JP4125225B2 - Diabetes preventive and improving agent containing Yamabushitake strain and Yamabushitake - Google Patents

Description

  The present invention relates to a Yamabushitake strain and an agent for improving and preventing diabetes including Yamabushitake, and in particular, to a Yamabushitake strain having an effect of improving diabetes prevention.

  Conventionally, diseases such as diabetes, heart disease, and stroke are called “adult diseases” and have been thought to develop and progress with aging. However, in recent years, it has become clear that these diseases are deeply related to the lifestyle of individuals regardless of age. For this reason, diseases caused by lifestyle habits such as eating habits, exercise habits, rest, smoking, and drinking have come to be called lifestyle-related diseases with reference to the disease name Life-Related Disease in the United States.

Diabetes mellitus, one of lifestyle-related diseases, is a complex disease caused by hyperglycemia, and when chronic, it leads to microvascular disorders such as retinopathy, nephropathy, neuropathy, and the promotion of arteriosclerosis, myocardial infarction, cerebral infarction It is also known to cause etc.
There are two types of diabetes: insulin-dependent diabetes that requires supplementation due to insulin deficiency (type I diabetes) and abnormalities in receptors and sugar transport carriers, despite the fact that a sufficient amount of insulin is produced. There is non-insulin dependent diabetes mellitus (type II diabetes) whose action is not expressed for the reason, and type II diabetes is mainstream in Japan.
Various types of exercise therapy, diet therapy, and pharmacotherapy have been implemented for the treatment of diabetes.
Japanese Laid-Open Patent Publication No.

However, it is difficult to continue exercise therapy and diet therapy. In addition, as drug therapy, there are a method by internal use and a method by insulin injection, but side effects may be a problem due to long-term administration of these drugs.
In addition, the number of pancreatic β cells in type II diabetic patients is clearly decreased compared to non-diabetic patients, and this decrease is considered to be an important factor in pancreatic β cell dysfunction. Was not expected to have a direct improvement effect on pancreatic β cells.
As the diabetic population is expected to increase in the future, appropriate prevention and improvement methods need to be established quickly.
The present invention has been made in view of the above-described problems of the prior art, and provides an agent for improving and preventing diabetes that has a low possibility of causing side effects and has an excellent effect.

In order to achieve the above object, as a result of intensive studies by the present inventors, it has been found that Yamabushitake, in particular Yamabushitake strain having a deposit number of FERM P-19600, has an excellent effect for improving diabetes prevention. It came to be completed.
That is, the first subject of the present invention is a Yamabushitake strain whose deposit number is FERM P-19600.
The second subject of the present invention is an agent for improving and preventing diabetes containing Yamabushitake as an active ingredient.
It is preferable that the agent for improving and preventing diabetes includes a dried powder of Yamabushitake and / or a hot water extract thereof.
In the agent for improving and preventing diabetes, it is preferable that Yamabushitake is a Yamabushitake strain having the deposit number FERM P-19600.

Yamabushitake according to the present invention, in particular, Yamabushitake strain with a deposit number of FERM P-19600, has a low possibility of causing side effects and has an excellent effect of improving diabetes prevention.
Moreover, since a direct improvement effect can be expected for pancreatic β cells, it is particularly effective for type II diabetic patients that are mainstream in Japan.

Hereinafter, preferred embodiments of the present invention will be described in detail.
Yamabushitake (Helicium erinaceum) is an edible moth belonging to the genus Basidiomycetes, Hydrangea, Coralhariaceae, and Haritake.
The fruit body has a needle-like shape, and the inside of the fruit body has a spongy structure. The size is about 5 to 10 cm, and the color is white to tan.
In the present invention, Yamabushitake may be a fruit body, an extract of the fruit body, or a dried product of the fruit body, but the fruit body may be either raw or dried, but handleability, storage stability, extraction efficiency, etc. From this point, a dried fruit body is desirable, and in order to efficiently take an effective amount, it is preferably used as an extract of a dried powder of the fruit body.

  Prior to obtaining the Yamabushitake extract, it is preferable to destroy the Yamabushitake tissue. This makes it possible to extract efficiently. As a means of destroying the tissue of Yamabushitake, pulverization processing by various pulverization mixers such as bead mill, Waring blender, homogenizer, impact crushing processing by blasting machine, etc., physical processing such as freezing processing, ultrasonic processing, sodium hydroxide, etc. There are chemical treatments such as alkaline treatment with an aqueous solution of the above, treatment with an enzyme having cell wall decomposing action such as cellulase and pectinase, and osmotic pressure treatment, and these can be carried out alone or in appropriate combination. Of these tissue disruption treatments, pulverization or enzyme treatment is preferred when the fruit body of Yamabushitake is used as the raw material, and enzyme treatment is preferred when the mycelium is used as the raw material.

In the enzyme treatment, a known cell wall degrading enzyme or polysaccharide degrading enzyme can be used, and one or two of cellulase, hemicellulase, chitinase, α- and β-glucuronidase, pectinase, xylanase, α- and β-glucanase, etc. The above can be used. In order to enzyme-treat Yamabushitake, the enzyme may be added as an aqueous solution to Yamabushitake, which has been appropriately shredded or crushed, and shaken or stirred.
In order to obtain the Yamabushitake extract of the present invention, an extraction solvent may be added to the Yamabushitake that has been subjected to tissue destruction treatment as described above, and the mixture may be appropriately stirred and extracted.
The extraction solvent is not particularly limited. For example, primary alcohol such as water, methanol and ethanol, polyhydric alcohol such as 1,3-butylene glycol and propylene glycol, lower alkyl ester such as ethyl acetate, carbonization such as benzene and hexane. Hydrogen, ethyl ether, acetone, etc. are mentioned, These can also be used in combination of 2 or more types. Preferable extraction solvents include water, methanol, ethanol, 1,3-butylene glycol, or a mixed solvent thereof. In particular, those obtained by extracting dried powder of Yamabushitake fruit body with hot water are preferred.

Moreover, although extraction operation may be performed under normal pressure and normal temperature, you may carry out under 1-5 atmospheres, 60-150 degreeC pressurization, and a heating. Specifically, after hot water extraction, it is preferably separated into an extract and a residue by centrifugation or vacuum filtration, and the residue is extracted with an aqueous ethanol solution and mixed with the previous hot water extract.
The extraction residue may be re-extracted under the same conditions as described above, and this extraction operation may be repeated several times.
The extract may be an extract or a dried product thereof. In the case of a dried product, the extract is removed by subjecting the extract to a treatment such as concentration under reduced pressure, sterilization, freeze drying, spray drying, or the like.
In the present invention, it is preferable to use fruit bodies, but an effect can be expected for the mycelium. As the mycelium, a raw or dried mycelium obtained by culturing inoculum in a medium containing a carbon source and a nitrogen source can be used. The dried one is convenient.
Yamabushitake according to the present invention was commissioned to the National Institute of Advanced Industrial Science and Technology on November 28, 2003, and the display for identification is called FEC201185 (TUHW Co.).

The scientific properties are as follows.
1. Scientific properties ... Fungus characteristics: Forms white colonies under nutrient medium containing carbon and nitrogen sources. In addition, a clamp connection (a faint connection) is observed under an optical microscope.
2. Taxonomic position ... basidiomycetes 3. Culture conditions (1) Medium name ... SMYA medium (S: Saccharose, M: Malt extract, Y: Yeast extract, A: Agar)
S, A → commercial product M, Y → Difco (Difco)
(2) Medium composition: per 1000 ml of medium
1% sucrose 10g
1% malt extract 10g
0.4% yeast extract 4g
2% agar 20g
(3) pH of medium: 5.0 to 7.0 (optimum pH 5.5)
(4) Sterilization condition of medium ... 121 ° C 20 minutes (5) Medium temperature ... 28 ° C
(6) Culture period: 10 days (7) Oxygen requirement ... Aerobic

4). Storage conditions Can be stored by freezing method.
(1) Freezing conditions: -80 ° C
(2) Protective agent: 10-20% glycerin aqueous solution (optimum 20%)
(3) Restoration rate after freezing: 100% in 1 year and 99% in 3 years
5. Conditions of survival test (1) Microorganism restoration ... 40 ℃
(2) Inoculation / culture / confirmation method: Same as the culture conditions.

Using the Yamabushitake of the present invention, an agent for improving and preventing diabetes can be produced. The agent for improving and preventing diabetes of the present invention is administered orally and applied for the prevention and improvement of diabetes. The amount of intake when used as an agent for improving diabetes prevention varies depending on the symptoms and is not particularly limited. However, 1-20 g, especially 5-15 g, optimally 10-15 g, in terms of dry weight of yamabushitake per adult, should be consumed. If it is effective, it can be expected.
The agent for improving and preventing diabetes of the present invention is usually administered as an internal medicine.
In the case of internal use, powders, tablets, capsules, granules, teas, suspending agents, flow extracts, liquids, syrups and the like can be prepared by conventional methods. In the formulation, usual formulation carriers such as excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, and flavoring agents may be used as necessary. it can. Moreover, a coating can also be given with a suitable coating agent etc. as needed.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to this. Further, the blending amount (%) is mass% unless otherwise specified.
The GOTO-KAKIZAKI rat (GK rat) is a specific obesity type II diabetic model animal, and is considered to be an animal model close to Japanese diabetic condition. Using this GK rat, the test was conducted focusing on the blood glucose lowering effect of Yamabushitake and the effect on pancreatic β cells.

[1] Test method Preliminary breeding and classification of test rats (5-6 weeks old)
Five-week-old male GOTO-KAKIZAKI / Crj rats (GK rats) were purchased from Nippon Charles River Co., Ltd. In addition, the glucose tolerance test was performed at the time of purchase, and only male rats (body weight 108 to 143 g) showing 1-hour values of 243 to 272 mg / dl were selected and purchased.
In a breeding room adjusted to room temperature 22 ± 1 ° C. and humidity 60 ± 10%, the light is adjusted for 12 hours a day (7-19 o'clock light period) with a white fluorescent lamp, and feed (CRF-1: Oriental Yeast Industry) Co., Ltd.) and tap water were freely ingested and pre-bred for 1 week.
After preliminary breeding, each individual was weighed and subjected to a glucose tolerance test, and classified into 5 groups of 8 per group so that the average value of body weight and the average value of 120 minutes in the glucose tolerance test were almost equal.

2. Preparation of Yamabushitake extract Yamabushitake (deposit number FERM P-19600) fruit body was dried in a drying apparatus at 40-60 ° C. variable mode, pulverized, and sieved to a 16-mesh sieve to obtain a powder. Further, 5 g, 10 g, and 15 g of the obtained powder and 600 ml of sterilized water were added to an Erlenmeyer flask equipped with a Liebig condenser, respectively, extracted for 1 hour in hot water at 80 ° C., and then allowed to cool to room temperature. The supernatant solution was filtered with a filter paper (Toyo Filter Paper, No. 2, diameter 90 mm) to obtain a hot water extract of Yamabushitake (yield: about 100%). Hereinafter, each extract is referred to as a 5 g extract, a 10 g extract, and a 15 g extract.

3. Extract administration method (6-17 weeks old)
In the following manner, each extract was administered to each group of rats and reared from 6 weeks of age to 17 weeks of age. Feed and tap water were freely given to each group.
Each extract was orally administered every day at 10 am, and sterilized water was freely ingested after the extract was administered. No oral administration was performed on the glucose tolerance test day. The environment of the breeding room was a temperature of 22 ± 1 ° C., a humidity of 60 ± 10%, and white fluorescent lighting for 12 hours a day (7-19 o'clock light period).

Group A: Non-administration group 1 ml of sterile tap water is administered per 100 g body weight per day (no extract is administered).
Group B: 5 g administration group 1 ml of the 5 g extract is administered per 100 g of body weight per day (corresponding to a person with a body weight of 60 kg ingesting 5 g of Yamabushitake dry powder extracted with 600 ml of hot water per day) .
Group C: 10 g administration group 1 ml of the 10 g extract is administered per 100 g of body weight per day (equivalent to a person with a body weight of 60 kg ingesting 10 g of Yamabushitake dry powder extracted with 600 ml of hot water per day) .
Group D: 15 g administration group 1 ml of the 15 g extract is administered per 100 g of body weight per day (equivalent to a person with a body weight of 60 kg ingesting 15 g of Yamabushitake dry powder extracted with 600 ml of hot water per day) .
Group E: Non-sugar load control group 1 ml of sterilized tap water is administered per 100 g of body weight per day (the sugar tolerance test and the extract are not administered).

4). Glucose tolerance test Glucose tolerance test was performed once a week in the morning.
As a method, all rats were fasted for about 20 hours (free drinking water) on the day before the glucose tolerance test, and 10 ml / kg of a 20% glucose solution was orally administered to each experimental animal.

[2] Analysis method Blood test value <total glucose and blood sugar>
The blood glucose level was measured immediately after the glucose tolerance test on the last day of the experiment, and then the 30, 60, 90, and 120 minute values were measured. The blood glucose level was measured by collecting blood (5 μl) at each time from the tail artery and using a blood glucose tester (Glucocard, Hoechst Marion Lucel Co., Ltd.). In addition, the E group did not perform glucose load. The 120 minute value was taken as the glucose value, and the sum of the immediately following value, the 30, 60, 90, and 120 minute values was determined as the total blood glucose value.
<Pancreatic insulin>
Pancreatic insulin levels were measured by a radioimmunoassay method with ¹²⁵ I insulin using a portion of the pancreas collected from each rat on the last day of the experiment.

<Others>
The day before the last day of the experiment, all rats were fasted, and the next day, deep anesthesia (Nembutal, 45 mg / kg, ip) was taken, and blood was collected from the left ventricle as much as possible with a 20 G blood collection needle.
Using the collected blood, a biochemical test (automatic chemical analyzer: Auto Lab, Radio lmmuno Assay method) was performed.
The results obtained were analyzed by Wilcoxon U-test for comparison between groups, and judged to have a significant difference with a risk rate within 5% (p <0.05). All grades were expressed as mean and standard error values.

[3] Test results Table 1 shows the results.
(Table 1)
Group A Group B Group C Group D Group E Group
No administration group 5 g administration group 10 g administration group 15 g administration group Non-sugar load control group
Glucose (mg / dl)
238.5 ± 9.6 223.6 ± 7.6 196.3 ± 9.1 ^* 191.4 ± 3.9 ^* 191.3 ± 1.9 ^*
Total blood sugar (mg / dl)
1320 ± 24 1268 ± 36 1033 ± 29 ^* 1005 ± 39 ^* 521 ± 2.3 ^*
Fructosamine (μmol / dl)
239 ± 24 224.8 ± 18 198 ± 18 ^* 194 ± 24 ^* 200 ± 12 ^*
1,5-anhydroglucitol (μg / ml)
5.6 ± 1.0 8.3 ± 0.9 11.9 ± 1.2 ^* 12.6 ± 0.8 ^* 5.4 ± 0.9
Pancreatic insulin (pg / ml)
38.9 ± 4.2 39.5 ± 3.0 41.8 ± 4.0 77.9 ± 2.4 ^* 42.9 ± 4.1
Total cholesterol (mg / dl)
108.2 ± 1.6 112.8 ± 1.6 121.5 ± 2.3 129.6 ± 1.6 ^* 115.8 ± 2.1
HDL cholesterol (mg / dl)
74.9 ± 0.3 76.4 ± 0.6 78.6 ± 0.9 ^* 81.3 ± 0.7 ^* 72.1 ± 0.6
Triglyceride (mg / dl)
24.9 ± 2.1 26.1 ± 1.3 33.6 ± 2.1 ^* 38.2 ± 1.9 ^* 31.6 ± 1.6 ^*
* : Significant difference

<Total glucose and blood sugar>
The glucose / blood glucose sum was markedly decreased according to the dose of the extract. In particular, in the 10 g administration group and the 15 g administration group (groups C and D), as compared with the non-administration group (group A), suppression of the increase was recognized with a significant difference, and the glucose load was not performed in the glucose level. It was almost the same as the value of the E group.
Therefore, even if a large amount of sugar was consumed, it was confirmed that the intake of the Yamabushitake extract suppresses the increase in blood glucose level to the same level as when no sugar was consumed.

<Fructosamine>
Fructosamine exists as a glycated protein in which glucose is bound to the amino group of plasma protein. Because the plasma protein has a half-life of 10-14 days, fructosamine reflects the glycemic state about two weeks ago. Therefore, fructosamine measurement is used as an index for short-term blood glucose control.
The fructosamine level was also significantly reduced according to the extract dose. In particular, the 10 g administration group and the 15 g administration group (groups C and D) have a significant difference as compared with the non-administration group (group A), and a decrease in fructosamine level was observed, and no glucose loading was performed. The value was lower (normal range).
Therefore, also in the fructosamine level, the blood glucose level increase inhibitory effect by ingestion of the Yamabushitake extract was confirmed.

<1,5-Anhydroglucitol>
1,5-Anhydroglucitol (1,5-AG) is a monosaccharide that is frequently present after glucose, and is widely present in the living world. Ingested primarily orally from food and thought to be mostly reabsorbed by the kidneys, 1,5-AG is specifically low when it is excreted in the urine due to the onset of diabetes . Therefore, measurement of 1,5-AG is considered to be useful for diagnosis and treatment effect determination of diabetes.
The 1,5-AG value was also markedly increased according to the extract dose. In particular, in the 10 g administration group and the 15 g administration group (groups C and D), an increase in the 1,5-AG value was observed with a significant difference compared to the non-administration group (group A).
Therefore, regarding 1,5-anhydroglucitol level, improvement of diabetes was confirmed by ingestion of Yamabushitake extract.

<Pancreatic insulin>
Insulin is a hormone secreted from the β-cells of the pancreas, and is necessary when glucose in the blood is taken into the cell and used as energy. Diabetes reduces the secreted amount of this insulin or the action of insulin. Is a disease in which glucose is not used and overflows into the blood.
The pancreatic insulin level was markedly increased according to the extract dose. In particular, in the 15 g administration group (Group D), an increase in pancreatic insulin level was observed with a significant difference compared to the non-administration group (Group A).
Therefore, it was confirmed that the intake of Yamabushitake extract suppresses the decrease in pancreatic insulin level due to diabetes.

<Total cholesterol>
Cholesterol is an essential component for maintaining cell morphology and function as a constituent of biological cell membranes and as a precursor for steroid hormones and bile acids. About 8% of the living body is distributed in the blood, and the cholesterol pool in the body is maintained in a steady state by turning over about 45% per day. 70% of blood cholesterol is in the ester form and the rest is in free form, but the sum is called total cholesterol. By measuring total cholesterol, it is possible to estimate a disease that causes abnormal lipid metabolism.
The total cholesterol level was markedly increased according to the extract dose. In particular, in the 15 g administration group (Group D), an increase in the total cholesterol level was recognized with a significant difference compared to the non-treatment group (Group A).

<HDL cholesterol>
High-density lipoprotein (HDL) plays an important role in reversely transferring cholesterol from the periphery to the liver and is involved in the removal mechanism of cholesterol accumulated in cells. Diabetes is mentioned as one of the diseases where the HDL cholesterol level becomes low.
A marked increase in HDL cholesterol level was observed according to the extract dose. In particular, in the 10 g administration group and the 15 g administration group (groups C and D), an increase in the HDL cholesterol level was recognized with a significant difference compared to the non-administration group (group A).

<Triglyceride>
Triglyceride is one of triglycerides together with diglyceride and monoglyceride, and measurement of triglyceride is performed as an index for diagnosis of disease causing lipid metabolism abnormality, prognosis determination, and the like.
A significant increase in triglyceride level was observed according to the dose of the extract. In particular, in the 15 g administration group (Group D), an increase in the triglyceride value was observed with a significant difference compared to the non-administration group (Group A).

From the above results, it became clear that diabetes was obviously prevented and improved by administration of Yamabushitake according to the present invention.
Further, administration of Yamabushitake according to the present invention did not cause any adverse side effects. For comparison, Wister Kyoto rats (WKY rats), which are normal rats, were also tested, but no abnormal decrease in blood glucose level due to administration of the extract was observed. From this, it can be seen that Yamabushitake extract does not abnormally lower blood sugar levels and does not cause any problematic side effects, so it can be safely ingested.

2. Pathological findings After excision of the pancreas on the last day of the experiment, it was infiltrated with 4% formalin / phosphate buffer to prepare a sample for tissue observation and β-cell count. Tissue sections were 4 μm, and the number of β cells in each group, foam degeneration, enlargement of pancreatic islets, and observation of vitreous substances were performed by hematoxylin-eosin staining method or Gomori staining method.
Foam degeneration, pancreatic islet enlargement, and vitreous substances all indicate pancreatic tissue degeneration.
A β cell is a cell that secretes insulin on the islets of Langerhans, and diabetes occurs when the secretory amount of insulin decreases due to a decrease in β cell function. The number of β cells was detected immunohistologically using an insulin monoclonal antibody. After staining with PCNA, the number of β cells in 5 islets was counted under a microscope.

Foam degeneration, pancreatic islet enlargement, and vitreous substances were evaluated by comparison with the non-administration group, and the evaluation criteria for the number of β cells were as follows.
-: 9 or less +: 10-29
++: 30 to 49
+++: 50 or more The results are shown in Table 2.
(Table 2)
Group A Group B Group C Group D
β-cell count ++ ++ ++++ ++
Foam modification No change Slight improvement Improvement
Pancreatic islet enlargement No change Slight improvement Improvement
Glass-like substance No change Suppression of deposition Suppression of deposition

A significant increase in the number of β cells was observed with the administration of the extract. That is, compared with the non-administration group (Group A), a marked suppression of the decrease in the number of β cells was observed in the 10 g administration group and the 15 g administration group (Groups C and D).
In addition, foam degeneration, pancreatic islet enlargement, and vitreous substances were significantly improved in the 10 g administration group and the 15 g administration group (Groups C and D) compared to the non-administration group (Group A). It was.
Therefore, in any of the items of foam degeneration, pancreatic islet enlargement, vitreous-like substance, and β-cell count, a significant prevention and improvement effect of diabetes was confirmed by ingestion of Yamabushitake in a dose-dependent manner.

From the above results, it was confirmed that the Yamabushitake according to the present invention has a low possibility of causing side effects and has an excellent effect for improving and preventing diabetes.
In addition, since the agent for improving and preventing diabetes according to the present invention can be expected to have a direct improvement effect on pancreatic β cells, it is particularly effective for type II diabetic patients that are mainstream in Japan.

Claims (3)

Yamabushitake strain with a deposit number of FERM P-19600. Diabetes prevention-improving agent comprising Yamabushitake strain having deposit number FERM P-19600 as an active ingredient. The diabetes prevention-improving agent according to claim 2, comprising a dried powder of Yamabushitake strain having deposit number FERM P-19600 and / or a hot water extract thereof.