JP4738053B2 - Oligosaccharide esterified compounds effective for obesity and diabetes - Google Patents

Description

  The present invention relates to an agent effective for obesity and diabetes, and an oligosaccharide esterified product.

  The number of diabetics continues to increase and is a serious problem. Diabetes includes type 1 (insulin-dependent) diabetes in which insulin secretion is insufficient and type 2 (non-insulin-dependent) diabetes in which the effect of insulin is reduced. In recent years, patients with type 2 (non-insulin dependent) diabetes have increased significantly due to lifestyle changes such as lack of exercise and a fatty diet, accounting for more than 80% of the total number of patients.

Insulin secreted from pancreatic β cells binds to the α subunit of the insulin receptor present on the surface of the liver, muscle and fat cells. The signal promotes the action of the tyrosine kinase of the β subunit of the insulin receptor and is transmitted into the cell to exert various insulin actions. It has been clarified that the activity of this tyrosine kinase is decreased in the non-insulin-dependent type, which is considered to be a part of the cause of insulin resistance. (Non-patent documents 1 to 3)
Currently, insulin-dependent diabetic patients are treated with insulin. In non-insulin-dependent diabetic patients, sulfonylurea drugs that promote insulin secretion from β cells, suppression of sugar release in the liver In addition, a biguanide agent that promotes the uptake of sugar in peripheral tissues such as fat and muscle, an insulin resistance improving drug that enhances the sensitivity of the receptor to insulin, suppresses gluconeogenesis in the liver, and absorbs carbohydrates from the digestive tract An α-glucosidase inhibitor or the like that improves postprandial hyperglycemia by being delayed has been used.

However, these drugs are known to cause hypoglycemic symptoms such as intense hunger, cold sweat, palpitation, trembling of hands and feet, and headache with dosage. Therefore, provision of a drug with higher effectiveness and superior safety and price is necessary for further improving the quality of life (QOL) of patients.
The human body is controlled by sympathetic nerves and parasympathetic nerves, but when stress is applied to the living body, the sympathetic nerve center in the hypothalamus is stimulated, and glucose is released from the pancreas by promoting glucagon secretion and liver glycogenolysis. Since it is released inside, blood sugar level rises. Furthermore, sympathetic nerve stimulation suppresses a decrease in blood glucose level by suppressing insulin secretion from the pancreas. Moreover, secretion of glucocorticoids from the adrenal cortex promotes glycogenolysis in the liver and suppresses glucose utilization in muscles. From the above, glucose remains in the blood, leading to an increase in blood sugar level. If glucocorticoids are present in the blood more than necessary, the blood glucose level is always high, resulting in cell damage to the pancreas, which may lead to diabetes, obesity, hypertension, osteoporosis, hyperlipidemia, etc. Sex is conceivable.
To further describe sympathetic nerves, there are adrenergic receptors on the surface of tissue cells that respond to sympathetic stimulation, and there are two types, α and β. The reaction through the α receptor is generally an excitatory reaction and causes contraction of smooth muscle and blood vessels.
On the other hand, there are three subtypes of β receptor, and the heart reaction caused by β1 receptor stimulation is an excitatory response such as an increase in heart rate, contractile force, and automaticity, and β2 receptor stimulation is Causes relaxation of smooth muscles such as tracheal smooth muscle, intestinal smooth muscle, and uterine muscles and dilation of blood vessels, glycogenolysis in the liver and promotion of glucagon secretion in the pancreas. Moreover, it has been clarified that stimulation of β3 receptor localized in adipocytes promotes lipolysis.
So far, the development of drugs targeting the β3 receptor has been intensively studied. However, although there are differences depending on individual differences and types of drugs, palpitation, hot flashes on the face, tachycardia, chest pain, arrhythmia, increased blood pressure, headache, dizziness, insomnia, excitement, tinnitus, trembling of fingers, numbness, limbs and fingers The problems and effectiveness of side effects such as guts, dry mouth, nausea, vomiting, abdominal pain, loss of appetite, stomach discomfort, constipation, irritability (rash, itching, etc.), malaise, swelling are still issues.
Therefore, there is a strong demand for the development of foods or medicines that are sufficiently satisfactory for safe and reliable treatment even after long-term use. To date, such problems have been solved. There are no medicines or foods as far as the inventors know.

  As a result of intensive studies to solve the above-mentioned problems and continuing to search for various plant extracts, we found allylatose B, an oligosaccharide ester, from germinated brown rice. Arilatose B has been isolated and identified from plants of the family Aceraceae (Non-patent Document 4), but it is a new finding that it has been isolated from germinated brown rice that is eaten daily.

Diabetes, 41 (4), 476-83,1992 Diabetologia 41,257,1998 Nippon Rinsho, 58 (2), 370-375,2000 J. Nat. Prod., 63, 1066-1069, 2000

An object of the present invention is to provide an oligosaccharide ester, allylatose B, a glucocorticoid receptor antagonist and an adrenergic β3 receptor agonist containing allylatose B, and pharmaceuticals and food compositions using them. It is. A glucocorticoid receptor antagonist and an adrenergic β3 receptor agonist have an effect of improving glucose metabolism and lipid metabolism, and are used for prevention or treatment of obesity and diabetes.

  The present inventor has conducted extensive research to solve the above problems, and as a result of continuing to search for various plant extracts, allylatose B separated from the germinated brown rice ethanol extract, the above problems, The inventors have found that there is an effective action and have completed the present invention.

That is, the present invention is summarized as follows.
(1) A glucocorticoid receptor antagonist, an adrenergic β3 receptor agonist, comprising allylatose B represented by the following chemical formula (I):

According to the present invention, a glucocorticoid receptor antagonist, adrenaline β3 receptor agonist, which has superior stability and safety, and is advantageous in terms of price, containing allylatose B, and a prophylactic or therapeutic agent for obesity and diabetes containing them can do.

  Hereinafter, allylatose B, a glucocorticoid receptor antagonist containing the same, an adrenergic β3 receptor agonist, and a medicine and food composition using them will be described.

First, allylatose B of the present invention is a compound represented by the chemical formula (I). For chemical formula (I), the IUPAC name is (E)-((2R, 3S, 4S, 5R, 6R) -6-((2S, 3S, 4S, 5R) -3,4-dihydroxy-2,5- bis (hydroxymethyl) -tetrahydroxy-3-methoxyphenyl) acrylate.
The allylatose B represented by the chemical formula (I) of the present invention can be obtained, for example, by separating it from an ethanol extract of germinated brown rice.
Germinated brown rice can be obtained, for example, by the following method. The brown rice is left as it is, or a part of the brown rice is crushed with a rice mill or a non-washing rice machine to peel and tear, and the resulting brown rice is immersed in a germination tank (germination tank), or water necessary for germination is added. To do. Milling can also be performed after immersion. The degree of germination is generally such that bulges of about 0.5 mm to 2.0 mm from the embryo part, or protrusions and young buds can be confirmed, but nutritional components such as γ-aminobutyric acid due to germination are added. Analysis and germination time can also be set to maximize nutrient content. After germination, heat treatment or the like is applied to stop germination. As the method, steaming may be performed, or temperature treatment or drying may be performed by an appropriate method such as hot air, microwave, or cooling. .

Germinated brown rice has blood cholesterol lowering action, blood pressure rise inhibiting action (Japanese Patent Laid-Open No. 2003-9810), blood sugar rise inhibiting action (Yukihiko Ito et al., Glycemic index (GI) by ingesting germinated brown rice, glycemic load (GL) and insulin reaction) It is known to have the 2nd Japan glycemic index study group program (2003).
In addition, oligosaccharides are known to have a blood glucose level lowering action by α-glucosidase inhibition, but such action has not been reported to allylose B of the oligosaccharide ester of the present invention.
As a method for producing allylatose B represented by the above chemical formula (I) of the present invention, first, koji (including the starch portion) is collected from the germinated brown rice as described above, and then hot water is used to expand the starch. After processing and leaving still for a while, ethanol of the density | concentration of 50-99.5 mass%, Preferably 70-99.5 mass% is added there, A supernatant is concentrated and dried and the germinated rice extract is obtained. Next, it can be obtained by separating and purifying allylatose B from the germinated brown rice extract by reverse phase HPLC or the like.

  The glucocorticoid receptor antagonist and adrenergic β3 receptor agonist of the present invention may use allylatose B thus obtained as it is as an active ingredient, or germinated brown rice obtained as described above. May be used as a composition containing the ethanol extract as it is. As such a composition, any various components that do not impair the effects of the present invention and do not adversely affect the composition as an auxiliary, medium, or carrier, and contain the allylatose B of the present invention. Things can be configured.

  When allylatose B of the present invention is used as a glucocorticoid receptor antagonist or an adrenergic β3 receptor agonist, its application amount is the age, body weight, symptom, application route, application schedule, formulation form, etc. of the intaker. For example, in the case of oral administration, the weight standard of allylatose B is usually about 0.0001 to 0.5 g / kg, more preferably 0.001 to 0.2 g / kg in terms of adult weight. The dose may be divided into several times a day at about kg.

  Moreover, it can be set as a glucocorticoid receptor antagonist and an adrenergic (beta) 3 receptor agonist by mix | blending the allylatose B of this invention with a suitable base, a support | carrier, a medium, and an excipient | filler.

  The allylatose B, glucocorticoid receptor antagonist, and adrenergic β3 receptor agonist of the present invention can be used as an oral composition for medicine or food.

  As an application method of allylatose B of the present invention for pharmaceutical use, either oral administration or parenteral administration can be employed. In administration, the active ingredient can be mixed with a solid or liquid non-toxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration and injection, and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, solutions such as solutions, suspensions and emulsions, freeze-dried preparations, and the like. It can be prepared by conventional means. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin , Water, physiological saline and the like. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents and the like can be appropriately added as necessary.

  As a composition for foods, treatment and prevention of hypertension, obesity, diabetes, allergies, kidney disorders, bronchial disorders by adding allylatose B of the present invention as it is, or by adding various nutritional components, or by including them in foods and drinks. It can be used as a health food or food material useful in For example, after adding a suitable auxiliary agent such as starch, lactose, maltose, vegetable oil powder, cacao butter powder, stearic acid, etc., using conventional means, a form suitable for oral use, such as granule, granule, tablet It may be formed into capsules, pastes, etc. and used for food, and various foods such as processed foods such as ham and sausage, fishery processed foods such as kamaboko and chikuwa, bread, confectionery, butter, milk powder, fermented milk It may be used by adding to dairy products. The amount of allylatose B of the present invention can be appropriately set depending on the type and state of the food or food material.

Next, the present invention will be described in more detail with reference to examples. The following production examples, examples and formulation examples show preferred examples of the present invention and are not limited thereto. Further, “%” in each example is based on mass.
1) Crude extraction of allylatose B 4.1 kg of koji (including starch) was prepared from 14 kg of germinated brown rice. Then, in order to expand the starch, 40 L of hot water was added and the mixture was boiled for about 30 minutes and allowed to stand until the liquid temperature dropped to 40 ° C. or lower, and then 40 L of 99.5% ethanol was added thereto and stirred. It was placed at 4 ° C. overnight and the supernatant was concentrated to dryness to obtain 100 g of germinated rice extract.
2) Purification and fractionation of allylatose B Using 5 g of the obtained germinated rice extract, it was separated and purified by the following method and conditions.

·Equipment configuration:
Medium pressure liquid chromatography system: Kronlab GmbH
Reversed phase column: Polyprep 60-60 RP-18 (Macheley & Nagel)
・ Eluent:
Purified water 100%, followed by purified water 100% → methanol 100% gradient, followed by isopropanol 100%
・ Preparation
Fraction P (0.15 g) eluting with an eluent of approximately 52% purified water and 48% methanol was obtained.

3) Separation and purification of fraction P by high-performance liquid chromatography using 0.15 g of fraction F according to the following method and conditions.

·Equipment configuration:
Fully automatic fractionation / purification / sorting system: SEPBOXLight
Preparative column: Merck Select B 250 × 16 mm, 10 μm
Detector: ELSD (Sedex75)
UV (Merck, 254nm)
・ Eluent:
Flow rate: 15 mL / min
Eluent: A ... 0.5 mM ammonium formate, 0.1% formic acid aqueous solution
B: 0.5 mM ammonium formate, 0.1% formic acid acetonitrile:
Methanol = 1: 1 solution Gradient:
Time (minutes) A (%) B (%)
0.0 72 28
30.0 62 38
30.10 100
38.0 0 100
-Fractionated substance 6.9 mg of sample (a) was fractionated from fraction P.

4) Confirmation of purity of sample (a) by high performance liquid chromatography, confirmation of retention time and equipment configuration:
HPLC system: Merck Hitachi
Analysis column: Merck Select B 250 × 4 mm, 5 μm
Detector: ELSD (Sedex75)
・ Eluent:
Flow rate: 1 mL / min
Eluent: A ... 0.5 mM ammonium formate, 0.1% formic acid aqueous solution
B: 0.5 mM ammonium formate, 0.1% formic acid acetonitrile:
Methanol = 1: 1 solution Gradient:
Time (minutes) A (%) B (%)
0.0 85 15
30.0 0 100
40.0 0 100

The purity and holding time of the sample (a) were confirmed under the analysis conditions. The retention time is 9.2 minutes.

5) Structure determination of sample (a) About the sample (a) fractionated by said 3), the structure of the compound was determined using the LC / MS analyzer and the NMR analyzer.

  The same eluent for LC / MS analysis was used as in 4), and the gradient conditions were appropriately adjusted. The MS system used PE-Sciex API150 (+ / (-)-ESI, Fast-Switching-Mode).

¹ H-NMR, HSQC, HMBC, and HH-COSY were measured at a resonance frequency of 400 MHz or 500 MHz. ^{The 13} C-NMR spectrum was assigned using the methods of HMBC and HSQC. The solvent was deuterated methanol and used for the chemical shift standard ( ¹ H-NMR 3.30 ppm, ¹³ C-NMR 49.0 ppm).
The LC / MS and NMR data of sample (a) are shown below.
LC / MS
(+)-ESI: 536 [M + N H ₄ ] ⁺ , 357 [M C ₆ H ₁₀ O ₅ + H] ⁺ , 177 [C ₁₀ H ₉ NO ₃ ]
⁺
(−)-ESI: 547 [M + HCOO], 517 [M−H] ^−
1 H-NMR
The spectral assignments and chemical shifts are shown in Table 1.

以上により、サンプル（ａ）はアリラトースBと同定された。

From the above, sample (a) was identified as allylatose B.

Next, the glucocorticoid receptor binding test was performed on the allylatose B obtained as described above by the method described below.
The glucocorticoid receptor binding test was conducted with reference to the method of Meshinchi, S et al. (J. Biol. Chem., 265, 4863, 1990). A cytosolic fraction was prepared from mouse fibroblasts (L-929), and allylatose B (final concentrations: 10 and 30 μg / ml) obtained in Example 1 and 10 nM.
[ ³ H] dexamethasone was used for the test (the reaction solution was Hepes-Tris buffer (pH 7.4) containing 0.1 mM EDTA). For nonspecific binding, 10 μM triamcinolone was used. After incubation at 4 ° C. for 4 hours, the labeled ligand bound to the receptor was suction filtered onto a Whatman GF / B glass fiber filter using a cell harvester (manufactured by Brandel), and the reaction was stopped immediately. Washed 3 times with 5 ml of Tris-HCl buffer (pH 7.7). Next, the radioactivity on the filter was measured with a scintillation counter to determine the total amount of binding. The amount of binding in the presence of 10 μM triamcinolone measured at the same time was defined as the amount of non-specific binding, and the amount of specific binding was determined by subtracting this from the total amount of binding.
The results are shown in Table 2. The affinity (%) shown here was calculated by the following formula.

Affinity (%) = 100 − [(C ₁ −B ₁ ) / (C ₂ −B ₁ )] × 100
(Wherein C ₁ represents the amount of [ ³ H] dexamethasone bound to the membrane fraction in the presence of a known amount of the test compound and [ ³ H] dexamethasone, and C ₂ represents the test compound. the represents a bond amount for ^[3 H] dexamethasone membrane fraction of the time, except, B ₁ represents an amount of binding ^[3 H] membrane fraction of dexamethasone in excess of triamcinolone (10μM) presence.) As a positive control in this reaction system, the IC ₅₀ value of dexamethasone was 2.9 nM.

(result)

Next, an adrenergic receptor binding test was performed on allylatose B obtained as described above by the method described below.

Adrenaline β ₃ receptor binding test
(Test method)
The binding test to the adrenergic β ₃ receptor was carried out by partially modifying the method of CURRANP. K. et al. (Cell. Signal., 8 (5), 355-364 (1996)). Membrane fractions were prepared from human neuroblasts (SK-N-MC) and tested using allylatose B (final concentrations: 10 and 30 μg / ml) obtained in Example 1 and 0.6 nM [ ¹²⁵ I] iodocyanopindolol. (The reaction solution was 50 mM Hepes-NaOH (pH 7.4) containing 4 mM MgCl ₂ , 0.04% BSA, 0.1 μM propranolol).
For nonspecific binding, 50 μM alprenolol was used. After reacting at 37 ° C. for 90 minutes, using a glass filter (Model 701, manufactured by Skatron Inc.), filtration was performed to separate the membrane preparation bound to [ ¹²⁵ I] iodocyanopindolol, and the above-mentioned Tris was performed twice. Washed with 5 ml of buffer. Place this glass filter in a vial, mix with aquasol (cocktail for liquid scintillation), measure the amount of [ ¹²⁵ I] iodocyanopindolol bound with a liquid scintillation counter, and calculate the affinity (%) from the following formula. It is shown in Table 3.
Affinity (%) = 100 − [(C ₁ −B) / (C ₀ −B)] × 100
(Wherein, C ₁ represents the amount of binding ^[125 I] iodocyanopindolol film in a state where a known amount of test compound is ^[125 I] iodocyanopindolol coexist, C ₀ is except the test compound [represents the amount of binding ^[125 I] iodocyanopindolol membrane, B represents a bond amount for ^[125 I] iodocyanopindolol membranes in the presence of an excess of alprenolol (50μM).] when the Note, in the present assay system The IC ₅₀ value of cyanopindolol, which is an adrenergic β ₃ receptor blocker as a positive control, was 30 nM, and as can be seen from the results, allylatose B of the present invention has an effect on the adrenergic β ₃ receptor. Recognize.

(result)

Next, formulation examples as pharmaceuticals and foods, which are specific usage forms of allylatose B of the present invention, are shown below.

Formulation Example 1: Composition (tablet) for the prevention and treatment of obesity and diabetes
Using allylatose B represented by the chemical formula (I) obtained in Example 1 above, tablets for prevention and treatment of obesity and diabetes having the following composition were prepared by a conventional method.

(Composition) (mass%)
Arilatose B
2.0
Lactose 77.0
Corn starch 20.0
Guar gum 1.0

Formulation Example 2: Juice Juice having the following composition was produced by the conventional method using allylatose B represented by the chemical formula (I) obtained in Example 1 above.

(Composition) (mass%)
Frozen and concentrated Wenzhou orange juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Perfume 0.2
Color element 0.1
Arilatose B
0.2
Water 83.28

Claims (2)

A pharmaceutical composition which is a glucocorticoid receptor antagonist comprising the oligosaccharide ester allylatose B represented by the following chemical formula (I):

A pharmaceutical composition which is an adrenergic β3 receptor agonist, comprising the compound represented by the above chemical formula (I) .