KR100781817B1 - Freeze Dryed Material, Ethanol Precipitates, Ethylacetate Fraction of Agaricus Blazei Mushroom Mycelia and Manufacturing Process Thereof - Google Patents

Abstract

The present invention relates to agaricus mushroom mycelium lyophilisate, ethanol precipitates, ethyl acetate fractions and a method for preparing the same, in particular culturing agaricus mushroom strains; Preparing hot water extract samples by extracting the cultured hot water; Separating the culture filtrate from which residue is removed from the hot water extract sample, and concentrating the culture filtrate under reduced pressure to obtain a concentrate; When the agaricus mushroom mycelium extract production method comprising a; and lyophilizing the concentrate; lyophilisate, ethanol precipitates, ethyl acetate fraction prepared therefrom has an excellent effect on the inhibitory activity of alphaglucosidase, Significantly reduced postprandial blood sugar increase in normal and type 2 diabetic patients has an excellent effect in preventing and / or treating diabetes in the human body.

Agaricus mushroom mycelium, lyophilization, ethanol precipitation,

Description

Freeze dried material, Ethanol Precipitates, Ethylacetate Fraction of Agaricus Blazei Mushroom Mycelia and Manufacturing Process Thereof}

1 is a flowchart showing the step of sequentially fractionating the Agaricus mushroom mycelium lyophilisate (HEAB-FD) according to an embodiment of the present invention with an organic solvent,

2 is a graph showing the effect of HEAB-FD administration on blood glucose content in STZ-induced type 1 diabetic rats according to an embodiment of the present invention,

Figure 3 is a graph showing the glycated hemoglobin content in blood after fasting in the control and HEAB-FD treated group according to an embodiment of the present invention,

Figure 4 is a graph showing the glucose content in the plasma after fasting in the control and HEAB-FD treated group according to an embodiment of the present invention,

Figure 5 is a graph showing the blood urea nitrogen content in the plasma after fasting in the control and HEAB-FD treated group according to an embodiment of the present invention,

Figure 6 is a graph showing the cholesterol and triglyceride content in the plasma after fasting in the control and HEAB-FD treated group according to an embodiment of the present invention,

7 is a graph showing liver TBARS content and catalase activity in the control group and HEAB-FD treated group according to an embodiment of the present invention (significance level; P <0.05),

8 is a graph showing the activity of GOT and GPT in plasma in the control and HEAB-FD treated group according to an embodiment of the present invention,

9 is a graph showing disaccharidase activity in the control and HEAB-FD treated group according to an embodiment of the present invention,

10 is a graph showing the expression of GLUT4 mRNA in liver and white adipocytes of the control group and HEAB-FD treated mice according to one embodiment of the present invention,

FIG. 11 is a graph showing the expression of insulin receptor mRNA in liver and white adipocytes of rats treated with the control group and HEAB-FD according to one embodiment of the present invention.

12 is a graph showing the effect of HEAB-FD on the content (%) of GLUT-1 protein in HepG-2 cells according to one embodiment of the present invention,

Figure 13 is a graph showing the effect of HEAB-FD on the content (%) of GLUT-4 protein in HepG-2 cells according to an embodiment of the present invention,

14 is a graph showing the effect of HEAB-FD on the content (%) of PEPCK1 protein in HepG-2 cells according to one embodiment of the present invention,

FIG. 15 is HEAB-FD and each solvent fraction according to an embodiment of the present invention in comparison with acarbose intestinal α-glucosidase inhibitory activity (Acabose; 25 and 50 ug, HEAB-FD (CE); 100 ㎍, EA; ethylacetate fraction, HX; hexane fraction, BT; butanol fraction, CL; chloroform fraction

16 is a graph showing the inhibitory effect of intestinal l α-glucosidase activity of rats by solvent fractions of HEAB-FD according to one embodiment of the present invention,

17 is a graph showing the effect of inhibiting the intestinal α-glucosidase activity of rat Isofalvones (Daidzein, Genistein, and Genistin) according to an embodiment of the present invention,

18 is a graph showing the effect of HEAB-FD intake on blood insulin concentration in type 2 diabetic patients according to an embodiment of the present invention (significance level; P <0.05),

19 is a graph showing the effect of HEAB-FD intake on the blood glucose concentration in type 2 diabetic patients according to an embodiment of the present invention (significance level; P <0.05).

The present invention relates to agaricus mushroom mycelium, and more particularly, to agaricus mushroom mycelium lyophilisate, ethanol precipitate, ethyl acetate fraction and a method for producing the same. More specifically, a method for producing Agaricus mushroom mycelium extract through the process of culturing Agaricus mushroom strain, hot water extraction, separation of the culture filtrate, concentration under reduced pressure, lyophilization.

Diabetes is on the rise due to the recent changes in dietary patterns of modern people, and according to statistics from the National Statistical Office, diabetes is the fourth leading cause of death among Korean adults. Diabetes mellitus is a chronic vaginal release of glucose into the urine due to a higher concentration of glucose (blood sugar) in the human body, which is caused by the secretion of beta (β) cells of the pancreatic Langerhans Island. Lack of insulin, anterior pituitary gland, adrenal gland, endocrine gland hyperactivity such as the thyroid gland, central nerves, especially the hypothalamic lesions, etc., these factors are thought to be combined.

To this end, insulin has a function of promoting the production of glycogen from glucose, oxidation of glucose, conversion to fat, and the like. Injecting an aqueous solution of insulin lowers blood sugar and thus is used to treat diabetes. Methods for treating diabetic patients include dietary intake of foods high in fiber, such as barley and legumes, and prevention and treatment effects of diabetes mellitus by inhibiting alpha amylase enzyme activity of mushroom extracts. -327), mulberry leaf extracts, silkworms and various diets, such as sulfonic urea, meglitinide, biuanide, alpha glucosidase, thiazolodinedione is used.

Compared to folk diets, pharmacotherapy has a rapid therapeutic effect, but has long-term side effects such as lactic acidosis, bloating, vomiting, abdominal pain and hepatotoxicity. For this reason, many diabetic patients control glycemic control through diet rather than drug treatment, but the scientific evidence on the food materials used in these diets is insufficient and the effect is weak. For this reason, there is an urgent need to discover food products derived from natural products that are effective in lowering blood sugar but have no side effects for the treatment of diabetes.

The present invention has been made to solve the above problems, culturing Agaricus mushroom mycelium, induces autolytic enzyme reaction, lyophilisate, ethanol precipitate, ethylacetate fraction prepared from the reactants and the preparation for obtaining them It is to provide a method.

In addition, through the Agaricus mushroom mycelium extract prepared by the present invention, that is, the Agaricus mushroom mycelium lyophilisate, ethanol precipitate, ethyl acetate fraction, has an excellent effect on the inhibitory activity of alpha glucosidase (maltase), normal people It is an object of the present invention to contribute to the prevention and / or treatment of diabetes in the human body by significantly reducing postprandial blood sugar increase in type 2 diabetes patients.

In addition, by ingesting the agaricus mushroom mycelium lyophilisate, ethanol sediment, ethyl acetate fraction according to the present invention, increasing the gene expression of GLUT4 and increasing glucose transport in peripheral tissues for preventing and treating diabetes mellitus can show the effect of improving blood glucose The purpose is to provide a composition.

The present invention for achieving the above object relates to agaricus mushroom mycelium, and more particularly to agaricus mushroom mycelium lyophilisate, ethanol precipitate, ethyl acetate fraction and a method for producing the same. More specifically, it is a method of producing Agaricus mushroom mycelium extract through culturing, hot water extraction, culture filtrate separation, reduced pressure concentration, lyophilization process.

The present invention is characterized by using the strain Agaricus (Agaricus blazei) to prepare an extract having a hypoglycemic effect. As for the case of gold mushrooms, the growing period (6 months-3 years) and the raw material itself are expensive, whereas the Agaricus mushroom mycelium has a short incubation period (3-7 days) and thus the cost itself is low, so it is continuously used by diabetics. It is a material that can reduce the burden. Therefore, the present inventors completed the present invention by culturing Agaricus mushroom mycelium, preparing a desired composition by using an autolytic enzyme reaction system, and then extracting a fraction having a greater hypoglycemic effect therefrom.

Here, Agaricus mushroom mycelium extract refers to all of the agaricus mushroom mycelium lyophilisate, ethanol precipitate, ethyl acetate fractions according to the present invention, the lyophilisate according to the present invention incubated agaricus mushroom mycelium, after hot water extraction , The culture filtrate is separated, and subjected to lyophilization through a concentrated process under reduced pressure, the ethanol precipitate is prepared by dissolving the lyophilisate in water and adding ethanol, followed by centrifugation to separate the precipitate. It is characterized by being manufactured.

In addition, the ethyl acetate fraction is a step of dissolving the lyophilisate in water and adding ethanol, followed by centrifugation to separate the first supernatant; and adding tricarboxylic acid (TCA) to the supernatant Separating the bisupernatant; And fractionating the second supernatant with an ethylacetate solution to obtain a fraction.

The present invention will be described in more detail below.

First, the present invention comprises the steps of culturing Agaricus mushroom strains; Preparing hot water extract samples by extracting the cultured hot water; Separating the culture filtrate from which residue is removed from the hot water extract sample, and concentrating the culture filtrate under reduced pressure to obtain a concentrate; And lyophilizing the concentrate; agaricus mushroom mycelium lyophilisate manufacturing method comprising a.

According to the present invention, a method for preparing agaricus mycelium extract having a hypoglycemic effect is first treated with protease and cerulase enzyme in soybean, and then inoculated with agaricus mycelium in a medium prepared by adding sulfuric acid, magnesium sulfate, and potassium phosphate. Incubate the liquid. Here, agaricus mushrooms were used in culture by HK Biotech Co., Ltd. received from Korea Seed Management Office, and the culture conditions were aerated at 15 to 35 ° C. for 3 to 7 days of incubation.

The culture may be performed by mixing a liquid medium and Agaricus mushroom strains in a Erlenmeyer flask and culturing in a small amount and culturing in a large amount, followed by a process of autoly degrading Agaricus mushroom mycelium culture. It is also possible to go further.

In addition, in the present invention, the step of preparing a hot water extract sample by extracting the cultured hot water culture, the hot water extract the culture at 100 ℃ to 150 ℃, then cooled to room temperature and concentrated the filtrate filtered by a hot water extract sample It is desirable to prepare. In the present invention, such a hot water extract sample was named SLAB. Subsequently, the hot water extract sample (SLAB) is filtered with a filter to separate the residue and the culture filtrate, and the culture filtrate is concentrated under reduced pressure to obtain a concentrate. Then, by lyophilizing the concentrate, to prepare agaricus mushroom mycelium lyophilisate according to the present invention.

Here, another embodiment for achieving the another object of the present invention is prepared by the above-described lyophilisate manufacturing method, it is preferable that the agaricus mushroom mycelium lyophilisate, characterized in that it has a hypoglycemic effect. The present inventors named such lyophilisate HEAB-FD (Freeze Drying), hereinafter, using HEAB-FD, it describes a method for producing agaricus mushroom mycelium ethanol precipitate and ethyl acetate fraction.

One of the agaricus mushroom mycelium extract according to the present invention, agaricus mushroom mycelium ethanol precipitate production method comprises the steps of culturing the agaricus mushroom strain; Preparing hot water extract samples by extracting the cultured hot water; Separating the culture filtrate from which residue is removed from the hot water extract sample, and concentrating the culture filtrate under reduced pressure to obtain a concentrate; Lyophilizing the concentrate to prepare a lyophilisate; And dissolving the lyophilisate in water and adding ethanol, followed by centrifugation to separate the precipitate.

That is, after preparing the agaricus mycelium lyophilisate described above, as shown in Figure 1, the lyophilisate is dissolved in water, ethanol is added, and then centrifuged to separate the precipitate; 1 is a flow chart showing the step of sequentially fractionating Agaricus mushroom mycelia lyophilisate (HEAB-FD) according to an embodiment of the present invention with an organic solvent.

As shown in FIG. 1, the lyophilisate is added to water or distilled water to dissolve the sample, and ethanol (EtOH) is added thereto, and then left to stand for about a day, followed by centrifugation to obtain a supernatant and a precipitate (Precipitation). : PPT). Agaricus mushroom mycelium ethanol precipitate refers to a precipitate prepared by following the flow chart on the left side of FIG. 1. Since the precipitate is characterized in that the lyophilisate is precipitated using ethanol, it was called ethanol precipitate, and this ethanol precipitate may be repeatedly precipitated by ethanol following the flowchart shown in FIG. 1, and then centrifuged to separate the precipitate. .

Here, another embodiment for achieving another object of the present invention is prepared by the above-described method for producing ethanol precipitates, agaricus mushroom mycelium ethanol precipitates, characterized in that it has a blood sugar lowering effect is preferred.

In addition, agaricus mushroom mycelium ethyl acetate fraction production method as one of the agaricus mushroom mycelium extract according to the present invention comprises the steps of culturing the agaricus mushroom strain; Preparing hot water extract samples by extracting the cultured hot water; Separating the culture filtrate from which residue is removed from the hot water extract sample, and concentrating the culture filtrate under reduced pressure to obtain a concentrate; Lyophilizing the concentrate to prepare a lyophilisate; Dissolving the lyophilisate in water and adding ethanol, followed by centrifugation to separate the first supernatant; Separating the second supernatant by adding tricarboxylic acid (TCA) to the first supernatant; And distilling the second supernatant into an ethylacetate solution to obtain a fraction.

That is, the method for preparing the ethyl acetate mycelium acetate fraction according to the present invention is as shown in the flowchart on the right side of FIG. 1. First, the above-mentioned lyophilisate is dissolved in water and ethanol is added, followed by centrifugation to separate the first supernatant, ethanol is evaporated from the first supernatant, and then 10% tricarboxylic acid. : TCA) is added and the second supernatant is separated by centrifugation one day later. Thereafter, the second supernatant is fractionated with an ethylacetate solution to obtain a fraction. Before the fractionation, the second supernatant may be fractionated into a hexane solution and a chloroform solution.

Here, another embodiment for achieving the other object of the present invention is prepared by the ethanol precipitate production method described above, the agaricus mycelium ethyl acetate fraction is characterized in that it has a hypoglycemic effect.

Hereinafter, a method for preparing Agaricus mushroom mycelium extract (freeze-dried composition, ethanol precipitate, ethylacetate fraction) having a hypoglycemic effect of the present invention will be described in detail, and the functional characteristics and hypoglycemic activity of the extract prepared therefrom And embodiments for related effects are described in detail. However, these examples are merely illustrative of the present invention, the present invention is not limited thereto.

Example : One Agaricus mushroom mycelium  Extract manufacturer

1) Erlenmeyer flask culture (500 ml)

Sodium lysate 0.4%, prolin 1%, xylose 1%, sulfur white 2%, MgSO _{4 ·} 7H ₂ O 0.05%, KH ₂ PO ₄ 0.05% of the liquid medium (300 mL) was prepared in an Erlenmeyer flask (500 mL). After addition and autoclaving (121 ℃, 30 minutes), and then cooled to room temperature, Agaricus blazei grown in PDA medium finely cut and inoculated to less than 7㎜ in diameter and shaking incubator (130 rpm, 25 ℃, 8 days)

2) Bulk culture of incubator

      The same liquid medium (350 L) as in the method of "1) is added to the mass culture tank (500 L), sterilized by autoclaving (121 DEG C, 60 minutes), and then cooled sufficiently using a cooling tank, followed by After inoculating 30 liters of Agaricus culture incubated by the method, the culture was carried out for 3 days (75 rpm, 25 ° C).

3) Autolysis of Agaricus Mushroom Mycelium Cultures

      Agaricus mycelium culture medium is reacted at 55 ± 5 ℃ for 3 hours.

4) Preparation of hot water extract sample

     The hydrolyzed Agaricus mushroom mycelium culture as described above was extracted with hot water at 121 ° C. for 60 minutes, cooled to room temperature and passed through a filter press containing diatomaceous earth. The filtrate was passed through the filter in this way and the concentrated solution was used as a hot water extract sample (SLAB).

5) Preparation of lyophilisate (HEAB-FD)

The SLAB 350L was filtered through an ultra-membrane filter to separate the residue and the culture filtrate. In addition, the separated culture filtrate was concentrated under reduced pressure at about 20 times (Brix 50) in a 1,000 L concentration tank (75 ° C) to obtain a 15 L concentrate. The freeze-dryer was used to concentrate the culture concentrate obtained by the above culture method and concentration technique. Freeze-drying was used to obtain the lyophilizate HEAB-FD according to the present invention, a sample of 596 g / 1 L dry weight HEAB-FD was used as an analytical and clinical sample.

6) Preparation of Ethanol Precipitate and Ethylene Acetate Fraction

50 g of the HEAB-FD sample was added to 150 ml of distilled water to dissolve the sample. After adding 800 ml of 95% EtOH (75% EtOH solution) to the 4 ℃ refrigerator for one day, centrifugation (10,000 rpm, 10 min) to separate the supernatant and sediment and each of them shown in Figure 1 Fractions were followed according to the flow chart.

That is, 10.4 g of ethanol (EtOH) precipitate (β-D-Glucan, containing some other polysaccharide) was obtained from 50 g of the lyophilizate, and 0.3 g of protein precipitate (PPT) was obtained by adding 10% TCA to the first supernatant. Thereafter, hexane, chlorofrom, ethylacetate, butanol, and water-soluble fractions were 0.6, 4.8, 7.9, 12.1, and 13.9 g, respectively, by sequential fractionation of organic solvents. The results are as shown in Table 1 below. The sample for analysis used the fraction obtained from 50 g of dry matter.

Table 1: Content of Solvent Fraction in Lyophilized Agaricus Mushroom Mycelium]

Experimental Example  One: Agaricus mushroom mycelium  Of extract Antidiabetic  Effect (α- glucosidase : maltase activity inhibitory effect)

Aluglucusidase (maltase, 50 μl, Sigma, USA; 0.7 U / mL) and Acarbose 5 mg / mL solution and 10 μl of Agaricus mushroom mycelium extract according to the present invention were placed in a 96-well plate and the Microplate reader (Model 550, Biorad) , USA) was used to measure OD ₄₀₅ . 5 minutes after the substrate solution (5 mM p-nitrophenyl-α -D- glucopyranoside in 0.1 M phosphate buffer, pH 7.0) was added to 50 ㎕ and inhibit enzyme from the change in absorbance by measuring the back, OD ₄₀₅ was reacted at room temperature for 5 minutes activity Was calculated.

As a result of the experiment, as shown in Table 2, when compared with the conventionally reported gold sand mushroom (Korean Patent Publication No. 2001-327), Ganoderma lucidum mushroom (cited in Korean Patent Publication No. 2001-327), akabosu, The inhibition rate of α-glucosidase was 0% in the control and 99 ± 0.7% in the acarbose treatment, 80 ± 1.3% in the lyophilized treatment of Agaricus mushroom mycelium, 104 ± 1.7% in the ethanol sediment, and 160 in the ethylacetate fraction. ± 1.5%. In other words, the freeze-dried food showed a higher inhibition rate than the gold sand mushroom and Ganoderma lucidum mushroom, but showed a lower inhibition rate than the akabos treatment. Ethanol precipitates and ethyl acetate fractions showed higher inhibition rates.

Table 2: Inhibitory effect of Agaricus mushroom mycelium composition on rat intestinal alphaglucosidase]

Experimental Example  2: Agaricus mushroom mycelium  Freeze-dried Antidiabetic  Effect (Type 1 Diabetic rats  Hypoglycemic effect in the model: Streptozotocin  ( STZ ) -induced diabetes rat experiment)

1) Short term experiment

To induce diabetes in diabetic animals, streptozotocin (STZ, Sigma Co., USA, 65 mg / kg) dissolved in 0.1 M citrate buffer (pH 4.5) was injected into the abdominal cavity of rats (SD) weighing 230 g to 270 g. Induced. One week after the administration of STZ, blood was collected from the tail vein of the fasted animal, and the blood glucose measured by the liver glucose level (Glucotrend, Germany) was considered to be diabetes when the blood glucose was 200 mg / dL or more. The experimental animal diet was prepared using the AIN-93M basal diet as shown in Table 3 below.

Table 3: Diet of Control and Lyophilized (HEAB-FD) Added Groups

Diabetic rats were divided into two groups by the ovarian method, and then fasted for 12 hours, and blood was collected from the tail vein. The control group (n = 7) was soluble starch (1 g / kg, Sigma Co., USA), and the sample administration group (n = 7) was soluble starch (1 g / kg) and lyophilized sample (HEAB-FD, 500 mg). / kg) was dissolved in physiological saline and gastric intubation. Blood glucose was measured by collecting blood from the tail vein at 30, 60, 90, 120, 180, and 240 minutes after administration. The blood glucose increase curve was calculated by calculating the blood glucose increase at each time point, and the area of the blood glucose increase curve (area under the curve, AUC) was calculated.

As a result, as shown in Figure 2 and Table 4, the blood glucose increase after starch (1 g / kg) to the fasting control group was 128.6 ± 14.0, 140.6 ± 15.7 mg / dL at 30 and 60 minutes, respectively . In the case of administration of HEAB-FD sample (500 mg / kg) with starch, blood glucose increase was 76.7 ± 5.1 and 101.0 ± 10.6 mg / dL at 30 and 60 minutes, respectively, which was significantly lower than the control group (P < 0.05).

The area under the curve (AUC) of the post-prandial blood glucose increase curve of HEAB-FD samples was 10,027 ± 944 mg · min / dL, which was significantly smaller than the control group (13,381 ± 1,306 mg? Min / dL) (P < 0.05). Therefore, it can be seen that the HEAB-FD sample has an effect of lowering postprandial blood sugar increase in diabetic rats.

Table 4. Effects of Lowering Postprandial Blood Glucose Levels in HEAB Treated Groups in STZ-Induced Type 1 Diabetic Rats

2) long-term experiment

Induced diabetes by injecting STZ (65 mg / kg) into the abdominal cavity in male rats weighing 220-250 g, and taking blood glucose from the tail vein of a fasting animal 1 week after STZ was measured above 200 mg / dL Diabetes was considered to be induced and used in the experiment. After the animals were divided into two groups by the ingot method, the control group received an AIN-93G basal diet and the experimental group received a free meal for 8 weeks with a diet containing 10% HEAB-FD sample. The carbohydrate, protein, fat, and dietary fiber contents of the control and experimental groups were the same, and the intake ratios of the complex and simple sugars were similar (Table 3).

Body weight and dietary intake of the test animals were measured twice weekly. At 8 weeks, the animals were fasted for 12 hours and sacrificed with heart puncture. Plasma was separated from the blood and used in the experiment. Blood glycated hemoglobin was measured by chromatographic method and plasma glucose was measured by enzyme method.

The experimental results showed that the glycated hemoglobin concentrations of the control and HEAB-FD sample groups were 6.9 ± 0.4% and 6.2 ± 0.3%, respectively. Low tendency was shown (FIG. 3). Glycosylated hemoglobin has been used as an important glycemic control indicator to determine the average blood sugar levels of 3-4 months ago in diabetics. Therefore, long-term intake of HEAB-FD decreased glycated hemoglobin concentration, which was effective for long-term blood glucose control.

The glucose concentration in plasma is shown in FIG. 4. Fasting glucose in the FD group of 10% HEAB was 295.7 ± 14.4 mg / dL, which was significantly lower than the control group (341.6 ± 14.6 mg / dL) (P <0.05). Therefore, HEAB-FD samples exhibiting α-glucosidase inhibitory activity in vitro and in vivo showed hypoglycemic effect in STZ-induced diabetes type 1 animal model. The most important goal in the treatment of diabetes is glycemic control, which is known to be the most important factor in the prevention and treatment of complications, which are the leading cause of death in diabetic patients. Therefore, long-term intake of agaricus active ingredient was shown to be effective in treating diabetes by reducing fasting blood sugar as well as postprandial blood sugar.

Experimental Example  3: type 1 Diabetic rats  Model (db / db mosue  Prevention and / or treatment of complications associated with diabetes mellitus in

 1) plasma triglyceride and cholesterol concentration

Plasma triglyceride and cholesterol concentrations obtained from the type 1 diabetic rats used in the test of Experimental Example 2 were measured by the enzyme method. Plasma triglyceride and cholesterol concentrations were lower in the HEAB-FD sample group (Table 5). Reducing lipid concentration in diabetes is known to help prevent diabetic complications such as kidney lesions and coronary artery disease. Therefore, ingestion of HEAB-FD may play an important role in preventing cardiovascular disease and kidney lesions.

Table 5: Serum Neutral Lipid Contents and Cholesterol Contents in a Type 1 Diabetic Rat Insulated with HEAB-FD Diet]

2) Alanine aminotransferase (GOT), Aspartate aminotransferase (GPT) activity

As a result of measuring the activity of GOT and GPT in plasma collected from the type 1 diabetic rat used in the test of Experimental Example 2, the GOT activity was 161.0 ± 7.6 U / L in the HED FD sample group (203.8). ± 18.4 U / L), significantly lower (Table 6). Plasma GPT activity tended to be lower in the FD sample group of HEAB than in the control group. GOT and GPT are enzymes that are present in a large amount in hepatocytes. When fatty liver is induced or the liver is damaged, it is leaked into the blood to increase enzyme activity. Ingestion of FD samples of HEAB has shown the possibility of inhibiting liver function damage due to diabetes.

Table 6: Plasma GOT (U / L) and GPT (U / L) Activities of Type 1 Diabetic Rats Fed HEAB-FD Diet]

3) plasma blood urea nitrogen (BUN)

The concentration of BUN in plasma collected from the type 1 diabetic rats used in the test of Experimental Example 2 was measured by the enzyme method. There was no significant difference in the plasma BUN concentration, which is a cardiac injury indicator of the control and HEAB FD sample groups (FIG. 5).

Experimental Example  4: Agaricus Mushroom Mycelium Extract  Freeze-dried Antidiabetic  Effect (Type 2 Diabetic Rat Model (db / db mosue  Investigation into hypoglycemic effect)

Four-week-old type 2 diabetic male db / db mice (n = 16) were divided into two groups and fed with AIN-93G basal diet and 10% HE lyophilized sample for 6 weeks. The carbohydrate, protein, fat, and dietary fiber content of each diet were the same, and the intake ratios of the complex sugars and the simple sugars were similar (Table 3). The animals were fed with ad libitum , and the temperature and humidity of the feeding room were maintained at 20-25 ℃ and 50-60%. The contrast was turned on and off every 12 hours.

Body weight and dietary intake of the test animals were measured twice a week. Animals fasted for 14 hours on the 6th week of the diet were sacrificed by cardiac sampling to collect blood and organs, and plasma was separated from the blood and used for the experiment. Blood glycated hemoglobin was measured by chromatography and plasma glucose was measured by enzyme method.

As a result, blood glycated hemoglobin, plasma glucose and plasma insulin concentrations are shown in Table 7 . Long-term intake of HEAB-FD samples in Db / db mice significantly reduced fasting glucose and plasma insulin levels (P <0.05). The glycated hemoglobin concentration tended to decrease in the HEAB-FD sample group. The most important goal in the treatment of diabetes is glycemic control, which shows that the active ingredient of HEAB-FD sample lowers fasting blood sugar, which is effective in treating diabetes, and the lowering of blood glucose due to the ingestion of the active ingredient of HEAB-FD sample reduces insulin demand. To reduce insulin concentration. Since blood glycated hemoglobin is an indicator of long-term changes in blood glucose, it is expected to show a significant change when the HEAB-FD sample active ingredient is taken for a longer period of time.

Table 7: Blood Glycosylated Hemoglobin, Plasma Glucose, and Plasma Insulin Concentrations in Type 2 Diabetic Rats Fed a HEAB-FD Diet]

Experimental Example  5: type 2 Diabetic rats  Model (db / db mosue  Prevent and / or treat effects of diabetes-related complications

1) Improvement of blood lipid profile

The concentration of triglyceride and cholesterol in plasma collected from the type 2 diabetic rats used in the test of Experimental Example 4 was measured by the enzyme method. The total cholesterol concentration was 178.0 ± 9.3 mg / dL, which was significantly lower than the control group (231.8 ± 9.9 mg / dL) (P <0.05). There was no significant difference in plasma triglyceride concentration between the control and HEAB-FD sample groups (FIG. 6). Long-term intake of HEAB-FD sample active ingredient may improve hypercholesterolemia and contribute to the prevention of cardiovascular complications including atherosclerosis.

2) plasma GOT, GPT activity

The activity of GOT and GPT in plasma collected from type 2 diabetic rats used in the test of Experimental Example 4 was measured by enzyme method, and the plasma GOT activity was 324.6 ± 12.8 U / L in the control group (399.4 ± 32.8). U / L) was significantly lower (P <0.05). Plasma GPT activity was lower in the HEAB-FD sample group (111.2 ± 14.0 U / L) than in the control group (146.6 ± 31.3 U / L) (FIG. 8). GOT and GPT are enzymes that are present in a large amount in liver cells. When fatty liver is induced or the liver is damaged, it is leaked into the blood to increase enzyme activity. Ingestion of HEAB-FD samples showed the possibility of inhibiting liver function damage due to diabetes.

3) Plasma BUN and Creatinine Concentration

As a result of measuring the concentrations of BUN and creatine in plasma collected from type 2 diabetic rats used in the test of Experimental Example 4, plasma BUN and creatinine concentrations of the control and HEAB-FD sample groups were not significantly different. There was no significant difference in renal function between the two groups (Table 8).

Table 8: Plasma BUN (urea nitrogen) and creatinine concentrations in type 2 diabetic rats fed the HEAB-FD diet]

4) Measurement of liver lipid concentration

The extraction of lipids from liver tissue was performed using Folch et al., And total cholesterol and triglyceride concentrations of liver lipid extracts were measured by enzyme method.

The cholesterol and triglyceride concentrations of liver tissues collected from the type 2 diabetic rats used in the test of Experimental Example 4 were 43.0 ± 1.9 mg / g protein and 73.1 ± 5.9 mg / g protein, respectively, which were not significantly different from the control group. (Table 9).

Table 9: Hepatic cholesterol and triglyceride contents of type 2 diabetic rats fed the HEAB-FD diet

5) Measurement of lipid peroxide and catalase activity in liver tissue

The lipid peroxide of liver tissue was measured by Ohkawa et al., And the content of malondialdehyde (MDA) reacting with thiobarbituric acid (TBA) was measured. The standard solution was 1,1,3,3, tetramethoxypropane (TMP). Was used. Catalase activity of liver tissue was measured by Abei method, and enzyme activity was 1 unit of enzyme that decomposes 1μ mole substrate for 1 minute.

As a result of measuring lipid peroxides and catalase of liver tissues collected from the type 2 diabetic rats used in the test of Experimental Example 4, the TBARS content (2.58 ± 0.09 nmol MDA / mg protein) of the liver tissues of the HEAB-FD sample group was Significantly reduced compared to the control (5.20 ± 0.18 nmol MDA / mg protein), the production of lipid peroxide was shown to be suppressed (Fig. 7, P <0.05). The catalase activity of the HEAB-FD sample group was 2.84 ± 0.18 U / mg protein, which was significantly increased compared to the control group (2.33 ± 0.04 U / mg protein) (FIG. 5, P <0.05).

In diabetic animals, the free radical production system is accelerated, thereby accelerating lipid peroxidation, thus easily causing peroxidative damage of tissues. Catalase converts lipid peroxides into oxygen and water to protect tissues from peroxidation. Therefore, ingestion of agaricus active ingredients in diabetic animals is thought to increase catalase activity, inhibit lipid peroxidation and improve diabetic complications. Long-term intake of agaricus active ingredient is thought to have the effect of improving diabetes by protecting diabetic animals from oxidative stress.

6) glycogen concentration and PEPCK measurement of liver tissue

Glycogen concentrations of liver tissues collected from type 2 diabetic rats used in the test of Experimental Example 4 were measured by Hassid and Abraham. PEPCK activity was measured by Chang and Lane's method, and enzyme activity was 1 unit of enzyme that degrades 1 μmole substrate for 1 minute.

There was no significant difference in liver tissue glycogen concentration and PEPCK activity of control and HEAB-FD samples (Table 10). In diabetic animals, glycogen breakdown is accelerated in the liver, and your life reaction is also accelerated, which leads to exacerbation of hyperglycemia. Ingestion of agaricus active ingredients does not have a significant effect on glycogen degradation and your life reaction.

Table 10: Hepatic glycogen content and PEPCK activity in type 2 diabetic rats fed the HEAB-FD diet]

Experimental Example  6: Study of Glucose Control Mechanism

To study the glycemic control mechanism of the active ingredient of the sample in the diabetic animal model, the male db / db mouse (n = 16), 4 weeks old, was divided into two groups, and the diet of AIN-93G basal and 10% supplemented diet was taken for 6 weeks. After 14 hours of fasting and sacrifice. Carbohydrate digestive enzyme activity was measured by small intestine mucosa, and liver and inguinal white adipose tissue (WAT) were collected to investigate the effect on gene expression of insulin receptor and glucose transporter (GLUT4).

1) Intestinal enzyme activity measurement

The α-amylase activity in the small intestine was measured by Bernfeld's method as the total reducing sugar content released by the enzyme from soluble starch. Enzyme activity was 1 unit of the amount of enzyme that liberates 1 μmole maltose for 1 minute. Α-glucosidase (maltase) and sucrase activity of the small intestine were measured by Dahiqvist's method. The enzyme activity was 1 unit of 1 μmole substrate digested for 1 minute.

As a result, α-amylase activity of fasting intestinal mucosa was 0.69 ± 0.15 U / mg protein in HEAB-FD sample group, which was significantly lower than control group (Table 11, P <0.05). Intestinal α-glucosidase (maltase) activity of the HEAB-FD sample group was 276.6 ± 11.7 U / g protein, which was significantly lower than that of the control group (304.4 ± 5.3 U / g protein) (P <0.05). The HEAB-FD sample group (157.1 ± 14.8 U / g protein) showed a lower tendency than the control group (190.5 ± 8.4 / g protein) (FIG. 9).

Carbohydrates in the diet are digested and absorbed into glucose by the action of amylase and disaccharide degrading enzymes. Therefore, long-term ingestion of HEAB-FD inhibits α-amylase and α-glucosidase activity, which slows carbohydrate digestion, thereby inhibiting post-prandial blood glucose, which in turn lowers insulin demand and also improves insulin sensitivity, leading to fasting glycemic control. It is feed.

Table 11. α-Amylase Activity of Small Intestinal Mucosa of Type 2 Diabetic Rats Fed HEAB-FD Diet]

2) Glucose transport and insulin receptor expression levels in liver and adipose tissue

The effect of long-term intake of the sample on gene expression of insulin receptor and glucose transporter (GLUT4) was investigated. Animal liver and adipose tissue (WAT) total RNA was isolated using TRI reagent (Sigma. St Louise, USA). Total RNA isolated was fractionated on 1.2% formaldehyde-agarose gel and then transferred to Hybond N + nylon membrane (Amersham pharmacia, USA). Northern blot hybridization was performed using ³² P-labeled cDNA of the target gene as a probe.

The results of investigating the effect of long-term intake of HEAB-FD samples on the gene expression of insulin receptor, glucose transporter (GLUT4) are as follows (Fig. 10). GLUT4 mRNA expression in the HEAB-FD sample group was 2.6 ± 0.2 in liver tissue, which was significantly increased compared to the control group (1.0 ± 0.1) (Fig. 12, P <0.001) and control group (1.0 ± 0.1) in adipose tissue (1.3 ± 0.1). ± 0.1) (Fig. 9, P <0.05). Gene expression of insulin receptors in hepatic and adipose tissues was not significantly different between HEAB-FD sample group and control group (FIGS. 11 and 13). Therefore, ingestion of HEAB-FD active ingredient increased GLUT4 gene expression and increased glucose transport in peripheral tissues.

Experimental Example  7: Blood Glucose Control Effects in Hepatocellular Models

Hepatic cells (HepG-2 cells) were used to measure the expression level of phosphoenolphyruvate carboxykinase (PEPCK) and glucose transporter in hepatocytes by western blot. HepG-2 cells were distributed from Korea Cell Line Bank and used DMEM medium. HepG-2 cells were transferred to 6-well and the samples were treated for 1 hour or 6 hours at concentrations of 10, 25, 50, 10 mg / ml.

Hepatocytes were 137 mM NaCl, 2.7 mL KCl, 10 mM Na ₂ PO ₄ 2 mM KH ₂ PO ₄ After washing with PBS containing (pH7.4), 50-200 uL of trition lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM β-blycerophosphate, pH 7.14, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na ₃ VO ₄ , 1% Triton X-100, 10% glycerol, 5 ug / mL leupeptin, 5 ug / ml aprotinin, 3 uM benzamidine, 0.4 mM DTT, 1 mM PMSF).

Thereafter, the protein for each sample was quantified, and the expression of 10 ug of the protein was confirmed using anti-GLUT1, anti-GLUT4 antibody and anti-PEPCK1. Membrane protein was electrophoresed on 12% SDS-PAGE followed by electrophoresis on PVDF membrane. To prevent nonspecific binding to the antibody, the membrane was reacted at room temperature for 1 hour in TBS buffer containing 3% BSA and 0.1% Tween 20 and then reacted at 4 ° C. for 16 hours. After washing the membrane, the polyclonal antibody specific for C-terminal of rat GLUT-4 was diluted 1: 1000 in Blocking buffer and reacted at room temperature for 30 minutes. After washing the membrane, the anti-rabbit IgG conjugated horseradish peroxidase was diluted at a ratio of 1 to 1000, treated at room temperature for 30 minutes, and washed again. Proteins attached to the membrane were measured using Western blotting detection system.

The results of investigating the effect of HEAB-FD sample active ingredient on the expression of glucose transporter 1 (GLUT1) are as follows (Fig. 12). When hepatocytes (HepG-2 cells) were exposed to HEAB-FD samples for 1 hour and 6 hours, GLUT1 protein expression of HEAB-FD sample group was higher than that of control group at concentrations of 10, 25 and 50 mg / mL. Although there was no significant difference.

The results of investigating the effect of HEAB-FD sample active ingredient on the expression of glucose transporter 4 (GLUT4) in HepG-2 cells (FIG. 13) are as follows. When exposed to HEAB-FD sample for 1 hour, GLUT4 protein expression was increased at 10, 25, 50 mg / mL compared to the control group, but there was no significant difference. After 6 hours of exposure to the active ingredient of HEAB-FD sample, GLUT4 protein expression was significantly increased compared to the control at concentrations of 25 and 50 mg / mL (p <0.05). Therefore, the active ingredient of HEAB-FD sample increases the expression of GLUT4 and is thought to have a blood glucose control effect.

The results of investigating the effect of HEAB-FD samples on the expression of PEPCK are as follows (Fig. 14). When hepatocytes (HepG-2 cells) were exposed to HEAB-FD sample active ingredient for 1 hour and 6 hours, PEPCK protein expression of HEAB-FD sample group decreased at 10, 25 and 50 mg / mL compared to the control group. There was no significant difference.

Experimental Example  8: menstruum Fractional sample Antidiabetic  Effect (α- glucosidase : maltase inhibition)

From 50 g of the lyophilisate, 10.4 g of EtOH precipitate (β-D-Glucan, containing some other polysaccharide) and 10% TCA was added to the supernatant to obtain 0.3 g of protein. Subsequent fractionation of organic solvents yielded 0.6, 4.8, 7.9, 12.1, and 13.9 g of hexane, chlorofrom, ethylacetate, butanol, and water-soluble fractions, respectively. The sample for analysis used the fraction obtained from 50 g of dry matter.

Inhibition rate of α-glucosidase of the fraction is shown in FIG. 0.5 mg of Hexane (HX), chlorofrom (CL), ethylacetate (EA), butanol (BT), 75% EtOH precipitate (EtOH), and 10% TCA precipitate, respectively, were compared with the inhibition rate of 0.5 mg of Acarbose (AC). It was. Acarose showed 34% inhibition, and Hexane (HX), chlorofrom (CL), ethylacetate (EA), butanol (BT), 75% EtOH precipitate (EtOH), and 10% TCA precipitates were 1.8 ± 0.2 and 2.9 ± 0.2, respectively. , 29.0 ± 3.4, 11.1 ± 1.7%, 18.7 ± 2.4, 3.8 ± 0.4. The inhibition rate of ethylacetate fraction was 29.0% and 75% EtOH precipitate was 19%. This is a high inhibition rate when comparing pure reagent acarbose with 34% inhibition of 0.5 mg treatment.

HEAB-FD sample (10 g) was dissolved in 100 mL of distilled water and then fractionated into hexane, chloroform, ethylacetate, butanol, and aqueous fraction according to polarity as shown in FIG. 1. In Table 9 and Table 12, the amount of fractions and the degree of α-glucosidase inhibition were compared. There was a big difference in the amount of each fraction. The largest fraction was the aqueous layer, about 80% of the total. Next, butanol and ethylacetate layers were 10% and 5%, respectively. The content of chloroform and hexane layers was very low. Acarbose 25 and 50 μg of inhibitory activity were 23 and 30%, respectively. The α-glucosidase activity inhibitory activity of these fractions was 62, 59, 38, and 17% of ethylacetate, hexane, butanol, and chlorform fractions, respectively.

15 is a result of comparing the activity of α-glucosidase activity of each fraction with Acarbose. 100 ㎍ of the thylacetate and hexane fractions were 187% and 170% more effective than 50 ㎍ of Acarbose, respectively, and 271% and 260% more effective than 25 ㎍ of Acarbose. The results showed that the activity of these fractions was almost the same as that of Acarbose when calculated in units. While these fractions contain the actual active ingredients, innocent enzyme inhibition assays do not inhibit the default result, ie, the actual enzymatic activity, but because they may contain substances that interfere with the enzymatic activity. Fractions other than the Ethylacetate and hexane fractions inhibited significant α-glucosidase activity but were much lower than those fractions.

Table 12: Inhibitory Effects of HEAB-FD and Solvent Fractions on Intestinal α-glucosidase Activity in Rats]

Experimental Example  9: Ethylacetate  Α- from fraction glucosidase  Isolation of Active Material

In order to separate the α-glucosidase inhibitor from the thylacetate fraction, the ethylacetate fraction was separated by a C ₁₈ reversed phase column and detected by a dual UV detector (220 and 280 nm), resulting in 10 peaks (Table 13). These were repeatedly collected and concentrated to measure their α-glucosidase activity inhibition. As a result, the highest activity peak was 3 times and inhibited about 70% of α-glucosidase activity. Other peaks showed slightly lower inhibition rates.

Therefore, the peak 3 was temporarily separated and identified by UV, TLC, and IR. As a result, it was found to be an isoflavone glycoside (multiple monosaccharides are combined). Therefore, the related isoflavone was purchased to inhibit the activity of α-glucosidase. It was measured (FIG. 17). As a result, daidzein was 33.9% genestein 85% and genistin 15%.

Table 13: Inhibitory Effects of α-glucosidase Activity on Peaks Isolated from Ethylacetate Fractions Using Reversed-phase HPLC ^{1 )}

Experimental Example  10: Clinical Trial (Effect of Glucose Improvement by Short-term Treatment of Type 2 Diabetic Patients)

To investigate the α-glucosidase inhibitory activity of the samples in diabetic patients, type 2 diabetic patients taking sulfonylurea-based drugs, oral hypoglycemic agents, were recruited. Nine patients (female) with an average age of 35-75 years and fasting blood glucose levels of 127-270 mg / dL and glycated hemoglobin levels of 6.5% or higher were selected.

To investigate the effect of a short-term dose of the sample on postprandial blood glucose, 4.6 g of glucose with rice (58.3 g of rice) was consumed (control), or lyophilized sample (15.2 g) with rice, or 1 hour before Intake. The intake of soluble carbohydrates (available carbohydrates) at each load test was 50 g. For the control group, lyophilized samples (7.6 g of solid) were taken simultaneously or 1 hour prior. When only rice was ingested, 4.6 g of glucose was added to rice in the same amount as sugar contained in 200 mL of HEAB. Intravenous blood was collected at 0, 60, and 120 minutes after eating, and centrifuged to separate plasma.

Glucose concentration was measured by enzyme method. The blood glucose increase curve was calculated by calculating the blood glucose increase at each time point, and the area (AUC) of the blood glucose increase curve was calculated. Insulin concentrations were measured by radioimmunoassay method. The experiment was conducted at least two weeks apart, diabetic patients stopped taking the drug for three days before the experiment.

General details of the test subjects are shown in Table 14. The mean age of the patients was 67.9 ± 3.0 years, disease duration was 9.0 ± 2.4 years, body mass index was 24.9 ± 1.2 kg / m ² _, In diabetic patients, fasting venous blood glucose was 139.2 ± 8.3 mg / dL, glycated hemoglobin level was 6.8 ± 0.4%, and insulin concentration was 13.4 ± 2.1 μU / mL.

Table 14: General Information of Type 2 Diabetic Patients in this Study

As a result, diabetic patients ingested only rice, or ingested HEAB-FD with rice first, followed by active ingredients 19 shows a change in blood glucose increase value when rice is ingested 1 hour after ingestion. When diabetic patients ingested HEAB-FD 1 hour before eating rice, the blood glucose increase of 60 minutes after eating was significantly lower than that of eating rice only or 1 hour before eating rice (P < 0.05).

The area of blood glucose increase after eating (AUC) was 14,700 ± 1,444 mg · min / dL with rice and 11,581 ± 1,038 mg · min / dl with HEAB-FD active ingredient at the same time, and 1 hour before HEAB-FD. Intake of 13,900 ± 1,725 mg · min / dL, AUC was significantly lower when the rice and HEAB-FD active ingredient at the same time (Table 15). Therefore, intake of HEAB-FD with meal in diabetic patients seems to have a large effect on postprandial hyperglycemia.

[Table 15. Effect of HEAB-FD Intake on Postprandial Blood Glucose Change in Type 2 Diabetic Patients]

As a result of investigating post-prandial insulin increase in diabetic patients (FIG. 18), the increase in insulin at 60 minutes after eating was significantly higher than the case of ingesting only rice and 1 hour before eating rice. (P <0.05). Therefore, in diabetic patients, ingestion of HEAB-FD active ingredient with diet is thought to have an effect on improving insulin resistance.

Experimental Example  11: Clinical Trial (Effect of Glucose Improvement by Long-Term Treatment of Type 2 Diabetic Patients)

To determine the glycemic control effect of the sample in diabetic patients, 20 patients with type 2 diabetes (12 males, 8 females) were randomly divided into two groups.

The experimental group was to take the sample for 12 weeks. Patients took 200 mL of HEAB-FD containing 7.6 g of dry weight of the active ingredient with three meals daily. As a control group, placebo (200 mL of liquid product containing 7.6 g of lactose) was taken three times daily. The body mass index (BMI, kg / m ² ) was calculated by measuring the height and weight of the patient before and after the start of the experiment, and blood pressure and body fat (Bioimpedance analyzer, TANICA, Japan) were measured. Blood was drawn from the vein on an empty stomach before and after the intake period.

Blood glycated hemoglobin concentration was measured by chromatography, and blood glucose and plasma fructosamine, cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, blood urea nitrogen (BUN), and creatinine concentration were measured by enzyme method. Plasma GOT (glutamate oxaloacetate transaminase) and GPT (glutamate pyruvate transaminase) activities were measured and insulin concentrations were measured by radioimmunoassay method. In the glucose loading test, 75 g of glucose was ingested on an empty stomach before and after the experiment period, and blood glucose and insulin were measured by intravenous blood collection at 0, 60, 120 and 240 minutes of glucose load.

Subjects were asked to maintain their daily diet and lifestyle during the experiment and report side effects such as nausea, vomiting and bloating.

The subject's body measurements are shown in Table 16. The mean age of the controls was 62.1 ± 1.7 years and the mean duration of disease was 6.3 ± 1.5 years. The mean age of HEAB-FD was 59.4 ± 2.3 years and the disease duration was 6.4 ± 1.5 years. All diabetic patients were taking sulfonylurea-based oral hypoglycemic agents. The mean body mass index (BMI) of subjects decreased to 23.8 ± 0.7 kg / m ² before ingestion of HEAB-FD active ingredient and 23.5 ± 0.8 kg / m ² after ingestion (0.3 ± 0.2 kg / m ² ) .

The amount of body fat before ingestion of HEAB-FD active ingredient was 26.3 ± 1.8% and showed a decrease of 25.9 ± 1.7% (-0.4 ± 0.2%). Body weight and waist circumference were 61.2 ± 3.3 kg and 85.5 ± 1.8 cm before ingestion, respectively, and decreased to 60.4 ± 3.3 kg and 85.0 ± 6.6 cm after ingestion, respectively. Body weight, body mass index, body fat and waist circumference of the control group were not significantly different before and after the experimental period.

Table 16: Physical Characteristics of Type 2 Diabetic Patients in this Study

In clinical trials, the effect of improving glucose tolerance was measured by the blood glucose increase curve area and insulin area result after oral glucose tolerance test performed before and after the experiment. It is shown in 17. Long-term intake of HEAB-FD active ingredient showed a tendency to decrease the area of increase in blood glucose and the area of increase in insulin during glucose loading.

Table 17: Changes in Serum Glucose and Insulin Levels after Ingestion of Glucose in Patients with Type 2 Diabetes

The results of blood glucose control are as follows. Table 18 shows the blood glycated hemoglobin and plasma glucose, fructosamine, and insulin concentrations before and after ingestion of HEAB-FD. Blood glycated hemoglobin concentration was 8.5 ± 0.5% before HEAB-FD intake but significantly decreased to 7.9 ± 0.5% after ingestion (-0.6 ± 0.1, P <0.01). Fasting plasma glucose in diabetic patients was 172.1 ± 14.0 mg / dL before HEAB-FD intake, but significantly decreased (-22.8 ± 2.8 mg / dL) after ingesting HEAB-FD for 12 weeks to 149.3 ± 12.4 mg / dL. (P <0.001).

The control of fasting blood glucose levels is also important in the control of blood glucose levels in diabetic patients, but the concentration of glycated hemoglobin, which represents the average blood glucose in the last 2-3 months, is also important. This is because glycated hemoglobin levels are highly correlated with the onset and progression of complications. Fructosamin was found to be 399.2 ± 7.4 μmol / L and 382.9 ± 6.9 μmol / L, respectively, before and after agaricus active ingredient, which was significantly decreased by the intake of HEAB-FD (-16.3 ± 3.0 μmol / L, P < 0.05). Fructosamin is a measure of glycemic control in the last two to three weeks. Plasma insulin concentration was 17.8 ± 0.8 uU / mL before HEAB-FD intake, but significantly decreased (-2.1 ± 0.4 uU / mL) after ingesting HEAB-FD for 12 weeks (15.7 ± 0.9 uU / mL). <0.05). Ingestion of agaricus active ingredient is thought to reduce blood glucose levels, thus reducing insulin demand and thus improving insulin resistance by controlling insulin secretion. Therefore, the intake of HEAB-FD active ingredient has been shown to be very helpful in improving blood sugar control and insulin resistance in diabetic patients. There were no significant differences in blood glycated hemoglobin and plasma glucose, fructosamine and insulin concentrations in the control group.

Table 18: Changes in serum glucose (mg / dL), glycated hemoglobin (%), fructosamine (μmol / L) and insulin (μU / mL) concentrations after ingestion of HEAB-FD in type 2 diabetic patients

Experimental Example  12: Preventing and / or treating the complications associated with diabetes in clinical trials (improving blood glucose by long-term treatment of type 2 diabetes patients)

1) Blood pressure lowering effect

The systolic blood pressure before HEAB-FD was significantly decreased to 148.5 ± 6.2 mmHg and 140.2 ± 4.9 mmHg after ingestion (-8.3 ± 2.2 mmHg, P <0.05). The diastolic blood pressure was 89.6 ± 6.3 mmHg before and after ingestion, There was a trend to decrease to 85.6 ± 5.2 mmHg (-4.0 ± 2.0 mmHg, Table 19). Systolic and diastolic blood pressure of the control group were not significantly different before and after the experimental period. In diabetics, a decrease in blood pressure has been reported to reduce cardiovascular complications. Therefore, ingestion of “HEAB-FD” may be helpful in controlling blood pressure and preventing and improving cardiovascular complications.

Table 19. Changes in blood pressure of type 2 diabetic patients after HEAB-FD intake

2) improved lipid metabolism

Lipid profiles before and after ingestion of HEAB-FD are shown in Table 20. Plasma triglyceride concentration was 169.2 ± 25.2 mg / dL before intake of HEAB-FD but significantly decreased to 154.4 ± 24.4 mg / dL after intake (-14.8 ± 3.7, P <0.05). Plasma cholesterol levels in diabetic patients were 213.8 ± 12.0 mg / dL before HEAB-FD intake, but significantly decreased (-22.1 ± 7.4 mg / dL) after ingestion of HEAB-FD for 12 weeks, reaching 191.7 ± 9.9 mg / dL. (P <0.05).

LDL-cholesterol concentrations were 146.8 ± 8.9 mg / dL and 138.4 ± 8.9 mg / dL before and after ingestion of HEAB-FD, respectively, which were significantly decreased due to ingestion of HEAB-FD (-8.4 ± 2.1 mg / dL, P <0.05). HDL-cholesterol concentrations tended to increase to 44.4 ± 1.8 mg / dL and 45.8 ± 1.5 mg / dL before and after ingestion, respectively. The lipid profile of the control group was not significantly different before and after the experiment. Type 2 diabetes suffers from dyslipidemia, an increase in triglycerides and LDL-cholesterol and a decrease in HDL-cholesterol, and this change is known to cause coronary artery disease, a diabetic complication. Therefore, ingestion of HEAB-FD is expected to help prevent diabetes complications by improving lipid profile.

Table 20: Changes in plasma triglyceride (mg / dL), cholesterol (mg / dL), HDL-cholesterol (mg / dL) and LDL-cholesterol (mg / dL) in type 2 diabetic patients after HEAB-FD intake

3) Improvement of liver function and kidney function

GOT and GPT concentrations, which are indicators of the level of liver function in diabetic patients, did not show significant difference before and after ingestion of HEAB-FD (Table 21). BUN and creatinine concentrations, which are indicators of renal function, were not significantly different before and after ingestion of HEAB-FD.

Table 21: Changes in GOT, GPT, BUN and creatinine in Type 2 Diabetic Patients after HEAB-FD Intake

Therefore, long-term intake of HEAB-FD in type 2 diabetic patients significantly reduced systolic blood pressure and decreased fasting blood glucose and glycated hemoglobin, fructozamin and insulin concentrations, indicating a strong glycemic control effect. In addition, ingestion of HEAB-FD significantly reduced blood triglycerides, cholesterol, and LDL-cholesterol levels, indicating an improvement in dislipidemia. Therefore, ingestion of HEAB-FD is thought to contribute to the regulation of diabetes mellitus and to improve diabetic complications.

The Agaricus mushroom mycelium extract (Agaricus mushroom mycelium lyophilisate, ethanol precipitate, ethyl acetate fraction) according to the present invention as described above is summarized as follows. 1) Lowers blood sugar and reduces insulin demand. 2) It reduces the Cholesterol level and prevents atherosclerosis. 3) Improves liver function by reducing GOT. 4) Reduce TBARS and increase catalase helps prevent diabetes complications. 5) It is not involved in glycogen degradation or your biosynthesis reaction. 6) Reduces the activity of amylase and maltase in the small intestine, thereby delaying carbohydrate absorption. 7) Promotes the expression of GLUT4 in the liver and adipose tissue, thereby promoting the absorption of sugar.

In the above description, it should be understood that those skilled in the present invention can only make modifications and changes to the present invention without changing the gist of the present invention as merely illustrative of a preferred embodiment of the present invention.

As described above, the present invention can provide a new agaricus mushroom mycelium lyophilisate, ethanol precipitate, ethyl acetate fraction and a method for preparing the same. In particular, culturing Agaricus mushroom strains; Preparing hot water extract samples by extracting the cultured hot water; Separating the culture filtrate from which residue is removed from the hot water extract sample, and concentrating the culture filtrate under reduced pressure to obtain a concentrate; It can provide a method for producing agaricus mushroom mycelium extract comprising a; and lyophilizing the concentrate.

According to the present invention, the lyophilisate, ethanol precipitate, and ethyl acetate fraction prepared therefrom have an excellent effect on the inhibitory activity of alpha glucosidase (maltase), and after eating in normal and type 2 diabetic patients. Significantly reduce blood sugar levels have an excellent effect on the prevention and / or treatment of diabetes in the human body.

In addition, the agaricus mushroom mycelium lyophilisate, ethanol precipitate, and ethyl acetate fraction according to the present invention reduced plasma cholesterol and GOT / GPT activity in diabetic patients and inhibited lipid peroxidation of liver tissues in type 2 diabetic rats. There is an excellent effect in preventing and / or treating complications caused in this regard. Therefore, the present invention is a very useful invention in the food and pharmaceutical industry because it can be developed as a food that is expected to have an excellent effect in preventing and / or treating complications in diabetic patients, as well as excellent effects in preventing and treating diabetes.

Claims (6)

delete delete Culturing Agaricus mushroom strains; Preparing hot water extract samples by extracting the cultured hot water; Separating the culture filtrate from which residue is removed from the hot water extract sample, and concentrating the culture filtrate under reduced pressure to obtain a concentrate; Lyophilizing the concentrate to prepare a lyophilisate; And Agalicus mushroom mycelium ethanol precipitate production method comprising the step of dissolving the lyophilisate in water and adding ethanol, and then separating the precipitate by centrifugation. Agaricus mushroom mycelium ethanol precipitate prepared by the manufacturing method according to claim 3, having a blood sugar lowering effect. Culturing Agaricus mushroom strains; Preparing hot water extract samples by extracting the cultured hot water; Separating the culture filtrate from which residue is removed from the hot water extract sample, and concentrating the culture filtrate under reduced pressure to obtain a concentrate; Lyophilizing the concentrate to prepare a lyophilisate; Dissolving the lyophilisate in water and adding ethanol, followed by centrifugation to separate the first supernatant; Separating the second supernatant by adding tricarboxylic acid (TCA) to the first supernatant; And Agaricus mushroom mycelium ethyl acetate fractions production method comprising the step of: obtaining a fraction by fractionating the second supernatant with ethyl acetate (ethylacetate) solution. Agaricus mushroom mycelium ethyl acetate fraction prepared by the manufacturing method according to claim 5, having a hypoglycemic effect.

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