KR20060020548A - Hypoglycemic effects of crude polysaccharides extracted from tremella fuciformis and the composts containing this extracts for antidiabetic effect - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of diabetes comprising the crude polysaccharide extract isolated from the baekryeok mushroom, the crude extract of the white-necked mushroom crude polysaccharide isolated through the present invention exhibits a therapeutic effect on hypoglycemia, preventing and treating diabetes It can be used as a medicine and health supplement food useful for.

Alder mushroom, crude polysaccharide, hypoglycemic effect

Description

Hypoglycemic effects of crude polysaccharides extracted from Tremella fuciformis and the composts containing this extracts for antidiabetic effect}

Figure 1. Schematic diagram of the preparation of crude polysaccharide extract of Albino mushroom

Figure 2. Monosaccharide Assay from Bio-LC of Crude Polysaccharide Extracts of Albinus Mushroom

Figure 3.Efficacy of hypoglycemic efficacy of the extract of Crude Mushroom Copolysaccharide in Streptozotocin-diabetic rats

A) Average dietary intake of individuals per group over 12 weeks (g)

B) Average weight change in each group over 12 weeks (g)

C) Average blood glucose level (mg / dL) per group for 12 weeks

Fig. 4. Experiment of hypoglycemic effect of white-headed mushroom copolysaccharide extract in KK / A ^y mice, a genetic diabetes obesity model

A) Average dietary intake of individuals in each group over 7 weeks (g)

B) Average weight change in each group over 7 weeks (g)

C) Average blood glucose level (mg / dL) per group for 7 weeks

Fig. 5. Experimental hypoglycemic effect of antidiabetic ('PMO product) containing an extract of mushroom copolysaccharide in KK / A ^y mice, a genetic diabetes obesity model

A) Average blood glucose level (mg / dL) per group for 12 weeks

The present invention relates to a white polysaccharide copolysaccharide extract and a composition comprising the same so that the bioactive fraction exhibiting excellent hypoglycemic effect from tremella fuciformis can be appropriately applied to the human body.

The mortality rate of Korean people due to diabetes increased significantly from 11.8 per 100,000 population in 1990 to 22.6 in 2000 over the past 10 years. Diabetes has become the fifth most dangerous disease among Korean deaths. There is an increasing trend. Type 2 diabetes (Adult Diabetes) accounts for over 90% of all diabetic patients and is statistically high in obese people after age 40. Type 2 diabetes mellitus is characterized by insulin resistance. Insulin resistance causes hyperinsulinemia by excessive secretion of insulin in the body, and eventually leads to necrosis of β cells, resulting in insulin deficiency, severe hyperglycemia, and weight loss. (Defronzo RA, Diabetes Rev. 5: 177-269. 1997; Polonsky KS et al., New Engl. J. Med. 334: 777-783, 1996; Reaven GM, Physiol. Rev. 75: 473-486,1995). Therefore, it is essential to improve insulin sensitivity in peripheral tissues for the prevention and treatment of type 2 diabetes mellitus, and three drugs of the troglitazone type (troglitazone, rosiglitazone, pioglitazone) Is developed. However, these drugs are not yet available for long-term safety and have been restricted in use, and troglitazone has been shown to cause severe liver toxicity and is currently banned from sale. Screening of active substances is being actively conducted in Korea as well as all over the world. Studies on natural products with antidiabetic activity revealed by previous studies include methanol extract fractions of lxeris dentata Nakai, Prunus davidiana Fr., and Phragmites communis Trin. (Coi JS et al .: Kor. J. Pharmacogn 21 (2), 153-157 (1990)) reported that the blood sugar levels of chickens were extracted from commeinae herba water in streptozotocin (STZ) diabetic rats. Fractions and Methanol Extract Fractions (Soo Young Park: Master's Thesis, Graduate School of Duksung Women's University, 1992), Methanol Extract of Bean Sprouts (Kim JI et al .: J. Korean Soc.Food Sci.Nutr. 32 (6), 921-925 ( 2003) has a hypoglycemic effect. There is also a report on the hypoglycemic effect of polysaccharides isolated from mushrooms. For example, β-glucan (Choi JM et al .: J. Korean Soc. Food Sci. Nutr. 32 (6), 1418-1425 (2000) of Agaricus mushroom. )) the SX-fraction (V of reports and Grifola frondosa shows a hypoglycemic effect in db / db mice Manohar et al .: Diabetes, Obesity and metabolism, 4:. 43-48 (2002)) the KK / a ^y There are reports of hypoglycemic effects in mice. The hypoglycemic effect of these mushrooms is thought to be due to the fiber present in the form of β-glucan in the mushrooms. Dietary fiber is a generic term for macromolecules that are not digested by human digestive enzymes, and are classified into water-soluble and insoluble dietary fiber (Trowell, 1972). It is known to have a reducing effect and a hypoglycemic effect, so the diet for diabetics recommends a food containing a lot of fiber.

The fungus is an edible mushroom belonging to the species Tremellales, Tremellaceae and Tremella, and is distributed in Korea, Japan, and China. (Park Wan-hee, Lee Ho-deuk Illustrated by Korean Medicinal Mushrooms: 632-63, Kyohaksa, Seoul). Existing studies on the biological activity of these white-eyed mushrooms include antitumor (Shigeo U et al .: Chem. Pharm. Bull., 20 (10): 2293-2294,1972), immunostimulation (Qipin G et al .: 288 (19): 135-142, 1996), serum cholesterol lowering (Peter CKC: Nutrition Research, 16 (10): 1721-1725,1996), but there are no studies in Korea.

A great feature of the fungus is that the fiber content is very high, about 63.5% of the dry matter (Hsing-Hsien C et al .: J. Agric. Food Chem. 50: 7438-7443, 2002). In addition to the previously reported physiological activity, anti-diabetic effects are expected. The present inventors extracted and isolated crude polysaccharides from mushrooms, analyzed the components of constituent monosaccharides, and verified the hypoglycemic effect in streptozotocin-diabetic rats and KK-A ^y mice, a genetic obesity-diabetes model, to prevent and treat diabetes. The present invention has been completed by revealing that it can be usefully used.

In the present invention, the pharmacological effect of the fraction obtained by extracting and separating crude polysaccharides from mushrooms in order to provide a new physiologically active material having no adverse effects on the living body and having an excellent hypoglycemic effect, by testing the pharmacological effect, diabetic rats using Streptozotocin It was confirmed that the hypoglycemic effect was excellent in KK / A ^y mice, which is a model of genetic obesity and diabetes. Therefore, an object of the present invention is to provide a crude polysaccharide isolated, extract from the white neck showing a hypoglycemic effect in type 2 diabetes model animals, and another object is to provide a composition containing the crude polysaccharide fraction extracted from the mushroom as an active ingredient By doing so it can be taken as a health functional food for blood sugar control.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention exhibits a hypoglycemic effect on type 2 diabetes, and relates to a crude polysaccharide extract and an effective functional food for glycemic control using the extract as an effective ingredient for preventing and treating diabetes.

In the present invention, to report the hypoglycemic effect of the white-necked mushroom crude polysaccharide extract through in vivo experiments, the white-necked mushroom crude polysaccharide extract obtained in the present invention is a diabetes-induced rat model using Streptozotocin and genetic obesity diabetes model KK / A ^y mice showed excellent hypoglycemic effect.

In order to verify the efficacy of the mushroom crude polysaccharide extract of the present invention, as a result of confirming the hepatotoxicity after a long-term diet (12 weeks) to the test animals, the hepatotoxicity of GPT, GOP values of the experimental group dietary crude polysaccharide extract It was concluded that the safety of mushroom crude polysaccharide extract was proven to be different from the control group. As such, the extract of cotyledon mycobacterium is a natural product with no hypoglycemic effect and other side effects or toxicity, and thus it can be used clinically for the prevention and treatment of complications caused by diabetes. In other words, it can be used as a functional food ingredient and active ingredient for blood sugar control.

1. Raw materials used in the present invention

The white mushrooms used in the present invention were imported from Fujian, China. Diabetes-induced diabetic rats were injected with streptozotocin intraperitoneally in male Sprague-Dawley rats and KK / A ^y mice, a genetic model of obese diabetes, were used.

2. Preparation of Crude Mushroom Copolysaccharide Extract

Extraction method of crude white polysaccharide extract was performed as follows. Dried white-wood mushrooms were ground with a grinder, 20 times hydrolyzed and soaked in refrigerated state for one day, followed by primary filtration. After filtration, the solution was further heated 30 times, followed by hot water extraction at 95 ° C. for 6 hours. The same amount was added to the mixture again and centrifuged at 5,000 rpm for 10 minutes to recover the supernatant. Three times the alcohol (95%, Daehan Alcohol Co., Ltd.) was added to the recovered supernatant, and white wood precipitated the crude polysaccharide, followed by centrifugation at 6,000 rpm for 10 minutes to separate only the precipitated crude polysaccharide. The isolated crude polysaccharide was lyophilized and used for the preparation of animal experiments.

3. Monosaccharide Analysis of Crude Mushroom Polysaccharide Extract

As described above, monosaccharides of crude polysaccharides prepared from mushrooms were analyzed using Bio-LC. Monosaccharide analysis conditions of the crude polysaccharide are as shown in [Table 1].

4. Biological activity test

1) Diabetes Induction Model Using Streptozotocin

Induced Diabetes

Sixteen-week-old male Sprague-Dawley rats were injected once with 45 mg / kg of streptozotocin dissolved in 0.01 M citrate buffer at pH 4.5 and adapted for 1 week with free feeding. After measuring the blood glucose, only individuals with a blood sugar value of 200 mg / dL or more were used for the experiment.

(B) Verification of hypoglycemic effect in diabetic rats using Streptozotocin

The design of the experiment was carried out between the control group of the non-dietary crude polysaccharide extracted from white agar and the experimental group in which the crude polysaccharide extracted from albino was fed to 1g per 1 kg of body weight and dissolved in drinking water, and the major factors related to blood glucose, weight and blood lipid metabolism. Through the difference, the white neck was intended to observe the antidiabetic effect of the crude polysaccharide, and the experimental period was conducted for a total of 12 weeks. Six experimental animals were designed for each group.

2) Genetic obesity diabetes model

A 10-week-old male KK-A ^y / Ta Jcl mouse was used by Clea (Tokyo, Japan). The temperature of the breeding room was raised in an air conditioning system maintained at 22 ± 2 ° C. : 08: 00 ～ 20: 00). The animals were acclimated for 1 week with free feeding, and then divided into two groups of eight animals with similar weight and blood sugar. The design of the experiment was similar to that of STZ-diabetic animals, and the control group did not feed the copolysaccharides on the whites and the whites were fed to drink 1 g per 1 kg of body weight and dissolved in drinking water, and the blood sugar, weight and glycated hemoglobin between the experimental groups. The purpose of this study was to investigate the antidiabetic effect of crude polysaccharides on white polysaccharides.

3) Collection of blood samples and measurement of body weight and blood sugar level

In the STZ diabetes-induced model, body weight and blood glucose were measured at a constant time once every 7 days. Blood glucose measurement was measured using a blood glucose meter (Rochu, Germany) using glucose oxidase reaction after fasting for 16 hours and collecting blood from the tail vein. At 12 weeks after the end of the diet, 16 hours after the fasting, the animals were sacrificed by spinal separation and a large amount of blood was obtained through heart blood collection. The collected blood was centrifuged at 5,000 rpm for 10 minutes to take the supernatant plasma and used as analytical sample. KK-Ay / Ta Jcl mice, a genetic obesity diabetic model, were measured for blood glucose and body weight at a constant time once every 7 days as in the case of STZ-diabetic model. The blood glucose measurement was measured using a blood glucose meter (Rochu, Germany) without the fasting time and the blood glucose measurement (Rochu, Germany) at the end of the dosing experiment to sacrifice a large amount of blood at the end of the diet to obtain a large amount of blood for analysis of glycated hemoglobin And plasma for insulin analysis were separated and used as analytical samples.

4) Biochemical Measurement

We measured the concentration of triglycerides, total cholesterol and HDL-cholesterol, which are important factors related to lipid metabolism, from plasma collected from STZ-induced diabetic model. The levels of triglyceride, total cholesterol and HDL-cholesterol in blood were determined by colorimetric method using analytical kits of Asan Pharmaceutical.

Glycosylated hemoglobin concentration was quantified by kit (BioSystem, Spain) using affinity chromatography from the starch from KK-Ay / Ta Jcl mice, a genetic obesity diabetes model. LINCO Research, USA).

5. Statistical Processing

The experimental results were expressed as mean and standard deviation, and the statistical significance of the mean values between the groups was verified by student t-test.

(Example 1): (Analysis of monosaccharides from Bio-LC of angiosperm mushroom polysaccharide fraction)

As in the process described in Figure 1, the crude polysaccharide prepared from the mushrooms were analyzed using the Bio-LC monosaccharide components.

As shown in FIG. 2, glucose was found in 12.04 components, mannose in 12.83 components, and xylose peaks in 13.34 components. The composition ratios were 5.9%, 27.7%, and 60.4%. In other words, xylose was the most abundant, followed by mannose and glucose. In general, mushroom-derived crude polysaccharides are known to have the highest content of glucose, but they showed different results and are considered to be the characteristics of this crude polysaccharide component.

Example 2 (Verification of Hypoglycemic Effect in Diabetic-Induced Rats Using Streptozotocin of Mushroom Crude Polysaccharide Fraction from Albinus)

A) Dietary Intake and Weight Change

The results of observing the dietary intake during the 12 weeks of breeding were as shown in (A) of FIG. 3, which shows that the experimental group fed the white crude mushroom copolysaccharide fraction had higher dietary intake in the first and second weeks than the control group. In addition, dietary intake continued to decrease from week 3 and continued until week 12 after the end of the experiment. This was thought to be because the satiety intake of drinking water containing crude polysaccharides was relatively higher than the control group, and there was no difference in drinking water intake between the two groups. Changes in body weight showed a tendency to decrease continuously until week 12 after the experiment. This is a representative symptom in STZ-induced diabetes. Insulin production is reduced due to the destruction of β-cells in the body following STZ administration, resulting in a lack of energy production due to glucose metabolism. This is explained as being reduced. As shown in FIG. 3 (B), when the weight change between the control group and the experimental group was examined in more detail, the control group showed a slightly higher weight than the experimental group, but there was no significant difference. Judging by the difference.

B) changes in blood glucose

Changes in blood glucose levels of the test subjects with diabetes induced by STZ in the experimental group and the control group are shown in FIG. The initial mean blood glucose levels of the control and experimental groups were 301.75 mg / dL and 316.12 mg / dL, respectively. However, in the case of the control group, the blood glucose increase continued from the start of the experiment to the end of the experiment, and thus the average blood sugar of the individual was 462.3 mg / dL. On the other hand, in the experimental group, a clear hypoglycemic effect was observed from the 9th week of the start of the diet and continued until the end of the experiment. At the end of the experiment, the average blood glucose level was 362 mg / dL, which was significantly lower than the control group (p < 0.05).

C) blood triglyceride and cholesterol concentration

The effect of white neck on triglyceride and cholesterol concentrations in blood of STZ-induced diabetic rats treated with crude polysaccharide for 12 weeks is shown in [Table 2]. Serum triglyceride content was 125.9 ± 11.8 mg / dL in the control group and 70 ± 24.5 mg / dL in the experimental group fed with white polysaccharides (p <0.5). In general, the most common symptom of lipid metabolism in diabetic patients is hypertriglyceridemia, which accelerates the development of cardiovascular complications of diabetes. Therefore, lowering triglyceride content in blood is also an important factor in the treatment of diabetes mellitus, and the white polysaccharide is thought to be helpful for the prevention and treatment of hypertriglyceridemia. The total cholesterol level in the control group was also 103.4 ± 11.9mg / dL in the control group and 92 ± 10.4mg / dL in the experimental group, showing a tendency to decrease.HDL cholesterol concentration was 48.9 ± 8.1mg / dL in the control group and 50.5 ± in the experimental group. 5.4 mg / dL was shown to increase in the experimental group.

Example 3 Genetic Obesity Diabetic Model Mice of Crude Mushroom Copolysaccharide Fraction (KK / A ^y Hypoglycemic effect in

A) Dietary Intake and Weight Change

The dietary intake for 7 weeks of experiment breeding is shown in (A) of FIG. 4, and there was no significant difference between the control group and the experimental group. However, the dietary intake of the experimental group was rather low. As shown in FIG. 4B, the change in body weight was similar to the change in dietary intake. In detail, the mean weights of the initial control group and the experimental group were 41.6 ± 1.8g and 42 ± 1.8g, respectively, and there was no significant difference, and the mean weight of 7 weeks after the experiment was also 44.7 ± 2g and 43.8 ± 3g. There was no difference.

B) changes in blood glucose levels

Experimental results of the white polysaccharide administration of KK-A ^y / Ta Jcl mice are shown in FIG. 4 (C). As shown in the figure, the initial blood glucose level of 475.5 mg / dL in the control group increased continuously until the end of the experiment, and rose to 544.4 mg / dL at the 7th week of the experiment, whereas the experimental group in which the white tree was fed the crude polysaccharide The blood glucose level decreased from 516.75mg / dL throughout the experimental period and significantly lower blood glucose level than the control group from the 5th week (p <0.01), and the mean blood glucose level was 409.25mg / dL at the 7th week of the experiment. Significantly decreased (p <0.001). As a result, the blood sugar level of the diet group fed with the white polysaccharide was decreased by 20.9%, 27.34%, and 24.9% at the 5th, 6th, and 7th week of the experiment, respectively, compared to the control group. It was found that there was an effect of reducing (p <0.01, p <0.001).

C) blood insulin and glycated hemoglobin concentration

Most patients with type 2 diabetes have hyperinsulinemia, which is an important common factor in the pathogenesis of lipid metabolism disorders, hypertension and coronary atherosclerosis, which are common in diabetic patients. Therefore, the reduction of insulin is a very important factor in the prevention and treatment of diabetic complications. As a result of measuring blood insulin concentration of KK-A ^y / Ta Jcl mice, the control and experimental groups showed significant difference of 2.02 ± 0.73ng / mL and 1.22 ± 0.59ng / mL, respectively. (p <0.05). These results are believed to be due to the decrease in insulin secretion stimulation due to the hypoglycemic effect of white polysaccharides diet. In diabetic patients, hyperglycemia promotes nonspecific glycosylation of proteins in the blood, and hemoglobin also undergoes nonspecific glycosylation, thus increasing glycated hemoglobin levels. Therefore, glycated hemoglobin levels are used as an indicator of long-term glycemic control in diabetic patients. In this study, the concentration of glycated hemoglobin was measured from KK-A ^y / Ta Jcl mice as an indicator to confirm the hypoglycemic effect of crude polysaccharides. . After 6 weeks of dieting the copolysaccharides in the white neck, the glycated hemoglobin concentrations of the control group and the experimental group were measured. These results indicate that the white neck through the blood sugar level has the same tendency as the hypoglycemic effect of the crude polysaccharide, and the white neck further supports the hypoglycemic effect of the crude polysaccharide.

(Example 4): (Preparation of hypoglycemic composition comprising a white polysaccharide fraction)

To prepare a blood sugar lowering composition comprising the crude polysaccharide fraction as a main ingredient. The compounding ratio of the prepared composition is shown in [Table 4]. Hypoglycemic effect of the leaf water mushroom hot water extract and cold water extract in the compounding ratio has also been verified by the present inventors. The blood glucose lowering composition comprising a crude polysaccharide fraction of white neck was quantified according to the blending ratio shown in Table 4, and then mixed using an experimental stirrer, and sterilized by boiling at 105 ° C. for 30 minutes to complete the preparation of the blood glucose lowering composition.

Example 5 KK / A of Hypoglycemic Composition ('PMO-product') ^y  Hypoglycemic efficacy in mice)

The hypoglycemic effect of PMO-product was verified using KK / A ^y mice. The design of the experiment was divided into 1% dietary group by adding 1% of dietary control and PMO product, and comparing the difference in blood glucose and the difference in plasma insulin and glycated hemoglobin concentration between these two groups. The purpose of this study was to verify the hypoglycemic effect of. The experimental animals used in this experiment were male KK-A ^y / Ta Jcl (genetic obesity type 2 diabetes model) mice, which were sold by Clea (Tokyo, Japan), were acclimated for 2 weeks, and weighed at 6 weeks of age. The blood sugar was divided into two groups of 10 animals each. Body weight and dietary intake were measured weekly, and blood glucose was collected from the tail vein without fasting and weekly blood glucose changes were measured. As shown in FIG. 5, the blood glucose change by PMO-product intake for 12 weeks was shown, and the results of this study showed that the intake of PMO product was significantly (p <0.05) compared to the control group in the 1% diet group from the 5th week of diet. Inhibition of blood sugar increase was found, and a 12-week dietary intake showed a significant (p <0.05) inhibitory effect.

The initial blood sugar level was not high at about 213 mg / dl, but the blood sugar level gradually increased, resulting in the maintenance or elevation of hyperglycemia from the 5th week in the control group to 500 mg / dl or higher in the control group, while the 1% dietary group at 7th week. Going on, blood sugar levels reached 500 mg / dl and then decreased.

The results of analysis of plasma insulin concentration and glycated hemoglobin after the conclusion of the empirical formula are shown in [Table 5]. In detail, the plasma insulin concentration and glycated hemoglobin concentration in the 1% diet group were significant (p < 0.05) showed a decreasing effect to support the hypoglycemic effect of the PMO product.

As described through the above embodiment, the crude polysaccharide isolated from the white agar mushroom has an excellent effect that can be used for the prevention and treatment of diabetes, the present invention is a very useful invention in the health food industry for the prevention and treatment of diabetes. .

Claims (2)

Crude mushroom polysaccharide extract effective in treating and preventing diabetes obtained by separating the precipitate obtained by filtration of Tremella fuciformis by cold water immersion and distilling the hot water extract with three times the volume of alcoholic solution Claim 1, white blood sugar lowering health functional food composition containing the extract of the mushroom polysaccharide as an active ingredient.

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