KR20220097318A - Composition for reducing endoplasmic reticulum stress - Google Patents

Abstract

The present invention relates to a composition with endoplasmic reticulum stress reducing functionality comprising allulose. According to one example of the present invention, there is an effect of effectively reducing endoplasmic reticulum stress and helping to control blood sugar only by ingesting allulose without changing dietary life or exercise life.

Description

Composition for reducing endoplasmic reticulum stress

The present invention relates to a composition comprising allulose having endoplasmic reticulum stress reducing functionality.

Intracellular stress includes oxidative stress, mitochondrial stress, heat shock stress, and endoplasmic reticulum stress (ER stress). The lumen of the endoplasmic reticulum (ER) is a specialized cellular environment for post-translational modification and protein folding. There are two types of ER, the rough endoplasmic reticulum with ribosomes and the smooth endoplasmic reticulum without ribosomes. About 1/3 of intracellular proteins are translated from mRNA to protein in the rough endoplasmic reticulum, followed by posttranslational modification, that is, folding and assembly, glycation, and disulfide bonds. through the active protein structure. In addition, smooth endoplasmic reticulum is a synthesis site for lipids and steroids and plays an important role in regulating intracellular calcium concentration as a calcium store.

As such, the ER stress response is an important compensatory mechanism for protecting cells by preserving ER function from various cellular stresses, but recently, excessive ER stress response is induced due to an incorrect signaling system, and there are diseases caused or caused by this. it is known

Recently, the incidence of diabetes mellitus is increasing, and the mortality rate due to cardiovascular disease, which is a accompanying complication, is also increasing. Untreated chronic diabetes complications include neurological and renal diseases, but among them, the incidence of cardiovascular diseases such as hypertension, arteriosclerosis, cerebral infarction, cerebral thrombosis, and myocardial infarction is high. As one of the main factors for frequent cardiovascular disorders, including arteriosclerosis, it is known that the tissues of diabetic patients are highly sensitive to oxidative stress and that lipid peroxidation is promoted by increased production of free radicals. Therefore, research related to strengthening the antioxidant defense system of the living body to protect the tissue from peroxidation is progressing.

Diabetes mellitus is a metabolic disease characterized by high blood sugar caused by a malfunction in the secretion or function of insulin required for blood sugar control in the body. Insulin resistance is a phenomenon commonly observed in most obese and type 2 diabetes patients, and the main cause is a postreceptor defect in insulin action. decrease occurs.

Although exercise and diet are important to control blood sugar, many people have difficulties because it is difficult to manage a steady diet or exercise. In addition, although people having difficulty in controlling blood sugar reduce blood sugar by drug therapy, the safety of drugs for long-term intake is a problem due to the nature of chronic diseases.

Therefore, unlike existing drugs, which are concerned about safety, they have been developing functional materials that reduce blood sugar by promoting endoplasmic reticulum stress reduction. This functional material is called allulose, and as an epimer of fructose carbon 3, it has a sweetness equivalent to 70% of sugar, so it has a sweet taste, and is excellent in reducing ER stress when ingested and provides a safe composition.

One embodiment of the present invention relates to a composition for preventing, improving or treating ER stress-related diseases containing allulose.

Another embodiment of the present invention relates to a composition for ameliorating, preventing, or treating blood sugar control, insulin sensitivity control, or diabetes by reducing endoplasmic reticulum stress containing allulose as an active ingredient.

Another example of the present invention comprising allulose as an active ingredient. To a composition for reducing the expression of one or more ER stress-related proteins selected from the group consisting of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, IRE1α and sulfonation (SO3H).

A further embodiment of the present invention relates to a composition for preventing, improving or treating oxidative stress-related diseases, including allulose.

One embodiment of the present invention relates to a composition for preventing, improving or treating ER stress-related diseases, including allulose. The ER stress-related disease may be an ER stress-related disease of muscle tissue, and may be an ER or sarcoplasmic reticulum of muscle tissue.

In the present invention, ER stress-related diseases include increased insulin resistance, increased blood sugar, 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 can The treatment of cancer may be a therapeutic means to induce apoptosis by increasing ER stress.

Another example of the present invention is one or more endoplasmic reticulum stress selected from the group consisting of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, IRE1-alpha and sulfonation (SO ₃ H) containing allulose as an active ingredient It relates to a composition for reducing the expression or activity of a related protein.

Another embodiment of the present invention relates to a composition for increasing or activating p-AMPK and SIRT1 protein expression, comprising allulose as an active ingredient.

Another embodiment of the present invention relates to a composition for improving, treating, or preventing diabetes mellitus, lowering blood sugar through endoplasmic reticulum stress reduction, insulin sensitivity reduction, or diabetes containing allulose as an active ingredient. The composition may be a pharmaceutical composition or a food composition. The endoplasmic reticulum stress may be generated in muscle tissue or pancreatic beta cells. The present invention also relates to a composition for reducing ER (muscular ER) stress in muscle tissue, lowering blood sugar through ER stress reduction, reducing insulin sensitivity, or improving, treating or preventing diabetes, comprising allulose as an active ingredient . Specifically, the composition for lowering blood sugar, reducing insulin sensitivity, or improving, treating or preventing diabetes according to the present invention is p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, IRE1-alpha and sulfonation (SO ₃ H). It may be to reduce the expression or activity of one or more endoplasmic reticulum stress-related proteins selected from the group consisting of, and/or to achieve increased or activation of the expression of p-AMPK and SIRT1 proteins.

According to the present invention, allulose has a blood sugar control ability, and in particular can reduce blood sugar after a meal. Specifically, allulose according to the present invention can reduce postprandial blood glucose by administering allulose during or after a meal in a healthy subject or subject whose blood glucose level is on the borderline of diabetes. Accordingly, the composition comprising allulose as an active ingredient according to the present invention can suppress or reduce the increase in blood sugar due to a meal and can lower the increased blood sugar, so that a healthy subject, a subject having a blood sugar level at the border of diabetes , or may inhibit an increase in blood sugar or induce a drop in blood sugar in a subject having diabetes. The subject may be a mammal including a human.

A further embodiment of the present invention relates to a composition for preventing, improving or treating oxidative stress-related diseases, including allulose. The allulose has antioxidant activity that reduces oxidative stress. The allulose may reduce oxidative stress by reducing Nox4 ((NADPH oxidase 4: NOX4) expression, reducing protein oxidation, or inhibiting superoxide anion, NADPH Oxidase activity, and lipid peroxidation.

The allulose may reduce oxidative stress by reducing 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. 5 to 50%, 10 to 50%, 5 to 30%, 10 to 30%, 5 to 20% of intramuscular superoxide anion, NADPH Oxidase activity, or lipid peroxidation through ingestion of the composition containing allulose as an active ingredient , 5 to 15%, or 10 to 20%.

The composition containing allulose as an active ingredient according to the present invention may be a composition formulated so that the daily intake is 10 to 80 g (10 to 80 g/60 kg/day) per 60 kg body weight of the ingested individual. The composition containing allulose as an active ingredient may be ingested before, after, or simultaneously with a meal. The composition containing allulose as an active ingredient may be ingested for a period of 4 to 20 weeks (weeks), preferably 8 to 12 weeks (weeks). An effective effect can be derived only by ingesting the composition according to the present invention once, but the effect can be further maximized when ingested during the above period.

In detail, the effect of reducing ER stress and oxidative stress was confirmed only in a composition containing allulose as an active ingredient through an intake test of various types of sweeteners in a high-fat diet mammal.

In addition, according to the present invention, allulose reduces insulin resistance, and in particular, increased insulin resistance (tolerance) in obesity and diabetes caused by a high-fat diet can be reduced by administration of allulose. Specifically, in the present invention, the decrease in NOX4 expression by the administration of allulose suppresses the generation of ROS in muscle cells, thereby regulating glucose metabolism, thereby reducing insulin resistance. Allulose promotes energy metabolism by activating AMPK-SIRT1-PGC-1α, contributing to the improvement of insulin resistance.

According to the present invention, allulose contributes to blood sugar control by reducing ER stress in muscle, specifically, by reducing ER stress in muscle cells or muscle tissue. More specifically, when carbohydrates are ingested, the glucose absorbed in the intestine is sent to each tissue in the body to be stored for later use or immediately oxidized to generate energy. When glucose is orally ingested, it is provided to the liver, muscle tissue, brain tissue or intestine (splanchnic bed), and adipose tissue, and allulose can particularly effectively lower blood sugar in the body by activating the storage of the muscle tissue.

Hereinafter, the present invention will be described in more detail.

One embodiment of the present invention relates to a composition for preventing, improving or treating ER stress-related diseases, including allulose. In the present invention, ER stress-related diseases include increased insulin resistance, increased blood sugar, 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 can

More particularly, the present invention relates to a composition for ameliorating, preventing, or treating blood sugar control, insulin sensitivity control, or diabetes mellitus through reduction of endoplasmic reticulum stress, comprising allulose as an active ingredient.

As used herein, the term "ER stress" refers to an immature protein exceeding the ability of the ER to process by a physiological or pathological environment is introduced into the ER, or calcium in the ER is depleted to cause a disorder in ER function. say

When ER stress occurs, cells have a defense mechanism for survival, which is called "ER stress response". The ER stress response involves 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). mediated by This ER stress response is particularly well observed in places such as plasma cells, pancreatic beta cells, hepatocytes, and osteoblasts, where the function of synthesizing and secreting proteins is active. , has been shown to act as a pathogenesis of various diseases such as hyperhomocysteinemia.

Another embodiment of the present invention is to use allulose as an active ingredient. To a composition for reducing the expression of one or more proteins selected from the group consisting of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, IRE1α and sulfonation (SO3H).

Specifically, as a result of analyzing the expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP and IRE1α sulfonation (SO ₃ H) in gastrocnemius samples from experimental animals, the diabetic control group (DC) compared to the normal control group (NC). ), 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). ), it was confirmed that the increased expression level of the gene decreased ( FIG. 8 ). Accordingly, allulose appears to improve insulin resistance by reducing ER stress, and can protect cells by inhibiting apoptosis (apoptosis) induced by ER stress.

In the present invention, allulose increases the expression of p-AMPK and SIRT1 proteins. Specifically, increasing the expression of p-AMPK and SIRT1 proteins or activating SIRT1 results in the deacetylation of PGC-1α (ie, the deacetylation of acetylated-PGC-1α). expression is decreased), increases the activity of GLUT4 present in the muscle, and moves glucose from the outside to the inside of the cell, contributing to lowering blood sugar, reducing insulin resistance, or improving or treating diabetes. Insulin-responsive glucose transporter 4 (GLUT4) is an insulin-dependent glucose transporter, mainly distributed in skeletal muscle and adipose tissue, and plays a role in moving glucose from the outside of the cell to the inside of the cell.

Specifically, it was confirmed that the expression levels of p-AMPK and SIRT1 proteins were increased in the group that consumed allulose powder and liquid form in a concentration-dependent manner compared to the diabetic control group (DC). Specifically, as a result of analyzing the expression levels of p-AMPK and SIRT1 proteins expressed in gastrocnemius of experimental animals, the expression levels of p-AMPK and SIRT1 were significantly reduced in diabetic control group (DC) compared to normal control group (NC). However, it was confirmed that the expression levels of p-AMPK and SIRT1 proteins were increased in the concentration-dependently intake group of allulose powder and liquid form compared to the diabetic control group (DC) (FIG. 9A).

As a result of confirming the expression of acetylated-PGC-1α protein in the present invention, acetylated-PGC-1α increased in the diabetic control group (DC) compared to the normal control group (NC), but in the group that consumed allulose powder and liquid in a concentration-dependent manner, The expression of acetylated-PGC-1α was reduced (FIGS. 9B and C). Therefore, it is expected that allulose can help reduce insulin resistance by activating AMPK-SIRT1-PGC-1α to enhance energy metabolism.

As used herein, the term "ER stress-related disease" refers to a disease that occurs or aggravates due to ER stress, for example, due to excessive accumulation of protein in the cell, the degradation system becomes difficult to operate anymore, and it is itself toxic to the cell. These may include diseases (such as neurodegenerative diseases), or diseases (such as diabetes) caused or caused by an excessive ER stress response caused by a faulty 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 ER stress-related diseases. Accordingly, the ER stress-related disease in the present specification may be increased insulin resistance, increased blood sugar, diabetes, Alzheimer's disease, Parkinson's disease, glutamine multimer-induced aggregation disease, Huntington's disease, ischemic disease, cardiovascular disease, hyperhomocysteinemia, arteriosclerosis or cancer have. 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, it can be seen that PERK and its underlying substrate, eIF2a pathway, are usually activated in the patient's tissue sample. It has been reported, and in the case of Huntington's disease, when one part of the gene is over-amplified by mutation, the glutamine polymers agglomerate with each other and form a protein aggregation that is difficult to handle with the decomposition capacity of the cell. It is known that endoplasmic reticulum stress occurs naturally, and neuronal cell death occurs.

One embodiment of the present invention provides a use for improving or treating insulin resistance, increased blood sugar, or diabetes by allulose, and the disease belongs to endoplasmic reticulum stress and / or oxidative stress-related diseases, for example, in muscle tissue. related to endoplasmic reticulum stress and/or oxidative stress.

Specifically, in relation to oxidative stress, the reduction of NOX4 expression by the administration of allulose suppresses the generation of ROS in muscle cells, thereby regulating glucose metabolism, thereby reducing insulin resistance. In relation to the endoplasmic reticulum stress, allulose increases AMPK expression or activation, and in muscle tissue and heart muscle, AMPK promotes muscle contraction to promote glucose absorption, which activates GLUT1 or Inducing the translocation of GLUT4 to the plasma membrane increases the transport of glucose into cells. In addition, it is known that allulose induces activation of SIRT1, which leads to deacetylation of PGC-1α, thereby increasing the activity of glucose transport 4 (GLUT4) in muscle. Insulin-responsive glucose transporter 4 (GLUT4) is an insulin-dependent glucose transporter, mainly distributed in skeletal muscle and adipose tissue, and plays a role in moving glucose from the outside of the cell to the inside of the cell.

Insulin secretion disorder observed in type 2 diabetes causes ER stress. In this study, it was confirmed whether allulose regulates ER stress induced in type 2 diabetes. Since insulin resistance is the most important cause of diabetes mellitus, the reduction of insulin resistance is effective in the treatment of diabetes, and is particularly important in type 2 diabetes. Pancreatic beta cells have a well-developed endoplasmic reticulum, and smooth function of the ER plays an important role in the function of beta cells, and dysfunction of beta cells due to ER stress contributes to diabetes induction.

Allulose increases phosphorylated AMPK (AMP-activated protein kinase) and activates AMPK signal to promote cellular absorption of glucose in the blood, thereby lowering blood sugar, exhibiting anti-obesity activity, and/or increasing AMPK activity. It can lower blood lipid levels. AMPK is known to be involved in glucose metabolism, fat metabolism, mitochondrial production and energy metabolism involved in blood sugar control by inhibiting the release of glucose from the liver regardless of insulin. AMPK is known to increase the gene expression of PGC-1α, which is known to play an important role in mitochondrial biogenesis.

AMPK is an important factor in the regulation of energy balance at most cellular and systemic levels, and is known as a serine/threonine kinase as a regulator of lipid and glucose metabolism. is being noticed in AMPK inhibits the anabolic action that consumes ATP and promotes the catabolic action (glucose uptake, glycogenolysis, glycogenolysis, fatty acid oxidation) that produces ATP. and increased glucose absorption. The degree 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 glucose uptake by promoting muscle contraction, which activates GLUT1 regardless of insulin action or induces GLUT4 transport to the plasma membrane to increase glucose delivery into cells make it

Sirtuin1 (SIRT1) is a factor required for AMPK activation and is known to be involved in PGC-1α metabolism. That is, AMPK activation increases the intracellular NAD(+) content, and SIRT1, an NAD(+)-dependent deacetylation enzyme, is activated to deacetylation and activation of PGC-1α. Activation of SIRT1 has been reported to increase the activity of glucose transport 4 (GLUT4) in muscle by deacetylation of PGC-1α. Insulin-responsive glucose transporter 4 (GLUT4) is an insulin-dependent glucose transporter, mainly distributed in skeletal muscle and adipose tissue, and plays a role in moving glucose from the outside of the cell to the inside of the cell. SIRT1 protein expression in cells or animal muscle can be measured by western blotting or ELISA.

Peroxisomal proliferator-activated receptor gamma coactivator-1α (PGC-1α) is responsible for glucose metabolism, mitochondrial biogenesis, muscle fiber specialization, and adaptive thermogenesis ( It is known to play an important role in adaptive thermogenesis. It is known that an increase in the expression level of PGC-1α increases the mitochondrial DNA copy number and promotes mitochondrial proliferation. Substances that promote the expression of PGC-1α promote the generation of mitochondria, which in turn promotes fatty acid oxidation by mitochondria and generates ATP energy, which can promote energy consumption in the body. Mitochondria play an important role in energy metabolism by activating glucose transport and fat oxidation.

Another embodiment of the present invention is a composition for preventing, improving, or treating oxidative stress-related diseases comprising allulose as an active ingredient.

In the present specification, a disease caused by oxidative stress or a disease related to oxidative stress is cancer, osteomyelitis, AIDS, cardiovascular disease, colorectal cancer, bladder cancer, coronary artery disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, chronic kidney disease, alcohol sexual liver disease, obstructive pulmonary disease, insulin resistance syndrome or diabetes, preferably coronary artery disease or diabetes.

Damage to cells or tissues from reactive oxygen species (ROS) is known to be related to not only diabetes, but also inflammation and aging. To confirm whether allulose has antioxidant activity, DHE tissue staining was performed on superoxide anion, NADPH Oxidase activity, and lipid peroxidation generated in the cell to measure the reactive oxygen species scavenging activity of allulose. Specifically, it has the effect of reducing the superoxide anion generated in the cell (FIG. 3). Peroxide containing hydrogen peroxide (H ₂ O ₂ ) is one of the major reactive oxygen species (ROS) that cause oxidative stress.

This experiment was performed to analyze the inhibitory effect of NADPH Oxidase activity and lipid peroxidation generated by a diabetic animal model (DC) by allulose powder and liquid form. That is, it was analyzed whether allulose had an activity to protect cells from oxidative stress caused by hydrogen peroxide (H ₂ O ₂ ). Superoxide anion, NADPH Oxidase activity, and lipid peroxidation produced by a diabetic animal model (DC) were concentration-dependently inhibited by allulose powder and liquid forms ( FIGS. 4 and 5 ). It was found that allulose powder and liquid form had a cytoprotective effect from oxidative stress caused by hydrogen peroxide (H ₂ O ₂ ).

It was confirmed that protein oxidation was significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, it was confirmed that the concentration-dependent intake of allulose powder and liquid form decreased protein oxidation compared to the diabetic control group (DC) (FIG. 6).

Specifically, as a result of analyzing the effect of D-allulose on the expression level of NOX4 protein, it was confirmed that the protein expression level of NOX4 was significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, it was confirmed that the NOX4 protein expression level was decreased in the group ingesting allulose powder and liquid form in a concentration-dependent manner compared to the diabetic control group (DC) (FIG. 7). Reduction of NOX4 expression by administration of allulose appears to reduce insulin resistance by inhibiting the generation of ROS in muscle cells, thereby regulating glucose metabolism.

Reduction of NOX4 expression by administration of allulose appears to reduce insulin resistance by inhibiting the generation of ROS in muscle cells, thereby regulating glucose metabolism. Excessive glucose metabolism in cells increases the expression of Nox4 in cells or tissues, and this increased expression of Nox4 causes excessive production of reactive oxygen species in cells and tissues. It is known that the reactive oxygen species produced at this time play an important role in the survival and death of cells and maintenance of the redox system.

The allulose applied to the present invention is not particularly limited, and includes liquid allulose including allulose, powder allulose, crystalline allulose and amorphous allulose. The allulo may be chemically synthesized or biologically prepared.

In the present specification, the amount of allulose administered to the subject is determined in an amount of 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, per 60 kg of body weight of the ingested subject per day. 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. Accordingly, the composition comprising allulose according to the present invention has a daily intake of 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 per 60 kg of body weight of the ingested individual. , 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 of allulose. It may be a composition comprising.

The subject or individual who administers or consumes the composition containing allulose as an active ingredient according to the present invention is an animal, including a human, and includes, for example, a human, a mouse, a rat, a monkey, and the like.

The composition containing allulose as an active ingredient according to the present invention can be ingested before, after, or simultaneously with a meal, and the intake conditions are not particularly limited.

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 (weeks), preferably 8 to 12 weeks (weeks). can be An effective effect can be derived only by ingesting the composition according to the present invention once, but it can be ingested twice or more.

The formulation unit for ingestion of the composition containing allulose as an active ingredient, for example, contains 1, 2, 3 or 4 times or 1/2, 1/3 or 1/4 times the individual intake amount. can be manufactured. The individual intake preferably contains an amount administered once a day of the active ingredient, which may be formulated in an amount corresponding to all, 1/2, 1/3 or 1/4 times the daily dose. have.

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

A suitable dosage of the composition according to the present invention is a formulation method, an administration method, the patient's age, weight, sex, pathological condition, food, administration time, administration route,. It can be prescribed in various ways depending on factors such as excretion rate and response sensitivity.

The composition containing allulose as an active ingredient according to the present invention 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" means a food manufactured (including processing) using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act. It refers to obtaining useful effects for health purposes such as maintaining and improving health by maintaining the normal function of the body or activating physiological functions. The allulose may be added to general foods, or may be prepared as encapsulation, powdering, suspension, or the like. When consumed, it has specific health effects, and unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time because it is made from food.

When the allulose of the present invention is used as a food additive, the allulose can be added as it is, used with other foods or food ingredients, or used appropriately according to other conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (prophylactic, health or therapeutic treatment).

The food is not particularly limited as long as it is a food to which allulose is applicable, for example, processed grain products, processed beans. Processed documents, processed sugar products, processed seafood, other processed products, confectionery, candy (eg, hard candy, jelly, gummi), bread, dumplings, processed meat products (eg, meat, sausage), dairy products (eg, fermented milk with lactic acid bacteria, ice cream), egg products, jelly, processed oils and fats, other noodles, fried noodles, solid tea, liquid tea, coffee, fruit and vegetable juice, fruit and vegetable beverages, other fermented beverages, ginseng and ginseng beverages, mixed beverages, beverage base, seasoned soybean paste, red pepper paste ， Cheonggukjang, sauces, complex seasonings, cabbage kimchi, seasoned salted fish, pickles, pickles, stewed agricultural products, stewed livestock products, seasoned dried fish and shellfish, dried fish and shellfish, peanut or nut processed products, processed fruits and vegetables, seasoned laver. Extracted foods, ready-to-eat foods, steamed rice, mushroom fruit body processed foods and beverages. It may be processed meat products, chocolate, confectionery, pizza, noodles (ramen, noodles, etc.), gum, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

Another embodiment of the present invention provides a composition for promoting body fat burning including allulose as an active ingredient in the form of tablets, powders, capsules, granules, syrups, jellies, bars, pastes, gels, beverages, and teas.

The food according to the present invention may contain normal food additives, and unless otherwise specified, whether it is suitable as a food additive is related to the item according to the general rules and general test methods of the Food Additives Code approved by the Food and Drug Administration. It is judged according to the standards and standards.

In one example, the weight (wt)% of allulose in the composition excluding the filler can be set variously, for example, about 0.1 to 99.9 weight% or 5 to 95 weight% of allulose based on the solid content of the composition. Or, the allulose is 80 (w / w) % or more, preferably 90 (w / w) % or more, more preferably 95 (w / w) % or more, 96 (w/w) % or more, based on total solids It may be a composition having a content of w/w)% or more, 97 (w/w)% or more, 98 (w/w)% or more, or 99 (w/w)% or more.

The allulose applied to the present invention is not particularly limited, and includes liquid allulose including allulose (allulose syrup, powdered allulose, crystalline allulose and amorphous allulose. The allulose may be chemically synthesized or It may be biologically manufactured.

It is in the form of a powder or syrup containing allolose contained in the composition according to the present invention, or, for example, allolose has a purity of 80 (w/w) % or more, preferably 90 (w/w) % or more, more preferably is 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 allolose powder, It may be a solution prepared at various concentrations using this, or a solution converted to allulose by a chemical or biological method as a fructose-containing solution.

The allulose syrup may be obtained through separation, retention and concentration processes from the allulose alone or mixed sugar. In an embodiment of the present invention, the allulose syrup that has undergone the separation and purification process may have an electrical conductivity of 1 to 50 μS/cm and may be a liquid allulose syrup having a colorless or pale yellow sweetness. Specific examples of the mixed sugar containing allulose include 5 to 95 parts by weight of allulose, 1 to 50 parts by weight of fructose and 1 to 55 parts by weight of glucose, and 1 to 10 parts by weight of oligosaccharide based on 100 parts by weight of the total solid content of the mixed sugar. and may not contain oligosaccharides. The allulose, fructose and glucose are preferably all form D-isomers.

According to an example of the present invention, without changing the diet or exercise life, only ingestion of allulose effectively reduces ER stress and has the effect of helping to control blood sugar.

1 is a graph showing the values of oral glucose tolerance test and insulin resistance test to confirm the effect of D-allulose on blood sugar reduction in db/db mice according to an example of the present invention.
2 is a graph showing the values after measuring the concentration of insulin in the serum to determine the effect of D-allulose on the concentration of insulin in hullcheong in db/db mice according to an example of the present invention.
3 is a confocal micrograph (FIG. 3A) of a tissue stained with DHE that was performed to confirm the effect of D-allulose on oxidative stress in db/db mice according to an example of the present invention, DHE fluorescence It is a graph (FIG. 3B) showing the intensity numerical result.
4 is a graph showing the measured NADPH oxidase activity concentration in the tissue to confirm the effect of D-allulose on NADPH oxidase activity in db/db mice according to an example of the present invention.
5 is a graph showing the measured values of the lipid peroxide content in order to confirm the effect of D-allulose on lipid peroxide in db/db mice according to an example of the present invention.
6 is a view showing the results of measuring the degree of oxidation of proteins in tissues in order to check the effect of D-allulose on the oxidation of proteins in db/db mice according to an example of the present invention.
7 shows the effect of D-allulose on the expression level of NOX4 protein in db/db mice according to an example of the present invention.
8 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.
9 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.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Diabetic animal model

(1) Normal diet-administered animal group (NCD)

For the experimental animals, a 5-week-old normal male group (C57BL/KsJ- db/+ mice, Normal Control, NC) was purchased from Orient Bio, acclimatized for 14 days, and then divided into 10 groups and tested for efficacy for 8 weeks. bred for consumption. The experimental diet for the efficacy evaluation test was a powdered feed for laboratory animals (Orient Bio, Inc.), and the powdered feed was bred so that it could be freely ingested with water.

(2) diabetic animal model (DC)

In db/db mice (C57BL/KsJ- db/db mice, Diabetic Control, DC), obesity and type 2 diabetes occur because leptin receptor mutation occurs and leptin, a hormone secreted by adipocytes, does not signal transduction. They were purchased from Orient Bio as a model and adapted for 14 days, then divided into 10 groups and bred to consume the efficacy evaluation product for 8 weeks. For the experimental diet for the efficacy evaluation test, powdered feed was supplied as a powder feed for laboratory animals (Orient Bio, Inc.), and water and feed were bred to be freely ingested.

Example 2: Liquid allulose administration group (AL)

In the diabetes model according to Example 1, a dose of 5.16 g/kg (high) of allulose liquid type was set based on the solid content. The normal diet was supplied so that it could be freely consumed along with water, and the liquid allulose form was orally administered twice a day (9:00 am, 5:00 pm). The normal diet was prepared based on the AIN-76A diet (Teklad.USA), and the weight ratio of carbohydrate:protein:lipid was adjusted to 60:20:15.

Example 3: Powder allulose administration group (AP)

In the diabetes model according to Example 1, a dose of 5.16 g/kg (high) of allulose powder was set based on the solid content. The normal diet was supplied so that it could be freely ingested along with water, and allulose powder was orally administered twice a day (9:00 am, 5:00 pm). The normal diet was prepared based on the AIN-76A diet (Teklad.USA), and the weight ratio of carbohydrate:protein:lipid was adjusted to 60:20:15.

Comparative Examples 1 and 2

In the diabetic animal model according to Example 1, 5.16 g/kg sucralose administered group as Comparative Example 1 and 5.16 g/kg erythritol as Comparative Example 2 instead of allulose used in Example 2 A comparative experiment was conducted by setting the administration group.

If the daily intake of allulose 5.16 g/kg is converted into a human, it is 25 g per 60 kg body weight of the ingested individual. At this time, sucrolose was administered orally according to the same sweetness level, and erythritol was administered in the same amount as allulose. Weight change was observed by administering a normal diet and sucralose and erythritol at the same time.

Test Example 1: Blood glucose and insulin analysis

(1) Oral Glucose Tolerance Test (OGTT)

The normal group and the diabetic animal model according to Example 1 were fasted for 12 hours, glucose was orally administered to mice at a dose of 2 g/kg, and blood was administered to the mouse tail at intervals of 0, 15, 30, 60, 90, and 120 minutes. Blood glucose concentration was measured by collecting from a vein. It was calculated by the area of the blood glucose curve according to the glucose tolerance test.

(2) Insulin resistance test (ITT analysis)

The normal group and the diabetic animal model according to Example 1 were fasted for 12 hours, and 1 unit of insulin solution per kg of body weight was injected intraperitoneally into the mouse, and then after 0, 15, 30, 60, 90, 120 minutes, blood was drawn from the tail vein of the mouse. was collected and blood sugar was measured.

(3) Measurement of serum insulin concentration

After administration of allulose, sucralose, or erythritol for 8 weeks according to Examples 1 to 3 and Comparative Examples 1 to 2, blood was collected from mice in each experimental group on the end of the experiment, and serum was obtained by centrifugation, The serum insulin concentration was measured using the Mouse Insulin ELISA Kit.

Specifically, 10 μL of animal serum was added to an anti-insulin coated 96 well plate, left at 20-25° C. for 2 hours, and washed 4 times with Washing Buffer. Add 100 μL of HRP-conjugated streptavidin to the wash, incubate at 20-25°C for 30 minutes, wash 4 times, add 100 μL of Substrate chromogen reagent, and incubate at 20-25°C for 20 minutes, 50 μL of a reaction stopper The absorbance was measured at 450 nm (reference wavelength, 620 nm) using a Multi Microplate Reader (infinite M200PRO, Tecan, Mannedorf, Switzerland).

Test Example 2: Evaluation of antioxidant activity of allulose

Damage to cells or tissues by reactive oxygen species (ROS) is known to be related to diabetes as well as inflammation and aging. To confirm whether allulose has antioxidant activity, DHE tissue staining was performed on superoxide anion, NADPH Oxidase activity, and lipid peroxidation generated in the cell to measure the reactive oxygen species scavenging activity of allulose.

(1) Inhibitory effect of superoxide formation by oxidative damage

In order to observe superoxide formation due to oxidative damage, dihydroethidium (DHE), an oxidative fluorescent dye, was stained. DHE staining was performed in a dark wet box in 1 μM DHE (in PBS; pH 7.4) solution for 30 minutes, dehydrated and sutured to prepare tissue specimens. , Carl Zeiss, Germany) was used for observation. The experimental results are shown as a confocal micrograph of the fluorescence-stained tissue in FIG. 3A, and the numerical results of DHE fluorescence intensity are shown as a graph in FIG. 3B. The graph shown in B of FIG. 3 is a graph showing the DHE fluorescence intensity values obtained as a result of DHE fluorescence staining performed to confirm the effect of D-allulose on oxidative stress in db/db mice.

(2) Protective effect on cell damage caused by oxidative stress

This experiment was performed to analyze the inhibitory effect of allulose powder and liquid form on NADPH Oxidase activity and lipid peroxidation generated by a diabetic animal model (DC). That is, it was analyzed whether allulose had an activity to protect cells from oxidative stress caused by hydrogen peroxide (H ₂ O ₂ ).

Absorbance was measured at 450 nm wavelength using NADPH Oxidase activity kit (Biovision). For lipid peroxidation, 1 mL of the sample is mixed 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, heated at 95°C for 1 hour, cooled and 2.5 mL. Add mL of n-butanol/pyridine (15:1, v/v) and 0.5 mL of distilled water and shake. This was centrifuged at 3,000 × g for 10 minutes to obtain a supernatant, and absorbance at 532 nm was measured. The measurement results are shown in FIGS. 4 and 5 .

4 is a graph showing the measured NADPH oxidase activity concentration in the tissue in order to confirm the effect of D-allulose on NADPH oxidase activity in db/db mice. 5 is a graph showing the measured values of the lipid peroxide content in order to confirm the effect of D-allulose on lipid peroxide in db/db mice.

4 and 5, superoxide anion, NADPH Oxidase activity, and lipid peroxidation produced by a diabetic animal model (DC) were concentration-dependently inhibited by allulose powder and liquid form (Fig. 4 and 5). It was found that allulose powder and liquid form had a cytoprotective effect from oxidative stress caused by hydrogen peroxide (H ₂ O ₂ ).

(3) reducing activity of protein oxidation

Carbonyl content using DNPH reagent is often used to measure the degree of oxidation of proteins together with thiol group content. Specifically, the carbonyl content analysis using the DNPH reagent was measured using the OxyBlot Protein Oxidation Detection Kit. The carbonyl content analysis result using the DNPH reagent is shown in FIG. 6 . 6 is a view showing the results of measuring the degree of oxidation of proteins in tissues in order to check the effect of D-allulose on the oxidation of proteins in db/db mice.

As shown in FIG. 6 , it was confirmed that protein oxidation was significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, it was confirmed that the concentration-dependent intake of allulose powder and liquid form decreased protein oxidation compared to the diabetic control group (DC) (FIG. 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 causes excessive production of reactive oxygen species in cells and tissues. It is known that the reactive oxygen species produced at this time play an important role in the survival and death of cells and maintenance of the redox system.

Specifically, protein expression level analysis was performed by Western blotting. According to Examples 1 to 3 and Comparative Examples 1 to 2, allulose, sucralose and erythritol were administered to the normal group and the diabetes-inducing group for 8 weeks, and then the gastrocnemius muscle was treated with a lysis buffer (10 mM Tris- Protein obtained by dissolving at 4℃ using HCl, pH 7.4, 0.1 M EDTA, 10 mM NaCl, 0.5% Triton X-100, pro-tease inhibitor Cocktail) and centrifugation (14,000 rpm, 10 min, 4℃) was measured. The quantified protein was mixed with sample buffer, heated at 95° C. for 5 minutes, and separated by 10-12% SDS-PAGE. After separation, SDS-PAGE was performed using a semi-dry transfer system to transfer the protein to the PVDF membrane at 15 V, 60 minutes, and reacted with blocking buffer (5% Skim milk in 1 X TBS-T) for 1 hour or more, and NOX4 After overnight reaction at 4°C, it was washed 5 times with 1 X TBS-T at intervals of 7 minutes. After the secondary antibody was reacted at room temperature for at least 1 hour, it was washed 5 times at 7 minute intervals with 1 X TBS-T, and the color was developed with ECL reagent, and then the X-ray film was exposed to glare. The results are shown in FIG. 7, and it shows the effect of D-allulose on the expression level of NOX4 protein in db/db mice.

As shown in FIG. 7 , it was confirmed that the protein expression level of NOX4 significantly increased in the diabetic control group (DC) compared to the normal control group (NC). However, it was confirmed that the NOX4 protein expression level was decreased in the group taking allulose powder and liquid form in a concentration-dependent manner compared to the diabetic control group (DC). Reduction of NOX4 expression by administration of allulose appears to reduce insulin resistance by suppressing the generation of ROS in muscle cells, thereby regulating glucose metabolism.

Test Example 4: Evaluation of the effect of allulose on endoplasmic reticulum stress

Insulin secretion disturbance observed in type 2 diabetes causes ER stress. In the present invention, it was attempted to determine whether allulose regulates ER stress induced in type 2 diabetes.

Specifically, according to Examples 1 to 3 and Comparative Examples 1 to 2, allulose, sucralose and erythritol were administered to the normal group and the diabetes-inducing group for 8 weeks, and then substantially as in Test Example 3 Expression levels of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, and IRE1α sulfonation (SO ₃ H) were analyzed in gastrocnemius muscle samples from experimental animals by the same Western blotting method. The analyzed results are shown in FIG. 8 .

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 level of the gene increased in the diabetic control group (DC) was decreased in the concentration-dependently ingested allulose powder and liquid form.

As shown in the experimental results of FIG. 8 , it appears that allulose reduces ER stress and thus reduces insulin resistance.

Test Example 5: Evaluation of allulose effect on AMPK-SIRT1-PGC-1α

According to Examples 1 to 3 and Comparative Examples 1 to 2, allulose, sucralose and erythritol were administered in a normal control group (NC) and a diabetic control group (DC) for 8 weeks, and then Test Example 3 and The expression levels of p-AMPK and SIRT1 proteins expressed in gastrocnemius muscle samples were analyzed using substantially the same Western blotting method. The analyzed results are shown in FIG. 9 .

It was confirmed that the protein expression levels of p-AMPK and SIRT1 were significantly decreased in the diabetic control group (DC) compared to the normal control group (NC). However, it was confirmed that the expression levels of p-AMPK and SIRT1 proteins were increased in the group that consumed allulose powder and liquid form in a concentration-dependent manner compared to the diabetic control group (DC) (FIG. 9A).

Activation of SIRT1 was reported to increase the activity of glucose transport 4 (GLUT4) in muscle by deacetylation of PGC-1α. Insulin-responsive glucose transporter 4 (GLUT4) is an insulin-dependent glucose transporter, mainly distributed in skeletal muscle and adipose tissue, and plays a role in moving glucose from the outside of the cell to the inside of the cell.

As a result of confirming the expression of acetylated-PGC-1α protein in this experiment, acetylated-PGC-1α increased in the diabetic control group (DC) compared to the normal control group (NC), but in the group that consumed allulose powder and liquid in a concentration-dependent manner, The expression of acetylated-PGC-1α was reduced (FIGS. 9B and C). Therefore, it is expected that allulose can help reduce insulin resistance by activating AMPK-SIRT1-PGC-1α to enhance energy metabolism.

Claims (14)

A composition for improving, preventing or treating endoplasmic reticulum stress-related diseases, comprising allulose as an active ingredient. The composition of claim 1, wherein the composition reduces the expression of one or more proteins selected from the group consisting of p-IRE1α, p-eIF2α, ATF4, GRP78, CHOP, IRE1-alpha, and sulfonation (SO ₃ H). . The composition of claim 1, wherein the composition increases the expression of one or more proteins selected from the group consisting of p-AMPK and SIRT1. According to claim 1, wherein the endoplasmic reticulum stress-related disease is, insulin resistance increase, blood sugar increase, diabetes, Alzheimer's disease, Parkinson's disease, glutamine multimer-induced aggregation disease, Huntington's disease, Alzheimer's disease, ischemic disease, cardiovascular disease, hyperhomocysteinemia or A composition that is arteriosclerosis. The composition of claim 1, wherein the composition alleviates or reduces ER stress in a tissue. The composition of claim 1 , wherein the composition reduces insulin resistance. The composition of claim 1, wherein the composition reduces postprandial blood sugar. The composition of claim 1, wherein the composition reduces blood sugar through reduction of ER stress in muscle tissue. The composition according to claim 1, wherein the daily intake of allulose is 10 to 80 g per 60 kg body weight of the ingested subject. The composition of claim 1, wherein the allulose is in liquid or powder form. The composition of claim 1, wherein the allulose is included in an amount of 0.1 to 99.9 wt% based on 100 wt% of the solid content of the composition. The composition of claim 1, wherein the composition is administered for 4 to 20 weeks. The composition according to claim 1, which is a food composition or a pharmaceutical composition. The composition of claim 1, wherein the composition comprises tablets, powders, capsules, granules, syrups, jellies, bars, pastes, gels, beverages, and teas.

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