JP2015038045A - Sirtuin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sirtuin derivative using hyaluronic acid oligosaccharide.SOLUTION: A sirtuin derivative contains, as an active ingredient, a combination of at least one kind of hyaluronic acid oligosaccharide selected from a size of 2 sugars or more to 5 sugars or less and a polyphenol. This sirtuin derivative is used for aging suppression, keratinocyte barrier functional promotion, or used for treatment and/or prevention of heart disease, arteriosclerosis, osteoporosis, inflammatory enteritis, dementia, cerebral apoplexy, metabolic syndrome, cancer, lung disease, renal disease, diabetes, osteoarthritis, rheumatism, progeria, radiation injury, myopathy, brain developmental disease, diseases of the nervous system, hypertension, or obesity.

Description

  The present invention relates to a sirtuin inducer using a hyaluronic acid oligosaccharide.

  Hyaluronic acid is known as a natural polymer that constitutes an extracellular matrix such as joints, skin, and brain, and has been conventionally used as a moisturizing ingredient in pharmaceuticals and cosmetics.

  Hyaluronic acid is a polysaccharide in which a disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosamine is linked, and is classified into high molecular weight hyaluronic acid, low molecular weight hyaluronic acid, or hyaluronic acid oligosaccharide depending on its molecular weight. The The molecular weight of high molecular weight hyaluronic acid is generally about 100,000 to 1,000,000. The molecular weight of the low molecular weight hyaluronic acid is about 10,000 to 100,000. The molecular weight of hyaluronic acid oligosaccharide is about 400 to 10,000 (Non-patent Document 1). And hyaluronic acid has various physiological activities in vivo, and the physiological activity varies greatly depending on the molecular weight (Non-patent Document 2).

Science, 228, 1324-1326 (1985). Medical Application of Hyaluronan, Akira Asari. Chapter 21, Chemistry and Biology of Hyaluronan, Elsevier, ed: Garg, HG and Hales CA, 457-473 (2004)

  In particular, the physiological activity of hyaluronic acid oligosaccharides is considered to be very different from high molecular weight hyaluronic acid and low molecular weight hyaluronic acid. However, its function and utility have not been clarified yet.

  An object of the present invention is to provide a sirtuin inducer using a hyaluronic acid oligosaccharide.

The inventors of the present invention have intensively studied to achieve the above object and have completed the present invention. That is, the present invention is as follows.
[1] A sirtuin inducer comprising a combination of at least one hyaluronic acid oligosaccharide selected from sizes of 2 to 5 sugars and a polyphenol.
[2] The sirtuin inducer according to the above [1], wherein the polyphenol is resveratrol.
[3] The sirtuin inducer according to [1] or [2], which is used for inhibiting aging.
[4] The sirtuin inducer according to [1] or [2], which is used for promoting keratinocyte barrier function.
[5] Heart disease, arteriosclerosis, osteoporosis, inflammatory bowel disease, dementia, stroke, metabolic syndrome, cancer, lung disease, kidney disease, diabetes, osteoarthritis, rheumatism, progeria, radiation disorder, muscle disease, brain development The sirtuin inducer according to the above [1] or [2], which is used for the treatment and / or prevention of disorders, neurological diseases, hypertension, or obesity.
[6] The sirtuin inducer according to any one of the above [1] to [5], which is used in the form of a pharmaceutical, a functional food, or a cosmetic.

  According to the sirtuin inducer of the present invention, the active ingredient is a combination of at least one hyaluronic acid oligosaccharide selected from a size of not less than 2 sugars and not more than 5 sugars and a polyphenol, thereby promoting the expression of a sirtuin protein. can do. Thus, for example, heart disease, arteriosclerosis, osteoporosis, inflammatory bowel disease, dementia, stroke, metabolic syndrome, cancer, lung disease, kidney disease, diabetes, osteoarthritis, rheumatism, progeria, radiation disorder, muscle disease, brain Used for the treatment and / or prevention of developmental disorders, neurological diseases, hypertension, or obesity. It is also used to suppress aging of cells, tissues, living organisms, etc. And it is possible to keep a living individual healthy and extend its life. At the cellular and biological tissue level, the aging can be suppressed, and for example, the activity can be maintained and the survival period can be extended. Furthermore, the expression of filaggrin protein can be promoted together with the sirtuin protein in keratinocytes. Thus, for example, protection against bacteria, viruses, etc., allergy protection against house dust, pollen, etc., protection of wounds due to physical, chemical or biological factors, other protections, infections, allergies, wounds, etc. Used to relieve symptoms and promote early healing.

It is a photograph which shows the dyeing | staining image which dye | stained the human epidermal keratinocytes with the anti-SIRT1 antibody about each test group of a test example. It is a graph which shows the result of the dyeing | staining intensity | strength which dye | stained the human epidermal keratinocytes with the anti-SIRT1 antibody about each test group of a test example. It is a photograph which shows the dyeing | staining image which dye | stained the human epidermal keratinocytes with the anti- Filaggrin antibody about each test group of a test example. It is a graph which shows the result of the dyeing | staining intensity | strength which dye | stained the human epidermal keratinocyte with the anti- Filaggrin antibody about each test group of a test example.

(Hyaluronic acid oligosaccharide)
The hyaluronic acid oligosaccharide used in the present invention contains at least a disaccharide unit formed by glycosidic bonding of D-glucuronic acid and N-acetyl-D-glucosamine, which is a structural unit of hyaluronic acid. The following sizes are meant. Specifically, it is a hyaluronic acid oligosaccharide having a structure represented by the following formula (1), formula (2), formula (3) or formula (4).
GlcA (-GlcNAc-GlcA) _n -GlcNAc (1)
(In formula (1), GlcA represents a glucuronic acid residue, GlcNAc represents an N-acetylglucosamine residue, − represents a glycosidic bond, and n represents an integer of 0 to 1.)
GlcA (-GlcNAc-GlcA) _n (2)
(In formula (2), GlcA represents a glucuronic acid residue, GlcNAc represents an N-acetylglucosamine residue, − represents a glycosidic bond, and n represents an integer of 1 to 2)
GlcNAc (-GlcA-GlcNAc) _n -GlcA (3)
(In formula (3), GlcA represents a glucuronic acid residue, GlcNAc represents an N-acetylglucosamine residue, − represents a glycosidic bond, and n represents an integer of 0 to 1.)
GlcNAc (-GlcA-GlcNAc) _n (4)
(In Formula (4), GlcA represents a glucuronic acid residue, GlcNAc represents an N-acetylglucosamine residue, − represents a glycosidic bond, and n represents an integer of 1 to 2)
In the above formula, the glycosidic bond in GlcA-GlcNAc is preferably a β1 → 3 bond, and the glycosidic bond in GlcNAc-GlcA is preferably a β1 → 4 bond.

  The hyaluronic acid oligosaccharide used in the present invention is not particularly limited as long as it has the above-mentioned basic structure, and its sugar moiety is modified. For example, some or all of the sugar hydroxyl groups may have undergone esterification, etherification or the like. Some or all of the carboxyl groups of glucuronic acid may be subjected to esterification, amidation, and the like. The sugar located at the non-reducing end can be a saturated sugar (one that does not contain a double bond in the carbon-carbon bond in a monosaccharide) or an unsaturated sugar (a double bond in the carbon-carbon bond in a monosaccharide). May be included).

  Since the hyaluronic acid oligosaccharide has a size of 2 to 5 sugars, it easily penetrates into tissues and cells. In addition, it is suitable for efficient application due to its low viscosity. Furthermore, the production of sirtuin protein and filaggrin protein can be significantly promoted by synergistic action with polyphenol.

  In the present invention, a pharmaceutically acceptable salt of hyaluronic acid oligosaccharide can also be used. Examples of the salts include sodium salts and potassium salts which are alkali metal salts. Further, not only anhydrous salts but also hydrated salts may be included. The salt may be derived from the raw material or may be brought in during the preparation of the hyaluronic acid oligosaccharide. These salts function in the same manner as hyaluronic acid oligosaccharides, for example, by ionization in solution or in vivo.

  In the present invention, a pharmaceutically acceptable solvate of hyaluronic acid oligosaccharide can also be used. Examples of solvates include hydrates. For example, when it is left in the air or recrystallized, it may absorb water or adsorb water and become a hydrate. Moreover, it may be brought in at the time of preparation of hyaluronic acid oligosaccharide. These solvates function in the same manner as hyaluronic acid oligosaccharides, for example, by ionization in solution or in vivo.

  As the hyaluronic acid oligosaccharide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, only one kind thereof may be used, or two or more kinds thereof may be used. That is, the size and molecular structure of the hyaluronic acid oligosaccharide may be single, or a combination of these different hyaluronic acid oligosaccharides or a composite containing a plurality of these different hyaluronic acid oligosaccharides may be used. Good. Further, the salt or solvate may be a single species, a combination of different types thereof, or a composite containing those different types.

(Method for producing hyaluronic acid oligosaccharide)
The hyaluronic acid oligosaccharide used in the present invention may be one extracted from a natural product such as an animal, one obtained by culturing a microorganism, or one chemically or enzymatically synthesized. May be. Preferably, hyaluronic acid that can be obtained from a living tissue such as a chicken crown, umbilical body, skin, and joint fluid by a known extraction method is subjected to a decomposition treatment such as enzymatic decomposition, acid decomposition, base decomposition, heat treatment, and ultrasonic treatment. Thereafter, it can be produced by purifying the hyaluronic acid oligosaccharide by a known purification method. Further, hyaluronic acid produced by a fermentation method using lactic acid bacteria, bacteria belonging to the genus Streptococcus, or the like can be similarly produced using a starting material. Furthermore, commercially available high molecular weight hyaluronic acid may be used as a starting material.

  When producing hyaluronic acid oligosaccharides by the enzymatic degradation method, the methods disclosed in International Publication No. 2002/4471, Glycobiology, 2002, Vol. 12, No. 7, pages 421-426 are used. Can be manufactured. High molecular weight hyaluronate is dissolved in a buffer solution (for example, 0.1 M phosphate buffer solution) so that the pH of the solution is around 5, and then hydrolyzed by adding hyaluronidase. The temperature for hydrolysis is preferably around 37 ° C., and the reaction is carried out for about 1 to 24 hours. After the reaction, the supernatant is collected by centrifugation at about 10,000 rpm, and the supernatant is fractionated by an ion exchange column or the like, whereby a hyaluronic acid oligosaccharide having a specific size can be obtained.

  When producing a hyaluronic acid oligosaccharide by acid hydrolysis, a high molecular weight hyaluronate is reacted in a 50% aqueous solution of concentrated hydrochloric acid at 40 ° C. to reflux temperature for about 1 to 5 hours. After the reaction, the hyaluronic acid oligosaccharide having a specific size can be obtained by fractionation using an ion exchange column or the like. Moreover, it can also hydrolyze on more moderate conditions by heating using about 0.05-1M hydrochloric acid in the solvent of DMSO.

  On the other hand, Walvoort MT, Volbeda AG, Reintjens NR, van den Elst H, Plante OJ, Overkleeft HS, van der Marel GA, Codee JD `` Automated Solid-Phase Synthesis of Hyaluronan Oligosaccharides. '' Org. Lett., 2012, 14 (14 ), p3776-3779 discloses a method for efficiently producing the following hyaluronic acid oligosaccharides for each sugar size by the solid-phase synthesis method. it can.

  Of the above, the hyaluronic acid oligosaccharide having the basic sugar structure of (a), (b), (c), (d) and (f) is decomposed using hyaluronic acid as a starting material. Some hyaluronic acid oligosaccharides are hardly obtained or have a very poor yield.

(Polyphenol)
As the polyphenol used in the present invention, a polyphenol known to have a sirtuin protein production promoting action may be appropriately selected and used, and the production of sirtuin protein and filaggrin protein is remarkable due to the synergistic action with the hyaluronic acid oligosaccharide. Can be promoted.

  For example, JP-T-2007-527418 reports resveratrol, fisetin, butein, piceatannol, quercetin, and the like as polyphenols having a sirtuin protein production promoting action.

  In addition, JP-T-2007-527418 discloses a polyphenol having a sirtuin protein production promoting action as resveratrol, butein, piceatannol, isoliquiritigenin, fisetin, luteolin, 3,6,3 ′, 4 ′. -Tetrahydroxyflavone, quercetin, etc. have been reported.

  In addition, it has been reported that phlorizin, an apple-derived polyphenol, promotes an anti-aging action via the Sir2 gene corresponding to sirtuin in yeast (Xiang L, Sun K, Lu J, Weng Y, Taoka A , Sakagami Y, Qi J “Anti-aging effects of phloridzin, an apple polyphenol, on yeast via the SOD and Sir2 genes.” Biosci Biotechnol Biochem. 2011; 75 (5): 854-8. Epub 2011 May 20.).

  As the polyphenol, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof can be used as in the case of the hyaluronic acid oligosaccharide. Moreover, the said polyphenol, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate may use only 1 type, and may use the 2 or more types. Further, the salt or solvate may be a single species, a combination of different types thereof, or a composite containing those different types.

(Dosage form etc.)
The sirtuin inducer according to the present invention is a hyaluronic acid oligosaccharide, a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof described above (hereinafter simply referred to as “hyaluronic acid oligosaccharide”). In combination with the above-described polyphenol, pharmaceutically acceptable salt thereof or pharmaceutically acceptable solvate thereof (hereinafter simply referred to as “polyphenol”). is there.

  The content of the hyaluronic acid oligosaccharide is not particularly limited as long as the effects of the present invention are exhibited, but may be 0.0001% by mass or more and 99% by mass or less with respect to the total mass of the sirtuin inducer. Preferably, it is more preferably 0.01% by mass or more and 20% by mass or less, and most preferably 0.5% by mass or more and 5% by mass or less.

  The content of polyphenol is not particularly limited as long as the effect of the present invention is exhibited, but is preferably 0.0001% by mass or more and 99% by mass or less based on the total mass of the sirtuin inducer. More preferably, the content is 0.01% by mass or more and 20% by mass or less, and most preferably 0.5% by mass or more and 5% by mass or less.

  Moreover, it is preferable that content ratio of hyaluronic acid oligosaccharide and polyphenol is 0.1: 10-10: 0.1 in terms of mass concentration, and is 0.5: 5-5: 0.5. Is more preferable.

  The sirtuin inducer according to the present invention can be used, for example, in the form of pharmaceuticals, functional foods, or cosmetics.

  Examples of the pharmaceutical form include tablets, liquids, dry powders, injections, drops, external preparations, patches, vaporized liquids, and inserts. In this case, it may be prepared in combination with one or more pharmaceutically acceptable ingredients or carriers. For example, additives, excipients, disintegrants, binders, lubricants, surfactants, alcohol, water, water-soluble polymers, sweeteners, corrigents, acidulants, and the like can be added. When used in a form for external use, for example, a lubricant such as calcium stearate or emulsifier, purified water for supplying moisture to the stratum corneum such as ion-exchanged water, moisturizing the stratum corneum such as glycerin, PEG, and ceramide. Moisturizers, ester oils, vegetable oils and other emollients (oil components that prevent moisture from evaporating), solubilizers to solubilize raw materials, and adjust pH Buffering agent, antimicrobial agent for inhibiting microorganisms and preventing decay, coloring agent for coloring, antifading agent for preventing discoloration and discoloration such as UV absorber, astringent, disinfectant, activator Can add anti-inflammatory agent.

Examples of the functional food include liquid food, tablet food, powdered food, and granular food. In this case, it may be prepared in combination with one or more components or carriers that are edible. For example, vitamins B ₁ , Vitamin B ₂ , Vitamin B ₆ , Vitamin B ₁₂ , vitamins such as folic acid, niacin, vitamin E, fats and oils, thickeners, preservatives, colorants, antioxidants, seasonings, acidity A sweetener, a sweetener, etc. can be added.

  Examples of the cosmetics include eye drops, gels, creams, spray solutions, spray solutions, foamed aerosol preparations, gel or sheet face masks, and the like. In this case, it may be prepared in combination with one or more cosmetically acceptable ingredients or carriers. For example, lubricants such as calcium stearate and emulsifiers, purified water for supplying moisture to the stratum corneum such as ion-exchanged water, moisturizers for moisturizing the stratum corneum such as glycerin, PEG and ceramide, moisturizers such as ester oil and vegetable oil Emollient (oil component that prevents moisture from evaporating), solubilizer to solubilize raw materials, buffer to adjust pH, control microorganisms, spoilage An anti-fading agent for preventing discoloration and discoloration, such as a preservative for preventing the coloration, a colorant for coloring, and an ultraviolet absorber, an astringent, a bactericidal agent, an activator, a flame retardant, and the like can be added.

  As a dosage form of the sirtuin inducer according to the present invention, it can be taken orally or administered by intravenous injection or perfusion in the form of an injection or infusion. Alternatively, it may be applied to the skin or absorbed through suction from the respiratory system, or applied locally. It may be ophthalmic administration. In any case, the dosage form is not particularly limited. It can also be applied to animals as well as humans.

The dose per administration of hyaluronic acid oligosaccharide and / or polyphenol, the administration interval, etc. should be determined individually according to the administration form, purpose of use, specific symptoms of patient, age, sex, body weight, etc. There is no particular limitation, but when taken orally, 1 mg to 7500 mg per adult is exemplified. Moreover, when apply | coating to skin, the quantity of about 0.01 microgram-1000 microgram / cm < ² > is illustrated once per unit area. In addition, in the case of ophthalmic administration, an application amount of 0.01 μg to 1000 μg per time is exemplified.

  Hyaluronic acid oligosaccharides have almost no toxicity or antigenicity, and therefore have very little risk of side effects.

(Sirtuin inducer)
The sirtuin protein is a translation product of the SIRT1 gene and an enzyme that deacetylates histones. In cells where the sirtuin protein is expressed, the acetyl group is removed from the histone, and the force of winding the DNA around the histone is increased. As a result of the strong winding of DNA around histones, the expression of genes in that region decreases, thereby regulating the expression of specific genes and transcription factors. DNA damage is also reduced. Sirtuin proteins are also involved in the determination of the lifespan of cells, tissues, or individual organisms by being responsible for regulating various cell functions such as stress resistance, fat / glucose metabolism, and neuronal differentiation.

  For example, it has been reported that when sirtuin expression is increased by genetic manipulation, cancer formation is suppressed, and conversely, knockdown of a sirtuin gene (SIRT1 gene) promotes cancer formation (Kabra N, Li Z, Chen). L, Li B, Zhang X, Wang C, Yeatman T, Coppola D, Chen J `` SirT1 is an inhibitor of proliferation and tumor formation in colon cancer. '' J Biol. Chem. 2009 Jul 3; 284 (27): 18210- 7.).

  There are also reports that sirtuin induction can treat myocardial infarction (Samuel SM, Thirunavukkarasu M, Penumathsa SV, Paul D, Maulik N of myocardial infarction: switching gears toward survival and longevity. "J Agric Food Chem. 2008 Oct 22; 56 (20): 9692-8.).

  There is also a report that sirtuin induction suppresses vascular endothelial cell aging and prevents arteriosclerosis (Ota H, Eto M, Ogawa S, Iijima K, Akishita M, Ouchi Y “SIRT1 / eNOS axis as a potential target against vascular senescence, dysfunction and atherosclerosis. "Journal of Atherosclerosis and Thrombosis Vol. 17 (2010) No. 5 p431-435.).

  In addition, there is a report that resistin, which is a factor causing arterial stenosis and atherosclerosis, is suppressed by induction of sirtuin (Carter S, Miard S, Roy-Bellavance C, Boivin L, Li Z, Pibarot P, Mathieu P, Picard F “Sirt1 inhibits resistin expression in aortic stenosis.” PLoS One. 2012; 7 (4): e35110.).

  There is also a report that sirtuin induction prevents osteoporosis by suppressing NF-kappaB activation in osteoclasts causing bone destruction (Shakibaei M, Buhrmann C, Mobasheri A “Resveratrol-mediated SIRT-1 interactions with p300 modulate receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB signaling and inhibit osteoclastogenesis in bone-derived cells. "J Biol Chem. 2011 Apr 1; 286 (13): 11492-505.).

  In addition, there is a report that sirtuin induction can suppress NF-kappaB, a factor that exacerbates inflammation, and reduce symptoms of inflammatory enteritis (Singh UP, Singh NP, Singh B, Hofseth LJ, Price RL, Nagarkatti M, Nagarkatti PS Resveratrol (trans-3,5,4'-trihydroxystilbene) induces silent mating type information regulation-1 and down-regulates nuclear transcription factor-kappaB activation to abrogate dextran sulfate sodium-induced colitis.J Pharmacol Exp Ther. 2010 Mar ; 332 (3): 829-39.).

  Sirtuin protein has also been reported to suppress amyloid β production, which causes dementia (Alzheimer's disease) (Donmez G, Wang D, Cohen DE, Guarente L “SIRT1 suppresses beta-amyloid production by activating the alpha-secretase gene ADAM10. "Cell 2010 Jul 23; 142 (2): 320-32.).

  In addition, there are reports that therapeutic effects were observed by sirtuin induction in various animal models of Alzheimer's disease (Donmez G “The Effects of SIRT1 on Alzheimer's Disease Models.” Int J Alzheimers Dis. 2012; 2012: 509529.) .

  There is also a report that sirtuin protein is involved in the cerebral damage-inhibiting action by nicotinamide phosphoribosyltransferase in stroke (cerebral ischemia) models (Wang P, Xu TY, Guan YF, Tian WW, Viollet B, Rui YC, Zhai QW, Su DF, Miao CY “Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphate-activated kinase pathway.” Ann Neurol. 2011 Feb; 69 (2): 360 -74.)

  In addition, in the sirtuin protein high expression model (by genetic engineering), there was a report that the metabolic syndrome disorder and its associated canceration were suppressed compared to normal animals (Herranz D, Munoz-Martin M, Canamero M, Mulero F, Martinez-Pastor B, Fernandez-Capetillo O, Serrano M “Sirt1 improves healthy ageing and protects from metabolic syndrome-associated cancer.” Nat Commun. 2010 Apr 12; 1: 3.).

  In addition, there is a report that when sirtuin protein expression is increased by gene manipulation, cancer formation is suppressed, and conversely, weakening sirtuin protein expression promotes cancer formation (Kabra N, Li Z, Chen L, Li B, Zhang X , Wang C, Yeatman T, Coppola D, Chen J “SirT1 is an inhibitor of proliferation and tumor formation in colon cancer.” J Biol Chem. 2009 Jul 3; 284 (27): 18210-7.).

  There are also reports that sirtuin protein is an inhibitor of the action of lung inflammation and thrombosis caused by fine air particles (Wu Z, Liu MC, Liang M, Fu J “Sirt1 protects against thrombomodulin down-regulation and lung coagulation after particulate matter exposure. "Blood. 2012 Mar 8; 119 (10): 2422-9.).

  In addition, smoking causes autophagy to lung cells through the action of sirtuin protein, which is a cause of COPD (chronic obstructive pulmonary disease), and there is a report that autophagy was suppressed by sirtuin induction. (Hwang JW, Chung S, Sundar IK, Yao H, Arunachalam G, McBurney MW, Rahman I “Cigarette smoke-induced autophagy is regulated by SIRT1-PARP-1-dependent mechanism: implication in pathogenesis of COPD.” Arch Biochem Biophys. 2010 Aug 15; 500 (2): 203-9.).

  There is also a report that administration of a sirtuin activator to the renal ischemia-reperfusion injury model suppressed renal cell death and reduced renal injury (Fan H, Yang HC, You L, Wang YY, He WJ, Hao CM “The histone deacetylase, SIRT1, contributes to the resistance of young mice to ischemia / reperfusion-induced acute kidney injury.” Kidney Int. 2013 Jan 9.).

  There are also reports that sirtuin induction reduced cisplatin-induced renal tubular toxicity and NF-kappaB activation (Jung YJ, Lee JE, Lee AS, Kang KP, Lee S, Park SK, Lee SY, Han MK, Kim DH, Kim W `` SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunit and cytotoxicity in renal proximal tubule cells. '' Biochem Biophys Res Commun. 2012 Mar 9; 419 (2): 206- Ten.).

  There are also reports that sirtuin proteins are involved in the mechanism of insulin resistance (Chen YR, Lai YL, Lin SD, Li XT, Fu YC, Xu WC “SIRT1 interacts with metabolic transcriptional factors in the pancreas of Insulin-resistant and calorie-restricted rats. "Mol Biol Rep. 2013 Jan 6.).

  There are also reports that sirtuin proteins are involved in the mechanism of insulin sensitivity improvement by the antihypertensive drug telmisartan (Shiota A, Shimabukuro M, Fukuda D, Soeki T, Sato H, Uematsu E, Hirata Y, Kurobe H, Maeda N, Sakaue H, Masuzaki H, Shimomura I, Sata M `` Telmisartan ameliorates insulin sensitivity by activating the AMPK / SIRT1 pathway in skeletal muscle of obese db / db mice. '' Cardiovasc Diabetol. 2012 Nov 8; 11: 139. ).

  There are also reports that sirtuin proteins regulate TNFα-induced inflammation in human chondrocytes (Moon MH, Jeong JK, Lee YJ, Seol JW, Jackson CJ, Park SY “SIRT1, a class III histone deacetylase, regulates TNF-α-induced inflammation in human chondrocytes. "Osteoarthritis Cartilage. 2013 Mar; 21 (3): 470-80.).

  In human chondrocytes, there is a report that overexpression of sirtuin protein suppresses the expression of degenerative joint factor even when treated with IL-1beta (Matsushita T, Sasaki H, Takayama K, Ishida K, Matsumoto T, Kubo S, Matsuzaki T, Nishida K, Kurosaka M, Kuroda R, “The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin-1β in human chondrocytes.” J Orthop Res. 2012 Nov 9.).

  There are also reports that sirtuin proteins are involved in the action of rheumatoid exacerbation factors. (Kok SH, Lin LD, Hou KL, Hong CY, Chang CC, Hsiao M, Wang JH, Lai EH, Lin SK “Simvastatin inhibits Cyr61 expression in rheumatoid arthritis synovial fibroblasts through the regulation of SIRT1 / FoxO3a signaling.” Arthritis Rheum. 2012 Dec 12.).

  There is also a report that a sirtuin activator has shown a disease improving effect in a Progeria model animal. (Liu B, Ghosh S, Yang X, Zheng H, Liu X, Wang Z, Jin G, Zheng B, Kennedy BK, Suh Y, Kaeberlein M, Tryggvason K, Zhou Z `` Resveratrol rescues SIRT1-dependent adult stem cell decline and alleviates progeroid features in laminopathy-based progeria. "Cell Metab. 2012 Dec 5; 16 (6): 738-50.).

  There is also a report that radiation damage is caused by cell death induced by transfer of GAPDH from the cytoplasm to the nucleus, and that the sirtuin protein suppresses the nuclear transfer of GAPDH and suppresses cell death. (Joo HY, Woo SR, Shen YN, Yun MY, Shin HJ, Park ER, Kim SH, Park JE, Ju YJ, Hong SH, Hwang SG, Cho MH, Kim J, Lee KH "SIRT1 interacts with and protects glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) from nuclear translocation: implications for cell survival after irradiation. "Biochem Biophys Res Commun. 2012 Aug 10; 424 (4): 681-6.).

  There are reports that sirtuin proteins not only strengthen muscle fibers but also protect muscles from various diseases. (Tonkin J, Villarroya F, Puri PL, Vinciguerra M “SIRT1 signaling as potential modulator of skeletal muscle diseases.” Curr Opin Pharmacol. 2012 Jun; 12 (3): 372-6.).

  Moreover, when sirtuin protein is highly expressed, dendrite formation of hippocampal neurons is promoted. Conversely, when a sirtuin inhibitor is acted on, dendrites atrophy, and when sirtuin protein is highly expressed, amyloid β There are also reports that this also means that sirtuin proteins are responsible for the development and maintenance of cranial nerve tissues such as the hippocampus because they also suppress dendritic atrophy due to treatment. (Codocedo JF, Allard C, Godoy JA, Varela-Nallar L, Inestrosa NC "SIRT1 regulates dendritic development in hippocampal neurons." PLoS One. 2012; 7 (10): e47073.).

  There are also reports that sirtuin protein suppresses angiotensin II, a factor that induces hypertension (Li L, Gao P, Zhang H, Chen H, Zheng W, Lv X, Xu T, Wei Y, Liu D, Liang C “SIRT1 inhibits angiotensin II-induced vascular smooth muscle cell hypertrophy.” Acta Biochim Biophys Sin (Shanghai). 2011 Feb; 43 (2): 103-9.).

  There are also reports that sirtuin induction suppresses insulin resistance and obesity (Li H, Xu M, Lee J, He C, Xie Z “Leucine supplementation increases SIRT1 expression and prevents mitochondrial dysfunction and metabolic disorders in high- fat diet-induced obese mice. "Am J Physiol Endocrinol Metab. 2012 Nov 15; 303 (10): E1234-44.).

  Therefore, the sirtuin inducer according to the present invention includes, in particular, heart disease, arteriosclerosis, osteoporosis, inflammatory bowel disease, dementia, stroke, metabolic syndrome, cancer, lung disease, kidney disease, diabetes, osteoarthritis, rheumatism, It is preferably used for the treatment and / or prevention of progeria, radiation disorders, muscle diseases, brain development disorders, neurological diseases, hypertension, or obesity.

(Aging inhibitor)
Sirtuin proteins are also involved in aging inhibition.

  For example, sirtuins have been reported to have anti-aging effects (Mousa SA, Gallati C, Simone T, Dier E, Yalcin M, Dyskin E, Thangirala S, Hanko C, Rebbaa A “Dual targeting of the antagonistic pathways mediated by Sirt1 and TXNIP as a putative approach to enhance the efficacy of anti-aging interventions. "Aging (Albany NY). 2009 Mar 31; 1 (4): 412-24.).

  Sirtuins have also been reported to improve aging inhibition and metabolic syndrome (Ghosh HS “The anti-aging, metabolism potential of SIRT1.” Curr Opin Investig Drugs. 2008 Oct; 9 (10): 1095-102. Review.).

  In addition, sirtuins have been reported to have an effect of improving progeria (one of progeria) by suppressing aging of tissue stem cells (Liu B, Ghosh S, Yang X, Zheng H, Liu X, Wang Z, Jin G, Zheng B, Kennedy BK, Suh Y, Kaeberlein M, Tryggvason K, Zhou Z `` Resveratrol rescues SIRT1-dependent adult stem cell decline and alleviates progeroid features in laminopathy-based progeria. '' Cell Metab. 2012 Dec 5; 16 ( 6): 738-50.).

  Therefore, the sirtuin inducer according to the present invention is preferably used particularly for suppressing aging of cells, tissues, living organisms and the like. And it is possible to keep a living individual healthy and extend its life. In addition, at the cell and biological tissue level, the aging can be suppressed, and for example, the activity can be maintained and the survival period can be extended.

(Keratinocyte barrier function promoter)
Filaggrin protein is a translation product of the Filagrin gene, and is present on the surface layer of the epidermis and forms a stratum corneum that is indispensable for the barrier function of the skin. If filaggrin is not made, keratin abnormalities occur, the skin barrier function is lowered, and dermatitis is caused.

  Therefore, the sirtuin inducer according to the present invention promotes the barrier function of keratinocytes existing in the skin, gums, cornea and the like by promoting the expression of sirtuin and / or filaggrin protein in keratinocytes, which will be described later in the Examples. be able to. In addition, “barrier function promotion” means not only infection protection against bacteria, viruses, etc., house dust, allergy protection against pollen, wound protection due to physical, chemical or biological factors, and other defenses, It also means relief of symptoms and promotion of early healing when the infection, allergy, or wound occurs.

  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.

  The test substances used in the following test examples are shown together.

(1) HA2
A disaccharide-sized hyaluronic acid oligosaccharide having the structure of GlcA-GlcNAc was prepared by hydrolyzing hyaluronic acid with an enzyme preparation “chondroitinase ABC” (manufactured by Sigma-Aldrich) and purifying it. It was.

(2) HA4
A tetrasaccharide-sized hyaluronic acid oligosaccharide having a structure of GlcA (-GlcNAc-GlcA) ₁ -GlcNAc obtained by enzymatic degradation of hyaluronic acid (trade name “HYA-OLIGO4EF-5”, Hyalose) Made).

(3) HA5
A 5-saccharide-sized hyaluronic acid oligosaccharide having a (GlcNAc-GlcA-) ₂ -GlcNAc structure (product name “HYA-NANO5EF-1”) produced by elongating a monosaccharide one molecule at a time by enzymatic chemistry. Hyalose) was used.

(4) HA6
Hyaluronic acid oligosaccharide having a structure of GlcA (-GlcNAc-GlcA) ₂ -GlcNAc obtained by enzymatic decomposition of hyaluronic acid (trade name “HYA-OLIGO6EF-1”, Hyalose) Made).

(5) HA10
A 10-saccharide-sized hyaluronic acid oligosaccharide having a structure of GlcA (-GlcNAc-GlcA) ₄ -GlcNAc, obtained by enzymatic degradation of hyaluronic acid (trade name “HYA-OLIGO10EF-1”, Hyalose) Made).

(6) HA10 kDa
Hyaluronic acid having an average molecular weight of 10 kDa manufactured by QP Corporation was used.

(7) HA50 kDa
Hyaluronic acid having an average molecular weight of 50 kDa manufactured by QP Corporation was used.

(8) HA900 kDa
Hyaluronic acid having an average molecular weight of 900 kDa manufactured by Seikagaku Corporation was used.

(9) Resveratrol (Res)
Resveratrol (product number R5010, purity 99% or more) manufactured by Sigma-Aldrich was used.

Moreover, the group structure of the test example was as follows.
・ No treatment ・ Resveratrol (final concentration: 5 μM, the same shall apply hereinafter)
HA2 (final concentration: 10 ng / ml, the same shall apply hereinafter)
・ HA2 + resveratrol ・ HA4 (final concentration: 10 ng / ml, the same applies hereinafter)
・ HA4 + resveratrol ・ HA5 (final concentration: 10 ng / ml, the same applies hereinafter)
-HA5 + resveratrol-HA6 (final concentration: 10 ng / ml, the same applies hereinafter)
-HA6 + resveratrol-HA10 kDa (final concentration: 10 ng / ml, the same applies hereinafter)
HA 10 kDa + resveratrol HA 50 kDa (final concentration: 10 ng / ml, the same applies hereinafter)
-HA 50 kDa + resveratrol-HA 900 kDa (final concentration: 10 ng / ml, the same applies hereinafter)
・ HA900 kDa + resveratrol

<Test example>
Human epidermal keratinocytes (normal human keratinocytes (derived from keratinocyte, Asian woman (27 years old)) prepared in DMEM medium at a cell concentration of 1 × 10 ⁵ / ml were seeded at 100 μL per well in a 96-well plate, Each test substance was added to this in the above group configuration and cultured for 72 hours.

  After culturing for 72 hours, the cells of each well were fixed with 4% paraformaldehyde and double-stained with an anti-SIRT1 antibody (Santa Cruz Biotechnology) and an anti-Flaggrin antibody (Santa Cruz Biotechnology). As the secondary antibody for each antibody, fluorescent dye-labeled antibodies having different wavelengths were used.

  A fluorescent stained image was taken with an inverted microscope (manufactured by Leica), and the staining intensity of the randomly selected four central visual fields was measured by J-Image.

[Evaluation 1]
FIG. 1 shows a photograph of an image stained with an anti-SIRT1 antibody. FIG. 2 shows the results of staining intensity with anti-SIRT1 antibody. In FIG. 2, the average value of the staining intensity for the four visual fields is represented by a bar graph, and the standard deviation (SD) is represented by an error bar. In addition, for each test group to which only hyaluronic acid oligosaccharide or high molecular hyaluronic acid was added, regarding the significant difference in the results when resveratrol was used in combination, it was determined that there was a significant difference by t-test. , The range of those risk factors (p <0.07, p <0.05, or p <0.01). Furthermore, there is a significant difference in Dunnett's multiple comparison test for the significant difference of each other test group with respect to no treatment or the significant difference of each other test group with respect to the test group to which only resveratrol was added. For those determined to be, the range of their risk factors (p <0.05 or p <0.01) is shown.

  As shown in FIG. 2, under the test conditions, resveratrol, HA2, HA6, HA10 kDa, HA50 kDa, HA900 kDa, HA6 + resveratrol, HA10 kDa + resveratrol, HA50 kDa + resveratrol, HA900 kDa + resveratrol In the group, no significant effect of promoting sirtuin protein production was observed compared to the untreated group.

  On the other hand, in each test group of HA4, HA5, HA2 + resveratrol, HA4 + resveratrol, HA5 + resveratrol, a significant sirtuin protein production promotion effect was observed compared to the untreated group. In particular, each of the HA2 + resveratrol, HA4 + resveratrol, and HA5 + resveratrol test groups has a more significant effect of promoting sirtuin protein production than the test group to which HA2, HA4, and HA5 are added alone. It was.

[Evaluation 2]
FIG. 3 shows a photograph of an image stained with an anti-Flaggrin antibody. FIG. 4 shows the results of staining intensity with anti-Flaggrin antibody. In FIG. 4, the average value of the staining intensity for the four visual fields is represented by a bar graph, and the standard deviation (SD) is represented by an error bar. In addition, for each test group to which only hyaluronic acid oligosaccharide or high molecular hyaluronic acid was added, regarding the significant difference in the results when resveratrol was used in combination, it was determined that there was a significant difference by t-test. , The range of those risk factors (p <0.05 or p <0.01). Furthermore, there is a significant difference in Dunnett's multiple comparison test for the significant difference of each other test group with respect to no treatment or the significant difference of each other test group with respect to the test group to which only resveratrol was added. For those determined to be, the range of those risk factors (p <0.05) is shown.

  As shown in FIG. 4, under the present test conditions, a significant effect of promoting filaggrin protein production was observed in the test group to which resveratrol was added compared to the non-treated group. On the other hand, in each test group of HA2, HA4, HA5, HA6, HA10 kDa, HA50 kDa, HA900 kDa, HA6 + resveratrol, HA10 kDa + resveratrol, HA50 kDa + resveratrol, HA900 kDa + resveratrol There was no significant effect of promoting filaggrin protein production compared to the group.

  In contrast, the HA2 + resveratrol, HA4 + resveratrol, and HA5 + resveratrol test groups showed a more significant effect of promoting filaggrin protein production than the test group to which resveratrol was added alone. It was.

Claims (6)

  A sirtuin inducer comprising a combination of at least one hyaluronic acid oligosaccharide selected from sizes of 2 to 5 sugars and a polyphenol.   The sirtuin inducer according to claim 1, wherein the polyphenol is resveratrol.   The sirtuin inducer according to claim 1 or 2, which is used for inhibiting aging.   The sirtuin inducer according to claim 1 or 2, which is used for promoting keratinocyte barrier function.   Heart disease, arteriosclerosis, osteoporosis, inflammatory bowel disease, dementia, stroke, metabolic syndrome, cancer, lung disease, kidney disease, diabetes, osteoarthritis, rheumatism, progeria, radiation disorder, muscle disease, brain development disorder, nerve The sirtuin inducer according to claim 1 or 2, which is used for the treatment and / or prevention of diseases, corneal diseases, presbyopia, spinal cord injury, peripheral nerve rupture, hypertension, or obesity.   The sirtuin inducer according to any one of claims 1 to 5, which is used in the form of a pharmaceutical, a functional food, or a cosmetic.