Calcium absorption enhancer and gene expression enhancer

Abstract

This invention provides a novel calcium absorption enhancer. It also provides a gene expression enhancer that can enhance the expression of the Cav1.3 gene and / or PMCA1 gene, which are genes involved in calcium transport. [Solution] A calcium absorption enhancer containing quinic acid and / or malic acid as active ingredients. Also, a gene expression enhancer containing quinic acid and / or malic acid as active ingredients that enhances the expression of the Cav1.3 gene and / or PMCA1 gene.

Description

Technical Field

[0001] The present invention relates to a calcium absorption promoter. It also relates to a gene expression enhancer that enhances the expression of the Cav1.3 gene and / or the PMCA1 gene.

Background Art

[0002] Calcium, which is one of the essential minerals, mostly binds to phosphate and is present in bones and teeth, and a very small part is present in blood, muscles, nerves, etc. It is known that calcium deficiency causes osteoporosis, neuralgia, etc. However, in the current diet, calcium tends to be deficient. Therefore, foods and supplements that promote its absorption have been developed for the purpose of preventing calcium deficiency and enhancing absorption (for example, Patent Document 1, etc.).

[0003] On the other hand, for calcium absorption, there are a pathway that passes through the intercellular space (intercellular space transport pathway) and a pathway that passes through the cell interior (transcellular transport pathway). The pathway that passes through the cell interior is composed of three steps: calcium influx, which is the initial process, cytoplasmic movement, which is the intermediate process, and calcium efflux, which is the final process. For example, the Cav1.3 gene is known as one of the genes related to calcium influx, and PMCA1 is known as one of the genes related to calcium efflux.

Prior Art Documents

Patent Documents

[0006] The inventors of this invention have conducted extensive research to solve the above problems and have found that the following invention is suitable for the above purpose, leading to the present invention. That is, the present invention relates to the following invention. <1> A calcium absorption enhancer containing quinic acid and / or malic acid as active ingredients. <2> The above-mentioned, containing quinic acid and malic acid as active ingredients <1> A calcium absorption enhancer as described above. <3> The method of promoting calcium absorption by enhancing the expression of the Cav1.3 gene and / or the PMCA1 gene, <1> or <2> A calcium absorption enhancer as described above. <4> A gene expression enhancer containing quinic acid and / or malic acid as active ingredients, which enhances the expression of the Cav1.3 gene and / or the PMCA1 gene. [Effects of the Invention]

[0007] The present invention provides a novel calcium absorption enhancer. It also provides a gene expression enhancer that can enhance the expression of the Cav1.3 gene and / or PMCA1 gene, which are genes involved in calcium transport. [Modes for carrying out the invention]

[0008] The embodiments of the present invention will be described in detail below, but the description of the constituent elements described below is just one example (representative example) of an embodiment of the present invention, and the present invention is not limited to the following unless its gist is changed. In this specification, when the expression "~" is used, it is used to include the numerical value or physical property value before and after it.

[0009] The present invention relates to a calcium absorption enhancer containing quinic acid and / or malic acid as active ingredients.

[0010] The inventors have discovered that quinic acid alone, malic acid alone, or a combination of quinic acid and malic acid have a calcium absorption-promoting effect. The calcium absorption enhancer of the present invention can promote calcium absorption by containing at least one of quinic acid or malic acid. Furthermore, the calcium absorption enhancer of the present invention can be taken before, during, or after meals. By taking the calcium absorption enhancer of the present invention with meals, the absorption of calcium contained in the meal can be promoted by containing at least one of quinic acid or malic acid, thereby increasing the absorption of calcium into the body.

[0011] Furthermore, as described above, the pathway through which calcium passes through cells consists of three stages: the initial process of calcium influx, the intermediate process of cytoplasmic transport, and the final process of calcium efflux. In the intestinal tract, calcium channels are expressed on the apical side of cells, through which calcium is taken up, and calcium ions are pumped into the bloodstream by calcium pumps on the basolateral side. The inventors have discovered that the expression of Cav1.3 and PMCA1, genes involved in calcium transport, is significantly increased in the use of quinic acid alone, malic acid alone, or a combination of quinic acid and malic acid.

[0012] Cav1.3 (L-type voltage-gated calcium channel α1D subunit) is Ca 2+ It is an ion-transporting protein belonging to the channel family, and is involved in calcium influx, which is the initial stage of the intracellular pathway in the active intestinal transport of calcium. PMCA1 (Cell Membrane Calcium Transport ATPase 1), also known as the calcium pump, is a membrane protein involved in calcium efflux, which is the final stage of the intracellular pathway.

[0013] Therefore, the present invention can also be a gene expression enhancer that enhances the expression of the Cav1.3 gene and / or the PMCA1 gene, containing quinic acid and / or malic acid as active ingredients (hereinafter sometimes referred to as "the gene expression enhancer of the present invention").

[0014] Gene expression enhancement refers to an increase in the transcript of a gene and / or the protein encoded by that gene. Specifically, this includes inducing or promoting the transcription of genes (DNA) into mRNA, and inducing or promoting the translation of mRNA into protein.

[0015] Hereinafter, the "calcium absorption enhancer of the present invention" and the "expression enhancer of the present invention" will be collectively referred to as the "agent of the present invention."

[0016] (Quinic acid) Quinic acid exists as L-isomers and D-isomers, but the quinic acid contained in the agent of the present invention may be either the L-isomer or the D-isomer, or it may be a racemic mixture. Furthermore, in the agent of the present invention, quinic acid may be in the form of a salt, and examples of quinic acid salts include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and ammonium salt.

[0017] Quinic acid can be obtained by isolating it from plants containing quinic acid or by chemical synthesis. Quinic acid can be incorporated into the agent of the present invention as pure quinic acid. Alternatively, plants (whole or organs) or processed products thereof containing quinic acid may be incorporated into the agent of the present invention, or processed products of plants containing quinic acid may be used as the agent of the present invention. Examples of processed products of plants containing quinic acid include pulverized products, crushed products, juices, purees, extracts, and dried products, which can be combined to form the agent of the present invention, or used as the agent of the present invention. Cranberry fruit is a known plant that contains quinic acid.

[0018] The content of quinic acid in the agent of the present invention is designed according to the intake method and the like so that the intake amount of quinic acid per adult per day reaches the target amount. For example, the agent of the present invention preferably contains 0.005 mg or more of quinic acid as the intake amount per adult per day, more preferably 0.01 mg or more, and even more preferably 0.05 mg or more. Also, considering the ease of formulation into the agent of the present invention and the like, it is preferable that the agent contains 0.1 mg or more, 1 mg or more, 10 mg or more, 100 mg or more, 200 mg or more of quinic acid as the intake amount per adult per day. There is no particular limitation on the upper limit of the content of quinic acid in the agent of the present invention, but as the intake amount per adult per day, it is preferable to contain 10,000 mg or less of quinic acid, more preferably 5,000 mg or less, and even more preferably 3,000 mg or less.

[0019] The agent of the present invention may be taken once or divided into multiple times so that the quinic acid reaches the above intake amount. The intake amount per day means the total amount of the amount taken or administered in a day. When taken in multiple times in a day, it is the total of these amounts. Also, the agent of the present invention can be taken continuously and can be taken continuously for several days to several weeks or several months.

[0020] (Malic acid) Malic acid has L-form and D-form optical isomers, but the malic acid contained in the agent of the present invention may be either the L-form or the D-form, or may be a racemic form. Also, in the agent of the present invention, malic acid may be in the form of a salt, and examples of the salt of malic acid include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and ammonium salt.

[0021] Malic acid can be obtained by isolating it from plants containing malic acid or by chemically synthesizing it. Malic acid can be formulated into the agent of the present invention as pure malic acid. Further, a plant (whole or an organ) containing malic acid or its processed product may be formulated into the agent of the present invention, or a processed product of a plant containing malic acid may be used as the agent of the present invention as it is. Examples of the form of the processed product of a plant containing malic acid include, for example, crushed products, crushed materials, juice, puree, extract, and dried products. These can be formulated to form the agent of the present invention, or these can be used as the agent of the present invention as they are. Known plants containing malic acid include apple fruits and the like.

[0022] The content of malic acid in the agent of the present invention is designed according to the intake method and the like so that the intake amount of malic acid per adult per day reaches the target amount. For example, the agent of the present invention preferably contains 0.005 mg or more of malic acid, more preferably 0.01 mg or more, and even more preferably 0.05 mg or more as the intake amount per adult per day. Also, considering the ease of formulation into the agent of the present invention and the like, it is preferable that the agent contains 0.1 mg or more, 1 mg or more, 10 mg or more, 100 mg or more, 200 mg or more of malic acid as the intake amount per adult per day. There is no particular limitation on the upper limit of the content of malic acid in the agent of the present invention, but it is preferably 10,000 mg or less, more preferably 5,000 mg or less, and even more preferably 3,000 mg or less of malic acid as the intake amount per adult per day.

[0023] The agent of the present invention may be taken once or divided into multiple times so that the amount of malic acid reaches the above intake amount. The intake amount per day means the total amount of the amount taken or administered in a day. When taken in multiple times in a day, it is the total of these amounts. Also, the agent of the present invention can be taken continuously and can be continuously taken over several days to several weeks or several months.

[0024] Furthermore, the agent of the present invention preferably contains quinic acid and malic acid. In this case, quinic acid and malic acid are not particularly limited, but for example, the mass ratio of quinic acid to malic acid (quinic acid / malic acid) can be 0.3 or more, 0.4 or more, 0.5 or more, or 3.0 or less, 2.5 or less, 2.0 or less, 1.5 or less, 1.0 or less, etc. Furthermore, if quinic acid is in the form of a salt, the amount of quinic acid shall be converted to the value equivalent to free acid, and if malic acid is in the form of a salt, the amount of malic acid shall be converted to the value equivalent to free acid.

[0025] The agent of the present invention may contain other components in addition to quinic acid and malic acid. These other components are not particularly limited as long as they are safe for humans. Examples of other components include excipients for forming the agent and various additives used in food and pharmaceuticals.

[0026] The agent of the present invention is intended for use in animals, including humans. Examples of animals other than humans include dogs and cats. Furthermore, it is preferable that the agent be used in healthy individuals (i.e., people who are not suffering from diseases or illnesses).

[0027] The agent of the present invention is preferably in the form of an oral composition.

[0028] (Food composition) The agent of the present invention can be made into a food composition. By making the agent of the present invention into a food composition, it can be easily taken on a daily, long-term basis, and the absorption of calcium, which is often deficient in daily life, can be enhanced. When made into a food composition, examples of such compositions include compositions that combine quinic acid alone and / or malic acid alone, food compositions that use processed plants containing quinic acid and / or malic acid as the food composition itself, and food compositions that combine processed plants containing quinic acid and / or malic acid. This food composition includes not only general foods, but also foods and / or beverages consumed for the purpose of maintaining and promoting health, such as foods for special dietary uses, foods for specified health uses, foods with functional claims, nutritional function foods, nutritional supplements, health supplements, and supplements. Among these, foods for specified health uses, foods with functional claims, and nutritional function foods, which are health function foods, are preferred embodiments.

[0029] The shape of the food composition is not particularly limited and may be solid, liquid, semi-solid, gel, sol, or any other form. The foods and beverages are not particularly limited; for example, foods include sausages, ham, processed seafood products, jelly, candy, and chewing gum. Beverages include various fruit juices, teas, soft drinks, alcoholic beverages, and nutritional drinks. The food composition may also contain additives such as colorants, preservatives, thickeners and stabilizers, antioxidants, bleaching agents, antibacterial and antifungal agents, acidulants, seasonings, emulsifiers, fortifiers, flavorings, and sweeteners.

[0030] (Pharmaceutical composition) The agent of the present invention can be a pharmaceutical composition. This pharmaceutical composition may be either a pharmaceutical or a quasi-drug. When used as a pharmaceutical composition, the agent of the present invention basically contains quinic acid and / or malic acid, in addition to a pharmaceutically acceptable carrier. Examples of such compositions include compositions in which quinic acid and / or malic acid alone are blended into a pharmaceutical composition, compositions in which processed plant products containing quinic acid and / or malic acid are used as the pharmaceutical composition itself, and compositions in which processed plant products containing quinic acid and / or malic acid are blended into a pharmaceutical composition.

[0031] The dosage form of the pharmaceutical composition is not particularly limited and may be a solid or liquid formulation. Specifically, examples include powders, granules, tablets, capsules, lozenges, suspensions, emulsions, syrups, elixirs, and injections. Examples of pharmaceutically acceptable carriers include binders, stabilizers, excipients, diluents, pH buffers, thickeners, colorants, dispersants, emulsifiers, suspending agents, preservatives, disintegrants, solubilizers, and solubilizers.

[0032] The agent of the present invention may be used as an animal food composition such as pet food or an animal pharmaceutical composition. Specifically, it can be used in the form of a combination of quinic acid alone and / or malic acid alone in an animal food composition or an animal pharmaceutical composition, or a combination of a processed plant containing quinic acid and / or malic acid itself in an animal food composition or an animal pharmaceutical composition, or a combination of a processed plant containing quinic acid and / or malic acid in an animal food composition or an animal pharmaceutical composition. [Examples]

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples unless its essence is changed.

[0034] To confirm whether quinic acid and malic acid have a calcium absorption-promoting effect, we used an intestinal absorption evaluation model utilizing Caco-2 cells to verify the calcium absorption-promoting effect.

[0035] <Test substance> Quinic acid (manufactured by Fujifilm Wako Pure Chemical Industries) Malic acid (manufactured by Fujifilm Wako Pure Chemical Industries)

[0036] <Cells and culture medium used> Cell: Caco-2 cell Medium: D-MEM medium containing 10% fetal bovine serum (FBS), HBSS buffer

[0037] <Example 1: Calcium Uptake Assay> A calcium uptake assay was performed using various organic acids (quinic acid, malic acid) and Caco-2 cells in a 3-hour intestinal absorption model.

[0038] 1. Test Method 1.1. Construction of a Caco-2 cell intestinal absorption model 1) Caco-2 cells were cultured in cell culture flasks until an appropriate number of passages were reached. 2) After culturing, detach the cells using trypsin, 2 × 10 5 cell / cm 2 The seeds were sown in Trans-well inserts to achieve this result. 3) A monolayer was formed in the Trans-well insert while changing the culture medium every other day (for approximately 20 days). After monolayer formation, the TER value (membrane resistance) was 500 Ω·cm. 2 The above items were used.

[0039] 1.2. Calcium Uptake Assay 1) After washing the monolayer-formed Trans-well insert with HBSS warmed to 37°C, 0.5 mL of HBSS was dispensed into the inside of the insert and 1.0 mL into the outside, and the mixture was allowed to stand for 30 minutes to equilibrate. 2) After equilibration, the HBSS inside was aspirated and removed. A test solution was prepared by dissolving quinic acid and malic acid in a 4 μmol / mL calcium chloride solution to their respective concentrations, and 0.5 mL of this solution was dispensed into the inside of the insert. 3) In addition, test groups were established with quinic acid 0.2 mmol / L and malic acid 0.4 mmol / L, quinic acid 0.4 mmol / L and malic acid 0.4 mmol / L, and quinic acid 0.4 mmol / L and malic acid 0.2 mmol / L. 4) After 3 hours of incubation, the amount of calcium that permeated into each well at the bottom was measured using a colorimetric method with o-Cresolphthalein Complexone (Calcium Colorimetric Assay Kit, Bio Vision).

[0040] 2.Results Table 1 shows the results of calcium uptake assays using quinic acid alone and malic acid alone. Table 2 shows the results of calcium uptake assays using a combination of quinic acid and malic acid. As shown in Tables 1 and 2, calcium absorption-promoting effects were observed with quinic acid alone, malic acid, and combinations thereof. Furthermore, an additive effect was observed in the calcium absorption-promoting effect of the combination of quinic acid and malic acid.

[0041] [Table 1]

[0042] [Table 2]

[0043] <Example 2: Measurement of gene expression related to the transcellular transport of calcium> Gene expression was measured by PCR for four genes involved in the transcellular transport of calcium (Cav1.3 and CaT1, which are related to the initial stage of calcium absorption, "calcium influx"; Calbindin-D9k, which is related to the intermediate stage, "cytoplasmic transport"; and PMCA1, which is related to the final stage, "calcium efflux"). For gene expression measurement, a Caco-2 cell intestinal absorption model was prepared in the same manner as in Example 1, and after culturing for 20 days, the number of days required for tight junction formation, the TER value (membrane resistance) was 500 Ω·cm. 2 I confirmed that the above conditions were met before proceeding.

[0044] 1. Gene expression measurement 1) Similar to the test groups used in the calcium uptake assay, quinic acid, malic acid, and combinations thereof were treated with a 4 μmol / mL calcium chloride solution and exposed to a Caco-2 cell intestinal absorption model for 48 hours. 2) After exposure, the cell model was washed twice with PBS, and total RNA was extracted using ISOGENII (Nippon Gene). 3) We performed expression analysis on four genes related to calcium absorption using a one-step RT-PCR method via Real-time PCR. 4) The experimental results were evaluated using relative values ​​obtained by dividing the measured gene expression results by the reference gene β-Actin and converting the control value to 1.

[0045] 2.Results Table 3 shows the results of gene expression ratios for quinic acid alone. Table 4 shows the results of gene expression ratios for malic acid alone. Table 5 shows the results of gene expression for combinations of quinic acid and malic acid. As shown in Tables 3 to 5, significant increases in "Cav1.3" and "PMCA1" were observed in all test groups, both those treated with quinic acid and malic acid alone, and those treated with a combination of quinic acid and malic acid.

[0046] [Table 3]

[0047] [Table 4]

[0048] [Table 5]

Claims

[Claim 1] A gene expression enhancer containing quinic acid and / or malic acid as active ingredients, which enhances the expression of the Cav1.3 gene and / or PMCA1 gene.

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