JP2018203678A - Composition for blood glucose elevation inhibition - Google Patents

Abstract

To provide a composition for blood glucose elevation inhibition useful for the prevention or the treatment of diabetes and lifestyle diseases and the like.SOLUTION: Provided is a composition for blood glucose elevation inhibition containing syringic acid or a pharmacologically acceptable salt or solvate thereof as an active ingredient. Preferably, the aforementioned composition is for promoting the sugar incorporation to cells. Preferably, the composition is for activating GLUT4. Preferably, the composition is for prevention/melioration of diabetes. Preferably, the composition is for preventing/improving insulin resistance.SELECTED DRAWING: None

Description

  The present invention relates to a composition for suppressing an increase in blood sugar, comprising as an active ingredient an ingredient identified from an extract of Ganoderma mycelium culture medium.

  Diabetes is a metabolic disease that exhibits a hyperglycemic state due to impaired insulin secretion or dysfunction, induces complications such as neuropathy, retinopathy and nephropathy, and reduces the patient's quality of life (QOL). Glycemic control before or after onset is important. Currently, the development of health foods and food ingredients intended to prevent lifestyle-related diseases including diabetes has been actively conducted.

  In the present applicants as well, as disclosed in Patent Document 1, Ganoderma mycelium is grown on a solid medium made from bagasse (a fibrous component of sugar cane) and defatted rice bran, and the whole medium immediately before the fruiting body is generated. In order to prevent and treat diabetes by discovering that the extract of reishi mushroom mycelium culture medium, which is pulverized, extracted with hot water and spray-dried, has maltase inhibitory activity and suppresses digestive absorption of carbohydrates. It is clarified that it can be used as a pharmaceutical, health food, etc.

  On the other hand, according to another study, for example, as disclosed in Patent Document 2 by the present applicants, syringic acid contained in the extract of the above-mentioned Ganoderma mycelium culture medium is a disorder of hypoxic cerebral ischemia. It has been clarified that it can be used as an active ingredient for prevention and improvement.

Japanese Patent No. 4602675 Japanese Patent No. 5919208

  If a new active ingredient of a composition for suppressing an increase in blood glucose can be provided, the range of treatment and treatment options is expanded. In addition, it is desirable that the active ingredient is a compound that has been isolated and identified, because it is easy to ensure its effectiveness stably regardless of the production batch.

  Accordingly, an object of the present invention is to provide a composition for suppressing an increase in blood sugar, which comprises an isolated and identified compound as an active ingredient and is useful for the prevention and treatment of diabetes and lifestyle-related diseases.

  The present inventors have isolated and identified a component having an action effect of suppressing blood sugar elevation from a culture medium extract obtained by culturing mycelium of Ganoderma fungi in a medium containing a plant fiber raw material. It came to complete.

  That is, the composition for suppressing blood sugar elevation according to the present invention is characterized by containing syringic acid or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

  It is preferable that the said composition contains 0.1-100 mass% of syringic acid or its pharmaceutically acceptable salt or solvate per dry part.

  Moreover, it is preferable to promote sugar uptake into cells.

  Moreover, it is preferable that the said composition is for GLUT4 activation.

  Moreover, it is preferable that the said composition is for the prevention and improvement of diabetes.

  Moreover, it is preferable that the said composition is for prevention and improvement of insulin resistance.

  Moreover, it is preferable that the said composition is a composition for food-drinks.

  Moreover, it is preferable that the said composition is a composition for pharmaceuticals.

  According to the present invention, syringic acid, which is an active ingredient, has an excellent effect of suppressing an increase in blood sugar. Therefore, by utilizing this, it is possible to provide a composition for suppressing an increase in blood glucose that is useful for the prevention and treatment of diabetes and lifestyle-related diseases.

It is a graph which shows the result of having identified the syringic acid and vanillic acid obtained in Test Example 1 from the extract of Ganoderma mycelium culture medium. It is a graph which shows the result of having tested about the influence which syringic acid and vanillic acid have on the uptake | capture of glucose of a cell in Experiment 2. FIG. It is a graph which shows the result of having tested about the influence which syringic acid gives in the uptake | capture of glucose of a cell in Experiment 3. It is a graph which shows the result tested about the influence which syringic acid gives to a body weight in the test example 4. It is a graph which shows the result of having tested about the influence which syringic acid has on food intake in Experiment 4. It is a graph which shows the result tested about the influence which syringic acid gives to a blood glucose level in Experiment 4. It is a graph which shows the result of having tested about the influence which syringic acid has on the blood insulin level in Experiment 5. FIG. It is a graph which shows the result tested about the influence which syringic acid has on the expression of GLUT4 protein in a skeletal muscle in Experiment 6.

  The composition for suppressing blood sugar elevation of the present invention comprises syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) represented by the following chemical formula (1), or a pharmaceutically acceptable salt or solvate thereof as an active ingredient. It contains.

  Since syringic acid can be synthesized by a known method and is also commercially available, such a chemically synthesized product can be used. Alternatively, it can be separated from acai, shiitake mushrooms, mannen mushrooms and the like, and those derived from such natural products may be used. There is no particular limitation on the form of the salt or solvate, and typically, for example, hydrochloride, phosphate, sulfate, sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, acetate salt, Acid salts, maleate salts, fumarate salts, ethanol solvates and the like.

  In the composition for suppressing blood sugar elevation of the present invention, in addition to the above active ingredients, there is no particular limitation on blending other materials, and a pharmaceutically acceptable base or carrier is added as necessary. In the form of tablets, granules, capsules, pills, powders, liquids, powders, jellies, rods, injections, inhalants, coatings, etc., by known formulation methods Can do. In that case, the active ingredient is preferably contained in the whole composition in an amount of 0.1 to 100% by mass, and 0.5 to 50 per dry matter, although it differs depending on the dosage form and is not unclear. More preferably, it is contained in an amount of 1% by mass, and most preferably 1-20% by mass per dry matter. In addition, the dose varies depending on the age, sex, health condition, etc. of the subject and the dosage form, and is not unclear. Typically, for example, in the case of oral administration, it is typically about 0.2 to 2 g per day for an adult. It is. If the dose is less than the range, it is difficult to obtain a sufficient effect, and if the dose is more than the range, the risk of causing some side effects increases. The composition for suppressing blood sugar elevation according to the present invention can naturally be applied to humans, but there is no particular limitation on the application of animals such as pets and livestock. Alternatively, it may be used in combination with other components having an inhibitory effect on the increase in sugar, such as acarbose, SU agent (sulfonylurea agent), pioglitazone, and there is no particular limitation.

  Regarding the administration form of the composition for suppressing blood sugar elevation of the present invention, for example, oral administration, intravenous administration, intracerebral administration, intraperitoneal administration, aspiration, nasal administration, transdermal administration, intracavitary administration, etc. Is mentioned. Among them, as shown in the examples described later, since the effect of suppressing blood sugar elevation can be sufficiently obtained by ingestion, from the viewpoint of reducing the burden on the intake and ease of taking, the form of oral administration Is preferred.

  There is no restriction | limiting in particular as a usage form of the said composition unless the effect is impaired. For example, pharmaceuticals, quasi-drugs, health foods, functional foods, dietary supplements, supplements, health-care foods, functional labeling foods, veterinary drugs, quasi-drugs for animals, veterinary health foods, for animals It can be used in various product forms such as functional foods, animal nutritional supplements, animal supplements, or in combination with these products. It can also be used in combination with various foods and drinks. In particular, in the form of products for health and health foods and functional labeling foods, for those who are concerned about their own blood sugar level, it is intended to prevent or improve diabetes and prevent or improve insulin resistance. It is preferable because it becomes easy to be taken as part of health management.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

<Test Example 1>
Uses Ganoderma mycelium culture medium extract (trade name “MAK”, Noda Shokuhin Kogyo Co., Ltd.) (hereinafter referred to as “MAK”) as a starting material, and its activity using DPPH free radical inhibitory activity as an index. Fractions were isolated and their active ingredients were identified. Specifically, the antioxidant activity test was performed as follows.

(DPPH free radical inhibitory capacity test)
(1) Test sample MAK is subjected to the fractionation means of MPLC of ultrafiltration, DIAION HP-20 column (adsorption), ODS column (reverse phase) and silica gel column (normal phase) sequentially to separate I drew it. The solvent of each fractionated fraction was dried, and the content (%) of the component of each fraction was calculated from the weight, and is shown in FIG.

(2) Evaluation of antioxidant activity
Measured from DPPH free radical inhibitory ability. The radical inhibitory effect was evaluated by the change in absorbance due to the decrease in DPPH. The same amount of DPPH solution was added to 1 mL of the sample solution in a light-shielded test tube, and after that, the mixture was reacted for 30 minutes in a 25 ° C. water bath and measured at 525 nm. Sample and DPPH were dissolved in 50% ethanol. The inhibition rate was determined by the ratio of absorbance between the solution containing the sample and the control solution.

(Identification of active ingredients)
A fraction showing high antioxidant activity after fractionation by the final silica gel column / MPLC was collected, and the compounds contained in the fraction were analyzed by ¹ H-NMR, ¹³ C-NMR analysis (Agilent Technology Co., Ltd.), and LC. / MS analysis (JEOL Ltd.) identified syringic acid (molecular weight 198.17) represented by the following chemical formula (2) and vanillic acid (molecular weight 168) represented by the following chemical formula (3). 1).

  Separately, the contents of syringic acid and vanillic acid in MAK were quantified by HPLC using chemical products syringic acid (Wako Pure Chemical Industries, Ltd.) and vanillic acid (Sigma-Aldrich Co., Ltd.) as standard. In 1 g of MAK, 0.511 mg of syringic acid and 0.868 mg of vanillic acid were contained.

<Test Example 2>
Using syringic acid (Chemicals, manufactured by Wako Pure Chemical Industries, Ltd.) and vanillic acid (Chemicals, manufactured by Sigma-Aldrich), the activity of promoting cellular glucose uptake was tested.

(Measurement test of glucose uptake)
(1) Cells used L6 cells, which are rat skeletal muscle-derived cultured cells, were used, and culture was carried out using Dulbecco's Modified Eagle's Medium supplemented with 10% bovine serum albumin as a liquid medium at 37 ° C. and 5% CO ₂ . Cultured in the environment.

(2) Measurement of glucose uptake L6 cells were seeded and cultured for 24 hours. Thereafter, using the above chemical product, syringic acid or vanillic acid having a final concentration of 100 μM was added and further cultured for 24 hours. At that time, a culture that was cultured for 30 minutes in the presence of insulin at a final concentration of 10 nM, or a culture that was cultured for 24 hours in the presence of Troglitazone at a final concentration of 10 mM was used as a positive control. Moreover, what was cultured for 24 hours without adding each sample was made into the negative control (Control). Subsequently, replacement with Krebs-Ringer's phosphate buffer [123 mM NaCl, 4.94 mM KCl, 1.23 mM MgSO4, 0.84 mM CaCl ₂ , 4.99 mM glutamine, 20 mM NaH ₂ PO ₄ , 15 mM Hepes (pH 7.4)] for 2 hours After culturing, 2- [ ³ H (G)]-Deoxy-D-glucose (hereinafter sometimes referred to as “[ ³ H] 2-DG”) (9.25 MBq, NET328A, PerkinElmer, Waltham, USA) Was added and incubated for 10 minutes. Thereafter, the culture solution was removed and washed once with ice-cold DMEM medium and ice-cold PBS. Furthermore, 0.1% SDS containing 1N NaOH was added to collect the cells, and then the radioactivity was measured using a liquid scintillation counter (LSC5100, Aroka, Tokyo, Japan).

  As a result, the result of each test group was expressed as a relative value with the glucose uptake amount of the Control group being 1 (n = 3).

  As a result, as shown in FIG. 2, the addition of syringic acid showed an increase in the amount of glucose uptake of cells compared to the Control group. On the other hand, no increase in glucose uptake was observed with vanillic acid.

<Test Example 3>
Using syringic acid (chemical product, manufactured by Wako Pure Chemical Industries, Ltd.), the activity of promoting cellular glucose uptake was tested in the same manner as in Test Example 2.

  Specifically, the glucose uptake-promoting activity was the same as in Test Example 2 except that the test sample was prepared using the above-described chemical product and the final concentration of syringic acid was 0.01 μM, 1 μM, and 100 μM. Was tested. At that time, a 30-minute culture in the presence of 10 nM insulin was used as a positive control.

  The result was expressed as a relative value with the glucose uptake amount of the Control group taken as 1 for each test group, and each value was expressed as an average value ± standard deviation. Statistical significance was analyzed by t-test after equal variance test. (N = 3).

  As a result, as shown in FIG. 3, the addition of syringic acid increased the amount of cellular glucose uptake by about 1.1 times at a final concentration of 0.01 μM as compared to the Control group, and the final concentration was 1 μM. At 100 μM, the increase was approximately 1.2 times and 1.3 times, respectively, and an increase in glucose uptake dependent on the syringic acid concentration was observed.

<Test Example 4>
Using syringic acid (a chemical product, manufactured by Wako Pure Chemical Industries, Ltd.), the effects of syringic acid on blood glucose levels were tested by animal experiments. Specifically, the animal test was performed as follows.

(Animal test)
A group of 6 week-old female KK-A ^y mice (CLEA Japan, Inc.) preliminarily raised for 1 week and then fed with a high fat diet (trade name “Quick Fat”, manufactured by CLEA Japan, the same applies hereinafter) (Control) Group), a group giving a high-fat feed mixed with 0.025% by mass of syringic acid (0.025% syringic acid group), a group giving a high-fat diet mixed with 0.25% by mass of syringic acid (0.25%) Syringic acid group), a group fed with a high-fat feed mixed with 0.5% by weight of MAK (0.5% MAK group), a group fed with a high-fat feed mixed with 1.0% by weight of MAK (1.0% MAK) The group was divided into 5 groups and reared for 8 weeks by free intake. During the breeding period, body weight and food intake were measured weekly every week. In addition, blood was collected from the tail vein of mice fasted for about 1.5 hours at 4 and 8 weeks, and blood glucose level was measured using a glucose determination kit (trade name “glucose CII test Wako”, manufactured by Wako Pure Chemical Industries, Ltd.). Each value was expressed as an average value ± standard deviation. Statistical significance was analyzed by t test after variance test (n = 4-7).

  FIG. 4 shows the results of body weight transition, and FIG. 5 shows the results of food intake transition.

  As shown in FIG. 4 and FIG. 5, regarding the body weight and the amount of food intake, no significant difference was observed in the MAK group and the syringic acid group until the last week of breeding compared to the Control group.

  FIG. 6 shows the results of blood glucose level transition.

  As shown in FIG. 6, blood glucose levels in the Control group were 223.4 ± 16.4 mg / dL at 0 weeks, 493.3 ± 64.0 mg / dL at 4 weeks, and 496.4 ± 98.0 mg / dL at 8 weeks. The blood glucose level increased with the progress. In the MAK group, 37% ± 74.2 mg / dL in the 0.5% MAK group at 4 weeks and 339.8 ± 100.3 mg / dL in the 1.0% MAK group, significantly suppressed compared to the Control group, and the blood glucose level was large until 8 weeks No change was observed. On the other hand, in the syringic acid group, no difference was observed at 4 weeks compared with the Control group, but at 8 weeks, the 0.025% syringic acid group was 291.5 ± 65.7 mg / dL, and the 0.25% syringic acid group was 281.0 ± 164.0 mg. The value was significantly low at / dL.

<Test Example 5>
The blood insulin level in the last week of the breeding was measured.

  Specifically, after the animal test of Test Example 4, plasma (5 μL) at the time of dissection of each group of mice was collected, and the insulin concentration was measured using an ultrasensitive rat insulin measurement kit (Morinaga Institute of Science). Manufactured). Each value is shown as an average value ± standard deviation. Statistical significance was analyzed by t test after variance test (n = 3-6).

  The results are shown in FIG.

  As a result, as shown in FIG. 7, it was 254.9 ± 93.2 ng / dL in the 1.0% MAK group and 165.4 in the 0.025% and 0.25% syringic acid groups, respectively, compared with the control group 457.3 ± 101.7 ng / dL. The values were significantly low at ± 45.0 ng / dL and 177.5 ± 110.1 ng / dL.

<Test Example 6>
The expression of GLUT4 (glucose transporter type 4) in skeletal muscle was analyzed by Western blotting. Specifically, after the animal test of Test Example 4, skeletal muscles (gastrocnemius and soleus) were collected at the time of dissection of each group of mice, and cell membrane fractions were prepared as follows.

(Preparation of cell membrane fraction)
Add 1.2 mL of buffer solution [10 mM Tris-HCl (pH 7.4), 1 mM phenylmethylsulphonyl fluoride (PMSF), protease inhibitor mixture (manufactured by Wako Pure Chemical Industries, Ltd.)] to 0.1 g of skeletal muscle, and polytron homogenizer Crushed with. The tissue suspension was centrifuged at 450 × g and 4 ° C. for 10 minutes, and the supernatant was used as a cell extract fraction. Further, after adding a buffer similar to the above to the supernatant and pulverizing with a polytron homogenizer, the tissue suspension was centrifuged at 450 × g for 10 minutes at 4 ° C., and the supernatant was subjected to 16,800 × g for 20 minutes. Centrifugation was performed, and 150 μL of a buffer solution (1% SDS, 50 mM DTT) was added to the resulting precipitate to obtain a cell membrane fraction.

  The prepared cell membrane fraction was developed on a 10% SDS polyacrylamide gel. After electrophoresis, the protein was transferred to a PVDF membrane, and the membrane protein was used as an anti-GLUT4 antibody (C-20; Santa Cruz Biotechnology) or as a control (Control). Western blot analysis was performed according to a conventional method using an anti-caveolin-1 antibody (N-20; manufactured by Santa Cruz Biotechnology), which is a specific antibody against a certain caveolin-1.

  The results are shown in FIG.

  As a result, as shown in FIG. 8, the amount of GLUT4 in the cell membrane increased 1.99 times in the 0.5% MAK group compared to the Control group. Furthermore, it increased 2.44 times and 3.04 times in the 0.025% and 0.25% syringic acid groups, respectively.

Claims (8)

  A composition for suppressing an increase in blood glucose, comprising syringic acid or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.   A composition for suppressing an increase in blood glucose, comprising 0.1 to 100% by mass of the syringic acid or a pharmaceutically acceptable salt or solvate thereof per dry matter.   The composition for suppressing an increase in blood glucose according to claim 1, which is for promoting sugar uptake into cells.   The composition for suppressing an increase in blood glucose according to claim 1, which is for GLUT4 activation.   The composition for suppressing an increase in blood glucose according to claim 1, which is for the prevention and / or improvement of diabetes.   The composition for suppressing an increase in blood glucose according to claim 1, which is for prevention / improvement of insulin resistance.   The composition for suppressing an increase in blood glucose according to any one of claims 1 to 6, which is a composition for food and drink.   The composition for suppressing an increase in blood glucose according to any one of claims 1 to 6, which is a pharmaceutical composition.