JP5307019B2 - Bone resorption inhibitor and beverages, foods and pharmaceuticals containing the same - Google Patents

Abstract

It is intended to provide a bone resorption inhibitor which comprises, as the active ingredient, a barley extract obtained by extracting barley flour (milled barley) with water or a buffer solution, a fraction obtained by fractionating the barley extract or a product obtained by enzymatically treating the barley extract or fraction thereof as described above. Thus, a bone resorption inhibitor, which has a high safety for the human body, is applicable not only to drugs but to foods, shows a different mechanism from that of cathepsin K inhibitors and enables the prevention and amelioration (healing and relief) of bone diseases such as osteoporosis and rheumatoid arthritis, is provided.

Description

  The present invention relates to a bone resorption inhibitor and beverages, foods and pharmaceuticals containing the same.

  In recent years, with the progress of an aging society and changes in lifestyle habits, osteoporosis patients are increasing, and the number of osteoporosis patients in Japan exceeds 10 million. Women account for 70% of osteoporosis patients, and the disease often occurs in women over 50 years old. For this reason, especially for women, it is said that it is important for prevention of osteoporosis to keep the bone mass as high as possible until about 30 years old when the bone mass reaches the maximum.

  Recently, however, there are an increasing number of potential osteoporosis patients in early life due to lack of exercise and nutrition. Regarding the prevention or improvement of osteoporosis, in addition to a medical approach, various studies from a genetic / nutritional viewpoint have been conducted (Non-patent Document 1).

  In bone metabolism, osteoclasts are responsible for bone resorption and osteoblasts are responsible for bone formation. Osteoporosis is thought to be caused by an imbalance between bone resorption and bone formation and bone resorption exceeding bone formation. In addition to osteoporosis, increased bone resorption is deeply involved in bone diseases such as rheumatoid arthritis. Thus, osteoclasts responsible for bone resorption are considered to be targets for preventing or ameliorating (treating or alleviating symptoms) bone diseases including osteoporosis and rheumatoid arthritis.

  Cathepsins B, H, K, and L are known as proteases involved in bone resorption. Among them, it has been reported that cathepsin K is specifically localized in osteoclasts and can suppress bone resorption by inhibiting cathepsin K (Non-patent Documents 2 and 3). For this reason, cathepsin K is attracting attention as a target molecule for preventing or ameliorating (treating or alleviating symptoms) bone diseases including osteoporosis and rheumatoid arthritis.

  Examples of cathepsin K inhibitors include dipeptidyl ketone inhibitors, diaminopyridinone inhibitors, diacyl carbohydrazide inhibitors, pyridoxalpropionate derivatives, cyclic ketone derivatives, allylaminoethylamide derivatives, 4-amino-azepane. -3-one inhibitors, 3-allylamino-azetidin-2-one inhibitors and ketoamide derivatives have been developed.

  Cathepsin K inhibitors originating from natural products include basic protein compositions contained in milk or milk-derived raw materials (Patent Document 1), oryzastatin-I and oryzasistatin-II contained in gramineous plants (patents) Reference 2) has been reported.

In addition, examples of materials used in health functional foods include CPP (casein phosphopeptide), CCM (calcium malate citrate), soy isoflavone, fructooligosaccharide, polyglutamic acid, MBP (milk basic protein) and vitamins. K ₂ (menaquinone-7) is known (Non-patent Document 4).

JP-A-9-191858 JP 2006-151843 A Yoneda, "New bone bioscience", Yodosha, 2002 Littlewood et al., Bone, 1997, 20, p. 81-86 Inui et al. Biol. Chem. 1997, 272, p. 8109-8112 Japan Health and Nutrition Foods Association, “Food for Specified Health Use, Tokuho Anzen 2005 Edition”

  However, since cathepsin K inhibitors that are currently widely used are artificially chemically synthesized compounds, there is a problem in terms of safety when taken without receiving a doctor's diagnosis.

  Cathepsin K inhibitors inhibit the degradation of bone matrix components (protein components) by cathepsin K, but cannot inhibit the degradation of inorganic components (hydroxyapatite) that play a role of cement around the matrix components. . For this reason, even if a cathepsin K inhibitor is administered, the decomposition of the inorganic component proceeds, and the decomposition of the substrate component by a protease other than cathepsin K (for example, MMP-9) cannot be effectively prevented.

  Therefore, the present invention is a bone resorption inhibitor having a mechanism different from that of cathepsin K inhibitors, which is excellent in safety to the human body and can be applied not only to pharmaceuticals but also to foods, including osteoporosis and rheumatoid arthritis. It is an object of the present invention to provide a bone resorption inhibitor capable of preventing and improving (treating and relieving symptoms) bone diseases.

  The present inventors inhibit cathepsin K by a certain barley extract, a fraction obtained by fractionating the barley extract, and an enzyme-treated product obtained by enzymatic treatment of the barley extract or fraction. The present invention has been completed by finding that the decomposition of the inorganic components of bone caused by osteoclasts is suppressed.

  That is, the present invention contains, as an active ingredient, a barley extract obtained by extracting barley flour obtained by pulverizing barley with water or a buffer solution, a fraction obtained by fractionating the same, or an enzyme-treated product of the barley extract or the fraction. A bone resorption inhibitor is provided.

  Barley has been used for human consumption as a major cereal since ancient times, and a bone resorption inhibitor derived from barley extract is excellent in safety to the human body. In addition, the bone resorption inhibitor of the present invention is a cathepsin K inhibitor that suppresses only the decomposition of the substrate component (protein component) in order to suppress the decomposition of the bone inorganic component (hydroxyapatite) that proceeds first in bone resorption. Bone resorption can be effectively suppressed through a different mechanism.

  The barley extract fraction is preferably a basic fraction obtained by bringing the barley extract into contact with a cation exchange resin and then separating the components adsorbed on the resin from the resin. If such a basic fraction is used, the decomposition | disassembly of the inorganic component of the bone caused by the osteoclast can be suppressed notably.

  As a fraction of the barley extract, for example, a precipitate fraction obtained by further fractionating the basic fraction with 75% saturated ammonium sulfate can be used. In addition, for example, a basic protein fraction (for example, a protein fraction having a molecular weight of 5,000 to 50,000) obtained by further fractionating the precipitate fraction with an ultrafiltration membrane can be used.

  The barley extract or the enzyme-treated product of the fraction is the barley extract or fraction (preferably, the basic fraction, the precipitated fraction or the basic protein fraction) of pepsin, pancreatin and trypsin. An enzyme-treated product obtained by enzymatic treatment (digestion) with at least one kind of enzyme is preferable.

  The enzyme-treated product may be obtained by digestion with an enzyme (eg, pepsin, pancreatin, trypsin) in digestive fluid such as gastric juice or intestinal juice, and the barley extract or fraction was taken orally. In some cases, bone resorption can be suppressed.

  The basic protein fraction and the enzyme-treated product thereof can be used as an active ingredient of the bone resorption inhibitor of the present invention. That is, the present invention is also a bone resorption inhibitor containing, as an active ingredient, a protein contained in the basic fraction of barley extract or an enzyme-treated product thereof, wherein the basic fraction pulverizes barley. The step of extracting barley flour with water or a buffer, the step of bringing the obtained extract into contact with a cation exchange resin, and the step of separating the components adsorbed on the resin from the resin are performed in this order. The bone resorption inhibitor which is a fraction obtained by is provided.

  Since the bone resorption inhibitor of this invention is excellent in bone resorption suppression activity and safety | security, it can be used as components, such as a drink, a foodstuff, food-drinks additives, feed, and a pharmaceutical.

  ADVANTAGE OF THE INVENTION According to this invention, the bone resorption inhibitor excellent in the safety | security with respect to a human body is provided. In addition, according to the present invention, the degradation of the bone inorganic component (hydroxyapatite) that progresses first in the bone resorption is suppressed, and the bone resorption is effectively performed by a mechanism different from the degradation of the bone matrix component (protein component). A bone resorption inhibitor capable of being suppressed is provided.

  The bone resorption inhibitor of the present invention maintains its activity even if it is treated with enzymes in digestive juices such as gastric juice and intestinal juice (eg, pepsin, pancreatin, trypsin). Absorption can be suppressed.

It is a graph which shows the bone resorption area in the Pit Formation assay performed in presence of the barley extract. It is a graph which shows the bone resorption area in the Pit Formation assay performed in presence of the basic fraction or acidic fraction of a barley extract. It is a graph which shows the cathepsin K activity of a barley extract and cystatin C. It is a graph which shows the bone resorption area in the Pit Formation assay performed in the presence of Ichibanboshi or Haruna Nijo barley extract. It is a graph which shows the bone resorption area in the Pit Formation assay performed in the presence of the enzyme-treated product of barley extract. It is a graph which shows the bone resorption area in the Pit Formation assay performed in the presence of cystatin C. It is a graph which shows the bone density of the ovariectomized mouse | mouth which administered the basic protein fraction of the barley extract. It is a graph which shows the uterine weight of the ovariectomized mouse | mouth which administered the basic protein fraction of the barley extract.

  Hereinafter, preferred embodiments of the present invention will be described.

  The bone resorption inhibitor of the present invention is a barley extract obtained by extracting barley flour obtained by pulverizing barley with water or a buffer solution, a fraction obtained by fractionating the same, or the barley extract or the fraction obtained by enzyme treatment. The obtained enzyme-treated product is contained as an active ingredient.

  As the barley, any kind of barley such as Nijo, Rojo, bare, and leather can be used, and more specifically, for example, Haruna Nijo, Ichibanboshi, and CDC Fibar. In addition, any tissue derived from barley seeds such as whole grains, wheat grains, and straw can be used. For example, the inner layer or outer layer of barley seeds may be used alone, or malt may be used. Furthermore, for example, barley of a B hordin deletion line or a protein Z4 deletion line can be used.

  In the step of obtaining barley flour by pulverizing barley, it is sufficient if barley can be efficiently pulverized into fine powders. For example, a pulverizer such as a pin mill, a hammer mill, a ball mill, or a mixer can be used.

  In the step of extracting barley flour with water or a buffer solution to obtain a barley extract, the barley flour is stirred in water or a buffer solution to extract water-soluble proteins contained in the barley. The temperature of water or the buffer is preferably more than 0 ° C. and not more than 30 ° C., more preferably more than 0 ° C. and not more than 10 ° C., more preferably 2 ° C. to 8 ° C. (particularly 3 ° C. to 7 ° C.).

  In addition, the buffer solution used for extraction preferably exhibits a buffering action so as to have a neutral to slightly acidic pH, and the pH of the buffer solution is preferably 4.0 to 7.0, preferably 5.0 to 6.0 is more preferable. Examples of the buffer include phosphoric acid, citric acid, acetic acid, phthalic acid, succinic acid, carbonic acid, HEPES, MES, and Tris. For example, 0.1 M sodium acetate buffer (pH 5.5), 20 mM sodium acetate A buffer solution (pH 5.0) and a 20 mM MES buffer solution (pH 6.0) are particularly preferable.

  In the step of fractionating the barley extract to obtain a fraction, the barley extract is first brought into contact with a cation exchange resin. Subsequently, an acidic fraction can be obtained by collecting the components that have not been adsorbed on the resin. Moreover, a basic fraction can be obtained by separating the component adsorbed on the resin from the resin. The contact between the barley extract and the cation exchange resin can be performed, for example, by passing the barley extract through the cation exchange resin. In order to separate the component adsorbed on the cation exchange resin from the resin, for example, the adsorbed component may be eluted by passing an eluent through the resin. Examples of the cation exchange resin include Hitrap SP FF, HiTrap SP HP, HiTrap CM FF, HiTrap 16/10 SP XL, HiTrap 16/10 SP FF, HiTrap 16/10 CM FF, Resource S, Source S30, Source S30. , Mono S, Mini S, SP Sepharose HP, SP Sepharose XL, SP Sepharose Fast Flow, CM Sepharose Fast Flow (above GE Healthcare Biosciences), AG 50W Resins, AG MP-50R , Macro-Prep high Q, Macro-Prep high S, Macro Prep CM (above, Bio-Rad), gel type Diaion (registered trademark) SK series, porous type Diaion (registered trademark) PK series, gel type uniform particle size for water treatment Diaion (registered trademark), industrial chromatography Cation exchange resin for separation Diaion (registered trademark) UBK500 series (Mitsubishi Chemical Corporation), Asahipak Column ES-502C 7C, Shodex Column IEC CM-825, Shodex Column IEC SP-825, Shodex Column IEC (825) As mentioned above, Showa Denko Co., Ltd.) and SP-Toyopearl (Tosoh Corporation) can be mentioned. Of the cation exchange resins, strongly acidic ion exchange resins are preferred.

  The fraction of the barley extract is preferably a basic fraction obtained by bringing the barley extract into contact with a cation exchange resin and then separating the components adsorbed on the resin from the resin. If such a basic fraction is used, the decomposition | disassembly of the inorganic component (hydroxyapatite) of the bone in the case of bone resorption can be suppressed notably.

  As a fraction of the barley extract, for example, a precipitate fraction obtained by further fractionating the basic fraction with 75% saturated ammonium sulfate can be used. In addition, for example, a basic protein fraction (for example, a protein fraction having a molecular weight of 5,000 to 50,000) obtained by further fractionating the precipitate fraction with an ultrafiltration membrane can be used.

  The barley extract or the enzyme-treated product of the fraction is the barley extract or fraction (preferably the basic fraction, the precipitated fraction or the basic protein fraction), which is at least one of pepsin, pancreatin and trypsin. An enzyme-treated product obtained by enzyme treatment (digestion) with one kind of enzyme is preferable.

  Pepsin, pancreatin, and trypsin are digestive enzymes contained in digestive fluids such as gastric juice and intestinal fluid, and can be easily obtained from reagent manufacturers. Conditions for treating the barley extract or fraction with an enzyme (enzyme amount, temperature, time, etc.) are not particularly limited as long as they are optimum conditions for the enzyme. Examples of the available pepsin, pancreatin and trypsin include porcine gastric mucosa-derived pepsin, porcine pancreatic pancreatin, bovine pancreatic trypsin, and porcine pancreatic trypsin. The enzyme treatment can be performed, for example, by adding an enzyme such that the volume ratio is 10% with respect to the barley extract or fraction and reacting at 37 ° C. for at least 2 hours.

  The bone resorption suppressing activity can be measured, for example, as follows. That is, first, osteoclast precursor cells are seeded in each well of a culture plate coated with an inorganic component of bone (hydroxyapatite) and cultured until the cells are completely adhered to the plate. Thereafter, a candidate for bone resorption inhibitor is added and cultured at 37 ° C. for 7 to 10 days. When the formation of bone resorption traces can be confirmed in one well in a control well to which no bone resorption inhibitor is added, the culture is performed. Stop. Then, the state of the surface of each well is observed with a digital microscope, and the bone resorption area or the number of bone resorption traces is measured. The inhibition of the formation of the bone resorption trace means that the bone resorption is suppressed, and the strength of the bone resorption suppressing activity can be determined by the degree of the inhibition.

  The bone resorption inhibitor of the present invention prevents and improves (treats, for example, osteoporosis, rheumatoid arthritis, osteoarthritis, periodontal disease, Paget's disease of bone, and bone loss / fracture in a microgravity environment. In particular, it is suitable for prevention and improvement of osteoporosis and rheumatoid arthritis.

  The bone resorption inhibitor of the present invention can be added to beverages and foods for the purpose of prevention and improvement (treatment, alleviation of symptoms) of bone diseases such as osteoporosis and rheumatoid arthritis, and prevention or improvement (treatment of these bone diseases) Alternatively, it can be used as a component of a symptom alleviating agent (pharmaceutical). The bone resorption inhibitor of the present invention may be in any form of solid, liquid (water-soluble or fat-soluble solution or suspension), paste, etc., and powder, granule, tablet, syrup, troche, Any dosage form such as a capsule or an injection may be used. It can also take the form of a controlled release formulation. The above beverages, foods and pharmaceuticals are barley extract obtained by extracting barley flour obtained by pulverizing barley with water or a buffer, or a fraction obtained by fractionating the same (preferably the aforementioned basic fraction, precipitated fraction or basic fraction). A protein fraction), or an enzyme-treated product obtained by subjecting the barley extract or fraction to an enzyme treatment (preferably an enzyme treatment obtained by subjecting at least one enzyme of pepsin, pancreatin and trypsin to an enzyme treatment) Thing), may consist only of.

  In addition, the bone resorption inhibitor of this invention can also be mix | blended with food-drinks, such as food for specific health, special nutrition food, nutritional supplement food, health food, functional food, and food for sick people. Moreover, it can also mix | blend with feed.

  The beverage, food and feed of the present invention may further contain additives usually used in the art. Examples of such additives include apple fiber, soybean fiber, meat extract, black vinegar extract, gelatin, corn starch, honey, animal and vegetable oils and fats; monosaccharides such as glucose; disaccharides such as sucrose, fructose and mannitol; dextrose, Examples thereof include polysaccharides such as starch; sugar alcohols such as erythritol, xylitol, and sorbitol; vitamins such as vitamin C. These additives may be used alone or in combination of two or more.

  The pharmaceutical product of the present invention may further contain a pharmaceutically acceptable additive. Examples of the additive include monosaccharides such as glucose; disaccharides such as sucrose, fructose and mannitol; polysaccharides such as dextrose and starch; sugar alcohols such as erythritol, xylitol and sorbitol; vitamins such as vitamin C; Acacia gum, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose derivatives, tragacanth, gelatin, syrup, methyl hydroxybenzoate, talc, magnesium stearate, water, mineral oil. These additives may be used alone or in combination of two or more.

  The bone resorption inhibitor of the present invention may be administered to humans or non-human mammals. Administration methods include oral administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intracisternal administration, intravaginal administration, intraperitoneal administration, intravesical administration, inhalation administration, etc., and ointments and gels Any of topical administration using a cream or the like may be used. The dosage and administration method can be appropriately determined according to the condition, age, etc. of the individual to be administered. Suitable administration methods include, for example, oral administration.

  The bone resorption inhibitor of the present invention maintains its activity even when it is treated with an enzyme (eg, pepsin, pancreatin, trypsin) in digestive fluid such as gastric juice and intestinal juice. Therefore, beverages, foods, feeds, pharmaceuticals and the like containing the bone resorption inhibitor of the present invention can effectively inhibit bone resorption even when these are taken orally.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1: Bone resorption inhibitory activity of barley extract]
(Preparation of barley extract)
The fully dried barley seeds (variety: Haruna Nijo) were pulverized with a mixer (Lab Miller, Iwatani Corp.) for about 10 minutes to obtain finely baked barley flour. 2 g of the obtained barley flour was transferred to a mortar, where it was buffered for homogenization (0.1 M sodium acetate, pH 5.5, 5 mM EDTA, 0.1% Tween-20, 5 mM DTT, 1 tablet complete, EDTA free, 10 mL of protease inhibitor cocktail tablets (Roche) were added and suspended with a pestle.

  Thereafter, the barley flour suspension was centrifuged at 10,000 × g for 10 minutes at 4 ° C., and the supernatant was filtered through a Miracloth filter (Calbiochem). The obtained filtrate was applied to a PD-10 column (GE Healthcare), and buffer exchanged with 20 mM MES (pH 6.0) to obtain a barley extract.

(Pit formation assay)
Bone resorption inhibitory activity of the obtained barley extract was measured by Pit Formation assay.

Specifically, first, rat osteoclast progenitor cells (1 × 10 ⁶ cells) were added to a differentiation-inducing medium (5 mL) (medium composition: α-MEM, included in a rat osteoclast culture kit (Cell Garage)). 10% FBS, 50 ng / mL RANKL & M-CSF), and 100 μL (2 × 10 ⁴ cells / well) was dispensed into each well of a calcium phosphate-coated OAAS plate (Oscotec). The cells were cultured in a CO ₂ incubator until they completely adhered to the plate.

Thereafter, a barley extract having a predetermined protein concentration was added to each well and cultured in a CO ₂ incubator at 37 ° C. for 7 to 10 days. Control wells (wells to which an equal amount of sterile water was added instead of barley extract) When the formation of bone resorption traces was confirmed on the whole surface, the state of the surface of each well was observed with a digital microscope (VHX-100F, Keyence), and the bone resorption area was measured. In addition, the intensity | strength of the bone resorption suppression activity of a barley extract can be determined with the grade of inhibition of bone resorption trace formation.

  FIG. 1 is a graph showing bone resorption area in a Pit Formation assay performed in the presence of barley extract. In FIG. 1, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.

  As is clear from FIG. 1, the barley extract almost completely suppressed bone resorption when the protein concentration was 1 μg / mL or more, and markedly suppressed bone resorption when the protein concentration was 0.2 μg / mL.

  Example 1 showed that the barley extract has an activity of suppressing bone resorption, particularly an activity of suppressing the decomposition of the bone inorganic component (hydroxyapatite).

[Example 2: Bone resorption inhibitory activity of basic fraction and acidic fraction]
(Preparation of basic and acidic fractions)
A barley extract was obtained in the same manner as in Example 1. The obtained barley extract was applied to HiTrap SP FF (strong cation exchange column, GE Healthcare), and components that were not adsorbed to the cation exchange resin were eluted with 5 mL of the starting buffer (20 mM MES buffer, pH 6.0). Thus, an acidic fraction of the barley extract was obtained. Subsequently, the component adsorbed on the cation exchange resin was eluted with 5 mL of elution buffer (20 mM MES buffer, pH 6.0, 1M NaCl) to obtain a basic fraction of barley extract. The basic fraction contained proteins, amino acids and nucleic acids.

(Pit formation assay)
The bone resorption inhibitory activity of the obtained basic fraction and acidic fraction was tested by the Pit Formation assay in the same manner as in Example 1.

  FIG. 2 is a graph showing the bone resorption area in a Pit Formation assay performed in the presence of a basic fraction or an acidic fraction of barley extract. In FIG. 2, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.

  As is apparent from FIG. 2, the basic fraction almost completely suppressed bone resorption when the protein concentration was 1 μg / mL or more, and markedly suppressed bone resorption when the protein concentration was 0.2 μg / mL. The acidic fraction also showed bone resorption inhibitory activity, although not as strong as the basic fraction.

  Example 2 showed that the basic fraction and the acidic fraction of the barley extract have bone resorption inhibitory activity, in particular, the activity of inhibiting the decomposition of bone inorganic components (hydroxyapatite). Moreover, it was shown that a basic fraction has especially strong bone resorption suppression activity.

[Reference Example 1: Cathepsin K inhibitory activity of barley extract]
A barley extract was obtained in the same manner as in Example 1. The barley extract obtained was tested for cathepsin K inhibitory activity as follows.

  First, a buffer solution for enzyme reaction (100 mM NaOAc, 20 mM L-Cystein, 5 mM EDTA, pH 5.5) 130 μL and 4 μg / mL cathepsin K (human recombinant procathepsin K, Calbiochem) solution in each well of a 96-well plate 10 μL of the barley extract having a predetermined protein concentration, or 10 μL of a 1 μg / mL cystatin C (human urine-derived cystatin C, Calbiochem) solution, or 10 μL of the enzyme reaction buffer (control), Was further added and pre-incubated at 37 ° C. for 30 minutes.

  Subsequently, 50 μL of a fluorescent substrate solution (20 μM Z-Phe-Arg-MCA (Benzyloxycarbonyl-L-phenylalanyl-L-argineline-4-methylcoumeric-7-amide), peptide laboratory) was added, and an enzyme reaction was performed at 37 ° C. . The degree of increase in fluorescence intensity was measured with a microplate reader MTP-800AFC (excitation wavelength 350 nm, absorption wavelength 450 nm, Corona) to evaluate cathepsin K activity.

  FIG. 3 is a graph showing the cathepsin K activity of barley extract and cystatin C. In FIG. 3, the cathepsin K activities of barley extract and cystatin C are shown as a relative value (%) to the control. The concentration (μg / mL) indicates the concentration of the protein in the reaction solution.

  As is clear from FIG. 3, the barley extract did not inhibit the activity of cathepsin K even when the protein concentration was 5 μg / mL. On the other hand, cystatin C, which is a cathepsin K inhibitor, completely suppressed cathepsin K activity even at 1 μg / mL.

  Reference Example 1 shows that the barley extract does not have cathepsin K inhibitory activity, that is, the bone resorption inhibitory action of the barley extract is via a mechanism different from that of the cathepsin K inhibitor.

[Example 3: Bone resorption inhibitory activity of barley extract]
A barley extract was obtained in the same manner as in Example 1 from two barley varieties Ichibanboshi and Haruna Nijo barley seeds. And the bone resorption inhibitory activity of each obtained barley extract was tested by the Pit Formation assay similarly to Example 1.

  FIG. 4 is a graph showing the bone resorption area in a Pit Formation assay performed in the presence of Ichibanboshi or Haruna Nijo barley extract. In FIG. 4, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.

  As is clear from FIG. 4, both Ichibanboshi and Haruna Nijo showed remarkable bone resorption inhibitory activity.

[Example 4: Bone resorption inhibitory activity of enzyme-treated product]
Bone resorption inhibitory activity was tested on enzyme-treated products obtained by treating barley extract with artificial gastric juice and artificial intestinal fluid.

  A barley extract was obtained in the same manner as in Example 1. Artificial gastric juice (2% NaCl, 10 kU / mL pepsin, 0.6N HCl) was added to the barley extract with a predetermined protein concentration so that the volume ratio was 10%, and the mixture was reacted at 37 ° C. for 2 hours. An aqueous solution was added for neutralization.

Subsequently, artificial intestinal fluid (4% pancreatin, 4% trypsin, 1M NaHCO ₃ ) was added so that the volume ratio would be 10%, reacted at 37 ° C. for 2 hours, and heat-treated at 90 ° C. for 2 minutes. An enzyme-treated product of the extract was obtained.

  Pit formation assay was performed on the enzyme-treated product of the obtained barley extract in the same manner as in Example 1.

  FIG. 5 is a graph showing the bone resorption area in a Pit formation assay performed in the presence of an enzyme-treated product of barley extract. In FIG. 5, the bone resorption area is shown as a relative value (%) to the control bone resorption area. Moreover, a density | concentration (microgram / mL) shows the density | concentration of the protein in a culture medium.

  As is clear from FIG. 5, the enzyme-treated product obtained by treating the barley extract with artificial gastric juice and artificial intestinal fluid completely inhibits bone resorption when the protein concentration is 1 μg / mL or more, and 0.2 μg / mL. In the case of mL, bone resorption was remarkably suppressed.

  Example 4 showed that the enzyme-treated product of barley extract has an activity to suppress bone resorption, particularly an activity to suppress the decomposition of bone mineral components (hydroxyapatite).

[Comparative Example 1: Bone resorption inhibitory activity of cathepsin K inhibitor]
The bone resorption inhibitory activity of cystatin C, which is a cathepsin K inhibitor, was tested in the same manner as in Example 1 by the Pit Formation assay.

  FIG. 6 is a graph showing the bone resorption area in a Pit formation assay performed in the presence of cystatin C. In FIG. 6, the concentration (μg / mL) indicates the concentration of cystatin C in the medium.

  As is apparent from FIG. 6, even when cystatin C was contained at 10 μg / mL in the medium, the bone resorption area was almost the same as that of the control, and the bone resorption was hardly suppressed by the addition of cystatin C.

  Comparative Example 1 shows that cystatin C does not have the activity of suppressing the decomposition of bone inorganic component (hydroxyapatite), and does not suppress bone resorption in a bone resorption system that does not involve cathepsin K.

[Example 5: Bone resorption inhibitory activity of basic protein fraction]
Using a 7-week-old female ddy mouse (Japan SLC), the bone resorption inhibitory activity of the basic fraction of the barley extract was confirmed as follows. In the following test, mice were selected from 24 animals with good general condition after one week of acclimatization, and randomly selected sham (sham surgery) group, control group, barley protein 0.5% group and Divided into 4 groups of barley protein 1.0% group (6 animals in each group). During the acclimation breeding period and the test period, the mice were kept at a temperature of 23 ± 1 ° C., a humidity of 60 ± 5%, and a light / dark cycle of 12 hours (light period: 8 hours to 20 hours; dark period: 20 hours to 8 hours). Reared in

(Preparation of basic protein fraction)
Fully dried barley seeds (variety: Ryofu) were pulverized for about 10 minutes with a mixer (Yamabiko No. L-S, Kokuko) to obtain finely baked barley flour. 5.4 kg of the obtained barley flour was immersed in 27 L of 20 mM aqueous sodium acetate solution (pH 5.0) overnight and centrifuged at 8000 rpm for 20 minutes at 4 ° C. to obtain a barley extract (supernatant). The obtained barley extract was passed through a strong cation exchange resin (SP-Toyopearl, Tosoh Corp.), and the components adsorbed on the resin were eluted with a 0.5 M aqueous sodium chloride solution to obtain a basic fraction of the barley extract. Got.

  Then, the basic fraction is further fractionated with 75% saturated ammonium sulfate, and the resulting precipitate fraction is further fractionated with an ultrafiltration membrane to obtain a basic protein fraction (a fraction of a protein having a molecular weight of 5000 to 50000). Min).

(Feed preparation)
The feed administered to the mice of the barley protein 0.5% group and barley protein 1.0% group was prepared by adding the above base to the powdered feed AIN93G so that the barley protein content would be 0.5% and 1.0%, respectively. It was prepared by blending the sex protein fraction. For mice in the sham group and the control group, AIN93G was used as it was.

  The composition of each feed is shown in Table 1 below. In the table, the unit of each component amount is g / kg feed.

(Food administration)
After the acclimation breeding described above, mice in the control group, barley protein 0.5% group and barley protein 1.0% group were subjected to ovariectomy, and mice in the sham group were subjected to sham surgery, and then for 4 weeks. Each group of mice was allowed to freely ingest a predetermined feed and water. It is known that when the ovaries are removed, estrogen is deficient and bone mass is reduced.

(Measurement of bone density)
After 4 weeks of feed administration, each group of mice was dissected and the bone density at the proximal part of the femur was measured by a dual energy X-ray absorption measurement method (DXA method) using DCS600-ER-X (Aloka). It was measured. The results (mean ± standard deviation) are shown in FIG. The significant difference test was performed by the Tukey HSD method. In FIG. 7, the alphabets attached above each data (graph) indicate that there is a significant difference (p <0.05) between the data with different alphabets.

  As is clear from FIG. 7, the bone density was significantly reduced in the control group compared to the sham group. And in the barley protein 0.5% group and the barley protein 1.0% group, the bone density fall of the femur proximal part was significantly suppressed compared with the control group.

(Measurement of uterine weight)
The uterus was removed from each group of dissected mice and weighed. The results (mean ± standard deviation) are shown in FIG.

  As is apparent from FIG. 8, the three groups (control group, barley protein 0.5% group, barley protein 1.0% group) that had undergone ovariectomy had a significantly lower uterine weight than the sham group. It was. In addition, there was almost no difference in uterine weight among the three groups subjected to ovariectomy.

  According to Example 5, the basic protein fraction of barley (protein in the basic fraction) or the enzyme-treated product obtained by digesting this with the enzyme in the digestive fluid affects the reproductive organs such as the uterus. In other words, it was shown that it is possible to suppress bone resorption and the accompanying decrease in bone density.

  According to the above examples, comparative examples and reference examples, barley extract, its basic fraction, acidic fraction and basic protein fraction (protein in the basic fraction), and these enzyme-treated products It was clarified that bone resorption can be suppressed by a mechanism different from the degradation of the substrate component (protein component).

Claims (5)

A bone resorption inhibitor containing a basic fraction of barley extract or an enzyme-treated product thereof as an active ingredient,
The basic fraction includes a step of extracting barley flour obtained by pulverizing barley with water or a buffer, a step of bringing the obtained extract into contact with a cation exchange resin, and a component adsorbed on the resin from the resin. The bone resorption inhibitor which is a fraction obtained by implementing the process to isolate | separate in this order.   The bone resorption inhibitor according to claim 1, wherein the enzyme-treated product is an enzyme-treated product obtained by subjecting the fraction to an enzyme treatment with at least one enzyme selected from pepsin, pancreatin, and trypsin. A bone resorption inhibitor containing a protein contained in the basic fraction of barley extract or an enzyme-treated product thereof as an active ingredient,
The basic fraction includes a step of extracting barley flour obtained by pulverizing barley with water or a buffer, a step of bringing the obtained extract into contact with a cation exchange resin, and a component adsorbed on the resin from the resin. The bone resorption inhibitor which is a fraction obtained by implementing the process to isolate | separate in this order.   The bone resorption inhibitor according to claim 3, wherein the enzyme-treated product is an enzyme-treated product obtained by subjecting the protein to an enzyme treatment with at least one enzyme selected from pepsin, pancreatin, and trypsin.   The bone resorption inhibitor as described in any one of Claims 1-4 by which bone resorption is suppressed through decomposition | disassembly suppression of the inorganic component of bone.

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