Composition for improving metabolism

Abstract

To provide a material that can inhibit metabolic abnormality due to high glucose load, and dyslipidemia due to excessive fatty acids.SOLUTION: A composition for improving metabolic abnormality contains a specific material.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a composition for improving metabolism. [Background technology]

[0002] It is known that high glucose loading enhances oxidative stress in cells and causes metabolic abnormalities. In particular, it is believed that high glucose loading in diabetic patients and prediabetic subjects leads to metabolic abnormalities and various symptoms. These symptoms may include symptoms in the oral cavity.

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

[0004] It is also known that insulin resistance occurs in diabetic patients. It is also believed that there is a relationship between diabetes and periodontal disease, and it has been reported that insulin resistance occurs in the gingiva of diabetic model animals. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] J Clin Periodontol 2017; 44: 463-471. IADR / PER General Session 2018; Presentation ID 1622 [Non-Patent Document 2] J Dent Res 2014: 93; 596-601. [Non-Patent Document 3] PLOS ONE 2017;12:e0189601. Summary of the Invention [Problem to be solved by the invention]

[0006] It is known that excessive administration of fatty acids (especially palmitic acid) can cause insulin resistance, but it has not been confirmed whether it also causes insulin resistance in gingival cells.

[0007] The present inventors found that insulin enhances Akt phosphorylation in periodontal ligament cells in a concentration-dependent manner, and that application of palmitic acid to periodontal ligament cells suppresses insulin-induced enhancement of Akt phosphorylation. Inhibition of Akt phosphorylation is widely known as one of the indicators of insulin resistance. This shows that fatty acids can also cause insulin resistance in gingival cells.

[0008] Furthermore, the inventors conducted similar studies on high concentrations of sugar (glucose) and LPS (Lipopolysaccharide), which are components other than fatty acids that are thought to have the potential to cause insulin resistance. However, treatment with high concentrations of sugar or LPS did not inhibit Akt phosphorylation, and therefore it was believed that these components do not cause insulin resistance.

[0009] Based on these findings, it can be assumed that the increase in free fatty acids (especially palmitic acid) caused by obesity and diabetes (especially type 2 diabetes) induces inflammation and monocyte adhesion in various cells, causing sustained bone resorption. In particular, regarding the relationship between diabetes and periodontal disease, it can be assumed that free fatty acids (especially palmitic acid) induce inflammation and monocyte adhesion in periodontal ligament cells, aggravating periodontitis accompanied by sustained bone resorption, which may ultimately lead to tooth loss.

[0010] We further examined whether free fatty acids (especially palmitic acid) could increase the expression of inflammatory cytokines, chemokines, and adhesion factors, as well as the expression of receptors located upstream of the inflammatory cytokine, chemokine, and adhesion factor expression cascade (Toll-like receptor 4 (TLR4) and CD36), and found that the expression of all of these genes was enhanced. TLR4 is a receptor protein that recognizes LPS, etc. CD36 is a major membrane protein involved in the uptake of fatty acids, and is thought to be involved in disorders of fatty acid and lipid metabolism that occur with the onset of pathophysiological conditions such as insulin resistance and diabetic cardiomyopathy caused by a high-fat diet.

[0011] Based on these findings, it is believed that by using the above-mentioned gene expression as an indicator, it is possible to search for components that suppress lipid metabolic disorders caused by an increase in free fatty acids that occur in obesity and diabetes.

[0012] Therefore, the inventors conducted further studies with the aim of finding a component that can suppress metabolic abnormalities caused by high glucose load and also suppress lipid metabolic abnormalities caused by an increase in free fatty acids that occur in obesity and diabetes. [Means for solving the problem]

[0013] The inventors have screened for a component that can suppress metabolic abnormalities caused by high glucose loading, and furthermore, can suppress lipid metabolic abnormalities caused by an increase in free fatty acids that occur in obesity and diabetes, and have further improved the component.

[0014] The present disclosure includes, for example, the subject matter described in the following sections: Section 1. Contains at least one selected from the group consisting of components listed in the table below: A composition for improving metabolism in oral cells.

[0015] [Table 1]

[0016] TIFF2024095197000002.tif231170

[0017] TIFF2024095197000003.tif231170

[0018] TIFF2024095197000004.tif111170

[0019] Section 2. Contains at least one selected from the group consisting of components listed in the table below: A composition for improving metabolism in oral cells.

[0020] [Table 2]

[0021] Section 3. The composition according to item 1 or 2, for a subject having glucose metabolism disorder and / or lipid metabolism disorder. Section 4. Item 4. The composition according to any one of Items 1 to 3, which is a composition for improving sugar metabolism and / or lipid metabolism in oral cells. Section 5. Item 4. The composition according to any one of Items 1 to 3, which is used to improve ATP metabolic abnormality caused by high glucose load in oral cells. Section 6. Item 4. The composition according to any one of Items 1 to 3, which is used for suppressing inflammation caused by high glucose load in oral cells. Section 7. Item 4. The composition according to any one of Items 1 to 3, which is used for suppressing inflammation caused by an increase in free fatty acids in oral cells. Section 8. Item 8. The composition according to any one of Items 4 to 7, wherein the oral cells are periodontal ligament cells. Section 9. Item 9. The composition according to any one of items 4 to 8, which is a food composition. Effect of the Invention

[0022] It can suppress metabolic disorders caused by high glucose load and lipid metabolic disorders caused by increased free fatty acids. In particular, it can suppress such metabolic disorders and inflammation caused in cells in the oral cavity. This makes it possible to prevent or treat periodontal disease. More specifically, it can be expected to suppress periodontitis accompanied by bone resorption, and thus to suppress tooth loss associated with the progression of periodontitis. [Brief description of the drawings]

[0023] [Figure 1] This paper presents an overview of an experiment in which high glucose loading was applied to human periodontal ligament fibroblasts (HPDLFs) to examine how this affects ATP metabolism and gene expression levels of inflammatory cytokines. [Diagram 2] This is an outline of an experiment in which a test substance (sample) was first applied to human periodontal ligament fibroblasts, and then high glucose load was applied to examine how ATP metabolism and gene expression levels of inflammatory cytokines changed. [Diagram 3] The results of intracellular ATP measurement in the study shown in FIG. 1 (without application of test substance) are shown. 3d indicates the results after 3 days, 6d indicates the results after 6 days, and 9d indicates the results after 9 days. The bar graphs showing the results after each number of days show, from left to right, the relative values ​​when the control is taken as 100%, for the results of the control (L-glucose), D-glucose 25 mM, D-glucose 50 mM, and D-glucose 100 mM. The control for D-glucose 25 mM is L-glucose 25 mM, the control for D-glucose 50 mM is L-glucose 50 mM, and the control for D-glucose 100 mM is L-glucose 100 mM. [Figure 4]The results of measuring the expression level of MCP-1 gene in the study shown in Figure 1 (without application of the test substance) are shown after correction to the expression level of RPS18. 3d shows the results after 3 days, and 6d shows the results after 6 days. The bar graphs showing the results after each number of days show the relative values, from left to right, of the control (L-glucose), D-glucose 25 mM, D-glucose 50 mM, and D-glucose 100 mM, with the control set at 1. The control for D-glucose 25 mM is L-glucose 25 mM, the control for D-glucose 50 mM is L-glucose 50 mM, and the control for D-glucose 100 mM is L-glucose 100 mM. [Figure 5A] The results show how the expression levels of inflammatory cytokines, chemokines, and adhesion molecules in human periodontal ligament fibroblasts (HPDLF) changed after treatment with palmitic acid and culturing for 6 or 24 hours. Palmitic acid was administered by conjugating it to bovine serum albumin (BSA). As a control, the cells were treated with the same amount of BSA that was not conjugated with palmitic acid. The results are values ​​obtained by correcting the expression levels of each gene with the expression level of ribosomal protein S18 (RPS18). [Figure 5B] The results show how the expression level of receptor genes changes after treating human periodontal ligament fibroblasts (HPDLF) with palmitic acid and culturing for 6 or 24 hours. Palmitic acid was administered by conjugating it to bovine serum albumin (BSA). As a control, the cells were treated with the same amount of BSA that was not conjugated with palmitic acid. The results are values ​​obtained by correcting the expression level of each gene by the expression level of ribosomal protein S18 (RPS18). [Figure 6]This is an outline of an experiment to examine how the gene expression levels of inflammatory cytokines, chemokines, adhesion factors, and receptors change when human periodontal ligament fibroblasts (HPDLF) are first treated with a test substance (sample) and then treated with palmitic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes a composition for improving metabolism in oral cells (particularly an oral composition), but is not limited thereto, and the present disclosure includes all that is disclosed in the present specification and that can be recognized by a person skilled in the art.

[0025] The composition for improving metabolism in oral cells included in the present disclosure contains the specific components listed in the following table, either singly or in combination of two or more. The composition for improving metabolism included in the present disclosure may be referred to as the composition of the present disclosure. In addition, the components having these characteristics may be referred to as the components of the present disclosure. The composition of the present disclosure may be particularly suitably used as a composition for preventing or treating periodontal disease.

[0026] [Table 3]

[0027] TIFF2024095197000007.tif231170

[0028] TIFF2024095197000008.tif231170

[0029] TIFF2024095197000009.tif111170

[0030] Among these components, the components listed in the following table are more preferred.

[0031] [Table 4]

[0032] The content of the components of the present disclosure in the composition of the present disclosure is not particularly limited as long as the effects are not impaired, and may be, for example, about 0.01 to 99.99% by mass.

[0033] The intake form of the composition of the present disclosure is not particularly limited, but is preferably oral intake. That is, the composition of the present disclosure is preferably an oral composition. By orally ingesting the composition of the present disclosure, the effect of suppressing metabolic abnormalities and lipid metabolic abnormalities caused by high glucose load in cells (particularly cells in the oral cavity) can be preferably achieved. The composition of the present disclosure is preferably, for example, an oral pharmaceutical composition or a food composition (including a beverage composition and a food additive composition).

[0034] The composition of the present disclosure includes the above-mentioned components and may further include other components. The other components may be appropriately selected depending on the field in which the composition is used. For example, a medicamentically or food hygienically acceptable carrier may be used.

[0035] When used as a pharmaceutical composition, other components include pharma- ceutically acceptable bases, carriers, and / or additives (e.g., solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, etc.), etc. The form of the pharmaceutical composition is not particularly limited, and examples thereof include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, creams, and poultices.

[0036] When used as a food composition, other ingredients include bases, carriers, additives that are acceptable from the viewpoint of food hygiene, and other ingredients and materials that can be used as foods. The form of the food composition is not particularly limited, and examples thereof include processed foods, health foods (nutritional supplements, nutritional functional foods, foods for the sick, foods for specified health uses, functional products, etc.), supplements, foods for the sick (hospital food, sick food, nursing food, etc.). These can be prepared by conventional methods. In particular, when preparing a food composition as a health food (nutritional supplements, nutritional functional foods, foods for the sick, foods for specified health uses, functional products, etc.) or supplements, it is preferable to prepare it in the form of, for example, granules, capsules, tablets (including chewable agents, etc.), beverages (drink powders, drinks, smoothies, etc.) so that it is easy to take continuously, and among them, the forms of capsules, tablets, tablets, drink powders, drinks, jellies, and gummies are preferable from the viewpoint of ease of intake, but are not particularly limited thereto. When the food composition is used as a food additive composition, the food composition may be in the form of, for example, liquid, powder, flake, granule, or paste.

[0037] From the viewpoint that the effect can be preferably exerted, the subject of taking the composition of the present disclosure is preferably a subject with metabolic abnormality (particularly glucose metabolic abnormality and / or lipid metabolic abnormality), more specifically, for example, a subject with metabolic abnormality caused by high glucose load or a subject with an increase in free fatty acid is more preferable. More specifically, metabolic abnormality caused by high glucose load includes, for example, ATP metabolic abnormality caused by high glucose load, and inflammation that can be caused by the metabolic abnormality (inflammation caused by high glucose load). In addition, a subject with an increased expression level of inflammatory cytokines, chemokines, and adhesion factors due to an increase in free fatty acids is particularly preferable. In addition, a subject with gingival inflammation is preferable. Although not particularly limited, inflammation in which the expression levels of inflammatory cytokines and chemokines are increased is preferred, and in particular inflammation in which the expression levels of at least one inflammatory cytokine or chemokine selected from the group consisting of MCP-1 (Monocyte chemoattractant protein-1), IL-8 (Interleukin-8), MMP1 (Matrix metallopeptidase 1), IL-6 (Interleukin-6), and CXCL1 (CXC Motif Chemokine Ligand 1) are increased is preferred.

[0038] The timing of taking the composition of the present disclosure is not particularly limited, but it can be taken, for example, before or after eating. The meal is preferably one that contains carbohydrates (particularly carbohydrates that can become glucose when absorbed into the body) and lipids. For example, it can be preferably used during or after periodontal disease treatment to prevent recurrence.

[0039] Furthermore, although not particularly limited, the cells in which metabolic abnormalities can be suppressed by ingestion of the composition of the present disclosure are preferably cells in the oral cavity, 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 therefore most preferred.

[0040] In addition, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

[0041] In addition, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter included in the present disclosure. In other words, the present disclosure includes all subject matter consisting of all combinations of each combinable characteristic described in this specification. EXAMPLES

[0042] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0043] Examination of high glucose loading in periodontal ligament cells We applied high glucose to human periodontal ligament fibroblasts (HPDLFs) to examine how ATP metabolism and gene expression levels of inflammatory cytokines would change as a result (Figure 1). Furthermore, we applied a test substance (sample) to human periodontal ligament fibroblasts in advance, and then examined how ATP metabolism and gene expression levels of inflammatory cytokines would change when high glucose was applied (Figure 2). More specifically, we performed the study as follows. The following detailed explanation is for the study shown in Figure 2 (with test substance), but the study shown in Figure 1 was performed in the same way, except that the test substance was not applied and the D-glucose concentration was varied.

[0044] [Measurement of intracellular ATP concentration] Intracellular ATP was measured using a Luminescent ATP detection assay kit (ab113849; Abcam). Human periodontal ligament fibroblasts (HPDLF) were cultured at 0.12 × 10 5 The cells were seeded in a 48-well plate at a density of cells / 100 μL / well and cultured for 48 hours. The cells were pretreated with each test substance (sample) (1 μg / mL) for 24 hours, and then treated with 50 mM D-glucose (Sigma Aldrich) for 72 hours. The same concentration of L-glucose was used as a control. Then, 50 μL of cell lysis solution 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. Chemiluminescence due to the luciferase reaction was measured using a Cytation 5 plate reader (BioTek Instruments), and ATP concentration (μM) was measured using a standard curve. The ATP reduction improvement rate (%) when each test substance (sample) was applied was calculated using the following formula.

[0045]

number

[0046] In other words, the ATP decrease improvement rate (%) indicates the percentage obtained by subtracting the chemiluminescence value when only D-glucose was treated from the chemiluminescence value when D-glucose was treated after sample treatment, divided by the value obtained by subtracting the chemiluminescence value when only D-glucose was treated from the chemiluminescence value when only L-glucose was treated.

[0047] [Gene expression of MCP-1, IL-8, and MMP-1] Human periodontal ligament fibroblasts (HPDLF) were cultured at 0.45 × 10 5The cells were seeded in 12-well plates at a density of cells / 1 mL / well and cultured for 72 hours. The cells were pretreated with each sample (1 μg / mL) for 24 hours, and then treated with 50 mM D-glucose (Sigma Aldrich) for 72 hours. The same concentration of L-glucose was used as a control. Total RNA was extracted using RNeasy Mini Kit (Qiagen). Single-stranded cDNA was synthesized from 0.5 μg of total RNA using PrimeScript RT reagent Kit (Takara Bio). The expression of each gene was quantified using the 7500 Fast real-time PCR system (Applied Biosystems) with specific primers and the intercalator method using TB Green Fast qPCR Mix (Takara Bio). The expression level of each gene was corrected by the expression level of ribosomal protein S18 (RPS18), and the relative value was calculated with the expression level after treatment with L-glucose set to 1. The gene expression inhibition rate (%) when each test substance (sample) was applied was calculated using the following formula.

[0048]

number

[0049] In other words, the gene expression inhibition rate (%) here indicates the percentage obtained by subtracting the relative value when D-glucose was treated after sample treatment from the relative value when D-glucose was treated only, divided by the relative value when L-glucose was treated only from the relative value when D-glucose was treated only.

[0050] The results of the study shown in Figure 1 (without application of the test substance) are shown in Figure 3 (measurement results of intracellular ATP concentration) and Figure 4 (measurement results of MCP-1 gene expression level). Figure 3 confirmed that intracellular ATP production decreased in a D-glucose concentration-dependent manner (i.e., ATP metabolism decreased). Figure 4 also showed that the gene expression level of the inflammatory cytokine (MCP-1) increased in a D-glucose concentration-dependent manner.

[0051] Furthermore, the results of the study shown in Figure 2 (with application of the test substance) are also shown in Table 5. Specifically, the ATP reduction improvement rate (%) and the inhibition rate of each inflammatory cytokine gene expression obtained as described above are shown.

[0052] Examination of periodontal ligament cells treated with palmitic acid Human periodontal ligament fibroblasts (HPDLF) were treated with palmitate to examine how the expression levels of various inflammatory cytokines, chemokines, adhesion molecules, and receptor genes changed (in Figure 6, the examination was performed under conditions without the addition of each test substance). The results are shown in Figures 5A and 5B. (The expression level of each gene was corrected by the expression level of ribosomal protein S18 (RPS18), and the relative value was calculated when the expression level after treatment with BSA was set to 1.) Furthermore, we investigated how the expression levels of various inflammatory cytokines, chemokines, adhesion molecules, and receptor genes changed when human periodontal ligament fibroblasts were treated with each test substance (sample) in advance and then treated with palmitic acid (Figure 6). More specifically, we conducted the study as follows. Note that the detailed explanation below is for the study described in Figure 6 (with test substance added), but the study described under conditions without test substance added, the results of which are shown in Figures 5A and 5B, was also conducted under the same conditions except for the test substance addition step.

[0053] [Examination of the expression of various inflammatory cytokines, chemokines, adhesion factor genes, and receptor genes] Human periodontal ligament fibroblasts (HPDLF) were added at a concentration of 0.45 × 10 5The cells were seeded in a 12-well plate at a density of cells / 1mL / well and cultured for 72 hours. The cells were pretreated with each test substance (sample) (5μg / mL) for 24 hours, and then treated with 100μM palmitic acid (Sigma Aldrich) conjugated to BSA (Bovine serum albumin; Sigma Aldrich) for 24 hours. The same concentration of BSA was used as a control. Total RNA was extracted using an RNeasy Mini Kit (Qiagen). Single-stranded cDNA was synthesized from 0.5μg of total RNA using a PrimeScript RT reagent Kit (Takara Bio). Quantification of each gene expression was performed using the 7500 Fast Real-time PCR System (Applied Biosystems) by the intercalator method using specific primers and TB Green Fast qPCR Mix (Takara Bio). The expression level of each gene was corrected by the expression level of ribosomal protein S18 (RPS18), and the relative value was calculated with the expression level after treatment with BSA set to 1. The gene expression inhibition rate (%) of each sample was calculated using the following formula.

[0054]

number

[0055] In other words, the gene expression inhibition rate (%) here indicates the percentage obtained by subtracting the relative value when palmitic acid was treated after sample treatment from the relative value when palmitic acid was treated alone, divided by the relative value when BSA was treated alone from the relative value when palmitic acid was treated alone.

[0056] The results are also shown in Table 5. In the study shown in Figure 6 (with test substance added), IL-6, CXCL1, ICAM-1, IL-8, TLR4, and CD36 were selected and used as various inflammatory cytokines, chemokines, adhesion factor genes, and receptor genes.

[0057]

Table 5

[0058] TIFF2024095197000015.tif253170

[0059] TIFF2024095197000016.tif253170

[0060] TIFF2024095197000017.tif253170

[0061] TIFF2024095197000018.tif253170

[0062] TIFF2024095197000019.tif253170

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