KR101074338B1 - Composition for lowering blood glucose and Preparing method of the same - Google Patents

Abstract

The present invention is a hypoglycemic food composition and a method for producing the same, characterized in that the fermentation by injecting a lactic acid bacteria culture solution into the extract of ganoderma lucidum, bitter buckwheat, and bitumen mixture containing a large amount of components such as alkaloids and terpenes known as antidiabetic components Unlike the single drug efficacy of chemical synthetic drugs, it is a natural product that minimizes the resistance and side effects of long-term use and solves the glycemic control mechanisms that can cause a variety of hyperglycemia. Alpha-glucosidase inhibitory action, promoting insulin response to increase insulin sensitivity in peripheral tissues such as muscle or adipocytes, insulin-like action, promoting glucose transporter expression to increase insulin sensitivity, pancreatic cells Extracts natural substances with efficacy, such as their protective effect, By fermenting it, it not only maximizes the functions contained in these extracts, but also improves the intestinal environment, reduces cholesterol, promotes digestion and absorption, prevents and improves constipation and diarrhea, and enhances immunity by unidentified fermented products produced during fermentation. One of the benefits is that we can expect a variety of additional benefits.

Ganoderma lucidum, bitter buckwheat, bitter gourd, antidiabetic effect, fermented product, calcium, lactic acid bacteria

Description

Composition for lowering blood glucose and a method of preparing the same {Composition for lowering blood glucose and Preparing method of the same}

The present invention by injecting fermentation of lactic acid bacteria culture medium into the extract of Ganoderma lucidum, bitter buckwheat, Yeoju mixture, inhibits the digestion and absorption of carbohydrates in the small intestine by alpha-glucosidase inhibition, promote the activity of insulin function, insulin The present invention relates to a food composition for lowering blood sugar and a method for producing the same, which are capable of regulating functions such as similar actions.

Diabetes mellitus is a disease characterized by hyperglycemia caused by lack of insulin in the pancreas or abnormal insulin action in each tissue. Diabetes mellitus, retinopathy, cataracts, nephropathy It is a disease that causes many serious complications. This type of diabetes is an autoimmune disease in which the body's immune system attacks and damages insulin-producing pancreatic beta cells at a young age. Insulin dependent diabetes mellitus is a type 1 insulin-dependent diabetes mellitus. It is about 5%.

In addition, non-insulin dependent diabetes mellitus, which accounts for 95% of domestic diabetics, has abnormal effects on insulin, such as fat cells, due to heredity, obesity, and lack of exercise. It is caused by increased insulin resistance, and the number of diabetics increases every year due to westernization, lack of exercise, and increased life expectancy.

Diabetes is known to have a different pattern of diabetes due to differences in body structure and food culture with our Asians whose grains are staple, unlike Westerners who eat meat and have a large body size and a relatively obese population. Koreans have only 1 to 5% of type 1 diabetes due to lack of insulin, but about 10% of Americans (Korea Economy 2005.5.24). In addition, 70.5% of all diabetics and 36.1% of people who are overweight and 36.1% are diabetic in the United States, while Korean diabetics are less than 35% and 20% are severely obese. It seems that the ability to cope with increasing insulin secretion in proportion to intake is weak. This is because the rapid economic growth of Korea caused malnutrition of pancreatic beta-cells due to malnutrition in infants and adolescents, and it became overnourished as an adult. It is soaring.

Therefore, it is urgent to develop new anti-diabetic agents tailored to our diabetics from indigenous crop resources in Korea due to Westerners and other aspects of diabetes. Most of the diabetic drugs used to date are imported and have characteristics that are suitable for Westerners, so it is common for Koreans to fail to control blood sugar due to their therapeutic effects.

Currently used diabetic drugs include sulfonylurea drugs that stimulate the pancreas to release stored insulin, biguanide drugs that delay the absorption of sugar in the small intestine, acarbose drugs that prevent carbohydrates from being broken down into sugars, It is divided into thiazolidinediones, which increase insulin sensitivity in peripheral tissues such as muscle and fat cells, and glycinide-based drugs, which are taken before meals to stimulate insulin release.

Sulfonylurea-based drugs that have been used most recently for the treatment of diabetes in the world include glypizide, glyquidone, glybenclamide, glyclazide, and insulin in the pancreas. Females of childbearing age who are not effective for type 1 diabetes patients who have a function of promoting secretion but have little insulin production, or type 2 diabetes patients who have relatively less ability to secrete insulin in the pancreas. It is not available to. In addition, sulfonylurea-based drugs are metabolized in the liver and then released into the kidneys, and therefore, patients with hepatic dysfunction and renal disease require considerable attention. In addition, biguanide-based drugs such as metroformin (Metformin) are drugs that increase insulin sensitivity and control blood glucose levels, compared to sulfonylurea-based drugs, but have a weaker blood glucose lowering effect, but are known to have relatively weak side effects such as hypoglycemia.

However, in recent years, government-funded national research funds have been supporting a huge amount of research funds for the development of diabetes treatments, but no significant research results and commercially available treatments have been made. These findings have recently been conducted to search for functional foods or various bioactive substances that have a hypoglycemic effect, but most of them inhibit enzyme activity related to glucose metabolism, increase insulin synthesis and secretion, inhibit your life in liver tissue, Despite the different glycemic control mechanisms such as increased insulin sensitivity in muscle or adipose tissue, most of them have been shown to be effective against a single mechanism like drugs. On the other hand, as diabetes progresses, it is difficult to control blood sugar due to a complex cause rather than one cause. Therefore, in order to effectively control blood sugar, it is necessary to purchase drugs for each of them, hyperlipidemia according to hyperglycemia, In order to simultaneously treat diabetic complications caused by blood circulation, such as retinopathy, foot ulcers, sexual dysfunction, and numbness of hands and feet, various treatments are needed to add to it. It is also true. Thus, with this in mind, pharmaceutical companies have been developed and marketed to enable at least two complex effects.

In the East Asian countries, including Korea, about 80% of the treatments have been made using plants and herbs.In the 1990s, western countries, including the United States, used traditional plants and herbs for reasons of efficiency, small side effects, and relatively inexpensive diseases. This has greatly increased and various technologies are being developed.

Yeomju ( Momordica charantia ), which is used as such a drug, is also known as a yearly vine of Medicinal herb, and recently Yeoju contains not only insulin-improving effect and vegetable insulin (Vegetable insulin) but also high-value coucu Triterpenes of the cucurbitane-type [Srihar MG et al. Bitter ground ( Momordica charantia ) improves insulin sensitivity by increasong skeletal muscle insulin-stimulated IRS-1 tyrosine phosphorylation in high-fat-fed rats. Br J Nutr 99, 806-812, 2008, Liu Y et al. Cucurbitane-type triterpene glycosides from the fruit of Momordica charantia . Planta Med 74, 1291-1294, 2008], which has alpha-glucosidase inhibitory activity, which is very effective in improving diabetes. It became known. In particular, the caranthine component contained in the pulp is known to activate beta cells, which play an important role in insulin secretion.

In addition, Ganoderma lucidum is a mushroom belonging to the Democratic Mushroom Bulgogi, and it is a red sack with red sack. It is the most beautiful and effective among Ganoderma lucidum. It is a year old and grows naturally in the decaying stump of hardwoods. Ganoderma lucidum mushroom has been found to have various effects such as cholesterol removal, antithrombosis, hepatitis treatment, anti-cancer, anti-cancer, blood sugar reduction, and rejuvenation. Ganoderma lucidum bioactive substances include high-flavor ingredients, steroids, nucleic acids, glycans, germanium, lectins, antithrombin components and the like. Cytotoxic terpenoids ganoderic R, T, U, W, X, Y, Z are known to have anti-cancer effects among physiologically active substances of Ganoderma lucidum, and ganoderic acid B, D, F, H, R, S, Y has a function of lowering blood pressure. Ganoderma lucidum polysaccharide beta-1-> 3 glucan (β- (1-> 3) D-glucan) protein complex is known to be a new non-toxic anticancer drug.

Therefore, as a patent developed by using such drugs such as Yeoju, Ganoderma lucidum mushroom, etc., Korean Patent No. 749944 of the blood glucose lowering saponin fraction prepared in Yeoju, and a pharmaceutical composition comprising the same, and the registered Korean Patent No. 481114 Lowering blood sugar level by lactic fermentation of a pharmaceutical composition for lowering blood sugar, which contains fermented product obtained by solid fermentation of Hobanaba extract, fenugreek extract and Yeoju extract, and a water-soluble extract of Ganoderma lucidum in Korea Patent No. 534066 Patents related to Lactobacillus Lactobacillus fermentation broth for treating diabetes, comprising an ingredient effective for.

In addition, as a patent using other mushrooms, Korean J. Patent No. 859984, pine berry mushroom culture carcass extract and a blood glucose lowering composition containing the same, and Korean Patent No. 852479, Cloudy mushroom having a blood glucose lowering effect ( Coriolus versicolor ) As those related to the composition for treating diabetes comprising a polysaccharide as an active ingredient have been disclosed, various techniques for lowering blood sugar using a herbal medicine have been developed.

On the other hand, in addition to the drugs listed above, buckwheat is rarely found in grains, containing up to 10-15% of protein. Especially, buckwheat has a high protein content of 93.1, because it contains a large amount of essential amino acids that other crops, such as lysine and tryptophan, tend to lack. It is an excellent food source of protein. According to a study published in the Journal of Agriculture and Food Chemistry, a single dose of buckwheat extract in diabetic rats resulted in a drop of up to 19% in blood glucose. Although it is rare in other foods, chiro-inositol, which is known as the main ingredient of buckwheat, plays an important role in glucose metabolism.

Buckwheat is divided into general buckwheat and bitter buckwheat (Daldan buckwheat), and general buckwheat is grown in Korea, but bitter buckwheat is cultivated in the Himalayas, mainly in Nepal, and Yunnan area of 2000m above sea level in southern China. The area is considered a botanical origin. Bitter buckwheat is also called bitter buckwheat because it has a stronger bitter taste than ordinary buckwheat. It is also known as Beijing Dong-in Clinic, Tianjin Medical Clinic, Clinic, and Beijing Aquaculture Research Institute. Nutrient analysis and clinical trials of bitter buckwheat, their staple food, have been conducted on selenium, blood sugar and urine glucose lowering, hypertension, and cerebral artery, which are essential for the prevention and treatment of cancer. It was found that it contains special nutrients such as rutin (vitamin P), which prevents adult diseases such as hardening, arteriosclerosis, pulmonary bleeding, and ulcer disease, and the secret of longevity of this minority group is based on the bitter buckwheat. After I learned it, I got a lot of attention.

Recently, the Rural Development Administration's Goryeong Agricultural Research Institute has succeeded in fostering the bitter buckwheat superior system corolla No. 3-3, which has an excellent effect on preventing vascular diseases such as diabetes and hypertension compared to buckwheat. In Daegwan 3-3, 'rutin', a preventive substance for adult diseases such as hypertension and cerebral arteriosclerosis, was 70 times higher in seeds and 20 times higher in young leaves than normal buckwheat. In addition, it contains a large amount of 'quasartin' and 'quatcirine', which are effective in preventing and treating vascular diseases, and contains rutin, an antioxidant substance of flavonoids, which is not found in other grains. This routine is called vitamin P by controlling the permeability of capillaries and vitamin C ₂ by enhancing the effects of vitamin C. It is mainly used for anti-inflammatory, anti-cancer, In addition to the thrombotic effect, it is known to have a cytoprotective effect against oxidative stress and prevent blood vessel damage.

Therefore, the present inventors can exhibit higher anti-diabetic effect when mixed and used with Ganoderma lucidum, bitter buckwheat, and bitter melon containing ingredients of alkaloids and terpenes as described above, and milk containing skim milk (defatted milk) The present invention is completed by developing a food composition for lowering blood sugar in consideration of the addition of fermented with lactic acid bacteria and enhancing minerals such as calcium and zinc, which are essential for the action of insulin, and at the same time, reducing the bitter taste caused by gourds. I can do it.

Therefore, the present invention uses natural products extracted from the mixture of Ganoderma lucidum, bitter buckwheat, and Yeoju, unlike the single drug efficacy of the chemical synthetic drugs can be a cause of various hyperglycemia while minimizing the resistance and side effects of long-term use It is an object of the present invention to provide a food composition for lowering blood glucose and a method of manufacturing the same, wherein the method of reducing blood sugar control mechanisms and diabetic complications such as blood circulation disorders can be improved.

In addition, the present invention delays the absorption of sugar in the small intestine after ingesting the food, unlike single-effect or two-mechanical effects having limitations as a diabetic agent when used alone, such as ganoderma lucidum, bitter buckwheat and yeast. In order to prevent carbohydrates from being broken down into sugars, it inhibits alpha-glycosidase, enhances insulin sensitivity in peripheral tissues such as muscle and fat cells, and the insulin entering each peripheral tissue cell. It provides a glucose lowering food composition and a method for producing the same, characterized in that it has an anti-diabetic effect that can simultaneously exert a glucose transporter (GLUT4) expression for increasing sensitivity and a protective effect of pancreatic cells. Do another task.

In addition, the present invention is mixed with fermented skim milk powder rich in calcium components in the mixed extract of Ganoderma lucidum, bitter buckwheat, Yeoju fermented calcium components that play an essential role in the secretion of insulin in the pancreas is more easily absorbed by the human body anti- In addition to increasing diabetes efficacy, milk acts as a coating, reducing the bitter taste of these glutamate substances, which reduces the resistance to taking them, and significantly increases the number of lactic acid bacteria during fermentation. It is another object of the present invention to provide a food composition for lowering blood sugar and a method of preparing the same, which are expected to improve various effects such as improving the intestinal environment, reducing cholesterol, promoting digestive absorption, preventing and improving constipation and diarrhea, and enhancing immunity. It is a task.

The present invention for achieving the above object is a mixture of 5 ~ 10 parts by weight of Ganoderma lucidum mushroom, 5 ~ 10 parts by weight of bitter buckwheat, 5 ~ 10 parts by weight of bitter melon 70% (v / v%) methanol extract solvent 70 ~ Into 85 parts by weight to extract the mixture extract by reflux cooling extraction method,
The mixed extract is deodorized by blowing off the extraction solvent and then added to the skim milk powder so that the total solid content of the milk component is 7 ~ 12% by weight and heated to sterilized powdered milk mixture extract Lactobacillus acidophilus 30 Lactobacillus culture medium cultured for 8-12 hours at ~ 37 ℃ 2% by weight (v / v) and fermented to separate the cells and the supernatant by centrifugation, the blood sugar drop characterized in that the supernatant was prepared by vacuum lyophilization The manufacturing method of the food composition for foods, and the blood sugar-lowering food composition manufactured by this method are made into the subject solving means.

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The present invention by the above-mentioned means to solve the problem by using a natural product extracted from the mixture of Ganoderma lucidum, bitter buckwheat, Yeoju, unlike the single drug efficacy of chemical synthetic drugs, while minimizing the resistance and side effects of long-term use, It helps to solve the glycemic control mechanisms that can cause hyperglycemia.Also inhibits alpha-glucosidase in the small intestine after eating, and increases insulin sensitivity in peripheral tissues such as muscle or fat cells. Maximizes the functions contained in these extracts by extracting natural ingredients that are effective, such as promoting insulin response, insulin-like action, promoting glucose transporter expression for increasing insulin sensitivity, and protecting pancreatic cells. Unidentified fermented acid produced during fermentation as well as saving Various additional effects, such as improving the intestinal environment by (reproduct), cholesterol reduction, promoting digestion, constipation, and diarrhea prevention and improvement, immunity enhancement, it is also an advantage to be expected.

The present invention for achieving the above effects, unlike chemical synthetic drugs, to obtain the anti-diabetic composition derived from natural products for minimizing side effects and resistance to long-term use and at the same time to solve the blood glucose control mechanism that can cause a variety of hyperglycemia Hypoglycemic food composition for which anti-diabetic effect is further increased by synergistic effect due to the production of fermentation metabolites during fermentation by extracting alkaloids and terpenes from yeast, ganoderma and bitter buckwheat Regarding the configuration and operation of the present invention in detail as follows.

The blood sugar-lowering food composition according to the present invention is characterized by being prepared by vacuum freeze-drying the supernatant of the fermented fermented lactic acid bacteria culture solution into a mixed extract extracted from a mixture of Ganoderma lucidum, bitter buckwheat, and Yeoju.

In the present invention, the mixture extract 5-10 parts by weight of Ganoderma lucidum mushroom, 5-10 parts by weight of bitter buckwheat, 5-10 parts by weight of the bitter gourd mixed in 70-85 parts by weight of the extraction solvent and extracted by reflux cooling extraction.

In the present invention, the anti-diabetic effect is increased by extracting the active ingredients of each raw material by the mixed use having the above mixing ratio rather than the use of each material from Ganoderma lucidum, bitter buckwheat, Yeoju, and also increase the anti-diabetic effect in the pancreas insulin Calcium component, which plays an essential role in secretion, is prepared in the form of fermented milk so that it can be more easily absorbed by the human body to further increase antidiabetic efficacy.

Ganoderma lucidum, bitter buckwheat and litchi which can be used in the present invention is preferably used to finely pulverized in order to facilitate the extraction by the extraction solvent, the size of the powder particles is not particularly limited.

At this time, reflux cooling extraction can be repeatedly performed twice for 3 hours to extract the active ingredient from the mixture as much as possible.

In addition, the extraction solvent used in the present invention, it is preferable to use the purity of 70% (v / v%) methanol in order to increase the extraction efficiency of the active ingredient.

The fermented product is deodorized by blowing off the extraction solvent, and then added skim milk powder so that the total solid content of the milk component is 7-12% by weight and heated to sterilized milk powder mixture extract 2% by weight (v / v) Injected into a culture for about 8 to 12 hours at 30 ~ 37 ℃ to prepare a fermented milk.

In this case, the added skim milk powder acts as a coating agent, which reduces the bitterness of these bitter substances, which reduces the rejection when taking them.In addition, the number of lactic acid bacteria is significantly increased during fermentation, which is the biggest advantage of lactic acid bacteria. A variety of additional benefits, such as improving the environment, reducing cholesterol, promoting digestive absorption, preventing and improving constipation and diarrhea, boosting immunity, reducing allergies, and preventing tooth decay, can be expected to be much cheaper and help health.

In addition to the above functions, skim milk powder is also regulated by calcium and zinc components when insulin is secreted from the beta cells on the island of Langelhans in the pancreas, thus supplementing minerals such as calcium and zinc that are essential for insulin action. Do it.

The fermentation product is centrifuged to separate the cells and the supernatant, and the supernatant is lyophilized to prepare a food composition for lowering blood sugar.

Thus, after obtaining methanol extract from the mixture of Ganoderma lucidum, bitter buckwheat, and Yeoju, deodorizing organic solvent, add skim milk powder to the remaining aqueous solution, sterilize at 80 ~ 100 ℃ for 30 minutes, and then cool down to room temperature before mixing with lactic acid bacteria starter The lactic acid bacteria were cultured at 30-37 ° C. and fermented to the point where the curd was completely formed by the acid produced by the lactic acid bacteria by milk casein protein to obtain a fermentation product. After the fermentation is completed, the supernatant is recovered by centrifugation and freeze-dried to prepare a powder.

The lactic acid bacteria used at this time is a Lactobacillus acidophilus strain that is commonly used in the manufacture of fermented milk.

 Hereinafter, the technical configuration and operation of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not necessarily limited to these Examples.

Example 1 Obtaining Fermentation Products Having Antidiabetic Efficacy

1. Preparation of Mixed Extract

8% by weight of Ganoderma lucidum, 8% by weight of bitter buckwheat, and 8% by weight of bitter gourd, put the sample in a well-pulverized state in 70% purity (v / v%) methanol extractant 76% by weight and reflux-cooled extraction. Repeated extraction twice each time to obtain a mixed extract. At this time, the extraction yield was 2.57%.

2. Lactic acid bacteria fermentation using mixed extract

Lactobacillus acidophilus was pre-cultivated in a culture solution sterilized at 100 ° C by adding skim milk powder to the total aqueous content of milk components to 10% by weight of the remaining aqueous solution by deodorizing the extraction solvent by blowing the extraction solvent ) Fermented milk was prepared by injecting the lactic acid bacteria culture medium at 2 wt% (v / v) and incubating at 35 ° C. for 10 hours. At this time, the lactic acid bacteria fermented product cultured only lactic acid bacteria were prepared and used as a test control group.

3. Acquisition of Lactic Acid Bacteria Fermentation Products

The lactic acid bacteria fermentation broth prepared by the method 2 was centrifuged to separate the cells and the supernatant, and the supernatant was vacuum-lyophilized to obtain a powder and used for the experiment. At this time, the control fermentation broth obtained the powder in the same manner and used in the experiment.

Example 2 Physiological Function Characteristics of Fermented Products Having Antidiabetic Efficacy

1. Alpha-glucosidase inhibitory activity that interferes with the digestion of carbohydrates in the small intestine

The lactic acid bacterium fermentation broth prepared by the method of Example 1 was tested for the inhibition of alpha-glucosidase (α-glucosidase) to reduce carbohydrate digestion and postprandial blood glucose.

The reaction solution for measuring activity inhibition of alpha-glucosidase was 250 μl of 0.1 M sodium phosphate buffer (pH 7.0), 20 mM piPenji (PNPG; p- nitrophenyl α-Dglucopyranoside, sigma). 250ul of substrate and 5ul of sample solution were added, and then reacted at 37 ° C for 5 minutes, and the absorbance was measured at 405 nm. Thereafter, 250ul of 0.075 U alpha-glucosidase (α-glucosidase derived from Bacillus strearothermophilus ) was added thereto and reacted at 37 ° C. for 15 minutes, and then 2 ml of 0.1 M sodium carbonate buffer was added to terminate the reaction. Absorbance was measured at the same wavelength (Thirunavukkarasu, M., Pries, FG, Synthesis of α-amylase and α-glucosidase by membrane bound ribosomes from Bacillus licheniformis . Biochem. Biophys. Res. Commun. 114, 677-682 (1983) ).

Alpha-glucosidase inhibitory activity test results are the same as in the graph of Figure 1, the accompanying drawings, the mixed extract (Mix) in the graph of Figure 1 is a mixed mixture of three kinds according to the present invention, fermentation C (Fermentation control) is a lactic acid bacteria fermentation control without adding the herbal extract according to the present invention, mixed fermentation is a mixed fermentation product fermented by lactic acid bacteria by adding three kinds of mixed extracts according to the present invention, Acarbose (Acarbose) is commercially available Commercial samples are shown.

In Figure 1, the mixed extract and fermentation C showed a low inhibitory activity of alpha-glucosidase as much as 20%, while the mixed fermentation showed an inhibitory activity of 40%. The mixed fermentation product is slightly lower than the commercially available inhibitory activity of Acarbose, but is considered to be of value as an inhibitor of activity of natural-derived alpha-glucosidase that can be used without side effects.

2. Toxicity test of samples by MTT assay

3T3-L1 cells, which are preadipocytes cultured in a 10 cm 2 plate, were washed with phosphate buffer solution (GIBCO, Dulbecco's phosphate saline) and then detached with 0.25% trypsin-EDTA mixture. Afterwards, 5 × 10 ³ cells were each dispensed into 96-well plates using a haemacytometer. Samples were added at each concentration. Cells were incubated for 2 days at 37 ° C. in a 5% CO ₂ incubator, then the medium was removed and replaced with 100 ul of fresh medium (phenol red-free DMEM). 10 ul of 12 mM MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltet-razolium bromide) solution was added to each well, followed by incubation at 37 ° C for 4 hours. After removing 75 ul of the medium into the well, 50 ul of DMSO was added to dissolve the insoluble material, and reacted at 37 ° C. for 10 minutes to measure absorbance at 540 nm (Garn H, Krause H, Enzmann V, Dr'ssler). K. An improved MTT assay using the electron-coupling agent menadione.) Immunol Methods 168, 253 (1994). Tada H, Shiho O, Kuroshima K, Koyama M, Tsukamoto K. An improved colorimetric assay for interleukin 2.J Immunol Methods 93, 157 (1986)).

      As a result of the experiment (Olympus CKS41 X100) as shown in Figure 2, the sample concentration of 100 ~ 500 ug / ml did not show toxicity to 3T3-L1 cells.

In Figure 2 A is control, B is DMSO 1%, C is DMSO 2%, D is ganoderma lucidum MeOH ext. (500ug / ml), E is bitter methanol extract ( Momordica charantia MeOH ext. (500ug / ml)), F is bitter buckwheat methanol extract ( Fagopyrum tataricum MeOH ext. ( 500ug / ml)), G is mixed methanol extract (Mix MeOH ext. (500ug / ml)), H is fermented control (500ug / ml)), I is a bitter fermented yogurt ext. (500 ug / ml).

3. Confirmation of Adipocyte Differentiation Capacity of 3T3-L1 Cells

3T3-L1 preadipocytes are well known for their biological properties, and when treated with similar substances such as insulin and differentiation-inducing substances, they differentiate into adipocytes (Gerhold DL, Liu F., Jiang G., Li Z., Xu J., Lu M., Sachs, JR, Bagchi A., Fridman A., Holder DJ, Doebber TW, Berger J., Elbrecht A., Moller DE and Ahang BB Gene expression profile of adipocyte differentiation and its regulation by peroxisome proliferation -activated receptor gamma agonists. Endocrinology 143, 2106-2118 (2002). Miki H, Yamauchi T, Suzuki R, Komeda K, Tsuchida A, Kubota N, Terauchi Y, Kamon J, Kaburagi Y , Matsui J, Akanuma Y, Nagai R, Kimura S, Tobe K, Kadowaki T. Essential role of insulin receptor substrate 1 (IRS-1) and IRS-2 in adipocyte differentiation.Mol . Cell. Biol. 21, 2521-2532 (2001)). It is sensitive to insulin because it has insulin receptors and GLUT4 on the cell membrane, without being involved in the complex problems associated with metabolic feedback loops that exist in vivo . Schematic diagram of the insulin signal shows that apoptosis signal-regulation with insulin treatment increases insulin receptor substrate-1 and increases the activity of phosphatidylinositol 3-kinase (PI3-kinase). Increasing the activity of apoptosis signal regulating kinase (Akt) increases GLUT4 translocation to the cell membrane, increasing glucose uptake. Therefore, 3T3-L1 is widely used as a model to search for substances that improve insulin action or to study insulin signaling system. Therefore, in this experiment, preadipocyte 3T3-L1 that did not treat differentiation-inducing substance was not differentiated into adipocytes during 10 days of incubation, and cultured by treating differentiation-inducing substance (IBMX, Dexamethasone, insuline). One preadipocyte 3T3-L1 induced differentiation. In order to confirm the differentiation, staining oil red O (Oil red O) to stain the adipocytes (red), as a result of the cells (A) cultured in a normal medium as shown in the photo (Olympus CKS41 X100) of Figure 3 On the other hand, a red fat drop was observed in cells treated with differentiation-inducing substances (B).

4. Exploring Insulin Enhancers

Preadipocyte 3T3-L1 cultured in a 10 cm 2 plate was washed with DPBS (GIBCO, Dulbecco's phosphate saline), desorbed with 0.25% trypsin-EDTA, and then 6-hole using a hemocytometer. 5 × 10 ⁴ cells / well were dispensed into the well plate. In order to search for substances, insulin was treated with other differentiation-inducing substances at concentrations of 0, 1, 2.5, 5, and 10 ug / ml, and the degree of adipocyte differentiation was observed. Among them, 2.5 ug / ml insulin concentrations having differentiation rate were found. The following experiment was performed by selecting. 3T3-L1 cells were cultured in a confluent stage, followed by a postconfluent process for 2 days, followed by ganoderma lucidum, yeast, bitter buckwheat, mixed extract, fermentation control, and high fermentation samples. , 10, 100, and 250 ug / ml was added to the concentration and cultured for 5 days or more to observe the degree of adipocyte differentiation. The control group was used as the culture by adding only the same amount of DMSO. In addition, the differentiation inducer 0.25 uM DEX (dexamethasone), 0.5 mM IBMX (1-methyl-3-isobutylxanthine), 2.5 ug / ml The samples were incubated for 5 days or more by adding 1, 10, 100, and 250 ug / ml to the medium containing insulin (DMEM), respectively. As a control, a medium containing differentiation inducer (DMEM) was used. Adipocyte differentiation was measured by staining with oil red O to determine fat content. A simple method of oil red O staining is to remove the medium, add to each well the final 3% formalin (formaline sigma) to wash the cells, and add the same amount of formalin to remove the cells. Fixed. Fixed cells were washed with 60% isopropanol, and then stained for 30 minutes by adding 1 ml of oil red O solution. The oil red O solution was removed, washed four times with tertiary sterile water for 5 minutes, dried completely, and extracted with 100% isopropanol (contained 5% SDS) for 10 minutes. Adipocyte measurements were calculated by measuring absorbance at 500 nm and comparing with controls (Gray, AM and Flatt, PR Insulin-reasing and insulin-like activity of the traditional antidiabetic plant Coriandurm sativum (coriander). Brit. J. Nutr. 81 , 203-209 (1999). McNeel, RL and Mersmann, HJ Effects of isomers of conjugated linoleic acid on porcine adipocyte growth and differentiation.J. Nutr. Biochem. 14, 266-274 (2003)).

As a result of the experiment, as shown in the graph of FIG. 4, when only the sample was treated without the differentiation material, the mixed extract and the fermentation C showed differentiation rates of 114.7 and 98.3%, respectively, compared to the control, but the mixed fermentation samples were 174%, respectively. , Or showed higher differentiation rate than the mixed extract sample, and it is judged to have insulin-like activity in the mixed fermentation sample. Reported increases in glucose uptake of 3T3-L1 cells by natural products have been reported in norpseudoephedrine (Takaku, T., Jiang, M., Okuda, H. and Maeda, N. Isolation of insulin lkie substance from Ephedra sinica STAPF J. Traditional Med ., 14, 358-359. (1996)), unsaturated fatty acids of royal jelly (Kameda, K., Chikaki, M, Morimoto, C., Jiang, M. and Okuda, H. Insulin like action of trans-10-hydroxy-2-decanoic acid and its related substance.J. Tranditional Med ., 13, 456-457. (1996)), bakuchiol ( Otholobium pubescens) from the Peruvian traditional medicinal plant bakuchiol) (Krenisky, JM, Luo, J. and Carney, JR Isolation and antihyperglycemic activity of bakuchiol from Otholobium pubsecens, a peruvian medicinal plant used for the tretment of diabetes.Biol . Pharm. Bull ., 22, 1137-1140), ginseng, cheonmundong golden jigolpi, yellowish, maekmundong mixing prescription (Hong, SJ, Fong, JC and Hwang, JH Effects of crude drugs on glucose uptake in 3T3-L1 adipocyte. Gaxio ng Yi Xue Ke Xue Za Zhi , 16, 445-4512, (2000)), oak extract (Ko, BS, Kim, HK and Park, S. Insulin sinsitizing and insulin-like effects of water extracts from Kalopanax pictus NAKA fractions in 3T3-L1 adipocytes. Kor. J. Agri. Chem. Biotech . 45, 42-46. (2002) and others have been reported to promote differentiation, contain insulinic substances, and reduce insulin resistance in fractions.

As a result of inducing adipocyte differentiation of 3T3-L1 by adding a sample to the differentiation-inducing substance, as shown in the graph of FIG. As a result of the differentiation rate, it was observed that adipocyte differentiation promoting effect of inducing adipocyte differentiation of 3T3-L1 together with other differentiation-inducing substances (IBMX, DEX) even if no insulin was present.

And the type of adipocyte differentiation induction of bitter fermented yogurt is hypertrophic obesity (hypertrophic obesity, Fig. 6A) and hyperplastic-hypertrophic obesity, as shown in the photograph of Fig. 6 (Olympus CKS41 X100). 6B) were identified.

5. Indirect experiment with glucose intake into fat cells

Preadipocyte 3T3-L1 cells cultured on a 75 cm 2 plate were incubated at the confluent stage, and after 2 days of induction differentiation, they were incubated for 3 days and then once every 2 days. After replacing the high-density glucose DMEM medium and adding new high-density glucose medium after 10 days of complete conversion to adipocytes, the glucose availability in the medium according to the sample treatment group was measured using a glucose assay kit. . As a result of the experiment, as shown in the graph of FIG. 7, when the mixed fermentation product was treated, the availability of glucose was slightly increased to about 117%. In other words, the mixed fermentation is believed to play a role in increasing glucose (glucose) availability in 3T3-L1 cells.

6. Measurement of GLUT4 Volume in Cell Membrane

Following the results of Example 2.5, the photograph (Olympus CKS41 X100) of the measurement result of the GLUT4 amount change to investigate the insulin signaling mechanism according to the mixed fermentation treatment is as shown in FIG. The adipocytes 3T3-L1 were transferred to a 10 cm 2 plate, and the adipocytes 3T3-L1 were incubated for 12 hours in a high concentration of glucose medium (DMEM) added with 3% BSA instead of plasma, followed by the addition of 100 ug / ml of the sample. After incubation for 5 hours, when treated with GLUT4 bitter fermented yogurt samples, the band of GLUT4 appeared darker than the band treated with 1 ng / ml insulin. The increase in GLUT4 levels is known to be closely related to the enhanced insulin signal by enhancing the insulin signal by mixed fermentation samples, and it is reported that the increase of GLUT4 in the cell membrane increases glucose uptake. Ciaraldi TP, Kong AP, Chu NV, Kim DD, Baxi S, Loviscach M, Plodkowski R, Reitz R, Caulfield M, Mudaliar S, Henry RR.Regulation of glucose transport and insulin signaling by troglitazone or metformin in adipose tissue of type 2 diabetic subjects. Diabetes. 51, 30-36 (2002)). Thiazolidinediones, sold as insulin-sensitive agents, have a role in increasing glucose uptake in both adipocytes and muscle cells, and increase the translocation of GLUT4 to the cell membrane. (CHOU SC, GOLDSTEIN A. Chromogenic groupings in the Lowry protein determination. Biochem. J. 75, 109-115 (1960)). In the case of bitter fermented yogurt samples, the effect of increasing the amount of GLUT4 is believed to contain insulin-sensitive substances.

7. Effect of Mixed Fermentation on Cytotoxicity Induced by Streptozotocin (STZ)

STZ and alloxan (alloxan) are widely known as a substance that induces the destruction of pancreatic beta cells to prevent insulin secretion from the pancreas. In this experiment, we investigated the effects of mixed extract, fermentation C, and mixed fermentation on the cytotoxicity caused by STZ treatment of pancreatic beta cell line RIN-m5F. In the experiment, RIN-m5F was inoculated with 1 × 10 ⁵ cells in a 24-well plate and incubated for 18 hours, and then replaced with fresh medium for 5 hours, followed by 1 mM STZ and each sample (mixture extract, fermentation C, mixed fermentation). Water) was treated at a concentration of 5 ug / ml for 1 hour. In addition, MTT experiments showing cell viability by replacing with fresh medium and incubating for 18 hours were performed (MM Sailaja Devi, Undurti Narasimha Das.Effect of prostaglandins against alloxan-induced cytotoxicity to insulin secreting insulinoma RIN cells in vitro.Prostaglandins, Leukotrienes and Essential Fatty Acids 71, 309-318. (2004)). As shown in the photograph of Fig. 9 (Olympus CKS41 X100), the STZ-control decreased cell number significantly by STZ treatment, while the number of cells increased significantly when additionally treated samples, extracts, fermentation C and mixed fermentation. . In particular, it was observed under a microscope that the number of cells similar to the control group when the mixed fermentation was further processed. In other words, it is believed that beta cell lines are destroyed by the toxicity of STZ by treating mixed fermentation products.

Therefore, the blood glucose-lowering food composition according to the present invention, as confirmed through the above examples, unlike blood glucose lowering chemical agents each exhibiting one pharmacological effect in various metabolic pathways related to diabetes, diabetes without any side effects In the fermentation product obtained by fermenting alkaloid and terpene-containing methanol extract derived from natural products with the effect of reducing stable blood sugar without side effects such as hypoglycemia in patients 1) by inhibiting alpha-glucosidase Glucose transporter (GLUT4) for suppressing digestive absorption of carbohydrates in the small intestine, 2) promoting insulin function, 3) insulin-like action, and 4) increasing insulin sensitivity in peripheral tissue cells such as muscle and adipose tissue 5) It has a protective effect on the pancreatic cells, thereby promoting the expression of both type 1 and type 2 diabetes patients. It is characterized by one to be used.

As described above, the food composition for lowering blood sugar according to the present invention has been confirmed for its superiority through the above-described examples, and the present invention is also provided to those skilled in the art by the spirit and scope of the present invention as set forth in the claims below. It will be understood that various modifications and variations can be made in the present invention without departing from the scope thereof.

       1 is a graph showing a comparison of alpha-glucosidase inhibitory activity measurement results using a lactic acid bacterium fermentation extract prepared according to the present invention and a commercially available commercial sample.

Figure 2 is a photograph of the toxicity test results of the sample through the MTT assay using the lactic acid bacteria fermentation extract prepared according to the present invention,

Figure 3 is a photo taken using the lactic acid bacteria fermentation extract prepared in accordance with the present invention induction of differentiation of cells 3T3-L1 cells and oil red o staining state,

Figure 4 is a graph showing the results of searching for insulin-like active material using 3T3-L1 cells using the lactic acid bacteria fermentation extract prepared according to the present invention,

5 is a graph showing the results of searching for an insulin activity enhancing substance using 3T3-L1 cells using the lactic acid bacteria fermentation extract prepared according to the present invention;

Figure 6 is a photograph taken using the type of lactic acid bacteria fermentation extract prepared according to the present invention to promote the differentiation of fat cells differentiation of the sample,

Figure 7 is a graph showing the results of measuring the glucose (glucose) usage according to the sample treatment in 3T3-L1 cells using the lactic acid bacteria fermentation extract prepared according to the present invention,

Figure 8 is a photograph of the measurement results of the cell membrane GLUT4 expression according to the sample processing using the lactic acid bacteria fermentation extract prepared in accordance with the present invention and other samples,

9 relates to a photograph of the effects of various samples on the cytotoxicity induced by STZ using the lactic acid bacteria fermentation extract prepared according to the present invention.

Claims (8)

A mixture of 5-10 parts by weight of Ganoderma lucidum, 5-10 parts by weight of bitter buckwheat, and 5-10 parts by weight of bitter gourd, was added to 70-85 parts by weight of a 70% purity (v / v%) methanol extractant and reflux-cooled. Extract the mixed extract, The mixed extract is deodorized by blowing off the extraction solvent and then added to the skim milk powder so that the total solid content of the milk component is 7 ~ 12% by weight and heated to sterilized powdered milk mixture extract Lactobacillus acidophilus 30 Lactobacillus culture medium cultured for 8-12 hours at ~ 37 ℃ 2% by weight (v / v) and fermented to separate the cells and the supernatant by centrifugation, the blood sugar drop characterized in that the supernatant was prepared by vacuum lyophilization Method for producing a food composition for. delete delete delete delete delete delete A food composition for lowering blood sugar, which is prepared by the method of claim 1.

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