WO2017146341A1 - Composition for treating wounds, containing daphnin, and use thereof - Google Patents

Composition for treating wounds, containing daphnin, and use thereof Download PDF

Info

Publication number
WO2017146341A1
WO2017146341A1 PCT/KR2016/012094 KR2016012094W WO2017146341A1 WO 2017146341 A1 WO2017146341 A1 WO 2017146341A1 KR 2016012094 W KR2016012094 W KR 2016012094W WO 2017146341 A1 WO2017146341 A1 WO 2017146341A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
daphnetin
wound
skin
glucoside
Prior art date
Application number
PCT/KR2016/012094
Other languages
French (fr)
Korean (ko)
Inventor
노주원
김명석
이희주
문길주
오상록
밧수렌둘람자브
Original Assignee
한국과학기술연구원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020160039331A external-priority patent/KR101769545B1/en
Application filed by 한국과학기술연구원 filed Critical 한국과학기술연구원
Publication of WO2017146341A1 publication Critical patent/WO2017146341A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • a pharmaceutical composition for treating wounds a pharmaceutical composition for treating an inflammatory disease, a method for treating an individual's wound, a method for treating an inflammatory disease and a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging .
  • the skin acts as a shield to protect the body from the outside.
  • the blood fills the wound site by the natural healing action of the living body, the granule reduction of platelets and activation of the hageman factor are initiated, and the wound healing process proceeds.
  • Blood clotting is a temporary protective action that protects exposed wound tissues and provides a base for cells to migrate during the healing process.
  • the Wound healing and / or healing occurs in three major stages.
  • the first stage is the inflammatory phase, characterized by inflammation at the traumatic site.
  • the inflammatory stages can usually progress briefly in the absence of a serious infection.
  • the second stage is the proliferative phase, which is characterized by epithelialization, angiogenesis, granulation tissue formation and collagen deposition.
  • Angiogenesis involving new capillary formation is used to deliver nutrients and maintain granulation tissue formation. Without the formation of new capillaries into the wound, essential nutrients do not reach the wound, creating a wound that is not chronically cured.
  • As the surface of the damaged wound is covered by a layer of keratinocytes, new epidermis is produced and re-epithelialization occurs where the epithelial layer is rebuilt.
  • a series of processes are performed in which the wound area is reduced through the increase and reorganization of connective tissue.
  • the final stage of wound healing is the maturational phase where fibroblasts differentiate into collagen.
  • the convoluted cells and capillaries of the recovering tissues gradually disappear, and scarring can occur if the cells and capillaries of these tissues are hyperplastic or do not degrade normally.
  • the first aspect provides a pharmaceutical composition for treating a wound.
  • the second aspect provides a pharmaceutical composition for treating an inflammatory disease.
  • the third aspect provides a method of treating a wound in a subject.
  • a fourth aspect provides a method of treating an inflammatory disease in a subject.
  • a fifth aspect provides a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging.
  • the sixth aspect provides a cosmetic method for improving wounds, improving skin wrinkles, or preventing skin aging.
  • the first aspect is at least one compound selected from the group consisting of daphine, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin, pharmaceutically acceptable It provides a pharmaceutical composition for wound treatment comprising a possible salt or solvate as an active ingredient.
  • a second aspect is at least one compound selected from the group consisting of daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin, pharmaceutically acceptable
  • pharmaceutical compositions for treating inflammatory diseases comprising possible salts, or solvates as active ingredients.
  • the compound may be to increase the transcriptional activity of ⁇ -catenin, and / or to increase the expression of the collagen gene.
  • Increasing the transcriptional activity of ⁇ -catenin is associated with promoting wound healing.
  • Fathke C et al., BMC Cell Biol. 2006 Jan 20; 7: 4 states that the ectopic activation of beta-catenin dependent Wnt signals by lithium chloride in wounds results in epithelial cysts and sometimes basic hair follicle structures in the dermis. Reported.
  • Bielefeld KA et al., Cell Mol Life Sci. 2013 Jun; 70 (12): 2059-81 report that the Wnt and / or beta-catenin signaling pathway plays an important role in wound healing.
  • the compound daphin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin each have the following structure: Can be.
  • Daphnetin-8- ⁇ -glucoside ,
  • the daphin may be 8-hydroxy-2-oxo-2H-chromen-7-yl- ⁇ -D-glucopyranoside.
  • Daphnetin may be 7,8-dihydroxycoumarin.
  • Rutarencin is 3-hydroxy-3-methyl-5-oxo-5-[[3,4,5-trihydroxy-6- [6-methoxy-2-oxo-3- (2-oxochromen) -7-yl) oxychromen-7-yl] oxyoxan-2-yl] methoxy] pentanoic acid.
  • Daphnoretin may be 7-hydroxy-6-methoxy-3- (2-oxochromen-7-yl) oxychromen-2-one.
  • Chamaecromone is (+)-3- [1- [bis (4-hydroxyphenyl) methyl] -2-oxo-2- (2,4,6-trihydroxyphenyl) ethyl] -5,7 -Dihydroxy-4H-1-benzopyran-4-one.
  • Isoquarcitrin is 2- (3,4-dihydroxyphenyl) -5,7-dihydroxy-3-[(2S, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy- 6- (hydroxymethyl) oxan-2-yl] oxychromen-4-oneyl.
  • the pharmaceutically acceptable salt is not particularly limited as long as it is pharmaceutically acceptable, but may preferably be an inorganic acid salt, an organic acid salt or a metal salt.
  • preferred inorganic acid salts include hydrochloride, phosphate, sulfate or disulfate
  • examples of preferred organic acid salts include maleate, maleic acid salt, citrate salt, fumarate salt, besylate salt, campylate salt or edyl salt salt.
  • examples include calcium salt, sodium salt, magnesium salt, strontium salt or potassium salt.
  • the term "wound” means that the tissue is cut, torn, broken, burned, or traumatized. Damage to a human body arising from a disorder or disease causing injury.
  • tissue refers to a mass of cells in the human body that collectively form a specific function. Tissues can include eyes, mucus, lungs, kidneys, heart, intestines, tendons, vascular tissues, bones, skin, connective tissue, and nerves such as the spinal cord.
  • the wound may be an open wound with an open surface or a closed wound with no open surface.
  • One example of such a wound may be an open wound in the skin.
  • the wound may include lesions, sores, necrosis, and ulcers.
  • the necrosis may be related to dead tissue resulting from infection, injury or infarction.
  • the ulcer may be a local defect or dent in the surface of an organ or tissue, produced by stripping of necrotic tissue.
  • wounds are an epidermis of the skin; Dermis; Epidermis and dermis; Or wounds with damaged epidermis, dermis and subcutaneous fat layers.
  • wounds include cuts, incisions (eg surgical incisions), abrasions, lacerations or lacerations, fractures, contusions, burns. Or amputations.
  • a “treat” provided by the present invention may be one that provides for the wound to heal at a shorter time compared to natural healing.
  • the treatment may include amelioration and / or alleviation of the wound.
  • the treatment may include both treatment of a wound and / or a disease associated with the wound. Said treatment may refer to healing and / or regeneration of damaged tissue resulting from the wound.
  • the wound treatment may include the meaning of skin regeneration.
  • the treatment may be to maintain the original composition of the damaged tissue.
  • the treatment may be to promote healing and / or regeneration of the damaged tissue while minimizing the complications and / or scarring of diseases associated with the wound.
  • the composition may be for promoting the previous stage of the wound healing process.
  • the composition may be to promote the proliferative and / or mature phase during the wound healing step.
  • the composition may be for treating a wound in the proliferative and / or mature phase.
  • the composition is 0.001% to 80% by weight, for example, 0.01% to 60%, 0.01% to 40%, 0.01% to 30%, 0.01% to 20% by weight relative to the total weight of the composition %, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% % To 10% by weight, or 0.1 to 5% by weight of the compound may be included.
  • the composition comprises 0.01 mg to 1,000 mg, for example, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 50 mg, 0.01 mg to 100 mg, 0.01 mg to 50 mg, 0.01 mg to 10 per unit of administration. mg, 0.01 mg to 1 mg, or 0.01 mg to 0.5 mg of the compound.
  • the composition may be one containing no compound other than the compound as an active ingredient.
  • the composition may comprise a pharmaceutically acceptable diluent or carrier.
  • the diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and magnesium stearate as a lubricant, talc, or a combination thereof.
  • the carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof.
  • the excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof.
  • the disintegrant may be calcium carboxymethyl cellulose, sodium starch glycolate, calcium dihydrogen phosphate, or a combination thereof.
  • the binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof.
  • the lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
  • the composition may be formulated in a parenteral dosage form.
  • the parenteral dosage form may be an injection or an external dermal preparation.
  • the topical skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, mask pack, drug containing bandage, lotion, or a combination thereof.
  • the external skin preparations are commonly used in external preparations for cosmetics and medicines, such as aqueous components, oily ingredients, powder ingredients, alcohols, humectants, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , Colorants, various skin nutrients, or combinations thereof, as appropriate.
  • the external skin preparations include metal blockers such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidine, and caline.
  • metal blockers such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidine, and caline.
  • Fruit hot water extract, various herbal medicines, drugs such as tocopherol acetate, glytilinic acid, tranexamic acid and derivatives thereof or salts thereof, vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, can also be mix
  • the composition may be for promoting expression of collagen gene in cells.
  • the composition can increase the expression of collagen or can increase the activity of collagen produced by the composition.
  • the composition may also increase the activity or expression of up-stream enzymes or proteins that produce collagen.
  • the composition may be one that has an effect of treating a wound by increasing expression and / or activity of collagen.
  • the collagen may be type 1 collagen (collagen, type I) or type 3 collagen (collagen, type III).
  • the gene encoding the collagen may be selected from the group consisting of COL1A1 and COL3A1.
  • the third aspect provides a method of treating a wound in an individual comprising administering to the individual an amount of the composition of the first aspect described above that is effective to treat the wound.
  • a fourth aspect provides a method of treating an inflammatory disease in an individual comprising administering to the individual an amount of the composition of the second aspect described above that is effective to treat the inflammatory disease.
  • composition is the same as described above.
  • Administration can be by any method known in the art. Administration can be administered directly to the subject by any means, for example by intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. .
  • the administration can be administered systemically or locally. The administration may be to be administered locally to the site where the wound is present.
  • the subject may be a mammal, for example a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.
  • the administration of the compound may comprise 0.1 mg to 1,000 mg, for example 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg per individual.
  • a fifth aspect is at least one compound selected from the group consisting of daphine, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin, pharmaceutically acceptable
  • a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging, including a possible salt or solvate as an active ingredient.
  • the compound is the same as described above.
  • the term "improving a wound” may include the use of lowering or alleviating the extent of a wound.
  • the wound amelioration may be to lower or alleviate the extent of a wound that has already occurred.
  • the compound promotes the production of collagen in fibroblasts.
  • Collagen is a major component of the dermal layer of the skin. It is an elastic protein that has a strong binding property with moisture. That is, collagen has a property of imparting elasticity to the skin, and thus has an effect of preventing wrinkles from occurring. As a person ages, the amount of collagen in the skin decreases, and the skin undergoes an aging process that causes loss of elasticity and wrinkles. Therefore, the composition of the claimed invention comprising the compound can be used as a cosmetic composition for improving skin wrinkles or preventing skin aging.
  • the cosmetic composition may be provided in any formulation suitable for topical application.
  • a formulation suitable for topical application for example, in the form of an emulsion obtained by dispersing an oil phase in a solution, an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a mask pack or a sheet, a foam, or an aerosol composition.
  • Compositions of such formulations may be prepared according to conventional methods in the art.
  • the cosmetic composition may further include a moisturizer, an emulsifier, a ultraviolet absorber, a preservative, a fungicide, an antioxidant, a pH adjuster, organic and inorganic pigments, fragrances, coolants or limiting agents.
  • a moisturizer an emulsifier, a ultraviolet absorber, a preservative, a fungicide, an antioxidant, a pH adjuster, organic and inorganic pigments, fragrances, coolants or limiting agents.
  • the blending amount of the additional components such as the moisturizing agent can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, the blending amount is 0.01 to 5% by weight, specifically 0.01 to 3 based on the total weight of the composition Weight percent.
  • the composition is 0.001% to 80% by weight, for example, 0.01% to 60%, 0.01% to 40%, 0.01% to 30%, 0.01% to 20% by weight relative to the total weight of the composition %, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% % To 10% by weight, or 0.1 to 5% by weight of the compound may be included.
  • a sixth aspect provides a method for making up skin, comprising applying a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging according to the fifth aspect to the skin of an individual.
  • the method may be to improve the wound, to improve skin wrinkles, or to prevent skin aging.
  • the cosmetic composition is applied in numerous treatments, in particular cosmetic treatments of the skin, lips and hair, including the scalp.
  • composition for treating a wound according to one aspect, it can be used to efficiently treat a wound in a subject.
  • composition for treating an inflammatory disease it can be used to effectively treat an inflammatory disease in an individual.
  • the individual's wound can be efficiently treated.
  • the method for making up the skin it is possible to efficiently improve the wound, improve the skin wrinkles, or prevent skin aging.
  • Figure 2 is an electrophoresis picture showing the effect of seven compounds on the expression of COL1A1 and COL3A1 gene in fibroblasts.
  • Figure 3 shows the effect of the seven compounds on the expression of the COL1A1 gene in fibroblasts.
  • FIG. 4 is a diagram showing the effect of the seven compounds on the expression of the COL3A1 gene in fibroblasts.
  • FIG. 6 shows the presence of seven compounds, namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin to promote migration of keratinocytes. This is the result of checking.
  • FIG. 7 shows the presence of seven compounds, namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin to the expression level of MMP-1. This is the result of checking the impact.
  • Figure 8 Expression of daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromone, and isoquarcitrin in type 1 procollagen in UVB irradiated human fibroblasts The result of measuring the extent to which it affects is shown.
  • DMSO dimethylsulfoxide
  • FIG. 1 shows the presence of seven compounds, namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin, affect beta-catenin activity in cells. It is a figure which shows the effect.
  • A, B, C, D, E, F, and G are daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromone, and iso, respectively. Quercitrin is shown.
  • Human fibroblast Hs68 was placed in a well of a 6-well microtiter plate containing 2 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5 (v / v) fetal calf serum. 2 ⁇ 10 6 cells / well were added and incubated under the same conditions as above until the confluency reached 80%.
  • DMEM Dulbecco's Modified Eagle's Media: Hyclone
  • Figure 2 is an electrophoresis picture showing the effect of seven compounds on the expression of COL1A1 and COL3A1 gene in fibroblasts.
  • Figure 3 shows the effect of the seven compounds on the expression of the COL1A1 gene in fibroblasts.
  • 4 and 5 are the results analyzed from the electrophoresis picture of FIG.
  • Figure 5 shows the effect of the seven compounds on the expression of the COL3A1 gene in fibroblasts.
  • daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin are involved in the expression of COL3A1 gene, a collagen synthesis-related factor. It was found that the effect of activating is remarkable.
  • macrophage (macrophage) was confirmed the effect of the production of prostaglandin E 2 which is an inflammation promoting factor.
  • RAW 264.7 ATCC TIB-71. These cells are made from Abelson murine leukemia virus-induced tumors of mice, have mononuclear and macrophage morphology, and attach and grow.
  • RAW 264.7 cells were placed in a well of a 24-well microtiter plate containing Dulbecco's Modified Eagle's Media: Hyclone (DMEM) medium containing 10 (v / v) Fetal Bovine Serum for 24 hours. Cultured under the same conditions as described above. Each of the seven compounds was then added to the medium at the indicated concentrations (uM) and incubated at the same conditions for 24 hours.
  • DMEM Dulbecco's Modified Eagle's Media
  • FIG. 9 shows the effect of seven compounds on prostaglandin E2 production in macrophages.
  • the vertical axis shows the amount of prostaglandin E 2 released into the culture.
  • seven compounds significantly inhibited the production of prostaglandin E2 compared to the control.
  • the inhibitory effect of daphnoretin, chamaechromone, and daphnetin was particularly excellent.
  • the seven compounds namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin, migrate to human keratinocytes. Whether to increase was confirmed as follows.
  • HaCaT human keratinocytes were placed in a well of a 24-well microtiter plate containing 1 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5 (v / v) Fetal Bovine Serum. Added to 2 ⁇ 10 5 cells / well. Next, the keratinocytes were incubated in a 37 ° C. and 5% CO 2 incubator until the confluency reached 80%, and then the cell monolayer in the medium was scraped with a p10 pipette tip to induce “scratch-damage”. .
  • DMEM Dynamic Eagle's Media
  • Hyclone Hyclone
  • FIG. 6 shows the presence of seven compounds, namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin to promote migration of keratinocytes. This is the result of checking.
  • Collagen degrading enzyme-1 of the seven compounds namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin (Matrix Metalloproteinase-1, The inhibitory efficacy of production of MMP-1) was measured.
  • the level of MMP-1 in the supernatant was measured using the collagen MMP-1 ELISA kit (QIA55, Merck & Co., USA) from the supernatant.
  • the supernatant was added to a well of a 96-well plate uniformly coated with a mouse anti-MMP-1 antibody as a primary antibody, and incubated at room temperature for 2 hours to form an antigen-antibody complex. .
  • anti-MMP-1 antibody conjugated with horseradish peroxidase as a chromophore was placed in a well of a 96-well plate and incubated for 1 hour.
  • FIG. 7 shows the presence of seven compounds, namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin to the expression level of MMP-1. This is the result of checking the impact.
  • daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, dafnoretin, daphnetin, chamaechromene, and isoquarcitrin are excellent in inhibiting MMP-1 production. It can be seen that it has an effect. In particular, isoquarcitrin was confirmed to be the most effective.
  • Epigallocatechin gallate (EGCG) was used as a positive control.
  • the seven compounds namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin are Type-1 procollagen. It was confirmed whether to promote the expression of.
  • DMEM Dulbecco's Modified Eagle's Media
  • mouse anti-PIP monoclonal antibody conjugated with horseradish peroxidase (POD) to a well of 96-hole plate uniformly coated with mouse anti-PIP monoclonal antibody.
  • antibody-POD conjugate conjugated with horseradish peroxidase (POD)
  • POD horseradish peroxidase
  • the cell culture and standard solution were added respectively and left at 37 ° C. for 3 hours.
  • the wells were then washed and a substrate solution containing hydrogen peroxide and tetramethylbenzidine was added and incubated for 15 minutes at room temperature. Thereafter, a stop solution was added to the well to stop the reaction, and the absorbance was measured at 450 nm using a plate reader.
  • Figure 8 Expression of daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromone, and isoquarcitrin in type 1 procollagen in UVB irradiated human fibroblasts The result of measuring the extent to which it affects is shown. As a result, as shown in FIG. 8, seven compounds promoted the expression of type 1 procollagen, and in particular, daphnin, isoquarcitrin, daphnetin-8- ⁇ -glucoside, and rutalensin were largely type 1 pro Collagen expression was promoted. Daphnetin-8- ⁇ -glucoside most greatly promoted type 1 procollagen expression.
  • seven compounds namely daphnin, daphnetin-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetin, chamaechromone, and isoquarcitrin, promote wound healing and inflammation
  • the inhibitory effect was excellent.
  • the seven compounds namely daphnin, daphnetine-8- ⁇ -glucoside, rutarencin, daphnoretin, daphnetine, chamaechromeone, and iso under UV-irradiated conditions Quercitrin has been shown to reduce the expression of MMP-1 and promote collagen expression from human fibroblasts. That is, the seven compounds promoted the expression of collagen while reducing the expression of collagenase under the condition of UV irradiation in human fibroblasts. Thus, the seven compounds can promote collagen synthesis in tissues including human fibroblasts, for example in the skin, thereby maintaining and promoting elasticity of the tissues.
  • the seven compounds are effective in improving tissues including human fibroblasts, such as skin wrinkles, or preventing skin aging.

Abstract

Provided are: a pharmaceutical composition for treating wounds, containing daphnin; a pharmaceutical composition for treating inflammatory diseases; a method for treating wounds of an individual; a method for treating inflammatory diseases; and a cosmetic composition for treating wounds, alleviating skin wrinkles, and preventing skin aging.

Description

다프닌을 포함하는 상처 치료용 조성물 및 그의 용도Composition for treating wounds comprising daphin and its use
상처 치료용 약학적 조성물, 염증성 질환을 치료하기 위한 약학적 조성물, 개체의 상처를 치료하는 방법, 염증성 질환을 치료하는 방법 및 상처의 개선용, 피부 주름 개선, 또는 피부 노화 방지용 화장료 조성물에 관한 것이다.A pharmaceutical composition for treating wounds, a pharmaceutical composition for treating an inflammatory disease, a method for treating an individual's wound, a method for treating an inflammatory disease and a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging .
피부는 외부로부터 신체를 보호하는 방어막으로서 역할을 한다. 피부에 상처가 생기면 생체의 자연 치유 작용에 의해 상처자리를 혈액이 채우게 되고 혈소판의 과립감소와 하게만 인자(hageman factor)의 활성화가 시작되어 상처 치유 과정이 진행된다. 혈액의 응고는 임시 방어 작용으로서, 노출된 상처 조직들을 보호하고 치유 과정 동안 세포들이 이동할 수 있는 기반을 제공한다. The skin acts as a shield to protect the body from the outside. When a wound occurs on the skin, the blood fills the wound site by the natural healing action of the living body, the granule reduction of platelets and activation of the hageman factor are initiated, and the wound healing process proceeds. Blood clotting is a temporary protective action that protects exposed wound tissues and provides a base for cells to migrate during the healing process.
상처 치료 및/또는 치유는 크게 3개의 단계로 일어난다. 첫 번째 단계는 외상 부위에서의 염증을 특징으로 하는 염증기(inflammatory phase)이다. 염증기는 중대한 감염이 없는 상태에서 일반적으로 짧게 진행될 수 있다. 두 번째 단계는 증식기(proliferative phase)이며, 이는 상피화, 혈관형성, 육아 조직 형성 및 콜라게 침착을 특징으로 한다. 새로운 모세관 형성을 수반하는 혈관형성은 영양소를 전달하고 육아조직 형성을 유지하는데 사용된다. 상처 내로 새로운 모세관의 형성 없이 필수 영양소가 상처에 도달하지 못하여 만성적으로 치유되지 않는 상처를 생성시킨다. 손상된 상처의 표면이 각질형성세포(keratinocytes) 층에 의해 덮이면서 새로운 표피가 생성되고 상피층이 재건되는 재상피화가 일어난다. 재상피화가 완료되면 결합조직의 증가와 재편성을 통해 상처 면적이 감소되는 일련의 과정이 진행된다. 상처 치유의 최종 단계는 섬유아세포(fibroblast)가 콜라겐으로 분화하는 성숙기(maturational phase)이다. 성숙기 동안 회복기 조직의 엉긴 세포와 모세혈관이 조금씩 사라지게 되는데, 이러한 조직의 세포와 모세혈관이 과형성되거나 정상적으로 분해되지 않는 경우 흉터가 생길 수 있다. Wound healing and / or healing occurs in three major stages. The first stage is the inflammatory phase, characterized by inflammation at the traumatic site. The inflammatory stages can usually progress briefly in the absence of a serious infection. The second stage is the proliferative phase, which is characterized by epithelialization, angiogenesis, granulation tissue formation and collagen deposition. Angiogenesis involving new capillary formation is used to deliver nutrients and maintain granulation tissue formation. Without the formation of new capillaries into the wound, essential nutrients do not reach the wound, creating a wound that is not chronically cured. As the surface of the damaged wound is covered by a layer of keratinocytes, new epidermis is produced and re-epithelialization occurs where the epithelial layer is rebuilt. Once re-epithelialization is complete, a series of processes are performed in which the wound area is reduced through the increase and reorganization of connective tissue. The final stage of wound healing is the maturational phase where fibroblasts differentiate into collagen. During maturation, the convoluted cells and capillaries of the recovering tissues gradually disappear, and scarring can occur if the cells and capillaries of these tissues are hyperplastic or do not degrade normally.
상처 치유과정에서 상처를 신속하게 치료할 뿐만 아니라 부작용이나 흉터 없이 치료하는 것이 중요하기 때문에, 이러한 효능을 가지는 물질을 찾고자 하는 노력이 계속되고 있다. In the wound healing process, it is important not only to cure wounds quickly but also to treat them without side effects or scars, and thus, efforts to find a substance having such an effect continue.
제1 양상은 상처 치료용 약학적 조성물을 제공한다. The first aspect provides a pharmaceutical composition for treating a wound.
제2 양상은 염증성 질환을 치료하기 위한 약학적 조성물을 제공한다. The second aspect provides a pharmaceutical composition for treating an inflammatory disease.
제3 양상은 개체의 상처를 치료하는 방법을 제공한다. The third aspect provides a method of treating a wound in a subject.
제4 양상은 개체의 염증성 질환을 치료하는 방법을 제공한다.A fourth aspect provides a method of treating an inflammatory disease in a subject.
제5 양상은 상처의 개선용, 피부 주름 개선, 또는 피부 노화 방지용 화장료 조성물을 제공한다. A fifth aspect provides a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging.
제6 양상은 상처의 개선, 피부 주름 개선, 또는 피부 노화 방지를 위한 화장 방법을 제공한다.The sixth aspect provides a cosmetic method for improving wounds, improving skin wrinkles, or preventing skin aging.
제1 양상은 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린으로 이루어진 군으로부터 선택된 하나 이상의 화합물, 그의 약학적으로 허용가능한 염, 또는 용매화물을 유효 성분으로서 포함하는 상처 치료용 약학적 조성물을 제공한다. The first aspect is at least one compound selected from the group consisting of daphine, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin, pharmaceutically acceptable It provides a pharmaceutical composition for wound treatment comprising a possible salt or solvate as an active ingredient.
제2 양상은 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린으로 이루어진 군으로부터 선택된 하나 이상의 화합물, 그의 약학적으로 허용가능한 염, 또는 용매화물을 유효 성분으로서 포함하는 염증성 질환을 치료하기 위한 약학적 조성물을 제공한다. A second aspect is at least one compound selected from the group consisting of daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin, pharmaceutically acceptable Provided are pharmaceutical compositions for treating inflammatory diseases comprising possible salts, or solvates as active ingredients.
제1 및 제2 양상에 있어서, 상기 화합물은 β-카테닌의 전사 활성을 증가시키는 것, 및/또는 콜라겐 유전자의 발현을 증가시키는 것일 수 있다. β-카테닌의 전사 활성의 증가는 상처 치유 촉진과 관련된다. 예를 들면, Fathke C 등, BMC Cell Biol. 2006 Jan 20;7:4는 상처에서 리튬 클로리드에 의하여 베타-카테닌 의존적 Wnt 신호를 이소성 활성화(ectopic activation)시키면 상피낭(epithelial cysts) 및 때때로 진피 내에서 기본적 모낭(hair follicle) 구조를 생기게 한다는 것을 보고하였다. 또한, Bielefeld KA 등, Cell Mol Life Sci. 2013 Jun;70(12):2059-81은 Wnt 및/또는 베타-카테닌 신호전달 경로가 상처 치유에 중요한 역할을 한다는 것을 보고하고 있다.In the first and second aspects, the compound may be to increase the transcriptional activity of β-catenin, and / or to increase the expression of the collagen gene. Increasing the transcriptional activity of β-catenin is associated with promoting wound healing. For example, Fathke C et al., BMC Cell Biol. 2006 Jan 20; 7: 4 states that the ectopic activation of beta-catenin dependent Wnt signals by lithium chloride in wounds results in epithelial cysts and sometimes basic hair follicle structures in the dermis. Reported. In addition, Bielefeld KA et al., Cell Mol Life Sci. 2013 Jun; 70 (12): 2059-81 report that the Wnt and / or beta-catenin signaling pathway plays an important role in wound healing.
제1 및 제2 양상에 있어서, 상기 화합물 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린은 각각 다음 구조를 갖는 것일 수 있다. In the first and second aspects, the compound daphin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin each have the following structure: Can be.
다프닌:
Figure PCTKR2016012094-appb-I000001
,Daphnin:
Figure PCTKR2016012094-appb-I000001
,
다프네틴-8-β-글루코시드:
Figure PCTKR2016012094-appb-I000002
,Daphnetin-8-β-glucoside:
Figure PCTKR2016012094-appb-I000002
,
루타렌신:
Figure PCTKR2016012094-appb-I000003
,Rutarencin:
Figure PCTKR2016012094-appb-I000003
,
다프노레틴:
Figure PCTKR2016012094-appb-I000004
,Daphnoretin:
Figure PCTKR2016012094-appb-I000004
,
다프네틴:.
Figure PCTKR2016012094-appb-I000005
,Daphnetin :.
Figure PCTKR2016012094-appb-I000005
,
차마에크로모네:
Figure PCTKR2016012094-appb-I000006
,Chamaecromone:
Figure PCTKR2016012094-appb-I000006
,
이소쿼르시트린:
Figure PCTKR2016012094-appb-I000007
.Isoquarcitrin:
Figure PCTKR2016012094-appb-I000007
.
상기 다프닌은 8-히드록시-2-옥소-2H-크로멘-7-일-β-D-글루코피라노시드일 수 있다. 다프네틴은 7,8-디히드록시쿠마린일 수 있다. 루타렌신은 3-히드록시-3-메틸-5-옥소-5-[[3,4,5-트리히드록시-6-[6-메톡시-2-옥소-3-(2-옥소크로멘-7-일) 옥시크로멘-7-일]옥시옥산-2-일]메톡시]펜탄산일 수 있다. 다프노레틴은 7-히드록시-6-메톡시-3-(2-옥소크로멘-7-일) 옥시크로멘-2-오네일 수 있다. 차마에크로모네는 (+)-3-[1-[비스(4-히드록시페닐)메틸]-2-옥소-2-(2,4,6-트리히드록시페닐)에틸]-5,7-디히드록시-4H-1-벤조피란-4-오네일 수 있다. 이소쿼르시트린은 2-(3,4-디히드록시페닐)-5,7-디히드록시-3-[(2S,3R,4S,5S,6R)-3,4,5-트리히드록시-6-(히드록시메틸)옥산-2-일]옥시크로멘-4-오네일 수 있다.The daphin may be 8-hydroxy-2-oxo-2H-chromen-7-yl-β-D-glucopyranoside. Daphnetin may be 7,8-dihydroxycoumarin. Rutarencin is 3-hydroxy-3-methyl-5-oxo-5-[[3,4,5-trihydroxy-6- [6-methoxy-2-oxo-3- (2-oxochromen) -7-yl) oxychromen-7-yl] oxyoxan-2-yl] methoxy] pentanoic acid. Daphnoretin may be 7-hydroxy-6-methoxy-3- (2-oxochromen-7-yl) oxychromen-2-one. Chamaecromone is (+)-3- [1- [bis (4-hydroxyphenyl) methyl] -2-oxo-2- (2,4,6-trihydroxyphenyl) ethyl] -5,7 -Dihydroxy-4H-1-benzopyran-4-one. Isoquarcitrin is 2- (3,4-dihydroxyphenyl) -5,7-dihydroxy-3-[(2S, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy- 6- (hydroxymethyl) oxan-2-yl] oxychromen-4-oneyl.
상기 조성물에 있어서, 상기 약학적으로 허용가능한 염은 약학적으로 허용가능한 한 특별히 제한되지는 않으나, 바람직하게는 무기산염, 유기산염 또는 금속염일 수 있다. 바람직한 무기산염의 예에는 염산염, 인산염, 황산염 또는 이황산염을 들 수 있으며, 바람직한 유기산염의 예에는 말산염, 말레인산염, 구연산염, 푸마르산염, 베실산염, 캄실산염 또는 에디실염을 들 수 있고, 금속염의 예에는 칼슘염, 나트륨염, 마그네슘염, 스트론튬염 또는 칼륨염을 들 수 있다. In the composition, the pharmaceutically acceptable salt is not particularly limited as long as it is pharmaceutically acceptable, but may preferably be an inorganic acid salt, an organic acid salt or a metal salt. Examples of preferred inorganic acid salts include hydrochloride, phosphate, sulfate or disulfate, and examples of preferred organic acid salts include maleate, maleic acid salt, citrate salt, fumarate salt, besylate salt, campylate salt or edyl salt salt. Examples include calcium salt, sodium salt, magnesium salt, strontium salt or potassium salt.
상기 조성물에 있어서, 용어 "상처(wound)"는 조직이 잘려지거나(cut), 찢어지거나(torn), 부서지거나(broken), 타거나(burned), 또는 외상을 입거나(traumatized), 또는 이러한 손상을 유발하는 장애 또는 질환으로부터 발생된 인체에 대한 손상(injury)을 의미한다. 용어 "조직"은 함께 집단을 이루어 특정 기능을 형성하는 인체 중의 세포의 덩어리를 나타낸다. 조직에는 눈, 점액, 폐, 신장, 심장, 내장, 힘줄(tendon), 혈관조직, 뼈, 피부, 결합조직, 및 신경, 예컨대 척수가 포함될 수 있다. 상기 상처는 표면이 개방된 개방된 상처(open wound), 또는 표면이 개방되지 않은 폐쇄된 상처(closed wound)일 수 있다. 상기 상처의 일예는 피부에 있는 개방된 상처일 수 있다. 상기 상처는 병변(lesion), 헌데(sore), 괴사(necrosis), 및 궤양(ulcer)을 포함할 수 있다. 상기 괴사는 감염, 손상 또는 경색으로부터 생성되는 죽은 조직과 관련된 것일 수 있다. 상기 궤양은 괴사 조직의 탈피에 의해 생성된, 기관 또는 조직의 표면의 국소적인 결함 또는 패임일 수 있다. In the composition, the term "wound" means that the tissue is cut, torn, broken, burned, or traumatized. Injury to a human body arising from a disorder or disease causing injury. The term "tissue" refers to a mass of cells in the human body that collectively form a specific function. Tissues can include eyes, mucus, lungs, kidneys, heart, intestines, tendons, vascular tissues, bones, skin, connective tissue, and nerves such as the spinal cord. The wound may be an open wound with an open surface or a closed wound with no open surface. One example of such a wound may be an open wound in the skin. The wound may include lesions, sores, necrosis, and ulcers. The necrosis may be related to dead tissue resulting from infection, injury or infarction. The ulcer may be a local defect or dent in the surface of an organ or tissue, produced by stripping of necrotic tissue.
상기 상처의 일 예는 피부의 표피(epidermis); 진피(dermis); 표피 및 진피; 또는 표피, 진피 및 피하지방층이 손상된 상처일 수 있다. One example of such a wound is an epidermis of the skin; Dermis; Epidermis and dermis; Or wounds with damaged epidermis, dermis and subcutaneous fat layers.
또한, 상기 상처의 예는 베임(cut), 절개(incisions)(예, 수술적 절개), 찰과상(abrasions), 열상 또는 찢김(lacerations), 골절(fracture), 타박상(contusions), 화상(burns), 또는 절단(amputations)을 포함할 수 있다. Also, examples of such wounds include cuts, incisions (eg surgical incisions), abrasions, lacerations or lacerations, fractures, contusions, burns. Or amputations.
본 발명에 의하여 제공되는 "치료(treat)"는 자연 치유에 비하여 단축된 시간에 상처가 치유되는 것을 제공하는 것일 수 있다. 상기 치료는 상처의 개선 및/또는 완화를 포함할 수 있다. 또한, 상기 치료는 상처 및/또는 상처와 관련된 질환의 치료를 모두 포함하는 것일 수 있다. 상기 치료는 상처로부터 유발되는 손상된 조직의 치유 및/또는 재생을 의미할 수 있다. 상기 상처 치료는 피부 재생의 의미를 포함할 수 있다. 또한, 상기 치료는 상기 손상된 조직의 원래 조성을 유지하는 것일 수 있다. 또한, 상기 치료는 상처와 관련된 질환의 합병증 및/또는 흉터를 최소화하면서 상기 손상된 조직을 치유 및/또는 재생을 촉진하는 것일 수 있다. A “treat” provided by the present invention may be one that provides for the wound to heal at a shorter time compared to natural healing. The treatment may include amelioration and / or alleviation of the wound. In addition, the treatment may include both treatment of a wound and / or a disease associated with the wound. Said treatment may refer to healing and / or regeneration of damaged tissue resulting from the wound. The wound treatment may include the meaning of skin regeneration. In addition, the treatment may be to maintain the original composition of the damaged tissue. In addition, the treatment may be to promote healing and / or regeneration of the damaged tissue while minimizing the complications and / or scarring of diseases associated with the wound.
상기 조성물은 상처 치유 과정의 전단계를 촉진하기 위한 것일 수 있다. 또한, 상기 조성물은 상처 치유 단계 중 증식기 및/또는 성숙기를 촉진하는 것일 수 있다. 따라서, 상기 조성물은 증식기 및/또는 성숙기에 있는 상처를 치료하기 위한 것일 수 있다. The composition may be for promoting the previous stage of the wound healing process. In addition, the composition may be to promote the proliferative and / or mature phase during the wound healing step. Thus, the composition may be for treating a wound in the proliferative and / or mature phase.
상기 조성물은 조성물 총 중량에 대하여 0.001 중량% 내지 80 중량%, 예를 들면, 0.01 중량% 내지 60 중량%, 0.01 중량% 내지 40 중량%, 0.01 중량% 내지 30 중량%, 0.01 중량% 내지 20 중량%, 0.01 중량% 내지 10 중량%, 0.01 중량% 내지 5 중량%, 0.05 중량% 내지 60 중량%, 0.05 중량% 내지 40 중량%, 0.05 중량% 내지 30 중량%, 0.05 중량% 내지 20 중량%, 0.05 중량% 내지 10 중량%, 0.05 중량% 내지 5 중량%, 0.1 중량% 내지 60 중량%, 0.1 중량% 내지 40 중량%, 0.1 중량% 내지 30 중량%, 0.1 중량% 내지 20 중량%, 0.1 중량% 내지 10 중량%, 또는 0.1 중량% 내지 5 중량%의 상기 화합물을 포함할 수 있다. 상기 조성물은 투여 1 단위당 0.01 mg 내지 1,000 mg, 예를 들면, 0.1 mg 내지 100 mg, 0.1 mg 내지 50 mg, 0.1 mg 내지 50 mg, 0.01 mg 내지 100 mg, 0.01 mg 내지 50 mg, 0.01 mg 내지 10 mg, 0.01 mg 내지 1 mg, 또는 0.01 mg 내지 0.5 mg의 상기 화합물을 포함할 수 있다. 상기 조성물은 상기 화합물 외의 다른 화합물을 유효 성분으로 포함하지 않는 것일 수 있다.The composition is 0.001% to 80% by weight, for example, 0.01% to 60%, 0.01% to 40%, 0.01% to 30%, 0.01% to 20% by weight relative to the total weight of the composition %, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% % To 10% by weight, or 0.1 to 5% by weight of the compound may be included. The composition comprises 0.01 mg to 1,000 mg, for example, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 50 mg, 0.01 mg to 100 mg, 0.01 mg to 50 mg, 0.01 mg to 10 per unit of administration. mg, 0.01 mg to 1 mg, or 0.01 mg to 0.5 mg of the compound. The composition may be one containing no compound other than the compound as an active ingredient.
상기 조성물은 약제학적으로 허용가능한 희석제 또는 담체를 포함할 수 있다. 상기 희석제는 유당, 옥수수 전분, 대두유, 미정질 셀룰로오스, 또는 만니톨, 활택제로는 스테아린산 마그네슘, 탈크, 또는 그 조합일 수 있다. 상기 담체는 부형제, 붕해제, 결합제, 활택제, 또는 그 조합일 수 있다. 상기 부형제는 미결정 셀룰로오즈, 유당, 저치환도 히드록시셀룰로오즈, 또는 그 조합일 수 있다. 상기 붕해제는 카르복시메틸셀룰로오스 칼슘, 전분글리콜산 나트륨, 무수인산일수소 칼슘, 또는 그 조합일 수 있다. 상기 결합제는 폴리비닐피롤리돈, 저치환도 히드록시프로필셀룰로오즈, 히드록시프로필셀룰로오즈, 또는 그 조합일 수 있다. 상기 활택제는 스테아린산 마그네슘, 이산화규소, 탈크, 또는 그 조합일 수 있다.The composition may comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and magnesium stearate as a lubricant, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethyl cellulose, sodium starch glycolate, calcium dihydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
상기 조성물은 비경구 투여 제형으로 제형화될 수 있다. 비경구 투여 제형은 주사제, 또는 피부외용제일 수 있다. 피부외용제는 크림, 겔, 연고, 피부 유화제, 피부 현탁액, 경피전달성 패치, 마스크 팩, 약물 함유 붕대, 로션, 또는 그 조합일 수 있다. The composition may be formulated in a parenteral dosage form. The parenteral dosage form may be an injection or an external dermal preparation. The topical skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, mask pack, drug containing bandage, lotion, or a combination thereof.
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.The external skin preparations are commonly used in external preparations for cosmetics and medicines, such as aqueous components, oily ingredients, powder ingredients, alcohols, humectants, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , Colorants, various skin nutrients, or combinations thereof, as appropriate.
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 external skin preparations include metal blockers such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidine, and caline. Fruit hot water extract, various herbal medicines, drugs such as tocopherol acetate, glytilinic acid, tranexamic acid and derivatives thereof or salts thereof, vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, can also be mix | blended suitably.
상기 조성물은 세포 중의 콜라겐 유전자의 발현을 촉진하기 위한 것일 수 있다. 상기 조성물은 콜라겐의 발현을 증가시킬 수 있거나, 또는 상기 조성물에 의해 생성된 콜라겐의 활성을 증가시킬 수 있다. 상기 조성물은 또한 콜라겐을 생성시키는 상류(up-stream)의 효소 또는 단백질의 활성 또는 발현을 증가시킬 수 있다. 상기 조성물은 콜라겐의 발현 및/또는 활성을 증가시켜 상처 치료 효능을 갖는 것일 수 있다. 상기 콜라겐은 1형 콜라겐(collagen, type I) 또는 3형 콜라겐(collagen, type III)일 수 있다. 상기 콜라겐을 코딩하는 유전자는 COL1A1 및 COL3A1로 구성된 군으로부터 선택될 수 있다. The composition may be for promoting expression of collagen gene in cells. The composition can increase the expression of collagen or can increase the activity of collagen produced by the composition. The composition may also increase the activity or expression of up-stream enzymes or proteins that produce collagen. The composition may be one that has an effect of treating a wound by increasing expression and / or activity of collagen. The collagen may be type 1 collagen (collagen, type I) or type 3 collagen (collagen, type III). The gene encoding the collagen may be selected from the group consisting of COL1A1 and COL3A1.
제3 양상은 상처를 치료하기에 유효한 양의 상기한 제1 양상의 조성물을 개체에게 투여하는 단계를 포함하는 개체의 상처를 치료하는 방법을 제공한다. The third aspect provides a method of treating a wound in an individual comprising administering to the individual an amount of the composition of the first aspect described above that is effective to treat the wound.
제4 양상은 염증성 질환을 치료하기에 유효한 양의 상기한 제2 양상의 조성물을 개체에게 투여하는 단계를 포함하는 개체의 염증성 질환을 치료하는 방법을 제공한다. A fourth aspect provides a method of treating an inflammatory disease in an individual comprising administering to the individual an amount of the composition of the second aspect described above that is effective to treat the inflammatory disease.
상기 조성물에 대해서는 상술한 바와 동일하다. The composition is the same as described above.
투여는 당업계에 알려진 방법에 의하여 투여될 수 있다. 투여는 예를 들면, 정맥내, 근육내, 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. 상기 투여는 상처가 존재하는 부위에 국소적으로 투여하는 것일 수 있다. Administration can be by any method known in the art. Administration can be administered directly to the subject by any means, for example by intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. . The administration can be administered systemically or locally. The administration may be to be administered locally to the site where the wound is present.
상기 개체는 포유동물, 예를 들면, 사람, 소, 말, 돼지, 개, 양, 염소, 또는 고양이일 수 있다.The subject may be a mammal, for example a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.
상기 투여는 상기 화합물을 개체당 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.The administration of the compound may comprise 0.1 mg to 1,000 mg, for example 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg per individual. 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 to 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.
제5 양상은 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린으로 이루어진 군으로부터 선택된 하나 이상의 화합물, 그의 약학적으로 허용가능한 염, 또는 용매화물을 유효성분으로 포함하는 상처의 개선용, 피부 주름 개선, 또는 피부 노화 방지용 화장료 조성물을 제공한다. 상기 화합물에 대해서는 상술한 바와 동일하다. A fifth aspect is at least one compound selected from the group consisting of daphine, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin, pharmaceutically acceptable Provided is a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging, including a possible salt or solvate as an active ingredient. The compound is the same as described above.
용어 "상처의 개선용"은 상처의 정도를 낮추어주거나 완화시키는 용도를 포함할 수 있다. 예를 들면, 상기 상처 개선은 이미 발생한 상처의 정도를 낮추거나 완화시키는 것일 수 있다. The term "improving a wound" may include the use of lowering or alleviating the extent of a wound. For example, the wound amelioration may be to lower or alleviate the extent of a wound that has already occurred.
상기 화합물은 섬유아세포에서 콜라겐의 생성을 촉진한다. 콜라겐은 피부 진피층의 주요 성분으로서 탄력 단백질로 수분과 결합하는 힘이 강한 성질을 가지고 있다. 즉, 콜라겐은 피부에 탄력성을 부여하는 성질을 가지고 있고, 그에 따라 주름이 생기는 것을 방지하는 효과를 가진다. 사람이 나이가 들어감에 따라서, 피부의 콜라겐의 양이 줄어들고 피부는 탄력성을 잃고 주름이 생기되는 노화 과정을 거치게 된다. 따라서, 상기 화합물을 포함하는 청구된 발명의 조성물은 피부 주름 개선, 또는 피부 노화 방지용 화장료 조성물로서 사용될 수 있다.The compound promotes the production of collagen in fibroblasts. Collagen is a major component of the dermal layer of the skin. It is an elastic protein that has a strong binding property with moisture. That is, collagen has a property of imparting elasticity to the skin, and thus has an effect of preventing wrinkles from occurring. As a person ages, the amount of collagen in the skin decreases, and the skin undergoes an aging process that causes loss of elasticity and wrinkles. Therefore, the composition of the claimed invention comprising the compound can be used as a cosmetic composition for improving skin wrinkles or preventing skin aging.
상기 화장료 조성물은 국소 적용에 적합한 모든 제형으로 제공될 수 있다. 예를 들면, 용액, 수상에 유상을 분산시켜 얻은 에멀젼, 유상에 수상을 분산시켜 얻은 에멀젼, 현탁액, 고체, 겔, 분말, 페이스트, 마스크 팩 또는 시트, 포말(foam) 또는 에어로졸 조성물의 제형으로 제공될 수 있다. 이러한 제형의 조성물은 당해 분야의 통상적인 방법에 따라 제조될 수 있다.The cosmetic composition may be provided in any formulation suitable for topical application. For example, in the form of an emulsion obtained by dispersing an oil phase in a solution, an aqueous phase, an emulsion obtained by dispersing an aqueous phase in an oil phase, a suspension, a solid, a gel, a powder, a paste, a mask pack or a sheet, a foam, or an aerosol composition. Can be. Compositions of such formulations may be prepared according to conventional methods in the art.
상기 화장료 조성물은 보습제, 에몰리언트제, 자외선 흡수제, 방부제, 살균제, 산화 방지제, pH 조정제, 유기 및 무기 안료, 향료, 냉감제 또는 제한제를 더 포함할 수 있다. 상기 보습제 등의 추가 성분의 배합량은 본 발명의 목적 및 효과를 손상시키지 않는 범위 내에서 당업자가 용이하게 선정 가능하며, 그 배합량은 조성물 전체 중량을 기준으로 0.01 내지 5 중량%, 구체적으로 0.01 내지 3 중량%일 수 있다.The cosmetic composition may further include a moisturizer, an emulsifier, a ultraviolet absorber, a preservative, a fungicide, an antioxidant, a pH adjuster, organic and inorganic pigments, fragrances, coolants or limiting agents. The blending amount of the additional components such as the moisturizing agent can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, the blending amount is 0.01 to 5% by weight, specifically 0.01 to 3 based on the total weight of the composition Weight percent.
상기 조성물은 조성물 총 중량에 대하여 0.001 중량% 내지 80 중량%, 예를 들면, 0.01 중량% 내지 60 중량%, 0.01 중량% 내지 40 중량%, 0.01 중량% 내지 30 중량%, 0.01 중량% 내지 20 중량%, 0.01 중량% 내지 10 중량%, 0.01 중량% 내지 5 중량%, 0.05 중량% 내지 60 중량%, 0.05 중량% 내지 40 중량%, 0.05 중량% 내지 30 중량%, 0.05 중량% 내지 20 중량%, 0.05 중량% 내지 10 중량%, 0.05 중량% 내지 5 중량%, 0.1 중량% 내지 60 중량%, 0.1 중량% 내지 40 중량%, 0.1 중량% 내지 30 중량%, 0.1 중량% 내지 20 중량%, 0.1 중량% 내지 10 중량%, 또는 0.1 중량% 내지 5 중량%의 상기 화합물을 포함할 수 있다. The composition is 0.001% to 80% by weight, for example, 0.01% to 60%, 0.01% to 40%, 0.01% to 30%, 0.01% to 20% by weight relative to the total weight of the composition %, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% % To 10% by weight, or 0.1 to 5% by weight of the compound may be included.
제6 양상은 제5 양상에 따른 상처의 개선용, 피부 주름 개선, 또는 피부 노화 방지용 화장료 조성물을 개체의 피부에 적용하는 단계를 포함하는, 피부를 화장하기 위한 방법을 제공한다. A sixth aspect provides a method for making up skin, comprising applying a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging according to the fifth aspect to the skin of an individual.
상기 방법은 상처를 개선시키거나, 피부 주름을 개선시키거나, 또는 피부 노화를 방지하는 것일 수 있다.The method may be to improve the wound, to improve skin wrinkles, or to prevent skin aging.
상기 방법에 있어서, 상기 화장료 조성물은 두피를 포함하여 피부, 입술 및 모발의 수많은 처리, 특히 미용 처리에서 적용된다.In the process, the cosmetic composition is applied in numerous treatments, in particular cosmetic treatments of the skin, lips and hair, including the scalp.
일 양상에 따른 상처를 치료하기 위한 조성물에 의하면, 개체의 상처를 효율적으로 치료하는데 사용될 수 있다.According to a composition for treating a wound according to one aspect, it can be used to efficiently treat a wound in a subject.
일 양상에 따른 상처를 치료하는 방법에 의하면, 개체의 상처를 효율적으로 치료할 수 있다.According to the method of treating a wound according to one aspect, it is possible to efficiently treat the wound of the individual.
일 양상에 따른 염증성 질환을 치료하기 위한 조성물에 의하면, 개체의 염증성 질환을 효율적으로 치료하는데 사용될 수 있다.According to a composition for treating an inflammatory disease according to one aspect, it can be used to effectively treat an inflammatory disease in an individual.
일 양상에 따른 개체의 상처를 치료하는 방법에 의하면, 개체의 상처를 효율적으로 치료할 수 있다.According to the method for treating an individual's wound according to one aspect, the individual's wound can be efficiently treated.
일 양상에 따른 상처의 개선용, 피부 주름 개선, 또는 피부 노화 방지용 화장료 조성물에 의하여, 효율적으로 상처의 개선, 피부 주름 개선, 또는 피부 노화 방지에 사용될 수 있다. By improving the wound, improving skin wrinkles, or skin anti-aging cosmetic composition according to one aspect, it can be effectively used for improving wounds, improving skin wrinkles, or preventing skin aging.
일 양상에 따른 피부를 화장하기 위한 방법에 의하면, 효율적으로 상처를 개선시키거나, 피부 주름을 개선시키거나, 또는 피부 노화를 방지할 수 있다.According to the method for making up the skin according to one aspect, it is possible to efficiently improve the wound, improve the skin wrinkles, or prevent skin aging.
도 1은 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 존재가 세포에서 베타-카테닌 활성에 미치는 영향을 나타낸 도면이다.1 shows the presence of seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin, affect beta-catenin activity in cells. It is a figure which shows the effect.
도 2는 7개 화합물이 섬유아세포에서 COL1A1 및 COL3A1 유전자의 발현에 미치는 영향을 나타낸 전기영동 사진이다.Figure 2 is an electrophoresis picture showing the effect of seven compounds on the expression of COL1A1 and COL3A1 gene in fibroblasts.
도 3은 7개 화합물이 섬유아세포에서 COL1A1 유전자의 발현에 미치는 영향을 나타낸 도면이다. Figure 3 shows the effect of the seven compounds on the expression of the COL1A1 gene in fibroblasts.
도 4는 7개 화합물이 섬유아세포에서 COL3A1 유전자의 발현에 미치는 영향을 나타낸 도면이다.4 is a diagram showing the effect of the seven compounds on the expression of the COL3A1 gene in fibroblasts.
도 5는 7개 화합물이 대식세포에서 프로스타글란딘 E2 생성에 미치는 영향을 나타낸다. 5 shows the effect of seven compounds on prostaglandin E2 production in macrophages.
도 6은 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 존재가 각질형성세포의 이동을 촉진함을 확인한 결과이다. FIG. 6 shows the presence of seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin to promote migration of keratinocytes. This is the result of checking.
도 7은 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 존재가 MMP-1의 발현 수준에 미치는 영향을 확인한 결과이다.FIG. 7 shows the presence of seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin to the expression level of MMP-1. This is the result of checking the impact.
도 8은 UVB 조사된 사람 섬유아세포에서 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린이 타입-1 프로콜라겐의 발현 미치는 정도를 측정한 결과를 나타낸다.Figure 8 Expression of daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromone, and isoquarcitrin in type 1 procollagen in UVB irradiated human fibroblasts The result of measuring the extent to which it affects is shown.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 7개 화합물의 상처 치유 관련 활성의 확인Example 1: Confirmation of Wound Healing Related Activity of Seven Compounds
본 실시예에서는 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린에 대하여 인 비트로 상처 치유 및 염증 억제 효능을 확인하였다. 상기 화합물은 상업적으로 구입하여 사용하였다.In this example, in vitro wound healing and inflammation inhibition for seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin Efficacy was confirmed. The compound was purchased commercially and used.
1. 베타-카테닌(β-catenin) 활성화 효능 확인1. Confirmation of β-catenin activation effect
HaCaT 인체 각질세포를 우태아 혈청이 2.5(v/v)%로 함유된 DMEM(Dulbecco's Modified Eagle's Media: Hyclone) 배지가 들어있는 24공 플레이트의 웰에서 37℃ 및 5% CO2 배양기에서 24시간 배양하였다. 그 후 배양된 세포에 TOPflash(optimal Tcf binding site reporter gene) 혹은 FOPFlash(far-from-optimal Tcf binding site reporter gene) 및 베타-카테닌 전사 벡터(pRL-CMV reporter 플라스미드)를 Fugene 6(Roche Applied Science, Indianapolis, IN)을 사용하여 형질감염(transfection)시키고 동일한 조건에서 24 시간 배양하였다. 상기한 7가지 화합물을 각각 농도별로 첨가하고 동일한 조건에서 48시간 동안 배양한 후, 세포를 파쇄(lysis)시킨 후 루시페린(luciferin)을 첨가하여 발생한 발광(luminescence)을 측정하였다. 대조군으로서, 동일한 양의 용매 즉, 디메틸술폭시드(DMSO)를 사용하였다.Incubate HaCaT human keratinocytes in wells of 24-hole plates with DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5 (v / v)% fetal calf serum at 37 ° C and 5% CO 2 incubator for 24 hours. It was. The cultured cells were then subjected to TOPflash (optimal Tcf binding site reporter gene) or FOPFlash (far-from-optimal Tcf binding site reporter gene) and beta-catenin transcription vector (pRL-CMV reporter plasmid) to Fugene 6 (Roche Applied Science, Indianapolis, IN) was used for transfection and incubation for 24 hours under the same conditions. Each of the seven compounds was added at different concentrations and incubated for 48 hours under the same conditions. Cells were then lysed and luciferin was added to measure luminescence. As a control, the same amount of solvent, dimethylsulfoxide (DMSO), was used.
그 결과를 표 1 및 도 1에 나타내었다.The results are shown in Table 1 and FIG.
시료sample 농도(uM)Concentration (uM) 루시퍼라제 활성 (대조군 대비 배수)Luciferase Activity (Drainage vs. Control)
대조군Control DMSO DMSO 1One
다프노레틴Daphnoretin 1010 1.21.2
다프노레틴Daphnoretin 2020 2.22.2
이소쿼르시트린Isoquarcitrin 1010 1.21.2
이소쿼르시트린Isoquarcitrin 2020 1.61.6
차마에크로모네Chamaecromone 1010 1.21.2
차마에크로모네Chamaecromone 2020 1.41.4
다프네틴Daphnetin 1010 1.11.1
다프네틴Daphnetin 2020 1.41.4
다프닌Daphnin 1010 1.71.7
다프닌Daphnin 2020 1.81.8
다프네틴-8-β-글루코시드Daphnetin-8-β-glucoside 1010 1.61.6
다프네틴-8-β-글루코시드Daphnetin-8-β-glucoside 2020 1.91.9
루타렌신Rutarencin 1010 1.21.2
루타렌신Rutarencin 2020 1.51.5
도 1은 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 존재가 세포에서 베타-카테닌 활성에 미치는 영향을 나타낸 도면이다. 도 1에서, A, B, C, D, E, F, 및 G는 각각 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린를 나타낸다. 1 shows the presence of seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin, affect beta-catenin activity in cells. It is a figure which shows the effect. In FIG. 1, A, B, C, D, E, F, and G are daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromone, and iso, respectively. Quercitrin is shown.
표 1 및 도 1에 나타낸 바와 같이, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 존재가 세포에서 베타-카테닌 전사 활성을 현저하게 증가시켰다. 이러한 결과는, 이들 7개 화합물이 상처 치유에 효과가 있다는 것을 나타낸다. As shown in Table 1 and FIG. 1, the presence of daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromeone, and isoquarcitrin is beta-catenin in the cells. Markedly increased transcriptional activity. These results indicate that these seven compounds are effective in wound healing.
2. 섬유아세포에서 COL1A1 및 COL3A1 유전자의 발현에 미치는 영향2. Effect on COL1A1 and COL3A1 Gene Expression in Fibroblasts
상기 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린이 인간 섬유아세포에서 콜라겐 유전자의 발현을 촉진하는지 여부를 아래와 같이 확인하였다. Whether the seven compounds, daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin, promote the expression of collagen genes in human fibroblasts Whether it was confirmed as follows.
우태아 혈청이 2.5(v/v)%로 함유된 DMEM(Dulbecco's Modified Eagle's Media: Hyclone) 배지 2 ml이 들어있는 6-웰 평판 배양기(6-well microtiter plate)의 웰에 인체의 섬유아세포 Hs68을 2×106 세포/웰이 되도록 넣고 컨플루언시가 80%가 될 때까지 상기한 바와 동일한 조건에서 배양하였다.Human fibroblast Hs68 was placed in a well of a 6-well microtiter plate containing 2 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5 (v / v) fetal calf serum. 2 × 10 6 cells / well were added and incubated under the same conditions as above until the confluency reached 80%.
이렇게 배양한 세포에 상기 화합물을 지정된 농도로 첨가하고 24 시간 동안 상기한 바와 동일한 조건에서 배양시켰다. 음성 대조군으로는 상기 추출물을 녹이는데 사용한 DMSO를 동량을 사용하였다. 24시간 후, RT-PCR 어세이를 통해 콜라겐 유전자 발현의 정도를 확인하였다. 구체적으로, 24시간 경과한 다음 세포로부터 TRIzol시약(Invitrogen, Carlsbad, CA, USA)을 사용하여 총 RNA를 수확하여 역전사시킨 후, RT-PCR을 다음과 같이 수행하였다. 먼저 cDNA 합성을 위해 상기 RNA를 역전사 효소(reverse transcriptase)를 사용하여 역전사시켰다. RT-PCR은 다음의 특정 프라이머를 프라이머로 사용하여 수행하였다. 각 유전자의 상대적인 mRNA 발현량 값은 β-액틴 값으로 표준화하였다.To the cells so cultured, the compound was added at the indicated concentrations and cultured under the same conditions as described above for 24 hours. As a negative control, the same amount of DMSO used to dissolve the extract was used. After 24 hours, the degree of collagen gene expression was confirmed by RT-PCR assay. Specifically, after 24 hours, total RNA was harvested and reverse-transcribed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from the cells, and then RT-PCR was performed as follows. First, the RNA was reverse transcribed using reverse transcriptase for cDNA synthesis. RT-PCR was performed using the following specific primers as primers. Relative mRNA expression values of each gene were normalized to β-actin values.
도 2는 7개 화합물이 섬유아세포에서 COL1A1 및 COL3A1 유전자의 발현에 미치는 영향을 나타낸 전기영동 사진이다.Figure 2 is an electrophoresis picture showing the effect of seven compounds on the expression of COL1A1 and COL3A1 gene in fibroblasts.
도 3은 7개 화합물이 섬유아세포에서 COL1A1 유전자의 발현에 미치는 영향을 나타낸 도면이다. 도 4 및 도 5의 결과는 도 2의 전기 영동 사진으로부터 분석한 결과이다.Figure 3 shows the effect of the seven compounds on the expression of the COL1A1 gene in fibroblasts. 4 and 5 are the results analyzed from the electrophoresis picture of FIG.
도 4에 나타낸 바와 같이, 차마에크로모네, 다프네틴, 다프닌, 및 다프네틴-8-β-글루코시드는 콜라겐 합성 관련 인자인 COL1A1 유전자의 발현을 활성화시키는 효과가 현저하다는 것을 알 수 있었다.As shown in FIG. 4, it was found that chamaechromone, daphnetine, daphine, and daphnetin-8-β-glucoside had a significant effect of activating the expression of the COL1A1 gene, a collagen synthesis-related factor.
도 5는 7개 화합물이 섬유아세포에서 COL3A1 유전자의 발현에 미치는 영향을 나타낸 도면이다. 도 5에 나타낸 바와 같이, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린은 콜라겐 합성 관련 인자인 COL3A1 유전자의 발현을 활성화시키는 효과가 현저하다는 것을 알 수 있었다.Figure 5 shows the effect of the seven compounds on the expression of the COL3A1 gene in fibroblasts. As shown in FIG. 5, daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin are involved in the expression of COL3A1 gene, a collagen synthesis-related factor. It was found that the effect of activating is remarkable.
β-액틴β-actin COL1A1COL1A1 COL3A1COL3A1
정방향 프라이머Forward primer 서열번호 1SEQ ID NO: 1 서열번호 3SEQ ID NO: 3 서열번호 5SEQ ID NO: 5
역방향 프라이머Reverse primer 서열번호 2SEQ ID NO: 2 서열번호 4SEQ ID NO: 4 서열번호 6SEQ ID NO: 6
3. 대식 세포에서 프로스타글란딘 E2(prostaglandin E2: PGE2) 생성에 미치는 영향3. Effects on Prostaglandin E2 ( PGE2) Production in Macrophages
상기 7개 화합물의 존재하에서 대식세포(macrophage)가 염증 생성 촉진 인자인 프로스타글란딘 E2의 생성 미치는 영향을 확인하였다.In the presence of the seven compounds, macrophage (macrophage) was confirmed the effect of the production of prostaglandin E 2 which is an inflammation promoting factor.
대식세포는 RAW 264.7(ATCC TIB-71)를 사용하였다. 이 세포는 생쥐의 아벨슨 생쥐 백혈병 바이러스-유도된 종양(Abelson murine leukemia virus-induced tumor)로부터 만들어진 것으로서, 단핵세포 및 대식세포 형상을 갖고 부착하여 성장하는 특성을 갖는다. RAW 264.7 세포를 우태아 혈청이 10(v/v)%로 함유된 DMEM(Dulbecco's Modified Eagle's Media: Hyclone) 배지가 들어있는 24-웰 평판 배양기(24-well microtiter plate)의 웰에 넣고 24 시간 동안 상기한 바와 동일한 조건에서 배양하였다. 그 후 상기 7개 각 화합물을 배지에 지정된 농도(uM)로 첨가하고, 24시간 동안 동일한 조건에서 배양하였다. 그 후, 세포를 배양한 배지를 수득하여 배지로 분비된 PGE2의 생성 정도를 PGE2 ELISA assay kit를 이용하여 측정하였다. 음성 대조군은 동일한 양의 DMSO를 사용하였고, 양성 대조군은 리포폴리사카리드(LPS)를 사용하였다. Macrophages were used RAW 264.7 (ATCC TIB-71). These cells are made from Abelson murine leukemia virus-induced tumors of mice, have mononuclear and macrophage morphology, and attach and grow. RAW 264.7 cells were placed in a well of a 24-well microtiter plate containing Dulbecco's Modified Eagle's Media: Hyclone (DMEM) medium containing 10 (v / v) Fetal Bovine Serum for 24 hours. Cultured under the same conditions as described above. Each of the seven compounds was then added to the medium at the indicated concentrations (uM) and incubated at the same conditions for 24 hours. Thereafter, the cultured cells were obtained, and the production of PGE 2 secreted into the medium was measured using a PGE2 ELISA assay kit. Negative controls used the same amount of DMSO and positive controls used lipopolysaccharide (LPS).
도 9는 7개 화합물이 대식세포에서 프로스타글란딘 E2 생성에 미치는 영향을 나타낸다. 도 9에서 세로축은 배양액으로 방출된 프로스타글란딘 E2의 양을 나타낸다. 도 9에 나타낸 바와 같이, 7개 화합물은 대조군에 비하여 프로스타글란딘 E2의 생성을 현저하게 억제하였다. 이들 중 다프노레틴, 차마에크로모네, 및 다프네틴의 억제 효과가 특히 우수하였다.9 shows the effect of seven compounds on prostaglandin E2 production in macrophages. In Figure 9, the vertical axis shows the amount of prostaglandin E 2 released into the culture. As shown in Figure 9, seven compounds significantly inhibited the production of prostaglandin E2 compared to the control. Among them, the inhibitory effect of daphnoretin, chamaechromone, and daphnetin was particularly excellent.
4. 인체 각질형성세포의 이동에 미치는 영향4. Effect on the migration of human keratinocytes
상기 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린이 인체 각질형성세포가 이동(migration)하는 것을 증가시키는지 여부를 아래와 같이 확인하였다. The seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin, migrate to human keratinocytes. Whether to increase was confirmed as follows.
우태아 혈청이 2.5(v/v)%로 함유된 DMEM(Dulbecco's Modified Eagle's Media: Hyclone) 배지 1 ml이 들어있는 24-웰 평판 배양기(24-well microtiter plate)의 웰에 HaCaT 인체 각질형성세포를 2×105 세포/웰이 되도록 넣었다. 다음으로, 상기 각질형성세포를 컨플루언시가 80%로 될 때까지 37℃ 및 5% CO2 배양기에서 배양시킨 후, 배지 중의 세포 단일층을 p10 피펫 팁으로 긁어서 "긁힘-손상"을 유도하였다. HaCaT human keratinocytes were placed in a well of a 24-well microtiter plate containing 1 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5 (v / v) Fetal Bovine Serum. Added to 2 × 10 5 cells / well. Next, the keratinocytes were incubated in a 37 ° C. and 5% CO 2 incubator until the confluency reached 80%, and then the cell monolayer in the medium was scraped with a p10 pipette tip to induce “scratch-damage”. .
다음으로, 상기 긁어진 세포 단일층 상에 무혈청 DMEM 200㎕와 상기 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린을 20 μM 배지의 농도로 첨가하였다. 그 후, 상기한 배양 조건과 동일한 조건에서 인체 각질형성세포를 24시간 동안 배양시켰다. 음성 대조군으로는 상기 추출물을 녹이는데 사용한 DMSO를 동량 처리하였다. 이 후, 크리스탈 비올렛(crystal violet) 시약으로 세포층을 염색한 후 긁힘 상처가 회복되는 정도를 확인하였다. Next, 200 μl of serum-free DMEM and the seven compounds on the scratched cell monolayer, that is, daphnin, daphnetine-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene , And isoquarcitrin were added at a concentration of 20 μM medium. Thereafter, human keratinocytes were cultured for 24 hours under the same conditions as described above. As a negative control, the same amount of DMSO used to dissolve the extract was treated. Thereafter, after staining the cell layer with a crystal violet (crystal violet) reagent was confirmed the extent to which the scratches are recovered.
도 6은 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 존재가 각질형성세포의 이동을 촉진함을 확인한 결과이다. FIG. 6 shows the presence of seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin to promote migration of keratinocytes. This is the result of checking.
도 6에 나타낸 바와 같이, 음성 대조군이 처리된 세포의 긁힘 손상은 24시간 후에 상대적으로 치유되지 않은 채로 남아 있었는데 반해, 다프닌, 이소쿼르시트린, 및 다프네틴-8-β-글루코시드가 처리된 세포에서는 상처 가장자리의 세포 증식과 이동으로 긁힘-손상이 치유되어 24시간 후 긁힌 면적이 감소되었다. As shown in FIG. 6, the scratch damage of the cells treated with the negative control remained relatively untreated after 24 hours, whereas daffinin, isoquarcitrin, and daphnetin-8-β-glucoside were treated. In cells, scratch-damage was healed by cell proliferation and migration at the edge of the wound, reducing the scratched area after 24 hours.
5. UV에 의하여 노화가 유도된 섬유아세포에서 콜라겐 분해효소-1 분비 억제 측정 실험 5. Measurement Experiment of Collagen Degrading Enzyme-1 Secretion in UV-Induced Aging Fibroblasts
상기 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 콜라겐 분해효소-1(Matrix Metalloproteinase-1, MMP-1)의 생성 억제 효능을 측정하였다.Collagen degrading enzyme-1 of the seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin (Matrix Metalloproteinase-1, The inhibitory efficacy of production of MMP-1) was measured.
먼저, 2.5%의 우태아 혈청이 함유된 DMEM 배지가 들어있는 24-공 평판배양기에 사람 섬유아세포를 1×105 세포/공(well)이 되도록 넣고, 90% 컨플루언시 정도 자랄 때까지 배양하였다. 그 후 무혈청 DMEM 배지에 녹여진 상기 7개 화합물을 각각 10 μM농도로 24시간 동안 첨가한 후, UV 조사기를 이용하여 UVB 즉 15 mJ을 조사하였다. 그 후 무혈청 DMEM 배지에 녹여진 상기 7개 화합물을 각각 10 μM 농도로 추가로 첨가하였다. 24시간이 경과한 다음 세포 배양액을 채취하여 원심분리하고 상등액만 수확하였다. First, put human fibroblasts into 1 × 10 5 cells / well in a 24-hole plate incubator containing 2.5% fetal calf serum, DMEM medium, until 90% confluency grows. Incubated. Thereafter, each of the seven compounds dissolved in serum-free DMEM medium was added at a concentration of 10 μM for 24 hours, and then irradiated with UVB, that is, 15 mJ, using a UV irradiator. Thereafter, the seven compounds dissolved in serum-free DMEM medium were further added at a concentration of 10 μM each. After 24 hours, the cell culture was collected, centrifuged, and only the supernatant was harvested.
이후, 채취한 상등액으로부터 콜라겐 MMP-1 ELISA 키트(QIA55, Merck & Co., 미국)를 이용하여 상기 상등액 중 MMP-1의 수준을 측정하였다. 먼저, 1차 항체로 마우스 항-MMP-1 항체가 균일하게 도포된 96-공 평판(96-well plate)의 웰에 상기 상등액을 넣고 2시간 동안 상온에서 인큐베이션하여 항원-항체 복합체가 형성되도록 하였다. 2시간 후 발색단으로서 호스래디스 퍼옥시다제(horseradish peroxidase)가 접합된 항-MMP-1 항체를 96-공 평판의 웰에 넣고 1시간 동안 인큐베이션시켰다. 1시간 후 기질인 테크라메틸벤지딘(tetramethylbenzidine)을 넣어 실온에서 30분간 인큐베이션시켜 발색을 유발시킨 후 다시 종결 버퍼를 넣어 반응을 중지시키면 반응액의 색깔은 노란색을 띄며 반응 진행의 정도에 따라 노란색의 정도가 다르게 나타났다. 노란색을 띠는 96-공 평판의 웰의 흡광도를 흡광계를 이용하여 450/540 nm에서 측정하였다. Then, the level of MMP-1 in the supernatant was measured using the collagen MMP-1 ELISA kit (QIA55, Merck & Co., USA) from the supernatant. First, the supernatant was added to a well of a 96-well plate uniformly coated with a mouse anti-MMP-1 antibody as a primary antibody, and incubated at room temperature for 2 hours to form an antigen-antibody complex. . After 2 hours, anti-MMP-1 antibody conjugated with horseradish peroxidase as a chromophore was placed in a well of a 96-well plate and incubated for 1 hour. After 1 hour, add tetramethylbenzidine as a substrate, incubate at room temperature for 30 minutes to induce color development, and then stop the reaction by adding the termination buffer again. The color of the reaction solution becomes yellow depending on the progress of the reaction. The degree was different. The absorbance of the wells of yellow 96-hole plates was measured at 450/540 nm using an absorbance meter.
도 7은 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린의 존재가 MMP-1의 발현 수준에 미치는 영향을 확인한 결과이다. 그 결과 도 7에 나타낸 바와 같이, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린이 MMP-1 생성 억제 효능에 뛰어난 효과를 가짐을 알 수 있었다. 특히 이소쿼르시트린이 가장 효능이 뛰어남을 확인하였다. 양성 대조군으로는 에피칼로카테킨(Epigallocatechin gallate: EGCG)을 사용하였다.FIG. 7 shows the presence of seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromene, and isoquarcitrin to the expression level of MMP-1. This is the result of checking the impact. As a result, as shown in FIG. 7, daphnin, daphnetin-8-β-glucoside, rutarencin, dafnoretin, daphnetin, chamaechromene, and isoquarcitrin are excellent in inhibiting MMP-1 production. It can be seen that it has an effect. In particular, isoquarcitrin was confirmed to be the most effective. Epigallocatechin gallate (EGCG) was used as a positive control.
6. UV에 의하여 노화가 유도된 섬유아세포에서 콜라겐 발현 촉진 측정 실험6. Measurement Test for Collagen Expression Promotion in Aging-induced Fibroblast Cells
상기 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린이 타입-1 프로콜라겐(Type-1 procollagen)의 발현을 촉진하는지를 확인하였다.The seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromene, and isoquarcitrin are Type-1 procollagen. It was confirmed whether to promote the expression of.
먼저, 2.5%의 우태아 혈청이 함유된 DMEM(Dulbecco's Modified Eagle's Media) 배지가 들어있는 24-공 평판배양기에 사람 섬유아세포를 1×105 세포/ 공(well)이 되도록 넣고, 90% 정도 컨플루언시로 자랄 때까지 배양하였다. 그 후 무혈청 DMEM 배지에 녹여진 상기 7개 화합물을 각각 10 μM 농도로 첨가한 24시간 동안 배양한 후, UV 조사기를 이용하여 UVB 즉 15 mJ을 조사하였다. 그 후 무혈청 DMEM 배지에 녹여진 상기 7개 화합물을 각각 10 μM 농도로 추가로 첨가하고 동일하게 배양하였다. UV 조사전 첨가는 전처리(pretreatment)이고 UV 조사 후 첨가는 후처리(posttreatment)로 시료의 예방 및 치료 효능을 확인하기 위해 두 번 첨가하였습니다. 24 시간 경과한 다음 세포 배양액을 채취하여 원심분리하고 상등액만 수확하고, 프로콜라겐 타입 I C-펩티드 EIA 키트 (Procollagen Type I C-Peptide (PIP)EIA Kit)(Cat. #MK101, Takara Bio Ink., 일본)를 사용하여 수확된 상등액 중 프로콜라겐 타입-I C-펩티드(procollagen type-I C-peptide, PIP)의 수준을 측정하였다. PIP는 생체 내에서 콜라겐을 합성하는 과정에서 프로콜라겐으로부터 콜라겐으로 전환되는 과정에서 잘려 나오는 펩티드로서 PIP의 수준은 콜라겐의 수준과 비례하기 때문에 당업계에서 콜라겐 수준을 확인하는데 지표로서 사용되고 있는 펩티드이다. First, put human fibroblasts into 1 × 10 5 cells / well in a 24-hole plate incubator containing Dulbecco's Modified Eagle's Media (DMEM) medium containing 2.5% fetal bovine serum. Incubate until grown to fluency. Thereafter, the seven compounds dissolved in serum-free DMEM medium were incubated for 24 hours with 10 μM concentration, respectively, and then irradiated with UVB, that is, 15 mJ, using a UV irradiator. Thereafter, the seven compounds dissolved in serum-free DMEM medium were further added at 10 μM concentrations, respectively, and cultured in the same manner. The addition before UV irradiation is pretreatment and the addition after UV irradiation is added twice to confirm the prevention and treatment efficacy of the sample by posttreatment. After 24 hours, the cell culture was collected, centrifuged, and only the supernatant was harvested, and the procollagen Type I C-Peptide (PIP) EIA Kit (Cat. # MK101, Takara Bio Ink. , Japan) was used to measure the level of procollagen type-I C-peptide (PIP) in the harvested supernatant. PIP is a peptide that is cut off in the process of synthesizing collagen in vivo and converted from collagen to procollagen. Since PIP is proportional to the level of collagen, it is a peptide used in the art as an indicator for checking collagen level.
구체적으로 보면, 마우스 항-PIP 단클론 항체가 균일하게 코팅된 96-공 평판의 웰에 호스래디쉬 퍼옥시다제(horseradish peroxidase)(POD)가 접합된 마우스 항-PIP 단클론 항체("항체-POD 접합체"라고도 함)를 첨가하고, 그 후 상기 세포 배양액 및 표준 용액(standard solution)을 각각 첨가하고 37℃에서 3시간 동안 두었다. 그 후 웰을 세척하고 히드로겐 퍼옥시드와 테트라메틸벤지딘(tetramethylbenzidine)을 포함한 기질 용액을 첨가하고 실온에서 15분 인큐베이션하였다. 그 후 웰에 반응 중지 용액(stop solution)을 넣어 반응을 중지시키고 흡광계(plate reader)를 이용하여 450 nm에서 흡광도를 측정하였다.Specifically, a mouse anti-PIP monoclonal antibody ("antibody-POD conjugate) conjugated with horseradish peroxidase (POD) to a well of 96-hole plate uniformly coated with mouse anti-PIP monoclonal antibody. ), And then the cell culture and standard solution were added respectively and left at 37 ° C. for 3 hours. The wells were then washed and a substrate solution containing hydrogen peroxide and tetramethylbenzidine was added and incubated for 15 minutes at room temperature. Thereafter, a stop solution was added to the well to stop the reaction, and the absorbance was measured at 450 nm using a plate reader.
도 8은 UVB 조사된 사람 섬유아세포에서 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린이 타입-1 프로콜라겐의 발현 미치는 정도를 측정한 결과를 나타낸다. 그 결과 도 8에 나타낸 바와 같이, 7개 화합물은 타입-1 프로콜라겐 발현을 촉진하였으며, 특히 다프닌, 이소쿼르시트린, 다프네틴-8-β-글루코시드, 및 루타렌신이 크게 타입-1 프로콜라겐 발현을 촉진하였다. 다프네틴-8-β-글루코시드는 가장 크게 타입-1 프로콜라겐 발현을 촉진하였다.Figure 8 Expression of daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromone, and isoquarcitrin in type 1 procollagen in UVB irradiated human fibroblasts The result of measuring the extent to which it affects is shown. As a result, as shown in FIG. 8, seven compounds promoted the expression of type 1 procollagen, and in particular, daphnin, isoquarcitrin, daphnetin-8-β-glucoside, and rutalensin were largely type 1 pro Collagen expression was promoted. Daphnetin-8-β-glucoside most greatly promoted type 1 procollagen expression.
상기한 실시예에 의하면, 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린은 상처 치유 촉진 및 염증 억제 효과가 우수하였다.According to the above examples, seven compounds, namely daphnin, daphnetin-8-β-glucoside, rutarencin, daphnoretin, daphnetin, chamaechromone, and isoquarcitrin, promote wound healing and inflammation The inhibitory effect was excellent.
또한, 상기한 실시예에 의하면, 자외선이 조사된 조건에서 7개 화합물 즉, 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린은 사람 섬유아세포로부터 MMP-1의 발현을 감소시키고 콜라겐 발현을 촉진시키는 것으로 확인되었다. 즉, 상기 7개 화합물은 사람 섬유아세포에서 자외선이 조사된 조건에서 콜라겐 분해 효소의 발현은 감소시키면서 콜라겐의 발현은 촉진하였다. 따라서, 상기 7개 화합물은 사람 섬유아세포를 포함하는 조직에서, 예를 들면 피부에서 콜라겐 합성을 촉진하여 그 조직의 탄력성을 유지하고 촉진할 수 있다. In addition, according to the above-described embodiment, seven compounds, namely daphnin, daphnetine-8-β-glucoside, rutarencin, daphnoretin, daphnetine, chamaechromeone, and iso under UV-irradiated conditions Quercitrin has been shown to reduce the expression of MMP-1 and promote collagen expression from human fibroblasts. That is, the seven compounds promoted the expression of collagen while reducing the expression of collagenase under the condition of UV irradiation in human fibroblasts. Thus, the seven compounds can promote collagen synthesis in tissues including human fibroblasts, for example in the skin, thereby maintaining and promoting elasticity of the tissues.
따라서, 상기 7개 화합물은 사람 섬유아세포를 포함하는 조직, 예를 들면, 피부 주름을 개선시키거나, 또는 피부 노화를 방지하는데 효과가 있다.Thus, the seven compounds are effective in improving tissues including human fibroblasts, such as skin wrinkles, or preventing skin aging.
명세서에 언급된 서열을 서열목록의 형태로 본 명세서에 첨부합니다.The sequences mentioned in the specification are attached to this specification in the form of sequence listing.

Claims (11)

  1. 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린으로 이루어진 군으로부터 선택된 하나 이상의 화합물, 그의 약학적으로 허용가능한 염, 또는 용매화물을 유효 성분으로서 포함하는 상처 치료용 약학적 조성물.At least one compound selected from the group consisting of daphine, daphnetin-8-β-glucoside, rutarencin, dafnoretin, daphnetin, chamaechromene, and isoquarcitrin, pharmaceutically acceptable salts thereof, or A pharmaceutical composition for treating wounds comprising a solvate as an active ingredient.
  2. 청구항 1에 있어서, 상기 화합물은 β-카테닌의 전사 활성을 증가시키는 것인 조성물.The composition of claim 1, wherein the compound increases the transcriptional activity of β-catenin.
  3. 청구항 1에 있어서, 상기 화합물은 콜라겐 유전자의 발현을 증가시키는 것인 조성물.The composition of claim 1, wherein the compound increases the expression of the collagen gene.
  4. 상처를 치료하기에 유효한 양의 청구항 1 내지 3 중 어느 하나의 조성물을 개체에게 투여하는 단계를 포함하는 개체의 상처를 치료하는 방법.A method of treating a wound in an individual comprising administering to the individual an effective amount of a composition of any one of claims 1-3 to treat the wound.
  5. 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린으로 이루어진 군으로부터 선택된 하나 이상의 화합물, 그의 약학적으로 허용가능한 염, 또는 용매화물을 유효 성분으로서 포함하는 염증성 질환을 치료하기 위한 약학적 조성물.At least one compound selected from the group consisting of daphine, daphnetin-8-β-glucoside, rutarencin, dafnoretin, daphnetin, chamaechromene, and isoquarcitrin, pharmaceutically acceptable salts thereof, or A pharmaceutical composition for treating an inflammatory disease comprising a solvate as an active ingredient.
  6. 청구항 5에 있어서, 상기 화합물은 β-카테닌의 전사 활성을 증가시키는 것인 조성물.The composition of claim 5, wherein the compound increases the transcriptional activity of β-catenin.
  7. 청구항 6에 있어서, 상기 화합물은 콜라겐 유전자의 발현을 증가시키는 것인 조성물.The composition of claim 6, wherein the compound increases the expression of the collagen gene.
  8. 염증성 질환을 치료하기에 유효한 양의 청구항 5 내지 7 중 어느 하나의 조성물을 개체에게 투여하는 단계를 포함하는 개체의 상처를 치료하는 방법.A method of treating a wound in an individual comprising administering to the individual an amount of a composition of any one of claims 5 to 7 effective to treat an inflammatory disease.
  9. 다프닌, 다프네틴-8-β-글루코시드, 루타렌신, 다프노레틴, 다프네틴, 차마에크로모네, 및 이소쿼르시트린으로 이루어진 군으로부터 선택된 하나 이상의 화합물, 그의 약학적으로 허용가능한 염, 또는 용매화물을 유효성분으로 포함하는 상처의 개선용, 피부 주름 개선, 또는 피부 노화 방지용 화장료 조성물. At least one compound selected from the group consisting of daphine, daphnetin-8-β-glucoside, rutarencin, dafnoretin, daphnetin, chamaechromene, and isoquarcitrin, pharmaceutically acceptable salts thereof, or A cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging, including a solvate as an active ingredient.
  10. 청구항 9의 화장료 조성물을 개체의 피부에 적용하는 단계를 포함하는, 피부를 화장하기 위한 방법.A method for making up skin, comprising applying the cosmetic composition of claim 9 to the skin of an individual.
  11. 청구항 10에 있어서, 상처를 개선시키거나, 피부 주름을 개선시키거나, 또는 피부 노화를 방지하는 것인 방법.The method of claim 10, which improves wounds, improves skin wrinkles, or prevents skin aging.
PCT/KR2016/012094 2016-02-24 2016-10-26 Composition for treating wounds, containing daphnin, and use thereof WO2017146341A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2016-0022153 2016-02-24
KR20160022153 2016-02-24
KR10-2016-0039331 2016-03-31
KR1020160039331A KR101769545B1 (en) 2016-02-24 2016-03-31 Composition for treating wound comprising daphnin and use thereof

Publications (1)

Publication Number Publication Date
WO2017146341A1 true WO2017146341A1 (en) 2017-08-31

Family

ID=59685443

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2016/012094 WO2017146341A1 (en) 2016-02-24 2016-10-26 Composition for treating wounds, containing daphnin, and use thereof

Country Status (1)

Country Link
WO (1) WO2017146341A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3666250A1 (en) * 2018-12-10 2020-06-17 ETH Zurich Cosmetic preparations comprising daphne extracts
US11110075B2 (en) * 2019-08-14 2021-09-07 Xi'an Jiaotong University Use of daphnetin in improving function of aortic endothelial cell

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060193789A1 (en) * 2002-10-25 2006-08-31 Foamix Ltd. Film forming foamable composition

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060193789A1 (en) * 2002-10-25 2006-08-31 Foamix Ltd. Film forming foamable composition

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AHN, J. Y. ET AL.: "Effect of Taxifolin Glycoside on Atopic Dermatitis-like Skin Lesions in NC/Nga Mice", PHYTOTHERAPY RESEARCH, vol. 24, no. 7, 29 December 2009 (2009-12-29), pages 1071 - 1077, XP018502987 *
SUNTAR, I. ET AL.: "Daphne Oleoides Schreber ssp. Oleoides Exhibits Potent Wound Healing Effect: Isolation of the Active Components and Elucidation of the Activity Mechanism", RECORDS OF NATURAL PRODUCTS, vol. 8, no. 2, 19 March 2014 (2014-03-19), pages 93 - 109, XP055409688 *
SUNTAR, I. ET AL.: "Efficacy of Daphne Oleoides Subsp. Kurdica Used for Wound Healing: Identification of Active Compounds through Bioassay Guided Isolation Technique", JOURNAL OF ETHNOPHARMACOLOGY, vol. 1, no. 3, 10 April 2012 (2012-04-10), pages 1058 - 1070, XP028502678 *
WITAICENIS, A. ET AL.: "Antioxidant and Intestinal Anti-inflammatory Effects of Plant-derived Coumarin Derivatives", PHYTOMEDICINE, vol. 21, no. 3, 2014, pages 240 - 246, XP055409690 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3666250A1 (en) * 2018-12-10 2020-06-17 ETH Zurich Cosmetic preparations comprising daphne extracts
WO2020120451A1 (en) 2018-12-10 2020-06-18 Eth Zurich Cosmetic preparations comprising daphne extracts
US11896707B2 (en) 2018-12-10 2024-02-13 Eth Zurich Cosmetic preparations comprising Daphne extracts
US11110075B2 (en) * 2019-08-14 2021-09-07 Xi'an Jiaotong University Use of daphnetin in improving function of aortic endothelial cell

Similar Documents

Publication Publication Date Title
WO2016167527A1 (en) Cosmetic composition for skin regeneration and pharmaceutical composition for wound treatment, containing sinapic acid, which is marker component of cynanchum atratum extracts, or cynanchum atratum extracts containing same
WO2017175909A1 (en) Pharmaceutical composition for wound healing containing substance p
WO2019190200A1 (en) Cosmetic composition comprising human adipocyte conditioned media extract and pdrn
WO2017146514A1 (en) Hair care cosmetic composition containing highly stable fibroblast growth factor-9 mutant as active ingredient
WO2011136434A1 (en) Liquid culture concentrate of human adipose-derived stem cells having a skin-regenerating or wrinkle-improving effect and a use therefor
WO2017146341A1 (en) Composition for treating wounds, containing daphnin, and use thereof
WO2016021934A1 (en) Composition containing asparagus officinalis extract for preventing or alleviating striae distensae
WO2011096688A2 (en) Composition for improving scalp and hair health comprising a dibenzo-p-dioxin derivative
KR20210093838A (en) Composition for Inducing Autophagy Activity Comprising 2-Fucosyllactose
KR101818734B1 (en) Composition for treating wound containing stellera chamaejasme extract or fraction thereof and method for treating wound in a subject
WO2014129818A1 (en) Pharmaceutical composition for preventing or treating diabetic wound
KR101769545B1 (en) Composition for treating wound comprising daphnin and use thereof
WO2019245332A1 (en) Pharmaceutical composition for treating wound or activating skin, containing beta-glucan, glycitin, and 4',6,7-trimethoxyisoflavone
CN111840560A (en) Application of calcium ionophore in leucoderma treatment
WO2019143145A1 (en) Composition for treating skin inflammation, comprising resolvin d2 as active ingredient
WO2021206335A1 (en) Peptide for preventing or treating hair loss, and use thereof
WO2021241780A1 (en) Composition for inhibiting hair graying, and use thereof
KR20150115186A (en) Cosmetic composition for anti-stretch mark effect on the skin with the extract of Olea europaea leaf
WO2010077093A9 (en) Composition for promoting hair growth or preventing hair loss comprising erythropoietin
WO2019124603A1 (en) Composition for preventing or treating keloid, containing iwr-1 as active ingredient
WO2021075929A1 (en) Composition, comprising an artemisia princeps leaf extract, for alleviation of skin damage
JP6545856B2 (en) Composition for treating wounds, comprising a venom extract or fraction thereof, and method of treating wounds in an individual
KR100981089B1 (en) Pharmaceutical composition for treatment of vitiligo containing histamine
WO2021215891A1 (en) Hepatic regeneration promoting composition comprising 6-o-trans-feruloyl catalpol as active ingredient
WO2012070888A2 (en) Composition for skin external application containing microbubbles

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16891745

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 16891745

Country of ref document: EP

Kind code of ref document: A1