KR101167678B1 - Composition for preventing and treating of diabetes containing glyceollin or glyceollin enhanced fermented soybean as an effective ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing and treating diabetes containing glycerol or fermented soybeans enhanced with glycerin as an active ingredient, wherein the glycerol according to the present invention has a hypoglycemic effect and increased neutral fat accumulation through increased glucose uptake in cells. Adipose cell differentiation inhibitory effect was shown, and insulin secretion increase effect, beta cell proliferation effect and GLP-1 secretion effect were shown. In addition, glycerol-enriched fermented soybeans have a maintenance effect on blood glucose homeostasis in vivo, increase insulin sensitivity, increase the content of glycogen in the liver, and lower the content of triglycerides. Compositions containing fermented soybeans enhanced with olin or glycerol as an active ingredient can be usefully used as a composition for preventing and treating diabetes.

Description

Composition for preventing and treating of diabetes containing glyceollin or glyceollin enhanced fermented soybean as an effective ingredient}

The present invention relates to a composition for preventing and treating diabetes comprising glycerol (glyceollin) or glycerol (glyceollin) enhanced fermented soybean as an active ingredient.

Diabetes mellitus is one of the leading causes of death worldwide (Lee SI et al. al ., J Med Food 11 (3): 518-24, 2008). The number of diabetics is increasing rapidly due to the rapid increase in elderly population, high calorie diet, obesity and inactive lifestyles (Rang, HP et. al ., "Pharmacology", Longman Groupt Ltd., UK, pp 504-508, 1991). In particular, the number of diabetic patients in Korea has increased rapidly over the past decade, making it the fourth largest cause of death in Korea (Shin, KO). meat al . , Food Sci Biotechnol 17 (2): 287-294, 2008). Diabetes is a severe metabolic disorder with vascular complications, with significant mortality (Vats, V. et. al ., J Ethnopharmacol 90 (1): 155-60, 2004). In diabetics, metabolism of carbohydrates, proteins and lipids is abnormal due to hormone imbalances (Gonuth SM, Ann Intern Med 79: 812-822, 1973).

Diabetes is classified into insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM), which are largely insulin dependent, depending on the cause of the disease and the method of treating the symptoms. Type 1 diabetes is a state in which the function of the pancreatic beta cells is reduced due to genetic causes, viral infections, etc., almost no secretion of insulin, and suddenly occurs in the 10s to 20s. Type 2 diabetes is caused by family history, obesity, stress, eating errors, or drugs that antagonize insulin, etc., and appears well after the age of 40, also called adult diabetes. In fact, the majority of all diabetics are Type 2 diabetics. Type 2 diabetes is associated with a decrease in insulin activity in peripheral tissues such as liver, muscle, and fat cells, resulting in decreased glucose utilization, and in particular, increased production of glucose in the liver, resulting in elevated blood glucose levels in the beta cells of the pancreas. It is a disease that occurs when there is not enough insulin secretion to control it. In other words, type 2 diabetes occurs because insulin secretion does not follow insulin resistance with reduced insulin action.

In type 2 diabetes, insulin is secreted, but in the peripheral tissues such as liver, muscle, and fat cells where insulin acts, insulin activity decreases and glucose utilization decreases, thereby increasing blood sugar and responding to the pancreatic beta cells continuously. It is accompanied by hyperinsulinemia with insulin secretion [Kadowaki T, Hara K, Yamauchi T, Terauchi Y, Tobe K, Nagai R. 2003. Molecular mechanism of insulin resistance and obesity. Exp Biol Med ( Maywood ) 228: 1111-1117.

However, even in type 2 diabetes, insulin secretion may not be sufficiently made to increase blood glucose levels in the beta cells of the pancreas when blood sugar levels rise. Type 2 diabetes in Korea corresponds to this case, and in this case, both insulin action and insulin secretion are low, and the symptoms of diabetes are more severe and obesity than in the West [Kim J, Choi S, Kong B, Oh Y, Shinn S. 2001. Insulin secretion and sensitivity during oral glucose tolerance test in Korean lean elderly women. J Korean Med Sci 16: 592-597. Therefore, for the prevention and treatment of type 2 diabetes in Korea, it is known that it is important to improve insulin action and increase insulin secretion ability by stimulation of glucose.

The first drugs used to treat type 2 diabetes are drugs that promote insulin secretion. This is a drug of the sulfonylurea family, which acts directly on ATP-regulated potassium (KATP) channels in the pancreatic beta cell membranes, closing the channels, thereby depolarizing the cell membranes. and an insulin secretagogue (insulin secretagogues) which promote secretion of insulin sikimeuro the Ca ^{2 +} influx into the cell [Krentz AJ, Bailey CJ. 2005. Oral antidiabetic agents: current role in type 2 diabetes mellitus. Drugs 65: 385-411; Quesada I, Soria B. 2004. Intracellular location of KATP channels and sulphonylurea receptors in the pancreatic beta-cell: new targets for oral antidiabetic agents. Curr Med Chem . 11: 2707-2716. However, this drug has a problem in that it can induce hypoglycemic shock by secreting insulin even if the blood sugar is low by closing the KATP channel regardless of the blood sugar level.

Recently, a new treatment for type 2 diabetes is insulin-sensitive substances that improve insulin action and help normalize blood sugar with small amounts of insulin. Insulin-sensitive substances are known to relieve insulin resistance, a fundamental problem of type 2 diabetes, to normalize blood sugar with a small amount of insulin, and ultimately reduce the amount of insulin required for blood sugar to eliminate hyperinsulinemia [Ciaraldi TP]. , Kong AP, Chu NV, Kim DD, Baxi S, Lovicach M, Plodkowski R, Reitz R, Caulfield M, Mudaliar S, Henry RR. 2002.Regulation of glucose transport and insulin signaling by troglitazone or metformin in adipose tissue of type 2 diabetic subjects. Diabetes 51: 30-36]. In other words, insulin-sensitive substances could alleviate the symptoms of insulin resistance syndrome as well as diabetes, helping to facilitate the signaling system that occurs in the cells after insulin binds to the insulin receptors. To improve. The secretion of insulin from pancreatic beta cells is indirectly related to insulin / IGF-1 (Insulin like Growth Factor-1) signaling, and the degradation of beta cells is associated with decreased beta cell production and apoptosis. Increasingly, the amount of beta cells is reduced, and as a result, diabetes mellitus does not meet the need for insulin secretion.

In addition, GLP-1 (Glucabon-like peptide-1) is a peptide secreted by L-cells of small intestine cells in vivo, and promotes insulin secretion by glucose stimulation in beta cells, and promotes growth and suppression of beta cell growth. It is known to improve the function of beta cells by increasing the amount of beta cells through.

A recently developed glucose-stimulated insulin secretagogue is the glucagon like peptide-1 (GLP-1) receptor agonist, exendin-4 (exendin-4, exenatide). There is).

Exendin-4, unlike drugs of the sulfonylurea family, does not act directly on the KATP channel, but binds to the GLP-1 receptor present in the cell membrane and raises the cAMP concentration in the cell, thereby enhancing protein kinase A (PKA). ) Is activated. Did not increase the Ca ^{2 +} influx into the only active PKA cells, when blood sugar is increased is glucose is absorbed into the beta-cell As via glycolysis (glycolysis) due to high concentrations of ATP / ADP is PKA is activated, Ca ^{2 +} Increased influx to promote insulin secretion [Drucker DJ. 2001. Development of glucagon-like peptide-1-based pharmaceuticals as therapeutic agents for the treatment of diabetes. Curr Pharm Res 7: 1399-1412]. Therefore, exendin-4 does not promote insulin secretion when blood sugar is normal, but acts as a glucose-stimulating insulin secretagogue to promote insulin secretion only when blood glucose is elevated. It is also known to improve insulin secretion as well as to improve insulin action.

As another drug, Rosiglitazone, a major component of Avandis, recently developed as an insulin enhancer and approved by the FDA, has been shown to have a high affinity for PPARγ in vitro and has no effect on liver function. It is known to induce the expression of lipoprotein lipase in adipocytes. It is known to have an effect of increasing insulin sensitivity in monkeys and humans, and also to reduce blood sugar and triglyceride levels. In addition, there are drugs of the pioglitazone line that enhance insulin action, and there are metformin lines that reduce your biosynthesis in the liver.

In addition, in Korea and other Asian countries, various herbal medicines have been used for the treatment of diabetes mellitus and various diseases as folk remedies. However, the effects of many herbal medicines used for folk medicine have not been revealed.

In general, phytonalexin is a low molecular antimicrobial substance produced by a plant to combat fungus when various pests or fungi invade. These substances are not found in plants before infection, Synthesized very quickly in time and works at the site of infection and is toxic to various bacteria and fungi. Such phytoalexin is produced in-house as a function of protecting the plant, but recently, the antimicrobial activity of this substance has been revealed, and there are active studies to use it for stable and inexpensive medicines, dyes, flavors and fragrances by mass culturing it. The trend is taking place.

Glycerollin (glyceollin) is a type of phytoalexin produced in soybean, and has been reported to inhibit angiogenesis, and recently, anti-estrogen activity and anti-estrogen activity of glycerol have been found to be anti-cancer effects, It is expected that the development of cancer prevention and treatment agents such as breast cancer and ovarian cancer is expected. However, the anti-diabetic effect of glycerin homeostasis is not known at all.

Thus, the present inventors prepared fermented soybeans using glycerol-enhanced fungal stress, and extracted them with an organic solvent to isolate glycerol, and then glycerin lowered blood glucose lowering effect and increased neutral fat accumulation by increasing glucose uptake in cells. Adipocyte differentiation inhibitory effect, insulin secretion increase effect, beta cell proliferation effect and GLP-1 secretion effect was confirmed, and the administration of fermented soybeans fortified with glycerin in diabetic mouse model to blood glucose homeostasis in vivo It has been shown to have a maintenance effect, increase insulin sensitivity, increase the liver glycogen content, and lower the content of triglycerides, thereby making the glycerol or glycerol-enriched fermented soybean effective for preventing and treating diabetes. The present invention has been completed by revealing that it can be used as a component.

An object of the present invention is to provide a composition for preventing and treating diabetes, or a health food for preventing and improving diabetes, comprising glycerol or glycerol-enhanced fermented soybean as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing and treating diabetes containing glycerol as an active ingredient.

In addition, the present invention provides a health food for preventing and improving diabetes containing glycerol as an active ingredient.

In addition,

1) inoculating the soybean precultured strains; And

2) It provides a health food for preventing and improving diabetes containing fermented soybean with increased glycerol content as an active ingredient prepared by the manufacturing method comprising the step of fermenting the inoculated soybean.

Glycerin according to the present invention exhibits a hypoglycemic effect through increased glucose uptake and inhibition of adipocyte differentiation through inhibition of triglyceride accumulation, and increases insulin secretion, beta-cell proliferation and GLP-1 secretion. Indicates. In addition, glycerol-enriched fermented soybeans have a maintenance effect on blood glucose homeostasis in vivo, increase insulin sensitivity, increase the content of glycogen in the liver, and decrease the content of triglycerides. Therefore, the composition containing glycerol or glycerol-enhanced fermented soybean according to the present invention as an active ingredient can be usefully used as a composition for preventing and treating diabetes.

1 is a diagram showing a chromatogram of fermented soybeans and non-fermented soybean of the present invention.
Figure 2 is a diagram showing the structural formula of glycerol (glyceollin).
3 is a diagram confirming the glucose uptake effect by insulin stimulation in 3T3-L1 adipocytes.
Figure 4 shows the effect of glucose uptake by glycerol on insulin stimulation in 3T3-L1 adipocytes:
* Statistically significant difference at P <0.05 between control and 0.5 uM glycerol and 5 uM glycerol treatment groups;
different alphabets of a, b, c indicate a statistically significant difference at P <0.05 between the three groups; And
+++ statistically significant difference between the control and P <0.001.
Figure 5 shows the effect of triglycerides on triglyceride accumulation in the process of differentiation of 3T3-L1 fibroblasts into adipocytes:
* Statistically significant difference at P <0.05 between control and 0.5 uM glycerol and 5 uM glycerol treatment groups;
different alphabets a, b indicate that there was a statistically significant difference at P <0.05 between groups; And
+++ statistically significant difference between the control and P <0.001.
Figure 6 shows the effect of PPAR-r agonist on glycerol by comparing the relative luciferase activity by glycerol administration in 293 cells:
+++ statistically significant difference between the control and P <0.001.
Figure 7 shows the effect of insulin secretion of glycerol in pancreatic beta cells:
* Statistically significant difference at P <0.05 between control and 0.5 uM glycerol and 5 uM glycerol treatment groups;
different alphabets of a, b, c indicate a statistically significant difference at P <0.05 between the three groups;
++ statistically significant difference between control and P <0.01; And
+++ statistically significant difference between the control and P <0.001.
8 is a diagram confirming the beta cell proliferation effect of glycerol:
* Statistically significant difference at P <0.05 between control and 0.5 uM glycerol and 5 uM glycerol treatment groups;
different alphabets a, b indicate that there was a statistically significant difference at P <0.05 between groups; And
++ Statistically significant difference between control and P <0.01.
Figure 9 is a comparison of the relative mRNA expression amount confirms the effect on the expression level change of ER stress-related genes by glycerol:
* Statistically significant difference at P <0.05 between control and 0.5 uM glycerol and 5 uM glycerol treatment groups;
different alphabets a, b indicate that there was a statistically significant difference at P <0.05 between groups;
++ statistically significant difference between control and P <0.01; And
+++ statistically significant difference between the control and P <0.001.
10 is a diagram confirming the effect of GLP-1 secretion of glycerol in colorectal cancer cell line.
* Statistically significant difference at P <0.05 between control and 0.5 uM glycerol and 5 uM glycerol treatment groups; And
Different alphabets of a, b, c indicate that there was a statistically significant difference between the three groups at P <0.05.
11 is a diagram confirming the change in blood glucose concentration over time after dietary intake of diabetic mice, diabetic mice ingested fermented soybeans and non-fermented soybeans of the present invention, and normal mice, respectively.
* Statistically significant difference in P <0.05 between diabetic mouse group and diabetic mouse fermented soybean intake group; And
+ Statistically significant difference at P <0.001 between diabetic and normal mouse groups.
12 is a diagram showing the oral glucose tolerance test results of diabetic mice, diabetic mice ingested fermented soybeans and non-fermented soybeans, and normal mice, respectively, by calculating an area under the curve of glucose and insulin. .
* Statistically significant difference in P <0.05 between diabetic mouse group and diabetic mouse fermented soybean intake group;
different alphabets of a, b, c indicate a statistically significant difference at P <0.05 between the three groups; And
+ Statistically significant difference at P <0.001 between diabetic and normal mouse groups.
FIG. 13 is a diagram showing the results of insulin resistance experiments of diabetic mice, diabetic mice fed fermented soybeans and non-fermented soybeans, and normal mice, respectively, through relative changes in basal blood glucose levels over time after insulin administration.
* Statistically significant difference in P <0.05 between diabetic mouse group and diabetic mouse fermented soybean intake group; And
+ Statistically significant difference at P <0.001 between diabetic and normal mouse groups.
14 is a diagram confirming the glycogen and triglyceride content changes in the liver by glycerol in diabetic mice, diabetic mice and normal mice ingested fermented soybean and non-fermented soybean of the present invention, respectively.
* Statistically significant difference in P <0.05 between diabetic mouse group and diabetic mouse fermented soybean intake group;
different alphabets of a, b, c indicate a statistically significant difference at P <0.05 between the three groups; And
+ Statistically significant difference at P <0.001 between diabetic and normal mouse groups.

The present invention provides a composition for preventing and treating diabetes containing glycerol as an active ingredient.

The glycerol is preferably any one selected from the group consisting of [Formula 1], [Formula 2] or [Formula 3], but is not limited thereto.

The glycerol is preferably extracted from soybeans, but is not limited thereto. The extraction is preferably extracted with water, a lower alcohol of C ₁ to C ₄ or a mixture thereof, but is not limited thereto, and the lower alcohol is preferably ethanol, but is not limited thereto.

Preferably, the separation is performed by HPLC, but is not limited thereto, and the HPLC apparatus uses a Jasco 1580 system and a Gemini 5 uM C18 column. However, all common compound separation methods known in the art may be used.

The diabetes is preferably type 2 diabetes, but is not limited thereto.

In a specific embodiment of the present invention, the beans are surface sterilized with ethanol, washed with deionized water, immersed in water and divided, and the divided beans are placed in a petri dish covered with a sterilized filter paper soaked. Fungi {Aspergillus dust sojae), and Aspergillus duck claim (Aspergillus oryzae )} was inoculated on the divided side and sealed, and then fermented by storing in a dark chamber (chamber) at 25 ℃ for 3 days to prepare fermented soybeans fortified with glycerol.

When boiled soybeans were inoculated with fungal bacteria, it was confirmed that glycerol was not produced, thereby confirming that glycerol was a secondary metabolite derived from living soybean rather than the released metabolite of fungal bacteria.

In addition, the fermented soybeans were extracted with ethanol, followed by centrifugation to obtain a supernatant liquid to obtain a fermented soybean extract, followed by HPLC and LC / Mass to separate glycerol contained in the fermented soybean extract (see FIG. 1). Was identified (see FIG. 2).

In addition, in order to differentiate 3T3-L1 fibroblasts into adipocytes, 1Tug / ml of insulin, 0.25 uM dexamethasone and 0.5 mM isomethylbutylxanthine were added to 3T3-L1 fibroblasts. The cells were cultured in the medium, and 3T3-L1 adipocytes were obtained by inducing differentiation into adipocytes. Glucose uptake effect according to insulin concentration was confirmed in the obtained 3T3-L1 adipocytes, and as a result, it was confirmed that the glucose uptake is increased proportionally when insulin is treated in 3T3-L1 adipocytes (see Fig. 3). ).

In addition, to determine whether cells cultured with insulin and glycerol in 3T3-L1 adipocytes increased glucose uptake compared to cells cultured with insulin alone, cell lysates were obtained and the content of ¹⁴ C contained therein. Glucose uptake was confirmed by measuring the glucose uptake rate. As a result, glucose uptake was increased in a concentration-dependent manner when 3T3-L1 adipocytes were treated with 1 ng / mL of insulin and glycerol. Was most increased (see FIG. 4).

Therefore, glycerol increased the glucose uptake in adipocytes by insulin stimulation, confirming that there is a blood glucose enhancing effect.

In addition, in order to confirm the effect on the accumulation of triglycerides of glycerol, the amount of triglycerides was measured by adding glycerol in the process of differentiating fibroblasts into adipocytes, and as a result, the control group was not treated with glycerol. Compared to the case of glycerol treatment, the amount of triglyceride was significantly decreased as its concentration was increased (see FIG. 5). Therefore, it was confirmed that glycerol has an effect of inhibiting the accumulation of triglycerides and inhibiting the differentiation of fibroblasts into adipocytes.

In addition, PPAR (Peroxisome Proliferator Activated Receptor) -r, a receptor that increases the expression of genes involved in sugar metabolism, is a receptor that increases the expression of genes involved in the metabolism of sugars and lipids in the cell nucleus. Agonist for not only shows the effect of lowering blood sugar, but also known to lower the concentration of fatty acids in the blood, in order to confirm the mechanism of hypoglycemic action through the increased glucose absorption effect of glycerol according to the present invention, PPAR of glycerol -r agonist activity was measured.

As a result, rosiglitazone, which is known as a representative agent of PPAR-r, exhibited an effect as a PPAR-r agonist by increasing its relative luciferase activity at higher concentrations.However, when glycerol is treated, it is relatively lucifer. Lase activity was not seen (see FIG. 6). Therefore, it was confirmed that the hypoglycemic effect through the increase of glucose uptake of glycerol was not the action of the PPAR-r agonist.

In addition, insulin secretion measurement experiments were performed to determine the effect of glycerin on insulin secretion by glucose treatment in the Min6 cell line, a pancreatic beta cell responsible for insulin secretion, and as a result, high concentration glucose (20 mM) in Min6 cells was performed. When glycerol was treated in the KRB solution containing), it was lower than the positive control exendin-4, but the insulin secretion was significantly increased compared to the negative control in proportion to the concentration (see FIG. 7). Therefore, it was confirmed that glycerin has an effect of increasing insulin secretion by glucose in the beta cells of the pancreas.

In addition, in order to confirm the beta cell proliferation effect of glycerol, Min6 cells were treated with glycerol, and after treatment, the cell number was measured at 4 and 8 hours to confirm the cell proliferation effect. As a result, after treatment with glycerol, beta cell proliferation increased according to the concentration compared to the DMSO treated group, which was a negative control, in both 4 hours and 8 hours (see FIG. 8). Therefore, it was confirmed that glycerol has a proliferative effect of pancreatic beta cells.

In addition, in order to confirm whether the antidiabetic effect of glycerol is related to ER stress of beta cells, beta cells were treated with glycerol, and thus the expression rate of ER stress related genes was confirmed. As a result, the expression rate of XBP-1 / GAPDH, AFT4 / GAPDH, CHOP / GAPDH, AFT6 / GAPDH, which are genes involved in ER stress, decreased significantly in a beta-cell administered glycerol in a dose-dependent manner compared to the negative control ( 9).

Therefore, it was confirmed that glycerol has an effect of reducing ER stress of beta cells.

In addition, in order to confirm the secretion effect of GLP-1 of glycerol, colon cancer cell line NCI-H716 cells were cultured in a dish coated with Matrigel (Becton Dickinson, Bedford, MA, USA) for GLP-1 secretion experiments. After differentiation into endocrine cells, the cells were treated with glycerol and the cells were lysed. The peptides in the lysed cells were extracted by alcohol extraction, followed by radioimmunoassay to determine the amount of GLP-1 secretion in the medium and GLP-1 in the cells. The amount was quantified. As a result, when glycerol was treated with KRB solution containing high glucose (20 mM) in NCI-H716 cell line, GLP-1 secretion was significantly increased in proportion to the concentration compared to the negative control (see FIG. 10).

Therefore, glycerol was confirmed to have an effect of increasing the secretion of GLP-1 in colorectal cancer cell line.

The composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to glycerol.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose is 10 to 500 mg / kg, preferably 100 mg / kg, in the amount of lyophilized glycerol according to the present invention, preferably administered once to several times a day.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of diabetes.

In addition, the present invention provides a health food for preventing and improving diabetes containing glycerol as an active ingredient.

The glycerol is preferably any one selected from the group consisting of [Formula 1], [Formula 2] or [Formula 3], but is not limited thereto.

[Formula 1]

;

;

[Formula 2]

; 및

; And

(3)

.

.

The glycerol is preferably extracted from soybeans, but is not limited thereto. The extraction is preferably extracted with water, a lower alcohol of C ₁ to C ₄ or a mixture thereof, but is not limited thereto, and the lower alcohol is preferably ethanol, but is not limited thereto.

Preferably, the separation is performed by HPLC, but is not limited thereto, and the HPLC apparatus uses a Jasco 1580 system and a Gemini 5 uM C18 column. However, all common compound separation methods known in the art may be used.

The diabetes is preferably type 2 diabetes, but is not limited thereto.

Glycerin of the present invention exhibits a hypoglycemic effect through increased glucose uptake and inhibition of adipocyte differentiation through inhibition of triglyceride accumulation, an effect of increasing insulin secretion, an effect of increasing beta cell proliferation, and a secretion effect of GLP-1. In addition, glycerol-enriched fermented soybeans reduced blood sugar, increased insulin sensitivity, increased liver glycogen content, and lowered triglyceride content. Therefore, the composition of the present invention containing glycerol according to the present invention as an active ingredient can be used as a health food for preventing and improving diabetes.

In addition,

1) inoculating the soybean precultured strains; And

2) It provides a health food for preventing and improving diabetes containing fermented soybean with increased glycerol content as an active ingredient prepared by the manufacturing method comprising the step of fermenting the inoculated soybean.

The beans can be used regardless of the species, and after surface sterilization in ethanol, washed with deionized water and immersed in water, preferably divided and used, but not limited thereto. Aspergillus sojae ) or Aspergillus oryzae , but is not limited thereto. The inoculation is preferably inoculated fungal spore suspension on the bean split surface, but is not limited thereto, the fermentation is preferably carried out at 20 to 30 ℃, more preferably carried out at 22 to 26 ℃, it is carried out at 25 ℃ This is the most preferred, but not limited to, it is preferable to store in a dark chamber (chamber) for 1 to 7 days, more preferably for 2 to 5 days, it is most preferable to carry out by storing for 3 days, but is not limited thereto. It doesn't work.

The diabetes is preferably type 2 diabetes, but is not limited thereto.

In a specific embodiment of the present invention, the beans are surface sterilized with ethanol, washed with deionized water, immersed in water and divided, and the divided beans are placed in a petri dish covered with a sterilized filter paper soaked. Fungi {Aspergillus dust sojae), and Aspergillus duck claim (Aspergillus oryzae )} was inoculated on the divided side and sealed, and then fermented by storing in a dark chamber (chamber) at 25 ℃ for 3 days to prepare fermented soybeans fortified with glycerol.

In addition, C57BL6J mice were treated with STZ (Streptozoin) and induced high-fat diet to prepare a diabetic model mouse, and the fermented soybeans and non-fermented soybeans of the present invention were ingested as protein sources to the diabetic mice, respectively. Antidiabetic effect of glycerol-enhanced fermented soybean of the present invention in diabetic mice was measured.

To investigate the glucose metabolism of glycerin in serum, oral glucose tolerance test after dietary intake of fasting blood glucose and serum insulin of diabetic mice, normal mice, and diabetic mice administered fermented soybeans and non-fermented soybeans of the present invention, respectively, was performed. It was.

As a result, diabetic mice had higher peak blood glucose levels than normal mice, resulting in high blood glucose, and significantly lower blood glucose levels after reaching high blood glucose, which means that the diabetic mouse group had higher insulin resistance than the normal mouse group. Fermented soybeans in the diabetic mouse group showed a lower blood sugar rise and lower peak blood sugar levels than in the diabetic mouse group or the non-fermented soybeans, and the blood sugar drop was also fast (see FIG. 11).

Also, when the oral glucose tolerance test result was calculated under the area under the curve, the diabetic mouse group had a larger area than the normal mouse group, indicating that the blood glucose level was higher for oral administration of glucose. Also, when the fermented soybeans were ingested in diabetic mouse group, the area under the curve was smaller than that of diabetic mice. When the non-fermented soybeans were ingested, the blood sugar lowering effect was lower than that of diabetic mice. I wrote it down. During oral glucose tolerance test, the diabetic mouse group was lower in the early and late stages compared to the normal mouse group, and the fermented soybeans group had the initial change from 0 to 40 minutes. Serum insulin concentrations were higher compared to the other groups, while late changes did not show a difference between the groups (see FIG. 12).

Therefore, when ingested fermented soybean glycerol enhanced diabetic mice, it was confirmed that there is a blood glucose homeostasis maintenance effect.

In addition, homeostasis model assessment of insulin resistance for diabetic mice, normal mice, diabetic mice to which the fermented soybean and the non-fermented soybean of the present invention, respectively, fasting blood glucose and serum insulin were tested for insulin resistance after dietary intake. HOMA _IR ) measured the change in insulin resistance. As a result, when the insulin was observed intraperitoneally, the change in blood glucose was observed in normal mice. After 60 minutes, it was reduced to 50%, confirming that normal mice are highly sensitive to insulin. However, diabetic mice showed significantly lower blood glucose after insulin administration compared to normal mice, resulting in a 10% reduction after 30 minutes, and a less than 20% after 90 minutes, and similar results with non-fermented soybeans. . However, when fermented soybeans were administered to diabetic mice, insulin sensitivity was improved and decreased by 25% after 30 minutes and by 30% or more after 90 minutes (see FIG. 13).

Therefore, when glycated glycerin enhanced fermented soybeans were administered to diabetic mice, insulin sensitivity was significantly increased compared to diabetic mice.

In addition, when administration of fermented soybeans fortified with glycerin to diabetic mice, the amount of glycogen and triglycerides stored by comparing the amount of stored glycogen and triglycerides was compared to determine whether there is a change in the stored glycogen and triglyceride accumulation in the liver due to diabetes. As a result, glycogen stored in liver was lower in diabetic mice than normal mice, and when fermented soybean containing glycerol was fed to diabetic mice, the amount of glycogen stored in liver was increased compared to the non-fermented soybean. It was. On the other hand, triglyceride content was higher in diabetic mice than normal mice. When fermented soybean containing glycerol was fed to diabetic mice, the amount of triglyceride was decreased compared to that of non-fermented soybean, which is opposite to glycogen. The trend was shown (see FIG. 14).

Therefore, it was confirmed that the glycogen content and elevated triglyceride content in liver due to diabetes can be changed to normal contents through ingestion of fermented soybean containing glycerol.

When glycerol or glycerol-enriched fermented soybean according to the present invention is used as a food additive, the glycerol or glycerol-enhanced fermented soybean can be added as it is or used with other foods or food ingredients, according to a conventional method. Can be used as appropriate. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. Is sure.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

< Example  1> using mold Fermented soybean  Produce

<1-1> mold Preculture

Two types of fungi (Aspergillus sojae) and Aspergillus oryzae (Korea Microorganism Conservation Center) were prepared by culturing in a dark chamber at 25 ° C.

<1-2> Mold inoculation to soybean

Soybean (Dept. of Agriculture, Gyeongsang National University) was surface sterilized in 70% ethanol for 3 minutes, washed with deionized water for 2 minutes, immersed in water for 5 hours, and then petri dish with sterilized filter paper moistened with 1 ml of water. petri dish, 90 X 15 mm), placed in half split soybean (in the method study for large inoculation, soybeans were split into 1 / 2-1 / 8 slices using a blender), Example 1-1 above Fungal spore suspensions (~ 10 ul) cultured at> were inoculated on the split cotton. The non-fermented soybeans, which were the control, were divided in half but did not inoculate the fungus. The inoculated Petri dishes were wrapped with parafilm and then fermented by storing them in a dark chamber at 25 ° C. for 3 days, thereby preparing fermented beans using mold.

< Example  2> From fermented soybean Glycerin  Isolation and identification

<2-1> Glycerol  Enhanced Fermented soybean  Preparation of extract

0.5 g of the fermented soybean prepared in Example 1 was homogenized in 80% ethanol (1.5 ml), incubated at 50 ° C. for 1 hour, and then cooled at room temperature, followed by centrifugation at 14,000 g for 10 minutes. The supernatant was taken to prepare fermented soybean extract.

<2-2> Glycerin  Isolation and identification

In order to isolate and identify the glycerol contained in the fermented soybean extract, HPLC and LC / Mass were performed. Specifically, HPLC was used for Jasco 1580 system (Japan), the column was Gemini 5um C18 (Phenomenex, 150x2.0 mm, USA). Acetonitrile (acetonitrile, HPLC grade, Sigma, USA) and distilled water were used as the mobile phase solvent, and the ratio of acetonitrile was controlled to increase sequentially from 90% to 65% and 90%, and the inoculation volume was 10 ul, developed at room temperature, and a flow rate of 0.8 ml per minute. Thereafter, the chromatogram was analyzed by absorbance at 280 nm using a UV / Visible detector (Jasco), and as a result, glycerol, which is a peak which is not seen in non-fermented soybeans and only confirmed in fermented soybeans, was separated (Fig. One).

In addition, in order to analyze the molecular weight and structure of the separated material, LC / Mass was performed. Specifically, a mass spectrometer is an ion trap having a built-in heated capillary electrospray interface. trap was used, the positive ion mode was 5000 v spray needle voltage, and the drying gas temperature was 300 ° C. Elution solvent system is 0.1% formic acid (A) and water (B) 90% A-10% B 2 minutes, 60% A-40% B at a flow rate of 0.2 mL / min. 10 minutes, 30% A to 70% 20 minutes, 10% A to 90% B 25 minutes, 10% A to 90% B 25 minutes, 10% A to 90% B 30 minutes, 90% to 10% B 33 minutes It was performed by setting, as a result, it was confirmed that the glycerin is confirmed only in fermented soybean not expressed in non-fermented soybeans, the structure was identified (Fig. 2).

< Experimental Example  1> Glycerin  Confirmation of glucose absorption effect by insulin stimulation

<1-1> 3 T3  Induction of Adipocyte Differentiation Using Fibroblasts

In order to differentiate 3T3-L1 fibroblasts (ATCC, Manassas, VA, USA) into adipocytes, 3T3-L1 fibroblasts were incubated at 10% CO2 using DMEM medium containing 10% fetal bovine serum. In a 175 mm ² flask. Two days after the bottom area of the flask was almost full of cells, the medium was incubated for 2 days with 1 ug / ml of insulin, 0.25 uM dexamethasone and 0.5 mM isomethylbutylxanthine. To induce differentiation into adipocytes to obtain 3T3-L1 adipocytes.

<1-2> Glucose Absorption Effect by Insulin Stimulation

In order to confirm the glucose uptake effect according to insulin concentration in 3T3-L1 adipocytes differentiated in Experimental Example 1-1, insulin was 0, 1, 3, 5, 10, 25 and 50 ng / After treatment at a concentration of mL, glucose uptake effect was measured.

As a result, glucose uptake was proportionally increased when insulin was applied to 3T3-L1 adipocytes by concentration (FIG. 3).

<1-3> Effect of Glucose Uptake by Glycerin on Insulin Stimulation

The 3T3-L1 adipocytes differentiated in Experimental Example 1-1 were replaced with a medium once every two days for one week, and then transferred to a 12 well plate, followed by incubation for two days, followed by phosphate buffer solution (PBS). After washing, the mixture was incubated for 8 hours by mixing glycerol at a concentration of 0.5 uM and 5 uM in DMEM medium containing 1% bovine serum albumin and without glucose, and then incubated for 30 minutes at 37 ° C. [ ¹⁴ C] 2 -1 uCi / mL of deoxyglucose and 1 mM of glucose were added, and then incubated at 22 ° C. for 30 minutes. In this step, when treated with and without glycerol, insulin was treated together at 0 ng / ml, 1 ng / ml and 50 ng / ml, respectively, and 1 ng / ml insulin after glycerol treatment. It was confirmed whether to increase glucose uptake compared to treatment with 50 ng / ml of insulin alone.

Cells cultured with insulin and glycerol were washed with phosphate buffer solution (PBS) containing 10 mM glucose, lysed cells with 0.5 N sodium hydroxide, neutralized the lysed cells with acetic acid, and then The content of ¹⁴ C contained was measured by a beta counter, and glucose uptake was confirmed by calculating Disintegrations per minute (dpm) per unit protein.

As a result, 3T3-L1 adipocytes increased glucose uptake in a concentration-dependent manner when treated with 1 ng / mL of insulin and glycerol, and when treated with 5 uM glycerol, glucose uptake was most increased (FIG. 4).

Therefore, glycerol increased the glucose uptake in adipocytes by insulin stimulation, confirming that there is a blood glucose enhancing effect.

< Experimental Example  2> Glycerin  Triglyceride Effect

In order to confirm the effect of glycerol on triglyceride accumulation, the amount of triglyceride was measured by adding glycerol in the process of differentiating fibroblasts into adipocytes. Specifically, 3T3-L1 fibroblasts were cultured using differentiation medium containing 0.5 uM and 5 uM of glycerol added to induce differentiation into adipocytes, followed by differentiation for 6 days, followed by triglycerides. The amount was measured. The amount of triglycerides was obtained by washing the differentiated cells with phosphate buffer solution (PBS) and lysing them with lysis buffer, and using a triglyceride kit (Trinder kit, Yeongdong Pharm., Korea). Measured. Through the amount of triglycerides measured, the degree of differentiation of adipocytes of 3T3-L1 fibroblasts was determined.

As a result, when treated with glycerol, the amount of triglyceride was significantly reduced as the concentration was increased compared to the control group not treated with glycerol (FIG. 5).

Therefore, it was confirmed that glycerol has an effect of inhibiting the accumulation of triglycerides and inhibiting the differentiation of fibroblasts into adipocytes.

< Experimental Example  3> Glycerin PPAR -r agent ( agonist ) effect

Peroxisome Proliferator Activated Receptor (PPAR) -r, a receptor that increases the expression of genes involved in sugar metabolism, is a receptor that increases the expression of genes involved in the metabolism of sugars and lipids in the cell nucleus. Agents are known to not only lower blood sugar, but also lower the concentration of fatty acids in the blood.

In order to confirm the mechanism of hypoglycemic action through the increased glucose uptake effect of glycerol according to the present invention, PPAR-r agonist activity of glycerol was measured. Specifically, 293 cells (ATCC, Manassas, VA, USA), which are human embryonic kidney (HEK) cells, were cultured and seeded at 1 × 10 ⁴ cells per well in 96 well cell culture plates 24 hours before DNA transduction. It was. The seeded cells were then PPRE-luciferase construct (construct, firefly pGL3-DR-1-luciferase vector; 0.12 ug DNA / well), pSV-SPORT-PPAR-expression vector (0.12 ug DNA / well) and The pSV-SPORT-Retinoid X Receptor (RXR) -α vector (0.08 ug DNA / well) was transduced with Lipofectamine Plus reagent (purchased from Invitrogen) and the level of transfection was investigated to investigate the efficiency of transduction. A renilla phRL-TK vector (10 ng DNA / well) was used and after 42 hours of transduction, the cells were incubated for 20 hours in serum-free DMEM medium containing 0.1% bovine serum albumin, followed by glycerol, DMSO used as negative control and rosiglitazone used as positive control were divided into low and high concentrations and treated for 8 hours. It is then washed with phosphate buffer solution (PBS) and IX passive lysis buffer solution (passive) Lysing the cells with lysis buffer (promega), followed by fireflies (PPRE-luciferase) and renilla luciferase (renilla) luciferase Activity in the Dual-Luciferase Reporter Assay System System, purchased from Promega), and the activity was calculated by the ratio of firefly luciferase activity and renilla luciferase activity to compare relative luciferase activity.

As a result, rosiglitazone, which is known as a representative agent of PPAR-r, exhibited an effect as a PPAR-r agonist by increasing its relative luciferase activity at higher concentrations.However, when glycerol is treated, it is relatively lucifer. Lase activity was not shown (FIG. 6).

Therefore, it was confirmed that the hypoglycemic effect through the increase of glucose uptake of glycerol was not the action of the PPAR-r agonist.

< Experimental Example  4> in pancreatic beta cells Glycerin  Check mechanism of action

<4-1> Pancreatic Beta Cell Culture

The pancreatic beta cells used in the experiment, the Min6 cell line (provided by Doctor Morris F. White, Harvard University Boston Pediatric Hospital), were treated with 15% fetal bovine serum, 2 mM el-glutamine, 100 IU / ml penicillin, and 100 μg / ml. Cultured in DMEM (Dulbecco's modified Eagle's medium) containing high glucose with streptomycin antibiotics.

<4-2> insulin secretion effect

In order to confirm the effect of glycerol on insulin secretion by glucose treatment in the beta cells of the pancreas responsible for insulin secretion function, insulin secretion measurement experiments were performed. Specifically, the Min6 cells cultured in Experimental Example 4-1 were transferred to a 12 well plate at 1 × 10 ⁶ cells / ml 2 days before the insulin secretion experiment, followed by 10% FBS, 2 mM el-glutamine, 100 IU / ml penicillin and 100 [mu] g / ml streptomycin were added in high concentration glucose DMEM medium, and the next day the culture was changed to low concentration glucose DMEM medium containing 0.2% BSA. The culture medium of the cell cultured plate was prepared with Krebs-Ringer buffer (KRB solution, pH 7.2), a solution for insulin measurement containing 0.2% (w / v) BSA and 0.5 uM and 5 uM glycerin. The change was incubated for 2 hours, and then KRB solutions were collected from each well and stored at −80 ° C. until insulin concentration was measured. Negative control was treated with DMSO, and positive control was treated with exendin (exendin-4), a persistent diabetes treatment, at a concentration of 2.5 nM.

Cells in the wells were scraped and lysed with grinding buffer (1 M HCl containing 5% (v / v) HCOOH, 1% (v / v) trifluoroacetic acid and 1% (w / v) NaCl) and dissolved in lysed cells. Peptides were extracted by alcohol extraction.

Radioimmunoassay using the insulin RIA kit (Linco Research Inc., St. Charles) was used to quantify the amount of insulin secreted from the KRB solution and the amount of insulin remaining in the cells, and to correct the amount of cells. The amount was quantified by Bradford Protein Assay (Bio-Rad).

As a result, when glycerol was treated with KRB solution containing high concentration of glucose (20 mM) in Min6 cells, insulin secretion was significantly increased compared to negative control in proportion to the concentration of exendin-4, which was lower than the positive control group. (FIG. 7).

Therefore, it was confirmed that glycerin has an effect of increasing insulin secretion by glucose in the beta cells of the pancreas.

<4-3> cell proliferation effect

In order to confirm the beta cell proliferation effect of glycerol, the Min6 cells cultured in Example 4-1 were transferred to 96 wells, and treated with glycerol at concentrations of 0.5 uM and 5 uM, and after glycerol treatment, for 4 hours. The cell number was measured using the WST-1 kit at 8 hours and the cell proliferation effect was confirmed.

As a result, after treatment with glycerol, beta cells proliferated with increasing concentrations compared to the DMSO treated group, which was a negative control, in both 4 and 8 hours (FIG. 8).

Therefore, it was confirmed that glycerol has a proliferative effect of pancreatic beta cells.

<4-5> ER  Changes in Stress-related Gene Expression

When immature proteins beyond the vesicle's ability to process due to physiological or pathological conditions are introduced into the endoplasmic reticulum or calcium depleted in the endoplasmic reticulum, endoplasmic reticulum dysfunction occurs. This condition is called ER stress. Pancreatic beta cells are one of the important features that the endoplasmic reticulum is well developed, the smooth function of the endoplasmic reticulum plays an important role in the function of the beta cells and beta cell dysfunction caused by endoplasmic reticulum stress is expected to cause diabetes.

In order to confirm whether the antidiabetic effect of glycerol is related to ER stress of beta cells, beta cells were treated with glycerol, and thus the expression rate of ER stress related genes was confirmed. Specifically, the cultured Min6 beta cell line was treated with 0.5 uM and 5 uM glycerol, and the obtained cells were lysed to extract total RNA and synthesized cDNA with reverse transcriptase, followed by real-time polymerase using primers of each gene. The chain reaction method was used to measure the mRNA levels of genes involved in ER stress. In order to correct the unequal amount of each cell, the mRNA level of GAPDH, a housekeeping gene, was measured and the expression rates of genes were compared with the relative mRNA levels of genes involved in ER stress.

 As a result, the expression rate of XBP-1 / GAPDH, AFT4 / GAPDH, CHOP / GAPDH, AFT6 / GAPDH, genes involved in ER stress, decreased significantly in concentration-dependently compared to the negative control group. 9).

Therefore, it was confirmed that glycerol has an effect of reducing ER stress of beta cells.

< Experimental Example  5> In colorectal cancer cell line Glycerin GLP -1 secretion measurement

GLP-1 (glucagon like peptide-1) is a hormone that is secreted from cells in the digestive tract after a meal and promotes the release of insulin from the pancreas.In addition to promoting insulin secretion, GLP-1 (glucagon like peptide-1) promotes the growth and protection of pancreatic beta cells that make insulin In addition to helping to slow down the movement of the digestive system and suppress appetite, as well as controlling blood sugar. Colorectal cancer cell line was used to confirm the secretion effect of GLP-1 of glycerol.

<5-1> Cell Line Culture

The NCI-H716 cell line (ATCC, Manassas, VA, USA), the colorectal cancer cells used in the experiment, was treated with 10% FBS (fetal bovine serum), 2 mM L-glutamine, and 100 IU / ml penicillin (penicillin). ) And suspension cultured in Roswell Park Memorial Institulete (RPMI) medium with 100 μg / ml streptomycin antibiotic. Cultured cells were transferred to dishes coated with Matrigel (Becton Dickinson, Bedford, Mass., USA) and differentiated into endocrine cells for GLP-1 secretion experiments. Two days before the experiment, 1 x 10 ⁶ cells / ml of differentiated cells were transferred to Matrigel coated 12-well culture plates, followed by 10% FBS, 2 mM el-glutamine, 100 IU / ml penicillin, The cells were cultured in high concentration DMEM medium to which 100 μg / ml of streptomycin antibiotic was added.

<5-2> GLP -1 secretion effect

The culture medium of the plate in which the differentiated cells were cultured was changed into KRB buffer solution (Krebs Ringer bicarbonate, pH 7.2) containing 0.2% (w / v) bovine Serum Albumin (BSA) and glycerin, and incubated for 2 hours. . Collect KRB solution from each well and add it to -80 ° C until 50 μg / ml of phenylmethylsulphonyl fluoride (PMSF) and 34 μg / ml of diprotin-A were added to measure GLP-1 concentration. Stored in.

The cells in the wells were scraped and scraped with 1 M hydrochloric acid (HCl), with 1% (v / v) trifluoroacetic acid, including homogenization buffer (5% (v / v) formic acid (HCOOH). ) And 1% (w / v) sodium chloride) and the lysed intracellular peptides were extracted by alcohol extraction method.

Radioimmunoassay using the Linco GLP-1 Kit (GLP-1 (7-36) Active RIA Kit, Linco Research Inc., St. Charles) to reveal the amount of GLP-1 secretion of KRB solution and GLP-1 in cells The amount of protein was quantified, and the protein amount of the cell homogenate was quantified by Bradford protein assay (Bio-Rad, Inc.) to correct the amount of cells.

As a result, when glycerol was treated with KRB solution containing high glucose (20 mM) in NCI-H716 cell line, GLP-1 secretion was significantly increased in proportion to the concentration compared to the negative control (Fig. 10).

Therefore, glycerol was confirmed to have an effect of increasing the secretion of GLP-1 in colorectal cancer cell line.

< Experimental Example  6> In diabetic animal model Glycerol  Enhanced Fermented soybean Anti-diabetic  effect

<6-1> Preparation and Dietary Conditions of Diabetic Animal Model Mice

To prepare a diabetic animal model, 8 to 9 weeks old C57BL6J mice (purchased from Daehan Biolink) were quarantined for about 2 weeks, followed by 20 mg of STZ per body weight (Streptozoin, purchased from Sigma) for 2 days. Diabetic mice that induce type 2 diabetes symptoms by injection through injection were prepared, and the comparison mouse group was injected with the same amount of citrate buffer solution. Diabetic mice treated with STZ induced diabetes with rapid hyperglycemia (approximately 11 mM) after 7 days of STZ administration, whereas comparative mice treated with citrate buffer solution did not show hyperglycemia, so they were classified into normal mouse groups and subsequently tested. Was performed.

High-fat diets increase insulin resistance and lose beta cell function, making diabetes symptoms worse, so all diabetic mice are modified diet formulations based on the AIN-93 formulation composition typically used in experimental diets for a period of eight weeks. Maintained a high fat diet consisting of 40 energy percent (En%) carbohydrates, 20 En% protein and 40% En% fat. In the case of the control group, starch, sugar, casein and shorting were used as the main sources of carbohydrate, protein and fat, respectively. The fermented soybean group and the non-fermented soybean group used 20% of the fermented soybean and non-fermented soybean of the present invention as protein sources, respectively. What is lacking in protein was filled with casein. Each group was subjected to experiments after feeding the diet for 8 weeks.

<6-2> oral Glucose tolerance  inspection

The ability to keep blood sugar constant, that is, the degree of homeostasis of blood glucose, can be measured by the oral glucose tolerance test. An oral glucose tolerance test measures how efficiently glucose is consumed after it is consumed so that it does not raise blood sugar in the liver and muscles.

To learn more about the glucose metabolism of glycerin in serum, fasting blood glucose and serum insulin in diabetic mice, normal mice, diabetic mice administered with fermented soybeans and non-fermented soybeans of the present invention, 2 g glucose / Oral glucose tolerance test was performed with kg body weight.

As a result, diabetic mice had higher peak blood glucose levels than normal mice, resulting in high blood glucose, and significantly lower blood glucose levels after reaching high blood glucose, which means that the diabetic mouse group had higher insulin resistance than the normal mouse group. Fermented soybeans in the diabetic mouse group showed a lower tendency in blood glucose and lower peak blood glucose than in the diabetic mouse group or the non-fermented soybeans, and also had a quick drop in blood glucose (FIG. 11).

Also, when the oral glucose tolerance test result was calculated under the area under the curve, the diabetic mouse group had a larger area than the normal mouse group, indicating that the blood glucose level was higher for oral administration of glucose. When the fermented soybeans were ingested in the diabetic mouse group of the present invention, the area under the curve was smaller than that of diabetic mice. There was little compared. During oral glucose tolerance test, the diabetic mouse group was lower in the early and late stages compared to the normal mouse group, and the fermented soybeans group had the initial change from 0 to 40 minutes. Serum insulin concentrations were higher compared to the other groups, while late changes did not show a difference between the groups (FIG. 12).

Therefore, when ingested fermented soybean glycerol enhanced diabetic mice, it was confirmed that there is a blood glucose homeostasis maintenance effect.

<6-3> Insulin Resistance Measurement

Homeostasis model assessment to measure insulin resistance by measuring fasting blood glucose and serum insulin of diabetic mice, normal mice, diabetic mice administered fermented soybeans and non-fermented soybeans of the present invention at 8 weeks after diet. of insulin resistance (HOMA _IR ) was measured for insulin resistance change. Blood glucose was measured in the serum glucose using the Beckman Glucose Analyzer II, and serum insulin concentration was measured using a rat insulin specific RIA kit (Lin Kit, Linco Research Inc. St. Charles, USA). Measurements were made using radioimmunoassay.

As a result, when the blood glucose level was observed after the administration of insulin to the abdominal cavity, the blood glucose was rapidly reduced and decreased to 40% of fasting blood sugar after 30 minutes, and to 50% after 60 minutes. It was confirmed that the high sensitivity to. However, diabetic mice showed significantly lower blood glucose after insulin administration compared to normal mice, resulting in a 10% reduction after 30 minutes, and a less than 20% after 90 minutes, and similar results with non-fermented soybeans. . However, when fermented soybeans were administered to diabetic mice, insulin sensitivity was improved and decreased by 25% after 30 minutes and by 30% or more after 90 minutes (FIG. 13).

Therefore, when glycated glycerin enhanced fermented soybeans were administered to diabetic mice, insulin sensitivity was significantly increased compared to diabetic mice.

<6-4> Determination of glycogen and triglycerides stored in the liver

Diabetics have a very low glycogen content in the liver, and even after eating excessive foods, glucose in the liver is hardly converted to glycogen and remains in the blood, leading to high blood sugar. In addition, insulin plays an important role in the synthesis of fatty acids, synthesis and decomposition of triglycerides, fatty acids are mostly synthesized in the liver and moved to the blood and stored in adipose tissue. In the liver, insulin promotes glucose conversion and fatty acid synthesis, Insulin deficiency decreases fatty acid degradation, leading to the accumulation of abnormally large amounts of triglycerides, a major cause of fatty liver complications of diabetes.

When administration of fermented soybeans fortified with glycerin to diabetic mice, the livers were extracted and compared to analyze the amount of glycogen and triglycerides stored. . Specifically, after the oral glucose tolerance test of diabetic mice, normal mice, diabetic mice administered fermented soybeans and non-fermented soybeans of the present invention for 2 days again, fasted for 16 hours, fasted and ketamine (100 mg / kg Body weight) and xylene (10 mg / kg body weight). After anesthesia, 100 nM insulin was injected into the inferior vena cava, and after 15 minutes, the white paper was killed and the liver was extracted to determine the amount of glycogen and triglycerides stored in the liver.

In order to measure the amount of glycogen stored in the liver, the liver was dissolved in RIPA buffer solution, centrifuged at 3,000 rpm for 10 minutes, and the supernatant obtained after centrifugation was treated with 1.5 N perchloric acid to remove proteins. Thereafter, the glucose concentration of the supernatant was measured before and after the addition of the α-amyloglucosidase to the supernatant from which the protein was removed. The amount of glycogen stored in the liver was calculated by subtracting the concentration before glucose from the concentration of glucose after adding α-amyloglucosidase to the supernatant.

In addition, in order to measure the amount of triglyceride stored in the liver, the liver was extracted by treating the liver with chloroform-methanol (2: 1, v / v), and triglycerides were separated by the Folch method. The contained triglycerides were extracted again with chloroform and measured using a Tinder kit (Yongdong Pharm.).

As a result, glycogen stored in the liver was lower in diabetic mice than normal mice, and when fermented soybean containing glycerol was fed to diabetic mice, the amount of glycogen stored in the liver was increased compared to the case of feeding non-fermented soybean. On the other hand, triglyceride content was higher in diabetic mice than normal mice. When fermented soybean containing glycerol was fed to diabetic mice, the amount of triglyceride was decreased compared to that of non-fermented soybean, which is opposite to glycogen. The trend was shown (FIG. 14).

Therefore, it was confirmed that the glycogen content and elevated triglyceride content in liver due to diabetes can be changed to normal contents through ingestion of fermented soybean containing glycerol.

The preparation examples for the compositions of the present invention are illustrated below.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Preparation of powders

2 g of glycerol of the present invention

1g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of glycerol of the present invention

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

100 mg of glycerol of the present invention

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

<1-4> Preparation of Injection Solution

10 μg / ml of glycerol of the present invention

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

The glycerol according to the invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume was adjusted with sodium chloride BP for injection and thoroughly mixed. . The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

< Manufacturing example  2> Manufacture of food

Food comprising a glycerol or glycerol-enhanced fermented soybeans according to the present invention was prepared as follows.

<2-1> Preparation of Flour Food

0.5 ~ 5.0% by weight of glycerol-enhanced fermented soybeans according to the present invention was added to the flour, and using this mixture to prepare bread, cakes, cookies, crackers and noodles to prepare health promoting foods.

<2-2> Preparation of Soups and Gravies

Glycerin-enhanced fermented soybeans according to the present invention were added to the soup and broth by 0.1 to 1.0 parts by weight, respectively, to prepare meat products for health promotion, soup of noodles and broth in a conventional manner.

<2-3> Preparation of Ground Beef

Glycerin-enhanced fermented soybeans according to the present invention were added to the ground beef each 10 parts by weight to prepare a ground beef for health promotion in a conventional manner.

<2-4> dairy products (dairy products )

5-10 wt% of glycerol-enriched fermented soybeans according to the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Preparation of Wire

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The glycerol-enriched fermented beans according to the present invention were concentrated under reduced pressure in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.

The dry grains of the grains, seeds and glycerol-enriched fermented soybeans according to the present invention were prepared by combining the following ratios:

Cereals (30 wt% brown rice, 15 wt% brisk, 20 wt% barley);

Seeds (7% perilla, 8% black beans, 7% black sesame seeds);

Dry powder of glycerol-enhanced fermented soybeans according to the present invention (3% by weight);

Ganoderma lucidum (0.5% by weight); And

Sulfur (0.5 wt%).

< Manufacturing example  3> Manufacturing of beverage

<3-1> Preparation of Carbonated Drink

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed, and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 Sterilizing at 20 ℃ for 180 seconds to mix with a cooling water 1: 1 ratio and then injected carbon dioxide gas 0.5 ~ 0.82% to prepare a carbonated beverage containing glycerol according to the present invention.

<3-2> Preparation of Health Beverage

After sterilization by homogeneously mixing the subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and glycerol according to the present invention This was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.

<3-3> Preparation of Vegetable Juice

5 g of glycerol-enriched fermented soybeans according to the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

<3-4> Preparation of Fruit Juice

1 g of glycerol-enriched fermented soybean according to the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

As described above, glycerol according to the present invention exhibits the effect of lowering blood glucose through increased glucose uptake and inhibiting fat cell differentiation through inhibiting triglyceride accumulation, increasing insulin secretion, increasing beta-cell proliferation, and Glycolin-enhanced fermented soybeans have a maintenance effect on blood glucose homeostasis, increase insulin sensitivity, increase liver glycogen content, and lower triglyceride content. Since the effect, the composition of the present invention containing glycerol or glycerol-enhanced fermented soybean of the present invention as an active ingredient can be usefully used as a composition for preventing and treating diabetes.

Claims (13)

Pharmaceutical composition for the prevention and treatment of diabetes containing glycerol (glyceollin) as an active ingredient.
The pharmaceutical composition for preventing and treating diabetes of claim 1, wherein the glycerol has a structure of the following [Formula 1], [Formula 2] or [Formula 3]:
[Formula 1]

;
(2)

; And
[French 3]

.
The pharmaceutical composition for preventing and treating diabetes of claim 1, wherein the glycerol is extracted from the beans and separated.
The pharmaceutical composition for preventing and treating diabetes of claim 3, wherein the extract is extracted with water, a lower alcohol of C ₁ to C ₄ , or a mixture thereof.
5. The pharmaceutical composition for preventing and treating diabetes of claim 4, wherein the lower alcohol is ethanol.
2. The pharmaceutical composition for preventing and treating diabetes of claim 1, wherein the diabetes is type 2 diabetes.
Health food for the prevention and improvement of diabetes containing glycerol as an active ingredient.
The method of claim 7, wherein the diabetes is diabetes prevention and improvement health food, characterized in that the type 2 diabetes.
1) inoculating the soybean precultured strains; And
2) Health foods for preventing and improving diabetes comprising fermented soybeans with increased glycerol content as an active ingredient prepared by a manufacturing method comprising fermenting inoculated soybeans.
The method of claim 9 wherein the fungal strain Aspergillus scavenger (Aspergillus sojae) and Aspergillus duck claim (Aspergillus oryzae ) health food for preventing and improving diabetes, characterized in that the strain.
10. The method for preventing and improving diabetes of claim 9, wherein the inoculation of the strain is inoculated with a fungal spore suspension on a split bean.
10. The method for preventing and improving diabetes of claim 9, wherein the fermentation is stored for 1 to 5 days in a dark chamber (chamber) of the strain inoculated soybean.
10. The method for preventing and improving diabetes foods of claim 9, wherein the diabetes is type 2 diabetes.

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