JP2016108301A - Osteoblast differentiation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an osteoblast differentiation promoter that can improve the balance of bone metabolism by promoting the differentiation of a bone cell, particularly an osteoprogenitor cell, and is easy to take continuously daily, and has no problem in safety; and to provide a composition containing the differentiation promotor for the prevention of osteopenic disease or for the improvement of symptoms thereof.SOLUTION: The invention provides an osteoblast differentiation promoter containing a solvent extract of the flower of Prunus, and a composition containing the extract, as an active ingredient, for the prevention of osteopenic disease or the improvement of symptoms thereof.EFFECT: The extract of the flower of Prunus accelerates the activity of alkaline phosphatase (ALP) depending on a concentration, and promotes the differentiation of the osteoblast.SELECTED DRAWING: None

Description

  The present invention relates to an osteoblast differentiation promoting agent from osteoprogenitor cells to osteoblasts and a composition for preventing / ameliorating a bone mass-reducing disease containing the same.

  Remodeling always occurs in bone tissue. In this remodeling, when a bone formation amount corresponding to the bone resorption amount cannot be obtained due to aging or the like, a bone loss-related disease accompanied by a decrease in bone weight represented by osteoporosis occurs. The decrease in bone mass causes osteoporosis in advance, and degeneration of alveolar bone locally, for example, in the oral cavity, and is one of the major factors that greatly impair QOL. It is an important issue.

  Representative methods for treating or preventing bone loss diseases include supplementation with inorganic compounds such as calcium; administration of hormonal preparations such as active vitamin D and estrogens. However, since the prevention and treatment of bone loss diseases cannot be sufficiently achieved unless they are continued for a long period of time, the administration of pharmaceuticals cannot be used as a prevention, and even when used for treatment, side effects Therefore, it is necessary to use the medicine under the strict management of a doctor. In addition, the intake of inorganic compounds alone does not have a sufficient effect on improving bone metabolism, so a sufficient effect cannot be obtained.

  Therefore, the development of treatments or prevention methods that do not involve the risk of side effects is being promoted. Since it is ingested continuously for a long period of time, many proposals have been studied by focusing on ingredients derived from animals and plants with experience of eating. For example, focusing on technologies contained in animals and plants [for example, isoflavones (Patent Literatures 1 to 3), xanthine compounds (Patent Literature 4), collagen proteins, etc. (Patent Literature 5)] and plant extracts Technology (patent documents 4 to 6) and the like. However, even with the above proposal, it is not possible to sufficiently prevent or treat a bone loss-related disease, and a more effective proposal is desired.

JP-A-6-46797 JP 2000-139411 A International Publication No. 2002/032441 JP 2008-303166 A JP 2013-124221 A JP 2002-363086 A JP 2006-008625 A JP 2014-043416 A

  The present invention can improve the balance of bone metabolism by promoting the differentiation of bone cells, particularly osteoprogenitor cells, and can promote the differentiation of osteoblasts that is easy to ingest daily and has no safety concerns. It is an object of the present invention to provide an agent and a composition containing the same for preventing or ameliorating symptoms of osteopenia.

  As a result of intensive studies in view of such circumstances, the present inventors have surprisingly found that a cherry blossom flower extract has an osteoblast differentiation promoting action, and has completed the present invention.

That is, this invention provides the oral composition of claim | item 1 thru | or claim | item 2.
Item 1. An osteoblast differentiation promoting agent containing a cherry blossom extract.
Item 2. An oral composition for the prevention or symptom improvement of osteopenic disease, which comprises the osteoblast differentiation promoting agent according to Item 1.

  According to the present invention, since it is an osteoblast differentiation promoter composed of a cherry blossom extract that has been eaten for a long time in Sakurayu etc. and is highly safe, there is a problem with safety even if it is taken daily. It is possible to provide an oral composition for preventing or ameliorating a bone loss-related disease that is easy to be taken.

  The cherry blossom extract used in the present invention refers to an extract (extract) of a deciduous tree flower belonging to the Rosaceae genus Sakura subgenus. Here, the flower may include other parts such as twigs as long as it contains a petal, and is preferably a part from a floral pattern to a petal. “Sakura” means a general term for plants belonging to the genus Sakura. Representative varieties of the cherry genus are the wild cherry groups represented by Yamazakura, Oyamazakura, Kasumi cherry, Oshimazakura, etc. Crested cherry group represented by bean cherry group, Okejizakura, Daisakuzakura, etc., Kanhizakura group represented by Kawazu-zakura, Tsubakikanzakura, etc. There is a group of cherry blossoms typified by Taizanfukun, but because of the incompatibility of cherry, there are many varieties other than the above because of natural crossing. In this invention, if it is a flower of the plant which belongs to a cherry tree subgenus, it will not specifically limit.

  The extraction solvent for the cherry blossom extract is not particularly limited as long as it is an aqueous solvent. For example, water, ethyl alcohol, sorbitol, carbon dioxide or nitrogen as a supercritical fluid can be used. Among these, water, ethyl alcohol, or a mixed solution thereof is preferable, and a mixed solution of water and ethyl alcohol is most preferable. Usually, purified water such as ion-exchanged water or reverse osmosis filtered water or distilled water is used, but reduced water (alkaline ionized water), acidic water, sodium chloride, sucrose, acetic acid, etc. that have undergone electrolysis are used. Dissolved water can also be used. When a water-ethyl alcohol mixture is used, the mixing ratio of the two can be arbitrarily changed. Among them, ethyl alcohol is preferably 15 to 60% by mass, more preferably 20 to 50% by mass, and most preferably 25 to 40% by mass. Moreover, it can also extract using the water- ethyl alcohol solvent from which alcohol content differs for the purpose of extracting various components or improving extraction efficiency.

  An extraction method will not be specifically limited if it is a method used for extraction of a plant body. For example, it is extracted by being immersed in a solvent and allowed to stand under standing conditions or stirring conditions, extracted under conditions in which the extraction solvent is circulated, extracted under pressurized / depressurized conditions, or extracted in a supercritical state, Alternatively, a method combining them can be used. Although it does not limit, extraction temperature is performed in the range of 0-130 degreeC. Since the optimum extraction temperature differs depending on the extraction method and the extraction solvent, the extraction temperature can be appropriately determined. In addition, from the viewpoint of the flavor and appearance (especially color) of the cherry blossom extract, the lower extraction temperature is generally preferable, and from the viewpoint of extraction efficiency, the higher temperature is generally preferable. The purpose of use can be determined in consideration. The extraction time greatly depends on the extraction method used and the solvent used. For example, when the solvent is a water-ethyl alcohol mixture, it is usually about 1 minute to 42 hours, preferably about several minutes to 24 hours, and more preferably about 20 minutes to 4 hours. When the solvent is water, it is usually about 1 to 106 hours, preferably about 10 minutes to 24 hours, and more preferably about 20 minutes to 6 hours. The ratio between the cherry blossom and the extraction solvent is not particularly limited and can be set as appropriate. For the bath ratio at the time of extraction, for example, supercritical extraction, an extraction method for circulating the solvent, an extraction method by standing still In such cases, the extraction conditions are completely different. In general, it is appropriate to set the extraction solvent in the range of 2 to 1000 parts by weight, preferably 2 to 50 parts by weight, and more preferably 3 to 30 parts per 1 part by weight of the cherry blossoms. About mass parts.

  The cherry blossom extract obtained as described above can be subjected to treatments such as concentration, purification, and drying as required. For example, concentration such as vacuum concentration treatment, heat concentration treatment, membrane concentration treatment, etc. for the purpose of improving storage stability, reducing transportation costs, removing or reducing ethyl alcohol, etc., improving appearance and smell, Filtration treatment, membrane separation treatment using ultrafiltration membrane or reverse osmosis membrane, centrifugal separation treatment, activated carbon adsorption treatment, resin column treatment, chemistry for the purpose of removing impurities and ethyl alcohol Purification such as treatment can be performed, and drying such as freeze-drying treatment, granulation drying treatment, and spray drying treatment can be performed. The obtained extract of cherry blossoms can be taken as it is, and can be mixed with other ingredients and used in various dosage forms. For example, after adding an excipient | filler to a dried material, it can be set as a tablet, a tablet, a granule, a powder, etc., or it can be set as the other food-drink form.

  The oral composition for prevention / amelioration of bone loss-related diseases containing the osteoblast differentiation promoter of the present invention is various forms such as liquid or liquid, viscous material, jelly, solid gel, powder, granule, block, etc. It can be. Among these, forms that can be ingested as beverages, for example, liquid, liquid, powder, and granule forms are preferable because they can be easily ingested in large quantities. In particular, it can be used as foods for specific uses such as foods for sick people, foods for people with difficulty swallowing, foods for specified health use, nutritional functional foods, and so-called nutritional supplements.

  In addition to the above-mentioned compounds, the composition for preventing / ameliorating a bone mass-decreasing disease may further contain other components as long as the effects of the present invention are not impaired. For example, other known food additives such as emulsifiers, sweeteners, antioxidants, colorants, seasonings, vitamins, minerals, fragrances, colorants, thickening polysaccharides, etc. Ingredients (including food additives as well as food additives) may be included.

  Hereinafter, the present invention will be specifically described, but the present invention is not limited to the following examples. In the following, “%” means “mass%” unless otherwise specified.

Alkaline phosphatase activation effect of cherry blossom extract

Mouse osteoblasts (MC3T3-E1) cultured using α-MEM medium supplemented with 10% FBS are suspended in α-MEM medium supplemented with 10% FBS at a concentration of 1.0 × 10 ⁵ cells / mL. 100 μl each of the suspension was seeded in a 96-well plate. The number of tests was 3 per sample. The culture was performed for 7 days, during which the medium was changed every 3 to 4 days. The alkaline phosphatase (hereinafter abbreviated as “ALP”) activity measurement cells obtained under each culture condition were stored frozen for each well plate.
The ALP activity was measured by thawing the cryopreserved cells in a well plate and adding a mixture solution prepared by adding PMSF, a protease inhibitor, to Cell-LyEX1, which is a cell lysing agent, to 2 mM. 40 μL was added and stirred at room temperature for 15 minutes. Thereafter, the supernatant obtained by centrifugation was used as a sample for measuring ALP activity.
LabAssay ALP (Wako Pure Chemical Industries) and Micro BCA Protein Assay Reagent Kit (PIERCE) were used for the ALP activity measurement and protein content measurement, respectively. Performed by the method shown.

(ALP activity measurement)
100 μl of a carbonate buffer containing p-nitrophenyl phosphate as a substrate buffer and 20 μl of each sample or ALP standard stock solution were added to the wells prepared as described above and stirred for 1 minute using a plate mixer. After stirring, color development is performed by incubating at 37 ° C. for 15 minutes. Then, 80 μl of reaction stop solution sodium hydroxide solution is added, mixed for 1 minute with a plate mixer, and the absorbance of each well using a plate reader (measurement wavelength: 405 nm) Was measured. A standard curve for ALP activity was prepared using 0.0625, 0.125, 0.25, and 0.5 mmol / L ALP standard solutions, and the ALP activity (mmol / min) in the sample was calculated from the measured values. did.

(Protein measurement)
100 μl of the reaction solution and 100 μl of each sample or BSA concentrated standard solution were added to the wells prepared by the above method and stirred for 1 minute using a plate mixer. After stirring, the color was developed by incubating at 37 ° C. for 15 minutes, and the absorbance (measurement wavelength 562 nm) of each well was immediately measured using a plate reader. The standard curve of BSA activity was prepared using 1.25, 2.55, 10, 20, 40, 80 μg / ml BSA standard solution, and the protein content (mg / mL) in the sample from the measured value. Was calculated.

The ALP activation effect was evaluated by calculating “ALP unit amount per protein mass” obtained by dividing the measured ALP activity (mmol / min) by the protein mass (mg / mL). The calculated results are shown in Table 1.

  As shown in Table 1, it was found that the cherry flower extract enhances ALP activity in a concentration-dependent manner. In particular, at 10 μg / ml, a significantly higher ALP specific activity was observed at a risk rate of 5% compared to the control. Since the expression of ALP in osteoblasts is known as an index of osteoblast differentiation, it is suggested that the extract of cherry blossoms promotes cell differentiation of osteoblasts.

Claims (2)

  An osteoblast differentiation promoting agent containing a cherry blossom extract.   An oral composition for the prevention or symptom improvement of osteopenia, comprising the osteoblast differentiation promoting agent according to claim 1.