JP6611212B2 - Acne-improving composition - Google Patents

Description

  The present invention relates to a method for selecting an antibacterial agent.

  Conventionally, parabens, phenoxyethanol, and polyhydric alcohols are widely used as antibacterial agents used for external preparations for skin such as cosmetics. In addition, chlorhexidine gluconate, benzalkonium chloride, and the like are used for medical purposes. On the other hand, various antibacterial peptides exist on the skin surface to prevent infection from the outside.

Antibacterial peptides are produced by living organisms themselves, and are therefore expected to replace chemical substances such as antibiotics (see, for example, Patent Documents 1 and 2). Moreover, it is thought that a drug resistant microbe does not appear easily. Antibacterial peptides prevent bacterial infection contamination to treat gram-negative bacterial contamination of foodstuffs, food processing equipment, food processing equipment, food contact surfaces, medical devices, hospital and operating room surfaces, or It is known that it is used for prevention (see, for example, Patent Document 3).
Defensin, an antibacterial peptide, is active against bacteria, fungi, and many enveloped and non-enveloped viruses, of which β-defensin is a cationic peptide with a broad antibacterial activity.
The defensin family includes hBD-1, hBD-2, hBD-3 and the like, and hBD-1 and hBD-2 are representative examples of human β-defensins. Salivary glands,
Expression of hBD-1 and hBD-2 mRNA in mucosal cells such as the tongue, gums and cheeks has been confirmed and, like β-defensin, plays an important role in preventing infection from microbial bacteria. (For example, see Patent Document 4).

On the other hand, acne occurs mainly in teens, but in recent years, cases that develop even after the 30s have increased, and various measures have been taken. Acne is caused by P. acnes, a Gram-positive bacterium, and in women it is said that the menstrual cycle is associated with acne, and there are reports that indicate a relationship with hormones ( For example, refer nonpatent literature 1 and 2.).

JP 2004-107275 A JP 2005-281225 A Special table 2013-502231 gazette JP2013-151442A Arch Dermatol. 2004; 140 (4): p423-424 Transfiguration 痤 pressure ulcer management (general editing: Masutaka Furue, professional editing: Shinkazu Hayashi Nakayama Shoten) P94-96

  The present invention has been made under such circumstances, and provides a method for selecting an antibacterial agent using the expression level of an antibacterial peptide as an index for the purpose of preventing or improving deterioration of the skin condition accompanying the menstrual cycle. This is the issue.

The present inventors examined the effect of hormones that change with the menstrual cycle on antibacterial peptides, and revealed that the expression of antibacterial peptides is reduced by progesterone, which is a luteinizing hormone. Research has been conducted to provide a method for improving the decrease in the expression of antibacterial peptides caused by progesterone, and hBD-3 has a high antibacterial activity against gram-positive bacteria among a plurality of antibacterial peptides. Using the expression level of 3 as an index, it was found that an antibacterial agent that improves the decrease in antibacterial peptide expression caused by progesterone can be selected, and the present invention has been completed. That is, the present invention is as follows.
<1> In a culture system of normal human epidermal keratinocytes, the expression level of an antimicrobial peptide in the presence of the test substance and progesterone and the expression level of the antimicrobial peptide in the presence of only progesterone are measured, and the test substance and A method for selecting an antibacterial agent, wherein the test substance is selected as an antibacterial agent when the expression level of the antibacterial peptide in the presence of progesterone exceeds the expression level of the antibacterial peptide in the presence of progesterone alone.
<2> The method for selecting an antibacterial agent according to <1>, wherein the antibacterial peptide is hBD-3.
<3> The expression level of the antimicrobial peptide is the expression level of hBD-3 mRNA,
The method for selecting an antibacterial agent according to <1>.
<4> In the culture system of normal human epidermal keratinocytes, the expression level (α) of the antimicrobial peptide in the presence of the test substance and progesterone, the expression level (β) of the antimicrobial peptide in the presence of only progesterone, and as a control The test substance is selected as an antibacterial agent when the value calculated from the following formula (1) is 50% or more using the test substance and the expression level (γ) of the antibacterial peptide when no progesterone is added. A method for selecting an antibacterial agent.
Formula (1) (α-β) / (γ-β) × 100 (%)
<5> The method for selecting an antibacterial agent according to <1> to <4>, which is an antibacterial agent exhibiting an antibacterial activity against Propionibacterium acnes (P. acnes).
<6> Antibacterial agent selected by the selection method according to <1> to <5>.

It is a graph which shows the inhibitory effect of the plant extract with respect to the fall of the hBD-3 mRNA expression level by a progesterone. It is a graph which shows the antimicrobial activity with respect to P.acnes of an antimicrobial peptide.

Hereinafter, the method for selecting the antibacterial agent of the present invention will be described.
An example of the procedure in the embodiment of the selection method of the present invention will be given below, but is not limited to the following contents without departing from the gist of the present invention, and can be implemented with appropriate modifications.

(1) Method for selecting antibacterial agent of the present invention The expression level of hBD-3 mRNA can be measured using any method. For example, PCR can be performed using a DNA fragment having a sequence that specifically binds to the gene sequence as a primer, and quantitative detection can be performed.
Specifically, normal human epidermal keratinocytes are seeded on a plate, and are subjected to a low Ca-containing medium at 37 ° C. and 5% CO ₂ . After 3 days of culture, progesterone, or progesterone and a test substance are added, and the medium is replaced with a high Ca-containing medium and cultured for 3 days. After completion of the culture, the cells are collected, total RNA is extracted, and cDNA is synthesized from the obtained total RNA. Real-time PCR can be performed using the synthesized cDNA as a template, and the expression level of hBD-3 mRNA can be quantified by a calibration curve method.
As a control, hBD-3 mRNA without progesterone and test substance added
When the expression level is measured and the value calculated from the following formula (1) is 50% or more, the test substance can be selected as an antibacterial agent.
Formula (1) (α-β) / (γ-β) × 100 (%)
α is the hBD-3 mRNA expression level when progesterone and the test substance are added β is the hBD-3 mRNA expression level when progesterone is added γ is the hBD-3 mRNA expression level when the progesterone and the test substance are not added (control)

Hereinafter, the antibacterial agent of the present invention will be described.
An example of the procedure in the embodiment of the selection method of the present invention will be given below, but is not limited to the following contents without departing from the gist of the present invention, and can be implemented with appropriate modifications.
(2) Antibacterial agent of the present invention Antibacterial peptides include hBD-1, hBD-2, hBD-3, hBD-4 and the like, and have a role of preventing infection from the outside on the mucous membrane and skin surface.
HBD-3 in the present invention exhibits an antibacterial action against Gram-positive bacteria, and particularly shows an antibacterial action against P. acnes.
The antibacterial agent of the present invention is an antibacterial agent selected based on the expression level of the antibacterial peptide.
Progesterone that increases or decreases depending on the menstrual cycle decreases the expression level of hBD-3 mRNA, and consequently reduces the antibacterial effect of hBD-3 against Gram-positive bacteria, particularly the antibacterial action against P. acnes.
According to the method for selecting an antibacterial agent of the present invention, an antibacterial agent that suppresses a decrease in the antibacterial action of such an antibacterial peptide against gram-positive bacteria is selected and used in cosmetics, quasi drugs, pharmaceuticals, foods, etc. I can do it. In particular, when antibacterial action against acne bacteria is expected, it is preferably used as a skin external preparation such as cosmetics and quasi drugs.

The test substance targeted by the selection method of the present invention may be a pure substance, an extract derived from animals or plants, or a mixture thereof.
The extracts derived from animals and plants mean not only animal or plant-derived extracts themselves, but also generic names of extract fractions, purified fractions, extracts or fractions, and solvent-removed products of purified products. Examples of plant-derived extracts include native or grown plants, extracts using products sold as herbal medicine ingredients, and commercially available extracts. Among them, the plant extract of the genus Murasaki belongs to the point of effect, and examples of the plant of the genus Murasaki belong to the following species: Is preferred.
For the extraction operation, the whole plant part can be used, and other parts such as the plant body, the above-ground part, the rhizome part, the tree trunk part, the leaf part, the stem part, the flower ear, and the flower bud can be used. It is preferable to improve the extraction efficiency by cutting. Examples of the extraction solvent include water, alcohols such as methanol, ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, diethyl ether, and tetrahydrofuran. One or two or more selected from polar solvents such as ethers can be exemplified as preferable ones. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a plant or the like used for extraction of a plant or the like. For example, there may be mentioned a method of immersing for several hours, cooling to room temperature, removing insoluble matters and / or solvent if desired, and fractional purification by column chromatography or the like.
In the present invention, after filtering an extract from animals and plants with a polar solvent, fractionation is performed by column chromatography, the concentration of active ingredients is increased, and a more effective fraction is selected by the selection method, thereby improving the skin. It is preferable to use as.
When the antibacterial agent of the present invention is used as a skin external preparation for cosmetics, quasi-drugs, etc., the antibacterial agent of the present invention is 0.0001% by mass to 10% by mass, more preferably 0.8% by mass relative to the total amount. It is preferable to contain 001 mass%-5 mass%, More preferably, 0.01 mass%-3 mass%. When using the solvent extract of a plant extract, it is 0.0000001 mass%-10 mass% with respect to the whole quantity, More preferably, it is 0.0001 mass%-5 mass%, More preferably, it is 0.00.
It is preferable to contain 001 mass%-3 mass%. This is because the effect of the external preparation for skin of the present invention is not exerted if it is less than the lower limit, and if it exceeds the upper limit, the effect reaches a peak, causing problems such as color and odor, and when used as an external preparation for skin, the degree of freedom is increased. This is because there is a case where it is damaged.

  When applied to cosmetics, it is possible to broadly blend the components normally used in cosmetics, and the dosage form and use are not limited at all. Hereinafter, the components that can be contained in the cosmetic when applied to the cosmetic will be described. For example, surfactants such as oils such as hydrocarbons, esters, triglycerides, fatty acids, higher alcohols, anionic surfactants, amphoteric surfactants, cationic surfactants, nonionic surfactants Polyhydric alcohols, thickening / gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders, and the like can be arbitrarily blended. Examples of active ingredients include whitening ingredients, wrinkle-improving ingredients, anti-inflammatory ingredients, animal and plant extracts. In addition, you may use together with the antibacterial agent of this invention demonstrated above.

The whitening component is not particularly limited as long as it is generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethylascorbic acid, tranexamic acid, arbutin, 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2- (triphenylmethyloxy) ethanol, 2- (triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane and aminodiphenylmethane, N- (p-toluyl) cysteic acid, N- (p -Methoxybenzoyl) cysteic acid and the like. Furthermore, as other whitening components, N-benzoyl-serine, N- (p-methylbenzoyl) serine, N- (p-ethylbenzoyl) serine, N- (p-methoxybenzoyl) serine, N- (p-fluorobenzoyl) ) Serine, N- (p-trifluoromethylbenzoyl) serine, N- (2-naphthoyl) serine, N- (4-phenylbenzoyl) serine, N- (p-methylbenzoyl) serine methyl ester, N- (p -Methylbenzoyl) serine ethyl ester, N- (2-naphthoyl) serine methyl ester, N-benzoyl-O-methylserine, N- (p-methylbenzoyl) -O-methylserine, N- (p-methylbenzoyl) -O -Acetylserine, N- (2-naphthoyl) -O-methylserine and the like.
Some of these whitening components are already on the market, or they can be obtained by synthesis. For example, 3-O-ethylascorbic acid can be synthesized by a known method described in JP-A-8-134055. There are also commercially available products (“VC ethyl” manufactured by Nippon Seika Co., Ltd.), and these can be obtained and used. 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2- (triphenylmethyloxy) ethanol, 2- (triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, triphenyl Phenylmethanol, triphenylmethane, and aminodiphenylmethane are disclosed in pamphlet of Patent Document WO2010-074052, N- (o-toluoyl) cysteic acid, N- (m-toluoyl) cysteic acid, N- (p-toluoyl) cysteic acid, N -(P-methoxybenzoyl) cysteic acid, N- (4-phenylbenzoyl) cysteic acid, N- (p-toluoyl) homocysteic acid are disclosed in WO 2011-087006, N-benzoyl-serine, N- (p −Me Rubenzoyl) serine, N- (p-ethylbenzoyl) serine, N- (p-methoxybenzoyl) serine, N- (p-fluorobenzoyl) serine, N- (p-trifluoromethylbenzoyl) serine, N- ( 2-naphthoyl) serine, N- (4-phenylbenzoyl) serine, N- (p-methylbenzoyl) serine methyl ester, N- (p-methylbenzoyl) serine ethyl ester, N- (2-naphthoyl) serine methyl ester N-benzoyl-O-methylserine, N- (p-methylbenzoyl) -O-methylserine, N- (p-methylbenzoyl) -O-acetylserine, N- (2-naphthoyl) -O-methylserine, etc. Since the methods for synthesizing them are disclosed in each pamphlet It is possible to follow synthesis.
Content of the whitening component in cosmetics is 0.001-10 mass% normally, 0.01-10 mass% is preferable and 0.1-5 mass% is more preferable.

  In addition to the antibacterial agent of the present invention, a wrinkle improving component can be further contained. The wrinkle improving component is not particularly limited as long as it is generally used in cosmetics. For example, vitamin A or a derivative thereof may be retinal, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoic acid, retinoic palmitate, retinoic acetate, ursolic acid benzyl ester, ursol Examples thereof include acid phosphate ester, betulinic acid benzyl ester, and benzyl acid phosphate ester. Content of the wrinkle improvement component in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 1-5 mass% is more preferable.

  The extracts derived from animals and plants are not particularly limited as long as they are generally used for pharmaceuticals, cosmetics, foods and the like. For example, akebi extract, asunaro extract, asparagus extract, avocado extract, achacha extract, almond extract, arnica extract, aloe extract, aronia extract, apricot extract, ginkgo biloba extract, Indian mushroom extract, fennel extract, udo extract, ages extract, sorghum extract , Enmezo extract, Ogon extract, Oren extract, Ginseng extract, Hypericum extract, Odori extract, Orange extract, Oyster extract, Cuckoo extract, Chamomile extract, Carrot extract, Kawara mugwort extract, Licorice extract, Kiwi extract, Cucumber extract, Guava extract, Kujin extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, Black rice extract, Chlorella extract, Mulberry extract Cayket extract, gentian extract, gentian extract, Gennosho extract, black tea extract, burdock extract, rice extract, rice fermented extract, rice bran fermented extract, rice germ oil, bilberry extract, salvia extract, salmon extract, sasa extract, hawthorn extract, sacha Extract, Salamander extract, Shiitake extract, Giant extract, Perilla extract, Linden extract, Shimotake extract, Peonies extract, Pepper extract, Pepper root extract, Birch extract, Horsetail extract, Stevia extract, Stevia fermented product, Papaver extract, Hawthorn extract , Elderberry extract, yarrow extract, mint extract, sage extract, mallow extract, nematode extract, assembly extract, saw Kuhi extract, Daio extract, Soybean extract, Taisou extract, Thyme extract, Dandelion extract, Clove extract, Chimpi extract, Sacha tea extract, Pepper extract, Toki extract, Toki-senka extract, Tonin extract, Spruce extract, Dokudami extract, Tomato extract, Natto extract, Carrot extract , Garlic extract, Novara extract, Hibiscus extract, Bacmond extract, Lotus extract, Lotus germ extract, Parsley extract, Birch extract, Hamelis extract, Hikikoshi extract, Hinoki extract, Fukitane poppo extract, Fukinoto extract, Bukkuri extract, Butcher bloom extract, Grape extract , Grape seed extract, loofah extract, safflower extract, peppermint extract, bodaiju extract, button extract, hop extract, matsuki , Maronnier extract, Citrus extract, Mukuroji extract, Melissa extract, Mozuku extract, Peach extract, Cornflower extract, Eucalyptus extract, Yukinoshita extract, Yuzu extract, Lily extract, Yakuinin extract, Artemisia extract, Lavender extract, Apple extract, Rooibos tea extract, Ganoderma extract , Lettuce extract, lemon extract, forsythia extract, forsythia extract, rose extract, rosemary extract, roman chamomile extract, royal jelly extract, bitumen extract and the like. In addition, when the antibacterial agent selected in the selection method of the present invention is a plant extract, it may be contained in a superimposed manner with the plant extract listed above. Content of the plant and animal origin extract in cosmetics is 0.0001-30 mass% normally, 0.001-10 mass% is preferable, and 0.01-5 mass% is more preferable.

Examples of the anti-inflammatory component include clarinone, glabrizine, glycyrrhizic acid, glycyrrhetinic acid, pantothenyl alcohol, and the like, and preferably glycyrrhizic acid and its salt, glycyrrhetinic acid alkyl and its salt, and glycyrrhetic acid and its salt.
Content of the anti-inflammatory component in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 1-5 mass% is more preferable.

Examples of the oil component include polar oil and volatile hydrocarbon oil.
Polar oils include synthetic ester oils such as isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, octyldodecyl oleate, dimethyloctane Hexyldecyl acid, cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearylate, ethylene glycol di-2-ethylhexylate, dipentaerythritol fatty acid ester, N-alkylglycol monoisostearate , Neopentyl glycol dicaprate, diisostearyl malate, glycerin di-2-heptylundecanoate, tri-2-ethylhexyl It can be exemplified Le trimethylolpropane, trimethylolpropane triisostearate, tetra-2-ethylhexyl acid pentane erythritol, tri-2-ethylhexyl acid glycerin, trimethylolpropane triisostearate.

  Furthermore, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, tri-2-heptylundecanoic acid glyceride, castor oil fatty acid methyl ester, oleic acid oil, cetostearyl alcohol, oleyl alcohol, stearyl alcohol, Octyldodecanol, acetoglyceride, 2-heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L-glutamic acid-2-octyldecyl ester, di-2-heptylundecyl adipate, ethyl laurate, sebacate diacid 2-ethylhexyl, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexyl succinate, vinegar Ethyl, butyl acetate, amyl acetate, triethyl citrate, may also be mentioned octyl methoxycinnamate and the like.

  Natural oils include avocado oil, flaxseed oil, eno oil, olive oil, kayak oil, beef tallow, sesame oil, wheat germ oil, rice bran oil, sasanqua oil, safflower oil, squalane, soybean oil, tea seed oil, camellia oil, Cinnagi oil, turtle oil, rapeseed oil, corn oil, germ oil, persic oil, castor oil, jojoba oil, Japanese kiri oil, macadamia nut oil, mink oil, cottonseed oil, coconut oil, peanut oil, egg yolk oil, carnauba wax, bird Examples include glycerin, glycerin trioctanoate, and glycerin triisopalmitate.

  Examples of the hydrocarbon oil include linear or branched hydrocarbon oils, which may be volatile hydrocarbon oils or non-volatile hydrocarbon oils. Specific examples of the hydrocarbon oil include synthetic squalane, vegetable squalane, squalene, liquid isoparaffin, light isoparaffin, hydrogenated polyisobutene, isododecane, stearic acid, light liquid isoparaffin, isohexadecane, liquid paraffin, pristane, α-olefin oligomer, Examples include ozokerite, ceresin, paraffin, paraffin wax, polyethylene wax, polyethylene / polypropylene wax, (ethylene / propylene / styrene) copolymer, (butylene / propylene / styrene) copolymer, polyisobutylene, microcrystalline wax, petrolatum and the like.

Surfactants include fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, alkylsulfuric triethanolamine ether, sulfosuccinic acid esters, polyoxyethylene alkyl sodium sulfate and other anionic surfactants, stearyl chloride Cationic surfactants such as trimethylammonium, benzalkonium chloride, laurylamine oxide, dialkylammonium salts, betaine surfactants (alkyl betaines, amide betaines, sulfobetaines, etc.), imidazoline amphoteric surfactants (2-cocoyl) -2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt), amphoteric surfactants such as acylmethyltaurine, sorbitan fatty acid esters (sorbitan monostearate) , Sorbitan sesquioleate, etc.), glycerin fatty acids (such as glyceryl monostearate), propylene glycol fatty acid esters (such as propylene glycol monostearate), hardened castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters ( POE sorbitan monooleate, polysoxyethylene sorbitan monostearate, etc.), POE sorbite fatty acid esters (POE-sorbite monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid ester (Polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE2-
Octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE / POP alkyl ethers (POE / POP2-decyltetradecyl ether, etc.), Tetronics, POE castor oil / cured Castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), nonionic surfactants such as sucrose fatty acid ester, alkyl glucoside, and the like.

  Polyhydric alcohols include polyethylene glycol, glycerin, 1,3-butanediol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4 -Hexylene glycol, 1,2-hexanediol, 1,2-octanediol and the like.

  Examples of thickeners include guar gum, quince seed, carrageenan, galactan, gum arabic, pectin, mannan, starch, xanthan gum, curdlan, methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, methylhydroxypropylcellulose, chondroitin sulfate, dermatan sulfate, glycogen, Heparan sulfate, hyaluronic acid, sodium hyaluronate, tragacanth gum, keratan sulfate, chondroitin, mucoitin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, kerato sulfate, locust bean gum, succinoglucan, caronic acid, chitin, chitosan, carboxymethyl Chitin, agar, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, al Le-modified carboxyvinyl polymers, sodium polyacrylate, polyethylene glycol, and bentonite.

  As powders, the surface may be treated, mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, etc. May be treated, bengara, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine, bitumen, titanium oxide, zinc oxide inorganic pigments, surface may be treated, titanium mica, fish phosphorus foil, Pearl agent such as bismuth oxychloride, red 202, red 228, red 226, yellow 4, blue 404, yellow 5, red 505, red 230, red 223 which may be raked No., Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204, organic dyes, polyethylene powder, polymeta Methyl acrylic acid, nylon powder, organic powders such as organopolysiloxane elastomers, and the like.

Examples of the UV absorber include paraaminobenzoic acid UV absorbers, anthranilic acid UV absorbers, salicylic acid UV absorbers, cinnamic acid UV absorbers, benzophenone UV absorbers, sugar UV absorbers, 2- (2 UV absorbers such as'-hydroxy-5'-t-octylphenyl) benzotriazole, 4-methoxy-4'-t-butyldibenzoylmethane, and the like.

  Moreover, the dosage form in the case of applying as cosmetics can take any of the conventionally known lotion dosage forms, emulsion dosage forms, essence dosage forms, cream dosage forms, and powder-containing dosage forms.

The skin external preparation which concerns on this invention can be prepared by processing the antibacterial agent of this invention, and arbitrary components by a conventional method.
Hereinafter, the present invention will be described in detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.

(Production Example 1: Production of external preparation for skin containing antibacterial agent of the present invention (lotion))
According to the following procedure, an external preparation for skin containing an antibacterial agent was prepared.
That is, the prescription components in Table 1 were dissolved by heating at 50 ° C. to obtain a skin external preparation 1 according to the present invention.

(Production Example 2: Production of an external preparation for skin containing the antibacterial agent of the present invention (essence))
According to the following procedure, an external preparation for skin containing an antibacterial agent was prepared.
That is, the prescription components in Table 1 were dissolved by heating at 50 ° C. to obtain a skin external preparation 2 according to the present invention.

(Test Example 1 Measurement of hBD-3 mRNA expression level)
Normal human epidermal keratinocytes were seeded in a 24-well plate at 6.0 × 10 ⁴ cells / well, and cultured at 37 ° C. and 5% CO ₂ in 0.15 mM-Ca-containing medium (humedia-KG2, Kurashiki Spinning Co., Ltd.). Cultured under. After 3 days of culture, the medium was replaced with 1.45 mM-Ca-containing Humedia-KG2 medium excluding phenol red, BPE, and cortisol. Progesterone and sicon extract were added (α) or only progesterone was added (β), and the cells were further cultured for 3 days. After completion of the culture, cells were collected, total RNA was extracted using RNeasy Mini Kit (QIAGEN), and cDNA was synthesized from the obtained total RNA using SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR was performed using QuantiFast SYBR Green PCR kit (QIAGEN) using the synthesized cDNA as a template, and the expression level of hBD-3 mRNA was relatively quantified by a calibration curve method. At this time, 18S rRNA was used as an endogenous control, and the initial gene dosage was corrected. Further, as a control, the hBD-3 mRNA expression level in the case where neither progesterone nor chicone extract was added was measured, and the degree of improvement in hBD-3 mRNA expression level decrease by progesterone was evaluated from the following formula (1). The results are shown in Table 3 and FIG.
Formula (1) (α-β) / (γ-β) × 100 (%)
α is the hBD-3 mRNA expression level when progesterone and the test substance are added β is the hBD-3 mRNA expression level when progesterone is added γ is the hBD-3 mRNA expression level when the progesterone and test substance are not added

コントロールのｈＢＤ−３ｍRNA発現量を１００としたときのα、βの相対量を示した。

The relative amounts of α and β when the control hBD-3 mRNA expression level is 100 are shown.

From the results shown in FIG. 1 and Table 3, it was confirmed that the addition of progesterone decreases the expression level of hBD-3 mRNA, but that the sicon extract has the effect of suppressing the decrease in hBD-3 mRNA caused by the progesterone.

(Test Example 2 Measurement of P.acnes viable count)
P. acnes (ATCC6919) was cultured in a GAM liquid medium (Nissui Pharmaceutical) for 3 days. P. acnes suspended in 20 mM phosphate buffer (pH 6.5, containing 100 nM NaCl) to 1 × 10 ⁶ CFU / mL is seeded in a 96-well plate, and hBD-1 and hBD-2 are added thereto. , HBD-3 and hBD-4 were added to a final concentration of 10 μg / mL, respectively. After incubating at 37 ° C. under anaerobic conditions for 5 hours, the culture solution was inoculated on a GAM agar medium (Nissui Pharmaceutical) and cultured at 37 ° C. under anaerobic conditions for 3 days. The viable count of acnes was evaluated. The results are shown in FIG.

From the results shown in FIG. 2, the antibacterial peptides hBD-1, hBD-2, and hBD-4 did not show an antibacterial activity against P. acnes, which is an acne bacterium, but hBD-3 is a positive control, benzalkco chloride. A strong antibacterial action against P. acnes, an acne bacterium, was observed to the same extent as that of nium.

ADVANTAGE OF THE INVENTION According to this invention, the antibacterial agent which improves the fall of hBD-3 mRNA expression level can be provided.

Claims (4)

The composition for acne improvement in the progesterone rising period in the menstrual cycle which contains a sicon extract as an active ingredient. The composition according to claim 1, wherein the acne improvement is due to suppression of hBD-3 expression reduction.   The composition according to claim 1 or 2, which is an external preparation for skin.   The composition according to claim 3, which is a cosmetic or a quasi drug.

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