WO2019139307A1 - Composition for skin care comprising flocculin - Google Patents

Composition for skin care comprising flocculin Download PDF

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WO2019139307A1
WO2019139307A1 PCT/KR2019/000184 KR2019000184W WO2019139307A1 WO 2019139307 A1 WO2019139307 A1 WO 2019139307A1 KR 2019000184 W KR2019000184 W KR 2019000184W WO 2019139307 A1 WO2019139307 A1 WO 2019139307A1
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skin
composition
saccharomyces
flocculin
usa
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PCT/KR2019/000184
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French (fr)
Korean (ko)
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유정진
김민지
김하영
강승현
김연준
박명삼
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코스맥스 주식회사
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Priority to CN201980000340.5A priority Critical patent/CN110267643B/en
Publication of WO2019139307A1 publication Critical patent/WO2019139307A1/en

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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17Amino acids, peptides or proteins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06Fungi, e.g. yeasts
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/178Lectin superfamily, e.g. selectins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64Proteins; Peptides; Derivatives or degradation products thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728Fungi, e.g. yeasts
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/08Anti-ageing preparations
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00Function of food ingredients
    • A23V2200/30Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat

Definitions

  • composition for skin care To a composition for skin care.
  • Flocculin is a lectin-like protein in the Lager yeast cell wall, which serves to precipitate lager yeast.
  • it has a strong binding property with mannose, binds with mannose of other yeast walls, produces precipitates of yeast cells, and also has a function of protecting yeast from stress factors such as nutrient depletion, temperature and UV in the culture.
  • yeast is used as a cosmetic ingredient for the purpose of skin improvement such as wrinkle improvement and whitening effect.
  • the yeast extract is used as a cosmetic ingredient by removing yeast cells from the cell wall by proteolytic enzymes, organic solvents or autolysis, and then removing the insoluble portions and concentrating them.
  • the thus-treated yeast extract is composed of a complex of various proteins, peptides, amino acids, minerals and vitamin B groups and is useful as a cosmetic additive.
  • Other yeast extracts are known in skin wound healing and collagen synthesis effects (US Patent 5,776,441, US Patent 6,177,105) and are used as cosmetic raw materials. However, to date, there has been no use of flocurin in cosmetic compositions for skin care purposes.
  • One aspect provides a cosmetic composition for skin care comprising floculline.
  • Another aspect provides a composition for a health functional food for skin cosmetics comprising flocurin.
  • Another aspect provides a pharmaceutical composition for the cosmetic of the skin comprising flocucrin.
  • Another aspect provides a method of treating or ameliorating skin damage or aging comprising administering to an individual an effective amount of a composition comprising flocurin.
  • Another aspect provides the use of a composition comprising flocurin for the manufacture of a skin damaging or aging therapeutic agent.
  • One aspect provides a cosmetic composition for skin care comprising floculline. Another aspect provides a composition for a health functional food for skin cosmetics comprising flocurin. Another aspect provides a pharmaceutical composition for preventing or treating skin damage, or skin aging, comprising flocurin. Another aspect provides a composition for external application for skin cosmetics.
  • Flocculin generally refers to the lectin-like protein in the cell wall that is expressed in microorganisms of the genus Saccharomyces. Marika H. (1994) Purification and partial characterization of a flocculin from brewer's yeast ), Or can be obtained commercially by isolating it from microorganisms of the genus Saccharomyces according to a known method.
  • the composition may further comprise a culture or an extract of the microorganism of the genus Saccharomyces.
  • cultures or extracts can be readily obtained by a person skilled in the art according to methods known in the art.
  • culture of microorganisms can be carried out in a commercially available medium such as LB medium, Ham's F10 (Sigma), MEM (Minimal Essential Medium, Sigma), RPMI-1640 (Sigma), and DMEM (Dulbecco's Modified Eagle's Medium, Sigma) Etc. may be used.
  • LB medium such as Ham's F10 (Sigma), MEM (Minimal Essential Medium, Sigma), RPMI-1640 (Sigma), and DMEM (Dulbecco's Modified Eagle's Medium, Sigma) Etc.
  • hormones or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements and glucose may be supplemented with known concentrations of known agents.
  • the culture conditions for example, temperature, pH and the like, can be appropriately determined by those skilled in the art depending on the selected microorganism.
  • the extract may be extracted with water, acetone, alcohol, for example, C1-C6 alcohol, or a mixture thereof as a solvent.
  • the C1-C6 alcohol may be methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, or hexanol.
  • the solvent may be, for example, a mixture of water and an alcohol, that is, an aqueous solution of an alcohol.
  • the alcohol concentration of the alcohol aqueous solution may be 1 to 100 (v / v)%, for example, 1 to 99.5 (v / v)%, 10 to 100 (v / (V / v)%, 40 to 100 (v / v)%, 50 to 100 (v / v)%, 60 to 100 (v / (V / v)%, 60 to 90 (v / v)%, 60 to 80 (v / v)%, 65 to 75 have.
  • the aqueous alcohol solution may be methanol, ethanol, or an aqueous butanol solution.
  • the composition may further comprise a fraction of the extract.
  • fraction refers to a substance, i.e., a fractioned substance, in which the extract is divided into its components.
  • the fraction may be obtained by solvent fractionation.
  • the solvent fractionation may refer to mixing the extract with a solvent and separating the substance present in the solvent.
  • skin aesthetics refers to returning a skin damaged by skin damage or aging of the skin to a condition prior to damage, such as by wrinkles, volume enhancement, elasticity improvement, or skin regeneration.
  • the skin includes all parts of the body including the face, hands, arms, legs, feet, chest, belly, back, buttocks, and scalp.
  • skin damage refers to the damage caused by external physical damage, penetration of chemicals, bacteria, fungi, viruses, exposure to ultraviolet rays, skin moisture loss, wrinkles due to aging, oxidation by free radicals And include damage from a clinical and cosmetic point of view of skin tissue or cells, including skin, skin inflammation, seborrheic dermatitis, redness (erythema, erythema), edema, psoriasis, eczema, pruritus (itching), and atopic dermatitis .
  • the term "skin aging” includes endogenous aging with time and extrinsic aging by external environment.
  • the aging of the skin may include skin wrinkles, dullness, stain and the like.
  • the wrinkles of the skin may be reduced due to the degeneration of the skin.
  • Skin wrinkles can be due to photoaging, age, facial expression, lack of moisture, or a combination thereof.
  • the photoaging may be skin aging by exposure to ultraviolet light (including UVA, UVB, and UVC).
  • the skin wrinkle improvement may inhibit or inhibit the generation of wrinkles on the skin, or may alleviate the wrinkles already generated.
  • the composition may be formulated into a parenteral dosage form.
  • the parenteral dosage form may be an injection or an external preparation for skin.
  • the external preparation for skin may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof.
  • the external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, or a combination thereof, and may be suitably blended as necessary.
  • the external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Saccharides such as trehalose can also be appropriately compounded.
  • a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose
  • the cosmetic composition may be a cosmetic composition (skin lotion), a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream,
  • the composition may be formulated into a formulation comprising an active ingredient, an essence, a nutritional essence, a pack, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a suspension, a gel, a powder, a paste, .
  • Compositions of such formulations may be prepared according to methods conventional in the art.
  • the cosmetic composition may contain at least one selected from the group consisting of preservatives, stabilizers, surfactants, thickeners, solubilizers, moisturizers, emollients, ultraviolet absorbers, preservatives, bactericides, emulsifiers, antioxidants, pH adjusting agents, organic and inorganic pigments, And may further contain additives commonly used in the art.
  • the amount of the additional ingredient such as the preservative may be easily selected by a person skilled in the art within the range not impairing the object and effect of the present invention, and the amount thereof may be 0.001 to 40% by weight based on the total weight of the composition.
  • the pharmaceutical composition may contain the effective amount or the effective component of the flocululin.
  • the effective amount may be appropriately selected depending on the individual.
  • the severity of the disease, the age, body weight, health, sex, sensitivity of the patient to the drug, time of administration, route of administration and rate of release, duration of treatment, factors involved in the combination or concurrent use with the composition Can be determined according to well-known factors.
  • Administration can be by any method known in the art. Administration may be by any means directly administered to a subject, such as by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration, .
  • the administration can be systemically or locally administered.
  • the subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.
  • the subject may be an individual in need of skin aesthetic improvement, for example skin moisturizing, barrier enhancement, or skin regeneration effect.
  • Such administration may comprise from 0.1 mg to 1000 mg, such as from 0.1 mg to 500 mg, from 0.1 mg to 100 mg, from 0.1 mg to 50 mg, from 0.1 mg to 25 mg, or from 0.1 mg to 100 mg per day of a composition for skin care comprising floculline , 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg Or 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg.
  • a composition for skin care comprising floculline , 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg Or 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg,
  • the dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, These factors can be taken into account to appropriately adjust the dosage.
  • the number of doses may be at least once per day or within the range of clinically acceptable side effects and may be administered at one or two or more doses at the site of administration and may be administered daily or every two to five days The number of days of administration can be administered from one day to 30 days at one time of treatment. If necessary, the same treatment may be repeated after the appropriate time.
  • the dosage may be the same as that of a human per kg, or the dosage may be converted into a dose in terms of a volume ratio (for example, an average value) One dose may be administered.
  • the composition When the composition is a composition for a health functional food, it can be formulated into a typical health functional food formulation known in the art.
  • the health functional food may be prepared by conventional formulations such as powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups, Beverages, tea, drinks, alcoholic beverages, and vitamins, such as, for example, beverages, snacks, confectionery, pizza, ramen, other noodles, gums, jellies, ice creams,
  • a pharmaceutically acceptable carrier or additive may be used for the formulation of the health food, and any carrier or additive known to be usable in the art for the preparation of the formulation to be prepared may be used.
  • additives there can be used various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Carbonating agents, and the like.
  • it may contain natural fruit juice, fruit juice beverage and flesh for the production of vegetable beverages.
  • These additive components can be used independently or in combination, and the proportion of the additive is not critical, but can be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition.
  • the content of the extract of floculin in the composition for health functional foods can be suitably determined according to the intended use (prevention or improvement). Generally, it may contain 0.01 to 15% by weight of the total food, and when it is prepared as a beverage, it may contain 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 mL.
  • the present inventors have found that, in the experiment using the composition containing the flocucrin, it is possible to improve the expression of pilar green, the expression of clodin 1, the expression of hyaluronic acid synthase, the expression of exfoliating enzyme, Melanin production inhibitory effect was confirmed. Therefore, a composition containing flocucurin can be usefully used for the treatment of skin cosmetics or skin-related diseases.
  • the composition containing flocurin exhibits skin barrier improvement, moisturizing, whitening, cell proliferation effect, etc., and promotes collagen synthesis and elastin synthesis, so that it can be used for skin beauty.
  • FIG. 1 is a graph showing the effect of increasing fila green expression of flocucrin in HaCat cells.
  • FIG. 2 is a graph showing the effect of increasing the expression of claudin 1 of flocucrin in HaCat cells.
  • FIG. 3 is a graph showing the effect of increasing HAS3 expression of flocucrin in HaCat cells.
  • 5 is a graph showing the effect of inhibiting the production of melanin of flocucrin in HaCat cells.
  • Example One Floculine Ready
  • Sakaromayses Paz Astoria Augustine (Saccharomyces Pastorianus ) was cultured according to the method of Marika H. (1994, Purification and partial characterization of a flocculin from brewer's yeast), and a solution containing flocucurin was isolated.
  • the separated flocucrin solution was prepared by powder freezing at -70 ° C and 5.0 Torr.
  • Sakaromayses Waves Story Janus were cultured according to the method of Marika H. (1994.Purification and partial characterization of a flocculin from brewer's yeast).
  • the yeast culture was centrifuged at 3500 rpm, and the supernatant was filtered with a 0.45 ⁇ m membrane filtration membrane.
  • the sterilized yeast culture was lyophilized at -70 ° C and 5.0 Torr to produce powder.
  • HaCat a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
  • ATCC American Type Culture Collection
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • DMEM Hyclone, Logan, UT, USA
  • human keratinocytes After the cells inoculated with 5 x 10 5 cells / well density in 6-well cell culture plate, with 10% of FBS with 1% AA added medium And cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. To the cultured cells, Comparative Example 1 or Example 1 was added, followed by culturing for 24 hours. Cells were recovered and RNA was isolated by adding 1 ml of trizol (RNA iso, TAKARA, Japan).
  • RT-PCR was performed using a PCR machine (Step One Plus, Applied Biosystems, USA) and CyberGreen supermix (Applied Biosystems, USA) Polymerization was carried out at 95 ° C for 30 seconds, 54 ° C for 1 minute, and 72 ° C for 1 minute for 40 cycles.
  • the nucleotide sequences of the primers are shown in Table 1 below.
  • Example 1 As a result, it was observed that the pilar green expression was improved 2.84 times in the Example 1 addition group compared to the no treatment group (Fig. 1). Thus, the skin barrier enhancement effect of the composition of Example 1 can be expected.
  • Pillar green primer sequence Name of the primer order Pillar Green Forward 5'-GGCACTGAAAGGCAAAAAGG-3 '(SEQ ID NO: 1) Reverse 5'-AAACCCGGATTCACCATAATCA-3 '(SEQ ID NO: 2) beta -actin Forward 5'-GGCCATCTCTTGCTCGAAGT-3 '(SEQ ID NO: 3) Reverse 5'-GACACCTTCAACACCCCAGC-3 '(SEQ ID NO: 4)
  • HaCat a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
  • ATCC American Type Culture Collection
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • DMEM Hyclone, Logan, UT, USA
  • RT-PCR was carried out using a PCR machine (Step One Plus, Applied Biosystems, USA) and CyberGreen supermix (Applied Biosystems, USA) with Claudin 1 primer and cDNA for 2 min at 50 ° C, At 95 ° C for 10 seconds and at 60 ° C for 1 minute for 40 cycles.
  • the nucleotide sequences of the primers are shown in Table 2 below.
  • the expression level of the gene was finally analyzed by correction for the ⁇ -actin gene.
  • HaCat a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
  • ATCC American Type Culture Collection
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • DMEM Hyclone, Logan, UT, USA
  • RT-PCR was performed using PCR machine (Step One Plus, Applied Biosystems, USA) and SYBR Green supermix (Applied Biosystems, USA) was added with HAS3 primer and cDNA to activate polymerase for 5 min at 94 ° C, 95 Deg.] C for 30 seconds, 54 [deg.] C for 1 minute, and 72 [deg.] C for 1 minute.
  • the nucleotide sequences of the primers are shown in Table 3 below.
  • the expression level of the gene was finally analyzed by correction for the ⁇ -actin gene.
  • Example 1 As a result, it was confirmed that flocoolin of lager yeast (Example 1) expressed a larger amount of hyaluronic acid synthase than the conventional yeast culture (Comparative Example 1) (Fig. 3). Therefore, since the increased hyaluronic acid synthase can enhance the water-trapping ability in the skin, excellent skin moisturizing effect of the composition of Example 1 can be expected.
  • HaCat a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
  • ATCC American Type Culture Collection
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • DMEM Hyclone, Logan, UT, USA
  • RT-PCR was performed using a PCR machine (Step One Plus, Applied Biosystems, USA) and CyGreen supermix (Applied Biosystems, USA) was added with KLK7 primer and cDNA for 2 min at 50 ° C, 10 min at 95 ° C After the reaction, polymerization was carried out at 95 ° C for 10 seconds and 40 ° C for 1 minute at 60 ° C.
  • the base sequences of the primers are shown in Table 4 below. The expression level of the gene was finally analyzed by correction for the ⁇ -actin gene.
  • Example 1 It was confirmed that flocucurin of lager yeast (Example 1) expressed a greater amount of keratolytic enzyme (KLK7) compared to the general yeast culture (Comparative Example 1) (Fig. 4). Therefore, a smooth and bright skin tone correction effect of the composition of Example 1 can be expected.
  • B16 F10 cells a murine melanoma
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • 0.1 ⁇ M ⁇ -MSH was added, and then added for each sample, followed by culturing for 3 days.
  • arbutin was treated at 100 ppm.
  • the cultured cells were washed with PBS and trypsin.
  • the recovered cells were counted with a hematocytometer and collected at 1 ⁇ 10 6 cells / mL for each treatment group, and centrifuged at 1000 rpm for 10 minutes to obtain cells.
  • the recovered cells were dried at 60 ° C for 1 hour, and 100 ⁇ l of a 1 M NaOH solution containing 10% DMSO was added to obtain melanin in the cells. This melanin solution was assayed for absorbance at 490 nm with a microplate reader to evaluate the inhibition of melanin formation.
  • Example 1 As a result, it was confirmed that flocoolin of lager yeast (Example 1) inhibited melanin production more than the conventional yeast culture (Comparative Example 1) (FIG. 5). Thus, the composition of Example 1 can have an excellent whitening effect by inhibiting the production of melanin in skin cells.

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Abstract

One aspect provides a composition for skin care comprising flocculin. The composition comprising flocculin exhibits the effects of an improvement of the skin barrier, moisturizing, whitening, cell proliferation, etc., and promotes collagen synthesis and elastin synthesis, thereby being able to be used for skin care.

Description

플록쿨린을 포함하는 피부 미용을 위한 조성물Composition for skin care comprising flocurin
피부 미용을 위한 조성물에 관한 것이다.To a composition for skin care.
플록쿨린(Flocculin)은 라거 효모(Lager yeast) 세포벽의 렉틴 유사 단백질(lectin like protein)로, 라거 효모를 침전시키는 역할을 한다. 특히 만노오스와 결합력이 우수하여, 다른 효모벽의 만노오스와 결합하고 효모세포의 침전물을 생성시키기도 하며, 배양 중의 영양 고갈, 온도, UV와 같은 스트레스 인자로부터 효모를 보호하는 기능도 가지고 있다. Flocculin is a lectin-like protein in the Lager yeast cell wall, which serves to precipitate lager yeast. In particular, it has a strong binding property with mannose, binds with mannose of other yeast walls, produces precipitates of yeast cells, and also has a function of protecting yeast from stress factors such as nutrient depletion, temperature and UV in the culture.
한편, 효모는 화장품 성분으로서 주름개선, 미백효과 등 피부 개선을 목적으로 사용되고 있다. 효모 추출액은 효모 세포를 원료로 단백질 분해 효소, 유기 용매 혹은 자가분해에 의해 세포벽을 파쇄시킨 후, 불용성 부분을 제거하고 농축시켜 화장품 성분으로서 이용된다. 이렇게 처리된 효모 추출물은 각종 단백질, 펩타이드, 아미노산, 미네랄 및 비타민 B군의 복합물로 구성되어 화장품 첨가물로서 유용하다. 그 밖에 효모추출물의 경우 피부 상처 치료 및 콜라겐 합성효과(US Patent 5,776,441, US Patent 6,177,105)에 알려져 있으며 여러 화장품 원료로 사용되고 있다. 하지만 현재까지 플록쿨린을 피부 미용 목적으로 화장료 조성물에 이용한 사례는 찾아볼 수 없다.On the other hand, yeast is used as a cosmetic ingredient for the purpose of skin improvement such as wrinkle improvement and whitening effect. The yeast extract is used as a cosmetic ingredient by removing yeast cells from the cell wall by proteolytic enzymes, organic solvents or autolysis, and then removing the insoluble portions and concentrating them. The thus-treated yeast extract is composed of a complex of various proteins, peptides, amino acids, minerals and vitamin B groups and is useful as a cosmetic additive. Other yeast extracts are known in skin wound healing and collagen synthesis effects (US Patent 5,776,441, US Patent 6,177,105) and are used as cosmetic raw materials. However, to date, there has been no use of flocurin in cosmetic compositions for skin care purposes.
일 양상은 플록쿨린을 포함하는 피부 미용을 위한 화장료 조성물을 제공한다.One aspect provides a cosmetic composition for skin care comprising floculline.
다른 양상은 플록쿨린을 포함하는 피부 미용을 위한 건강기능식품용 조성물을 제공한다.Another aspect provides a composition for a health functional food for skin cosmetics comprising flocurin.
다른 양상은 플록쿨린을 포함하는 피부 미용을 위한 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for the cosmetic of the skin comprising flocucrin.
다른 양상은 플록쿨린을 포함하는 조성물의 유효량을 개체에 투여하는 단계를 포함하는 피부 손상 또는 노화를 치료 또는 개선시키는 방법을 제공한다.Another aspect provides a method of treating or ameliorating skin damage or aging comprising administering to an individual an effective amount of a composition comprising flocurin.
다른 양상은 피부 손상 또는 노화 치료제의 제조를 위한, 플록쿨린을 포함하는 조성물의 용도를 제공한다.Another aspect provides the use of a composition comprising flocurin for the manufacture of a skin damaging or aging therapeutic agent.
일 양상은 플록쿨린을 포함하는 피부 미용을 위한 화장료 조성물을 제공한다. 다른 양상은 플록쿨린을 포함하는 피부 미용을 위한 건강기능식품용 조성물을 제공한다. 다른 양상은 플록쿨린을 포함하는 피부 손상, 또는 피부 노화를 예방 또는 치료하기 위한 약학적 조성물을 제공한다. 또 다른 양상은 피부 미용을 위한 피부 외용제 조성물을 제공한다. One aspect provides a cosmetic composition for skin care comprising floculline. Another aspect provides a composition for a health functional food for skin cosmetics comprising flocurin. Another aspect provides a pharmaceutical composition for preventing or treating skin damage, or skin aging, comprising flocurin. Another aspect provides a composition for external application for skin cosmetics.
"플록쿨린(Flocculin)"은 일반적으로 사카로마이세스 속 미생물에서 발현되는 세포벽의 렉틴 유사 단백질(lectin like protein)을 의미하는 것으로, Marika H.(1994. Purification and partial characterization of a flocculin from brewer's yeast) 등의 문헌에서 알려진 방법에 따라 사카로마이세스 속 미생물로부터 분리하거나, 상업적으로 구매하여 얻을 수 있다. 구체적으로 상기 사카로마이세스 속 미생물로 알려진 것들은 사카로마이세스 바야누스(Saccharomyces bayanus), 사카로마이세스 보울라디(Saccharomyces boulardii), 사카로마이세스 불데리(Saccharomyces bulderi), 사카로마이세스 카리오카누스(Saccharomyces cariocanus), 사카로마이세스 카리오커스(Saccharomyces cariocus), 사카로마이세스 세레비지에(Saccharomyces cerevisiae), 사카로마이세스 시바리에리(Saccharomyces chevalieri), 사카로마이세스 다이레넨시스(Saccharomyces dairenensis), 사카로마이세스 엘립소이더스(Saccharomyces ellipsoideus), 사카로마이세스 유바야누스(Saccharomyces eubayanus), 사카로마이세스 엑시구스(Saccharomyces exiguus), 사카로마이세스 플로렌티누스(Saccharomyces florentinus), 사카로마이세스 클루이베리(Saccharomyces kluyveri), 사카로마이세스 마르티니에(Saccharomyces martiniae), 사카로마이세스 모나센시스(Saccharomyces monacensis), 사카로마이세스 노르벤시스(Saccharomyces norbensis), 사카로마이세스 파라독수스(Saccharomyces paradoxus), 사카로마이세스 파스토리아누스(Saccharomyces pastorianus), 사카로마이세스 스펜세로럼(Saccharomyces spencerorum), 사카로마이세스 츄리센시스(Saccharomyces turicensis), 사카로마이세스 유니스포러스(Saccharomyces unisporus), 사카로마이세스 우바럼(Saccharomyces uvarum), 및 사카로마이세스 조나투스(Saccharomyces zonatus) 등이 있다. 알려진 플록쿨린의 아미노산 서열은, 예를 들면, NCBI Accession No: AIC64115.1, AFJ20718.1, BAG49462.1, 또는 ABS87371.1 등을 포함한다."Flocculin" generally refers to the lectin-like protein in the cell wall that is expressed in microorganisms of the genus Saccharomyces. Marika H. (1994) Purification and partial characterization of a flocculin from brewer's yeast ), Or can be obtained commercially by isolating it from microorganisms of the genus Saccharomyces according to a known method. Specifically, those known as microorganisms of the genus Saccharomyces are Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces cerevisiae, Saccharomyces < / RTI > < RTI ID = 0.0 > florentinus, < / RTI > Saccharomyces < RTI ID = 0.0 > exiguus, < / RTI & ), Saccharomyces kluyveri, Saccharomyces martiniae, Saccharomyces cerevisiae, But are not limited to, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spensorum, Saccharomyces spp., Saccharomyces spiculorum, Saccharomyces spicensis, Saccharomyces unisporus, Saccharomyces uvarum, and Saccharomyces zonatus. . The amino acid sequence of a known flocucurin includes, for example, NCBI Accession No: AIC64115.1, AFJ20718.1, BAG49462.1, or ABS87371.1 and the like.
상기 조성물은, 상기 사카로마이세스 속 미생물의 배양물 또는 추출물을 더 포함할 수 있다. 상기 배양물 또는 추출물은 당해 분야에 알려진 방법에 따라 통상의 기술자가 쉽게 얻을 수 있다. 예를 들어, 미생물의 배양은 시판되는 배지, 예를 들면 LB 배지, Ham's F10 (Sigma), MEM (Minimal Essential Medium, Sigma), RPMI-1640 (Sigma), 및 DMEM (Dulbecco's Modified Eagle's Medium, Sigma)등이 이용될 수 있다. 필요한 경우, 호르몬 또는 기타 성장 인자, 염, 완충제, 뉴클레오티드, 항생제, 미량 원소 및 글루코오스 등이 공지된 적합한 농도로 보충될 수 있다. 배양 조건, 예를 들어 온도, pH 등은 선택된 미생물에 따라 당업자에 의해 적절하게 결정될 수 있다. 또한, 상기 추출물은 물, 아세톤, 알콜, 예를 들면, C1-C6 알콜, 또는 이들의 혼합물을 용매로 이용하여 추출한 것일 수 있다. 상기 C1-C6 알콜은 메탄올, 에탄올, 프로판올, 이소프로판올, 1,3-프로판디올, 부탄올, 펜탄올, 또는 헥산올 등일 수 있다. 상기 용매는 예를 들면, 물과 알콜의 혼합물 즉 알콜 수용액일 수 있다. 알콜 수용액의 알콜 농도는 1 내지 100(v/v)%, 예를 들면, 1 내지 99.5(v/v)%, 10 내지 100(v/v)%, 20 내지 100(v/v)%, 30 내지 100(v/v)%, 40 내지 100(v/v)%, 50 내지 100(v/v)%, 60 내지 100(v/v)%, 70 내지 100(v/v)%, 75 내지 100(v/v)%, 60 내지 90(v/v)%, 60 내지 80(v/v)%, 65 내지 75(v/v)%, 또는 70(v/v)%일 수 있다. 상기 알콜 수용액은 메탄올, 에탄올, 또는 부탄올 수용액일 수 있다. 상기 조성물은 상기 추출물의 분획물을 더 포함할 수 있다. 용어 "분획물(fraction)"은 추출물이 그 일부의 성분으로 나누어진 물질, 즉 분획된 물질을 나타낸다. 상기 분획물은 용매 분획화 (fractionation)에 의하여 얻어진 것일 수 있다. 상기 용매 분획화는 추출물을 용매와 혼합하고 상기 용매에 존재하는 물질을 분리하는 단계를 지칭하는 것일 수 있다.The composition may further comprise a culture or an extract of the microorganism of the genus Saccharomyces. Such cultures or extracts can be readily obtained by a person skilled in the art according to methods known in the art. For example, culture of microorganisms can be carried out in a commercially available medium such as LB medium, Ham's F10 (Sigma), MEM (Minimal Essential Medium, Sigma), RPMI-1640 (Sigma), and DMEM (Dulbecco's Modified Eagle's Medium, Sigma) Etc. may be used. If desired, hormones or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements and glucose may be supplemented with known concentrations of known agents. The culture conditions, for example, temperature, pH and the like, can be appropriately determined by those skilled in the art depending on the selected microorganism. In addition, the extract may be extracted with water, acetone, alcohol, for example, C1-C6 alcohol, or a mixture thereof as a solvent. The C1-C6 alcohol may be methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, or hexanol. The solvent may be, for example, a mixture of water and an alcohol, that is, an aqueous solution of an alcohol. The alcohol concentration of the alcohol aqueous solution may be 1 to 100 (v / v)%, for example, 1 to 99.5 (v / v)%, 10 to 100 (v / (V / v)%, 40 to 100 (v / v)%, 50 to 100 (v / v)%, 60 to 100 (v / (V / v)%, 60 to 90 (v / v)%, 60 to 80 (v / v)%, 65 to 75 have. The aqueous alcohol solution may be methanol, ethanol, or an aqueous butanol solution. The composition may further comprise a fraction of the extract. The term "fraction" refers to a substance, i.e., a fractioned substance, in which the extract is divided into its components. The fraction may be obtained by solvent fractionation. The solvent fractionation may refer to mixing the extract with a solvent and separating the substance present in the solvent.
용어 "피부 미용"은 피부 손상 또는 피부 노화로 인해 손상된 피부를 피부 주름 개선, 볼륨 강화, 탄력 개선, 또는 피부 재생 등의 방식으로 손상되기 이전의 상태로 되돌리는 것을 의미한다. 상기 피부는 얼굴, 손, 팔, 다리, 발, 가슴, 배, 등, 엉덩이, 및 두피를 포함하는 신체의 모든 피부 부위를 포함한다. The term "skin aesthetics" refers to returning a skin damaged by skin damage or aging of the skin to a condition prior to damage, such as by wrinkles, volume enhancement, elasticity improvement, or skin regeneration. The skin includes all parts of the body including the face, hands, arms, legs, feet, chest, belly, back, buttocks, and scalp.
용어 "피부 손상"은 외부의 물리적 손상, 화학 물질, 세균, 곰팡이, 바이러스 등의 침투, 자외선에 대한 노출, 피부의 수분 손실, 노화 등에 의한 주름, 자유라디칼(활성산소) 등에 의한 산화, 피부 색소 침착, 피부 염증, 지루성 피부염, 붉어짐(발적, 홍반), 부종, 태선화, 습진, 소양감(가려움), 및 아토피 피부염 등을 포함하는 피부 조직 또는 세포의 임상적 및 미용적 관점에서의 손상을 포함한다. The term "skin damage" refers to the damage caused by external physical damage, penetration of chemicals, bacteria, fungi, viruses, exposure to ultraviolet rays, skin moisture loss, wrinkles due to aging, oxidation by free radicals And include damage from a clinical and cosmetic point of view of skin tissue or cells, including skin, skin inflammation, seborrheic dermatitis, redness (erythema, erythema), edema, psoriasis, eczema, pruritus (itching), and atopic dermatitis .
용어 "피부 노화"는 시간의 흐름에 따른 내인성 노화 및 외부 환경에 의한 외인성 노화를 포함한다. 상기 피부 노화는 피부 주름, 잡티, 기미 등을 포함할 수 있다. 상기 피부 주름은 피부가 쇠하여 생긴 잔줄일 수 있다. 피부 주름은 광노화, 연령, 얼굴 표정, 수분 부족, 또는 이들의 조합에 의한 것일 수 있다. 상기 광노화는 자외선(UVA, UVB, 및 UVC 포함) 노출에 의한 피부 노화일 수 있다. 상기 피부 주름 개선은 피부에 주름이 생성되는 것을 억제 또는 저해하거나, 이미 생성된 주름을 완화시키는 것일 수 있다. The term "skin aging" includes endogenous aging with time and extrinsic aging by external environment. The aging of the skin may include skin wrinkles, dullness, stain and the like. The wrinkles of the skin may be reduced due to the degeneration of the skin. Skin wrinkles can be due to photoaging, age, facial expression, lack of moisture, or a combination thereof. The photoaging may be skin aging by exposure to ultraviolet light (including UVA, UVB, and UVC). The skin wrinkle improvement may inhibit or inhibit the generation of wrinkles on the skin, or may alleviate the wrinkles already generated.
상기 조성물은 비경구 투여 제형으로 제형화될 수 있다. 비경구 투여 제형은 주사제, 또는 피부외용제일 수 있다. 상기 피부외용제는 크림, 겔, 연고, 피부 유화제, 피부 현탁액, 경피전달성 패치, 약물 함유 붕대, 로션, 또는 그 조합일 수 있다. 상기 피부외용제는 통상 화장품이나 의약품 등의 피부외용제에 사용되는 성분, 예를 들면 수성성분, 유성성분, 분말성분, 알코올류, 보습제, 증점제, 자외선흡수제, 미백제, 방부제, 산화방지제, 계면활성제, 향료, 색제, 각종 피부 영양제, 또는 이들의 조합과 필요에 따라서 적절하게 배합될 수 있다. 상기 피부외용제는, 에데트산이나트륨, 에데트산삼나트륨, 시트르산나트륨, 폴리인산나트륨, 메타인산나트륨, 글루콘산 등의 금속봉쇄제, 카페인, 탄닌, 벨라파밀, 감초추출물, 글라블리딘, 칼린의 과실의 열수추출물, 각종생약, 아세트산토코페롤, 글리틸리틴산, 트라넥삼산 및 그 유도체 또는 그 염등의 약제, 비타민 C, 아스코르브산인산마그네슘, 아스코르브산글루코시드, 알부틴, 코지산, 글루코스, 프룩토스, 트레할로스 등의 당류도 적절하게 배합할 수 있다.The composition may be formulated into a parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin. The external preparation for skin may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, or a combination thereof, and may be suitably blended as necessary. The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Saccharides such as trehalose can also be appropriately compounded.
상기 조성물이 화장료 조성물인 경우, 화장료 조성물은 화장수(스킨로션), 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐 로션, 영양 로션, 마사지크림, 영양 크림, 모이스쳐 크림, 핸드크림, 파운데이션, 에센스, 영양 에센스, 팩, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션, 바디클렌저, 현탁액, 겔, 분말, 페이스트, 팩트, 마스크팩 또는 시트 또는 에어로졸 조성물을 포함하는 제형으로 제조될 수 있다. 이러한 제형의 조성물은 당해 분야에서 통상적인 방법에 따라 제조될 수 있다. 상기 화장료 조성물은 보존제, 안정화제, 계면활성제, 점증제, 용해제, 보습제, 에몰리언트제, 자외선 흡수제, 방부제, 살균제, 유화제, 산화 방지제, pH 조정제, 유기 및 무기 안료, 향료, 냉감제 또는 제한제 등 당해 분야에서 통상적으로 이용되는 첨가물을 더 포함할 수 있다. 상기 보존제 등의 추가 성분의 배합량은 본 발명의 목적 및 효과를 손상시키지 않는 범위 내에서 당업자가 용이하게 선정 가능하며, 그 배합량은 조성물 전체 중량을 기준으로 0.001 내지 40 중량%일 수 있다.When the composition is a cosmetic composition, the cosmetic composition may be a cosmetic composition (skin lotion), a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, The composition may be formulated into a formulation comprising an active ingredient, an essence, a nutritional essence, a pack, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a suspension, a gel, a powder, a paste, . Compositions of such formulations may be prepared according to methods conventional in the art. The cosmetic composition may contain at least one selected from the group consisting of preservatives, stabilizers, surfactants, thickeners, solubilizers, moisturizers, emollients, ultraviolet absorbers, preservatives, bactericides, emulsifiers, antioxidants, pH adjusting agents, organic and inorganic pigments, And may further contain additives commonly used in the art. The amount of the additional ingredient such as the preservative may be easily selected by a person skilled in the art within the range not impairing the object and effect of the present invention, and the amount thereof may be 0.001 to 40% by weight based on the total weight of the composition.
상기 조성물이 약학적 조성물인 경우, 약학적 조성물은 상기 플록쿨린을 유효한 양, 또는 유효 성분으로서 포함할 수 있다. 상기 유효한 양은 개체에 따라 적절하게 선택할 수 있다. 질환의 중증도, 환자의 연령, 체중, 건강, 성별, 환자의 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 상기 조성물과 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 투여는 당업계에 알려진 방법에 의하여 투여될 수 있다. 투여는 예를 들면, 정맥내, 근육내, 경구, 경피 (transdermal), 점막, 코안 (intranasal), 기관내 (intratracheal) 또는 피하 투여와 같은 경로로, 임의의 수단에 의하여 개체로 직접적으로 투여될 수 있다. 상기 투여는 전신적으로 또는 국부적으로 투여될 수 있다. When the composition is a pharmaceutical composition, the pharmaceutical composition may contain the effective amount or the effective component of the flocululin. The effective amount may be appropriately selected depending on the individual. The severity of the disease, the age, body weight, health, sex, sensitivity of the patient to the drug, time of administration, route of administration and rate of release, duration of treatment, factors involved in the combination or concurrent use with the composition, Can be determined according to well-known factors. Administration can be by any method known in the art. Administration may be by any means directly administered to a subject, such as by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration, . The administration can be systemically or locally administered.
상기 개체는 포유동물, 예를 들면, 사람, 소, 말, 돼지, 개, 양, 염소, 또는 고양이일 수 있다. 상기 개체는 피부미용 개선, 예를 들어 피부 보습, 장벽 강화, 또는 피부 재생 효과를 필요로 하는 개체일 수 있다.The subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The subject may be an individual in need of skin aesthetic improvement, for example skin moisturizing, barrier enhancement, or skin regeneration effect.
상기 투여는 플록쿨린을 포함하는 피부 미용을 위한 조성물을 개체당 일당 0.1 mg 내지 1,000 mg, 예를 들면, 0.1 mg 내지 500 mg, 0.1 mg 내지 100 mg, 0.1 mg 내지 50 mg, 0.1 mg 내지 25 mg, 1 mg 내지 1,000 mg, 1 mg 내지 500 mg, 1 mg 내지 100 mg, 1 mg 내지 50 mg, 1 mg 내지 25 mg, 5mg 내지 1,000 mg, 5 mg 내지 500 mg, 5 mg 내지 100 mg, 5 mg 내지 50 mg, 5 mg 내지 25 mg, 10mg 내지 1,000 mg, 10 mg 내지 500 mg, 10 mg 내지 100 mg, 10 mg 내지 50 mg, 또는 10 mg 내지 25 mg을 투여하는 것일 수 있다. 다만, 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있고, 당업자라면 이러한 요인들을 고려하여 투여량을 적절히 조절할 수 있다. 투여 횟수는 1일 1회 또는 임상적으로 용인가능한 부작용의 범위 내에서 2회 이상이 가능하고, 투여 부위에 대해서도 1개소 또는 2개소 이상에 투여할 수 있으며, 매일 또는 2 내지 5일 간격으로 총 투여 일수는 한번 치료 시 1일에서 30일까지 투여될 수 있다. 필요한 경우, 적정 시기 이후에 동일한 치료를 반복할 수 있다. 인간 이외의 동물에 대해서도, kg당 인간과 동일한 투여량으로 하거나, 또는 예를 들면 목적의 동물과 인간과의 기관(심장 등)의 용적비(예를 들면, 평균값) 등으로 상기의 투여량을 환산한 양을 투여할 수 있다.Such administration may comprise from 0.1 mg to 1000 mg, such as from 0.1 mg to 500 mg, from 0.1 mg to 100 mg, from 0.1 mg to 50 mg, from 0.1 mg to 25 mg, or from 0.1 mg to 100 mg per day of a composition for skin care comprising floculline , 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg Or 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg. However, the dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, These factors can be taken into account to appropriately adjust the dosage. The number of doses may be at least once per day or within the range of clinically acceptable side effects and may be administered at one or two or more doses at the site of administration and may be administered daily or every two to five days The number of days of administration can be administered from one day to 30 days at one time of treatment. If necessary, the same treatment may be repeated after the appropriate time. For an animal other than a human, the dosage may be the same as that of a human per kg, or the dosage may be converted into a dose in terms of a volume ratio (for example, an average value) One dose may be administered.
상기 조성물이 건강기능식품용 조성물인 경우, 당해 기술분야에 공지되어 있는 통상적인 건강기능식품의 제형으로 제제화될 수 있다. 상기 건강기능식품은 예를 들어 산제, 과립제, 정제, 환제, 캅셀제, 현탁액, 유제, 시럽제, 침제, 액제, 엑스제 등의 일반적인 제형으로 제조될 수도 있고, 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 젤리, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등의 임의의 건강식품 형태로 제조될 수도 있다. 상기 건강식품의 제제화를 위해 식품학적으로 허용 가능한 담체 또는 첨가제를 사용할 수 있으며, 제조하고자 하는 제형의 제조에 당해 기술분야에서 사용 가능한 것으로 공지되어 있는 임의의 담체 또는 첨가제가 이용될 수 있다. 상기 첨가제로서 각종 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 이외에도 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 첨가제 성분은 독립적으로 또는 조합하여 사용할 수 있으며, 첨가제의 비율은 크게 중요하진 않지만 조성물 100 중량부 당 0.01 ~ 0.1 중량부의 범위에서 선택될 수 있다. 상기 건강기능식품용 조성물 중의 상기 플록쿨린의 추출물의 함량은 사용 목적(예방 또는 개선)에 따라 적합하게 결정될 수 있다. 일반적으로, 전체 식품 중량의 0.01 내지 15 중량%로 포함할 수 있으며, 음료로서 제조될 경우 100 mL를 기준으로 0.02 내지 10 g, 바람직하게는 0.3 내지 1 g의 비율로 함유할 수 있다. When the composition is a composition for a health functional food, it can be formulated into a typical health functional food formulation known in the art. The health functional food may be prepared by conventional formulations such as powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups, Beverages, tea, drinks, alcoholic beverages, and vitamins, such as, for example, beverages, snacks, confectionery, pizza, ramen, other noodles, gums, jellies, ice creams, A pharmaceutically acceptable carrier or additive may be used for the formulation of the health food, and any carrier or additive known to be usable in the art for the preparation of the formulation to be prepared may be used. As the above-mentioned additives, there can be used various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Carbonating agents, and the like. In addition, it may contain natural fruit juice, fruit juice beverage and flesh for the production of vegetable beverages. These additive components can be used independently or in combination, and the proportion of the additive is not critical, but can be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition. The content of the extract of floculin in the composition for health functional foods can be suitably determined according to the intended use (prevention or improvement). Generally, it may contain 0.01 to 15% by weight of the total food, and when it is prepared as a beverage, it may contain 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 mL.
본 발명자들은 상기 플록쿨린을 포함하는 조성물을 이용한 실험에서, 일반 효모 배양물 또는 무처리군에 대비하여 현저한 필라그린 발현 향상, 클라우딘 1 발현 향상, 히아루론산 합성효소 발현 향상, 각질탈각효소 발현 향상, 및 멜라닌 생성 억제 효과를 확인하였다. 따라서, 플록쿨린을 포함하는 조성물은 피부 미용, 또는 피부 관련 질환의 치료 등에 유용하게 이용될 수 있다.The present inventors have found that, in the experiment using the composition containing the flocucrin, it is possible to improve the expression of pilar green, the expression of clodin 1, the expression of hyaluronic acid synthase, the expression of exfoliating enzyme, Melanin production inhibitory effect was confirmed. Therefore, a composition containing flocucurin can be usefully used for the treatment of skin cosmetics or skin-related diseases.
플록쿨린을 포함하는 조성물은 피부의 장벽 개선, 보습, 미백, 세포 증식 효과 등을 나타내고, 콜라겐 합성 및 엘라스틴 합성을 촉진함으로써 피부 미용에 유용하게 이용될 수 있다.The composition containing flocurin exhibits skin barrier improvement, moisturizing, whitening, cell proliferation effect, etc., and promotes collagen synthesis and elastin synthesis, so that it can be used for skin beauty.
도 1은 HaCat 세포에서 플록쿨린의 필라그린 발현 증가 효과를 나타내는 그래프이다. FIG. 1 is a graph showing the effect of increasing fila green expression of flocucrin in HaCat cells.
도 2는 HaCat 세포에서 플록쿨린의 클라우딘 1 발현 증가 효과를 나타내는 그래프이다.2 is a graph showing the effect of increasing the expression of claudin 1 of flocucrin in HaCat cells.
도 3은 HaCat 세포에서 플록쿨린의 HAS3 발현 증가 효과를 나타내는 그래프이다.FIG. 3 is a graph showing the effect of increasing HAS3 expression of flocucrin in HaCat cells.
도 4는 HaCat 세포에서 플록쿨린의 KLK7 발현 증가 효과를 나타내는 그래프이다.4 is a graph showing the effect of increasing the expression of KLK7 by flocucrin in HaCat cells.
도 5는 HaCat 세포에서 플록쿨린의 멜라닌 생성 억제 효과를 나타내는 그래프이다.5 is a graph showing the effect of inhibiting the production of melanin of flocucrin in HaCat cells.
이하, 본 발명을 구체적으로 설명하기 위해 실시예 및 실험예를 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예 및 실험예는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 아래에서 상술하는 실시예 및 실험예에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예 및 실험예는 당 업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되는 것이다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the embodiments and experimental examples according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the above-described embodiments and examples. The embodiments and experimental examples of the present invention are provided to enable those skilled in the art to more fully understand the present invention.
실시예Example 1:  One: 플록쿨린Floculine 준비 Ready
사카로마이세스 파스토리아누스(Saccharomyces pastorianus)를 Marika H.(1994. Purification and partial characterization of a flocculin from brewer's yeast)의 방법에 따라 배양하고, 플록쿨린이 포함된 용액을 분리하였다. 분리한 플록쿨린 용액은 -70℃, 5.0 Torr 조건에서 동결 건조하여 파우더로 제조하였다. Sakaromayses Paz Astoria Augustine (Saccharomyces Pastorianus ) was cultured according to the method of Marika H. (1994, Purification and partial characterization of a flocculin from brewer's yeast), and a solution containing flocucurin was isolated. The separated flocucrin solution was prepared by powder freezing at -70 ° C and 5.0 Torr.
비교예Comparative Example 1: 일반 효모 배양물 준비 1: Preparation of general yeast culture
사카로마이세스 파스토리누스를 Marika H.(1994.Purification and partial characterization of a flocculin from brewer's yeast)의 방법에 따라 배양하였다. 효모배양물을 3500 rpm에서 원심분리하고, 상등액을 0.45 μm 멤브레인 여과막으로 여과하여 제균하였다. 제균된 효모배양물은 -70℃, 5.0 Torr 조건에서 동결 건조하여 파우더로 제조하였다. Sakaromayses Waves Story Janus were cultured according to the method of Marika H. (1994.Purification and partial characterization of a flocculin from brewer's yeast). The yeast culture was centrifuged at 3500 rpm, and the supernatant was filtered with a 0.45 μm membrane filtration membrane. The sterilized yeast culture was lyophilized at -70 ° C and 5.0 Torr to produce powder.
실험예Experimental Example 1:  One: 피부장벽Skin barrier 인자  factor 필라그린(Filaggrin)의Filaggrin's 발현 확인 Confirmation of expression
사람 각질형성세포주인 HaCat는 America type culture collection (ATCC, Rockville, MD, USA)에서 구매하였으며 10%의 소태아 혈청 (FBS, ATCC, Rockville, MD, USA)과 1%의 antibiotic/antimycotic (AA, ATCC, Rockville, MD, USA)을 첨가한 DMEM (Hyclone, Logan, UT, USA)을 이용하여 37℃, 5%, CO2 조건의 인큐베이터에서 배양하였다. HaCat, a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
사람 각질형성세포에서 장벽강화 효과를 확인하기 위해, 사람 각질형성세포를 6 웰 세포 배양 접시에 5 x 105cells/웰 밀도로 접종한 후, 10%의 FBS와 1%의 AA가 첨가된 배지를 이용하여 37℃, 5% CO2 배양기에서 24시간 배양하였다. 배양된 세포에 비교예 1, 또는 실시예 1을 첨가하여 24시간 동안 배양하고 세포를 회수하여 트리졸 (RNA iso, TAKARA, 일본) 1 ㎖을 첨가하여 RNA를 분리하였다. Nanodrop 2000 (Thermo, USA)를 이용해 RNA를 정량 한 후, 42℃에서 55분, 70℃에서 15분 동안 반응시켜 cDNA를 합성하였다 (Reverse Transcriptase Mix, ELPIS biotech, 한국). RT-PCR은 PCR 기계 (Step One Plus, Applied Biosystems, 미국)를 이용하였고 사이버그린 (SYBRGreen supermix, Applied Biosystems, 미국)을 필라그린 프라이머 및 cDNA와 함께 첨가하여 94℃에서 5분 동안 중합효소 활성화, 95℃ 30초, 54℃ 1분, 72℃ 1분간 40 사이클로 중합반응 하였다. 프라이머의 염기서열은 아래의 표 1와 같다. 유전자의 발현양은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였다.To determine enhanced barrier effect in human keratinocytes, human keratinocytes after the cells inoculated with 5 x 10 5 cells / well density in 6-well cell culture plate, with 10% of FBS with 1% AA added medium And cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. To the cultured cells, Comparative Example 1 or Example 1 was added, followed by culturing for 24 hours. Cells were recovered and RNA was isolated by adding 1 ml of trizol (RNA iso, TAKARA, Japan). RNA was quantified using Nanodrop 2000 (Thermo, USA), and cDNA was synthesized by reacting at 42 ° C for 55 minutes and 70 ° C for 15 minutes (Reverse Transcriptase Mix, ELPIS biotech, Korea). RT-PCR was performed using a PCR machine (Step One Plus, Applied Biosystems, USA) and CyberGreen supermix (Applied Biosystems, USA) Polymerization was carried out at 95 ° C for 30 seconds, 54 ° C for 1 minute, and 72 ° C for 1 minute for 40 cycles. The nucleotide sequences of the primers are shown in Table 1 below. The expression level of the gene was finally analyzed by correction for the β-actin gene.
그 결과, 무처리군 대비 실시예 1 첨가군에서 필라그린 발현이 2.84배 향상됨을 관찰하였다(도 1). 따라서, 실시예 1의 조성물의 우수한 피부 장벽 강화 효과를 기대할 수 있다.As a result, it was observed that the pilar green expression was improved 2.84 times in the Example 1 addition group compared to the no treatment group (Fig. 1). Thus, the skin barrier enhancement effect of the composition of Example 1 can be expected.
필라그린 프라이머 염기 서열Pillar green primer sequence
프라이머 이름Name of the primer 서열order
필라그린Pillar Green 정방향 5'-GGCACTGAAAGGCAAAAAGG-3' (서열번호 1)Forward 5'-GGCACTGAAAGGCAAAAAGG-3 '(SEQ ID NO: 1)
역방향 5'-AAACCCGGATTCACCATAATCA-3' (서열번호 2)Reverse 5'-AAACCCGGATTCACCATAATCA-3 '(SEQ ID NO: 2)
β-액틴beta -actin 정방향 5'-GGCCATCTCTTGCTCGAAGT-3' (서열번호 3)Forward 5'-GGCCATCTCTTGCTCGAAGT-3 '(SEQ ID NO: 3)
역방향 5'-GACACCTTCAACACCCCAGC-3' (서열번호 4)Reverse 5'-GACACCTTCAACACCCCAGC-3 '(SEQ ID NO: 4)
실험예Experimental Example 2:  2: 피부세포Skin cell 결속 인자  Binding factor 클라우딘Claudin 1( One( ClaudinClaudin 1)의1) of 발현 확인 Confirmation of expression
사람 각질형성세포주인 HaCat는 America type culture collection (ATCC, Rockville, MD, USA)에서 구매하였으며 10%의 소태아 혈청 (FBS, ATCC, Rockville, MD, USA)과 1%의 antibiotic/antimycotic (AA, ATCC, Rockville, MD, USA)을 첨가한 DMEM (Hyclone, Logan, UT, USA)을 이용하여 37℃, 5%, CO2 조건의 인큐베이터에서 배양하였다. HaCat, a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
사람 각질형성세포를 6 웰 세포 배양 접시에 5 x 105cells/웰 밀도로 접종하여 10%의 FBS와 1%의 AA가 첨가된 배지를 이용하여 37℃, 5% CO2 배양기에서 24시간 배양하였다. 배양된 세포에 비교예 1, 또는 실시예 1를 첨가하여 24시간 동안 배양하고 세포를 회수하여 트리졸 (RNA iso, TAKARA, 일본) 1 ㎖을 첨가하여 RNA를 분리하였다. Nanodrop 2000 (Thermo, USA)를 이용해 RNA를 정량한 후, 42℃에서 55분, 70℃에서 15분 동안 반응시켜 cDNA를 합성하였다 (Reverse Transcriptase Mix, ELPIS biotech, 한국). RT-PCR은 PCR 기계 (Step One Plus, Applied Biosystems, 미국)를 이용하였고 사이버그린 (SYBRGreen supermix, Applied Biosystems, 미국)을 클라우딘 1 프라이머 및 cDNA와 함께 첨가하여 50℃에서 2분, 95℃ 10분에서 반응 후 95℃ 10초와 60℃ 1분간 40 사이클로 중합반응 하였다. 프라이머의 염기서열은 하기의 표 2와 같다. 유전자의 발현양은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였다.From keratinocytes 6-well cell cultured for 24 hours in Petri dishes 5 x 10 5 cells / inoculation as well density by using a of 10% FBS and 1% AA added medium 37 ℃, 5% CO 2 incubator in Respectively. To the cultured cells, Comparative Example 1 or Example 1 was added and cultured for 24 hours. Cells were recovered and RNA was isolated by adding 1 ml of trizol (RNA iso, TAKARA, Japan). RNA was quantified using Nanodrop 2000 (Thermo, USA), and cDNA was synthesized by reacting at 42 ° C for 55 minutes and 70 ° C for 15 minutes (Reverse Transcriptase Mix, ELPIS biotech, Korea). RT-PCR was carried out using a PCR machine (Step One Plus, Applied Biosystems, USA) and CyberGreen supermix (Applied Biosystems, USA) with Claudin 1 primer and cDNA for 2 min at 50 ° C, At 95 ° C for 10 seconds and at 60 ° C for 1 minute for 40 cycles. The nucleotide sequences of the primers are shown in Table 2 below. The expression level of the gene was finally analyzed by correction for the β-actin gene.
그 결과, 무처리군 대비 실시예 1 첨가군에서 클라우딘 1 발현이 1.45배 증가하는 것을 확인하였다(도 2). 따라서 실시예 1 조성물의 우수한 피부 보습 효과를 기대할 수 있다.As a result, it was confirmed that the expression of Claudin 1 increased by 1.45 times in the group to which Example 1 was added compared to the untreated group (Fig. 2). Therefore, excellent skin moisturizing effect of the composition of Example 1 can be expected.
클라우딘 1 프라이머 염기 서열 Clone 1 primer base sequence
프라이머 이름Name of the primer 서열order
Claudin 1Claudin 1 정방향 5'-GCTCTAGAATTCCGAGCGAGTCATGGCCAACGC-3' (서열번호 5)5'-GCTCTAGAATTCCGAGCGAGTCATGGCCAACGC-3 '(SEQ ID NO: 5)
역방향 5'-GCTCTAGAATTCTCACACGTAGTCTTTCCCGCT-3' (서열번호 6)Reverse 5'-GCTCTAGAATTCTCACACGTAGTCTTTCCCGCT-3 '(SEQ ID NO: 6)
β-actinβ-actin 정방향 5'-GGCCATCTCTTGCTCGAAGT-3' (서열번호 7)Forward 5'-GGCCATCTCTTGCTCGAAGT-3 '(SEQ ID NO: 7)
역방향 5'-GACACCTTCAACACCCCAGC-3' (서열번호 8)Reverse 5'-GACACCTTCAACACCCCAGC-3 '(SEQ ID NO: 8)
실험예Experimental Example 3: 히아루론산 합성 효소  3: Hyaluronic acid synthase HAS3의HAS3 발현 확인 Confirmation of expression
사람 각질형성세포주인 HaCat는 America type culture collection (ATCC, Rockville, MD, USA)에서 구매하였으며 10%의 소태아 혈청 (FBS, ATCC, Rockville, MD, USA)와 1%의 antibiotic/antimycotic (AA, ATCC, Rockville, MD, USA)을 첨가한 DMEM (Hyclone, Logan, UT, USA)을 이용하여 37℃, 5%, CO2 조건의 인큐베이터에서 배양하였다. HaCat, a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
사람 각질형성세포를 6 웰 세포 배양 접시에 5 x 105cells/웰 밀도로 접종하여 10%의 FBS와 1%의 AA가 첨가된 배지를 이용하여 37℃, 5% CO2 배양기에서 24시간 배양하였다. 배양된 세포에 비교예 1 또는 실시예 1을 첨가하여 24시간 동안 배양하고 세포를 회수하여 트리졸 (RNA iso, TAKARA, 일본) 1 ㎖을 첨가하여 RNA를 분리하였다. Nanodrop 2000 (Thermo, USA)를 이용해 RNA를 정량 한 후, 42℃에서 55분, 70℃에서 15분 동안 반응시켜 cDNA를 합성하였다 (Reverse Transcriptase Mix, ELPIS biotech, 한국). RT-PCR은 PCR 기계 (Step One Plus, Applied Biosystems, 미국)를 이용하였고 사이버그린 (SYBRGreen supermix, Applied Biosystems, 미국)을 HAS3 프라이머 및 cDNA와 함께 첨가하여 94℃에서 5분 동안 중합효소 활성화, 95℃ 30초, 54℃ 1분, 72℃ 1분간 40 사이클로 중합반응 하였다. 프라이머의 염기서열은 하기의 표 3와 같다. 유전자의 발현양은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였다.From keratinocytes 6-well cell cultured for 24 hours in Petri dishes 5 x 10 5 cells / inoculation as well density by using a of 10% FBS and 1% AA added medium 37 ℃, 5% CO 2 incubator in Respectively. Comparative Example 1 or Example 1 was added to the cultured cells, followed by culturing for 24 hours. Cells were recovered and RNA was isolated by adding 1 ml of trizol (RNA iso, TAKARA, Japan). RNA was quantified using Nanodrop 2000 (Thermo, USA), and cDNA was synthesized by reacting at 42 ° C for 55 minutes and 70 ° C for 15 minutes (Reverse Transcriptase Mix, ELPIS biotech, Korea). RT-PCR was performed using PCR machine (Step One Plus, Applied Biosystems, USA) and SYBR Green supermix (Applied Biosystems, USA) was added with HAS3 primer and cDNA to activate polymerase for 5 min at 94 ° C, 95 Deg.] C for 30 seconds, 54 [deg.] C for 1 minute, and 72 [deg.] C for 1 minute. The nucleotide sequences of the primers are shown in Table 3 below. The expression level of the gene was finally analyzed by correction for the β-actin gene.
그 결과, 일반 효모배양물(비교예 1) 대비 라거 효모의 플록쿨린이(실시예 1) 더 많은 양의 히아루론산 합성효소를 발현시킴을 확인하였다(도 3). 따라서, 증가된 히아루론산 합성효소가 피부 내 수분 포집 능력을 향상시킬 수 있으므로, 실시예 1 조성물의 우수한 피부 보습 효과를 기대할 수 있다.As a result, it was confirmed that flocoolin of lager yeast (Example 1) expressed a larger amount of hyaluronic acid synthase than the conventional yeast culture (Comparative Example 1) (Fig. 3). Therefore, since the increased hyaluronic acid synthase can enhance the water-trapping ability in the skin, excellent skin moisturizing effect of the composition of Example 1 can be expected.
HAS 3 프라이머 염기 서열HAS 3 primer base sequence
프라이머 이름Name of the primer 서열order
HAS3HAS3 정방향 5'- CTTAAGGGTTGCTTGCTTGC -3' (서열번호 9)Forward 5'-CTTAAGGGTTGCTTGCTTGC -3 '(SEQ ID NO: 9)
역방향 5'- GTTCGTGGGAGATGAAGGAA -3' (서열번호 10)Reverse 5'-GTTCGTGGGAGATGAAGGAA -3 '(SEQ ID NO: 10)
β-actinβ-actin 정방향 5'-GGCCATCTCTTGCTCGAAGT-3' (서열번호 11)Forward 5'-GGCCATCTCTTGCTCGAAGT-3 '(SEQ ID NO: 11)
역방향 5'-GACACCTTCAACACCCCAGC-3' (서열번호 12)Reverse 5'-GACACCTTCAACACCCCAGC-3 '(SEQ ID NO: 12)
실험예Experimental Example 4: 각질 탈각 효소  4: exfoliating enzyme KLK7의Of KLK7 발현 확인 Confirmation of expression
사람 각질형성세포주인 HaCat는 America type culture collection (ATCC, Rockville, MD, USA)에서 구매하였으며 10%의 소태아 혈청 (FBS, ATCC, Rockville, MD, USA)와 1%의 antibiotic/antimycotic (AA, ATCC, Rockville, MD, USA)을 첨가한 DMEM (Hyclone, Logan, UT, USA)을 이용하여 37℃, 5%, CO2 조건의 인큐베이터에서 배양하였다. HaCat, a human keratinocyte cell line, was purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA), supplemented with 10% fetal bovine serum (FBS, ATCC, Rockville, MD, USA) and 1% antibiotic / antimycotic Were incubated in DMEM (Hyclone, Logan, UT, USA) supplemented with ATCC, Rockville, MD, USA at 37 ° C and 5% CO 2 .
사람 각질형성세포를 6 웰 세포 배양 접시에 5 x 105 cells/웰 밀도로 접종하여 10%의 FBS와 1%의 AA가 첨가된 배지를 이용하여 37℃, 5% CO2 배양기에서 24시간 배양하였다. 상기 세포에 비교예 1, 또는 실시예 1을 첨가하여 24시간 동안 배양하고 세포를 회수하여 트리졸 (RNA iso, TAKARA, 일본) 1 ㎖을 첨가하여 RNA를 분리하였다. Nanodrop 2000 (Thermo, USA)를 이용해 RNA를 정량 한 후, 42℃에서 55분, 70℃에서 15분 동안 반응시켜 cDNA를 합성하였다 (Reverse Transcriptase Mix, ELPIS biotech, 한국). RT-PCR은 PCR 기계 (Step One Plus, Applied Biosystems, 미국)를 이용하였고 사이버그린 (SYBRGreen supermix, Applied Biosystems, 미국)을 KLK7 프라이머 및 cDNA와 함께 첨가하여 50℃에서 2분, 95℃ 10분에서 반응 후 95℃ 10초와 60℃ 1분간 40 사이클로 중합반응 하였다. 프라이머의 염기서열은 하기의 표 4와 같다. 유전자의 발현양은 β-actin 유전자에 대한 보정을 통해 최종적으로 분석하였다.From keratinocytes 6-well cell cultured for 24 hours in Petri dishes 5 x 10 5 cells / inoculation as well density by using a of 10% FBS and 1% AA added medium 37 ℃, 5% CO 2 incubator in Respectively. Comparative Example 1 or Example 1 was added to the cells, followed by culturing for 24 hours. The cells were recovered and RNA was isolated by adding 1 ml of trizol (RNA iso, TAKARA, Japan). RNA was quantified using Nanodrop 2000 (Thermo, USA), and cDNA was synthesized by reacting at 42 ° C for 55 minutes and 70 ° C for 15 minutes (Reverse Transcriptase Mix, ELPIS biotech, Korea). RT-PCR was performed using a PCR machine (Step One Plus, Applied Biosystems, USA) and CyGreen supermix (Applied Biosystems, USA) was added with KLK7 primer and cDNA for 2 min at 50 ° C, 10 min at 95 ° C After the reaction, polymerization was carried out at 95 ° C for 10 seconds and 40 ° C for 1 minute at 60 ° C. The base sequences of the primers are shown in Table 4 below. The expression level of the gene was finally analyzed by correction for the β-actin gene.
일반 효모배양물(비교예 1) 대비 라거 효모의 플록쿨린이(실시예 1) 더 많은 양의 각질 탈각 효소(KLK7)를 발현시킴을 확인하였다(도 4). 따라서, 실시예 1 조성물의 매끈하고 밝은 피부톤 보정 효과를 기대할 수 있다.It was confirmed that flocucurin of lager yeast (Example 1) expressed a greater amount of keratolytic enzyme (KLK7) compared to the general yeast culture (Comparative Example 1) (Fig. 4). Therefore, a smooth and bright skin tone correction effect of the composition of Example 1 can be expected.
KLK7 프라이머 염기 서열KLK7 primer base sequence
프라이머 이름Name of the primer 서열order
KLK 7KLK 7 정방향 5'- GCATCCCCGACTCCAAGAA-3' (서열번호 13)Forward 5'-GCATCCCCGACTCCAAGAA-3 '(SEQ ID NO: 13)
역방향 5'- CAGGGTACCTCTGCACACCAA-3 (서열번호 14)Reverse 5'-CAGGGTACCTCTGCACACCAA-3 (SEQ ID NO: 14)
β-actinβ-actin 정방향 5'-GGCCATCTCTTGCTCGAAGT-3' (서열번호 15)Forward 5'-GGCCATCTCTTGCTCGAAGT-3 '(SEQ ID NO: 15)
역방향 5'-GACACCTTCAACACCCCAGC-3' (서열번호 16)Reverse 5'-GACACCTTCAACACCCCAGC-3 '(SEQ ID NO: 16)
실험예 5: 멜라닌 생성 억제 효과 확인Experimental Example 5: Confirmation of inhibitory effect on melanin formation
쥐의 색소세포(murine melanoma)인 B16 F10 세포를 10% FBS를 포함하는 DMEM에 현탁시켜 웰당 1 Х 105 cells으로 6-웰 플레이트에 접종하여 웰 바닥에 부착될때까지 배양하였다. 멜라닌 생성 유도를 위하여, 0.1 μM α-MSH를 처리하고, 이후 시료별로 첨가한 후 3일간 배양하였다. 멜라닌 생성능 실험의 양성대조군으로 알부틴 100 ppm 을 처리하였다. 배양한 세포는 PBS로 씻고 트립신(trypsin)으로 회수하였다. 회수한 세포를 혈구계(hematocytometer)로 계수하여 각 처리 그룹별로 1Х106 cells/mL으로 동일한 세포수가 되도록 모아, 1000 rpm에서 10분간 원심분리하여 세포만을 얻었다. 회수된 세포를 60에서 1시간 건조시켜 10% DMSO가 포함된 1M NaOH용액 100 ㎕을 넣어 세포내의 멜라닌을 얻었다. 이 멜라닌 액을 마이크로플레이트 리더로 490 nm에서 흡광도를 측정하여 멜라닌 생성 저해율을 평가하였다.B16 F10 cells, a murine melanoma, were suspended in DMEM containing 10% FBS and inoculated into 6-well plates at 1 × 10 5 cells per well and cultured until adhered to the bottom of the wells. For induction of melanogenesis, 0.1 μM α-MSH was added, and then added for each sample, followed by culturing for 3 days. As a positive control for melanin production, arbutin was treated at 100 ppm. The cultured cells were washed with PBS and trypsin. The recovered cells were counted with a hematocytometer and collected at 1 × 10 6 cells / mL for each treatment group, and centrifuged at 1000 rpm for 10 minutes to obtain cells. The recovered cells were dried at 60 ° C for 1 hour, and 100 μl of a 1 M NaOH solution containing 10% DMSO was added to obtain melanin in the cells. This melanin solution was assayed for absorbance at 490 nm with a microplate reader to evaluate the inhibition of melanin formation.
그 결과, 일반 효모배양물(비교예 1) 대비 라거 효모의 플록쿨린이(실시예 1) 멜라닌 생성을 더 많이 억제 시킴을 확인하였다(도 5). 따라서, 실시예 1 조성물은 피부 세포 내의 멜라닌 생성을 억제함으로써 우수한 미백 효과를 가질 수 있다.As a result, it was confirmed that flocoolin of lager yeast (Example 1) inhibited melanin production more than the conventional yeast culture (Comparative Example 1) (FIG. 5). Thus, the composition of Example 1 can have an excellent whitening effect by inhibiting the production of melanin in skin cells.

Claims (8)

  1. 플록쿨린(flocculin)을 포함하는 피부 미용을 위한 화장료 조성물.A cosmetic composition for skin care comprising floculin.
  2. 청구항 1에 있어서, 상기 플록쿨린은 사카로마이세스(Saccharomyces) 속 균주로부터 분리된 것인 화장료 조성물.The method of claim 1, wherein the floculline is selected from the group consisting of Saccharomyces , Lt; / RTI > is isolated from the parent strain.
  3. 청구항 1에 있어서, 상기 사카로마이세스 속 균주는 사카로마이세스 파스토리아누스(Saccharomyces pastorianus)인 화장료 조성물.[Claim 2] The method according to claim 1, wherein the Saccharomyces sp. Strain is Saccharomyces Paz thoria Taunus (Saccharomyces pastorianus) of the cosmetic composition.
  4. 청구항 1에 있어서, 상기 화장료 조성물은 사카로마이세스(Saccharomyces) 속 균주의 배양물 또는 그의 추출물을 더 포함하는 것인 화장료 조성물.The method according to claim 1, wherein the cosmetic composition is my process as Saccharomyces (Saccharomyces) Lt; RTI ID = 0.0 > 1, < / RTI > or an extract thereof.
  5. 플록쿨린(flocculin)을 포함하는 피부 미용을 위한 건강기능식품용 조성물.Composition for a health functional food for skin care comprising floculin.
  6. 플록쿨린(flocculin)을 포함하는 피부 손상 또는 피부 노화를 예방 또는 치료하기 위한 약학적 조성물.A pharmaceutical composition for preventing or treating skin damage or aging of the skin comprising flocculin.
  7. 플록쿨린(flocculin)을 포함하는 조성물의 유효량을 개체에 투여하는 단계를 포함하는 피부 손상 또는 노화를 치료 또는 개선시키는 방법.A method of treating or ameliorating skin damage or aging comprising administering to an individual an effective amount of a composition comprising flocculin.
  8. 피부 손상 또는 노화 치료제의 제조를 위한, 플록쿨린(flocculin)을 포함하는 조성물의 용도. Use of a composition comprising flocculin for the manufacture of a skin damaging or aging treatment agent.
PCT/KR2019/000184 2018-01-12 2019-01-07 Composition for skin care comprising flocculin WO2019139307A1 (en)

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