JP2009269900A - Accelerator for forming tight junction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent or improve wrinkle, dry skin, tanned skin, and aging skin while maintaining skin firmness and moisture by promoting the formation of tight junctions of epidermal cells. Provided is a skin preparation for external use.
[Solution] Kinginga (Holy honeysuckle, Scientific name: Lonicera japonica Thunberg
(Tight junction formation accelerator characterized by containing an extract obtained from (Caprifoliaceae)), and a tight junction formation promoter characterized by containing an extract obtained from the above goldfish and niacinamide, and An external composition for skin.
[Selection] Figure 2

Description

  The present invention relates to a tight junction formation promoter that promotes the formation of tight junctions in cultured cells or living bodies, and also relates to a tight junction formation promoter that can be added to cosmetics, pharmaceuticals, and the like to enhance the barrier function of the skin.

  The skin is an organ located in the outermost layer of the human body, and one of its important roles is to prevent moisture from evaporating excessively from the inside of the body and to prevent foreign substances such as bacteria from entering the body. There is a barrier function that controls the permeation of substances. It is known that the barrier function of the skin is destroyed by drying in the winter or exposure to excessive ultraviolet rays. Tight junctions exist in the form of a belt around cells and control the permeation of substances by bringing adjacent cells into close contact and sealing. Tight junctions are well developed on the inner walls of the intestinal tract, renal tubules, blood vessels, and the like, and tight junctions bring cells into close contact with each other, thereby controlling the entry and exit of the gap between cells. Since the barrier function of the stratum corneum has developed in the skin, it has been considered that tight junctions do not exist. However, tight junction constituent proteins such as occludin, claudin 1 and claudin 4 exist in the epidermis / granule layer, and the continuous tight junction composed of these plays an important role in substance permeability and can act as a permeation barrier. It became clear (Non-Patent Document 1). Furthermore, due to the activation of low molecular weight G protein Rac1 and polar protein aPKC, tight junction constituent proteins are lined up on a continuous line (Non-patent Document 2), and the amount of claudin 4 decreases between cells. It has also been reported that substance permeability increases (Non-patent Document 3).

  In recent years, attempts have been made to search for materials that enhance the tight junction function from such a viewpoint. For example, ganglioside promotes the formation of tight junctions and prevents invasion of allergens (Patent Document 1), gastrointestinal mucosa function maintaining agent containing gastrointestinal hormones as an active ingredient (Patent Document 2), nucleic acids and degradation products thereof Tight junction formation accelerators (Patent Document 3) as active ingredients, peptides and amino acids (Patent Document 4) having allergen absorption inhibitory activity, and the like are disclosed. However, it has not yet been satisfactory from the viewpoints of stability and economical efficiency in the preparation, and development of a material having no side effects has been desired.

JP-A-8-109133 JP-A-8-268908 Japanese Patent Laid-Open No. 9-30978 JP 2002-257814 A Experimental Dermatology, 16, p324, 2007 J. Invest. Dermatol., 127, p782, 2007 J. Cell Biol., 145, p195, 1999

  Accordingly, an object of the present invention is to provide a material that promotes the formation of tight junctions in epidermal cells and a material that promotes protein expression of claudin 4 and phospho-aPKC involved in the formation of tight junctions.

  Another object of the present invention is to provide an external composition for skin having an effect of improving the barrier function by promoting the formation of tight junctions of epidermal cells.

  The above-mentioned object is a tight junction formation promoter, claudin 4 expression promoter characterized by containing an extract (Kinginka extract) obtained from goldfish (Ronicera japonica Thunberg (Caprifoliaceae)) And a phospho-aPKC expression promoter, and by combining a goldfish extract and niacinamide in combination, a particularly excellent tight junction formation promoting effect, claudin 4 and phospho-aPKC expression promoting effect are obtained. be able to.

  The goldfish extract of the present invention promotes the expression of claudin 4 and phospho-aPKC in human epidermal cells and promotes the formation of tight junctions. In addition, the combination of the goldfish extract and niacinamide promotes the formation of tight junctions particularly remarkably (see test examples described later). Therefore, when the goldfish extract of the present invention is applied to a composition for external application to skin, tight junction formation in epidermal cells is promoted, skin firmness and moisture are maintained, and wrinkles, dry skin, tanned skin, aging skin are prevented or An improvement effect can be obtained.

  Hereinafter, the configuration of the present invention will be described in detail.

  The goldfinch used in the present invention is a flower that blooms in early summer, is white at the beginning of blooming, changes to yellow as time passes, and is mixed with white and yellow flowers and blooms. The Kinginga extract can be obtained by solvent extraction using a known extraction solvent using the Kinginga plant as an extraction raw material.

  Examples of the extraction solvent for obtaining the goldfish extract used in the present invention include water or alcohols such as methanol, ethanol and isopropyl alcohol, polyhydric alcohols such as ethylene glycol, propylene glycol and 1,3-butylene glycol, and acetone. Such as ketones, esters such as ethyl acetate, ethers such as diethyl ether, and aromatic compounds such as benzene.

  Further, the goldfish extract used in the present invention may be an extracted solution, or may be concentrated, dried, filtered, re-extracted, etc. by a conventional method. Kinginga extracts are commercially available and can be used.

  The tight junction formation promoter, claudin 4 expression promoter and phospho-aPKC expression promoter of the present invention can be applied to cultured cells or living cells by themselves to promote tight junction formation. The composition may be used by applying to an external preparation composition for skin, such as a paint or a pharmaceutical composition.

  The form of the tight junction formation accelerator, claudin 4 expression accelerator and phospho-aPKC expression accelerator of the present invention, and their skin external preparation composition may be any of liquid, solid or semi-solid, preferably Examples include ointments, gels, creams, sprays, patches, lotions, packs, emulsions, powders, toiletries, foaming agents, perfumes, bathing agents, etc. The skin to be applied to all skin on the human body including the scalp Can be used.

In addition, the tight junction formation promoter, claudin 4 expression promoter and phospho-aPKC expression promoter of the present invention are further combined with niacinamide to further promote tight junction formation and maintain skin firmness and moisture. It is possible to provide a more excellent skin external preparation composition that can prevent or improve dry skin, tanned skin, and aging skin.

  In addition to the above, the tight junction formation accelerator, claudin 4 expression accelerator and phospho-aPKC expression accelerator of the present invention, and their skin external preparation compositions include coloring pigments such as tar dyes and iron oxides. , Parabens, phenoxyethanol and other preservatives, dimethylpolysiloxane, methylphenylpolysiloxane, silicone oils such as cyclic silicon, hydrocarbons such as paraffin and petrolatum, olive squalane, rice squalane, rice germ oil, jojoba oil, castor oil, Vegetable oil such as safflower oil, olive oil, macadamia nut oil, sunflower oil, waxes such as beeswax, mole, carnauba wax, ester oils such as octyldodecyl myristate, cetyl palmitate, isostearyl isostearate, isopropyl myristate, etc. Arco , Cetanol, behenyl alcohol, stearyl alcohol, higher alcohols such as long-chain branched aliphatic alcohols, sterols and derivatives such as cholesterol, phytosterols, branched fatty acid cholesterol esters, macadamia nut fatty acid phytosteryl esters, and processing oils such as hardened oils , Stearic acid, myristic acid, isostearic acid, oleic acid, higher fatty acids such as iso-type long-chain fatty acids and anteiso-type long-chain fatty acids, terpenes such as limonene and hydrogenated bisabolol, glyceryl tricapryl / caprate, 2-ethylhexanoic acid Triglycerides such as glyceryl, triiso type long chain fatty acid glyceryl, glyceryl tripalmitate, anionic surfactants such as sodium cetyl sulfate, N-stearoyl-L-glutamate, polyoxy Nonionic surfactants such as ethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene polyhydric alcohol fatty acid ester, polyoxyethylene hydrogenated castor oil, polyhydric alcohol fatty acid ester, polyglycerin fatty acid ester, modified silicon, sucrose ester , Cationic surfactants such as tetraalkylammonium salts, amphoteric surfactants such as betaine type, sulfobetaine type, sulfoamino acid type, natural surfactants such as lecithin, lysophosphatidylcholine, ceramide, cerebroside, Pigments such as titanium oxide and zinc oxide, antioxidants such as dibutylhydroxytoluene, inorganic salts such as sodium chloride, magnesium chloride, sodium sulfate, potassium nitrate, sodium sulfate, sodium metasilicate, calcium chloride, sodium citrate Organic acid such as potassium acetate, sodium oxalate, sodium aspartate, sodium lactate, dichloroacetic acid, mevalonic acid, glycyrrhizic acid and its salts, ethanolamine hydrochloride, ammonium nitrate, arginine hydrochloride, diisopropylamine salt, urea, decarboxycarnosine Organic amines and salts thereof, chelating agents such as edetic acid, xanthan gum, carboxyvinyl polymer, carrageenan, pectin, alkyl-modified carboxyvinyl polymer, thickener such as agar, potassium hydroxide, diisopropanolamine, triethanolamine Neutralizing agents such as, UV absorbers such as hydroxymethoxybenzophenone sulfonate, dipropylene glycol, 1,3-butylene glycol, glycerin, propylene glycol, sorbitol, mal Polyols such as Toll, Diglycerin and Raffinose, various amino acids, vitamins such as ascorbic acid, biotin and tocopherol and vitamin derivatives such as ascorbic acid sulfate, ascorbic acid phosphate and nicotinic acid tocopherol, etc. It can mix | blend suitably within the range which achieves the objective.

  The content in the composition when applying the goldfish extract used in the present invention to various compositions varies depending on the form and cannot be defined unconditionally. The range of 0.0001 to 1% by mass in terms of conversion is preferable, and 0.001 to 0.01% by mass is more preferable. However, in the case of what is diluted at the time of use like a bath agent, the range of 0.01-5 mass% is preferable.

  When promoting the formation of tight junctions on cultured cells, it is preferable to contain the goldfish extract in the cell culture fluid in an amount of 0.001% by mass or more in terms of dry solid content, and more preferably, 0.001% by mass. It is 01-0.03 mass%.

  In the present invention, in the case where niacinamide is applied to various compositions in combination with the kingfisher extract, the content is preferably in the range of 0.0001 to 10% by mass based on the total amount of the composition. Preferably it is 0.001-1 mass%.

  As an Example, the test example and formulation example which show the tight junction formation promotion effect of this invention are described. The cells, media and evaluation methods used in each test are as follows. Unless otherwise stated,% indicates mass%.

-Cultured epidermal cells As human normal epidermal keratinocytes, commercially available products (Epidercell / newborn foreskin: manufactured by Kurabo Industries) were used.

Epidermal cell culture medium Based on MCDB153 medium (manufactured by Wako Pure Chemical Industries, Ltd.), hydrocortisone (0.5 μmol / L), ethanolamine (0.1 mmol / L), phosphoethanolamine (0.1 mmol / L) , Modified MCDB153 medium supplemented with insulin (5 μg / mL) and EGF (epidermal growth factor: 10 ng / mL) was used. At the time of use, 4 μL of BPE (bovine pituitary extract, Kurabo Industries Co., Ltd.) was added per 1 mL of the medium, and this was used as the epidermal cell culture medium.

(Test Example 1) Tight junction formation promoting action Test compound (concentration in culture medium added in parentheses)
Kinginga extract (0.01-0.03%, converted to dry solids) and niacinamide (0.001%)

2. Test Method Human normal epidermal keratinocytes were seeded at 1.5 × 10 ⁵ cells / well per millicell cell culture insert (MILLIPORE, diameter 12 mm, diameter hole 0.4 μm, polycarbonate filter), 0.5 mL upper layer, The cells were cultured for 3 days at 37 ° C. in an atmosphere of 95% (V / V) air-5% (V / V) carbon dioxide gas in 2 mL of the lower layer epidermal cell culture medium. On day 4 of the culture, the medium was replaced with an epidermal cell culture medium containing 1.8 mM of calcium ions to which a predetermined concentration of the test compound was previously added. Further, after culturing for 3 to 6 days, a TER (Transientical Electrical Resistance) value (Ωm) was measured using an electric resistance measuring device (Millicell-ERS (manufactured by MILLIPORE)). In addition, the sample addition tested in each group n = 4, and the result was shown by each average value.

3. Test result As a result of measuring the TER value when the goldfish extract was added, the TER value of epidermal cells was increased by adding the goldfish extract compared to the non-addition group (FIG. 1- (1)) ( FIG. 1- (2) (3)).
Moreover, compared with the non-addition group (FIG. 2- (1)), the TER value further increased by simultaneously adding the goldfish extract and niacinamide (FIG. 2- (3)).

(Test Example 2) Protein expression analysis of claudin 4 and phospho-aPKC Test compound (inside parenthesis, medium addition concentration)
Kinginga extract (0.03%, in terms of dry solids) and niacinamide (0.001%)

2. Culture Method Human normal epidermal keratinocytes are seeded at 1.5 × 10 ⁵ cells / well in a 24-well plate, and 95% (V / V) air-5% (V) in 1 mL of epidermal cell culture medium. / V) The cells were cultured at 37 ° C. for 3 days in an atmosphere of carbon dioxide gas. On day 4 of the culture, the medium was replaced with an epidermal cell culture medium containing 1.8 mM of calcium ions to which a predetermined concentration of the test compound was previously added. After further culturing for 4 days, the cells were lysed with Tris buffer containing SDS, sonicated and then centrifuged, and the supernatant was used as a cell extract. Western blotting of claudin 4 and phospho-aPKC on the cell extract was performed by the following method.

3. Western blot 3-1. SDS polyacrylamide gel electrophoresis 8 μL of cell extract, 4-fold concentrated SDS Sample Buffer (manufactured by Invitrogen) 4 μL, 10-fold concentrated Sample Reducing Agent (manufactured by Invitrogen) 1.6 μL, and 2.4 μL of water were mixed and mixed at 70 ° C. After heating for minutes, it was subjected to electrophoresis. Electrophoresis was performed using 4-12% Bis-Tris Gel (manufactured by Invitrogen) and in accordance with the instructions attached to NuPAGE GelSystem (manufactured by Invitrogen).

3-2. Transfer of protein to PVDF membrane Transfer of protein to PVDF membrane (Immobilon 1 (manufactured by MILLIPORE)) was performed using Xcell II Blot Module (manufactured by Invitrogen) according to the attached instructions.

3-3. Immunological detection The PVDF membrane after transfer was prepared by using a blocking reagent (a reagent in which skim milk was dissolved in claudin 4: 5% concentration in PBS containing 0.05% Tween-20, phospho-aPKC: BSA in a concentration of 5%, 0. (Reagent dissolved in PBS containing 05% Tween-20) for 30 minutes at room temperature, human claudin 4 antibody (manufactured by ZYMED Laboratories) diluted with blocking reagent 40,000 times, or phospho-aPKC diluted 1000 times The antibody (manufactured by Cell Signaling) was reacted at 4 ° C. for 24 hours. The dilution ratio of each antibody was set in advance in order to obtain an appropriate detection signal. Next, the PVDF membrane was washed by shaking in PBS containing 0.05% Tween-20 and anti-mouse IgG antibody (ICN) diluted 5000 times with PBS containing 0.05% Tween-20, or 2000 times. The diluted anti-rabbit IgG antibody (manufactured by DAKO) was reacted at room temperature for 45 minutes. Finally, the PVDF membrane was washed with shaking in PBS containing 0.05% Tween-20, and SuperSignal WestPico Chemiluminous Substrate (manufactured by PIERCE) or SuperSignal West Dura Extended DurSent Inc. was used as the instruction manual of SuperSignal Dura Extended SubC A signal was detected.

4). Test results:
As a result of analyzing the protein expression of claudin 4 and phospho-aPKC when the goldfish extract was added, the goldfish extract was compared with the non-addition group (FIGS. 3- (1) and 4- (1)). Addition increased the expression of claudin 4 and phospho-aPKC (FIGS. 3- (2) and 4- (2)). Moreover, the expression of claudin 4 was further increased by the simultaneous addition of the goldfish extract and niacinamide (FIGS. 3- (3) and 4- (3)).

  Next, according to the following compounding ingredients, cosmetic liquids, creams and milky lotions were produced by conventional methods.

(Example-1) Cosmetic liquid (component name) (Blending amount)
Ethanol 6
PEG-60 hydrogenated castor oil 1
Dipropylene glycol 10
Glycerin 3
Dimethicone copolyol methyl 0.4
Glycosyl trehalose 5
Hydrolyzed hydrogenated starch 5
Carrageenan 2
Raffinose 3
Niacinamide 1
Methylserine 0.1
Aminobutyric acid 1
Hydrolyzed silk 1
Hydrolyzed conchiolin 1
Kinginga extract (honeysuckle flower extract)
1
Kyonin extract 1
Apricot juice 2
Avocado extract 1
Disodium phosphate 0.1
Potassium phosphate 0.1
EDTA-2Na 0.05
Phenoxyethanol 0.5
Perfume 0.02
Total remaining water 100

(Example-2) Cream (component name) (Blending amount)
Squalane 10
Maltitol 10
Macadamia nut oil 5
1,3-butylene glycol 4
Behenyl alcohol 5
Dimethicone 1
Glyceryl stearate 2
PEG-6 sorbite beeswax 2
Polysorbate 65 2
Stearic acid 1
Cetyl palmitate 1
Beeswax 1
Vaseline 1
Sodium stearoyl glutamate 1
Polysorbate 80 0.5
Niacinamide 0.5
Xanthan gum 0.3
Methylserine 0.5
Acetylglucosamine 0.1
Aminobutyric acid 1
Tocopherol nicotinate 0.001
Hydrogenated lecithin 0.1
Apricot juice 0.1
Hydrolyzed silk 0.1
Kinginga extract (honeysuckle flower extract)
0.001
Kyonin extract 1
Yuzu Extract 1
Avocado extract 5
Hydrolyzed conchiolin 0.1
EDTA-2Na 0.1
Phenoxyethanol 0.5
Methylparaben 0.25
Total remaining water 100

(Example-3) Emulsion (component name) (blending amount)
Ethanol 10
Glycerin 10
Dipropylene glycol 5
PEG-75 5
Cyclomethicone 2
Niacinamide 1
PEG-60 hydrogenated castor oil 1
Phenyltrimethicone 2
Macadamia nut oil 2
Polysorbate 20 1
Ammonium glycyrrhizinate 0.2
Methylserine 0.001
Aminobutyric acid 0.001
Tocopherol nicotinate 0.1
Apricot juice 1
Xanthan gum 0.05
Octyl methoxycinnamate 1
Kinginga extract (honeysuckle flower extract)
0.1
Hydrolyzed silk 0.1
Kyonin extract 0.1
Silane root extract 0.001
Avocado extract 0.1
Hydrolyzed conchiolin 0.1
Carbomer 0.5
Potassium hydroxide 0.3
EDTA-3Na 0.05
Phenoxyethanol 0.1
Fragrance 0.05
Total remaining water 100

  By using the cosmetics of Examples 1 to 3, the firmness and moisture of the skin were improved, and the effect of improving wrinkles, dry skin, tanned skin, and aging skin was seen.

  Ointment, gel, cream that can prevent or improve wrinkles, dry skin, sunburn skin, and aging skin by promoting tight junction formation of epidermal cells according to the present invention and maintaining skin firmness and moisture It is possible to provide various skin external preparation compositions such as sprays, patches, lotions, packs, emulsions, powders, toiletries, foaming agents, perfumes and bathing agents.

It is a figure which shows the tight junction formation promotion effect of the goldfish extract in a human normal epidermal keratinocyte. In the figure, (1) shows the non-added group, (2) shows the 0.01% Kinginga extract added group, and (3) shows the 0.02% Kinginga extract added group. It is a figure which shows the tight junction formation promotion effect of the goldfish extract and niacinamide in a human normal epidermal keratinocyte. In the figure, (1) is the non-added group, (2) is the group added with 0.03% goldfish extract, (3) is the group added 0.03% goldfish extract + 0.001% niacinamide. It is a figure which shows the expression increasing effect of the goldfish extract and the niacinamide claudin 4 in a human normal epidermal keratinocyte. In the figure, (1) is the non-added group, (2) is the group added with 0.03% goldfish extract, (3) is the group added 0.03% goldfish extract + 0.001% niacinamide. It is a figure which shows the expression increase effect of the goldfish extract and phospho-aPKC of niacinamide in a human normal epidermal keratinocyte. In the figure, (1) is the non-added group, (2) is the group added with 0.03% goldfish extract, (3) is the group added 0.03% goldfish extract + 0.001% niacinamide.

Claims (7)

Tight junction formation promoter characterized by containing the extract obtained from goldfish (Rhizophaceae honeysuckle). A tight junction formation promoter characterized by containing an extract obtained from goldfish (a honeysuckle family honeysuckle) and niacinamide. A claudin 4 expression promoter characterized by containing an extract obtained from goldfish (Rhizopus honeysuckle). A claudin 4 expression promoter characterized by containing an extract obtained from goldfish (Rhizophoraceae honeysuckle) and niacinamide. A phospho-aPKC expression promoter characterized by containing an extract obtained from kingfisher (honeysuckle family honeysuckle). A phospho-aPKC expression promoter characterized by containing an extract obtained from goldfish (Rhizopus honeysuckle) and niacinamide. A composition for external use on the skin, comprising an extract obtained from Kinginga (honeysuckle honeysuckle) and niacinamide.

Images