JP6147322B2 - Sirtuin 1 activator - Google Patents

Description

  The present invention relates to a new sirtuin 1 activator.

  A sirtuin (SIRT) is also called a longevity gene, and its activation is said to extend the lifespan of an organism. Among the sirtuin families, sirtuin 1 (SIRT1) has been studied in various ways, and it is known that activation of SIRT1 can suppress neurodegenerative diseases such as Alzheimer's disease, arteriosclerosis, osteoporosis, and skeletal muscle loss. (Non-Patent Document 1).

  As a SIRT1 activator, resveratrol which is a kind of polyphenol contained in red wine is widely known (Non-patent Document 2). Moreover, it has been reported that N-acetylglucosamine, which is an amino sugar constituting hyaluronic acid, activated SIRT1 in an experimental system using human chondrosarcoma cells (SW1353) (Non-patent Document 2).

JP 2013-199639 A

Experimental Medicine, Vol.31, No.20 (extra number), p45-52 (2013) Glucosamine Research, Vol.9, p21-24 (2013)

  An object of the present invention is to provide a new SIRT1 activator.

  Therefore, the present inventor examined the SIRT1 activation action of various components using normal chondrocytes instead of cancer cell lines such as SW1353 used in Non-Patent Document 2, and surprisingly, low molecular weight Hyaluronic acid, N-acetylglucosamine, glucosamine hydrochloride, proteoglycan, chondroitin sulfate, and N-acetylgalactosamine, which is a constituent amino sugar of chondroitin sulfate, have a molecular weight of 500 to 6,000 at concentrations that did not show SIRT1 activation action. The chondroitin sulfate oligosaccharide of the present invention was found to have an excellent SIRT1 activation action, and the present invention was completed.

  That is, the present invention provides the following [1] to [3].

[1] A SIRT1 activator comprising a chondroitin sulfate oligosaccharide having a molecular weight of 500 to 6,000 as an active ingredient.
[2] The SIRT1 activator according to [1], wherein the chondroitin sulfate oligosaccharide has at least a glucuronic acid-N-acetylgalactosamine-sulfuric acid structure.
[3] The SIRT1 activator according to [1] or [2], wherein the chondroitin sulfate oligosaccharide is a high-temperature high-pressure decomposition product of chondroitin sulfate.

  The chondroitin sulfate oligosaccharide having a molecular weight of 500 to 6,000 of the present invention is known to have excellent oral absorbability as described in Patent Document 1, but it is reported whether or not it exhibits the same pharmacological action as chondroitin sulfate. It has not been. Such chondroitin sulfate oligosaccharides having a molecular weight of 500 to 6,000 have excellent SIRT1 activation action, and various uses such as Alzheimer's disease, dementia and other neurodegenerative diseases, osteoporosis, diabetes, etc. In particular, it is useful for the prevention and treatment of diseases such as type 2 diabetes, mitochondrial diseases, reduced keratinocyte barrier function, lung diseases, corneal diseases, presbyopia and obesity.

The effect | action which has on the SIRT1 gene expression of the various components in a human normal chondrocyte is shown.

The active ingredient of the SIRT1 activator of the present invention is chondroitin sulfate oligosaccharide having a molecular weight of 500 to 6,000.
Such chondroitin sulfate oligosaccharides are known to have excellent oral absorbability as described in Patent Document 1, but it is not known whether or not they have the same pharmacological action as chondroitin sulfate.

The molecular weight of the chondroitin sulfate oligosaccharide is 500 to 6,000, more preferably 500 to 5,000, still more preferably 500 to 4,500, and still more preferably 500 to 3,000. Here, the molecular weight can be measured by gel filtration HPLC using a chondroitin sulfate oligosaccharide having a known molecular weight as a standard product. The average molecular weight of the chondroitin sulfate oligosaccharide is preferably 500 to 3,500, more preferably 1,000 to 3,000, and further preferably 1,500 to 2,500.
The chondroitin sulfate oligosaccharide preferably has at least a glucuronic acid-N-acetylgalactosamine-sulfate structure.
That is, those having 1 to 12 glucuronic acid-N-acetylgalactosamine-sulfuric acid structures are preferable, those having 1 to 10 such structures are more preferable, those having 1 to 9 such structures are more preferable, and those structures Those having 1 to 6 are more preferred. Here, glucuronic acid-N-acetylgalactosamine-sulfuric acid includes glucuronic acid-N-acetylgalactosamine 4-sulfate structure, glucuronic acid-N-acetylgalactosamine 6-sulfate structure, glucuronic acid-N-acetylgalactosamine 4,6- The sulfuric acid structure is included.

The chondroitin sulfate oligosaccharide can be obtained by decomposing chondroitin sulfate under various conditions. Examples of the degradation conditions include various methods such as degradation with acids and alkalis, degradation with enzymes, degradation under high temperature and high pressure conditions, and methods for obtaining a degradation product having at least glucuronic acid-N-acetylgalactosamine-sulfuric acid structure. preferable. More preferable decomposition methods include hydrothermal treatment (supercritical water or subcritical water treatment) under conditions of high temperature (175 to 220 ° C.) and high pressure (5 MPa to 25 MPa) as described in Patent Document 1.
Further, as the raw material chondroitin sulfate, those derived from mammals, fish and birds, for example, those derived from cattle, pigs, whales, rays, sharks, salmon, squids, chickens and the like can be used.

  The chondroitin sulfate oligosaccharide has an excellent SIRT1 activation action in normal chondrocytes as shown in Examples described later. In normal chondrocytes, hyaluronic acid-related substances such as low molecular weight hyaluronic acid, N-acetylglucosamine, and glucosamine hydrochloride did not show SIRT1 activation action. In addition, proteoglycan, chondroitin sulfate and N-acetylgalactosamine did not show SIRT1 activation action. Therefore, the excellent SIRT1 activation action of chondroitin sulfate oligosaccharide in the present invention was completely unexpected.

  The SIRT1 activator of the present invention has various uses based on the SIRT1 activation action, such as Alzheimer's disease, neurodegenerative diseases such as dementia, osteoporosis, diabetes such as type 2 diabetes, mitochondrial disease, and keratinocyte barrier function decrease. It is useful as a preventive or therapeutic agent for lung disease, corneal disease, presbyopia, obesity and the like. Here, examples of the form of the SIRT1 activator of the present invention include pharmaceuticals, foods and drinks (food for specified health use, functional display foods and the like), and cosmetics. Examples of pharmaceuticals include oral preparations, external preparations for skin, eye drops, injections and the like.

  Examples of the dosage form of an orally administered preparation when used as a pharmaceutical include, for example, tablets, capsules, granules, dragees, pills, fine granules, powders, powders, sustained-release preparations, suspensions, emulsions, A syrup agent etc. are mentioned.

  The above-mentioned preparation can be produced by a conventional method, and the chondroitin sulfate oligosaccharide may be used alone or in combination with a pharmaceutically acceptable carrier. Examples of the carrier include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid; starch, dextrin, gum arabic powder, gelatin, methyl cellulose, hydroxypropyl Binding agents such as cellulose, crystalline cellulose, ethyl cellulose, polyvinyl pyrrolidone, macrogol; disintegrating agents such as hydroxypropyl starch, sodium carboxymethylcellulose, carboxymethylcellulose calcium, carboxymethylcellulose, low-substituted hydroxypropylcellulose; sodium lauryl sulfate, soybean lecithin, Surfactants such as sucrose fatty acid ester, polysorbate 80; talc, waxes, hydrogenated vegetable oil, magnesium stearate, calcium stearate Lubricants such as aluminum, aluminum stearate, polyethylene glycol; fluidity promoters such as light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate; distilled water for injection, physiological saline, glucose Examples include diluents such as aqueous solutions, olive oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, and polyethylene glycol. If necessary, conventional additions such as flavoring agents, coloring agents, fragrances, bactericides, osmotic pressure regulators, pH regulators, emulsifiers, absorption aids, antioxidants, thickeners, tonicity agents, etc. An agent can also be added as appropriate.

Eye drops include other medicinal ingredients such as anti-inflammatory agents such as dipotassium glycyrrhizinate, epsilon aminocaproic acid, allantoin, sodium azulene sulfonate, zinc sulfate, vitamins such as flavin adenine dinucleotide sodium, pyridoxine hydrochloride, panthenol , Amino acids such as potassium L-aspartate, L-glutamic acid, and the like.
Examples of eye drops include inorganic salts such as potassium chloride, calcium chloride, sodium chloride, sodium hydrogen carbonate, sodium carbonate, dry sodium carbonate, magnesium sulfate, sodium hydrogen phosphate, sodium dihydrogen phosphate, diphosphate diphosphate. Buffering agents such as potassium hydrogen and citric acid, polyvinyl alcohol, polyvinyl pyrrolidone, hydroxyethyl cellulose, sodium hyaluronate, methyl cellulose, dextran and other thickeners, glycerin, glucose, D-mannitol, D-sorbitol, trehalose, etc. Agents, solubilizing agents such as ethanol and propylene glycol, stabilizers such as sodium bisulfite, sodium sulfite and sodium thiosulfate, and cooling agents such as L-menthol may be added.

  Examples of cosmetics and skin external preparations include ointments, aqueous solutions, emulsions, creams, lotions, liniments and the like. In order to obtain these preparations, various oils, water, emulsifiers and the like can be blended.

  As the form of food and drink, additives that can be used as food and drink are used as appropriate, and it can be formed into a form suitable for food using conventional means, for example, granular, granular, tablet, capsule, paste, etc. Well, used in addition to various foods such as processed meat products such as ham and sausage, processed fishery products such as kamaboko and chikuwa, bread, confectionery, butter, powdered milk, fermented food and drink, or water, fruit juice, You may add and use for drinks, such as milk, a soft drink, and a tea drink.

  The dose of the SIRT1 activator of the present invention varies depending on the administration route, symptoms, etc., but the chondroitin sulfate oligosaccharide is preferably 5 to 800 mg per adult day.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.

Example 1
(1) Specimen / Proteoglycan: Proteoglycan HG80 (manufactured by Nippon Yakuhin Co., Ltd.) with a purity of 80% extracted from salmon nasal cartilage is used.
-Chondroitin sulfate: An external standard "chondroitin sulfate" (manufactured by ZPD) extracted from porcine tracheal cartilage is used (average molecular weight 13,000).
Chondroitin sulfate oligosaccharides: Chondroitin sulfate oligosaccharides (manufactured by Marukyo Bio Foods) obtained by reducing the molecular weight of chondroitin sulfate extracted from porcine tracheal cartilage by a microchemical process method (JP 2013-199639 A) are used (average molecular weight 2 , 500, molecular weight 500-5,000, at least with glucuronic acid-N-acetylgalactosamine sulfate).
・ Low molecular weight hyaluronic acid: “Hyaro Nano” (manufactured by Kewpie), which has been obtained by reducing the molecular weight of hyaluronic acid by fermentation to an average molecular weight of 2,000, is used.
N-acetylgalactosamine: A reagent “N-Acetyl-D-galactosamine” (manufactured by SIGMA) having a purity of 98% is used.
-N-acetylglucosamine: “Marine Sweet” (manufactured by Yaizu Suisan Chemical Co., Ltd.) having a purity of 100% obtained by enzymatic degradation of chitin is used.
Glucosamine hydrochloride: 100% pure “glucosamine hydrochloride” (manufactured by Bayir Chemicals) obtained by hydrolyzing chitin with hydrochloric acid is used.

(2) Method When normal human chondrocytes are cultured in a 10 cm dish using a chondrocyte growth medium, the medium until now is aspirated and removed, and a chondrocyte growth medium containing each specimen is obtained. In addition, it was cultured overnight.
Thereafter, normal human chondrocytes were collected from a 10 cm dish, the cell density was measured in a suspended state, and only the cells were collected by centrifugation.
Next, the amount of SIRT1 in each cell was measured using an ELISA kit (Sirtuin1 (human) (Intracellular) ELISA Kit manufactured by Adipogen), and the amount of SIRT1 per recovered cell was determined.
A similar evaluation was performed using a chondrocyte growth medium to which no specimen was added as a negative control.

(3) Results Various materials were added at various concentrations to normal human chondrocyte cultures, and the SIRT1 gene amount was measured by ELISA.
As a result, as shown in FIG. 1, it was revealed that SIRT1 gene expression greatly increased only when 1% of chondroitin sulfate oligosaccharide was added.
In contrast, the addition of proteoglycan, chondroitin sulfate, low molecular weight hyaluronic acid, N-acetylgalactosamine, N-acetylglucosamine and glucosamine hydrochloride did not increase the expression of SIRT1 gene.

Claims (3)

A sirtuin 1 activator comprising a chondroitin sulfate oligosaccharide having a molecular weight of 500 to 6,000 as an active ingredient (excluding a therapeutic agent for osteoporosis and a therapeutic agent for diabetes or Alzheimer's disease caused by peritoneal dialysis ).   The sirtuin 1 activator according to claim 1, wherein the chondroitin sulfate oligosaccharide has at least a glucuronic acid-N-acetylgalactosamine-sulfate structure.   The sirtuin 1 activator according to claim 1 or 2, wherein the chondroitin sulfate oligosaccharide is a high-temperature high-pressure decomposition product of chondroitin sulfate.