JP2007126453A - Hyaluronan production promotor and hyaluronan decomposition suppressor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hyaluronan production promotor, a hyaluronan decomposition suppressor and a skin-care preparation. <P>SOLUTION: The medicine contains hyaluronan as an active ingredient. The hyaluronan is preferably a tetrose (HA4), wherein -D-glucuronic acid-β-1,3-D-N-acetylglucosamine-β-1,4- is made one unit and two units thereof are contained in the tetrose. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a hyaluronan production promoter and hyaluronan degradation inhibitor containing hyaluronan as an active ingredient.

  Hyaluronan is a long-chain polysaccharide composed of disaccharide repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, while oligosaccharides are also known. Hyaluronan is produced by extraction from biological tissues such as chicken crowns, corpses, skin, joint fluid, or fermentation methods using bacteria belonging to the genus Streptococcus, and has no toxicological or immunological effects. For example, treatment of arthritis by intraarticular injection of hyaluronan is well known. In the following description, the tetrasaccharide hyaluronan is referred to as HA4.

  HA4 has been reported to have therapeutic / suppressive effects on organ preservation, liver damage, and gastric ulcers (see Patent Document 1). Moreover, it is known that HA4 has a stress protein expression enhancing action and a cell death suppressing action (see Non-Patent Document 1).

  In general, the expression “hyaluronic acid” includes tetrasaccharide hyaluronan, which means that hyaluronan is widely included regardless of the number of sugars. To date, the application of hyaluronan to industry has been extensively studied. However, in reality, methods for promoting the synthesis of hyaluronan itself and suppressing the degradation of hyaluronan have not been sufficiently studied.

WO2002 / 004471 Xu H, Ito T, Tawada A, Maeda H, Yamanokuchi H, Isahara K, Yoshida K, Uchiyama Y, Asari A. Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72. J Biol Chem. 2002 10; 277 (19 ): 17308-14.

  Therefore, an object of the present invention is to provide a novel hyaluronan production promoter and hyaluronan degradation inhibitor, and to provide a skin care agent utilizing hyaluronan production promoting action and hyaluronan degradation inhibiting action.

  The hyaluronan production promoter, hyaluronan degradation inhibitor and skin skin agent according to the present invention that have achieved the above-mentioned object contain tetrasaccharide hyaluronan as an active ingredient. The hyaluronan production promoter according to the present invention promotes intracellular hyaluronan production and suppresses the degradation of hyaluronan. In addition, the skin care agent according to the present invention improves skin moisturization by the intracellular hyaluronan production promoting action and hyaluronan degradation inhibiting action by the active ingredient.

  Since the hyaluronan production promoter, hyaluronan degradation inhibitor and skin care agent according to the present invention comprise tetrasaccharide hyaluronan as an active ingredient, there is an advantage that it can be easily mass-produced relatively inexpensively. Further, tetrasaccharide hyaluronan is expected to be an agent with extremely few side effects because it has almost no toxicity and antigenicity, and enhances the therapeutic action and disease prevention action inherent in the living body.

  Hereinafter, the present invention will be described in detail. The hyaluronan production promoter according to the present invention is a drug having a function of promoting hyaluronan production by cells. Moreover, the hyaluronan degradation inhibitor according to the present invention is a drug that suppresses the degradation of produced hyaluronan. Furthermore, the skin care agent according to the present invention is a drug that improves skin moisture retention through hyaluronan production promoting action and hyaluronan degradation inhibitory action in cells.

  In the following description, the hyaluronan production promoter, hyaluronan degradation inhibitor and skin care agent according to the present invention are simply referred to as drugs. The hyaluronan contained in the drug according to the present invention basically includes at least one disaccharide unit in which the 1-position of β-D-glucuronic acid and the 3-position of β-D-N-acetylglucosamine are bonded. As long as it is more than sugar and is basically composed of β-D-glucuronic acid and β-DN-acetylglucosamine, those elements are combined with one or more disaccharide units combined. May be used, and derivatives thereof such as those having a hydrolyzable protecting group such as an acyl group can also be used. The sugar may be an unsaturated sugar, and examples of the unsaturated sugar include non-reducing terminal sugars, usually those in which the 4- and 5-position carbons of glucuronic acid are unsaturated. As the hyaluronan used in the present invention, specifically, those extracted from natural products such as animals, those obtained by culturing microorganisms, those synthesized chemically or enzymatically, etc. should be used. Can do. For example, it can be obtained by known extraction and purification methods from biological tissues such as chicken crown, corpse, skin, and joint fluid. It can also be produced by a fermentation method using Streptococcus bacteria or the like.

  In the present invention, hyaluronan oligosaccharides are also included in hyaluronan, and low molecular weight hyaluronan such as disaccharides and derivatives thereof consisting of one disaccharide unit are also included. More preferred examples include hyaluronan having about 2 to 20 sugars.

  In particular, in the present invention, hyaluronan includes -D-glucuronic acid-β-1,3-DN-acetylglucosamine-β-1,4-- as one unit, a disaccharide containing one of these, 2 It is preferable that it is a mixture containing 4 saccharides including 3, 6 saccharides including 3, 8 saccharides including 4, and 10 saccharides including 5.

  Specifically, hyaluronan having a low molecular weight can be obtained by known methods such as enzymatic decomposition, alkaline decomposition, heat treatment, and ultrasonic treatment (Biochem., 33 (1994) p6503-6507). It is preferably produced by a method of reducing the molecular weight, a method of chemically or enzymatically synthesizing (Glycoconjugate J., (1993) p435-439, WO93 / 20827). For example, hyaluronan degrading enzymes (hyaluronidase (derived from testicles), hyaluronidase (derived from Streptomyces), hyaluronidase SD, etc.), chondroitinase AC, chondroitinase ACII, chondroitinase ACIII, chondroitinase ABC, etc. A method to produce hyaluronan oligosaccharides by causing an enzyme that degrades hyaluronan to produce hyaluronan oligosaccharides (see Shinsei Chemistry Laboratory "Carbohydrate II-Proteoglycans and Glycosaminoglycans" p244-248, published in 1991, Tokyo Chemical Dojin) Can be mentioned.

  As an alkali decomposition method, for example, a base such as about 1N sodium hydroxide is added to a hyaluronan solution, heated for several hours to lower the molecular weight, and then neutralized by adding an acid such as hydrochloric acid. And a method for obtaining a low molecular weight hyaluronan. Hyaluronan used in the present invention includes a salt form, and a pharmaceutically acceptable salt thereof can be used as necessary in the preparation. For example, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, amine salts such as tri (n-butyl) amine salt, triethylamine salt, pyridine salt and amino acid salt Can do.

  The drug of the present invention can be used without particular limitation, such as hyaluronan having a certain molecular weight alone or a combination of hyaluronan having various molecular weights. The agent of the present invention comprises hyaluronan as an active ingredient, and promotes the production of hyaluronan without adversely affecting the living body by administering an effective amount thereof to mammals including humans. Decomposition can be suppressed.

  The agent of the present invention is administered hyaluronan or a salt thereof, as it is or as necessary, with a carrier, excipient, or other additive, orally or parenterally (intra-articular administration, intravenous, intramuscular, subcutaneous). As a pharmaceutical for administration (injection), enteral administration, transdermal administration, etc.), it can be formulated into any dosage form and administered to a patient by any administration method. In particular, the drug according to the present invention is preferably an oral preparation.

  Examples of oral preparations include solid preparations such as powders, granules, capsules and tablets; liquid preparations such as syrups, elixirs and emulsions. The powder can be obtained by mixing with excipients such as lactose, starch, crystalline cellulose, calcium lactate, calcium hydrogen phosphate, magnesium aluminate metasilicate, and silicic anhydride. In addition to the above excipients, the granule is optionally combined with a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, a binder such as carboxymethylcellulose, carboxymethylcellulose calcium, carboxymethylrose, carboxymethylcellulose calcium, etc. Further, a disintegrant may be added and granulated by wet or dry method. Tablets can be obtained by compressing the powder or granules as they are or by adding a lubricant such as magnesium stearate or talc. The tablet or granule is coated with an enteric base such as hydroxypropylmethylcellulose phthalate or methyl methacrylate copolymer, or coated with ethylcellulose, carnauba wax, hardened oil, etc. can do. A hard capsule can be obtained by filling a hard capsule with the above powder or granule. Soft capsules can be obtained by mixing hyaluronan or a salt thereof with glycerin, polyethylene glycol, sesame oil, olive oil or the like and coating it with a gelatin film. A syrup can be obtained by dissolving a sweetener such as sucrose, sorbitol, and glycerin and hyaluronan or a salt thereof in water. In addition to sweeteners and water, essential oils, ethanol and the like can be added to make elixirs, or gum arabic, tragacanth, polysorbate 80, sodium carboxymethylcellulose and the like can be added to make emulsions or suspensions. Moreover, a corrigent, a coloring agent, a preservative, etc. can be added to these liquid preparations as needed.

  Examples of parenteral preparations include injections, rectal administration agents, pessaries, external preparations for skin, inhalants, aerosols, eye drops and the like. Injections include hyaluronan or salts thereof, pH regulators such as hydrochloric acid, sodium hydroxide, lactic acid, sodium lactate, sodium monohydrogen phosphate, sodium dihydrogen phosphate; isotonic agents such as sodium chloride and glucose; and It can be obtained by adding distilled water for injection, sterilizing and filtering and then filling ampoules. Further, mannitol, dextrin, cyclodextrin, gelatin and the like can be added and lyophilized in a vacuum to obtain an injection that can be dissolved at the time of use. Moreover, after adding an emulsifier, such as lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, to hyaluronan or its salt, it can also be set as the emulsion for injection emulsified in water.

  For rectal administration, after adding a suppository base such as mono-, di- or triglyceride of cacao fatty acid or polyethylene glycol to hyaluronan or a salt thereof, dissolve by heating, and pour it into a mold to cool, Alternatively, hyaluronan or a salt thereof can be mixed with polyethylene glycol, soybean oil or the like and then coated with a gelatin film. The external preparation for skin can be obtained by adding white petrolatum, beeswax, liquid paraffin, polyethylene glycol or the like to hyaluronan or a salt thereof, and heating and kneading as necessary. The tape agent can be obtained by kneading hyaluronan or a salt thereof with a pressure-sensitive adhesive such as rosin or an alkyl acrylate polymer and spreading it on a nonwoven fabric or the like. The inhalant can be obtained by, for example, dissolving or dispersing hyaluronan or a salt thereof in a propellant such as a pharmaceutically acceptable inert gas and filling the pressure-resistant container.

  In particular, the specific dosage form of the skin care agent according to the present invention is not particularly limited, and examples thereof include emulsions, creams, lotions, and sheet-like hydrogels. The skin care agent according to the present invention can contain other components generally contained as a skin care agent in addition to the above-described active ingredients. Examples of other components include a base, a solubilizer, a humectant, an emulsifier, a dissolution stabilizer, an antiseptic, an antioxidant, a pH adjuster, a saponifier, and a solvent. The skin care agent according to the present invention can contain one or more components selected from these other components.

  The solubilizing agent is not particularly limited. For example, decaglyceryl monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene lanolin alcohol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxy Examples thereof include propylene decyl tetradecyl ether and polyoxyethylene oleyl ether. Although it does not specifically limit as a moisturizer, For example, glycerin, xylit, sorbit, dipropylene glycol, butylene glycol, propylene glycol, polyethylene glycol (200, 400, 600, 1000, 2000, etc.), highly polymerized polyethylene glycol, methyl glue, Examples thereof include D-mannitol and hydroxyproline. Although it does not specifically limit as a dissolution stabilizer, For example, potassium chloride, sodium chloride, a citric acid, sodium sulfite etc. can be mentioned. Although it does not specifically limit as antiseptic | preservative, For example, p-hydroxybenzoic acid ester, chlorhexidine hydrochloride, orthophenylphenol, chlorhexidine gluconate, triclosan, triclocarban, phenoxyethanol can be mentioned. Examples of the antioxidant include, but are not limited to, vitamin C (ascorbic acid), vitamin E (dl-α-tocopherol), citric acid, sodium citrate, and dibutylhydroxytoluene. Although it does not specifically limit as a pH adjuster, For example, a succinic acid, a citric acid, anhydrous citric acid, sodium citrate, arginine, sodium hydroxide, potassium hydroxide, sodium phosphate can be mentioned. The base is not particularly limited. For example, lanolin, ceresin, paraffin, microcrystalline wax, petrolatum, jojoba oil, tri (capryl / capric acid) glycerin, isostearic acid, oleic acid, stearic acid, palmitic acid, behenic acid , Myristic acid, higher alcohols (isostearyl alcohol, oleyl alcohol, octyldodecanol, cholesterol, cetanol, stearyl alcohol, decyltetradecanol, phytosterol, hexyldecanol, etc.), cetyl palmitate, octyl palmitate, isopropyl lanolinate Can do. The emulsifier is not particularly limited. For example, hydrogenated soybean phospholipid, glyceryl distearate, polyoxyethylene glyceryl monostearate, sorbitan monostearate, polyoxyethylene stearyl ether, polyoxyethylene lauryl ether, polyethylene monostearate Examples include glycol, polyethylene glycol distearate, sodium N-stearoyl-L-glutamate, sodium N-acyl-L-glutamate, self-emulsifying glyceryl monostearate, and oleophilic glyceryl monostearate. Although it does not specifically limit as a saponifying agent, For example, potassium hydroxide, sodium hydroxide, and a triethanolamine can be mentioned.

  In addition, as other ingredients, ingredients used in ordinary pharmaceuticals, quasi drugs, cosmetics, etc., for example, conditioning ingredients, coloring agents such as pigments, pigments, viscosity modifiers, pH regulators, salts, vitamins, fragrances , UV absorbers, UV scattering agents, metal sequestering agents, viscosity modifiers, emulsion stabilizers, and the like.

  (Administration method) The administration method of the drug comprising the hyaluronan of the present invention as an active ingredient is not particularly limited, and examples thereof include intravenous administration, oral administration, intra-articular administration and skin application.

  (Dose) The dose is appropriately determined according to the target disease, patient age, health condition, body weight, etc. In the case of intravenous administration, oral administration and intra-articular administration, generally 0.05 to 50 mg Administer / kg once a day or in portions. In the case of skin application, a concentration of 0.01 to 10% is generally administered several times a day.

  (Toxicity) The hyaluronan used in the present invention showed little or no cytotoxicity at doses showing biological activity as a drug.

  Hereinafter, although the chemical | medical agent which concerns on this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

[Example 1]
In this example, the presence state of hyaluronan inside and outside the cell by the drug according to the present invention was measured using a substance that binds to 10 or more sugars of hyaluronan. As the substance, biotin-labeled hyaluronan binding protein (biotin-labeled HABP; manufactured by Seikagaku Corporation) was used.

In this example, HT1080 (human fibrosarcoma cell) was used as the measurement target cell. HT1080 is available from RIKEN.

Preparation of test substance In this example, 1 μg / ml of HA4 was prepared . Specifically, HA4 is produced by the method of Tawada et al. (Tawada A, Masa T, Oonuki Y, Watanabe A, Matsuzaki Y, Asari A. Large-scale preparation, purification, and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers. Glycobiology. 2002; 12 (7): 421-6.) And the concentration was adjusted with physiological saline.

Experimental method The obtained HT1080 cells were detached by trypsin treatment, seeded in a MEM medium under the following conditions, and cultured (1 × 10 ⁵ cells / ml, 96 wells).
Group 1 No addition Group 2 HA4 was added to the medium (1 μg / ml) and incubated for 24 hours.

  After incubation, the cells were fixed with cold methanol. Subsequently, the plate was washed with PBS containing 0.5% BSA (calf serum) (BSA-PBS) and incubated with biotin-labeled HABP. The incubation conditions were 24 hours at 4 ° C. Then, after washing with BSA-PBS, it was incubated with FITC-labeled streptavidin (30 minutes at room temperature (24 ° C.)). After incubation, the plate was washed with BSA-PBS.

  The images were taken after observation with a fluorescence microscope (Nikon). In addition, under a microscope, cells having a diffuse HA-stained image on the cell surface were counted in three randomly selected visual fields (200 times).

Results Images taken for each group are shown in FIG. 1, and the results of measuring the number of cells with diffusible HA are shown in FIG. From the results shown in FIG. 1, hyaluronan having 10 or more sugars was stained granular in the cytoplasm in any group. In addition to such stained images, more cells with diffusible HA stained images were observed in the HA4-treated group (group 2) than in the non-added group (FIGS. 1 and 2). Moreover, as shown in FIG. 1, many granular HA was recognized by the additive-free group (1 group).

Consideration
HA is synthesized by hyaluronan synthase (HAS) present in the cell membrane and secreted extracellularly from the pores of the cell membrane. Therefore, the granular hyaluronan found in the cells is not HA in the process of synthesis, but is taken up from the outside into intracellular endosomes, phagosomes and lysosomes, and is considered to follow the fate of degradation. On the other hand, diffusive HA is HA anchored on the cell surface by CD44 or HAS. The HA moored by the HAS is the HA just produced. Thus, cells rich in diffusible HA indicate that HA production is active.

  The above results show that there were many cells containing diffusible HA in the HA4-treated group. Therefore, it was shown that HA4 has a hyaluronan production promoting effect. In the above results, the HA4 treatment group showed that the amount of granular HA indicating HA in the process of decomposition was low. Therefore, it was shown that HA4 has a hyaluronan degradation inhibitory action.

[Example 2]
In this example, the hyaluronan production promoting action by the drug according to the present invention was demonstrated using human skin tissue.

Skin tissue TESTSKIN ^™ LSE manufactured by Toyobo Co., Ltd. was used. TESTSKIN ^™ LSE can be used to construct a human skin tissue model by culturing using the attached assay medium and using the attached 6-well plate. A schematic diagram of one well in the 6-well plate is shown in FIG. As shown in FIG. 3, each well in the 6-well plate is composed of a transwell 2 having a permeable membrane 1 on the bottom surface. TESTSKIN ^™ LSE is cultured on the bottom surface of the transwell by filling the inside of the transwell 2 and the lower space 3 of the bottom surface of the transwell 2 with the assay medium to form a human skin tissue model 4.

Preparation of test substance HA4 prepared in the same manner as in Example 1 was used.

Experimental Method First, TESTSKIN ^™ LSE was cultured in a 6-well plate at 37 ° C. and 5% CO ₂ for 24 hours.
Group 1 [HA4 (−)]: A transwell containing TESTSKIN ^™ LSE was placed in a 6-well plate to which 1.2 ml of assay medium was added, and 1 ml of assay medium was added to the transwell.
Group 2 [HA4 (+)]: A transwell containing TESTSKIN ^™ LSE was placed in a 6-well plate to which 1.2 ml of assay medium containing HA4 was added at a concentration of 1 μg / ml, and a concentration of 1 μg / ml was placed in the transwell. 1 ml of assay medium containing HA4 was added.

  Next, the transwell was cut out and embedded in an OCT compound to prepare a frozen section (thickness 10 μm). Next, hyaluronic acid staining with hyaluronic acid binding protein (HABP) was performed in the same manner as in Example 1.

Specifically, the frozen section was fixed with 30% for 4 minutes using 4% paraformaldehyde / PBS, and washed three times with PBS. Further, treatment with 0.3% H ₂ O ₂ / methanol was performed at 4 ° C. for 30 minutes to block endogenous peroxidase, and the plate was washed three times with PBS. Next, endogenous biotin was blocked according to the protocol using an endogenous avidin biotin blocking kit (Nichirei). Next, after adding 100 mM Sodium acetate buffer and incubating at 37 ° C. for 15 minutes, 20 TRU actinomycete-derived hyaluronidase (Seikagaku Corporation) was added to the negative control group selected from each group at 60 ° C. Incubated for 2 hours. After washing with PBS three times, 1% BSA / PBS was added and blocked at room temperature for 1 hour. The blocking agent was removed, biotin-labeled HABP (Seikagaku Corporation) diluted to 2 ug / ml with 1% BSA / PBS was added, incubated at room temperature for 1 hour, and washed three times with PBS. Streptavidin-HRP (Calbiochem) diluted to 200 ng / ml with 1% BSA / PBS was added, incubated at room temperature for 1 hour, washed 3 times with PBS, developed with DAB (Zymed), and observed under a microscope did.

Results Images taken for each group are shown in FIG. 4A is a human skin tissue model of Group 1 [HA4 (−)], and B) is a human skin tissue model of Group 2 [HA4 (+)]. In addition, C) in FIG. 4 is a sample obtained by treating hyaluronidase derived from 20 TRU actinomycetes on the human skin tissue model of group 1 [HA4 (-)], and D) human skin of group 2 [HA4 (+)]. 20 TRU actinomycete-derived hyaluronidase treated sample of tissue model.

  As shown in FIG. 4, in the human skin tissue model of Group 2 [HA4 (+)], many cells positive for HABP staining were detected as indicated by arrows. In contrast, in the group 1 [HA4 (−)] human skin tissue model, there are very few cells positive for HABP staining. Hyaluronan was also observed around the cells. On the other hand, as shown in C) and d) in FIG. 4, the stainability was lost by the hyaluronidase treatment.

Discussion From the above results, it was demonstrated that HA4 has an action of promoting hyaluronan production in a human skin tissue model.

Example 3
In this example, the skin care effect when the drug according to the present invention was orally administered was demonstrated using experimental animals.

Experimental animals In this example, C57BL6 mice were used.

Preparation of test substance In this example, a hyaluronan oligosaccharide mixture (hereinafter, HA oligosaccharide mixture) having a composition as shown in FIG. 5 was prepared by hydrochloric acid hydrolysis. The hyaluronan oligosaccharide mixture was prepared by the so-called hydrochloric acid hydrolysis method as shown below. As a raw material in the hydrochloric acid hydrolysis method, Na hyaluronic acid (manufactured by Kibun Food Chemifa Corporation, standard name: FCH-SU) was used. First, 450 ml of distilled water was accurately measured in a 1 L beaker and heated to 70 ° C. Next, 50 g of the raw material was added to the distilled water (70 ° C.) in several portions under stirring with a stirrer bar. After confirming dissolution of the powder and a solution temperature of 70 ° C., 50 ml of 6N hydrochloric acid was accurately added. The time when hydrochloric acid was added was defined as the reaction start time. While maintaining the solution at 70 ° C., the reaction was stirred for 16 hours after the reaction was disclosed. Immediately after 16 hours, the mixture was poured into about an equal volume (500 ml) of distilled water and neutralized with 2N NaOH (pH 6-7). Thereafter, the solution (about 1 L) was concentrated to 200 ml using an evaporator. The resulting concentrated solution (10 ml) was desalted (Sephadex G25). Thereafter, desalted fractions containing HA oligosaccharides were collected and lyophilized. The HA oligosaccharide was confirmed by carbazole reaction and HPLC. As a result, 1.96 g of HA oligo was obtained as a lyophilized powder (composition shown in FIG. 5).

Experimental Method In this example, first, distilled water containing an HA oligosaccharide mixture was placed in a water supply bottle and allowed to be drunk to C57BL6 mice for 2 weeks (in order to ingest the HA oligosaccharide mixture at a rate of 200 mg / kg body weight per day). In addition, the concentration of the HA oligosaccharide mixture was adjusted in terms of the average water intake per day).

  After ingestion for 2 weeks, the back skin of C57BL6 mice was ingested in a size of 8 mm in diameter, and the weight immediately after collection was measured. The resulting weight was taken as the wet weight. Next, freeze-drying was performed for 48 hours, and the weight after freeze-drying was measured. The resulting weight was taken as the dry weight. Then, the amount of water in the back skin was calculated by subtracting the dry weight from the wet weight.

  In this example, HA4 prepared in the same manner as in Example 1 was orally administered to C57BL6 mice (100 mg / kg), blood was collected (portal blood and arterial blood) 30 minutes later, and the presence of HA4 in the blood was confirmed. Detection was by HPLC. In addition, the sample was treated with chondroitinase ABC (manufactured by Seikagaku Corporation), which is an enzyme that degrades HA4 (1 unit / ml, pH 6.0, 37 ° C. and 1 hour treatment conditions). It was confirmed that the peak that appeared was indeed HA4.

result
FIG. 6 shows the results of measuring the water content in the back skin of C57BL6 mice orally administered with the HA oligosaccharide mixture and C57BL6 mice administered only with distilled water as a control. As shown in FIG. 6, in the C57BL6 mouse orally administered with the HA oligosaccharide mixture, both the wet weight and the dry weight increased as compared with the control mouse, and the water content also increased. did.

  Further, FIG. 7 shows a profile obtained as a result of subjecting portal vein blood and arterial blood collected from C57BL6 mice orally administered with HA4 to HPLC. Fig. 7 (a) shows the result of HPLC for portal vein blood, (b) shows the result of HPLC for arterial blood, and (c) shows the result of chondroitinase ABC treatment on the collected arterial blood. It is a result. As shown in FIG. 7, a prominent peak was observed at the position indicated by the arrow in the arterial blood of mice administered orally with HA4 (FIGS. 7 (a) and (b)). This peak coincided with the appearance position of HA4, and disappeared by chondroitinase ABC treatment as shown in FIG. 7 (c), and thus was found to be a peak corresponding to HA4.

From the above results, when an HA oligosaccharide mixture containing HA4 is ingested orally, it moves to the skin through the blood and increases water and other skin components in the skin, that is, the skin becomes fresh (skin care effect) ).

Example 4
In this example, the skin care effect when the drug of the present invention was applied to the skin was demonstrated using experimental animals.

Experimental Animal In this example, a 7-month-old female pig for meat (LWD breed) was used. The experimental animals were bred in a breeding room set in a natural environment of temperature and humidity and with a lighting time of 12 hours (8 am to 8 pm). During breeding, drinking water was freely consumed and feed (hybrid 70) was restricted to twice a day. In addition, the pork chamber was washed in the morning and evening.

Preparation of test substance In this example, an HA oligosaccharide mixture prepared in the same manner as in Example 3 was prepared as a coating cream prepared by mixing 0.1% by weight with a substrate having the composition shown in Table 1. . In addition, as a comparative example, a coating cream and a coating cream consisting only of a base material were prepared in the same manner except that the HA oligosaccharide mixture was changed to high molecular hyaluronan (average molecular weight 800,000).

Experimental Method The dorsal skin of the laboratory animals being reared was divided into 3 cm squares with 2 cm intervals, and 0.1 g of the above applied cream was applied once a day. The application cream was applied for 7 days. The day after the final application, blood was lethal in the anesthesiology department, the back skin was removed together with the fat layer, a part was embedded in an OCT compound, and a part was frozen.

(Hyaluronan binding experiment with hyaluronan-binding protein)
Frozen sections were prepared (thickness 18 μm) using pig back skin embedded in OTC compound. The sections were fixed in 30% at room temperature using 4% paraformaldehyde / PBS and washed three times with PBS. Further, treatment with 0.3% H ₂ O ₂ / methanol was performed at 4 ° C. for 30 minutes to block endogenous peroxidase, and the plate was washed three times with PBS. Next, endogenous biotin was blocked according to the protocol using an endogenous avidin biotin blocking kit (Nichirei). Next, after adding 100 mM Sodium acetate buffer and incubating at 37 ° C. for 15 minutes, 20 TRU actinomycete-derived hyaluronidase (Seikagaku Corporation) was added to the negative control group and incubated at 60 ° C. for 2 hours. After washing with PBS three times, 1% BSA / PBS was added and blocked at room temperature for 1 hour. The blocking agent was removed, biotin-labeled hyaluronic acid-binding protein (Seikagaku Corporation) diluted to 2 ug / ml with 1% BSA / PBS was added, incubated for 1 hour at room temperature, and washed three times with PBS. Streptavidin-HRP (Calbiochem) diluted to 200 ng / ml with 1% BSA / PBS was added, incubated at room temperature for 1 hour, washed 3 times with PBS, developed with DAB (Zymed), and observed under a microscope did.

(Measurement experiment of hyaluronan content in skin)
Pig skin was dried at 60 ° C. overnight, 100 ul of 1% pronase (Merck) was added per mg, and enzyme digested at 40 ° C. for 17 hours. After inactivating pronase at 100 ° C. for 10 minutes, the mixture was centrifuged at 15000 rpm for 10 minutes, and the supernatant was used as the hyaluronic acid fraction.

  HA having a molecular weight of 750,000 was prepared with 50 mM citrate-phosphate buffer (pH 5) to a concentration of 0.1 μg / ml, 50 μl each was added to an amino plate (Sumitomo Bakelite), and left at room temperature for 15 minutes. 10 μl of sodium cyanoborohydride prepared to 10 mg / ml with distilled water was added to the plate and left at room temperature for 1 hour. After washing 3 times with TBS, it was blocked with 200 μl Block Ace (Dainippon Sumitomo Pharma) at 4 ° C. for 3 days. Wash with PBS / 0.05% Tween three times, add 50 ul of porcine skin hyaluronic acid fraction diluted 75-fold with Block Ace diluted 10-fold, and then use Block Ace diluted 10-fold to 100 ng / ml. 50 μl of the prepared biotin-labeled hyaluronic acid binding protein (Seikagaku) was added and incubated at 37 ° C. for 1 hour. After washing 3 times with PBS / 0.05% Tween, streptavidin-HRP (Calbiochem) diluted 4000 times with 100 μl of 10-fold diluted Block Ace was added and incubated at 37 ° C. for 1 hour. After washing three times with PBS / 0.05% Tween, 100 μl of TMB (MOSS) was added to develop color, and the reaction was stopped with 1N hydrochloric acid. A450 / 630nm was measured.

Results The results of the “hyaluronan staining experiment with hyaluronan-binding protein” described above are shown in FIG. As shown in FIG. 7, when the application cream containing only the base material and the application cream containing polymer HA were used, production of hyaluronan was not observed in the back skin. On the other hand, when the application cream containing the HA oligosaccharide mixture was used, hyaluronan staining was detected on the back skin. In addition, as shown in FIG. 7, since the staining property disappeared by the hyaluronidase treatment, it was proved that the production of hyaluronan was promoted when the applied cream containing the HA oligosaccharide mixture was used. .

  Moreover, the result of the above-mentioned “measurement experiment of hyaluronan content in skin” is shown in FIG. As shown in FIG. 8, the hyaluronan content was higher when the application cream containing the HA oligosaccharide mixture was used than when the application cream consisting only of the base material and the application cream containing the polymer HA were used. Increased significantly. From this result, it was demonstrated that the production of hyaluronan was promoted when the applied cream containing the HA oligosaccharide mixture was used.

2 is a photograph of each group of cells prepared in Example 1. FIG. It is a characteristic view which shows the result of having measured the number of the cells which have diffusible HA on a cell surface in each group prepared in Example 1. FIG. 6 is a schematic diagram of one well in the 6-well plate used in Example 2. FIG. 2 is a photograph of sections of a human skin tissue model in each group of Example 2. FIG. FIG. 4 is a characteristic diagram showing the composition of the HA oligosaccharide mixture prepared in Example 3. FIG. 6 is a characteristic diagram showing wet weight, dry weight, and moisture content of mouse back skin in each group of Example 3. FIG. 6 is a characteristic diagram obtained as a result of subjecting portal blood and arterial blood collected from mice in each group of Example 3 to HPLC. It is a photograph which shows the result of the hyaluronan dyeing | staining experiment by the hyaluronan binding protein using the pig back skin in each group of Example 4. FIG. It is a characteristic view which shows the result of the measurement experiment of the hyaluronan content in skin using the pig back skin in each group of Example 4.

Claims (9)

  A hyaluronan production promoter comprising hyaluronan as an active ingredient.   The hyaluronan is a tetrasaccharide containing two units of -D-glucuronic acid-β-1,3-DN-acetylglucosamine-β-1,4-. 1. The hyaluronan production promoter according to 1.   A hyaluronan decomposition inhibitor containing hyaluronan as an active ingredient.   The hyaluronan is a tetrasaccharide containing two units of -D-glucuronic acid-β-1,3-DN-acetylglucosamine-β-1,4-. 3. The hyaluronan degradation inhibitor according to 3.   A skin care agent containing hyaluronan as an active ingredient.   The hyaluronan is a tetrasaccharide containing two units of -D-glucuronic acid-β-1,3-DN-acetylglucosamine-β-1,4-. 5. A skin care agent according to 5.   The hyaluronan is composed of -D-glucuronic acid-β-1,3-DN-acetylglucosamine-β-1,4- as one unit, a disaccharide containing one, a tetrasaccharide containing two, 6. The skin care agent according to claim 5, which is a mixture comprising 6 saccharides comprising 3 of these, 8 saccharides comprising 4 of them and 10 saccharides comprising 5 of them.   6. The skin care agent according to claim 5, wherein the skin care agent is applied to the skin.   6. A skin care agent according to claim 5, wherein the skin care agent is suitable for oral administration.

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