WO2014203883A1 - Hyaluronic acid production promoter - Google Patents

Hyaluronic acid production promoter Download PDF

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Publication number
WO2014203883A1
WO2014203883A1 PCT/JP2014/066000 JP2014066000W WO2014203883A1 WO 2014203883 A1 WO2014203883 A1 WO 2014203883A1 JP 2014066000 W JP2014066000 W JP 2014066000W WO 2014203883 A1 WO2014203883 A1 WO 2014203883A1
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WIPO (PCT)
Prior art keywords
hyaluronic acid
lipocalin
acid production
production promoter
degradation product
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PCT/JP2014/066000
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French (fr)
Japanese (ja)
Inventor
高野　義彦
晴彦 加藤
宏 上野
敏也 小林
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雪印メグミルク株式会社
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Publication of WO2014203883A1 publication Critical patent/WO2014203883A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/66—Proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists

Definitions

  • the present invention relates to a hyaluronic acid production promoter, a hyaluronic acid production promoting supplement, a food and drink, and a hyaluronic acid production promoting cosmetic useful for preventing rough skin, wrinkles, reduced elasticity, reduced joint function, and the like.
  • the skin is roughly composed of the epidermis present on the skin surface and the dermis present in the lower part of the epidermis.
  • the epidermis mainly has a barrier function that protects against foreign substances, pathogenic bacteria, and ultraviolet rays, and a function that protects the leakage of substances from the living body.
  • the dermis is about 15-40 times thicker than the epidermis and plays a role in maintaining the mechanical strength of the skin.
  • the dermis is a fiber component comprising 70% of the dry weight of collagen, but a substrate such as glycoprotein or proteoglycan exists between dermis fibers and skin fibroblasts.
  • Proteoglycans are macromolecules with a molecular weight of 105-106 or more, in which glycoproteins and mucopolysaccharides such as glycosaminoglycans are bound. Since dermal glycosaminoglycans are mainly composed of hyaluronic acid and dermatan sulfate, hyaluronic acid It can be said that it is the main matrix component in the dermis.
  • Hyaluronic acid mainly has a function of retaining the moisture of the skin, so it is considered that the decrease of the moisture content of the skin can be prevented by increasing the hyaluronic acid content in the dermis.
  • Many cosmetics containing hyaluronic acid have been proposed as an improving agent for the retention function.
  • the hyaluronic acid applied to the skin surface only exhibits a moisturizing effect on the skin surface, and cannot essentially improve the skin functional deterioration.
  • Patent Document 1 proposes a complex of hyaluronic acid and phospholipid.
  • no method for increasing hyaluronic acid in the body without ingesting hyaluronic acid is disclosed.
  • hyaluronic acid production promoters extracts obtained from hamachal, kanranized boswellia genus frankincense, sandalwood family sandalwood genus sandalwood, rose family rose genus damask rose, perilla family mannenro genus rosemary are disclosed. (Patent Documents 1 and 2)
  • hyaluronic acid in synovial fluid covers the surface of articular cartilage and helps smooth operation of joint functions.
  • the concentration of hyaluronic acid in normal human joint fluid is about 2.3 mg / mL.
  • the concentration of hyaluronic acid in joint fluid decreases to about 1.2 mg / mL, and at the same time, The viscosity is also significantly reduced.
  • hyaluronic acid content decreases in pyogenic arthritis and gouty arthritis as in the case of rheumatoid arthritis (Non-patent Document 1).
  • the amount of hyaluronic acid in the joint fluid is increased in order to improve the lubrication function, cover / protect articular cartilage, suppress pain, and improve the properties of pathological joint fluid.
  • Non-patent Document 2 when rheumatoid arthritis patients are given joint injection therapy with sodium hyaluronate, the above improvement has been observed (Non-patent Document 2).
  • Non-patent Document 3 the improvement effect by joint injection therapy of hyaluronic acid has been reported (Non-patent Document 3).
  • promotion of hyaluronic acid production is effective for the prevention and treatment of skin diseases such as rough skin, joint diseases such as rheumatoid arthritis, traumatic arthropathy, osteoarthritis, and osteoarthritis. Therefore, a supplement, cream or food or drink containing a hyaluronic acid production promoter that can be easily treated in daily life has been desired.
  • Lipocalin 2 (LCN2) and ⁇ -lactoglobulin are proteins belonging to the lipocalin family having eight ⁇ -barrel structures.
  • Lipocalin 2 (LCN2) also called human neutrophil gelatinase-binding lipocalin (NGAL)
  • NGAL human neutrophil gelatinase-binding lipocalin
  • Lipocalin 2 secreted from the liver has an antibacterial action by selectively binding to diderophore-Fe3 + to inhibit bacterial iron utilization and growth.
  • lipocalin 2 in plasma and blood can be used as a marker for inflammation and infection
  • lipocalin 2 in urine can be used as a marker for malignant tumors such as breast cancer.
  • Lipocalin 2 is known to be also contained in milk, but it has been reported that it is more contained in colostrum than in mature milk.
  • ⁇ -lactoglobulin is the major whey protein in milk. It has the ability to bind hydrophobic substances such as retinol, which is a useful physiologically active substance, and has excellent functional properties (emulsifying property, gelling property, foaming property).
  • Patent Literatures 1 and 2 because plant-derived raw materials are used, stable supply such as cultivation of hyaluronic acid production promoter raw materials is limited to a specific region, and fluctuations in supply due to the season are large. There is a problem that cannot be done. Therefore, the present invention provides a hyaluronic acid production promoter that can be taken orally, and provides a supplement for promoting hyaluronic acid production, a food and drink, and a cosmetic for promoting hyaluronic acid production containing such substances. Let it be an issue.
  • the present inventors diligently searched for a substance exhibiting hyaluronic acid production promoting action.
  • lipocalin family protein degradation obtained by degrading lipocalin family protein or lipocalin family protein was investigated.
  • the product has been found to increase the amount of hyaluronic acid produced, and the present invention has been completed. That is, the present invention includes the following aspects.
  • the hyaluronic acid production promoter according to (1) wherein the lipocalin family and / or lipocalin family degradation product is lipocalin 2 and / or lipocalin 2 degradation product.
  • a cosmetic comprising the hyaluronic acid production promoter according to any one of (1) to (6).
  • a hyaluronic acid production promoter with improved safety by using a lipocalin family protein and / or a lipocalin family protein degradation product as an active ingredient.
  • the hyaluronic acid production promotion effect by lipocalin 2 is shown. This compares the effects of lipocalin 2 on mRNA expression of the hyaluronic acid synthase gene (Has2).
  • the hyaluronic acid production promoter of the present invention is characterized in that lipocalin 2 and / or lipocalin 2 degradation product obtained by degrading lipocalin 2 with a proteolytic enzyme is an active ingredient.
  • the lipocalin 2 of the present invention can be used from any origin.
  • the lipocalin 2 derived from human and bovine has already been clarified in gene sequence and can be produced by gene recombination, but in the present invention, lipocalin 2 produced by genetic engineering techniques can also be used.
  • cells derived from the cell culture medium can also be used. Lipocalin 2 is contained in bovine colostrum and may be recovered from milk.
  • the lipocalin 2 degradation product is a lipocalin 2 purified by an arbitrary method so as to have a molecular weight of 8,000 or less with a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. It is possible to use limited peptide mixtures. However, the lower limit of the molecular weight is preferably 300 or more.
  • the hyaluronic acid production promoter of the present invention exhibits hyaluronic acid production promoting effect by oral administration or application.
  • the active ingredient lipocalin 2 or lipocalin 2 degradation product can be used as it is, but according to conventional methods, powders, granules, tablets, capsules It can also be used in the form of pharmaceutical preparations, drink preparations and the like.
  • oral preparations such as powders, granules, tablets and capsules are formulated by conventional methods using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.
  • a binder a disintegrant, a surfactant, a lubricant, a fluidity promoter, a colorant, a fragrance, and the like may be used as appropriate in addition to the excipient.
  • the binder include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, sodium carboxymethylcellulose, methylcellulose, crystalline cellulose, ethylcellulose, and polyvinylpyrrolidone.
  • the disintegrant include starch, hydroxypropyl starch. Carboxymethyl cellulose, sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose and the like.
  • lipocalin 2 and lipocalin 2 degradation products can be blended in supplements, nutrients, foods and drinks, etc. as they are or after preparation. Since lipocalin 2 or a lipocalin 2 degradation product is relatively stable to heat, it is possible to heat sterilize the raw material containing the lipocalin 2 or the lipocalin 2 degradation product under conditions that are usually performed.
  • various dosage forms such as liquids, solids, semisolids, etc. are prepared by blending with commonly known components according to the purpose of use.
  • Preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like.
  • the hyaluronic acid production promoter of the present invention is a hydrocarbon such as petrolatum, higher fatty acid lower alkyl esters such as stearyl alcohol, isopropyl myristate, animal fats such as lanolin, polyhydric alcohols such as glycerin, glycerin fatty acid esters, mono Cosmetics for promoting hyaluronic acid production by mixing with surfactants such as stearic acid and polyethylene glycol, inorganic salts, waxes, resins, water and, if necessary, preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate And can produce medicines.
  • surfactants such as stearic acid and polyethylene glycol
  • inorganic salts such as stearic acid and polyethylene glycol
  • waxes such as stearic acid and polyethylene glycol
  • preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate And can produce medicines.
  • the effective amount of the hyaluronic acid production promoter of the present invention by oral administration is appropriately defined by the formulation form, administration method, purpose of use, and age, weight, and medical condition of the patient to which it is applied.
  • lipocalin 2 and / or lipocalin 2 degradation products exhibit hyaluronic acid production promoting action when ingested at least 10 ⁇ g per kg of rat body weight. Therefore, according to the extrapolation method, ingestion of 10 ⁇ g or more of lipocalin 2 and / or lipocalin 2 degradation product per adult per day can be expected to promote hyaluronic acid production.
  • Or may be administered as a medicine. The administration can be divided into several times a day as necessary.
  • the effective amount by application of the hyaluronic acid production promoter of the present invention varies depending on the dosage form, but is preferably 0.001 to 2% by weight based on the total amount of the composition to be applied. What is necessary is just to mix
  • a column packed with 3,000 g of S Sepharose cake is thoroughly washed with deionized water, passed through 10,000 L of skimmed milk, washed thoroughly with deionized water, and then a straight line of 0.1 to 1.0 M sodium chloride. Elute with a gradient. Thereafter, the eluted fraction containing lipocalin 2 was fractionated again by phenyl S Sepharose hydrophobic column chromatography. Furthermore, this fraction was sequentially treated with C4 and C8 reverse phase chromatography and gel filtration chromatography using an HPLC system to obtain 2400 mg of lipocalin (fraction A). In addition, the lipocalin 2 obtained in this way can be used as a hyaluronic acid production promoter as it is.
  • Example 1 Fraction A 25 mg obtained in Example 1 was suspended in 100 ml of water, pancreatin was added to a final concentration of 1%, and enzyme treatment was performed at 37 ° C. for 5 minutes to 6 hours. And after heat-processing at 90 degreeC for 5 minute (s), the enzyme was deactivated, it lyophilized
  • the average molecular weight of the lipocalin 2 degradation product thus obtained was about 8,000 for B, about 500 for C, and about 300 for D.
  • fraction A obtained in Example 1 25 mg was suspended in 100 ml of water, pepsin was added so that the final concentration was 0.01%, the pH was adjusted to 2 to 3, and then 12 ° C at 37 ° C. After carrying out enzyme treatment for a time and adjusting the pH to 8, trypsin was added so that the final concentration was 0.01%, and the enzyme treatment was carried out at 37 ° C. for 12 hours. And after heat-processing at 90 degreeC for 5 minute (s) and deactivating an enzyme, it lyophilized
  • Example 1 The sample A obtained in Example 1 and the samples B to E obtained in Example 2 were examined for hyaluronic acid production promoting action by animal experiments using rats.
  • a 7-week-old Wistar male rat was obtained in the physiological saline-administered group (control group), the sample A obtained in Example 1 was administered in 10 ⁇ g per kg body weight of the rat (Group A-1), and in Example 1.
  • skin tissues (each 300 mg) collected immediately after sacrifice of the shaved rat on the day before the test were subjected to measurement.
  • Example 2 Experiments using sample A obtained in Example 1 and samples B, C, and D obtained in Example 2 using a normal human fibroblast cell line [CCD45SK (ATCCRL 1506) collected from white female skin] The hyaluronic acid production promoting effect was examined by the above. Using a modified Eagle's medium (MEM, 10-101, manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 10% by volume fetal bovine serum (hereinafter abbreviated as FBS), 4 ⁇ 10 4 normal human fibroblast cell lines / well / 0 After seeding in a 24-well plate so as to be 4 ml and culturing at 37 ° C.
  • MEM modified Eagle's medium
  • FBS fetal bovine serum
  • hyaluronic acid production in the group to which lipocalin 2 and / or lipocalin 2 degradation product was added was more than twice that of the group to which lipocalin 2 and / or lipocalin 2 degradation product was not added (control). It showed the ability to promote. This reveals that lipocalin 2 and / or lipocalin 2 degradation products have an action of acting on dermal fibroblasts and promoting hyaluronic acid production, and are shown to be useful as hyaluronic acid production promoters. It was.
  • the amount of hyaluronic acid in the culture solution thus obtained was measured with a hyaluronic acid measurement kit QnE Hyaluronic Acid (HA) Quantitative ELISA assay (manufactured by Biotech Trading Partners). The result is shown in FIG.
  • lipocalin 2 promotes hyaluronic acid production in the sample to which NGAL was added as compared with the control because the amount of hyaluronic acid was significantly increased.
  • RNA expression of the hyaluronic acid synthase gene was examined using a real-time PCR method. Specifically, NHDF (NB) cells were seeded in a 24-well plate at 0.5 ⁇ 10 5 cells / well, and were placed in a MEDIUM 106 medium (GIBCO) at 37 ° C. in a 5% CO 2 environment. For 3 days. After 3 days of culture period, NGAL dissolved in MEDIUM106 medium to 10 nM was added to and retained in cells for 2 hours, 4 hours, 6 hours, 9 hours, 12 hours and 24 hours, respectively. Total RNA was recovered by the procedure described above and cDNA was synthesized.
  • RNA extractant 0.5 ml of ISOGEN (manufactured by Nippon Gene) as an RNA extractant was added to the cultured cells and allowed to stand for 5 minutes, and then the cell solution solubilized by pipetting was collected in a 1.5 ml tube. After 0.1 ml of chloroform was added to the cell solution and stirred sufficiently, the upper layer (aqueous layer) separated into two layers was collected in a new 1.5 ml tube. 0.25 ml of 2-propyl alcohol was added to the recovered solution, left standing for 10 minutes, and then centrifuged at 15,000 rpm and 4 ° C. for 15 minutes to obtain a total RNA precipitate. The obtained precipitate was washed with 70% ethanol and then dissolved in DEPC water to obtain an RNA solution.
  • ISOGEN manufactured by Nippon Gene
  • CDNA was synthesized from 1 ⁇ g of RNA using Takara PrimeScript TM RT reagent Kit.
  • Real-time PCR using SYBR Green (Takara SYBR Prime Ex TaqII) was performed using the obtained cDNA as a template.
  • the reaction conditions were 95 ° C., 30 seconds of initial denaturation, 95 ° C., 5 seconds of denaturation, 57 ° C., 15 seconds of annealing, 72 ° C., 20 seconds of extension, for a total of 40 cycles.
  • Primers for confirming Has2 gene expression shown in Table 3 were used as primers. The results are shown in FIG.
  • Beverages for promoting hyaluronic acid production with the formulation shown in Table 4 were produced by conventional methods.
  • the flavor of the produced beverage was good and there were no problems such as precipitation.
  • a dough having the composition shown in Table 5 was produced by a conventional method, molded, and then baked to produce hyaluronic acid production promoting biscuits.
  • the hyaluronic acid production promoter having the composition shown in Table 6 was produced by a conventional method.
  • a lotion having the composition shown in Table 7 was produced by a conventional method.
  • a cream having the composition shown in Table 8 was produced by a conventional method.
  • Example 5 Using the lotion obtained in Example 6 and the cream obtained in Example 7, an actual use test was conducted.
  • a comparative product the same formulation as in Examples 6 and 7 was used except that lipocalin 2 and / or lipocalin 2 degradation product was excluded.
  • the skin lotion of the present invention has a significant improvement in dry feeling and rough skin compared with the skin lotion of the comparative product, and is excellent in hyaluronic acid production promoting effect. It was.
  • the cream of the present invention also has an improvement in dry feeling and a marked improvement in rough skin, as compared with the comparative cream, and has been found to have an effect of suppressing natural deterioration such as rough skin.
  • Test Example 6 For 20 patients with mild pain due to osteoarthritis, 100 g of the beverage of Example 3 was drunk once a day, and a one-year clinical trial was conducted. Assess joint pain and function with visual analog scale (VAS) for pain and with the Western Ontario and McMaster Universities (WOMAC) index for pain, function and stiffness in arthritic joints It was. The results are shown in Table 10.
  • the present invention relates to a hyaluronic acid production promoter, a hyaluronic acid production promoting supplement, a food and drink, and a hyaluronic acid production promoting cosmetic useful for preventing rough skin, wrinkles, reduced elasticity, reduced joint function, and the like.

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Abstract

In the present invention, a lipocalin family protein and/or a lipocalin family proteolytic product obtained by using a protease such as pepsin or pancreatin to decompose a lipocalin family protein is used as the active ingredient in a hyaluronic acid production promoter, a collagen production promoter, a food/beverage, and a collagen production promoting cosmetic. This lipocalin family protein and/or lipocalin family proteolytic product has the effect of increasing the amount of hyaluronic acid.

Description

ヒアルロン酸産生促進剤Hyaluronic acid production promoter
　本発明は、皮膚の荒れ、シワ、弾性低下、関節機能低下等を防止するのに有用なヒアルロン酸産生促進剤、ヒアルロン酸産生促進用サプリメント、飲食品及びヒアルロン酸産生促進用化粧料に関する。 The present invention relates to a hyaluronic acid production promoter, a hyaluronic acid production promoting supplement, a food and drink, and a hyaluronic acid production promoting cosmetic useful for preventing rough skin, wrinkles, reduced elasticity, reduced joint function, and the like.
　近年、光老化という、長年において肌に紫外線が照射され続けることによる、しみ、皺、たるみなどの肌の障害を防止することに関して、多くの研究がなされている。
　皮膚はおおまかに皮膚表面に存在する表皮と表皮の下部に存在する真皮からなる。表皮は主に外部からの異物や病原菌、紫外線から保護するバリアー機能や、生体内からの物質の漏洩を防護する機能を有する。また真皮は表皮の約１５～４０倍の厚さを持ち、皮膚の力学的強度を保つ役割を担っている。真皮はその乾燥重量の７０％がコラーゲンからなる繊維成分であるが、真皮の線維と皮膚線維芽細胞との間には、糖蛋白質や、プロテオグリカンといった基質が存在する。プロテオグリカンは、糖蛋白質とグリコサミノグリカンといったムコ多糖とが結合した分子量１０５～１０６以上の巨大分子であり、真皮のグリコサミノグリカンは、主にヒアルロン酸やデルマタン硫酸からなるため、ヒアルロン酸は真皮における主要なマトリックス成分であると言える。老化などの生理的要因や、太陽光などの紫外線、乾燥、酸化等などの外的環境の変化により皮膚の水分含量や真皮のヒアルロン酸量が低下すると、皮膚に皺が発生し、たるんだ状態を引き起こす。ヒアルロン酸は、主に皮膚の水分を保持する機能を有していることから、真皮中のヒアルロン酸含量を高めることによって、皮膚の水分含量の低下を防ぐことができると考えられ、皮膚における水分保持機能の改善剤として、ヒアルロン酸を配合した化粧料が数多く提案されている。しかしながら、これらは皮膚表面に塗布されたヒアルロン酸は、皮膚表面の保湿効果を発揮するのみであり、肌の機能低下を本質的に改善し得るものではない。
　体内のヒアルロン酸を増加させる為に、ヒアルロン酸を経口摂取する方法がある。しかし、ヒアルロン酸は、高い相対分子量や低い脂溶性、生体膜障壁の通過困難性、大量の多糖分解酵素の存在といった理由から、経口投与した際の消化管からの吸収が悪い。ヒアルロン酸の吸収増進のために、特許文献１では、ヒアルロン酸とリン脂質の複合体が提案されている。しかし、ヒアルロン酸を経口摂取せずに体内のヒアルロン酸を増加させる方法はなんら開示されていない。
　そこで、肌の機能低下を本質的に改善するために、真皮層のヒアルロン酸の生合成を促進させる必要があり、これによって皮膚のシワやたるみを防止できるヒアルロン酸産生促進剤が望まれていた。
　ヒアルロン酸産生促進剤としてはアマチャヅル、カンラン化ボスウェリア属フランキンセンス、ビャクダン科ビャクダン属ビャクダン、バラ科バラ属ダマスクロ－ズ、シソ科マンネンロウ属ロ－ズマリ－から得られる抽出物が開示されている。（特許文献１，２） In recent years, much research has been conducted on photoaging, which is to prevent skin disorders such as spots, wrinkles, and sagging caused by long-term irradiation of ultraviolet rays on the skin.
The skin is roughly composed of the epidermis present on the skin surface and the dermis present in the lower part of the epidermis. The epidermis mainly has a barrier function that protects against foreign substances, pathogenic bacteria, and ultraviolet rays, and a function that protects the leakage of substances from the living body. The dermis is about 15-40 times thicker than the epidermis and plays a role in maintaining the mechanical strength of the skin. The dermis is a fiber component comprising 70% of the dry weight of collagen, but a substrate such as glycoprotein or proteoglycan exists between dermis fibers and skin fibroblasts. Proteoglycans are macromolecules with a molecular weight of 105-106 or more, in which glycoproteins and mucopolysaccharides such as glycosaminoglycans are bound. Since dermal glycosaminoglycans are mainly composed of hyaluronic acid and dermatan sulfate, hyaluronic acid It can be said that it is the main matrix component in the dermis. If the skin moisture content or the amount of hyaluronic acid in the dermis decreases due to physiological factors such as aging, or changes in the external environment such as ultraviolet rays such as sunlight, dryness, oxidation, etc., wrinkles occur in the skin and it becomes dull cause. Hyaluronic acid mainly has a function of retaining the moisture of the skin, so it is considered that the decrease of the moisture content of the skin can be prevented by increasing the hyaluronic acid content in the dermis. Many cosmetics containing hyaluronic acid have been proposed as an improving agent for the retention function. However, the hyaluronic acid applied to the skin surface only exhibits a moisturizing effect on the skin surface, and cannot essentially improve the skin functional deterioration.
In order to increase hyaluronic acid in the body, there is a method of ingesting hyaluronic acid orally. However, hyaluronic acid is poorly absorbed from the digestive tract when administered orally because of its high relative molecular weight, low fat solubility, difficulty in passage through biological membrane barriers, and the presence of large amounts of polysaccharide-degrading enzymes. In order to enhance the absorption of hyaluronic acid, Patent Document 1 proposes a complex of hyaluronic acid and phospholipid. However, no method for increasing hyaluronic acid in the body without ingesting hyaluronic acid is disclosed.
Therefore, in order to essentially improve the functional deterioration of the skin, it is necessary to promote the biosynthesis of hyaluronic acid in the dermis layer, and thus a hyaluronic acid production promoter that can prevent skin wrinkles and sagging has been desired. .
As hyaluronic acid production promoters, extracts obtained from hamachal, kanranized boswellia genus frankincense, sandalwood family sandalwood genus sandalwood, rose family rose genus damask rose, perilla family mannenro genus rosemary are disclosed. (Patent Documents 1 and 2)
　一方、関節液中のヒアルロン酸は、関節軟骨の表面を覆い、関節機能の円滑な作動に役立っている。正常人関節液中のヒアルロン酸濃度は約２．３ｍｇ／ｍＬであるが、例えば、関節リウマチの場合、関節液中のヒアルロン酸濃度は約１．２ｍｇ／ｍＬへと低下し、同時に関節液の粘度も著しく低下する。また、化膿性関節炎や痛風性関節炎などでも関節リウマチの場合と同様、ヒアルロン酸含量の低下が起こることが知られている（非特許文献１）。
　上記疾患において、潤滑機能の改善、関節軟骨の被覆・保護、疼痛抑制及び病的関節液の性状改善をするために、関節液中のヒアルロン酸量を増加させることが行われている。例えば、関節リウマチ患者にヒアルロン酸ナトリウムの関節注入療法を行うと、上記の改善が認められている（非特許文献２）。同様に、外傷性関節症、骨関節炎や変形性関節症においても、ヒアルロン酸の関節注入療法による改善効果が報告されている（非特許文献３）。
　以上のことから、ヒアルロン酸産生の促進は、肌荒れ等の皮膚疾患、関節リウマチや外傷性関節症、骨関節炎、変形性関節症といった関節疾患の予防、治療に有効である。したがって、日常の生活の中で手軽に治療できるヒアルロン酸産生促進剤を含有するサプリメント、クリームあるいは飲食品が望まれていた。 On the other hand, hyaluronic acid in synovial fluid covers the surface of articular cartilage and helps smooth operation of joint functions. The concentration of hyaluronic acid in normal human joint fluid is about 2.3 mg / mL. For example, in the case of rheumatoid arthritis, the concentration of hyaluronic acid in joint fluid decreases to about 1.2 mg / mL, and at the same time, The viscosity is also significantly reduced. Also, it is known that hyaluronic acid content decreases in pyogenic arthritis and gouty arthritis as in the case of rheumatoid arthritis (Non-patent Document 1).
In the above diseases, the amount of hyaluronic acid in the joint fluid is increased in order to improve the lubrication function, cover / protect articular cartilage, suppress pain, and improve the properties of pathological joint fluid. For example, when rheumatoid arthritis patients are given joint injection therapy with sodium hyaluronate, the above improvement has been observed (Non-patent Document 2). Similarly, also in traumatic arthropathy, osteoarthritis and osteoarthritis, the improvement effect by joint injection therapy of hyaluronic acid has been reported (Non-patent Document 3).
From the above, promotion of hyaluronic acid production is effective for the prevention and treatment of skin diseases such as rough skin, joint diseases such as rheumatoid arthritis, traumatic arthropathy, osteoarthritis, and osteoarthritis. Therefore, a supplement, cream or food or drink containing a hyaluronic acid production promoter that can be easily treated in daily life has been desired.
　リポカリン２（ＬＣＮ２）及びβラクトグロブリンは、８本のβ－バレル構造を有するリポカリンファミリーに属するタンパク質である。リポカリン２（ＬＣＮ２）は、ヒト好中球ゼラチナーゼ結合性リポカリン（ＮＧＡＬ）とも呼ばれ、９２ｋＤａのヒト好中球タイプIVコラゲナーゼに結合するタンパク質として同定されている。肝臓から分泌されるリポカリン２は、ジデロフォア－Ｆｅ３＋と選択的に結合することで、バクテリアの鉄利用を阻害し増殖を阻害するといった抗菌作用を持つ。また、血漿、血中のリポカリン２が炎症や感染のマーカーとして、尿中のリポカリン２が乳がんなどの悪性腫瘍のマーカーとして利用できるとの報告がある。リポカリン２は、牛乳中にも含まれることが知られているが、成熟乳に比べて初乳中に多く含まれるとの報告がある。しかしながら牛乳中のリポカリン２の機能についての報告はない。
　βラクトグロブリンは牛乳中の主要な乳清タンパク質である。有用生理活性物質であるレチノール等の疎水性物質の結合能を有しており、優れた機能特性（乳化性、ゲル化性、起泡性）を有する。 Lipocalin 2 (LCN2) and β-lactoglobulin are proteins belonging to the lipocalin family having eight β-barrel structures. Lipocalin 2 (LCN2), also called human neutrophil gelatinase-binding lipocalin (NGAL), has been identified as a protein that binds to a 92 kDa human neutrophil type IV collagenase. Lipocalin 2 secreted from the liver has an antibacterial action by selectively binding to diderophore-Fe3 + to inhibit bacterial iron utilization and growth. In addition, it has been reported that lipocalin 2 in plasma and blood can be used as a marker for inflammation and infection, and lipocalin 2 in urine can be used as a marker for malignant tumors such as breast cancer. Lipocalin 2 is known to be also contained in milk, but it has been reported that it is more contained in colostrum than in mature milk. However, there is no report about the function of lipocalin 2 in milk.
β-lactoglobulin is the major whey protein in milk. It has the ability to bind hydrophobic substances such as retinol, which is a useful physiologically active substance, and has excellent functional properties (emulsifying property, gelling property, foaming property).
特開　２００７－５１０９１JP 2007-51091 特開　２０１３－２３４３７JP 2013-23437
　特許文献１，２に開示されている発明では、植物由来の原材料を用いる為、ヒアルロン酸産生促成剤の原料の栽培が特定の地域に限られていたり、季節による供給の変動が大きいなど安定供給が出来ないという課題がある。そこで、本発明は、経口摂取可能なヒアルロン酸産生促進剤を提供すること及び、そのような物質を配合したヒアルロン酸産生促進用サプリメント、飲食品及びヒアルロン酸産生促進用化粧料を提供することを課題とする。 In the inventions disclosed in Patent Literatures 1 and 2, because plant-derived raw materials are used, stable supply such as cultivation of hyaluronic acid production promoter raw materials is limited to a specific region, and fluctuations in supply due to the season are large. There is a problem that cannot be done. Therefore, the present invention provides a hyaluronic acid production promoter that can be taken orally, and provides a supplement for promoting hyaluronic acid production, a food and drink, and a cosmetic for promoting hyaluronic acid production containing such substances. Let it be an issue.
　本発明者らは、これらの課題を解決するために、ヒアルロン酸産生促進作用を示す物質について、鋭意、探索を進めたところ、リポカリンファミリータンパク質あるいはリポカリンファミリータンパク質を分解して得られるリポカリンファミリータンパク質分解物が、ヒアルロン酸産生量を増加させることを見出し、本発明を完成するに至った。すなわち本発明は、以下の態様を含むものである。
（１）リポカリンファミリー及び／又はリポカリンファミリー分解物を有効成分とするヒアルロン酸産生促進剤。
（２）上記リポカリンファミリー及び／又はリポカリンファミリー分解物が、リポカリン２及び／又はリポカリン２分解物である、（１）記載のヒアルロン酸産生促進剤。
（３）上記リポカリンファミリー及び／又はリポカリンファミリー分解物が、βラクトグロブリン及び／又はβラクトグロブリン分解物である、（１）記載のヒアルロン酸産生促進剤。
（４）前記リポカリン２分解物が、分子量３００以上、８０００以下である、（２）に記載のヒアルロン酸産生促進剤。
（５）前記リポカリン２分解物が、リポカリン２をタンパク質分解酵素で分解して得られたものである、（２）記載のヒアルロン酸産生促進剤。
（６）前記タンパク質分解酵素が、トリプシン、パンクレアチン、キモトリプシン、ペプシン、パパイン、カリクレイン、カテプシン、サーモライシン、Ｖ８プロテアーゼから選択されるいずれか１種以上である、（５）記載のヒアルロン酸産生促進剤。
（７）（１）～（６）のいずれかに記載のヒアルロン酸産生促進剤を配合したサプリメント。
（８）（１）～（６）のいずれかに記載のヒアルロン酸産生促進剤を配合した飲食品。
（９）（１）～（６）のいずれかに記載のヒアルロン酸産生促進剤を配合した化粧料。 In order to solve these problems, the present inventors diligently searched for a substance exhibiting hyaluronic acid production promoting action. As a result, lipocalin family protein degradation obtained by degrading lipocalin family protein or lipocalin family protein was investigated. The product has been found to increase the amount of hyaluronic acid produced, and the present invention has been completed. That is, the present invention includes the following aspects.
(1) A hyaluronic acid production promoter containing a lipocalin family and / or a lipocalin family degradation product as an active ingredient.
(2) The hyaluronic acid production promoter according to (1), wherein the lipocalin family and / or lipocalin family degradation product is lipocalin 2 and / or lipocalin 2 degradation product.
(3) The hyaluronic acid production promoter according to (1), wherein the lipocalin family and / or lipocalin family degradation product is β-lactoglobulin and / or β-lactoglobulin degradation product.
(4) The hyaluronic acid production promoter according to (2), wherein the lipocalin 2 degradation product has a molecular weight of 300 or more and 8000 or less.
(5) The hyaluronic acid production promoter according to (2), wherein the lipocalin 2 degradation product is obtained by degrading lipocalin 2 with a proteolytic enzyme.
(6) The hyaluronic acid production promoter according to (5), wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. .
(7) A supplement containing the hyaluronic acid production promoter according to any one of (1) to (6).
(8) A food and drink containing the hyaluronic acid production promoter according to any one of (1) to (6).
(9) A cosmetic comprising the hyaluronic acid production promoter according to any one of (1) to (6).
　本発明により、リポカリンファミリータンパク質及び／またはリポカリンファミリータンパク質分解物を有効成分とすることで安全性を向上させたヒアルロン酸産生促進剤を提供することができる。 According to the present invention, it is possible to provide a hyaluronic acid production promoter with improved safety by using a lipocalin family protein and / or a lipocalin family protein degradation product as an active ingredient.
リポカリン２によるヒアルロン酸産生促進効果を示す。The hyaluronic acid production promotion effect by lipocalin 2 is shown. リポカリン２がヒアルロン酸合成酵素遺伝子（Ｈａｓ２）のｍＲＮＡ発現へ及ぼす影響について比較したものである。This compares the effects of lipocalin 2 on mRNA expression of the hyaluronic acid synthase gene (Has2).
　本発明のヒアルロン酸産生促進剤の特徴は、リポカリン２及び／またはリポカリン２をタンパク質分解酵素で分解して得られるリポカリン２分解物を有効成分とすることにある。
　本発明のリポカリン２はどのような由来のものであっても使用可能である。たとえば、ヒト及びウシ由来のリポカリン２はすでにその遺伝子配列が明らかになっており、遺伝子組換えによる生産が可能であるが、本発明では、遺伝子工学的手法により生産されたリポカリン２も使用可能であり、細胞培養の培養液から回収した細胞由来のものも使用可能である。また、リポカリン２はウシ初乳中に含有されており、乳から回収したものであっても良く、生乳や粉乳、脱脂乳、還元乳等から、加熱処理、加塩処理、エタノール処理、イオン交換クロマトグラフィーやゲル濾過クロマトグラフィー等の各種クロマト処理、限外濾過処理等によって取得することも可能である。 The hyaluronic acid production promoter of the present invention is characterized in that lipocalin 2 and / or lipocalin 2 degradation product obtained by degrading lipocalin 2 with a proteolytic enzyme is an active ingredient.
The lipocalin 2 of the present invention can be used from any origin. For example, the lipocalin 2 derived from human and bovine has already been clarified in gene sequence and can be produced by gene recombination, but in the present invention, lipocalin 2 produced by genetic engineering techniques can also be used. In addition, cells derived from the cell culture medium can also be used. Lipocalin 2 is contained in bovine colostrum and may be recovered from milk. From raw milk, powdered milk, skim milk, reduced milk, etc., heat treatment, salting treatment, ethanol treatment, ion exchange chromatography can be used. It can also be obtained by various chromatographic treatments such as chromatography and gel filtration chromatography, ultrafiltration treatment and the like.
　リポカリン２分解物は、任意の方法で精製したリポカリン２をトリプシン、パンクレアチン、キモトリプシン、ペプシン、パパイン、カリクレイン、カテプシン、サーモライシン、Ｖ８プロテアーゼ等のタンパク質分解酵素で分子量が８，０００以下となるように限定分解したペプチド混合物を使用することが可能である。但し、分子量の下限は３００以上であることが好ましい。 The lipocalin 2 degradation product is a lipocalin 2 purified by an arbitrary method so as to have a molecular weight of 8,000 or less with a protease such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease. It is possible to use limited peptide mixtures. However, the lower limit of the molecular weight is preferably 300 or more.
　本発明のヒアルロン酸産生促進剤は、経口投与あるいは塗布することにより、ヒアルロン酸産生促進効果を発揮する。本発明のヒアルロン酸産生促進剤を経口投与するに際しては、有効成分であるリポカリン２あるいはリポカリン２分解物をそのままの状態で用いることもできるが、常法に従い、粉末剤、顆粒剤、錠剤、カプセル剤、ドリンク剤等に製剤化して用いることもできる。本発明において、粉末剤、顆粒剤、錠剤、カプセル剤等の経口剤は、例えば、澱粉、乳糖、白糖、マンニット、カルボキシメチルセルロース、コーンスターチ、無機塩類等の賦形剤を用いて常法によって製剤化することが可能である。この種の製剤には、前記賦形剤の他に、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、着色料、香料等を適宜使用してもよい。結合剤としては、例えば、澱粉、デキストリン、アラビアガム、ゼラチン、ヒドロキシプロピルスターチ、カルボキシメチルセルロースナトリウム、メチルセルロース、結晶性セルロース、エチルセルロース、ポリビニルピロリドンが挙げられ、崩壊剤としては、例えば、澱粉、ヒドロキシプロピルスターチ、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、架橋カルボキシメチルセルロースナトリウム、結晶性セルロース等が挙げられる。また、界面活性剤としては、大豆レシチン、蔗糖脂肪酸エステル等、滑沢剤としては、タルク、ロウ、蔗糖脂肪酸エステル、水素添加植物油等、流動性促進剤としては無水ケイ酸、乾燥水酸化アルミニウム、ケイ酸マグネシウム等が挙げられる。
　さらには、これらのリポカリン２やリポカリン２分解物をそのままあるいは製剤化した後、これをサプリメント、栄養剤や飲食品等に配合することも可能である。なお、リポカリン２あるいはリポカリン２分解物は、比較的熱に対して安定であるので、リポカリン２あるいはリポカリン２分解物を含む原料を通常行われるような条件で加熱殺菌することも可能である。 The hyaluronic acid production promoter of the present invention exhibits hyaluronic acid production promoting effect by oral administration or application. When the hyaluronic acid production promoter of the present invention is orally administered, the active ingredient lipocalin 2 or lipocalin 2 degradation product can be used as it is, but according to conventional methods, powders, granules, tablets, capsules It can also be used in the form of pharmaceutical preparations, drink preparations and the like. In the present invention, oral preparations such as powders, granules, tablets and capsules are formulated by conventional methods using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts. It is possible to In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a colorant, a fragrance, and the like may be used as appropriate in addition to the excipient. Examples of the binder include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, sodium carboxymethylcellulose, methylcellulose, crystalline cellulose, ethylcellulose, and polyvinylpyrrolidone. Examples of the disintegrant include starch, hydroxypropyl starch. Carboxymethyl cellulose, sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose and the like. In addition, as surfactant, soybean lecithin, sucrose fatty acid ester, etc., as lubricant, talc, wax, sucrose fatty acid ester, hydrogenated vegetable oil, etc., as fluidity promoter, silicic anhydride, dry aluminum hydroxide, Examples include magnesium silicate.
Furthermore, these lipocalin 2 and lipocalin 2 degradation products can be blended in supplements, nutrients, foods and drinks, etc. as they are or after preparation. Since lipocalin 2 or a lipocalin 2 degradation product is relatively stable to heat, it is possible to heat sterilize the raw material containing the lipocalin 2 or the lipocalin 2 degradation product under conditions that are usually performed.
　本発明のヒアルロン酸産生促進剤を塗布するに際しては、その使用目的に応じて、通常用いられる公知の成分に配合することによって、液剤、固形剤、半固形剤等の各種剤形に調製することが可能で、好ましい組成物として軟膏、ゲル、クリーム、スプレー剤、貼付剤、ローション、粉末等が挙げられる。例えば、本発明のヒアルロン酸産生促進剤をワセリン等の炭化水素、ステアリルアルコール、ミリスチン酸イソプロピル等の高級脂肪酸低級アルキルエステル、ラノリン等の動物性油脂、グリセリン等の多価アルコール、グリセリン脂肪酸エステル、モノステアリン酸、ポリエチレングリコール等の界面活性剤、無機塩、ロウ、樹脂、水及び、要すればパラオキシ安息香酸メチル、パラオキシ安息香酸ブチル等の保存料に混合することによって、ヒアルロン酸産生促進用化粧料や医薬品を製造することができる。 When applying the hyaluronic acid production promoter of the present invention, various dosage forms such as liquids, solids, semisolids, etc. are prepared by blending with commonly known components according to the purpose of use. Preferred compositions include ointments, gels, creams, sprays, patches, lotions, powders and the like. For example, the hyaluronic acid production promoter of the present invention is a hydrocarbon such as petrolatum, higher fatty acid lower alkyl esters such as stearyl alcohol, isopropyl myristate, animal fats such as lanolin, polyhydric alcohols such as glycerin, glycerin fatty acid esters, mono Cosmetics for promoting hyaluronic acid production by mixing with surfactants such as stearic acid and polyethylene glycol, inorganic salts, waxes, resins, water and, if necessary, preservatives such as methyl paraoxybenzoate and butyl paraoxybenzoate And can produce medicines.
　本発明のヒアルロン酸産生促進剤の経口投与による有効量は、その製剤形態、投与方法、使用目的、及びこれを適用される患者の年齢、体重、病状により適宜規定され一定でないが、ラットを用いた動物実験の結果、リポカリン２及び／またはリポカリン２分解物は、ラット体重１ｋｇ当たり１０μｇ以上摂取させることでヒアルロン酸産生促進作用を示すことが明らかとなった。したがって、外挿法によると、通常、成人一人当たり一日１０μｇ以上のリポカリン２及び／またはリポカリン２分解物を摂取すればヒアルロン酸産生促進効果が期待できるため、この必要量を確保できるよう飲食品に配合するか、あるいは、医薬として投与すれば良い。なお、投与は必要に応じて一日数回に分けて行うことも可能である。 The effective amount of the hyaluronic acid production promoter of the present invention by oral administration is appropriately defined by the formulation form, administration method, purpose of use, and age, weight, and medical condition of the patient to which it is applied. As a result of animal experiments, it has been clarified that lipocalin 2 and / or lipocalin 2 degradation products exhibit hyaluronic acid production promoting action when ingested at least 10 μg per kg of rat body weight. Therefore, according to the extrapolation method, ingestion of 10 μg or more of lipocalin 2 and / or lipocalin 2 degradation product per adult per day can be expected to promote hyaluronic acid production. Or may be administered as a medicine. The administration can be divided into several times a day as necessary.
　本発明のヒアルロン酸産生促進剤の塗布による有効量は、剤形により異なるが、適用する組成物全量を基準として、好ましくは、０．００１～２重量％となるように、リポカリン２及び／またはリポカリン２分解物を配合すれば良い。ただし、入浴剤のように使用時に希釈されるものは、さらに配合量を増やすことができる。 The effective amount by application of the hyaluronic acid production promoter of the present invention varies depending on the dosage form, but is preferably 0.001 to 2% by weight based on the total amount of the composition to be applied. What is necessary is just to mix | blend lipocalin 2 decomposition product. However, what is diluted at the time of use like a bath agent can increase a compounding quantity further.
　以下、実施例および試験例を示し、本発明をさらに詳しく説明する。実施例および試験例における「％」は断らない限り「重量％」を意味するものとする。
　以下に、実施例及び試験例を示して本発明を詳細に説明するが、これらは単に本発明の実施態様を例示するのみであり、本発明はこれらによって何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples and test examples. “%” In Examples and Test Examples means “% by weight” unless otherwise specified.
EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples and test examples. However, these are merely examples of the present invention, and the present invention is not limited to these examples.
　Ｓセファロース ３,０００ｇ を充填したカラムを脱イオン水で充分洗浄し、脱脂乳１０,０００Ｌを通液して、脱イオン水で充分洗浄した後、０．１～１．０Ｍの塩化ナトリウムの直線濃度勾配で溶出した。その後、リポカリン２を含む溶出画分を再度フェニルＳセファロース疎水性カラムクロマトグラフィーで分画した。さらに、この画分をＨＰＬＣシステムにてＣ４およびＣ８逆相クロマトグラフィー、ゲルろ過クロマトグラフィーで順次処理し、リポカリン２４００ｍｇ（画分Ａ）を得た。なお、このようにして得られたリポカリン２は、そのままヒアルロン酸産生促進剤として使用可能である。 A column packed with 3,000 g of S Sepharose cake is thoroughly washed with deionized water, passed through 10,000 L of skimmed milk, washed thoroughly with deionized water, and then a straight line of 0.1 to 1.0 M sodium chloride. Elute with a gradient. Thereafter, the eluted fraction containing lipocalin 2 was fractionated again by phenyl S Sepharose hydrophobic column chromatography. Furthermore, this fraction was sequentially treated with C4 and C8 reverse phase chromatography and gel filtration chromatography using an HPLC system to obtain 2400 mg of lipocalin (fraction A). In addition, the lipocalin 2 obtained in this way can be used as a hyaluronic acid production promoter as it is.
　実施例１で得られた画分Ａ２５ｍｇを、水１００ｍｌに懸濁し、最終濃度が１％となるようにパンクレアチンを加え、３７℃で５分から６時間、酵素処理を行った。そして、９０℃で５分間加熱処理して酵素を失活させた後、凍結乾燥して、リポカリン２分解物２４ｍｇ（画分Ｂ、Ｃ、Ｄ）を得た。なお、このようにして得られたリポカリン２分解物の平均分子量は、Ｂが約８，０００、Ｃが約５００、Ｄが約３００であった。また、実施例１で得られた画分Ａ２５ｍｇを、水１００ｍｌに懸濁し、最終濃度が０．０１％となるようにペプシンを添加し、ｐＨを２～３に調整した後、３７℃で１２時間酵素処理を行い、さらにｐＨを８に調整した後、最終濃度が０．０１％となるようにトリプシンを添加し、３７℃で１２時間酵素処理を行った。そして、９０℃で５分間加熱処理して酵素を失活させた後、凍結乾燥し、リポカリン２分解物２４ｍｇ（画分Ｅ）を得た。画分Ｂ、Ｃ、Ｄ、Ｅは、そのままヒアルロン酸産生促進剤として使用可能である。 Fraction A 25 mg obtained in Example 1 was suspended in 100 ml of water, pancreatin was added to a final concentration of 1%, and enzyme treatment was performed at 37 ° C. for 5 minutes to 6 hours. And after heat-processing at 90 degreeC for 5 minute (s), the enzyme was deactivated, it lyophilized | freeze-dried and 24 mg (fractions B, C, D) of lipocalin 2 degradation products were obtained. The average molecular weight of the lipocalin 2 degradation product thus obtained was about 8,000 for B, about 500 for C, and about 300 for D. Further, 25 mg of the fraction A obtained in Example 1 was suspended in 100 ml of water, pepsin was added so that the final concentration was 0.01%, the pH was adjusted to 2 to 3, and then 12 ° C at 37 ° C. After carrying out enzyme treatment for a time and adjusting the pH to 8, trypsin was added so that the final concentration was 0.01%, and the enzyme treatment was carried out at 37 ° C. for 12 hours. And after heat-processing at 90 degreeC for 5 minute (s) and deactivating an enzyme, it lyophilized | freeze-dried and 24 mg (fraction E) of lipocalin 2 degradation products were obtained. Fractions B, C, D, and E can be used as hyaluronic acid production promoters as they are.
［試験例１］
実施例１で得られた試料Ａ及び実施例２で得られた試料Ｂ乃至Ｅについて、ラットを用いた動物実験によりヒアルロン酸産生促進作用を調べた。７週齢のＷｉｓｔａｒ系雄ラットを、生理食塩水投与群（対照群）、実施例１で得られた試料Ａをラット体重１ｋｇ当たり１０μｇ投与する群（Ａ－１群）、実施例１で得られた試料Ａをラット体重１ｋｇ当たり１００μｇ投与する群（Ａ－２群）、実施例２で得られた試料Ｂ乃至Ｄをラット体重１ｋｇ当たり１０μｇ投与する群（Ｂ－１～Ｅ－１群）、実施例２で得られた試料Ｂ乃至Ｄをラット体重１ｋｇ当たり１００μｇ投与する群（Ｂ－２～Ｅ－２群）の１１試験群（ｎ＝６）に分け、それぞれを毎日１回ゾンデで経口投与して１０週間飼育した。皮膚のヒアルロン酸量については、試験前日に剃毛したラットを屠殺後速やかに回収した皮膚組織（各３００ｍｇ）を測定に供した。加熱によりタンパク変性させた皮膚組織をアクチナーゼによりタンパク質分解した。得られた分解物を、更にヒアルロニダーゼにてヒアルロン酸に分解した。ヒアルロン酸をＨＰＬＣ法にて測定した。
　その結果を表１に示す。 [Test Example 1]
The sample A obtained in Example 1 and the samples B to E obtained in Example 2 were examined for hyaluronic acid production promoting action by animal experiments using rats. A 7-week-old Wistar male rat was obtained in the physiological saline-administered group (control group), the sample A obtained in Example 1 was administered in 10 μg per kg body weight of the rat (Group A-1), and in Example 1. The group in which 100 μg of the obtained sample A was administered per kg of rat body weight (Group A-2), and the samples B to D obtained in Example 2 were administered in the amount of 10 μg per kg of rat body weight (Groups B-1 to E-1) The samples B to D obtained in Example 2 were divided into 11 test groups (n = 6) in a group (B-2 to E-2 group) administered with 100 μg per kg of body weight of rats, and each sample was subjected to a sonde once a day. Orally administered and reared for 10 weeks. Regarding the amount of hyaluronic acid in the skin, skin tissues (each 300 mg) collected immediately after sacrifice of the shaved rat on the day before the test were subjected to measurement. Skin tissue denatured by heating was proteolyzed with actinase. The obtained decomposition product was further decomposed into hyaluronic acid with hyaluronidase. Hyaluronic acid was measured by HPLC method.
The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　この結果、１０週間後の可溶性画分中ヒアルロン酸量は、対照群に比べ、すべての試験群で有意に高い値を示した。このことから、リポカリン２及び／又はリポカリン２分解物には、ヒアルロン酸産生促進作用があることが明らかとなり、ヒアルロン酸産生促進剤として有用であることが示された。また、このヒアルロン酸産生促進作用はリポカリン２及び／又はリポカリン２分解物をラット体重１ｋｇ当たり少なくとも１０．０μｇ投与した場合に認められることが明らかとなった。 As a result, the amount of hyaluronic acid in the soluble fraction after 10 weeks was significantly higher in all test groups than in the control group. From this, it was clarified that lipocalin 2 and / or lipocalin 2 degradation products have a hyaluronic acid production promoting action, indicating that it is useful as a hyaluronic acid production promoter. In addition, it has been clarified that this hyaluronic acid production promoting action is observed when lipocalin 2 and / or lipocalin 2 degradation product is administered at least 10.0 μg / kg body weight of the rat.
［試験例２］
　実施例１で得られた試料Ａ及び実施例２で得られた試料Ｂ、Ｃ、Ｄについて、正常ヒト線維芽細胞株〔白人女性の皮膚より採取されたＣＣＤ４５ＳＫ（ＡＴＣＣＲＬ １５０６）〕を用いた実験によりヒアルロン酸産生促進作用を調べた。１０容量％ウシ胎児血清（以下ＦＢＳと略記）含有変法イーグル培地（ＭＥＭ、１０‐１０１、大日本製薬社製）を用いて、正常ヒト線維芽細胞株を４×１０４個／ウエル／０．４ｍｌとなるように２４ウエルプレートに播種して、５％炭酸ガス、飽和水蒸気下、３７℃で２４時間培養した後、０．６容量％ＦＢＳ含有ＭＥＭ培地に置換した。そして、実施例１で得られた試料Ａ及び実施例２で得られた試料Ｂ、Ｃ、Ｄを、各ウエルに０．１容量％となるように添加（ｎ＝６）して、７２時間培養して培養液を得た。このようにして得られた培養液中の、ヒアルロン酸量（バイオテック トレーディング パートナーズ社製）を測定した。なお、対照として、リポカリン２又はリポカリン２分解物を添加しないで同様の試験を行った。その結果を表２に示す。 [Test Example 2]
Experiments using sample A obtained in Example 1 and samples B, C, and D obtained in Example 2 using a normal human fibroblast cell line [CCD45SK (ATCCRL 1506) collected from white female skin] The hyaluronic acid production promoting effect was examined by the above. Using a modified Eagle's medium (MEM, 10-101, manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 10% by volume fetal bovine serum (hereinafter abbreviated as FBS), 4 × 10 4 normal human fibroblast cell lines / well / 0 After seeding in a 24-well plate so as to be 4 ml and culturing at 37 ° C. under 5% carbon dioxide gas and saturated steam for 24 hours, the medium was replaced with a 0.6 volume% FBS-containing MEM medium. Then, the sample A obtained in Example 1 and the samples B, C, and D obtained in Example 2 were added to each well so as to be 0.1% by volume (n = 6), and 72 hours Culture was obtained by culturing. The amount of hyaluronic acid (manufactured by Biotech Trading Partners) in the thus obtained culture broth was measured. As a control, the same test was performed without adding lipocalin 2 or lipocalin 2 degradation product. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２の結果、リポカリン２及び／又はリポカリン２分解物を添加した群は、リポカリン２及び／又はリポカリン２分解物を添加していない群（対照）に比べていずれも２倍以上のヒアルロン酸産生促進能を示した。このことから、リポカリン２及び／又はリポカリン２分解物には、皮膚線維芽細胞に働きかけ、ヒアルロン酸産生を促進する作用があることが明らかとなり、ヒアルロン酸産生促進剤として有用であることが示された。 As a result of Table 2, hyaluronic acid production in the group to which lipocalin 2 and / or lipocalin 2 degradation product was added was more than twice that of the group to which lipocalin 2 and / or lipocalin 2 degradation product was not added (control). It showed the ability to promote. This reveals that lipocalin 2 and / or lipocalin 2 degradation products have an action of acting on dermal fibroblasts and promoting hyaluronic acid production, and are shown to be useful as hyaluronic acid production promoters. It was.
［試験例３］
　市販のヒトリポカリン２リコンビナントタンパク質（ＮＧＡＬ）（ＰＲＯＳＰＥＣ社）、及び正常ヒト新生児包皮皮膚線維芽細胞（ＮＨＤＦ（ＮＢ））を用いた細胞実験により、リポカリン２によるヒアルロン酸産生促進作用について調べた。ＮＨＤＦ（ＮＢ）細胞を１２穴プレートに０．５×１０5cells/wellになる様に播種し、ＭＥＤＩＵＭ１０６培地(ＧＩＢＣＯ社製)にて３７℃、５％ＣＯ2環境下にて３日間培養した。ＮＧＡＬが１０ｎＭなるようにＭＥＤＩＵＭ１０６培地に溶解したものを培養した細胞に添加し、２４時間培養した。このようにして得られた培養液のヒアルロン酸量をヒアルロン酸測定キットＱｎＥ　　Ｈｙａｌｕｒｏｎｉｃ　Ａｃｉｄ（ＨＡ）Ｑｕａｎｔｉｔａｔｖｅ　ＥＬＩＳＡ　ａｓｓａｙ（Ｂｉｏｔｅｃｈ　Ｔｒａｄｉｎｇ　Ｐａｒｔｎｅｒｓ社製）にて測定した。その結果を図１に示す。 [Test Example 3]
The hyaluronic acid production promoting action of lipocalin 2 was examined by a cell experiment using a commercially available human lipocalin 2 recombinant protein (NGAL) (PROSPEC) and normal human neonatal foreskin skin fibroblasts (NHDF (NB)). NHDF (NB) cells were seeded in a 12-well plate at 0.5 × 10 5 cells / well and cultured in MEDIUM 106 medium (GIBCO) at 37 ° C. in a 5% CO 2 environment for 3 days. . What was melt | dissolved in MEDIUM106 culture medium so that NGAL might be 10 nM was added to the cultured cell, and it culture | cultivated for 24 hours. The amount of hyaluronic acid in the culture solution thus obtained was measured with a hyaluronic acid measurement kit QnE Hyaluronic Acid (HA) Quantitative ELISA assay (manufactured by Biotech Trading Partners). The result is shown in FIG.
　この結果、コントロールと比較し、ＮＧＡＬを添加したサンプルは、ヒアルロン酸量が有意に増加したことから、リポカリン２がヒアルロン酸産生を促進することか確認された。 As a result, it was confirmed that lipocalin 2 promotes hyaluronic acid production in the sample to which NGAL was added as compared with the control because the amount of hyaluronic acid was significantly increased.
［試験例４］
　リポカリン２がヒアルロン酸合成酵素遺伝子（Ｈａｓ２）のｍＲＮＡ発現へ及ぼす影響について、リアルタイムＰＣＲ法を用いて検討した。具体的には、ＮＨＤＦ（ＮＢ）細胞を２４穴プレートに０．５×１０5cells/wellになる様に播種し、ＭＥＤＩＵＭ１０６培地(ＧＩＢＣＯ社製)にて３７℃、５％ＣＯ2環境下にて３日間培養した。３日間の培養期間のうち、ＮＧＡＬを１０ｎＭになるようにＭＥＤＩＵＭ１０６培地に溶解したものを、それぞれ２時間、４時間、６時間、９時間、１２時間、２４時間細胞に添加・保持した後、以下の手順でｔｏｔａｌ　ＲＮＡを回収しｃＤＮＡを合成した。培養した細胞にＲＮＡ抽出剤であるＩＳＯＧＥＮ(ニッポンジーン社製)を０．５ｍｌ添加し５分間静置した後、ピペッティングにて可溶化させた細胞液を１．５ｍｌ容のチューブに回収した。細胞液に０．１ｍｌのクロロホルムを添加し、十分に攪拌した後、二層に分離した上層(水層)を新たな１．５ｍｌ容のチューブに回収した。回収液に０．２５ｍｌの２－プロピルアルコールを添加し、１０分間静置後、１５,０００ｒｐｍ、４℃の条件にて１５分間遠心分離し、ｔｏｔａｌ ＲＮＡの沈殿物を得た。得られた沈殿物を、７０％エタノールにて洗浄した後、ＤＥＰＣ水に溶解しＲＮＡ液とした。１μg分のＲＮＡからＴａｋａｒａ　ＰｒｉｍｅＳｃｒｉｐｔＴＭ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔを用いてｃＤＮＡを合成した。得られたｃＤＮＡをテンプレートとして、ＳＹＢＲ　Ｇｒｅｅｎ（Ｔａｋａｒａ　ＳＹＢＲ　Ｐｒｉｍｅ　Ｅｘ　ＴａｑII）を使用したリアルタイムＰＣＲを行った。反応条件は、９５℃、３０秒の初期変性後、９５℃、５秒の変性、５７℃、１５秒のアニーリング、７２℃、２０秒の伸張であり、合計４０サイクル反応させた。プライマーは表３に記載のＨａｓ２遺伝子発現確認用プライマーを使用した。結果を図２に示す。 [Test Example 4]
The effect of lipocalin 2 on mRNA expression of the hyaluronic acid synthase gene (Has2) was examined using a real-time PCR method. Specifically, NHDF (NB) cells were seeded in a 24-well plate at 0.5 × 10 5 cells / well, and were placed in a MEDIUM 106 medium (GIBCO) at 37 ° C. in a 5% CO 2 environment. For 3 days. After 3 days of culture period, NGAL dissolved in MEDIUM106 medium to 10 nM was added to and retained in cells for 2 hours, 4 hours, 6 hours, 9 hours, 12 hours and 24 hours, respectively. Total RNA was recovered by the procedure described above and cDNA was synthesized. 0.5 ml of ISOGEN (manufactured by Nippon Gene) as an RNA extractant was added to the cultured cells and allowed to stand for 5 minutes, and then the cell solution solubilized by pipetting was collected in a 1.5 ml tube. After 0.1 ml of chloroform was added to the cell solution and stirred sufficiently, the upper layer (aqueous layer) separated into two layers was collected in a new 1.5 ml tube. 0.25 ml of 2-propyl alcohol was added to the recovered solution, left standing for 10 minutes, and then centrifuged at 15,000 rpm and 4 ° C. for 15 minutes to obtain a total RNA precipitate. The obtained precipitate was washed with 70% ethanol and then dissolved in DEPC water to obtain an RNA solution. CDNA was synthesized from 1 μg of RNA using Takara PrimeScript ™ RT reagent Kit. Real-time PCR using SYBR Green (Takara SYBR Prime Ex TaqII) was performed using the obtained cDNA as a template. The reaction conditions were 95 ° C., 30 seconds of initial denaturation, 95 ° C., 5 seconds of denaturation, 57 ° C., 15 seconds of annealing, 72 ° C., 20 seconds of extension, for a total of 40 cycles. Primers for confirming Has2 gene expression shown in Table 3 were used as primers. The results are shown in FIG.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　図２により、ＮＧＡＬをＮＨＤＦ（ＮＢ）細胞に添加した時に、Ｈａｓ２遺伝子のｍＲＮＡ発現量は、４時間で有意に亢進し、その後１２時間までゆるやかに減弱し、２４時間で対照群との差は見られなくなった。 As shown in FIG. 2, when NGAL was added to NHDF (NB) cells, the mRNA expression level of Has2 gene was significantly increased at 4 hours and then gradually attenuated to 12 hours, and at 24 hours, the difference from the control group was I can no longer see it.
　表４に示す配合のヒアルロン酸産生促進用飲料を常法により製造した。製造した飲料の風味は良好で沈殿等の問題もなかった。 Beverages for promoting hyaluronic acid production with the formulation shown in Table 4 were produced by conventional methods. The flavor of the produced beverage was good and there were no problems such as precipitation.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表５に示す配合のドウを常法により作製し、成形した後、焙焼してヒアルロン酸産生促進用ビスケットを製造した。 A dough having the composition shown in Table 5 was produced by a conventional method, molded, and then baked to produce hyaluronic acid production promoting biscuits.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表６に示す配合のヒアルロン酸産生促進剤を常法により製造した。 The hyaluronic acid production promoter having the composition shown in Table 6 was produced by a conventional method.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表７に示す配合の化粧水を常法により製造した。 A lotion having the composition shown in Table 7 was produced by a conventional method.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　表８に示す配合のクリームを常法により製造した。 A cream having the composition shown in Table 8 was produced by a conventional method.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
［試験例５］
　実施例６で得られた化粧水及び実施例７で得られたクリームを用いて、実使用テストを行った。比較品としては、リポカリン２及び／又はリポカリン２分解物を除いた以外は実施例６及び７と同じ配合のものを用いた。顔面のたるみや小ジワが認められる乾燥肌を有する成人女性２０人を、それぞれ１０人ずつ無作為に２群（Ａ、Ｂ群）に、また、手に肌荒れが認められる女性２０人を、それぞれ１０人ずつ無作為に２群（Ｃ、Ｄ群）に分け、Ａ群の顔面には本発明品の化粧水２ｇを、Ｂ群の顔面には比較品の化粧水２ｇを、Ｃ群の手指には本発明品のクリーム２ｇを、Ｄ群の手指には比較品のクリーム２ｇを、それぞれ１日２回通常の使用状態と同様に１０日間塗布した。結果を表９に示す。 [Test Example 5]
Using the lotion obtained in Example 6 and the cream obtained in Example 7, an actual use test was conducted. As a comparative product, the same formulation as in Examples 6 and 7 was used except that lipocalin 2 and / or lipocalin 2 degradation product was excluded. 20 adult women with dry skin where facial sagging and fine wrinkles are recognized, 10 people each randomly into 2 groups (Groups A and B), and 20 women with rough skin on the hands, respectively 10 people randomly divided into 2 groups (Group C, D), 2g of the product of the present invention was applied to the face of Group A, 2g of the comparison product was applied to the face of Group B, and fingers of Group C 2 g of the product of the present invention and 2 g of the comparative product were applied to the fingers of group D twice a day for 10 days in the same manner as in normal use. The results are shown in Table 9.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　表９の結果より、本発明品の化粧水は、比較品の化粧水に比べて、乾燥感の改善、肌荒れ等の改善が顕著であり、ヒアルロン酸産生促進効果に優れていることが実証された。また、本発明品のクリームについても、比較品のクリームに比べて、乾燥感の改善、肌荒れに顕著な改善がみられ、肌荒れ等の自然増悪抑制効果を有することが明らかとなった。
［試験例６］
　変形性関節炎による軽度の痛みを有する患者２０名を対象に、実施例３の飲料を１日１回１００ｇ飲用し、１年間の臨床試験を行った。関節の疼痛および機能の評価を、疼痛に対するビジュアルアナログスケール（ＶＡＳ）、及び、関節炎の関節における疼痛、機能、および硬直に関するＷｅｓｔｅｒｎ　Ｏｎｔａｒｉｏ　ａｎｄ　ＭｃＭａｓｔｅｒ　Ｕｎｉｖｅｒｓｉｔｉｅｓ（ＷＯＭＡＣ）指標にて変形性関節症の評価を行った。結果を表１０に示す。 From the results in Table 9, it was demonstrated that the skin lotion of the present invention has a significant improvement in dry feeling and rough skin compared with the skin lotion of the comparative product, and is excellent in hyaluronic acid production promoting effect. It was. In addition, the cream of the present invention also has an improvement in dry feeling and a marked improvement in rough skin, as compared with the comparative cream, and has been found to have an effect of suppressing natural deterioration such as rough skin.
[Test Example 6]
For 20 patients with mild pain due to osteoarthritis, 100 g of the beverage of Example 3 was drunk once a day, and a one-year clinical trial was conducted. Assess joint pain and function with visual analog scale (VAS) for pain and with the Western Ontario and McMaster Universities (WOMAC) index for pain, function and stiffness in arthritic joints It was. The results are shown in Table 10.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　本発明は、皮膚の荒れ、シワ、弾性低下、関節機能低下等を防止するのに有用なヒアルロン酸産生促進剤、ヒアルロン酸産生促進用サプリメント、飲食品及びヒアルロン酸産生促進用化粧料に関する。 The present invention relates to a hyaluronic acid production promoter, a hyaluronic acid production promoting supplement, a food and drink, and a hyaluronic acid production promoting cosmetic useful for preventing rough skin, wrinkles, reduced elasticity, reduced joint function, and the like.

Claims (9)

  1. 　リポカリンファミリー及び／又はリポカリンファミリー分解物を有効成分とするヒアルロン酸産生促進剤。 Hyaluronic acid production promoter containing lipocalin family and / or lipocalin family degradation product as an active ingredient.
  2. 　前記リポカリンファミリー及び／又はリポカリンファミリー分解物が、リポカリン２及び／又はリポカリン２分解物である、請求項１記載のヒアルロン酸産生促進剤。 The hyaluronic acid production promoter according to claim 1, wherein the lipocalin family and / or lipocalin family degradation product is lipocalin 2 and / or lipocalin 2 degradation product.
  3. 　前記リポカリンファミリー及び／又はリポカリンファミリー分解物が、βラクトグロブリン及び／又はβラクトグロブリン分解物である、請求項１記載のヒアルロン酸産生促進剤。 The hyaluronic acid production promoter according to claim 1, wherein the lipocalin family and / or lipocalin family degradation product is β-lactoglobulin and / or β-lactoglobulin degradation product.
  4. 　前記リポカリン２分解物が、分子量３００以上、８０００以下である、請求項２に記載のヒアルロン酸産生促進剤。 The hyaluronic acid production promoter according to claim 2, wherein the lipocalin 2 degradation product has a molecular weight of 300 or more and 8000 or less.
  5. 　前記リポカリン２分解物が、リポカリン２をタンパク質分解酵素で分解して得られたものである、請求項２記載のヒアルロン酸産生促進剤。 The hyaluronic acid production promoter according to claim 2, wherein the lipocalin 2 degradation product is obtained by degrading lipocalin 2 with a proteolytic enzyme.
  6. 　前記タンパク質分解酵素が、トリプシン、パンクレアチン、キモトリプシン、ペプシン、パパイン、カリクレイン、カテプシン、サーモライシン、Ｖ８プロテアーゼから選択されるいずれか１種以上である、請求項５記載のヒアルロン酸産生促進剤。 The hyaluronic acid production promoter according to claim 5, wherein the proteolytic enzyme is at least one selected from trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, and V8 protease.
  7. 　請求項１～６のいずれかに記載のヒアルロン酸産生促進剤を配合したサプリメント。 A supplement containing the hyaluronic acid production promoter according to any one of claims 1 to 6.
  8. 　請求項１～６のいずれかに記載のヒアルロン酸産生促進剤を配合した飲食品。 A food or drink containing the hyaluronic acid production promoter according to any one of claims 1 to 6.
  9. 　請求項１～６のいずれかに記載のヒアルロン酸産生促進剤を配合した化粧料。 A cosmetic comprising the hyaluronic acid production promoter according to any one of claims 1 to 6.
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