Composition for reducing endoplasmic reticulum stress

Abstract

The present invention relates to a composition containing allulose and having the functional effect of reducing endoplasmic reticulum stress.

Description

[Technical Field]

[0001] The present invention relates to a composition containing allulose that has the functionality of reducing endoplasmic reticulum stress. [Background technology]

[0002] Intracellular stresses include oxidative stress, mitochondrial stress, heat shock stress, and endoplasmic reticulum (ER) stress. The lumen of the endoplasmic reticulum (ER) is a specialized cellular environment for post-translational transformation and protein folding. There are two types of ER: the rough endoplasmic reticulum, which has ribosomes, and the smooth endoplasmic reticulum, which does not have ribosomes. Approximately one-third of intracellular proteins undergo post-translational modification, i.e., folding, assembly, glycation, and disulfide bonding, to form active protein structures after translation from mRNA to proteins in the rough endoplasmic reticulum. The smooth endoplasmic reticulum is also the site of lipid and steroid synthesis, and as a calcium reservoir plays an important role in regulating intracellular calcium concentration.

[0003] Thus, the endoplasmic reticulum stress response is an important compensatory mechanism that preserves the function of the endoplasmic reticulum and protects cells from various cellular stresses. However, it has recently become known that an excessive endoplasmic reticulum stress response can be induced by an incorrect signaling system, resulting in diseases that are either caused or triggered by this.

[0004] Recently, the incidence of diabetes has been increasing, and the mortality rate from cardiovascular disease, a complication of diabetes, has also been rising. Untreated chronic diabetes complications include various conditions, including neurological and renal disorders, but the incidence of cardiovascular diseases such as hypertension, arteriosclerosis, cerebral infarction, cerebral thrombosis, and myocardial infarction is particularly high. One of the main factors behind the frequent occurrence of cardiovascular disorders, including arteriosclerosis, is the increased susceptibility of the tissues of diabetic patients to oxidative stress, which promotes lipid peroxidation due to increased production of free radicals. For this reason, research is progressing on strengthening the body's antioxidant defense system to protect tissues from peroxidation.

[0005] Diabetes is a metabolic disease characterized by hyperglycemia caused by impaired secretion and function of insulin, which is necessary for regulating blood sugar in the body. Insulin resistance is a phenomenon commonly observed in most obese and type 2 diabetes patients, and is primarily caused by a postreceptor defect in insulin action, resulting in excessive expansion of insulin storage and a reduced ability to process nutrients in the blood after a meal. Although exercise and diet are important for regulating blood sugar, many people experience difficulties due to the difficulty of consistently managing their diet and exercise. Additionally, people who have difficulty regulating blood sugar can lower their blood sugar through drug therapy, but due to the nature of chronic diseases, the safety of drugs taken over a long period of time is an issue. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, unlike existing drugs with safety concerns, we have been developing a functional material that reduces blood sugar by promoting the reduction of endoplasmic reticulum stress. This functional material is called allulose, which is an epimer of the carbon-3 position of fructose and has a sweetness equivalent to 70% of that of sugar, while providing a sweet taste and exhibiting excellent endoplasmic reticulum stress reduction effects upon ingestion, and we provide a safe composition. [Means for solving the problem]

[0007] One example of the present invention relates to a composition containing allulose for preventing, ameliorating, or treating an endoplasmic reticulum stress-related disease.

[0008] One example of the present invention relates to the use of allulose for the prevention, amelioration or treatment of endoplasmic reticulum stress-related diseases.

[0009] One example of the present invention relates to a method for preventing, ameliorating, or treating an endoplasmic reticulum stress-related disease, comprising the step of administering a composition comprising allulose to a subject in need thereof.

[0010] Another example of the present invention relates to a composition for regulating blood glucose, regulating insulin sensitivity, or improving, preventing, or treating diabetes by reducing endoplasmic reticulum stress, comprising allulose as an active ingredient.

[0011] A further example of the present invention relates to the use of allulose as an active ingredient for regulating blood sugar, regulating insulin sensitivity, or improving, preventing, or treating diabetes by reducing endoplasmic reticulum stress.

[0012] A further example of the present invention relates to a method for regulating blood glucose, regulating insulin sensitivity, or improving, preventing, or treating diabetes by reducing endoplasmic reticulum stress, comprising the step of administering a composition comprising allulose as an active ingredient to a subject in need thereof.

[0013] Yet another example of the present invention is a pharmaceutical composition comprising allulose as an active ingredient, p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α The present invention relates to a composition that reduces the expression of one or more endoplasmic reticulum stress-related proteins selected from the group consisting of sulfonation (SO3H).

[0014] Yet another example of the present invention is a pharmaceutical composition comprising allulose as an active ingredient, p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α The present invention relates to a method for reducing the expression of one or more endoplasmic reticulum stress-related proteins selected from the group consisting of sulfonation (SO3H).

[0015] Further examples of the present invention relate to compositions, uses or methods for preventing, ameliorating or treating oxidative stress-related diseases, comprising allulose. [Effects of the Invention]

[0016] According to one example of the present invention, endoplasmic reticulum stress can be effectively reduced by simply taking allulose, without changing dietary or exercise habits, and this has the effect of helping to regulate blood sugar. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a graph showing the values obtained by conducting an oral glucose tolerance test and an insulin tolerance test to confirm the effect of D-allulose on blood glucose reduction in db / db mice according to an example of the present invention. [Figure 2] 1 is a graph showing the values obtained after measuring serum insulin concentrations in db / db mice to confirm the effect of D-allulose on serum insulin concentrations in accordance with one example of the present invention. [Figure 3] According to one example of the present invention, confocal micrographs (Figure 3A) of DHE fluorescently stained tissue were taken to confirm the effect of D-allulose on oxidative stress in db / db mice, and a graph (Figure 3B) shows the numerical results of DHE fluorescence intensity. [Figure 4] 1 is a graph showing the values obtained by measuring the concentration of NADPH oxidase activity in tissues in db / db mice to confirm the effect of D-allulose on NADPH oxidase activity according to one example of the present invention. [Figure 5] 1 is a graph showing the values obtained by measuring the lipid peroxide content in db / db mice to confirm the effect of D-allulose on lipid peroxides in accordance with an example of the present invention. [Figure 6]1 shows the results of measuring the degree of protein oxidation in tissues in db / db mice to confirm the effect of D-allulose on protein oxidation, according to one example of the present invention. [Figure 7] FIG. 1 shows the effect of D-allulose on the protein expression level of NOX4 in db / db mice, according to an example of the present invention. [Figure 8] FIG. 1 shows the effects of D-allulose on endoplasmic reticulum (ER) stress and irreversible oxidation of IRE1α in db / db mice, according to an example of the present invention. [Figure 9] FIG. 1 shows the supplementation effect of D-allulose on p-AMPK-SIRT1-PGC-1α in db / db mice, according to an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] One example of the present invention relates to a composition containing allulose for preventing, ameliorating, or treating an endoplasmic reticulum stress-related disease, which may be an endoplasmic reticulum stress-related disease in muscle tissue, or in the endoplasmic reticulum or sarcoplasmic reticulum in muscle tissue.

[0019] In the present invention, the ER stress-related disease may be increased insulin resistance, increased blood glucose, diabetes, Alzheimer's disease, Parkinson's disease, glutamine multimer-induced aggregation disease, Huntington's disease, Alzheimer's disease, ischemic disease, cardiovascular disease, hyperhomocysteinemia, arteriosclerosis, or cancer. In the treatment of cancer, increasing ER stress to induce cell death may be a therapeutic means.

[0020] Yet another example of the present invention is a pharmaceutical composition comprising allulose as an active ingredient, p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1-alpha The present invention relates to a composition that reduces the expression or activity of one or more endoplasmic reticulum stress-related proteins selected from the group consisting of sulfonation (SO3H).

[0021] Yet another example of the present invention relates to a composition for increasing or activating the expression of p-AMPK and SIRT1 proteins, which comprises allulose as an active ingredient.

[0022] Yet another example of the present invention relates to a composition for reducing endoplasmic reticulum (ER) stress, thereby lowering blood glucose, reducing insulin sensitivity, or ameliorating, treating, or preventing diabetes, comprising allulose as an active ingredient. The composition may be a pharmaceutical composition or a food composition. The ER stress may occur in muscle tissue or pancreatic beta cells. The present invention also relates to a composition for reducing ER (sarcoplasmic reticulum) stress in muscle tissue, for reducing blood glucose, reducing insulin sensitivity, or ameliorating, treating, or preventing diabetes, comprising allulose as an active ingredient. Specifically, the composition for reducing blood glucose, reducing insulin sensitivity, or ameliorating, treating, or preventing diabetes according to the present invention may comprise p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1-alpha The compound may reduce the expression or activity of one or more endoplasmic reticulum stress-related proteins selected from the group consisting of sulfonation (SO3H), and / or increase the expression or activation of p-AMPK and SIRT1 proteins.

[0023] According to the present invention, allulose has the ability to regulate blood glucose, and can particularly reduce postprandial blood glucose. Specifically, allulose according to the present invention can reduce postprandial blood glucose by administering it at or after a meal to healthy subjects or subjects whose blood glucose levels are on the borderline of diabetes. Therefore, a composition containing allulose according to the present invention as an active ingredient can suppress or reduce blood glucose increases caused by meals, lower increased blood glucose levels, and suppress blood glucose increases or induce blood glucose reduction in healthy subjects, subjects whose blood glucose levels are on the borderline of diabetes, or subjects with diabetes. The subject may be a mammal, including a human.

[0024] Another embodiment of the present invention relates to a composition containing allulose for preventing, ameliorating, or treating oxidative stress-related diseases. The allulose has antioxidant activity that reduces oxidative stress. The allulose can reduce oxidative stress by reducing Nox4 (NADPH oxidase 4: NOX4) expression, reducing protein oxidation, or inhibiting superoxide anion, NADPH oxidase activity, and lipid peroxidation.

[0025] Allulose can reduce oxidative stress by inhibiting one or more of the following in muscle cells or muscle tissues: decreased expression of Nox4, decreased protein oxidation, superoxide anion, NADPH oxidase activity, and lipid peroxidation. Ingestion of a composition containing allulose as an active ingredient can reduce intramuscular superoxide anion, NADPH oxidase activity, and lipid peroxidation by 5 to 50%, 10 to 50%, 5 to 30%, 10 to 30%, 5 to 20%, 5 to 15%, or 10 to 20%.

[0026] The composition according to the present invention, which contains allulose as an active ingredient, may be formulated so that the daily intake is 10 to 80 g per 60 kg of body weight of the consuming individual (10 to 80 g / 60 kg / day). The composition containing allulose as an active ingredient may be ingested before meals, after meals, or simultaneously with meals. The composition containing allulose as an active ingredient may be ingested for a period of 4 to 20 weeks, preferably 8 to 12 weeks. Although effective effects can be obtained by ingesting the composition according to the present invention only once, the effects can be further maximized by ingesting it for the above period.

[0027] Specifically, in a test in which mammals on a high-fat diet were given various types of sweeteners, it was confirmed that only the composition containing allulose as an active ingredient was effective in reducing endoplasmic reticulum stress and oxidative stress.

[0028] Specifically, in the present invention, administration of allulose reduces NOX4 expression, suppressing the production of ROS in muscle cells, regulating glucose metabolism, and reducing insulin resistance. Allulose activates AMPK-SIRT1-PGC-1α, promoting energy metabolism and contributing to the improvement of insulin resistance.

[0029] According to the present invention, allulose, which is included as an active ingredient, contributes to blood glucose regulation by reducing endoplasmic reticulum stress in muscles, specifically, by reducing endoplasmic reticulum stress in muscle cells or muscle tissue. More specifically, when carbohydrates are ingested, glucose is absorbed in the intestine and delivered to various tissues of the body, where it is stored for later use or immediately oxidized to generate energy. When glucose is orally ingested, it is delivered to the liver, muscle tissue, brain tissue, visceral tissue (splanchnic bed), and adipose tissue, and allulose activates storage in muscle tissue, in particular, thereby very effectively lowering blood glucose levels in the body.

[0030] The present invention will now be described in further detail. One example of the present invention relates to a composition containing allulose for preventing, ameliorating, or treating an endoplasmic reticulum stress-related disease, which may be increased insulin resistance, increased blood glucose, diabetes, Alzheimer's disease, Parkinson's disease, glutamine multimer-induced aggregation disease, Huntington's disease, Alzheimer's disease, ischemic disease, cardiovascular disease, hyperhomocysteinemia, arteriosclerosis, or cancer.

[0031] More specifically, the present invention relates to a composition for regulating blood sugar, regulating insulin sensitivity, or improving, preventing, or treating diabetes by reducing endoplasmic reticulum stress, which contains allulose as an active ingredient.

[0032] As used herein, the term "endoplasmic reticulum stress" refers to a condition in which the endoplasmic reticulum functions are impaired due to a physiological or pathological environment, which causes immature proteins to enter the endoplasmic reticulum in excess of its capacity to be processed, or due to a depletion of calcium in the endoplasmic reticulum. When ER stress occurs, cells mount a defense mechanism for survival, known as the ER stress response. The ER stress response is mediated by three signaling pathways present in the ER membrane: pancreatic ER kinase (PERK), inositol-requiring 1α / XBP-1 (inositol-requiring 1α / X-box binding protein 1), and activating transcription factor (ATF6). This ER stress response is particularly common in cells that actively synthesize and secrete proteins, such as plasma cells, pancreatic beta cells, hepatocytes, and osteoblasts. Recent studies have demonstrated that ER stress plays a role in the pathogenesis of various diseases, including ischemia, diabetes, viral infection, and hyperhomocysteinemia.

[0033] Yet another example of the present invention is a method for treating p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α The present invention relates to a composition that reduces the expression of one or more proteins selected from the group consisting of sulfonation (SO3H).

[0034] Specifically, we analyzed the expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO3H) in non-abdominal muscle samples from experimental animals. Compared to the normal control group (NC), the diabetic control group (DC) showed significantly increased expression of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO3H). However, the groups receiving allulose powder and liquid in a concentration-dependent manner showed a decrease in the expression levels of these genes, which were increased in the diabetic control group (DC) (Figure 8). Therefore, allulose appears to reduce endoplasmic reticulum stress and improve insulin resistance, and it can protect cells by inhibiting endoplasmic reticulum stress-induced apoptosis (cell death).

[0035] In the present invention, allulose increases the expression of p-AMPK and SIRT1 proteins; specifically, increased expression of p-AMPK and SIRT1 proteins or activation of SIRT1 deacetylates PGC-1α (i.e., decreases the expression of acetylated PGC-1α), which increases the activity of GLUT4 present in muscles and moves glucose from the outside to the inside of cells, contributing to blood sugar reduction, reduction of insulin resistance, or improvement or treatment of diabetes. Insulin-responsive glucose transporter 4 (GLUT4) is an insulin-dependent glucose transporter that is distributed mainly in skeletal muscle and adipose tissue and plays a role in moving glucose from the outside to the inside of cells.

[0036] Specifically, we confirmed that the groups that ingested allulose powder and liquid in a concentration-dependent manner had increased levels of p-AMPK and SIRT1 protein expression compared to the diabetic control group (DC). Specifically, analysis of the expression levels of p-AMPK and SIRT1 protein in the non-abdominal muscles of the experimental animals revealed that the expression levels of p-AMPK and SIRT1 protein were significantly reduced in the diabetic control group (DC) compared to the normal control group (NC), but that the groups that ingested allulose powder and liquid in a concentration-dependent manner had increased levels of p-AMPK and SIRT1 protein expression compared to the diabetic control group (DC) (Figure 9A).

[0037] In the present invention, the expression of acetylated-PGC-1α protein was confirmed and found to be increased in the diabetic control group (DC) compared to the normal control group (NC), but the expression of acetylated-PGC-1α decreased in the groups that ingested allulose powder and liquid in a concentration-dependent manner (Figure 9, B and C). Therefore, it is expected that allulose activates AMPK-SIRT1-PGC-1α, promoting energy metabolism and reducing insulin resistance.

[0038] As used herein, the term "endoplasmic reticulum stress-related disease" refers to a disease caused or exacerbated by endoplasmic reticulum stress. Examples of such diseases include those caused by excessive intracellular protein accumulation, which inhibits the functioning of the degradation system and is itself toxic to cells (e.g., neurodegenerative diseases), and those caused or induced by an excessive endoplasmic reticulum stress response induced by an incorrect signaling system (e.g., diabetes). Currently, diabetes, Parkinson's disease, glutamine multimer-induced aggregation disease, Huntington's disease, Alzheimer's disease, ischemic disease, cardiovascular disease, hyperhomocysteinemia, and arteriosclerosis are known as endoplasmic reticulum stress-related diseases. Therefore, as used herein, endoplasmic reticulum stress-related diseases may include increased insulin resistance, increased blood glucose, diabetes, Alzheimer's disease, Parkinson's disease, glutamine multimer-induced aggregation disease, Huntington's disease, ischemic disease, cardiovascular disease, hyperhomocysteinemia, arteriosclerosis, or cancer. Specific examples of the endoplasmic reticulum stress-related disease may be increased insulin resistance, increased blood sugar, or diabetes.

[0039] Specifically, in the case of Alzheimer's disease, PERK and its lower substrate, the eIF2a pathway, appear to be largely activated in patient tissue samples. In Parkinson's disease, a representative degenerative disease, the E3 ligase protein Parkin has been reported to be closely related to endoplasmic reticulum stress. In the case of Huntington's disease, it is known that when part of a gene is over-amplified due to mutation, glutamine polymers become entangled and form protein aggregates that the cell's ability to degrade them cannot withstand, resulting in spontaneous endoplasmic reticulum stress and the death of neurons.

[0040] One example of the present invention provides the use of allulose to improve or treat increased insulin resistance, increased blood sugar, or diabetes, wherein the disease belongs to endoplasmic reticulum stress and / or oxidative stress-related diseases, for example, diseases associated with endoplasmic reticulum stress and / or oxidative stress in muscle tissue.

[0041] Specifically, in relation to oxidative stress, administration of allulose reduces NOX4 expression, inhibiting intracellular ROS production and regulating glucose metabolism, thereby reducing insulin resistance. In relation to endoplasmic reticulum stress, allulose increases AMPK expression or activation. In muscle tissue and cardiac muscle, AMPK promotes muscle contraction and glucose absorption, which increases glucose transport into cells by activating GLUT1 and inducing GLUT4 translocation to the plasma membrane, regardless of insulin action. Allulose also induces SIRT1 activation, which deacetylates PGC-1α and increases the activity of muscle glucose transporter 4 (GLUT4). Insulin-responsive glucose transporter 4 (GLUT4) is an insulin-dependent glucose transporter primarily found in skeletal muscle and adipose tissue, transporting glucose from the outside to the inside of cells.

[0042] Impaired insulin secretion observed in type 2 diabetes induces endoplasmic reticulum stress, and this study investigated whether allulose modulates endoplasmic reticulum stress induced by type 2 diabetes. Because insulin resistance is the most important cause of diabetes, reducing insulin resistance is effective in diabetes treatment, and is particularly important in type 2 diabetes. Pancreatic beta cells have well-developed endoplasmic reticulum, and smooth function of the endoplasmic reticulum plays an important role in beta cell function, and beta cell dysfunction due to endoplasmic reticulum stress contributes to the induction of diabetes.

[0043] Allulose can lower blood glucose by increasing phosphorylated AMPK (AMP-activated protein kinase) and activating AMPK signaling, thereby promoting cellular glucose uptake, exhibiting anti-obesity activity, and / or lowering blood lipid levels by increasing AMPK activity. AMPK is known to be involved in glucose metabolism, fat metabolism, mitochondrial biogenesis, and energy metabolism, which are involved in blood glucose regulation by suppressing hepatic glucose release independent of insulin. AMPK is known to increase the gene expression of PGC-1α, which is thought to play an important role in mitochondrial biogenesis.

[0044] AMPK is a key factor in regulating energy balance at the cellular and systemic levels. As a serine / threonine kinase, it is known to regulate lipid and glucose metabolism. Its regulatory role has attracted attention in the research fields of obesity, diabetes, and various metabolic syndromes. AMPK inhibits ATP-consuming anabolic processes and promotes ATP-producing catabolic processes (glucose uptake, glycogenolysis, and fatty acid oxidation). Therefore, activation of AMPK in tissues such as the liver and skeletal muscle increases fatty acid oxidation and glucose uptake. The level of AMPK phosphorylation in cells, animal liver, and muscle tissue can be measured by Western blotting or ELISA. In muscle tissue and cardiac muscle, AMPK promotes muscle contraction and glucose uptake, which occurs independently of insulin action by activating GLUT1 and inducing GLUT4 translocation to the plasma membrane, increasing glucose transport into cells.

[0045] Sirtuin 1 (SIRT1) is a factor required for AMPK activation and is known to be involved in the metabolism of PGC-1α. Specifically, AMPK activation increases intracellular NAD(+), which activates the NAD(+)-dependent deacetylase SIRT1, leading to the deacetylation and activation of PGC-1α. SIRT1 activation has been reported to deacetylate PGC-1α and increase the activity of muscle glucose transporter 4 (GLUT4). Insulin-responsive glucose transporter 4 (GLUT4) is an insulin-dependent glucose transporter primarily found in skeletal muscle and adipose tissue, where it transports glucose from the outside to the inside of cells. SIRT1 protein expression can be measured in cells or animal muscles by Western blotting or ELISA.

[0046] Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is known to play an important role in glucose metabolism, mitochondrial biogenesis, muscle fiber specialization, and adaptive thermogenesis. Increased expression of PGC-1α is known to promote increased mitochondrial DNA replication and mitochondrial proliferation. Substances that promote PGC-1α expression promote mitochondrial biogenesis, which ultimately promotes mitochondrial fatty acid oxidation, generating ATP energy and promoting bodily energy expenditure. Mitochondria play an important role in energy metabolism by activating glucose transport and fat oxidation.

[0047] Yet another example of the present invention is a composition for preventing, ameliorating, or treating oxidative stress-related diseases, comprising allulose as an active ingredient.

[0048] As used herein, diseases caused by oxidative stress or diseases associated with oxidative stress include, but are not limited to, cancer, osteomyelitis, acquired immune deficiency, cardiovascular disease, colorectal cancer, bladder cancer, coronary artery disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, chronic kidney disease, alcoholic liver disease, obstructive pulmonary disease, insulin resistance syndrome or diabetes, preferably coronary artery disease or diabetes.

[0049] Cell and tissue damage from reactive oxygen species (ROS) is known to be related not only to diabetes but also to inflammation, aging, and other conditions. To confirm whether allulose has antioxidant properties, we measured the reactive oxygen species scavenging activity of allulose by DHE tissue staining to measure intracellularly generated superoxide anion, NADPH oxidase activity, and lipid peroxidation. Specifically, it has the effect of reducing intracellularly generated superoxide anion (Figure 3). Peroxides, including hydrogen peroxide (H2O2), are one of the main reactive oxygen species (ROS) that cause oxidative stress.

[0050] This experiment was conducted to analyze the inhibitory effects of powder and liquid allulose on NADPH oxidase activity and lipid peroxidation produced by a diabetic animal model (DC). Specifically, we analyzed whether allulose has the ability to protect cells from oxidative stress caused by hydrogen peroxide (H2O2). The powder and liquid forms of allulose exhibited a concentration-dependent inhibitory effect on the superoxide anion, NADPH oxidase activity, and lipid peroxidation produced by a diabetic animal model (DC) (Figures 4 and 5). This indicates that both powder and liquid forms of allulose have a protective effect on cells from oxidative stress caused by hydrogen peroxide (H2O2).

[0051] We confirmed that protein oxidation was significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, we confirmed that protein oxidation decreased in the groups that ingested allulose powder and liquid in a concentration-dependent manner compared to the diabetic control group (DC) (Figure 6).

[0052] Specifically, we analyzed the effect of D-allulose on NOX4 protein expression levels and confirmed that NOX4 protein expression levels were significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, we confirmed that NOX4 protein expression levels were reduced in the groups that ingested allulose powder and liquid in a concentration-dependent manner compared to the diabetic control group (DC) (Figure 7). The decrease in NOX4 expression due to allulose administration is thought to suppress the production of ROS in muscle cells, regulate glucose metabolism, and reduce insulin resistance.

[0053] The decrease in NOX4 expression caused by administration of allulose appears to suppress the production of ROS within muscle cells, regulate glucose metabolism, and reduce insulin resistance. Excessive intracellular glucose metabolism increases Nox4 expression within cells or tissues, and this increased expression of Nox4 induces excessive production of reactive oxygen species within cells and tissues. The reactive oxygen species produced in this process are known to play an important role in cell survival and death and the maintenance of the redox system.

[0054] The allulose applicable to the present invention is not particularly limited and includes liquid allulose, powdered allulose, crystalline allulose, and amorphous allulose. The allulose may be chemically synthesized or biologically produced.

[0055] In the present specification, the amount of allulose administered to a subject may be 10 to 80 g, 10 to 70 g, 10 to 65 g, 10 to 60 g, 10 to 55 g, 10 to 50 g, 20 to 80 g, 20 to 70 g, 20 to 65 g, 20 to 60 g, 20 to 55 g, 20 to 50 g, 30 to 80 g, 30 to 70 g, 30 to 65 g, 30 to 60 g, 30 to 55 g, or 30 to 50 g per 60 kg of body weight of the consuming individual. Therefore, the composition containing allulose according to the present invention may be a composition containing allulose such that the daily intake is 10 to 80 g, 10 to 70 g, 10 to 65 g, 10 to 60 g, 10 to 55 g, 10 to 50 g, 20 to 80 g, 20 to 70 g, 20 to 65 g, 20 to 60 g, 20 to 55 g, 20 to 50 g, 30 to 80 g, 30 to 70 g, 30 to 65 g, 30 to 60 g, 30 to 55 g, or 30 to 50 g per 60 kg body weight of the consuming individual.

[0056] The subjects or individuals to which the composition containing allulose as an active ingredient according to the present invention is administered or ingested are animals including humans, such as humans, mice, rats, monkeys, etc.

[0057] The composition according to the present invention containing allulose as an active ingredient can be taken before, after, or simultaneously with a meal, and there are no particular limitations on the conditions for ingestion.

[0058] The composition containing allulose as an active ingredient can be ingested for a period effective for achieving a reduction in body fat, for example, 4 to 20 weeks, preferably 8 to 12 weeks. Although a single intake of the composition according to the present invention can derive an effective effect, it can also be ingested two or more times.

[0059] The dosage unit for ingestion of the composition containing allulose as an active ingredient is prepared to contain, for example, 1, 2, 3, or 4 times the individual intake amount, or 1 / 2, 1 / 3, or 1 / 4 times the individual intake amount. The individual intake amount preferably contains the amount of the active ingredient to be administered once a day, which is usually formulated in an amount equivalent to the entire daily dose, 1 / 2, 1 / 3, or 1 / 4.

[0060] The composition comprising allulose according to the present invention may be a food composition or a pharmaceutical composition.

[0061] The suitable dosage of the composition of the present invention can be variously formulated depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient.

[0062] The composition according to the present invention, which contains allulose as an active ingredient, may be a food, a food additive, a beverage, a beverage additive, a health food, or a functional food. In the present invention, "health functional food" refers to a food produced (including processed) using raw materials or ingredients that have functional properties beneficial to the human body in accordance with the Health Functional Food Act, and "functionality" refers to obtaining beneficial effects for health purposes, such as maintaining the normal functions of the human body or activating physiological functions to maintain and improve health. The allulose can be added to general foods or prepared as encapsulated, powdered, or suspension. When ingested, it provides specific health benefits, and unlike general medicines, it is made from food ingredients, which has the advantage of eliminating side effects that can occur with long-term drug use.

[0063] When the allulose of the present invention is used as a food additive, it can be added as it is, used together with other foods or food ingredients, or used appropriately by other conventional methods. The amount of the active ingredient to be mixed can be suitably determined depending on the purpose of use (prevention, health, or therapeutic treatment).

[0064] The food is not particularly limited as long as it is a food to which allulose can be applied, and examples thereof include processed grain products, processed beans products, processed potatoes products, processed sugar products, processed seafood products, other processed products, sweets, candies (e.g., hard candies, jellies, gummies), breads, gyoza dumplings, processed meat products (e.g., meat, sausages), dairy products (e.g., lactic acid bacteria fermented milk, ice cream), processed eggs, jelly-like products (soba, acorns, etc., powdered and boiled into a jelly), processed oils and fats, other noodles, oily noodles, solid tea, liquid tea, coffee, fruit and vegetable juices, fruit and vegetable drinks, and other foods. Fermented drinks, ginseng drinks, mixed drinks, beverage bases, seasoned miso, chili pepper miso, natto soup, sauces, complex seasoned foods, Chinese cabbage kimchi, seasoned salted fish, salted pickles, candied pickles, pickled agricultural products, pickled livestock products, seasoned dried fish, dried fish, processed peanuts or nuts, processed fruit and vegetable products, seasoned seaweed and extract foods, ready-to-eat foods, steamed rice, processed mushroom fruiting body foods and drinks, processed meat products, chocolate, confectionery, pizza, noodles (ramen, soba, etc.), gums, ice cream, alcoholic drinks, vitamin complexes and health supplements.

[0065] Yet another example of the present invention provides a composition for promoting body fat burning, which contains allulose as an active ingredient and is in the form of a tablet, powder, capsule, granule, syrup, jelly, bar, paste, gel, beverage, or tea.

[0066] The food according to the present invention may contain conventional food additives, and whether or not a food additive is suitable for use as a food additive is determined in accordance with the specifications and standards for the relevant item, such as the general provisions and general test methods in the Food Additives Code approved by the Food and Drug Administration, unless otherwise specified.

[0067] In one example, the weight (wt) % of allulose in the composition excluding the filler can be set in various ways. For example, the allulose can be contained in an amount of about 0.1 to 99.9 wt % or 5 to 95 wt % based on the solid content of the composition. Alternatively, the allulose may be contained in an amount of 80 (w / w)% or more, preferably 90 (w / w)% or more, more preferably 95 (w / w)% or more, 96 (w / w)% or more, 97 (w / w)% or more, 98 (w / w)% or more, or 99 (w / w)% or more based on the total solid content.

[0068] The allulose applicable to the present invention is not particularly limited, and includes liquid allulose containing allulose (allulose syrup, powdered allulose, crystalline allulose, and amorphous allulose. The allulose may be chemically synthesized or biologically produced.

[0069] The allulose contained in the composition according to the present invention may be in the form of a powder or syrup, or for example, the allulose may be in the form of a powder of allulose with a purity of 80 (w / w)% or more, preferably 90 (w / w)% or more, more preferably 95 (w / w)% or more, 96 (w / w)% or more, 97 (w / w)% or more, 98 (w / w)% or more, or 99 (w / w)% or more, or may be a solution prepared using this at various concentrations, or a solution converted into allulose by chemical or biological methods in a fructose-containing solution.

[0070] The allulose syrup may be obtained by separating, purifying, and concentrating allulose alone or from a sugar mixture. In one example of the present invention, the allulose syrup after the separation and purification steps may be a liquid allulose syrup with an electrical conductivity of 1 to 50 μS / cm and a colorless or pale yellow, sweet taste. Specific examples of the allulose-containing sugar mixture may contain 5 to 95 parts by weight of allulose, 1 to 50 parts by weight of fructose, 1 to 55 parts by weight of glucose, and 1 to 10 parts by weight of oligosaccharides, based on 100 parts by weight of the total solid content of the sugar mixture, or may not contain oligosaccharides. The allulose, fructose, and glucose are preferably all D-isomers.

[0071] The present invention will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. [Example]

[0072] Example 1: Diabetic Animal Model (1) Normal diet group (NCD) The experimental animals were 5-week-old male normal mice (C57BL / KsJ-db / + mice, normal control, NC) purchased from Orient Bio Co., Ltd. and after 14 days of adaptation, they were divided into 10 groups and kept consuming the efficacy evaluation test product for 8 weeks. The experimental diet for the efficacy evaluation test was powdered feed for laboratory animals (Orient Bio Co., Ltd.), and the mice were allowed to have free access to the powdered feed and water.

[0073] (2) Diabetic animal model (DC) db / db mice (C57BL / KsJ-db / db mice, diabetic control, DC) are an animal model in which a mutation in the leptin receptor prevents signaling from leptin, a hormone secreted from adipocytes, leading to obesity and type 2 diabetes. They were purchased from Orient Bio Co., Ltd. and allowed to adapt for 14 days. After that, they were divided into 10 groups and bred to consume the efficacy evaluation test products for 8 weeks. The experimental diet for the efficacy evaluation test was powdered feed for laboratory animals (Orient Bio Co., Ltd.), and the mice were allowed free access to water and food.

[0074] Example 2: Liquid allulose administration group (AL) In the diabetic model of Example 1, allulose liquid was administered at a dose of 5.16 g / kg (high) based on the solid content. Normal diet was provided ad libitum along with water, and allulose liquid was orally administered twice daily (9:00 AM and 5:00 PM). The normal diet was prepared based on the AIN-76A diet (Teklad, USA), with a carbohydrate:protein:lipid weight ratio of 60:20:15.

[0075] Example 3: Powdered allulose administration group (AP) In the diabetic model of Example 1, allulose powder was administered at a dose of 5.16 g / kg (high) based on the solid content. Normal diet was provided ad libitum along with water, and allulose powder was orally administered twice daily (9:00 AM and 5:00 PM). The normal diet was prepared based on the AIN-76A diet (Teklad, USA), with a carbohydrate:protein:lipid weight ratio of 60:20:15.

[0076] Comparative Examples 1 and 2 In the diabetic animal model of Example 1, a comparative experiment was conducted by setting up a group administered with 5.16 g / kg of sucralose as Comparative Example 1 and a group administered with 5.16 g / kg of erythritol as Comparative Example 2 instead of allulose used in Example 2. The daily intake of allulose, 5.16g / kg, is equivalent to 25g per 60kg body weight of the individual taking it. Sucralose was orally administered with the same sweetness level, and erythritol was administered in the same amount as allulose. Normal diet, sucralose, and erythritol were simultaneously administered, and changes in body weight were observed.

[0077] Test Example 1: Blood Glucose and Insulin Analysis (1) Oral glucose tolerance test (OGTT) The normal group and diabetic animal model mice from Example 1 were fasted for 12 hours, and then glucose was orally administered to the mice at a dose of 2 g / kg. Blood samples were collected from the tail vein of the mice at intervals of 0, 15, 30, 60, 90, and 120 minutes to measure blood glucose concentrations. Blood glucose concentrations were calculated based on the blood glucose curve area in a glucose tolerance test.

[0078] (2) Insulin tolerance test (ITT analysis) The normal group and diabetic animal model of Example 1 were fasted for 12 hours, and the mice were intraperitoneally injected with 1 unit of insulin solution per kg of body weight. Blood was then collected from the tail vein of the mice 0, 15, 30, 60, 90, and 120 minutes later to measure blood glucose levels.

[0079] (3) Serum insulin concentration measurement In Examples 1 to 3 and Comparative Examples 1 and 2, allulose, sucralose, or erythritol was administered for 8 weeks, and then blood was collected from the mice in each experimental group on the final day of the experiment, centrifuged to obtain serum, and serum insulin concentrations were measured using a Mouse Insulin ELISA Kit. Specifically, 10 μL of serum from experimental animals was placed in an anti-insulin-coated 96-well plate and incubated at 20-25°C for 2 hours, followed by four washes with washing buffer. 100 μL of HRP-conjugated streptavidin was added to the washed well and incubated at 20-25°C for 30 minutes. After washing four times, 100 μL of substrate chromogen reagent was added and incubated at 20-25°C for 20 minutes. 50 μL of reaction stopper was added, and the absorbance at 450 nm (reference wavelength, 620 nm) was measured using a multi-microplate reader (infinite M200PRO, Tecan, Mannedorf, Switzerland).

[0080] Test Example 2: Evaluation of the antioxidant activity of allulose Damage to cells or tissues caused by reactive oxygen species (ROS) is known to be related not only to diabetes but also to inflammation, aging, etc. To confirm whether allulose has antioxidant properties, we measured the superoxide anion produced in cells, NADPH oxidase activity, and lipid peroxidation, and the reactive oxygen species scavenging activity of allulose using DHE tissue staining.

[0081] (1) Inhibitory effect of superoxide formation due to oxidative damage To observe superoxide formation due to oxidative damage, staining with the oxidative fluorescent dye dihydroethidium (DHE) was performed. DHE staining was performed by incubating the tissue in a 1 μM DHE solution (in PBS, pH 7.4) for 30 minutes in a dark, wet chamber. The tissue was then dehydrated and sutured to prepare tissue specimens. The fluorescently stained tissue was observed using a confocal microscope (LSM 510 META laser-scanning microscope, Carl Zeiss, Germany). Figure 3A shows confocal micrographs of the fluorescently stained tissue, and Figure 3B shows the numerical values of DHE fluorescence intensity. Figure 3B shows the numerical values of DHE fluorescence intensity obtained from DHE fluorescent staining performed to examine the effect of D-allulose on oxidative stress in db / db mice.

[0082] (2) Protective effect against cell damage caused by oxidative stress This experiment was conducted to analyze the inhibitory effects of powdered and liquid allulose on NADPH oxidase activity and lipid peroxidation produced by a diabetic animal model (DC). That is, we analyzed whether allulose has the ability to protect cells from oxidative stress caused by hydrogen peroxide (H2O2). Absorbance at 450 nm was measured using an NADPH Oxidase activity kit (Biovision). Lipid peroxidation was performed by mixing 1 mL of sample with 0.5 mL of 0.8% thiobarbituric acid (TBA) dissolved in 0.1 mL of 8.1% sodium dodecyl sulfate and 20% sodium acetate (pH 3.5) and 0.15 mL of distilled water, heating the mixture at 95°C for 1 hour, cooling it, and then adding 2.5 mL of n-butanol / pyridine (15:1, v / v) and 0.5 mL of distilled water and shaking. The mixture was centrifuged at 3,000 × g for 10 minutes, and the supernatant was collected and its absorbance at 532 nm was measured. The results are shown in Figures 4 and 5.

[0083] Figure 4 is a graph showing the values measured for the NADPH oxidase activity concentration in tissues to confirm the effect of D-allulose on NADPH oxidase activity in db / db mice. Figure 5 is a graph showing the values measured for the lipid peroxide content in db / db mice to confirm the effect of D-allulose on lipid peroxide.

[0084] As shown in Figures 4 and 5, the powder and liquid forms of allulose exhibited a concentration-dependent inhibitory effect on superoxide anion, NADPH oxidase activity, and lipid peroxidation produced by a diabetic animal model (DC) (Figures 4 and 5). This indicates that the powder and liquid forms of allulose have a protective effect on cells from oxidative stress caused by hydrogen peroxide (H2O2).

[0085] (3) Protein oxidation reduction activity Carbonyl content using DNPH reagent is often used to measure the degree of protein oxidation, along with the content of thiol groups. Specifically, carbonyl content analysis using DNPH reagent was performed using the OxyBlot Protein Oxidation Detection Kit. The results of the carbonyl content analysis using the DNPH reagent are shown in Figure 6. Figure 6 shows the results of measuring the degree of protein oxidation in tissues to confirm the effect of D-allulose on protein oxidation in db / db mice.

[0086] As shown in Figure 6, protein oxidation was significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, it was confirmed that protein oxidation decreased in the groups that ingested allulose powder and liquid in a concentration-dependent manner compared to the diabetic control group (DC) (Figure 6).

[0087] Test Example 3: Evaluation of the effect of allulose on NOX4 expression Excessive glucose metabolism in cells increases the expression of Nox4 in cells or tissues, and this increased expression of Nox4 induces the excessive production of reactive oxygen species in cells and tissues. The reactive oxygen species produced in this way are known to play an important role in cell survival and death and the maintenance of the redox system. Specifically, protein expression levels were analyzed using Western blotting. In Examples 1-3 and Comparative Examples 1-2, allulose, sucralose, and erythritol were administered to normal and diabetic groups for 8 weeks. Abdominal muscles were then lysed at 4°C using lysis buffer (10 mM Tris-HCl, pH 7.4, 0.1 M EDTA, 10 mM NaCl, 0.5% Triton X-100, protease inhibitor cocktail) and centrifuged (14,000 rpm, 10 min, 4°C) to measure protein concentration. The quantified proteins were mixed with sample buffer, heated to 95°C for 5 minutes, and separated by 10-12% SDS-PAGE. After separation, proteins were transferred to a PVDF membrane by SDS-PAGE using a semi-dry transfer system at 15V for 60 minutes and then incubated with blocking buffer (5% skim milk in 1X TBS-T) for over 1 hour. NOX4 was incubated overnight at 4°C and then washed five times with 1X TBS-T at 7-minute intervals. The secondary antibody was incubated at room temperature for over 1 hour, then washed five times with 1X TBS-T at 7-minute intervals. After color development with ECL reagent, the fluorescence was exposed to X-ray film. The results are shown in Figure 7, which illustrates the effect of D-allulose on NOX4 protein expression levels in db / db mice.

[0088] As shown in Figure 7, we confirmed that NOX4 protein expression levels were significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, we confirmed that NOX4 protein expression levels were reduced in the groups that received allulose powder and liquid in a concentration-dependent manner compared to the diabetic control group (DC). The decrease in NOX4 expression due to allulose administration is thought to suppress the production of ROS in muscle cells, regulate glucose metabolism, and reduce insulin resistance.

[0089] Test Example 4: Evaluation of the effect of allulose on endoplasmic reticulum stress Impaired insulin secretion observed in type 2 diabetes induces endoplasmic reticulum stress, and in the present invention, we attempted to determine whether allulose regulates endoplasmic reticulum stress induced by type 2 diabetes. Specifically, in Examples 1 to 3 and Comparative Examples 1 and 2, allulose, sucralose, and erythritol were administered for 8 weeks to normal and induced-diabetic groups, and then the expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO3H) in non-abdominal muscle samples from the experimental animals were analyzed using Western blotting methods essentially identical to those used in Test Example 3. The analysis results are shown in Figure 8.

[0090] Figure 8 shows the effects of D-allulose on endoplasmic reticulum (ER) stress and irreversible oxidation of IRE1-alpha in db / db mice. Compared to the normal control group (NC), the diabetic control group (DC) showed significantly increased expression of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO3H). However, the groups receiving allulose powder and liquid in a concentration-dependent manner showed a decrease in the expression of the above genes, which were increased in the diabetic control group (DC).

[0091] As shown in the experimental results in Figure 8, allulose appears to reduce endoplasmic reticulum stress and reduce insulin resistance.

[0092] Test Example 5: Evaluation of the effect of allulose on AMPK-SIRT1-PGC-1α In Examples 1 to 3 and Comparative Examples 1 and 2, allulose, sucralose, and erythritol were administered to a normal control group (NC) and a diabetic control group (DC) for 8 weeks, and then the expression levels of p-AMPK and SIRT1 proteins expressed in non-abdominal muscles of the experimental animals were analyzed using Western blotting methods essentially identical to those in Test Example 3. The analysis results are shown in Figure 9. We confirmed that the protein expression levels of p-AMPK and SIRT1 were significantly reduced in the diabetic control group (DC) compared to the normal control group (NC). However, we confirmed that the protein expression levels of p-AMPK and SIRT1 increased in the groups that ingested allulose powder and liquid in a concentration-dependent manner compared to the diabetic control group (DC) (Figure 9A). Activation of SIRT1 has been reported to deacetylate PGC-1α and increase the activity of muscle glucose transporter 4 (GLUT4). GLUT4 is an insulin-dependent glucose transporter that is distributed mainly in skeletal muscle and adipose tissue and plays a role in moving glucose from the outside to the inside of cells.

[0093] In this experiment, we confirmed the expression of acetylated-PGC-1α protein and found that acetylated-PGC-1α was increased in the diabetic control group (DC) compared to the normal control group (NC), but the expression of acetylated-PGC-1α decreased in the groups that ingested allulose powder and liquid in a concentration-dependent manner (Figure 9, B and C). Therefore, we expect that allulose activates AMPK-SIRT1-PGC-1α, promoting energy metabolism and reducing insulin resistance.

Claims

1. Contains allulose as an active ingredient The allulose is used so that the daily intake amount is 10 to 80 g per 60 kg of body weight of the user, Increases the expression of one or more proteins selected from the group consisting of p-AMPK and SIRT1; A food composition for reducing endoplasmic reticulum stress in muscle tissue and inhibiting lipid peroxidation.

2. The composition comprises p-IRE1α, ATF4, GRP78, and IRE1-alpha sulfonation (SO 3 The composition of claim 1, which reduces the expression of one or more proteins selected from the group consisting of:

3. The composition of claim 1 , wherein the composition reduces insulin resistance.

4. The composition of claim 1 , wherein the composition reduces postprandial blood glucose.

5. The composition of claim 1 , wherein the allulose is in liquid or powder form.

6. The composition according to claim 1, wherein the allulose is contained in an amount of 0.1 to 99.9% by weight based on 100% by weight of the solid content of the composition.

7. The composition of claim 1, wherein the composition is administered for 4 to 20 weeks.

8. 10. The composition of claim 1, wherein the composition comprises a tablet, powder, capsule, granule, syrup, jelly, bar, paste, gel, beverage, or tea form.

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