrozoline hydrochloride 0.5 Neostigmine methylsulfate 0.05 Benzalkonium chloride solution 0.1 Glycerin 25.0 Bone metabolism improver 1.0 pH adjuster (appropriate amount) Purified water remainder 100.0wt% [Industrial Applicability]

[0095] As described above, the present invention can provide a novel bone metabolism improving agent.

Claims

1. An osteoclast differentiation inhibitor containing at least one of the following active ingredients: alpha-lipoic acid, Coprinus comatus extract, Orobanchaceae plant extract, cherry blossom extract, gamma-oryzanol, a polyamine composition derived from plants and / or processed plant products, and butterbur extract.

2. An osteoblast differentiation promoter containing alpha-lipoic acid and an extract of a plant from the Orobanchaceae family as its active ingredient.

3. An agent that promotes BSPII expression in osteoblast precursor cells, with plant extract from the Orobanchaceae family as its active ingredient.

4. An agent that promotes type 1 collagen expression in osteoblast precursor cells, with plant extract from the Orobanchaceae family as its active ingredient.

5. A bone metabolism improving agent comprising at least one agent selected from the group consisting of those listed in claims 1 to 4 as an active ingredient.

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