Composition for mitochondrial activation

Abstract

To provide a technique that enables metabolic activation.SOLUTION: A composition for mitochondrial activation contains specific components.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a composition for activating mitochondria, etc.

Background Art

[0002] It is known that oxidative stress in cells is enhanced and metabolic abnormalities are caused by various factors (for example, by high glucose load). In particular, in diabetic patients and subjects at risk of diabetes, it is considered that a high glucose load is applied, metabolism becomes abnormal, and various symptoms are caused. Among such symptoms, symptoms in the oral cavity may also be included.

[0003] For example, it has been reported that in a diabetic state, the metabolic function of the periodontal tissue is impaired, and when the metabolic function of the gingiva of diabetic rats is activated, alveolar bone resorption is suppressed.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present inventors have searched for means capable of activating metabolism.

Means for Solving the Problems

[0006] For example, even when oxidative stress in cells is enhanced, if metabolism can be activated, it can be expected that metabolic abnormalities will be less likely to occur. For this reason, means capable of activating metabolism are considered useful.

[0007] The inventors focused on the function of mitochondria, which play a major role in metabolism within cells, particularly the ATP production ability and oxygen consumption ability in cells, and conducted research. Then, they searched for materials that could improve the ATP production and oxygen consumption rates, found the possibility of improving the ATP production and oxygen consumption abilities in cells by using specific materials, and further refined them.

[0008] This disclosure includes, for example, the subject matter described in the following items. Item 1. A composition for activating mitochondria, comprising at least one selected from the group consisting of pantothenic acid or a salt thereof, protamine or a salt thereof, and methionine or a salt thereof. A composition for activating mitochondria. Item 2. The composition according to Item 1, wherein the mitochondria are mitochondria in periodontal ligament cells. Item 3. The composition according to Item 1 or 2, wherein the mitochondrial activation is at least one selected from the group consisting of an increase in oxygen consumption in mitochondria, an improvement in the ATP production ability in mitochondria, and an increase in mitochondria. Item 4. The composition according to any one of Items 1 to 3, which is used for improving periodontal disease in diabetic patients. Item 5. A composition for improving symptoms caused by diabetes, prediabetes, or metabolic disorders due to aging, comprising at least one selected from the group consisting of pantothenic acid or a salt thereof, protamine or a salt thereof, and methionine or a salt thereof. A composition for improving symptoms caused by diabetes, prediabetes, or metabolic disorders due to aging, comprising at least one selected from the group consisting of pantothenic acid or a salt thereof, protamine or a salt thereof, and methionine or a salt thereof. Item 6. The composition according to Item 5, wherein the symptoms caused by diabetes, prediabetes, or metabolic disorders due to aging are obesity, renal dysfunction, retinopathy, neuropathy, cardiovascular disease, brain dysfunction, dementia, frailty, muscle weakness, or osteoporosis. Item 7. The composition according to any one of Items 1 to 6, which is an oral composition or an oral care composition. Item 8. The composition according to any one of claims 1 to 6, which is a food composition. Item 9. The oral composition according to claim 7, which is an ointment, a paste, a pasta, a gel, a liquid, a spray, a mouthwash, a liquid dentifrice, a dentifrice paste, or a gum.

Advantages of the Invention

[0009] It is possible to activate mitochondria and improve metabolic functions related to mitochondria (in particular, improve the ATP production and oxygen consumption ability of mitochondria). For example, even when oxidative stress in cells is enhanced, by improving the metabolic function, it is possible to suppress the occurrence of metabolic abnormalities.

[0010] In addition, it is possible to improve metabolic abnormalities caused by a high glucose load, particularly a decrease in metabolic functions related to mitochondria. In particular, it is possible to improve a decrease in ATP production and a decrease in oxygen consumption rate caused by a high glucose load.

[0011] In addition, by improving the metabolism of mitochondria in oral cells (for example, gingival cells and periodontal ligament cells), it becomes possible to activate the metabolic function of oral cells or suppress a decrease in the metabolic function, and thus it is expected to keep the oral cavity healthy (for example, prevent periodontal disease).

Brief Description of the Drawings

[0012]

Figure 1a

Figure 1b

Figure 1c

Figure 2a

Figure 2b

Figure 2c

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0013] Hereinafter, each embodiment included in the present disclosure will be described in more detail. The present disclosure preferably includes a composition for activating mitochondria and the like, but is not limited thereto, and the present disclosure includes all that is disclosed herein and can be recognized by those skilled in the art.

[0014] The composition for activating mitochondria included in the present disclosure contains pantothenic acid or a salt thereof, protamine or a salt thereof, and methionine or a salt thereof, alone or in combination of two or more. The composition for activating mitochondria included in the present disclosure may be referred to as the composition of the present disclosure. Also, these specific components may be referred to as the components of the present disclosure.

[0015] As the salt of pantothenic acid, an alkali metal salt or an alkaline earth metal salt is preferred. More specifically, a lithium salt, a sodium salt, a potassium salt, a calcium salt, a barium salt, or a magnesium salt is more preferred, a sodium salt or a calcium salt is even more preferred, and a calcium salt is particularly preferred. These salts can be used alone or in combination of two or more.

[0016] As the salt of protamine, an alkali metal salt or an alkaline earth metal salt is preferred. More specifically, a lithium salt, a sodium salt, a potassium salt, a calcium salt, a barium salt, or a magnesium salt is more preferred, a sodium salt or a calcium salt is even more preferred, and a calcium salt is particularly preferred. Also, as the salt of protamine, a sulfate salt or a hydrochloride salt is also preferably mentioned, and a sulfate salt is particularly preferred. These salts can be used alone or in combination of two or more.

[0017] As the salt of methionine, an alkali metal salt or an alkaline earth metal salt is preferred. More specifically, a lithium salt, a sodium salt, a potassium salt, a calcium salt, a barium salt, or a magnesium salt is more preferred, a sodium salt or a calcium salt is even more preferred, and a calcium salt is particularly preferred. Also, as the salt of methionine, a sulfate salt or a hydrochloride salt is also preferably mentioned. These salts can be used alone or in combination of two or more.

[0018] The content of the component of the present disclosure in the composition of the present disclosure is not particularly limited as long as the effect is not impaired, and for example, about 0.01 to 99.99% by mass can be mentioned.

[0019] The intake form of the composition of the present disclosure is not particularly limited, but oral intake or topical application (e.g., coating) to the oral mucosa is preferred. That is, the composition of the present disclosure is preferably an oral composition or an oral composition. By orally ingesting the composition of the present disclosure, mitochondria can be activated in cells (especially cells in the oral cavity), and the metabolic functions related to mitochondria can be improved (in particular, the ATP production and / or oxygen consumption ability of mitochondria can be improved). The composition of the present disclosure is preferably, for example, an oral pharmaceutical composition, a food composition (including a beverage composition and a food additive composition), or an oral composition.

[0020] The composition of the present disclosure contains the above components and may further contain other components. The other components can be appropriately selected according to the field in which the composition is used. For example, a pharmaceutically or food hygienically acceptable carrier can be used.

[0021] When used as a pharmaceutical composition, examples of other components include pharmaceutically acceptable bases, carriers, and / or additives (e.g., solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, etc.). Also, the form of the pharmaceutical composition is not particularly limited, and examples include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, creams, patches, etc.

[0022] When used as a food composition, examples of other components include bases, carriers, additives that are acceptable in food hygiene, and other components and materials that can be used as foods. Also, the form of the food composition is not particularly limited, and examples include processed foods, health foods (dietary supplements, foods with nutrient functions, foods for patients, foods for specified health uses, foods with functional claims, etc.), supplements, foods for patients (hospital foods, foods for patients, or nursing foods, etc.). These can be prepared by conventional methods. In particular, when preparing a food composition as a health food (dietary supplement, food with nutrient function, food for patients, food for specified health use, food with functional claim, etc.) or a supplement, it is preferably prepared in forms such as granules, capsules, tablets (including chewable tablets, etc.), beverages (beverage powders, drink agents, smoothies, etc.) so that continuous intake is easy. Among these, forms such as capsules, tablets, pills, beverage powders, drink agents, jelly, and gummies are preferable from the viewpoint of ease of intake, but are not particularly limited thereto. When used as a food additive composition among food compositions, examples of its form include liquid, powder, flake, granule, and paste forms.

[0023] When used as an oral composition, for example, it can be made into forms (dosage forms) such as ointments, pastes, pastilles, gels, liquids, sprays, mouthwashes, liquid dentifrices, paste dentifrices, and gums.

[0024] Also, as other components, components known to be formulated in oral compositions can be used.

[0025] For example, as the surfactant, a nonionic surfactant, an anionic surfactant, or an amphoteric surfactant can be blended. Specifically, for example, as the nonionic surfactant, sugar fatty acid esters such as sucrose fatty acid ester, maltose fatty acid ester, lactose fatty acid ester; fatty acid alkanolamides; glycerin fatty acid ester; sorbitan fatty acid ester; fatty acid monoglyceride; polyoxyethylene alkyl ether with a polyoxyethylene addition coefficient of 8 to 10 and an alkyl group carbon number of 13 to 15; polyoxyethylene alkyl phenyl ether with a polyoxyethylene addition coefficient of 10 to 18 and an alkyl group carbon number of 9; diethyl sebacate; polyoxyethylene hydrogenated castor oil; fatty acid polyoxyethylene sorbitan, etc. can be mentioned. As the anionic surfactant, sulfate esters such as sodium lauryl sulfate, sodium polyoxyethylene lauryl ether sulfate; sulfosuccinates such as sodium lauryl sulfosuccinate, sodium polyoxyethylene lauryl ether sulfosuccinate; acyl amino acid salts such as sodium cocoyl sarcosinate, sodium lauroyl methyl alaninate; sodium cocoyl methyl taurine, etc. can be mentioned. As the amphoteric ion surfactant, betaine type surfactants such as lauryldimethylaminoacetic acid betaine, coconut oil fatty acid amide propyldimethylaminoacetic acid betaine; imidazoline type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethyl ethylenediamine sodium; amino acid type surfactants such as N-lauryl diaminoethyl glycine, etc. can be mentioned. These surfactants can be blended alone or in combination of two or more. The blending amount is usually 0.1 to 5% by mass based on the total amount of the composition.

[0026] In addition, as flavoring agents, for example, menthol, carboxylic acid, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronellal, α-terpineol, methyl acetate, citronellyl acetate, methyleugenol, cineole, linalool, ethyllinalool, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, galbanum oil, perilla oil, wintergreen oil, clove oil, eucalyptus oil, pimento oil, d-camphor, d-borneol, star anise oil, cinnamon oil, mint oil, vanilla, etc. can be used. These can be blended, alone or in combination of two or more, in an amount of, for example, 0.001 to 1.5% by mass based on the total amount of the composition.

[0027] In addition, as sweetening agents, for example, sodium saccharin, acesulfame potassium, stevioside, neohesperidin dihydrochalcone, perillartine, thaumatin, aspartylphenylalanyl methyl ester, p-methoxycinnamic aldehyde, etc. can be used. These can be blended in an amount of, for example, 0.01 to 1% by mass based on the total amount of the composition.

[0028] Furthermore, as wetting agents, sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylitol, maltitol, lactitol, polyoxyethylene glycol, etc. can be blended alone or in combination of two or more.

[0029] As preservatives, parabens such as methylparaben, ethylparaben, propylparaben, butylparaben, sodium benzoate, phenoxyethanol, alkyldiaminoethyl glycine hydrochloride, etc. can be blended.

[0030] As coloring agents, legal dyes such as Blue No. 1, Yellow No. 4, Red No. 202, Green No. 3, mineral dyes such as ultramarine, fortified ultramarine, dark ultramarine, titanium oxide, etc. may be blended.

[0031] As a pH adjuster, citric acid, phosphoric acid, malic acid, pyrophosphoric acid, lactic acid, tartaric acid, glycerophosphoric acid, acetic acid, nitric acid, or chemically possible salts thereof such as sodium hydroxide may be blended. These can be blended alone or in combination of two or more so that the pH of the composition is in the range of 4 to 8, preferably 5 to 7. The blending amount of the pH adjuster may be, for example, 0.01 to 2% by weight.

[0032] As medicinal ingredients, vitamin Es such as dl-α-tocopherol acetate, tocopherol succinate, or tocopherol nicotinate, amphoteric bactericides such as dodecyldiaminoethyl glycine, nonionic bactericides such as triclosan, isopropylmethylphenol, and hinokitiol, anionic bactericides such as sodium lauroyl sarcosinate, cationic bactericides such as cetylpyridinium chloride, chlorhexidine hydrochloride, benzalkonium chloride, and benzethonium chloride, enzymes such as dextranase, amylase, protease, mutanase, lysozyme, and lysing enzyme (Lytech Enzyme), alkali metal monofluorophosphates such as sodium monofluorophosphate and potassium monofluorophosphate, fluorides such as sodium fluoride and stannous fluoride, tranexamic acid and epsilon-aminocaproic acid, aluminum chlorhydroxyl allantoin, dihydrocholesterol, glycyrrhetinic acid, sodium copper chlorophyllin, glycerophosphate, chlorophyll, sodium chloride, caropeptide, carbazochrom, hinokitiol, potassium nitrate, paratinit, etc. can be blended alone or in combination of two or more.

[0033] Also, as a base, it is possible to add alcohols, silicon, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, plastic base, etc.

[0034] The compositions of the present disclosure can be preferably used for activating mitochondria in cells. More specifically, the compositions of the present disclosure can preferably improve, for example, the decrease in oxygen consumption rate caused by high glucose or oxidative stress load. Therefore, it can be preferably used not only for healthy individuals but also, for example, in people with diabetes or pre-diabetes (and cells derived from such people) to improve metabolic abnormalities. For example, in a diabetic state, the metabolic function of the periodontal tissue is impaired, and this is considered to enhance the progression of periodontal disease in diabetic patients. Therefore, it can be preferably used, in particular, to improve metabolic abnormalities in cells in the periodontal tissue (for example, periodontal ligament cells), and thus to suppress and / or improve the progression of periodontal disease. In addition, not only can it improve the metabolism of mitochondria in cells in the oral cavity (for example, gingival cells and periodontal ligament cells), but it can also activate the metabolic function of various cells or suppress the decrease in metabolic function. Therefore, the compositions of the present disclosure can be used not only to suppress and / or improve the progression of periodontal disease, but also, for example, to improve the metabolic function of other tissues impaired by diabetes, pre-diabetes, or aging. For example, it can be used to suppress and / or improve obesity, renal dysfunction, retinopathy, neuropathy, cardiovascular disease, brain dysfunction, dementia, frailty, muscle weakness, and osteoporosis.

[0035] The subjects for ingestion or application of the compositions of the present disclosure are not particularly limited. For example, they can be preferably ingested or applied to healthy individuals. Also, from the perspective that the effects can be preferably achieved, subjects with metabolic abnormalities (especially subjects with reduced mitochondrial metabolism, such as those with enhanced oxidative stress in cells) are preferred. More specifically, for example, subjects with metabolic abnormalities caused by high glucose load are more preferred. Also, as described above, by improving the metabolism of mitochondria in cells in the oral cavity (for example, gingival cells and periodontal ligament cells), it becomes possible to activate the metabolic function of cells in the oral cavity or suppress the decrease in metabolic function. Therefore, it is expected that it is also possible to keep the oral cavity healthy (for example, prevent periodontal disease). Therefore, subjects with gingival inflammation or periodontal disease are also preferred.

[0036] Also, the timing of ingesting or applying the composition of the present disclosure is not particularly limited. For example, it can be used to ingest or apply before or after a meal (e.g., within about 0.5 or 1 hour before or after a meal). Also, for example, it can be preferably used for preventing recurrence during or after periodontal disease treatment.

[0037] Also, although not particularly limited, as cells in which metabolic abnormalities can be suppressed by ingesting or applying the composition of the present disclosure, oral cells are preferred, and among them, gingival cells and periodontal ligament cells (particularly gingival fibroblasts and periodontal ligament fibroblasts) are preferred. Periodontal ligament cells are known to be strongly involved in immune responses, inflammatory responses, and alveolar bone resorption in periodontal disease, and are most preferred.

[0038] In addition, as used herein, "comprising" includes "consisting essentially of" and "consisting of". Also, the present disclosure encompasses any combination of the constituent elements described herein.

[0039] Also, the various characteristics (properties, structures, functions, etc.) described for each of the above-described embodiments of the present disclosure may be combined in any manner in identifying the subject matter encompassed by the present disclosure. That is, the present disclosure encompasses all subject matters consisting of any combination of the combinable characteristics described herein.

Examples

[0040] Hereinafter, the embodiments of the present disclosure will be described more specifically with examples, but the embodiments of the present disclosure are not limited to the following examples.

[0041] After subjecting human periodontal ligament fibroblasts (HPDLF) to the test sample application treatment, the intracellular ATP concentration and the oxygen consumption rate (OCR) in the cells were measured. More specifically, it was carried out as follows.

[0042] Calcium pantothenate, protamine sulfate, and methionine were used as the test substances (samples). All test substances were dissolved in the medium used in the test and used as solutions.

[0043] Measurement of intracellular ATP in periodontal ligament fibroblasts Intracellular ATP was measured using a Luminescent ATP detection assay kit (ab113849; Abcam). Human periodontal ligament fibroblasts (HPDLF) were seeded in a 48-well plate at a density of 0.12×10 5 cells / 100μL / well and cultured for 48 hours. Then, after pretreatment with each test substance (sample) for 24 hours, they were further cultured for 72 hours in the presence of 50 mM D-glucose (Sigma Aldrich). As a control, the same treatment was performed with 50 mM L-glucose. Then, 50 μL of cell lysate was added for 5 minutes to lyse the cells and stabilize ATP. Furthermore, D-luciferase reagent was added and incubated for 10 minutes in the dark. The chemiluminescence due to the luciferase reaction was measured using a Cytation 5 plate reader (BioTek Instruments), and the ATP concentration (μM) was measured using a standard curve. The ATP reduction improvement rate (%) of each sample was determined using the following formula.

[0044]

Equation

[0045] That is, the ATP reduction improvement rate (%) is the ratio (%) obtained by dividing the value obtained by subtracting the ATP value when treated with only D-glucose from the ATP value when treated with D-glucose after sample treatment by the value obtained by subtracting the ATP value when treated with only D-glucose from the ATP value when treated with only L-glucose.

[0046] The results are shown in Figs. 1a to 1c. In the figures, pantothen is calcium pantothenate, and protamine is protamine sulfate. Also, the concentrations described in the figures indicate the concentrations of each test substance in the culture medium. The same applies to the following figures.

[0047] Measurement of oxygen consumption rate (OCR) in periodontal ligament fibroblasts OCR was measured using an Extracellular OCR plate assay kit (E297, DOJINDO). Human periodontal ligament fibroblasts (HPDLF) were seeded in a 96-well plate at a density of 2×10 5 cells / 100 μL / well and cultured overnight. Then, after pretreatment with each test substance (sample) for 24 hours, they were further cultured for 24 hours in the presence of 100 mM D-glucose (Sigma Aldrich). As a control, the same treatment was performed with 100 mM L-glucose. 100 μL of an oxygen probe reaction solution having the property that the phosphorescence intensity increases when the oxygen concentration in the culture medium decreases was added to each well. Then, mineral oil drops were added to each well to prevent the inflow of oxygen from the air. The phosphorescence intensity was measured using a Cytation 5 plate reader (BioTek Instruments), and the OCR of the cells was calculated by the Stern-Volmer equation. The OCR improvement rate (%) by each sample was calculated by the following formula.

[0048]

Equation

[0049] That is, the OCR reduction improvement rate (%) is the ratio (%) obtained by dividing the value obtained by subtracting the OCR value when only D-glucose is treated from the OCR value when D-glucose is treated after sample treatment by the value obtained by subtracting the OCR value when only D-glucose is treated from the OCR value when only L-glucose is treated.

[0050] The results are shown in Figs. 2a to 2c.

[0051] Measurement of oxygen consumption rate (OCR) in periodontal ligament fibroblasts (without high glucose load) OCR was measured using an Extracellular OCR plate assay kit (E297, DOJINDO). Human periodontal ligament fibroblasts (HPDLF) were seeded in a 96-well plate at a density of 2×10 5 cells / 100 μL / well and cultured overnight. Then, each test substance (sample: final concentration 1 μg / mL) was used to treat the cells for 24 hours.

[0052] The measurement of OCR was performed as follows. 100 μL of an oxygen probe reaction solution having the property that the fluorescence intensity increases when the oxygen concentration in the culture solution decreases was added to each well. Then, mineral oil drops were added to each well to prevent the inflow of oxygen from the air. The fluorescence intensity was measured using a Cytation 5 plate reader (BioTek Instruments), and the OCR of the cells was calculated by the Stern-Volmer equation. The OCR value (relative value) of each test substance is shown as a numerical value when the steady OCR value when nothing is administered is set to 1. That is, in order to set the steady OCR value when nothing is administered to 1, when the OCR value doubles due to the administration of the test substance, the OCR value is 2.

[0053] The results are shown in Figure 3. The uncoupling agent FCCP was used as the positive target substance at a final concentration of 2 μM. Note that FCPP is Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Also, only the medium was used as a control.

[0054] Method for measuring mitochondrial DNA in periodontal ligament fibroblasts Human Periodontal Ligament Fibroblast (HPDLF) was seeded in a 12-well plate at a density of 0.9×10 5 cells / well), and after 2 days, each test substance (sample: final concentration 1 μg / mL) was used to treat for 24 hours. Then, it was cultured for 3 days in the presence of 50 mM D-glucose (Sigma Aldrich, USA). The control was treated in the same manner with 50 mM L-glucose. DNA was isolated using the NucleoSpin Tissue extraction kit (Takara, Japan). Quantitative PCR of the isolated DNA was performed using the TB Green Fast qPCR Mix (Takara, Japan) (ABI7500fast (Thermo Fisher Scientific)), and the copy number of mitochondrial DNA (mtDNA) was calculated relatively based on nuclear DNA (nDNA).

[0055] The results are shown in Figure 4. In this figure, HG indicates that only 50 mM D-glucose was applied (without applying the test substance). Also, the control indicates that only 50 mM L-glucose was applied (without applying the test substance).

[0056] An increase in the relative copy number of mtDNA to nDNA is considered to mean that mitochondria increase, and consequently, the metabolic functions related to mitochondria are improved.

Claims

1. A composition for activating mitochondria, comprising at least one selected from the group consisting of pantothenic acid or a salt thereof, protamine or a salt thereof, and methionine or a salt thereof. A composition for activating mitochondria.

2. The composition according to claim 1, wherein the mitochondria are mitochondria in periodontal ligament cells.

3. The composition according to claim 1, wherein the mitochondrial activation is at least one selected from the group consisting of an increase in oxygen consumption in mitochondria, an improvement in ATP production ability in mitochondria, and an increase in mitochondria.

4. The composition according to claim 3, which is used for improving periodontal disease in diabetic patients.

5. A composition for improving symptoms caused by diabetes, prediabetes, or metabolic disorders due to aging, containing at least one selected from the group consisting of pantothenic acid or a salt thereof, protamine or a salt thereof, and methionine or a salt thereof. A composition for improving symptoms caused by diabetes, prediabetes, or metabolic disorders due to aging.

6. The composition according to claim 5, wherein the symptoms caused by diabetes, prediabetes, or metabolic disorders due to aging are obesity, renal dysfunction, retinopathy, neuropathy, cardiovascular disease, brain dysfunction, dementia, frailty, muscle strength decline, or osteoporosis.

7. The composition according to any one of claims 1 to 6, which is an oral composition or an oral cavity composition.

8. The composition according to any one of claims 1 to 6, which is a food composition.

9. The oral cavity composition according to claim 7, which is an ointment, a paste, a pasta, a gel, a liquid, a spray, a mouthwash, a liquid dentifrice, a kneaded dentifrice, or a gum.

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