JP7724290B2 - Composition for reducing endoplasmic reticulum stress - Google Patents

Description

The present invention relates to a composition containing allulose that has the functionality of reducing endoplasmic reticulum stress.

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: rough endoplasmic reticulum, which has ribosomes attached, and smooth endoplasmic reticulum, which does not have ribosomes. Approximately one-third of intracellular proteins are translated from mRNA into proteins in the rough endoplasmic reticulum, where they undergo posttranslational modification, such as folding, assembly, glycation, and disulfide bonding, to form active protein structures. The smooth endoplasmic reticulum is also a site for the synthesis of lipids and steroids, and as a calcium reservoir, it plays an important role in regulating intracellular calcium levels.

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

Recently, the incidence of diabetes has been on the rise, and the mortality rate from cardiovascular disease, a complication of diabetes, is also gradually increasing. Untreated chronic diabetes complications include a variety of conditions, including nerve and kidney disease, 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 that the tissues of diabetic patients are highly sensitive to oxidative stress, and increased production of free radicals promotes lipid peroxidation. For this reason, research is progressing on strengthening the body's antioxidant defense system to protect tissues from peroxidation.

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.

Therefore, unlike existing drugs that have 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 level equivalent to 70% of that of sugar, yet has an excellent effect of reducing endoplasmic reticulum stress when ingested, providing a safe composition.

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

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

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 containing allulose to a subject in need thereof.

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

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

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 containing allulose as an active ingredient to a subject in need thereof.

Yet another example of the present invention is a pharmaceutical composition for p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α, each containing allulose as an active ingredient. 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 (SO ₃ H).

Yet another example of the present invention is a pharmaceutical composition for p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α, each containing allulose as an active ingredient. 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 (SO ₃ H).

A further example of the present invention relates to a composition, use, or method for preventing, ameliorating, or treating oxidative stress-related diseases, which comprises allulose.

According to one example of the present invention, endoplasmic reticulum stress can be effectively reduced and blood sugar regulation can be achieved simply by taking allulose, without changing dietary or exercise habits.

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. 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 an example of the present invention. According to one example of the present invention, confocal micrographs (Figure 3A) of DHE fluorescently stained tissues 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. 1 is a graph showing 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. 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 according to an example of the present invention. 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. 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. FIG. 1 shows the effect 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. FIG. 1 shows the supplementary effect of D-allulose on p-AMPK-SIRT1-PGC-1α in db/db mice according to an example of the present invention.

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

In the present invention, the endoplasmic reticulum 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. Increasing endoplasmic reticulum stress to induce cell death may be a therapeutic means for treating cancer.

Yet another example of the present invention is a method for producing p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1-alpha, each containing allulose as an active ingredient. 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 (SO ₃ H).

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

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 comprises 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 (SO ₃ H) and/or increase the expression or activation of p-AMPK and SIRT1 proteins.

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 due to 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.

A further example 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.

Allulose can reduce oxidative stress by decreasing Nox4 expression in muscle cells or muscle tissue, reducing protein oxidation, or inhibiting one or more of superoxide anion, NADPH oxidase activity, or lipid peroxidation. Ingestion of a composition containing allulose as an active ingredient can reduce intramuscular superoxide anion, NADPH oxidase activity, or lipid peroxidation by 5-50%, 10-50%, 5-30%, 10-30%, 5-20%, 5-15%, or 10-20%.

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 individual taking it (10 to 80 g/60 kg/day). The composition containing allulose as an active ingredient may be taken before, after, or simultaneously with a meal. The composition containing allulose as an active ingredient may be taken for a period of 4 to 20 weeks, preferably 8 to 12 weeks. Although effective effects can be obtained by taking the composition according to the present invention just once, the effects can be further maximized by taking it for the above period.

In more detail, in a test in which mammals on a high-fat diet were given various types of sweeteners, only the composition containing allulose as an active ingredient was found to be effective in reducing endoplasmic reticulum stress and oxidative stress.

Specifically, in the present invention, the administration of allulose reduces NOX4 expression, suppressing the production of ROS within 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.

According to the present invention, allulose, contained as an active ingredient, contributes to blood sugar 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 ingested orally, it is provided to the liver, muscle tissue, brain tissue, visceral beds, and adipose tissue, and allulose activates storage in muscle tissue, in particular, thereby very effectively lowering blood sugar levels in the body.

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.

More specifically, the present invention relates to a composition containing allulose as an active ingredient for regulating blood glucose, regulating insulin sensitivity, or improving, preventing, or treating diabetes by reducing endoplasmic reticulum stress.

As used herein, the term "endoplasmic reticulum stress (ER stress)" refers to a condition in which the function of the endoplasmic reticulum is impaired due to a physiological or pathological environment, which causes immature proteins to enter the endoplasmic reticulum in excess of the endoplasmic reticulum's capacity to process them, or due to calcium depletion within 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: PERK (pancreatic ER kinase), IRE-1α/XBP-1 (inositol-requiring 1α/X-box binding protein 1), and ATF6 (activating transcription factor 6). 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 shown that ER stress plays a role in the pathogenesis of various diseases, including ischemia, diabetes, viral infection, and hyperhomocysteinemia.

Yet another example of the present invention is a pharmaceutical composition for p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α, each containing allulose as an active ingredient. The present invention relates to a composition that reduces the expression of one or more proteins selected from the group consisting of sulfonation (SO ₃ H).

Specifically, analysis of the expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO ₃ H) in non-abdominal muscle samples from experimental animals revealed that the expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO ₃ H) were significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, the expression levels of the above genes, which were increased in the diabetic control group (DC), were reduced in the groups that received allulose powder and liquid in a concentration-dependent manner (Figure 8). Therefore, allulose appears to reduce endoplasmic reticulum stress and improve insulin resistance, and can protect cells by inhibiting endoplasmic reticulum stress-induced apoptosis (cell death).

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 leads to the deacetylation of PGC-1α (i.e., decreased expression of acetylated-PGC-1α), which increases the activity of GLUT4 present in muscles, moving glucose from the outside to the inside of cells and 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 distributed mainly in skeletal muscle and adipose tissue, and plays a role in moving glucose from the outside to the inside of cells.

Specifically, it was confirmed that the groups that received 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 received 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).

In this invention, the expression of acetylated-PGC-1α protein was confirmed. Compared to the normal control group (NC), the diabetic control group (DC) showed increased acetylated-PGC-1α, but the expression of acetylated-PGC-1α decreased in the groups that ingested powdered and liquid allulose in a concentration-dependent manner (Figure 9, B and C). Therefore, it is expected that allulose activates AMPK-SIRT1-PGC-1α, enhances energy metabolism, and helps reduce insulin resistance.

As used herein, "endoplasmic reticulum stress-related diseases" refers to diseases caused or exacerbated by endoplasmic reticulum stress. Examples include diseases (e.g., neurodegenerative diseases) caused by excessive intracellular protein accumulation, which inhibits the degradation system and is itself toxic to cells, and diseases (e.g., diabetes) caused by an excessive endoplasmic reticulum stress response induced by an incorrect signaling system. 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.

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 a portion of a gene is over-amplified due to mutation, glutamine polymers become entangled and form protein aggregations that the cell's ability to degrade them cannot withstand, resulting in spontaneous endoplasmic reticulum stress and the death of neurons.

One example of the present invention provides the use of allulose to improve or treat increased insulin resistance, increased blood sugar, or diabetes, where 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.

Specifically, in relation to oxidative stress, administration of allulose reduces NOX4 expression, suppressing the production of ROS within muscle cells 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 activates GLUT1 and induces the movement of GLUT4 to the plasma membrane, regardless of the action of insulin, thereby increasing the transport of glucose into cells. Allulose is also known to induce the activation of SIRT1, thereby deacetylating PGC-1α and increasing the activity of glucose transporter 4 (GLUT4) in muscles. 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.

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 functioning 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.

Allulose increases phosphorylated AMPK (AMP-activated protein kinase) and activates AMPK signaling, thereby promoting cellular absorption of blood glucose and thereby lowering blood glucose levels, 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 glucose release from the liver, regardless of insulin. AMPK is known to increase gene expression of PGC-1α, which is thought to play an important role in mitochondrial biogenesis.

AMPK is an important factor in regulating energy balance at the most cellular and whole-body levels. It is known as a serine/threonine kinase and a regulator of lipid and glucose metabolism. Its regulatory role has attracted attention in the research fields of obesity, diabetes, and various metabolic syndromes. AMPK inhibits anabolic processes that consume ATP and promotes catabolic processes that produce ATP (glucose absorption, related processes, glycogenolysis, and fatty acid oxidation). Therefore, activation of AMPK in tissues such as the liver and skeletal muscle increases fatty acid oxidation and glucose absorption. The level of AMPK phosphorylation in cells, animal liver, and muscle tissues can be measured by Western blotting or ELISA. AMPK in muscle tissue and cardiac muscle promotes muscle contraction and glucose absorption, which occurs independently of the action of insulin by activating GLUT1 and inducing the movement of GLUT4 to the plasma membrane, thereby increasing the transport of glucose into cells.

Sirtuin 1 (SIRT1) is a factor required for AMPK activation and is known to be involved in the metabolism of PGC-1α. Specifically, activation of AMPK increases intracellular NAD(+), activating the NAD(+)-dependent deacetylase SIRT1, which deacetylates and activates 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 distributed primarily in skeletal muscle and adipose tissue, where it transports glucose from the outside to the inside of cells. SIRT1 protein expression in cells or animal muscles can be measured by Western blotting or ELISA.

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 consumption. Mitochondria play an important role in energy metabolism by activating glucose transport and fat oxidation.

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.

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.

Cell or tissue damage from reactive oxygen species (ROS) is known to be associated with not only diabetes but also inflammation, aging, and other conditions. To confirm whether allulose has antioxidant properties, the reactive oxygen species scavenging activity of allulose was measured by DHE tissue staining to measure intracellularly generated superoxide anion, NADPH oxidase activity, and lipid peroxidation. Specifically, allulose 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.

This experiment was conducted to analyze the inhibitory effects of allulose powder and liquid 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 superoxide anion, NADPH oxidase activity, and lipid peroxidation produced by the diabetic animal model (DC) were inhibited by allulose powder and liquid in a concentration-dependent manner (Figures 4 and ₅ ). This indicates that both allulose powder and liquid have a protective effect on cells from oxidative stress caused by hydrogen peroxide ( _H2O2 ).

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 was reduced in the groups that ingested allulose powder and liquid in a concentration-dependent manner compared to the diabetic control group (DC) (Figure 6).

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

The decrease in NOX4 expression caused by the 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 in maintaining the redox system.

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.

In the present specification, the amount of allulose administered to a subject may be a daily intake of 10-80g, 10-70g, 10-65g, 10-60g, 10-55g, 10-50g, 20-80g, 20-70g, 20-65g, 20-60g, 20-55g, 20-50g, 30-80g, 30-70g, 30-65g, 30-60g, 30-55g, or 30-50g per 60kg of body weight of the individual taking the allulose. Therefore, the composition containing allulose according to the present invention may contain allulose so 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.

The subjects or individuals to whom 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.

The composition of the present invention containing allulose as an active ingredient can be taken before, after, or simultaneously with meals, and there are no particular restrictions on the conditions for ingestion.

The composition containing allulose as an active ingredient can be ingested for a period effective to achieve body fat reduction, for example, 4 to 20 weeks, preferably 8 to 12 weeks. While effective effects can be achieved by ingesting the composition of the present invention just once, it can also be ingested two or more times.

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

The composition containing allulose according to the present invention may be a food composition or a pharmaceutical composition.

The appropriate dosage of the composition of the present invention can be formulated in various ways depending on factors such as the formulation method, mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

The composition according to the present invention containing allulose as an active ingredient may be a food, food additive, beverage, beverage additive, health food, or functional food. In the present invention, "health functional food" refers to a food manufactured (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 maintaining and improving health by activating physiological functions. The allulose can be added to general foods or manufactured into capsules, powders, suspensions, etc. When ingested, it provides specific health benefits, and, unlike general medicines, because it is made from food ingredients, it has the advantage of being free of side effects that can occur with long-term drug use.

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

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 bean products, processed potato products, processed sugar products, processed seafood products, other processed products, sweets, candies (e.g., hard candy, jellies, gummies), bread, gyoza, processed meat products (e.g., meat, sausage), dairy products (e.g., lactic acid bacteria fermented milk, ice cream), processed eggs, jelly-like solids (soba, acorn, etc. powder boiled into a jelly-like substance), 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 miso, natto soup, sauces, compound 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.), gum, ice cream, alcoholic drinks, vitamin complexes, and health supplements.

Yet another example of the present invention provides a composition for promoting body fat burning, containing allulose as an active ingredient, in the form of tablets, powder, capsules, granules, syrup, jelly, bars, paste, gel, beverage, or tea.

Food products according to the present invention may contain conventional food additives, and unless otherwise specified, their suitability as food additives 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.

In one example, the weight (wt) percentage of allulose in the composition, excluding the filler, can be varied. For example, the composition may contain about 0.1 to 99.9 wt% or 5 to 95 wt% of allulose based on the solid content of the composition. Alternatively, the composition may contain 80 (w/w)% or more of allulose, 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 of allulose based on the total solid content.

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.

The allulose contained in the composition according to the present invention may be in the form of a powder or syrup, or, for example, 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 may be in the form of a powder, a solution prepared using this at various concentrations, or a solution obtained by converting a fructose-containing solution into allulose by chemical or biological methods.

The allulose syrup may be obtained by separating, purifying, and concentrating the allulose alone or from a sugar mixture. In one example of the present invention, the allulose syrup that has undergone the separation and purification processes may be a colorless or pale yellow liquid allulose syrup with a sweet taste and an electrical conductivity of 1 to 50 μS/cm. 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 solids content of the sugar mixture, or may not contain oligosaccharides. The allulose, fructose, and glucose are preferably all D-isomers.

The present invention will now be described in more detail with reference to the following examples. However, these examples are intended merely to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Diabetic Animal Model (1) Normal Diet (NCD)
The experimental animals were 5-week-old normal male 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 for 8 weeks while consuming the efficacy evaluation test product. The experimental diet for the efficacy evaluation test was powdered feed for laboratory animals (Orient Bio Co., Ltd.), and the mice were allowed to freely access the powdered feed and water.

(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. The mice 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.

Example 2: Liquid allulose administration group (AL)
The diabetic model of Example 1 was administered allulose liquid 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 AIN-76A diet (Teklad, USA), with a carbohydrate:protein:lipid weight ratio of 60:20:15.

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 AIN-76A diet (Teklad, USA), with a carbohydrate:protein:lipid weight ratio of 60:20:15.

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, 5.16 g/kg, corresponds to 25 g per 60 kg of body weight in humans. Sucrolose was orally administered at the same sweetness level, and erythritol was administered in the same amount as allulose. Sucralose and erythritol were simultaneously administered to normal diets, and changes in body weight were observed.

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 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 from the blood glucose curve area in a glucose tolerance test.

(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.

(3) Measurement of serum insulin concentration In Examples 1 to 3 and Comparative Examples 1 and 2, allulose, sucralose, or erythritol was administered for 8 weeks. On the final day of the experiment, blood was collected from the mice in each experimental group and centrifuged to obtain serum. The serum insulin concentration was measured using a Mouse Insulin ELISA Kit.
Specifically, 10 μL of serum from the experimental animal was placed in an anti-insulin-coated 96-well plate, left to stand at 20-25° C. for 2 hours, and then washed four times with washing buffer. 100 μL of HRP-conjugated streptavidin was added to the washed sample 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 a 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).

Test Example 2: Evaluation of antioxidant activity of allulose It is known that cell or tissue damage caused by reactive oxygen species (ROS) is related not only to diabetes but also to inflammation, aging, etc. To confirm whether allulose has antioxidant activity, the superoxide anion, NADPH oxidase activity, and lipid peroxidation generated in cells were measured by DHE tissue staining, and the reactive oxygen species scavenging activity of allulose was measured.

(1) Inhibitory Effect of Superoxide Formation Due to Oxidative Damage To observe superoxide formation due to oxidative damage, staining with dihydroethidium (DHE), an oxidative fluorescent dye, was performed. DHE staining was performed by incubating the tissue in a 1 μM DHE solution (in PBS; pH 7.4) in a dark, wet chamber for 30 minutes, dehydrating, and sutured to prepare tissue specimens. The fluorescently stained tissues were observed using a confocal microscope (LSM 510 META laser-scanning microscope, Carl Zeiss, Germany). Figure 3A shows a confocal micrograph of the fluorescently stained tissue, and Figure 3B shows the numerical results of DHE fluorescence intensity. The graph shown in Figure 3B is a graph showing the DHE fluorescence intensity values obtained as a result of DHE fluorescent staining performed to confirm the effect of D-allulose on oxidative stress in db/db mice.

(2) Protective effect against cell damage caused by oxidative stress This experiment was conducted to analyze the inhibitory effect of allulose powder and liquid on NADPH oxidase activity and lipid peroxidation produced in a diabetic animal model (DC). That is, we analyzed whether allulose has the activity 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 0.1 mL of 8.1% sodium dodecyl sulfate, 0.5 mL of 0.8% thiobarbituric acid (TBA) dissolved in 20% sodium acetate (pH 3.5), and 0.15 mL of distilled water. The mixture was heated at 95°C for 1 hour, cooled, and then 2.5 mL of n-butanol/pyridine (15:1, v/v) and 0.5 mL of distilled water were added and shaken. The mixture was centrifuged at 3,000×g for 10 minutes, and the supernatant was collected and measured for absorbance at 532 nm. The measurement results are shown in FIGS. 4 and 5.

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

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 ).

(3) Protein Oxidation Reducing Activity Carbonyl content using DNPH reagent is often used to measure the level of protein oxidation, along with the content of thiol groups. Specifically, carbonyl content analysis using DNPH reagent was performed using an 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 level of protein oxidation in tissues in db/db mice to confirm the effect of D-allulose on protein oxidation.

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

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, and then the abdominal muscles were 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 the protein concentration. The quantified protein was mixed with sample buffer, heated to 95°C for 5 minutes, and separated by 10-12% SDS-PAGE. After separation, the protein was transferred to a PVDF membrane using a semi-dry transfer system at 15 V for 60 minutes and then reacted with blocking buffer (5% skim milk in 1X TBS-T) for over 1 hour. NOX4 was reacted overnight at 4°C, and then washed five times with 1X TBS-T at 7-minute intervals. After incubation with the secondary antibody at room temperature for at least 1 hour, the cells were washed five times with 1X TBS-T at 7-minute intervals, and then developed with ECL reagent, followed by fluorescent imaging on an X-ray film. The results are shown in Figure 7, which shows the effect of D-allulose on NOX4 protein expression levels in db/db mice.

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 administration of allulose is thought to suppress the production of ROS within muscle cells, regulate glucose metabolism, and reduce insulin resistance.

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. 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 diabetes-induced groups, and then the expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO ₃ H) were analyzed for non-abdominal muscle samples of the experimental animals using Western blotting methods essentially identical to those used in Test Example 3. The analysis results are shown in FIG.

8 shows the effect of D-allulose on endoplasmic reticulum (ER) stress and irreversible oxidation of IRE1-alpha in db/db mice. It was confirmed that the expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO ₃ H) were significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, it was confirmed that the expression levels of the above genes, which were increased in the diabetic control group (DC), were reduced in the groups that received allulose powder and liquid in a concentration-dependent manner.

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

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 the same Western blotting method as 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 received 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 increase the activity of muscle glucose transporter 4 (GLUT4) through the deacetylation of PGC-1α. GLUT4 is an insulin-dependent glucose transporter that is distributed mainly in skeletal muscle and adipose tissue and plays a role in transporting glucose from the outside to the inside of cells.

In this experiment, the expression of acetylated-PGC-1α protein was confirmed. Compared to the normal control group (NC), the diabetic control group (DC) showed increased acetylated-PGC-1α, but the expression of acetylated-PGC-1α decreased in the groups that received powdered and liquid allulose in a concentration-dependent manner (Figure 9, B and C). Therefore, it is expected that allulose activates AMPK-SIRT1-PGC-1α, enhances energy metabolism, and helps reduce insulin resistance.

Claims (8)

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 ,
p -AMPK and SIRT1, which increase the expression of one or more proteins selected from the group consisting of SIRT1 and SIRT1.
A food composition for reducing endoplasmic reticulum stress in muscle tissue and inhibiting lipid peroxidation . The composition according to claim 1, wherein the composition reduces the expression of one or more proteins selected from the group consisting of p-IRE1α, ATF4, GRP78, and IRE1-alpha sulfonation (SO ₃ H). The composition of claim 1, wherein the composition reduces insulin resistance. The composition of claim 1, wherein the composition reduces postprandial blood glucose. The composition of claim 1, wherein the allulose is in liquid or powder form. 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. The composition described in claim 1, wherein the composition is administered for 4 to 20 weeks. The composition of claim 1, wherein the composition is in the form of a tablet, powder, capsule, granule, syrup, jelly, bar, paste, gel, beverage, or tea.