WO2014112666A1 - Method for preparing extract from culture medium of ceriporia lacerata and pharmaceutical composition prepared thereby for preventing or treating diabetic diseases and diabetic complications, which contains extract from culture medium of ceriporia lacerata as active ingredient - Google Patents

Abstract

The present invention relates to a method for preparing an extract from the mycelial culture medium of Ceriporia lacerata and a pharmaceutical composition for preventing or treating diabetic diseases and diabetic complications prepared by the preparation method. According to the method for preparing an extract from the mycelial culture medium of Ceriporia lacerata, the extract contains a relatively high content of expolysaccharides in comparison with the conventional preparation method according to a prior document by these inventors and thus will be capable of being used as an ingredient for a pharmaceutical composition for diabetic diseases or a relevant functional food.

Description

A pharmaceutical composition for the prevention or treatment of diabetic diseases and complications of diabetes mellitus containing the method of preparing the extract of the seriporia racerata culture medium and the extract of the seriporia racerata culture solution prepared therefrom

The present invention relates to a method for producing Ceriporia lacerata mycelium culture extract and a pharmaceutical composition for preventing or treating diabetic diseases and diabetic complications prepared by the method.

Ceriporia lacerata is a type of white fungus. In order to use carbon sources such as cellulose and hemicellulose in the ecosystem, it performs a joint metabolism called lignin decomposition.

Very few studies have been conducted on the industrialization of Ceriporia lacerata , probably because the presence of Ceriporia lacerata was first reported to academia in 2002. And only two studies on bleaching.

The study of food and pharmaceutical formulations is Korean Patent No. 10-1031605 filed by the present inventors "Method of preparing a extract of Ceriporia laccerata culture for the prevention and treatment of the disease of diabetes and Ceriporia lacserata accordingly Culture extract ”is internationally unique. However, the document only mentions the effect on type 1 diabetes.

Diabetes treatment drugs that have been developed up to now include hypoglycemic agents and insulin injections, but they only delay the worsening of diabetes and have no meaning as a preventive and cure agent for diabetic complications.

In order to treat diabetes and diabetic complications, the first step is to develop a substance that stops the progression of the disease, and secondly, to develop or promote the regeneration of pancreatic beta cells responsible for insulin secretion regulation.

The results of the present study indicate that Ceriporia lacerata mycelium culture medium and its indicators and extracellular polysaccharides are very effective in blocking the progression of diabetes and promoting the regeneration of pancreatic beta cells. As a result, there is an urgent need for researches to identify the structure of the extracellular polysaccharide of Ceriporia lacerata and to find an optimal culture method for increasing its content.

The present invention has been made to solve the problems of the prior art as described above, the problem to be solved in the present invention is improved Ceriporia lacerata ( Ceriporia lacerata ) mycelium culture medium that can enhance the content of extracellular polysaccharides It is to provide a method for producing an extract.

The problem to be other solutions of the invention are three reports Ria la sera other (Ceriporia lacerata) three reports of the amount of extracellular polysaccharide produced from the manufacturing method of the mycelia culture extract increased Ria la sera other (Ceriporia lacerata) mycelium of the present invention It is to provide a pharmaceutical composition for preventing or treating diabetic diseases and diabetic complications containing the culture extract as an active ingredient.

In order to solve the above problems, the present invention is Ceriporia lacerata ( Ceriporia lacerata) comprising a liquid culture of Ceriporia lacerata mycelium, dry powdering of the culture solution and the preparation of a solvent extract ( Ceriporia lacerata) ) In the method for producing mycelium culture extract, Ceriporia lacerata mycelium culture medium 1 ~ 2% by weight of sugar, 0.2 ~ 1% by weight of glucose, 0.2 ~ 1% by weight of starch, 0.1 ~ 0.5% by weight, wheat flour 0.1-0.5% by weight, soy flour 0.2-2% by weight, magnesium sulfate (MgSO ₄ ) 0.05-0.1% by weight, potassium monophosphate (KH ₂ PO ₄ ) 0.05-0.1% by weight, potassium diphosphate (K ₂ HPO ₄ ) Preparation of Ceriporia lacerata mycelium culture extract containing 0.05 ~ 0.1% by weight and 92 ~ 98% by weight of water, characterized in that the hydrogen ion concentration is pH 4.5 ~ 6.0 Provide a method.

The culturing is preferably performed under a blue LED light source.

The culturing is preferably carried out by maintaining the concentration of carbon dioxide at 1,000 ~ 2,000ppm.

In addition, the present invention is a diabetic disease containing Ceriporia lacerata mycelium culture medium extract prepared by the method for producing a Ceriporia lacerata mycelium culture medium extract of the present invention as an active ingredient And it provides a pharmaceutical composition for the prevention or treatment of diabetes complications.

The diabetic disease may be type 2 diabetes.

The diabetic complications may be selected from the group consisting of chronic hyperglycemia, atherosclerosis, microangiopathy, diabetic retinopathy and kidney disease.

According to the method for producing a Ceriporia lacerata mycelium culture extract of the present invention, compared to the preparation method according to the prior art by the present inventors, the extracellular polysaccharide is contained in the extract in a higher content than the diabetic It may be applied as a material for the pharmaceutical composition of the disease or related functional foods.

Figures 1a and 1b shows the results of analyzing the ITS-5.8S rDNA sequence of the Ceriporia lacerata strain of the present invention.

2A and 2B are graphs showing the results of cultures and results showing residual sugar content according to pH and types of sugars in culture of a Ceriporia lacerata strain.

Figure 3 is a graph showing the mycelia growth and the content of extracellular polysaccharides according to the type of sugar.

Figure 4 is a graph showing the mycelia growth and the content of extracellular polysaccharides according to glucose concentration.

5 is a graph showing the mycelia growth and the content of extracellular polysaccharide according to the nitrogen source.

6 is a graph showing the mycelial growth and the content of extracellular polysaccharides according to the concentration of soy flour as a nitrogen source.

7 is a graph showing the mycelia growth and the extracellular polysaccharide content according to the trace elements.

8 is a graph showing the mycelia growth and the content of extracellular polysaccharides according to the concentration of trace elements MgSO ₄ .

9 is a graph showing the mycelial growth and the content of extracellular polysaccharides over time in a 5L incubator.

10 is a graph measuring the molecular weight of the extracellular polysaccharide according to the culture purification.

Figure 11 schematically shows an experimental procedure for testing the activity against diabetes of the Ceriporia lacerata mycelium culture extract of the present invention.

12 is a graph showing the food intake by treatment of Ceriporia lacerata mycelium culture extract in type 2 diabetic rats.

FIG. 13 is a graph showing water intake by treatment of Ceriporia lacerata mycelium culture extract in type 2 diabetic rats. FIG.

Figure 14 is a graph showing the weight gain by the treatment of Ceriporia lacerata mycelium culture extract in type 2 diabetic rats.

Figure 15 is a photograph showing the liver status of normal mice, type 2 diabetic mice and mice treated with Ceriporia lacerata mycelium culture extract.

16 is a graph showing the blood glucose concentration over time on an empty stomach.

17 is a graph showing blood glucose concentrations over time after oral administration of glucose.

FIG. 18 is a graph showing blood glucose concentrations after sacrifice of mice supplemented with Ceriporia lacerata mycelium culture extract for 6 weeks.

19A and 19B are graphs and micrographs showing that Ceriporia lacerata mycelium culture extracts promote adipocyte differentiation in a manner similar to insulin.

20 is a graph showing the degree of adipocyte differentiation of Ceriporia lacerata mycelium culture extract with or without insulin.

21A and 21B are data showing insulin signaling by Ceriporia lacerata mycelium culture extract in adipocytes.

22 is a graph showing the expression level of GLUT4 by the extract of Ceriporia lacerata mycelium culture medium in adipocytes.

The present invention provides a method for preparing a Ceriporia lacerata mycelium culture extract comprising liquid culture of Ceriporia lacerata mycelium, dry powdering of the culture broth and preparing a solvent extract. , Ceriporia lacerata ( Ceriporia lacerata ) mycelium culture medium 1 ~ 2% by weight sugar, 0.2 ~ 1% by weight of glucose, 0.2 ~ 1% by weight starch, 0.1 ~ 0.5% by weight, 0.1 ~ 0.5 wheat flour % By weight, soy flour 0.2-2% by weight, magnesium sulfate (MgSO ₄ ) 0.05-0.1% by weight, potassium monophosphate (KH ₂ PO ₄ ) 0.05-0.1% by weight, potassium diphosphate (K ₂ HPO ₄ ) 0.05-0.1 It provides a method for producing a Ceriporia lacerata mycelium culture medium extract comprising a wt% and 92-98 wt% of water, characterized in that the hydrogen ion concentration is pH 4.5 ~ 6.0.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The inventors of the present invention found that the indicator material excellent in blocking the progression of diabetes and diabetic complications and promoting the regeneration of beta cells is an extracellular polysaccharide of Ceriporia lacerata mycelium culture extract. In addition, the present invention has been completed for a method for preparing a Ceriporia lacerata mycelium culture extract which can increase the content of polysaccharide and a pharmaceutical composition for preventing or treating diabetes and diabetic complications.

Accordingly, the present invention provides a method for preparing a Ceriporia lacerata mycelium culture extract comprising liquid culture of Ceriporia lacerata mycelium, dry powdering of the culture broth and preparing a solvent extract. In the Ceriporia lacerata mycelium culture medium 1 ~ 2% by weight of sugar, 0.2 ~ 1% by weight of glucose, 0.2 ~ 1% by weight of starch, 0.1 ~ 0.5% by weight of wheat, 0.1% of wheat flour 0.5 wt%, soy flour 0.2-2 wt%, magnesium sulfate (MgSO ₄ ) 0.05-0.1 wt%, potassium monophosphate (KH ₂ PO ₄ ) 0.05-0.1 wt%, potassium diphosphate (K ₂ HPO ₄ ) 0.05 It provides a method for producing a Ceriporia lacerata mycelium culture extract, comprising ~ 0.1% by weight and 92-98 % by weight of water, characterized in that the hydrogen ion concentration is pH 4.5 ~ 6.0.

The culturing is preferably performed under a blue LED light source.

The culturing is preferably carried out by maintaining the concentration of carbon dioxide at 1,000 ~ 2,000ppm.

As a preferred embodiment, the method for producing a Ceriporia lacerata mycelium culture extract of the present invention can be prepared by the following method.

(a) three other reports Ria la sera (Ceriporia lacerata) three reports cultured mycelium Ria la sera other (Ceriporia lacerata) obtaining a mycelium culture liquid;

(b) powdering the culture by vacuum drying or lyophilization; And

(c) extracting said powder with at least one solvent selected from the group consisting of water, ethanol and methanol.

(A) the three reports in step Ria la sera other (Ceriporia lacerata) three reports Ria la sera other (Ceriporia lacerata) mycelium to obtain a liquid culture of the mycelium is the extracellular polysaccharide (Exopolysaccharide) by culturing in a liquid The medium composition for the liquid culture is 1 to 2% by weight of sugar, 0.2 to 1% by weight of glucose, 0.2 to 1% by weight of starch, 0.1 to 0.5% by weight of water, 0.1 to 0.5% by weight of wheat flour, 0.2 to 2% of soy flour. Weight%, magnesium sulfate (MgSO ₄ ) 0.05 to 0.1 weight%, potassium monophosphate (KH ₂ PO ₄ ) 0.05 to 0.1 weight%, potassium diphosphate (K ₂ HPO ₄ ) 0.05 to 0.1 weight% and water 92 to 98 weight It may include%.

At this time, liquid culture maintains 20 ~ 25 ℃ of hydrogen ion concentration (pH) 4.5 ~ 6.0, light source maintains blue LED, illuminance 0.5LUX, injects air at 0.5 ~ 1.5 (kgf / cm ² ) and carbon dioxide concentration is 1,000 It is preferable to perform 8 to 13 days while maintaining at ~ 2,000PPM, and 10 days at 22 ℃, pH 5, 1.0 (kgf / cm ² ), 1,500PPM conditions is most likely because of the high content of extrapolysaccharide (Exopolysaccharide) desirable.

The parent strain in step (a) is one of the superior strains stored at 4 ℃ in the PDA medium, using a PDB medium in the Erlenmeyer flask to maintain a constant temperature of 25 ℃ in a shaker incubator for 7 to 9 days Use rough ones. At this time, the amount of mycelia to be added to the inoculum is most preferably 0.5% of the solution to be cultured. Since the high mycelial mass (% / 100ml) does not increase the content of extracellular polysaccharides, the media composition is a selective culture condition that forms the highest content of extracellular polysaccharides, not the best nutritional ratio and environmental conditions for the growth of mycelia. Should apply.

The culture solution is separated and purified into a mycelium and an aqueous solution. The separation tablet was repeatedly purified to remove the mycelium with a centrifuge with a multi-sheet filter press and a vibration centrifugal separator (PALLSEP), and then irradiated with ultraviolet (UV) light for 1 minute. It should also be kept sealed after removing oxygen. This is because the presence of mycelia in the solution causes a change in the content of the active ingredient by the growth of the mycelia.

In step (b), the mycelia culture solution prepared in step (a) is powdered by vacuum drying or lyophilization. The drying is preferably carried out for 48 hours to 96 hours at a low temperature of 40 ° C or less, preferably 30 ° C or less, since a substantial portion of the effective substance may be lost when the drying is carried out at a high temperature. And, in the step (b), it is more preferable to use the vacuum freeze dryer than the vacuum dryer which sets the evaporation temperature relatively high because the change of the effective substance content is minimized.

In step (c), the mycelium culture broth obtained in step (b) is extracted with a solvent, and the seriporia racerata according to the present invention (Ceriporia lacerata) Mycelium An extracellular polysaccharide, which is a culture extract, is isolated and prepared.

The process is well suspended by adding 100 mL of distilled water to 5 g of dry powder, followed by centrifugation (8,000 rpm, 20 min) to add a cold alcohol corresponding to 2 to 3 times the amount of the supernatant thereof and a refrigerator (4 ° C). ) And let stand for 12 hours.

After centrifugation (8,000 rpm, 20 min) again only the supernatant from the stationary material, the precipitate is recovered to prepare crude Exopolysaccharide. The extract is preferably vacuum freeze dried at 30 ℃ or less.

Thus Ceriporia racerata produced by the present invention (Ceriporia lacerata) Mycelium The extract of the culture solution has a remarkably high content of active ingredients effective in the treatment of steroid-induced diabetes, and is very effective in stopping and treating the progress of related diseases and complications. More specifically, the seriporia racerata according to the present invention (Ceriporia lacerata) Mycelium The culture extract contains extracellular polysaccharides known to have antidiabetic effects in a very high content of 0.3 ± 0.03% / 1L in culture and 5.00 ± 0.02% / 100g in dry extract.

In addition, the present invention is a diabetic disease containing Ceriporia lacerata mycelium culture medium extract prepared by the method for producing a Ceriporia lacerata mycelium culture medium extract of the present invention as an active ingredient And it provides a pharmaceutical composition for the prevention or treatment of diabetes complications.

The diabetic disease may be type 2 diabetes.

The diabetic complications may be selected from the group consisting of chronic hyperglycemia, atherosclerosis, microangiopathy, diabetic retinopathy and kidney disease.

The pharmaceutical composition containing the Ceriporia lacerata mycelium culture extract prepared by the method for producing the Ceriporia lacerata mycelium culture extract of the present invention as an active ingredient is commonly used. Suitable carriers, excipients and diluents may further be included.

For example, in the preparation of powders, the extract 200mg, fine powder 100mg, talc 10mg is prepared by mixing and then filled in airtight cloth.

For example, the tablet is prepared by mixing the extract 100mg, fine powder 50mg, lactose 10mg, magnesium stearate 2mg and then tableting.

For example, the preparation of the liquid preparation is prepared by suspending 100 ml of the extract, 5 g of isomerized sugar, a suitable amount of scent of fragrance, and a suitable amount of preservative to fill the brown bottle. In this case, instead of the extract, the resultant of step (a) may be used directly.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

EXAMPLE

1. Preparation of Ceriporia lacerata Culture Extract

1-1. Preparation of Ceriporia lacerata Cultures

Ceriporia lacerata was isolated from the oak reimbursement section, and then frozen and stored at -80 ° C in the embryos grown by subculture, and the stored strain was stored in PDA medium (87 plastic culture). After ˜3 passages, only complete strains of sufficient quantity were stored and used in a 4 ° C. refrigerator. Then, after preparing 600ml of PDB medium in the Erlenmeyer flask, one PDA culture strain was added and shaken for 8 days. Then, 1.5% by weight of sugar, 0.5% by weight of glucose, 0,5% by weight of potato starch, 0.25% by weight of wheat flour, 0.25% by weight of water, magnesium sulfate (MgSO ₄ ) 0.05% by weight, potassium monophosphate (KH ₂ PO ₄ ) A liquid culture medium containing 0.05% by weight, 0.05% by weight of potassium diphosphate (K ₂ HPO ₄ ) and 96.85% by weight of water was sterilized for 20 minutes at 121 ° C., 1.5 kgf / cm ² in an 800 l fermenter and then cooled to 23 ° C. Inoculate 600 ml of PDB culture strain to be used as a starter in one state, and ventilate air at 0.5 to 1.5 (kgf / cm ² ), while the concentration of carbon dioxide is 1,000 to 2,000 PPM, and the Ceriporia lacerata mycelium is Ceriporia lacerata mycelium culture was prepared by liquid culture at a constant temperature of 23 ° C. for 10 days.

1-2. Preparation of Ceriporia lacerata Culture Extract

The prepared Ceriporia lacerata mycelium culture was powdered by lyophilization for 72 hours at a low temperature of 25 ℃ using a vacuum freeze dryer. Suspend well by adding 100 ml of distilled water to 5 g of dry powder, centrifugation (8,000 rpm, 20 minutes), and add a 2-fold to 3 times the amount of cold alcohol to the supernatant thereof and place in a refrigerator (4 ° C). Let time stand. After centrifugation (8,000rpm, 20 minutes) again only the supernatant from the stationary material, the precipitate was recovered to extract crude Exopolysaccharide. Crude extracellular polysaccharide was dried in a freeze dryer for 72 hours to obtain complete Exopolysaccharide.

2 . Optimization Conditions for Ceriporia lacerata Liquid Culture

2-1. Experiment method

2-1-1. Culture condition

Physicochemical characteristics and mycelium and extracellular polysaccharide content of carbohydrate and protein trace elements were measured to optimize Ceriporia lacerata liquid culture conditions according to the shaking flask culture conditions. The type of carbon source was evaluated by glucose, sucrose, lactose, fructose, and galactose according to the type and concentration (3 ~ 5%). The nitrogen source was tryptone, yeast extract, soy flour, L-glutamic acid, ammonium persulfate, malt extract. We evaluated the characteristics according to the type and concentration of peptone (0.25%). As the trace element type, the characteristics of KH ₂ PO ₄ , MgSO ₄ , ZnSO ₄ , CuSO ₄ , FeSO ₄ , and CaCl ₂ were evaluated according to the concentration of 0.1-0.5%. As a culture condition, a total volume of 800 mL in a 1,000 mL Erlenmeyer flask was incubated at 25 ° C. at a speed of 120 rpm for 8 days, and then analyzed. In the 5 L jar fermenter, the total volume was 3 L and the physicochemical characteristics of the culture days were analyzed.

2-1-2. pH, acidity and sugar determination

pH was measured using a pH meter, and sugar content using an electronic sugar meter. Acidity was measured by taking 10 mL of the culture solution and measuring the pH meter. After adding 0.1 N NaOH until the pH of the culture solution was 8.3, the amount of 0.1 N NaOH consumed was measured. It was calculated by comparing the tartaric acid content.

2-1-3. Method for measuring mycelia and extracellular polysaccharide content

The culture was centrifuged at 12,000 x g for 20 minutes, the precipitated precipitate was washed three times in distilled water, filtered and the filtrate was lyophilized and weighed to determine the mycelial yield. Centrifuge the culture at 12,000 x g for 20 minutes and add cold iso-propyl alchol corresponding to twice the volume to the supernatant. After overnight at 4 ° C, centrifuged at 12,000 x g for 20 minutes to dissolve the settled precipitate in distilled water and freeze-dried to weigh the mycelia.

2-1-4. Tyrosine content, protease, α-amylase Activity measurement, thrombolytic enzyme activity measurement

In order to measure the degree of peptide production in the culture medium of seriporia racerata, the amount of tyrosine in the culture medium was measured using folin phenol reagent. 0.7 mL of 0.44 M TCA (trichloroacetic acid) was added to 0.7 mL of the culture solution and reacted at 37 ° C. for 30 minutes, followed by centrifugation at 15,000 rpm for 10 minutes to remove the precipitate. 2.5 mL of 0.55 M Na ₂ CO ₃ and 0.5 mL of phenol reagent were sequentially added to 1 mL of the collected supernatant, followed by reaction for 30 minutes in a 37 ° C. constant temperature water bath. After cooling at room temperature, the absorbance of the reaction solution was measured at 660 nm with a spectrophotometer (UNION, Kontron instruments, France).

The enzyme activity was measured by dividing α-amylase and protease and the enzyme activity was measured using the culture medium as the enzyme solution. 1 mL of 1% soluble starch (0.02 M phosphate buffer, pH 7.0) was used as a substrate of α-amylase. 1 mL of the prepared enzyme solution was added and reacted at 37 ° C. for 30 minutes. The reaction was stopped with 10 mL of 1 M acetic acid, followed by iodide solution (0.005% I₂+ 0.05% KI) 2 mL was added and developed. After absorbance was measured at 660 nm, 10% of the blank OD value was reduced to 1 g. In the blank test as a control, a sample solution in which the previously prepared enzyme solution was boiled at 100 ° C. for 30 minutes was inactivated.

Protease activity was measured by adding 0.35 mL of casein solution and 0.35 mL of enzyme solution to the e-tube as a substrate and reacting in a constant temperature water bath (37 ℃, 10 min), and stopping the reaction by adding 0.7 mL of 0.44 M TCA solution. It was left to stand at 30C for 30 minutes. Centrifuge the reaction solution (15,000 rpm, 15 min), add 2.5 mL of 0.55 M Na ₂ CO ₃ and 0.5 mL of 3-fold folin reagent in 1 mL of the filtrate, and react for 30 minutes at 37 ° C. Was measured. Under this reaction condition, the amount of enzyme that liberates 1 g of tyrosine in 1 minute was 1 unit.

Thrombolytic enzyme activity was measured using the Astrup and Mllerz method, a kind of fibrin plate method. Fibrin plates were dissolved in 0.067 M sodium phosphate buffer (pH 7.4) in 0.5% fibrinogen and 10 mL was added to a 9 cm diameter petri dish. 0.1 mL of thrombin (100 unit / mL) dissolved in 0.067 M sodium phosphate buffer (pH 7.4) was added thereto, mixed quickly, and left at room temperature for 30 minutes to solidify. 20 L of the culture solution was added to each marked position on the fibrin plate and reacted at 37 ° C. for 2 hours. After preparing the standard curve of the standard plasmin enzyme activity, the thrombolytic enzyme in the culture medium was converted to the plasmin unit and showed the thrombolytic enzyme activity (%) compared with the standard curve. The control was calculated using plasmin (5 units / mL), a purified thrombolytic enzyme.

2-1-5. Sugar, protein content measurement

Sugar content was measured by phenol-sulfuric acid method. 25 mL of 80% phenol was added to 1 mL of the diluted sample, 2.5 mL of sulfuric acid was added, cooled at room temperature, and absorbance was measured at 425 nm. Protein content was measured by the BCA method and bovine serum albumin was used as a standard.

2-1-6. Molecular weight measurement of Exopolysaccharide (EPS) using GPC

The dried viscous material was dissolved in 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ (glacial acetic acid adjusted to pH 4) solution to 1%, and after centrifugation, only the supernatant was filtered with a 0.45 m syringe filter to obtain GPC (Gel Permeation). Chromatography). Analytical conditions were RI as a detector, and the GPC column was a mobile phase of 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ (adjusted pH to 4 with glacial acetic acid) using Shodex SB 805 HQ (Japan). Flow was at a rate of 1.0 mL / min. Standard curves were prepared using dextran (American Polymer Corporation, USA) with different molecular weights (130, 400, 770, 1200 kDa), and the molecular weight of EPS was measured using a refractive index meter (Table 1).

Table 1

	Determination of Molecular weight
HPLC System	Knauer K-501 system
Column	OHpak SB 805 HQ (Shodex, Japan)
Mobile phase	0.1 M Na 2 So 4 /0.05 M NaN 3 / pH 4
Flow rate	1.0 mL / min
Detector	RI (Knauer K-2310)

2-2. Experiment result

2-2-1. Ceriporia lacerata ITS-5.8S rDNA Sequence Assay

ITS-5.8S rDNA sequencing of the Ceriporia lacerata strain showed 92% homology with Ceriporia lacerata FJ462746 (FIG. 1).

2-2-2. Evaluation of Physicochemical Properties According to Sugar Type and Concentration

To evaluate the physicochemical properties of sugars, 3% of lactose, sucrose, glucose, fructose, and galactose were added to 3% of the sugars and cultured for 7 days. As a result, there was no significant difference in pH, and the residual sugar content was found to be slightly smaller. In addition, the shape and size of mycelium pellets were different depending on the type of carbon source, and it was confirmed that the effect of the carbon source on the mycelial growth was very large. In addition, mycelial content and EPS content were highest in glucose-treated groups, followed by fructose and sucrose. As a result of measuring mycelia and EPS content by adding Glucose by concentration (15%), concentration increased up to 3% and there was no significant change after that.

2-2-3. Evaluation of Physicochemical Characteristics by Type and Concentration of Nitrogen Sources

In order to evaluate the physicochemical characteristics according to the nitrogen source, 3% of 7 nitrogen sources such as tryptone, yeast extract, soy flour, L-glutamic acid, ammonium persulfate, malt extract, and peptone were incubated for 7 days. Soy flour was the highest in mycelial content and EPS content was similar to tryptone, yeast extract, soy flour, and L-glutamic acid, but in terms of economic and industrial aspects, soybean powder with high mycelium and EPS content was selected as nitrogen source. It was. When soybean powder was added at 0.25% concentration, pH did not change significantly and sugar content increased depending on soybean powder concentration. Tyrosine content was also increased by soybean powder concentration, and protease and alpha amylase activity tended to be high in 23% soybean flour, and slightly decreased at high concentration. However, thrombolytic enzyme activity was increased depending on soybean powder concentration. Mycelium and EPS content tended to increase to 3%, similar to the carbon source concentration, but after 3%, there was no significant change, so the optimal concentration was set to 3%. As a result of measuring the sugar and protein content of EPS in cultured soybean powder, the sugar content was about 40% and the protein content was about 33%. Table 2 shows the chemical properties and enzyme activity according to the content of soybean powder, Table 3 shows the composition of the extracellular polysaccharide according to the content of soybean powder.

TABLE 2

Soy flour	pH	˚Brix	Tyrosine content (mg%)	Protease activity (unit / mL)	α-amylase activity (unit / mL)	Fibrinolytic enzyme activity (unit / mL)
0.2	4.25	3.0	7.46	0.04	5.77	0.60
One	4.47	3.9	30.12	0.17	3.64	0.60
2	4.59	4.8	48.37	0.75	6.78	0.75
3	4.74	5.5	64.21	1.02	1.68	0.85
4	4.91	6.3	69.39	0.94	1.10	0.75
5	4.84	7.0	82.32	0.75	0.60	1.25

TABLE 3

Soy flour	Total sugar content (%)	Total protein content (%)
0.2	50.24 ± 1.06	33.13 ± 0.30
One	47.94 ± 0.15	32.49 ± 1.01
2	42.78 ± 0.08	37.91 ± 0.01
3	40.57 ± 0.68	33.34 ± 1.41
4	38.46 ± 0.09	34.34 ± 0.20
5	32.63 ± 0.30	36.20 ± 0.81

2-2-4. Evaluation of Physicochemical Properties According to Trace Elements and Their Concentrations

In order to evaluate the physicochemical properties according to the trace elements, 0.5% of 5 elements of KH ₂ PO ₄ , MgSO ₄ , ZnSO ₄ , CuSO ₄ , FeSO ₄ , and CaCl ₂ were added and cultured for 7 days. As a result, the mycelium content of CuSO _4, but the highest pointed household was selected for the EPS production MgSO ₄ MgSO ₄ Attachment households increased the most in trace elements. As a result of measuring the mycelium and EPS content by adding trace element concentration at 0 ~ 0.25% concentration, it increased up to 0.15% concentration but there was no big difference at 0.22%, so the optimal concentration was set to 0.15%.

2-2-5. Evaluation of Physicochemical Properties by Culture Period (5 L jar fermenter)

Mycelial and EPS contents were measured in the 5 L jar fermenter according to the incubation period using the selected optimal medium. There was no significant change in mycelium content until 8 days of cultivation, but decreased after 10 days. Although there was a tendency to increase, there was no significant difference. The optimal culture days were selected as 8 days.

2-2-6. Exopolysaccharide (EPS) Characterization

As a result of measuring the protein and sugar content by the second purification and proteolytic enzyme treatment of EPS, the sugar content was lowered by purification and the protein content tended to be higher. As a result of measuring the molecular weight of EPS using GPC, it was found that it was about 120 kDa, and the molecular weight was slightly decreased by proteolytic enzyme treatment.

3. Anti-diabetic effect of CLD and EPS using type 2 diabetes model

3-1. Experiment method

3-1-1. Ceriporia lacerata culture extract

Ceriporia lacerata culture extract used in this experiment was the one prepared in item 1.

3-1-2. Method for measuring mycelia and extracellular polysaccharide content

Lyophilized culture was prepared as a 10% solution, centrifuged (8,000 rpm, 20 min), and the supernatant was separated. Add cold isopropyl alcohol corresponding to 4 times the volume of the separated supernatant and overnight at 4 ℃. Centrifugation (10,000 rpm, 20 min) was again carried out to recover the settled precipitate, lyophilized and weighed to indicate the exopolysaccharide content as an indicator. The major component of Ceriporia laccerata culture was carbohydrate, containing 79% crude carbohydrate and 15% crude protein.

3-1-3. Experimental Animals and Experimental Design

In order to investigate the hypoglycemic effect of Ceriporia racerata culture and extracellular polysaccharides, we tested C57BL / Ksj (BL / Ls) homozygous diabetic ( db / db ) mice (SPF), a representative animal model of type 2 diabetes. ) Was used. db / db mice are animals that cause diabetes due to point mutations in lepr, the leptin receptor gene of chromosome 4, and as the leptin receptor decreases, signal transduction decreases and blood glucose increases, and it is established as an insulin-independent diabetes model. The animal was selected because of its abundance of basic data and suitable for evaluation and comparison of test results. The db / db mice used in this study were 3040 g 6-week-old males, produced by Japan SLC Inc., and supplied through a central laboratory animal. The body weight and blood glucose levels were measured after 7 days of quarantine purification and acclimatization. Thirty healthy animals were selected that were suitable for the test run and had no symptoms.

The experimental animals were divided into four groups of blood sugars: negative control group, extracellular polysaccharide low dose group (150 mg / kg), extracellular polysaccharide high dose group (300 mg / kg) and positive control group (metformin-300 mg / kg). And the weight was evenly divided to breed for 6 weeks. In addition, six normal and control groups were kept for 6 weeks under the same conditions. All test substances and positive control substances were orally administered at the same time every day, and the normal, negative control group was orally administered with water (FIG. 11). Changes in body weight were measured once a week during the six-week breeding period, and 12-hour fasting microvenous blood glucose was measured once a week using a blood glucose meter (ACCU-CHEK Sensor, Germany). The diet of experimental animals was supplied with commercial experimental animal solid feed (Samtaco co. Ltd., Korea), and water was freely ingested. The breeding conditions of the animal breeding room were adjusted to have a 12 hour contrast cycle (8 am to 8 pm illumination), a temperature of 23 ± 3 ° C., and a relative humidity of 50 ± 10%.

3-1-4. Oral Glucose Tolerance Test (OGTT)

After fasting for more than 12 hours and measuring fasting blood glucose, five or three samples of each group dissolved in distilled water of Ceriporia racerata powder and EPS were first administered orally. At this time, the control group was administered the same amount of physiological saline. Then, 40% glucose was orally administered to the other groups except the control group at a concentration of 2 g / kg bw, and then blood samples were collected from US veins at 30, 60, 90 and 120 minutes to observe changes in blood glucose. The blood glucose increase curve was calculated by calculating the blood glucose increase at each time point.

3-1-5. Collection of Experimental Animal Sacrificial Samples

Blood glucose should be measured in the vein at 12 hours on an empty stomach, and serum for biochemical analysis should be taken on an empty stomach for more than 12 hours. Therefore, the sacrifice of experimental animals was measured at the end of 6 weeks after breeding for 6 weeks. After this had elapsed.

At the sacrifice of experimental animals, all animals were anesthetized with ether after 12 hours of fasting, blood was collected from the abdominal vein, divided into serum separation tubes, centrifuged at 3,000 rpm for 20 minutes, and serum was used as a sample for biochemical index analysis. Immediately after sacrifice, all animals were weighed by removing liver, abdominal fat, etc. and rapidly frozen at -80 ° C for storage for further experiments. Part of the liver and pancreas were extracted, fixed in 4% paraformaldehyde solution, and used for tissue analysis.

3-1-6. Serum c-peptide, insulin and leptin concentrations

Serum c-peptide and insulin content, blood glucose-related functional markers, were collected and isolated from the abdominal vein using double antibody C-peptide (DPC, USA) and insulin RIA kit (DPC, USA), respectively. The method was measured using a mouse leptin RIA kit (LINCO, USA).

3-2. Experiment result

3-2-1. Dietary Intake and Weight Changes

The body weight change, diet and negative intake of experimental animals for 6 weeks are shown in Figs. 14, 15 and 16. The initial weights of the diabetic control group and the EPS-ingestion group were similar at 32 g. After 6 weeks, there was no significant change in the body weight between the diabetic control group and the experimental group. The diabetic control group showed higher tendency in the diet and negative intake than in the normal control group. The positive control group, MET300 group, was significantly lower than the other groups.

3-2-2. Long-term weight

The weights of the liver, kidney, spleen, kidney fat and abdominal fat of the test animals are shown in Table 4.

Table 4

	Liver (W / BW)	Kidney (W / BW)	Spleen (W / BW)	Kidney fat (W / BW)	Abdominal fat (W / BW)
NC	0.94 ± 0.14	0.29 ± 0.03	0.06 ± 0.01	0.12 ± 0.04	0.43 ± 0.17
DM	2.89 ± 0.04	0.41 ± 0.06	0.04 ± 0.02	0.82 ± 0.17	2.34 ± 0.28
DM-EX0150	2.50 ± 0.29	0.39 ± 0.05	0.06 ± 0.03	0.63 ± 0.12	2.48 ± 0.14
DM-EX0300	2.65 ± 0.05	0.40 ± 0.04	0.05 ± 0.02	0.55 ± 0.17	2.55 ± 0.15
DM-MET300	2.60 ± 0.19	0.39 ± 0.03	0.03 ± 0.02	0.89 ± 0.13	2.564 ± 0.33
DM-ALL300	2.50 ± 0.27	0.40 ± 0.03	0.06 ± 0.01	0.48 ± 0.12	2.41 ± 0.21

Liver weight tended to increase rapidly in the diabetic group and decreased significantly in the group receiving the sample. The tendency is consistent with reports that fat accumulates at the time of diabetes-induced hypertrophy. In addition, the kidney volume increased with the increase of glomerular filtration rate at the onset of diabetes. In this study, the size of kidney in diabetic group was increased but there was no significant difference. The spleen also showed no significant difference between the groups. Kidney fat increased rapidly in the diabetic group, but decreased significantly by the administration of EPS and Ceriporia powder, but there was no significant difference in abdominal fat.

3-2-3. Effect on blood sugar change

The result of measuring the change in blood glucose at 12 hours fasting is shown in FIG. 16. Initial blood glucose levels were similar at around 150 mg / dL in all groups, but a week after the administration of the sample, blood glucose levels started to rise slightly. After three weeks, blood glucose increased rapidly, and the diabetic control group had about 400 mg / dL of glucose. On the other hand, the metformin group did not raise blood sugar levels. Thereafter, blood glucose levels increased continuously, but the group treated with EPS and Ceriporia powder showed a lower tendency of blood glucose than the diabetic control group.

3-2-4. Oral glucose tolerance

The glucose tolerability of the EPS and Ceriporia powder was measured at 6 weeks of sample intake, and the results are shown in FIG. 17. In the diabetic control group, the blood glucose level was 600 mg / dL, the highest value of blood glucose meter, and hyperglycemia was maintained during the glucose loading weight measurement period. On the other hand, the initial fasting blood glucose of the EPS and Ceriporia powder-ingested group was 500 mg / dL, which was significantly lower than that of the diabetic control group. The measured value was 600 mg / dL, and gradually decreased thereafter to 520 mg / dL after 180 minutes, which was similar to the initial blood glucose level.

3-2-5. Blood sugar

Blood glucose levels in DM group rose to about 900 mg / dL after 6 weeks of oral EPS and sacrificial sacrifice, whereas blood glucose levels decreased in dose-dependent groups in the EPS 300 group. Decreases to about 700 mg / dL, suggesting that EPS plays a positive role in blood glucose reduction.

3-2-6. Effect on Serum Lipid Levels

Serum lipid levels were measured after oral administration of EPS for 6 weeks, and total cholesterol and triglyceride content tended to be about 2 times higher in the DM group than in the NC group. It was confirmed that the cholesterol and neutral lipid content was lowered. In addition, HDL cholesterol content was significantly increased and LDL-cholesterol content was significantly decreased in EPS group, EPS was found to improve serum lipid level although there was no significant change in weight loss in type 2 diabetes model. .

3-2-7. Effect on insulin, C-peptide and leptin levels

After oral administration of EPS for 6 weeks, serum levels of insulin, C-peptide, and leptin were measured, and there was no significant difference in insulin content between diabetic groups and C-peptide was higher than DM group in the sample administration group. This was confirmed to increase. It is assumed that the insulin secretion ability in the pancreas is active and leptin content is also increased to play a positive role in insulin resistance.

* 4. Effect of Exopolysaccharide on Insulin Signaling Mechanism in 3T3-L1 Cells

4-1. Experiment method

4-1-1. Cell culture

Preadipocytes, 3T3-L1 fibroblasts, are well known in their biological properties, have a property of differentiating into adipocytes when cultured under appropriate conditions, and inhibit or synthesize lipolysis in adipocyte metabolism. In addition to their use in research, adipocytes are insulin target cells that are widely used to study insulin signaling. The 3T3-L1 fibroblasts used in the experiment were distributed from Korea Cell Line Bank, 10% fetal bovine serum (FBS, GibcoBRL), 200mM glutaMAX (GibcoBRL), penicillin (10,000units / ml, Sigma), streptomycin (10mg / ml, Sigma ) Was added to Dulbecco's Modified Eagle's Medium (DMEM, GibcoBRL) high glucose at 3-day intervals and incubated at 37 ° C. and 10% CO ₂ . When subcultured, when 3T3-L1 fibroblasts were 60-80% confluent, wash the surface of the culture cells with Dulbecco's phosphate buffered saline (PBS, GibcoBRL) solution, and add 500μL of 2.5% trypsin (GibcoBRL) per 75 cm ² flask. After incubation for 5 minutes, cells were detached from the flask and passaged. The detached cells were transferred to a new flask suspended in 15 ml of DMEM high glucose culture with 10% FBS.

4-1-2. Glucose Intake into Adipose Cells

The 3T3-L1 fibroblasts were cultured until they were confluent in the same manner as the cell culture, and were confluent and after 2 days, 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX, Sigma), 25 μM dexamethasone (DEX, Sigma) and insulin (Sigma) were added to DMEM high glucose, and then incubated for 3 days, and then changed into a new culture solution every 2 days to change to fat cells. In addition, glucose intake experiments were conducted between 10 and 15 days of complete conversion to adipocytes. The glucose intake experiment, which reflects the degree of action of insulin, showed 20 × 10 ⁴ cells / ml of cells counted by a hemocytometer after 2.5% typsine treatment of 3T3-L1 adipocytes, which were completely transformed into adipocytes the day before. Was transferred to a 24 well plate. The medium was changed to DMEM low glucose and starvated. The fat cells transferred to the well plate were washed with PBS, and then extracted with Ceriporia lacerata culture solution 10 / ml and 1 / ml in HEPES solution containing 0.1% bovine serum albumin (BSA, Roche). Insulin was added and incubated at 37 ° C. for 1.5 hours. In addition, 10Ci / ml 2-deoxy-D- [3H] glucose (2-DG), a non-metabolized glucose analogue, was added to the cells and cultured again at 37 ° C. for 10 minutes. After 10 minutes, washed 5 times with cold PBS, lysed the cells with 1N NaOH, neutralized with 1N HCl, and measured the content of 3H intake of glucose into cells for 5 minutes (Tri-Carb 2100TR, Packard Bioscince, IL). Was measured. In order to exclude nonspecific glucose intake, incubation with cytochalasin B (sigma), which inhibits the action of glucosetransporter 4 (GLUT4), was also measured. The low concentration used in the experiment was 1 ng / ml, because the highest concentration of intracellular glucose intake was 50 ng / ml for investigating whether Ceriporia lacerata culture extract contained insulin-sensitive agents. The high concentration was chosen as insulin 25ng / ml. In this experiment, the fraction of Cordyceps sinensis with 1 ng / ml of insulin in 3T3-L1 adipocytes was incubated for 1.5 hours, and the glucose intake was compared with that of 1 ng / ml of insulin. All experiments were repeated three times, and glucose intake was measured along with the substance isolated from 0 ng / ml of insulin to rule out that the isolated substance acts as a detergent regardless of the action of insulin to destroy cell membranes and increase glucose uptake. When the value was higher than the basal value, it was considered that there was no action as an insulin-sensitive agent even though it increased glucose intake.

4-1-3. Investigation of protein expression involved in insulin signaling

For the experiment, 3T3-L1 adipocytes were treated with 2.5% typsine and the cells were transferred to 24 well plates. 24 hours before the experiment, Dulbecco's Modifiend Eagle's Medium (DMEM) low glucose culture with 10% fetel bovine serum was added and starvated. In addition, after washing with PBS, the extract of Ceriporia lacerata (Ceriporia lacerata) culture solution and insulin in HEPES and incubated at 37 ℃ for 1 hour, 50 unit aprotinin, 1mM Na ₃ VO ₄ , 1mM PMSF Cells were removed from the well plate on ice using RIPA buffer containing. The detached cells were centrifuged at 10,000 rpm for 20 minutes at 4 ° C. After diluting the centrifuged sublayer with Laemmli sample buffer, electrophoresing with SDS-PAGE, and then moving to nitrocellulose using rabbit GLUT4 antibody (Chemicon, Temecula, CA), IR, PI3-Kinase, Akt, MAPK, AMPK antibody Western blot and the content was determined by laser densitometer.

4-2. Experiment result

4-2-1. Insulin sensitivity

Insulin is the most commonly used drug for people with type 1 diabetes or type 2 diabetes. Insulin is a factor that promotes differentiation in adipocytes, and insulin acts to produce many adipocytes. Ceriporia racerata culture extract (EPS) has been shown to promote differentiation of adipocytes in a concentration-dependent manner as an insulin-like substance. The potential as a natural substitute for insulin was identified.

4-2-2. Glucose uptake

All-cell 3T3-L1 fibroblasts are well known for their biological characteristics and have the property of differentiating into adipocytes when cultured under appropriate conditions. Therefore, they are used for the inhibition or synthesis of lipolysis in adipocyte metabolism. In this experiment, the presence of an inducer such as insulin was used to investigate the presence of insulin-sensitive agents by using a characteristic that promotes differentiation by rapidly increasing enzymatic activity. That is, 3T3-L1 fibroblasts were converted to 3T3-L1 adipocytes by adding induced differentiators such as insulin, IBMX, and dexsamethasone. Glucose uptake into cells was measured by the amount of 2-deoxy-D- [3H] -glucose, a glucose analog that was transferred into cells by the glucose carrier GLUT4. For glucose intake experiments, the levels of intracellular glucose uptake were determined by starvation with DMEM low glucose culture and treatment with HEPES and EPS in starvation 3T3-L1 adipocytes. The experimental control group showed that glucose uptake increased when both basal and insulin were not treated with insulin, but EPS was acted as a sensitizer in the presence of insulin to increase the absorption.

4-2-3. Protein expression involved in insulin signaling

Intracellular signaling of insulin is complicated by several processes. The mechanism by which insulin acts on target cells is linked to insulin receptors in the plasma membrane, resulting in the action of various insulins. In other words, the insulin receptor is composed of two α-subunits and β-subunits. The action of insulin first begins when insulin in the blood binds to the α-subunit of the insulin receptor of the target cell. The activated α-subunit activates the tyrosine kinase of β-subunit inside the cell membrane. Generally, tyrosinekinase activity of β-subunit is considered to be essential for many physiological actions of insulin as an early stage of insulin action.

When the tyrosine kinase of the β-subunit is activated, it phosphorylates IRS-1, IRS-2, IRS-3, IRS-4, Shc, p60, and other binding proteins in the insulin signaling process. Signaling of insulin occurs through the dog's downward signaling pathway. Among them, tyrosine phosphorylation of IRS eventually leads to activation of phosphatidylinositol 3-kinase (PI3-Kinase).

 PI3-kinase is a heterodimer consisting of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Phosphorylated IRS-1 and IRS-2 bind to the P85 subunits of PI3-Kinase and then activate p110 subunits to activate phosphatidylinositide 4, 5 Convert biphosphate to phosphatidylinositide 3,4,5 triphosphate. These phosphoinositides are thought to be signaling agents that play an important role in the biological action of various growth promoters, but the exact function of each of these phosphoinositides in hormonal signaling is not known. Through these PI3-kinase pathways, various proteins and kinase, such as p70S6 kinase, are activated, undergo phosphorylation and dephosphorylation signaling. Activation of PI3-Kinase is important for many actions after insulin stimulation, from glucose transport, lipolysis inhibition, glycogen synthesis, protein synthesis to mitogenesis, but it is not yet clear how PI3-Kinase is involved in causing this response.

On the other hand, tyrosine kinase activity and tyrosine phosphorylation are not always considered necessary in all cells and in all cases for the action of insulin. It is known that there is a process that is independent of tyrosine phosphorylation. One of the condensation pathways that is independent of tyrosine phosphorylation is the pathway through G protein. One of the most active studies of G protien is Ras, a GTP binding protein that induces a variety of biological signals. Ras is regulated by SOS and GTPase actiating protein (GAP), and activation of Ras is performed by MAP kinase kinase (MAPKK), Raf-1 MAPK / E kinase (MAPKK or MEK), and p90 ribosomal S6 kinase. To occur. In addition, ARF and Rho proteins, G proteins, play an important role in the recycling of sugar transporters by activating phospholipase D. Rab 4 protein is thought to play an important role in the pathways involved in GLUT4 secretion. . Some of these signaling pathways independently have some interregulatory activity, resulting in the expression of the final biological effects of insulin, such as glucose transport, enzyme-activated protein and synthesis of nucleic acids. In the insulin signaling process, there are many signaling mechanisms and many protein substrates. Among them, IRS-1 plays a central role in the linkage of insulin signals, and IRS-1 carries the signals of insulin receptor PI3-kinase, GRB-2. , SOS, Ras, Rab 4, ARF, SYP, Nck, etc. are expected to play a role in the transmission. In this experiment, we examined the effect of Ceriporia lacerata mycelium culture extract with increased expression of GLUT4, a sugar transporter, on insulin signaling system. In other words, the IR of the central role of the insulin signaling system was examined for the concentration of IRS-1 in the cells according to the Ceriporia lacerata mycelium culture extract, and combined with IRS-1 to signal insulin to the cells. The concentration of PI3-kinase delivered was examined. In addition, the intracellular concentration of GLUT4, which transfers glucose upon receiving IRS-1 and PI3-Kinase signals, was also examined. As a result, it was confirmed that all proteins were phosphorylated by insulin and the expression level was increased by EPS treatment. EPS can be thought to be a substance that can improve insulin resistance by promoting glucose uptake through IR, PI3K, and Akt pathways and by increasing AMPK protein expression.

5. Analysis of Indicators and Antidiabetic Functional Substances

5-1. Experiment method

100 g of CLD is suspended in 1.5 L of water, and then 1.5 L of hexane is added. The upper portion was collected by separating into a separatory funnel and partitioning into a hexane insoluble layer and a soluble layer. The same volume of the hexane solvent as the lower layer was repeated twice in the same manner to obtain the maximum amount of material soluble in the hexane layer until the color of the solution became light, fractionated and dried. Subsequently, methylene chloride, ethyl acetate and butanol were carried out in the same manner as above to obtain 15 g of hexane soluble extract, 25 g of methylene chloride soluble extract, 30 g of ethyl acetate soluble extract, and 15 g of butanol soluble extract, respectively.

30 g of ethyl acetate soluble extract was subjected to silica gel column chromatography (12 × 60 cm Merck, ASTM7734 Germany) using an eluent (hexane: ethyl acetate: methanol = 10: 3: 1), and 15 fractions were purified by TLC pattern. Obtained.

5-2. Isolation of 2,5-dihydroxybenzoic acid compounds

The eluate (toluene: ethylacetate: acetic acid = 5: 3: 1) of the sixth fraction of the fractions was separated by silica gel column chromatography (1.5 × 12 cm, ASTM7734), and finally TLC (development) for separation and purification. 7 mg of a compound having an Rf of 0.58 in solvent / toluene: ethyl acetate: acetic acid = 5: 4: 1) was identified by EI-MASS and 1H-NMR analysis to identify 2,5-dihydroxybenzoic acid of the formula Used.

5-3. Isolation of Protocatechualdehyde Compounds

The eluate (toluene: ethyl acetate: acetic acid = 2.5: 1: 0.5) of the seventh fraction of the fractions was separated by silica gel column chromatography (1.5 × 15 cm, ASTM7734), and finally TLC (development) for separation and purification. 2 g of a compound having an Rf of 0.52 was obtained on a solvent / toluene: ethyl acetate: acetic acid = 5: 4: 1) and identified as protocatechualdehyde having the following chemical formula by EI-MASS and 1H-NMR analysis.

In order to measure the content of the two sugar-resistant substances, LC / MS / MS was used as Agilent Technologies Agilent 6410. The ion souce was negative and the fragmentor was 150. Gas temperature was 320 ℃ and gas flow was analyzed at a rate of 35mL / min. The capillary volt was 4000. The column of HPLC used Epic C18 and the column temperature was kept at 40 degreeC. As mobile phase, distilled water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid were used.

Trace amounts of 2,5-dihdroxybenzoic acid and protocatechualdehyde reported to have antidiabetic effects were detected and the results are shown in Table 5 below.

Table 5

Sample	Protocatechualdehyde	2.5-dihdroxybenzoic acid
Example 5	18.79 ± 0.87 (μg / g)	37.65 ± 1.32 (μg / g)

5-3. Isolation of Exopolysaccharide

In order to measure the content of Exopolysaccharide reported to have an excellent effect on blood sugar drop, 100 ml of distilled water was added to 5 g of Ceriporia lacerata mycelium culture extract, and then suspended well, followed by centrifugation (8,000 rpm, 20 min). To this supernatant was added cold alcohol corresponding to 2-3 times the amount, and placed in a refrigerator (4 ° C.) for 12 hours. After centrifugation (8,000 rpm, 20 min) only from the supernatant, the precipitate was recovered and crude crude Exopolysaccharide was extracted. The result of vacuum freeze drying was the content of Exopolysaccharide as 5.5 ± 0.5% / 100mg. appear. As a result of measuring the sugar and protein content of EPS, the sugar content was about 40% and the protein content was about 33%, indicating that the sugar and protein were polysaccharide combined.

As described above, according to the present invention, 5.5 ± 0.5% / 100mg of exopolysaccharide, 18.79 ± 0.87 (㎍ / g), and 2,5-dihdroxybenzoic acid, which are known to have an excellent effect on the treatment of diabetes and diabetic complications and hypoglycemia Ceriporia lacerata mycelium culture extract containing more than 37.65 ± 1.32 (μg / g) can be provided.

6. Rodent Toxicity and Effect Test

The safety and efficacy test of Ceriporia lacerata mycelium culture extract was conducted by KLP, a KLP accredited organization, by Good Laboratory Practice (GLP). Beta cells were increased to statistically significant levels (the weight of the pancreas, spleen, and thymus), and test results were found to be safer and better than Pfizer's metroformin (Table 6).

Table 6

general toxicity
a single dose toxicity test on rodents	7 weeks
toxisity test on rodents for 4 weeks repeat oral administration-DRF	8 weeks
toxisity test on rodents for 13 weeks repeated oral administration (including recovered group)	27 weeks
a single dose toxicity test on non rodents	8 weeks
result	nontoxic reaction
heredity toxicity test
back mutation test (includging preliminary test)	4 weeks
chromosomal anomaly test (including preliminary test)	8 weeks
micro nucleus test (including preliminary test)	8 weeks
result	negative reaction
effect test
Oral Glucose Tolenance Test	4 weeks
effect test on blood sugar dropping after being caused type 1 diabetes by STZ	8 weeks
curing diabetes using db-db mice	8 weeks
result	β-cell in pancreas with type 2 diabetes rats increased meaningfully in statistics, and it is judged that it is a fundamental diabetes medicine being recorded with safe and excellent effect compared to Metformin by pfizer used as a comparison medicine.

The present invention relates to a method for producing a Ceriporia lacerata mycelium culture extract and a pharmaceutical composition for the prevention or treatment of diabetic diseases and diabetic complications prepared by the production method is very useful industrially It is high.

Claims (6)

1. In the method for producing a Ceriporia lacerata mycelium culture extract comprising liquid culture of Ceriporia lacerata mycelium, dry powdering of the culture medium and preparing a solvent extract

   Ceriporia lacerata mycelium culture medium is 1 to 2% by weight of sugar, 0.2 to 1% by weight of glucose, 0.2 to 1% by weight of starch, 0.1 to 0.5% by weight of water, 0.1 to 0.5% of wheat flour %, Soy flour 0.2 ~ 2 wt%, Magnesium sulfate (MgSO ₄ ) 0.05 ~ 0.1 wt%, Potassium phosphate (KH ₂ PO ₄ ) 0.05 ~ 0.1 wt%, Potassium diphosphate (K ₂ HPO ₄ ) 0.05 ~ 0.1 wt % And water 92-98 % by weight, hydrogen ion concentration pH 4.5 ~ 6.0 characterized in that the production method of Ceriporia lacerata ( Ceriporia lacerata ) mycelium culture extract.
2. The method of claim 1,

   The culturing is a method for producing a Ceriporia lacerata ( Ceriporia lacerata ) mycelium culture extract, characterized in that carried out under a blue LED light source.
3. The method of claim 1,

   The culturing is a method for producing a Ceriporia lacerata ( Ceriporia lacerata ) mycelium culture extract, characterized in that the concentration is carried out by maintaining a concentration of 1,000 ~ 2,000ppm .
4. A pharmaceutical composition for preventing or treating diabetic diseases and diabetic complications comprising the extract of Ceriporia lacerata mycelium culture medium prepared by the method of any one of claims 1 to 3 as an active ingredient. .
5. The method of claim 4, wherein

   The diabetic disease is a pharmaceutical composition, characterized in that the type 2 diabetes.
6. The method of claim 4, wherein

   The diabetes complications are selected from the group consisting of chronic hyperglycemia, atherosclerosis, microangiopathy, diabetic retinopathy and kidney disease. Composition.

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