KR20080093196A - Exopolysaccharides for type 2 diabetes therapy from phellinus baumii mycellium and a preparation method thereof - Google Patents

Abstract

Exopolysaccharides produced from Phellinus baumii mycelium are provided to reduce the glucose content in blood, and blood lipid, so that they control blood sugar level of a patient suffering from diabetes including type 2 diabetes, or improve or treat hyperlipidemia caused by obesity. Exopolysaccharides produced from Phellinus baumii mycelium have 75-85% of carbohydrates including galactose and mannose and 15-25% of protein and are obtained by culturing Phellinus baumii mycelium, filtering the cultured Phellinus baumii mycelium solution, adding ethanol into the filtered solution and storing the mixture at 4 deg. C. A food composition for treating diabetes or improving hyperlipidemia comprises the exopolysaccharides produced from Phellinus baumii mycelium as effective ingredient.

Description

Anti-diabetic activated extracellular polysaccharide derived from mushroom mycelium and its preparation method {2: 1) Exopolysaccharides for type 2 diabetes therapy from Phellinus baumii mycellium and a preparation method

1 is a situation mushroom ( Phellinus) for the concentration of glucose in the plasma in type 2 diabetic mice ( ob / ob ) baumii ) is a graph showing the hypoglycemic effect of mycelia derived extracellular polysaccharides.

2 is a situation mushroom ( Phellinus ) for the glucose concentration in plasma in type 2 diabetic mice ( ob / ob ) baumii ) is a graph showing the results of a glucose tolerance test on mycelia derived extracellular polysaccharides.

FIG. 3 is a graph showing molecular biological effects of hypoglycemic effect by examining the expression level of PPARγ gene in adipocytes isolated from type 2 diabetic mice ( ob / ob ).

4 is a graph showing molecular biological effects of hypoglycemic effect by investigating the expression of PPARγ gene in blood isolated from type 2 diabetic mice ( ob / ob ).

The present invention relates to an anti-diabetic active extracellular polysaccharide derived from S. mushroom mycelium and a method for preparing the same, and more particularly, to separate the extracellular polysaccharide from the immersion medium of Phellinus baumii mycelium and to lower the glycemic effect of the polysaccharide. The present invention relates to an antidiabetic active extracellular polysaccharide derived from a situation mushroom mycelium and a method for producing the same.

Type 2 diabetes is currently affected by 135 million people, 5-7% of the world's population, and an explosive increase is projected to 300 million in the next 20 years. Various types of oral diabetes treatments have been used to treat type 2 diabetes, but more and more patients are trying to use anti-diabetic substances derived from natural products without side effects. Continuous use of synthetic antidiabetics can cause side effects and toxicity. Therefore, even though biologically active ingredients are not revealed due to efficiency, limitation of side effects, and relatively low cost, natural substances such as herbal medicines are widely prescribed. Through long treatments, many useful experiences for treating diabetes have been accumulated in oriental medicine. Today, there are approximately 33 types of herbal medicines that have been used mainly in folk medicine prescriptions to clinically treat diabetes and its complications. Antidiabetic active substances in herbal medicines include polysaccharides, terpenoids, flavonoids, sterols and alkanoids. In many countries, efforts have been made to find natural antidiabetics derived from various medicinal plants.

Mushrooms and Cordyceps sinensis are representative natural medicinal sources with antidiabetic activity. Despite the large lack of scientific evidence, many researchers have found Tremella aurantia ), Cordyceps sinensis ) and Lentinus edodes have been working to study antidiabetic effects from fruiting bodies or mycelium of various edible and medicinal fungi. (Lo, HC et al., Life Sciences 74, 2897-2908, 2004) reported that Cordyceps Sinensis may be used as a functional food for the treatment of diabetes. Kiho, T. et al., Carbohydrate research 251, 81-87, 1994, described Agrocybe cylindracea ) fruit water extracts were claimed to have antidiabetic effects. Yuan, Z. et al., Bioscience, Biotechnology, and Biochemistry 62, 1898-1903, 1998, reported that Auricularia The antidiabetic activity of water-soluble polysaccharides from fruiting bodies of auricula - judae Que) was investigated. Nevertheless, for this purpose, efforts to obtain extracellular polysaccharides from mycelium culture medium, which are not fruiting bodies of medicinal mushrooms, have been greatly lacked until now.

Meanwhile, the situation mushroom ( Phellinus baumii ) is a mushroom that has been used as a medicinal herb for various human diseases in some Asian countries. Zhang et al., Biotechnology Letters 26, 31-33, 2004, describe Phellinus baumii ) extract may be useful for suppressing acute pneumonia in humans. In addition, Shon et al., Food Chemistry 82, 593-597, 2003, described Phellinus baumii ) extracts were characterized for their antioxidant activity and free radical scavenging ability. In order to investigate the biological activity, most researchers used Phellinus rather than the extracellular polysaccharide obtained from the mycelial immersion medium. baumii fruiting body was mainly used, but there are no reports of antidiabetic effects on extracellular polysaccharides produced by mycelial culture.

However, in Korean Patent No. 10-0663712, which the inventors have applied for a patent, the immersion culture liquid of the situation mushroom mycelium was cultured in a culture medium containing fructose, yeast extract, peptone, and calcium chloride at 30 ° C. for 4 days for shaking culture. Thereafter, antidiabetic active extracellular polysaccharides prepared by centrifugation, filtration and filtration of the culture filtrate obtained by adding ethanol, left to stand, and centrifugation are disclosed, but the extracellular polysaccharide is mainly composed of mannose 48.7. % And arabinose 38.4%, and the protein portion mainly relates to polysaccharides consisting of 14.1% arginine and 12.0% glycine.

In view of the above, the inventors of the present invention consisted of carbohydrate (78.9%) and protein (21.1%), which have strong antidiabetic activity from the mycelium immersion medium of Phellinus baumii , and the protein part is glutamic acid. It consists of 17 amino acids, including the carbohydrate portion was completed by confirming that the extracellular polysaccharide consisting of mannose and galactose can be separated.

Therefore, an object of the present invention is a situation mushroom ( Phellinus It is to provide an extracellular polysaccharide having antidiabetic activity produced from the mycelium liquid immersion medium of baumii ).

Another object of the present invention is a situation mushroom ( Phellinus It is to provide a method for producing an extracellular polysaccharide from mycelium immersion culture solution of baumii ).

The object of the present invention is to immerse the mycelium ( Phellinus baumii ) mycelium culture to obtain a mycelium culture broth, and to obtain the culture filtrate by filtering the mycelium culture broth and then to obtain the extracellular polysaccharide by extraction with ethanol added to type 2 diabetes Plasma glucose, cholesterol, and triglyceride levels in mice ( ob / ob ) were orally administered to confirm the antidiabetic activity of the extracellular polysaccharides and to separate the fat cells of the mouse PPARγ gene, a key factor in hypoglycemic activity By analyzing the molecular mechanism.

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

The present invention is a situation mushroom ( Phellinus obtaining extracellular polysaccharide from the mycelium solution immersion medium of baumii ); Confirming antidiabetic activity of the extracellular polysaccharide by orally administering the extracellular polysaccharide to type 2 diabetic mice ( ob / ob ) to measure glucose, cholesterol and triglyceride concentrations in plasma; And isolating mouse fat cells to analyze the PPARγ gene, which is a key factor in hypoglycemic activity, to identify molecular mechanisms.

The present invention has anti-diabetic activity and is composed of 75-85% carbohydrate and 15-25% protein, and the protein part is composed of 17 amino acids including aspartic acid, that is, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, Extracellular derived from mycelium culture of Phellinus baumii consisting of valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine, and the major sugars in the carbohydrate fraction are characterized by mannose and galactose Provides polysaccharides.

In the present invention, the anti-diabetic activity situation mushroom ( Phellinus baumii ), the protein part constituting the extracellular polysaccharide derived from the mycelium culture medium is aspartic acid 13 ~ 17%, threonine 5 ~ 9%, serine 6 ~ 10%, glutamic acid 12 ~ 16%, proline 4 ~ 8%, glycine 7 ~ 11%, alanine 3-7%, cysteine 1-2%, valine 2-6%, methionine 1-2%, isoleucine 1-4%, leucine 1-4%, tyrosine 1-2%, phenylalanine 1-4 %, Histidine 1-3%, lysine 8-12%, arginine 2-6%, the major sugars in the carbohydrate portion is characterized by mannose 88-94% and galactose 5-10%.

Invention situation mushroom ( Phellinus baumii ) The most preferred composition of the mycelial culture-derived antidiabetic active extracellular polysaccharide is composed of 78.9% carbohydrate and 21.1% protein, and the carbohydrate is composed of mannose 92.18% and galactose 6.24%.

The present invention is also characterized by providing an antidiabetic food composition comprising the extracellular polysaccharide as an active ingredient.

The present invention is also characterized by providing a food composition for improving hyperlipidemia comprising the extracellular polysaccharide as an active ingredient.

Invention situation mushroom ( Phellinus Extracellular Polysaccharide of mycelial culture of origin baumii) is Phellinus (Phellinus baumii ) was obtained by adding 4 times the volume of ethanol to the mycelial immersion filtrate and standing at 4 ° C.

Situation mushroom of the present invention ( Phellinus baumii ) Extracellular polysaccharide produced in mycelial culture is administered orally at 200 mg per kg body weight of the mouse on a daily basis. On the last day of the experiment, mouse cells were harvested and the entire RNA of the adipocytes was isolated to perform molecular biology experiments.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example  1: situation mushroom ( Phellinus baumii In the mycelia culture medium Extracellular polysaccharide  Produce

Situation Mushroom ( Phellinus baumii ) is used to obtain extracellular polysaccharides with antidiabetic effect ( Phellinus baumii ) was directly isolated from fresh fruiting bodies by the present inventors (Daegu University Department of Biotechnology and Functional Materials Laboratory).

To prepare the inoculated microorganism, Phellinus was firstly placed in Petri dish in PDA medium (2.4% potato dextrose broth and 2% agar). The mycelium of baumii ) was incubated, and then the agar plate was punched about 5 mm in diameter with a sterilized cutter and transferred to the seed culture medium. Seed cultures were placed in 250 mL-flasks containing 50 mL of PMP medium (2.4% potato dextrose broth, 1% malt extract, 0.1% peptone) and shaken for 4 days at 28 ° C. at 150 rpm.

To produce exopolysaccharides ( EPs ), immersion cultures of Phellinus baumii were placed in a 5 liter-stirred fermentation tank under the following culture conditions and fermented: fructose 30 g / L, yeast extract 10 g / L, peptone 10g / l, calcium chloride 0.55 g / L, magnesium sulfate 0.5 g / L; Incubation temperature: 30 ° C .; Abandonment speed: 2 vvm; Stirring speed: 200 rpm; Initial pH: 6.0; Culture volume: 3 L.

To prepare the extracellular polysaccharide, the culture was centrifuged at 10,000 x g for 20 minutes, and then the supernatant was filtered with Whatman filter paper No. 2 (Watman International, UK-made Stone, Inc.). Four times of ethanol was added to the culture filtrate, the mixture was stirred vigorously, and left overnight at 4 ° C. The extracellular polysaccharide of the present invention was prepared by centrifugation of the precipitated extracellular polysaccharide at 10,000 x g for 20 minutes, and then the supernatant was discarded and lyophilized.

Situation mushrooms as above ( Phellinus The total carbohydrate content of the extracellular polysaccharide produced in baumii ) was measured by phenol-sulfuric acid method using glucose as a standard. Total protein was measured according to the Lowry method using albumin of fetal calf serum as a standard. The protein portion of the extracellular polysaccharide was measured at 280 nm and the carbohydrate portion was measured at 480 nm.

In mycelial growth and extracellular polysaccharide production in a 5 liter-stirred fermentation tank, the maximum mycelial concentration reached about 15 g / L on day 12, whereas the maximum extracellular polysaccharide production (about 3.5 g / L) was 13 Achieved on day 1 (not shown).

The carbohydrate and protein contents of the extracellular polysaccharide were 78.9% and 21.1%, respectively, and the average molecular weight (Mw) was determined to be 1.3 ~ 1.7 × 10 ⁶ Daltons (g / mol). In the extracellular polysaccharide, the protein part is composed of 17 amino acids including glutamic acid, and the carbohydrate part is mainly composed of 92.18% of mannose and 6.24% of galactose (Table 1).

carbohydrate(%) Mannose 92.18 ± 0.33 Galactose 6.24 ± 0.27 Etc 1.59 ± 0.07 amino acid(%) Aspartic acid 15.81 ± 1.14 Threonine 7.92 ± 0.45 Serine 8.82 ± 0.32 Glutamine acid 14.40 ± 0.60 Proline 5.99 ± 0.23 Glycine 9.74 ± 0.51 Alanine 5.08 ± 0.01 Cysteine 1.16 ± 0.02 Valine 4.72 ± 0.02 Methionine 0.43 ± 0.07 Isoleucine 2.51 ± 0.10 Leucine 2.91 ± 0.14 Tyrosine 0.86 ± 0.32 Phenylalanine 2.54 ± 0.04 Histidine 1.86 ± 0.18 Lysine 10.96 ± 1.22 Arginine 4.34 ± 0.14

Example  2: situation mushrooms ( Phellinus baumii Derived from mycelium Extracellular polysaccharide Hangang Urine effect investigation

Situation mushroom ( Phellinus) obtained in Example 1 Type 2 diabetic mice ( ob / ob ) were used to investigate the antidiabetic effect of extracellular polysaccharide of baumii ). ob / ob mice are genetically deficient in leptin and have similar metabolic disorders such as obesity and hyperglycemia in insulin-independent diabetes (type 2 diabetes) patients. It is an experimental animal.

C57BL / 6 J Ham SI- ob / ob male rats (Japan, SLC) weighing 25-35 g each were housed in mouse-only cases, and 12: in an air-conditioned room (23 ± 2 ° C, 55 ± 5% humidity): The feed and water were allowed to freely diet for at least 2 weeks under a 12 hour light cycle. During the experiment, rats were fed with old food purchased from Samyang.

Because ob / ob mice are transgenic mice, hyperglycemia and obesity occur after 7 weeks of age. Glucose in the blood of mice fasted at 7 weeks of age was measured, and mice with a glucose content of> 300 mg / dl were included in the diabetic group.

All animals were randomized into two groups, 6 per group: control group, type 2 diabetic mice administered 0.9% sodium chloride saline; Treatment group, type 2 diabetic mouse treated with extracellular polysaccharide (EPS), situation mushroom ( Phellinus) baumii ) extracellular polysaccharide is administered orally daily for 54 days at a concentration of 200 mg / 1 kg body weight. On day 54, the glucose tolerance test was performed. After fasting the mice for 15 hours, the mice were orally administered at a concentration of 2 g of glucose and 1 kg of body weight, and then blood glucose levels were examined at intervals of -30, 0, 30, 60, and 120 minutes.

Based on the different experimental designs (normal dose concentration: 100-300 mg / kg) of some researchers using natural substances for the treatment of diabetes, 200 mg / kg was chosen as the appropriate dose for this experiment.

Next, gain weight and feed intake were measured by time. Blood samples of experimental animals were taken in tubes treated with 0.1 M EDTA as anticoagulant and then centrifuged at 3000 x g for 10 minutes to separate plasma. Plasma glucose, cholesterol and triglyceride concentrations were measured according to the enzyme colorimetric method (Korea, YD Diagnostic Company).

As a result, the hypoglycemic effect of the situation mushroom-derived extracellular polysaccharide on plasma glucose concentration in diabetic mice is shown graphically in FIG. 1 and the results of the glucose loading test are shown in FIG. 2.

1 is a situation mushroom ( Phellinus ) for 54 days in type 2 diabetic mice ( ob / ob ) baumii ) shows the effect of extracellular polysaccharide on plasma glucose concentration. In both diabetic groups, the initial glucose concentration in plasma was about the same within the margin of error. However, in the control group, plasma glucose concentration continued to increase during the experiment to reach a final 442 mg / L. In contrast, the extracellular polysaccharide mice of Phellinus baumii significantly lowered the plasma glucose concentration to 34% on day 52 compared with the control group.

Figure 2 is a situation mushroom Phellinus glumii ) glucose tolerance test (glucose tolerance test) was performed to re-examine the hypoglycemic effect of the extracellular polysaccharide. As a result, both control and treatment groups showed hyperglycemia after 30 minutes of glucose administration, but the treatment group showed a rapid drop in blood glucose from 60 minutes and dropped to initial blood glucose 120 minutes after glucose administration. However, the control group was found to maintain a high glucose level of more than 500 mg / dl in the blood glucose, and based on these results the situation mushroom ( Phellinus baumii ) was able to reconfirm the hypoglycemic effect of the extracellular polysaccharide.

Example  3: ob / ob  In mouse fat cells PPAR Examine the amount of γ gene expression

Adipocytes from ob / ob mice were isolated on the last day of animal testing. After separating the RNA from the adipocytes using a reagent that separates the total RNA separation, the PPARγ expression of the adipocytes was examined using RT-PCR.

Statistical significance was analyzed by one-way analysis of ANOVA test using SPSS program. All data are expressed as mean ± standard deviation (P <0.05). When measured according to the technique of protective LSD, the mean value was considered to be significant difference at P <0.05.

The effects of the administration of extracellular polysaccharide (EPS) of Phellinus baumii on gain weight and feed intake in type 2 diabetic mice ( ob / ob ) for 52 days are shown in Table 2 below.

Military ^a Weight gained (g / day) Feed intake (g / day) Feed efficiency ^b Control 0.17 ± 0.03 ^c 6.93 ± 0.17 ^* 0.01 ± 0.00 ^* Treatment group 0.21 ± 0.03 3.66 ± 0.26 ^* 0.03 ± 0.01 ^* Note: a: classification b: weight gain / feed intake c: mean ± standard deviation (n = 6). *: Significant difference compared to control, P <0.05

Situation Mushroom ( Phellinus ) for Total Cholesterol and Triglycerides in Plasma in Type 2 Diabetic Mice ( ob / ob ) for 52 Days baumii ) effect of extracellular polysaccharide (EPS) is shown in Table 3 below.

Military ^a Total cholesterol (mg / dl) Triglycerides (mg / dl) Control 154 ± 22 ^b 217 ± 73 Treatment group 210 ± 17 69 ± 1.8 ^* NOTE: Class B: Mean ± Standard Deviation (n = 6). *: Significant difference compared to NC group, P <0.05

As shown in Table 3, the total cholesterol concentration in plasma between the experimental groups during the experimental period was in the normal range. Plasma triglyceride levels, however, were significantly reduced in oral administration of Phellinus baumii oral extracellular polysaccharide (EPS) mice, but the control group recorded higher values than normal values.

3 and 4 is a situation mushroom ( Phellinus Adipose cells were isolated from mice treated with the extracellular polysaccharide of baumii ) and control mice, and the gene expression levels of PPARγ in the blood and PPARγ in the blood were examined. TZD-based drugs, type 2 diabetes drugs, act as ligands on PPARγ of adipocytes, activate PPARγ, and increase insulin sensitivity, leading to hypoglycemic effects. As shown in Figures 3 and 4 the situation mushroom ( Phellinus baumii ) also increased PPARγ gene expression compared to the control. Therefore, it can be seen that the previous hypoglycemic effect was caused by this molecular mechanism.

From the above experimental results, the present invention situation mushroom ( Phellinus baumii ) mycelium-derived extracellular polysaccharides can significantly reduce the degree of diabetes in type 2 diabetic mice ( ob / ob ), and these results suggest that the cells of the present invention mushroom ( Phellinus baumii ) mycelium derived It was confirmed that the exogenous polysaccharide may be useful in suppressing diabetes.

As described above, the present invention relates to an extracellular polysaccharide having an anti-diabetic activity obtained from the mycelium of Phellinus baumii and a method for preparing the same, Phellinus When the extracellular polysaccharide produced from the mycelium is administered to type 2 diabetic mice ( ob / ob ), not only can the glucose content in the plasma be significantly reduced, but also the excellent hyperlipidemic effect can be achieved by reducing the blood lipids. It can be obtained because it has a very excellent effect to control the blood sugar of diabetic patients or improve hyperlipidemia due to obesity, etc. It is a very useful invention in the food and pharmaceutical industry.

Claims (3)

Situation Mushroom ( Phellinus baumii ), isolated from mycelium culture medium, consisting of 75-85% carbohydrate and 17-25 % protein, the main sugars of the carbohydrate fraction are mannose and galactose , and the mannose is 88-94 %, galactose Is an antidiabetic active extracellular polysaccharide, characterized in that 5 to 10%. An antidiabetic food composition comprising the extracellular polysaccharide of claim 1 as an active ingredient. A food composition for improving hyperlipidemia comprising the extracellular polysaccharide of claim 1 as an active ingredient.

Images