KR101769545B1 - Composition for treating wound comprising daphnin and use thereof - Google Patents

Abstract

Provided are a medicinal composite for wound treatment containing daphnin, a medicinal composite for the treatment of an inflammatory disease, a method to treat a wound of a unity, a method to treat an inflammatory disease, and a cosmetic composite for the improvement of a wound, the improvement of skin wrinkles, or the prevention of skin aging. The medicinal composite includes: daphnin and its pharmacologically allowable salt or solvate; daphnoretin and its pharmacologically allowable salt or solvate; and daphnetin and its pharmacologically allowable salt or solvate as effective components.

Description

[0001] The present invention relates to a daphnin-containing wound healing composition,

A pharmaceutical composition for treating an inflammatory disease, a method for treating a wound of an individual, 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. When a wound occurs in the skin, the wound is filled with blood by the natural healing action of the body, and the granule of the platelet is reduced, and the activation of the hageman factor starts and the wound healing process proceeds. Coagulation of blood is a temporary defensive action that protects exposed wound tissues and provides a basis for cells to move during the healing process.

Wound healing and / or healing occurs in three major stages. The first step is an inflammatory phase characterized by inflammation at the traumatic site. Inflammation can be generally short-lived in the absence of significant infection. The second stage is the proliferative phase, which is characterized by epithelialization, angiogenesis, granulation tissue formation and collagen deposition. Angiogenesis with 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 healed. The surface of the injured wound is covered with a layer of keratinocytes, resulting in a new epidermis and a re-epithelialization in which the epithelium is reconstructed. When the re-epithelization is completed, a series of processes are performed in which the wound area is reduced through an increase in connective tissue and a reorganization. The final stage of wound healing is the maturational phase in which fibroblasts differentiate into collagen. During maturation, the junk cells and capillaries of the convalescent tissue gradually disappear, and scarring can occur if the cells and capillaries of these tissues are hyperplasia or not normally degraded.

Since it is important not only to treat wounds quickly during wound healing, but also to treat them without side effects or scars, efforts are being made to find substances with this effect.

1. Korean Patent Application Publication No. KR 10-2015-0121492A 2. PCT International Application Publication WO 2012-102456 A1

1. Ipepk Suntar et al., Journal of Ethnopharmacology, 141, 1058-1070, 2012 2. Lee, Tae-hoon et al., International Journal of Molecular Medicine, 34, 145-152, 2014

The first aspect provides a pharmaceutical composition for treating wounds.

The second aspect provides a pharmaceutical composition for treating inflammatory diseases.

The third aspect provides a method of treating a wound in an individual.

The fourth aspect provides a method of treating an inflammatory disease of an individual.

The 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 a pharmaceutical composition comprising at least one compound selected from the group consisting of daphinine, daphnetin-8-? -Glucoside, rutarnine, daphnetrine, daphenetin, chamaecromone, and isoquercitrin, Or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

The second aspect is a pharmaceutical composition comprising at least one compound selected from the group consisting of daphnin, daphnetin-8-? -Glucoside, rutarnins, daphnoetin, daphenetin, chamaecromone, and isoquercitrin, The present invention provides a pharmaceutical composition for treating an inflammatory disease comprising, as an active ingredient, a salt or a solvate.

In the first and second aspects, the compound may be one that increases the transcriptional activity of &bgr; -catenin and / or increases the expression of collagen gene. Increased transcriptional activity of &bgr; -catenin is associated with promoting wound healing. For example, Fathke C et al., BMC Cell Biol. 2006 Jan 20; 7: 4 suggests that ectopic activation of the beta-catenin-dependent Wnt signal by lithium chloride in the wound leads to epithelial cysts and occasionally hair follicle structures within the dermis Respectively. Bielefeld KA et al., Cell Mol Life Sci. 2013 Jun; 70 (12): 2059-81 report that Wnt and / or beta-catenin signaling pathways play important roles in wound healing.

In the first and second aspects, the compound daphnin, daphnetin-8-? -Glucoside, rutalin, daprenetin, daphenetin, chamaecromone, and isoquercitrin are each represented by the following structure .

,Daphinin:

,

,Daphenetin-8- [beta] -glucoside:

,

,Rutarinsin:

,

,Daphne Norretin:

,

,Daphne Teen:.

,

,Charmone Croton:

,

.Isoquercitrin:

.

The daphnine may be 8-hydroxy-2-oxo-2H-chromen-7-yl -? - D-glucopyranoside. Daphenetin may be 7,8-dihydroxycoumarin. Rutarinsin is 3-hydroxy-3-methyl-5-oxo-5- [[3,4,5-trihydroxy-6- [6-methoxy- -7-yl) oxychromen-7-yl] oxyoxan-2-yl] methoxy] pentanoic acid. Daphnoletin may be 7-hydroxy-6-methoxy-3- (2-oxochromen-7-yl) oxychromen-2-one. Chromone is a (+) - 3- [1- [bis (4-hydroxyphenyl) methyl] -2-oxo-2- (2,4,6-trihydroxyphenyl) Dihydroxy-4H-1-benzopyran-4-one. Isoquercitrin 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-one.

In the above composition, the pharmaceutically acceptable salt is not particularly limited as far as it is pharmaceutically acceptable, but it may preferably be an inorganic acid salt, an organic acid salt or a metal salt. Examples of the preferred inorganic acid salt include hydrochloride, phosphate, sulfate or disulfate. Examples of preferable organic acid salts include malate, maleate, citrate, fumarate, besylate, camylate or eddylate. Examples include calcium salts, sodium salts, magnesium salts, strontium salts or potassium salts.

The term "wound" as used herein refers to a tissue that is cut, torn, broken, burned, traumatized, Means injury to the human body resulting from a disorder or disease that causes injury. The term "tissue" refers to a mass of cells in the body that together form a particular function. Tissues can include eyes, mucus, lungs, kidneys, heart, viscera, tendons, vascular tissues, bones, skin, connective tissue, and nerves, such as spinal cord. The wound may be an open wound whose surface is open, or a closed wound whose surface is not open. An 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 associated with dead tissue resulting from infection, injury or infarction. The ulcer may be a local defect or exfoliation of the surface of the organ or tissue produced by the dissection of the necrotic tissue.

An example of such a wound is the epidermis of the skin; Dermis; Epidermis and dermis; Or the damaged skin of the epidermis, dermis and subcutaneous fat layer.

In addition, examples of such wounds include cuts, incisions (e.g., surgical incisions), abrasions, lacerations, fractures, contusions, burns, , ≪ / RTI > or amputations.

The "treat " provided by the present invention may be to provide wound healing at a reduced time compared to natural healing. The treatment may include improvement and / or alleviation of the wound. In addition, the treatment may include treatment of a wound and / or a wound-related disease. Such treatment may mean healing and / or regeneration of damaged tissue caused by a 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 complications and / or scarring of wound related diseases.

The composition may be for promoting the pre-stage of the wound healing process. In addition, the composition may be one that promotes the proliferative and / or mature phase during the wound healing step. Thus, the composition may be for treating wounds in the proliferative and / or maturation phase.

The composition may be present in an amount of from 0.001% to 80%, such as from 0.01% to 60%, from 0.01% to 40%, from 0.01% to 30%, from 0.01% to 20% %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% % To 10 wt%, or 0.1 wt% to 5 wt% of the compound. The composition may be administered in an amount of 0.01 mg to 1000 mg, such as 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, mg, 0.01 mg to 1 mg, or 0.01 mg to 0.5 mg of the above compound. The composition may be one which does not contain a 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, the lubricant may be magnesium stearate, talc, or combinations 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 hydroxy cellulose, or a combination thereof. The disintegrant may be carboxymethylcellulose calcium, starch glycolate sodium, calcium monohydrogen phosphate anhydrate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The composition may be formulated into a parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin. Skin external agents may be creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, mask packs, drug containing dressings, lotions, or combinations 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.

The composition may be one for promoting expression of a collagen gene in a cell. The composition may increase the expression of collagen, or may increase the activity of the collagen produced by the composition. The composition may also increase the activity or expression of an up-stream enzyme or protein that produces collagen. The composition may be one that increases the expression and / or activity of collagen and has wound healing efficacy. The collagen may be collagen (type I) or collagen (type III). The gene encoding the collagen may be selected from the group consisting of COL1A1 and COL3A1.

A third aspect provides a method of treating a wound in an individual comprising administering to the subject a composition of the first aspect as described above in an amount effective to treat the wound.

The fourth aspect provides a method of treating an inflammatory disease of an individual comprising administering to the subject a composition of the above-described second aspect in an amount effective to treat the inflammatory disease.

The composition is the same as described above.

Administration can be by any method known in the art. Administration can be by direct administration to an individual by any means, for example, by routes such as intravenous, intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration . The administration can be systemically or locally administered. The administration may be topical administration to the site where the wound is present.

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.

Such administration may be in the range of 0.1 mg to 1000 mg, such as 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 1000 mg, 1 mg From 5 mg to 500 mg, from 1 mg to 100 mg, from 1 mg to 50 mg, from 1 mg to 25 mg, from 5 mg to 1000 mg, from 5 mg to 500 mg, from 5 mg to 100 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.

The fifth aspect is a pharmaceutical composition comprising at least one compound selected from the group consisting of daphnin, daphnetin-8-? -Glucoside, rutarnins, daphnoetin, daphenetin, chamaecromone, and isoquercitrin, The present invention provides a cosmetic composition for improving wounds, improving skin wrinkles, or preventing skin aging, which comprises a salt or a solvate as an active ingredient. The above-mentioned compounds are the same as described above.

The term "for the improvement of a wound" may include a use to lower or alleviate the degree of a wound. For example, the wound improvement may be to lower or alleviate the extent of the wound that has already occurred.

The compound promotes the production of collagen in fibroblasts. Collagen is a major component of the skin's dermis and is a resilient protein with strong binding properties to moisture. That is, collagen has a property of imparting elasticity to the skin and has an effect of preventing wrinkles from occurring. As a person gets older, the amount of collagen in the skin decreases, and the skin undergoes an aging process that loses elasticity and wrinkles. Accordingly, the composition of the claimed invention comprising the above compound can be used as a cosmetic composition for skin wrinkle improvement or skin aging prevention.

The cosmetic composition may be provided in all formulations suitable for topical application. For example, as a solution, an emulsion obtained by dispersing an oil phase in an aqueous phase, an emulsion obtained by dispersing an oil phase in water, 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 comprise a moisturizing agent, an emollient agent, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a perfume, a cold agent or a limiting agent. The amount of the additional component such as the above-mentioned moisturizing agent can be easily selected by those skilled in the art within a range not to impair the purpose and effect of the present invention, and the amount thereof is 0.01 to 5% by weight, specifically 0.01 to 3% % ≪ / RTI >

The composition may be present in an amount of from 0.001% to 80%, such as from 0.01% to 60%, from 0.01% to 40%, from 0.01% to 30%, from 0.01% to 20% %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% % To 10 wt%, or 0.1 wt% to 5 wt% of the compound.

A sixth aspect provides a method for creasing skin comprising applying a cosmetic composition for the improvement of wounds, the improvement of wrinkles of skin, or the aging of skin according to the fifth aspect to the skin of an individual.

The method may be to improve wound, improve skin wrinkles, or prevent skin aging.

In this method, the cosmetic composition is applied in numerous treatments, especially cosmetic treatments, of the skin, lips and hairs, including the scalp.

According to a composition for treating a wound according to one aspect, it can be used to treat an individual's wounds efficiently.

According to a method of treating a wound according to one aspect, it is possible to effectively treat a wound of the individual.

According to a composition for treating an inflammatory disease according to one aspect, it can be used to efficiently treat an inflammatory disease of an individual.

According to a method of treating a wound of an individual according to one aspect, it is possible to effectively treat a wound of the individual.

The present invention can be effectively used for improving wounds, improving skin wrinkles, or preventing skin aging by using a cosmetic composition for improving wounds, improving wrinkles of skin, or preventing skin aging according to one aspect.

According to the method for creasing the skin according to one aspect, it is possible to efficiently improve the wound, improve the wrinkles of the skin, or prevent skin aging.

Figure 1 shows the presence of seven compounds: daphnin, daphnetin-8- [beta] -glucoside, rutarnine, daphnoetin, daphnetin, chamaecromone, and isoquercitrin, Fig.
Fig. 2 is an electrophoresis image showing the effect of seven compounds on the expression of COL1A1 and COL3A1 genes in fibroblasts.
Figure 3 shows the effect of seven compounds on the expression of the COL1A1 gene in fibroblasts.
Figure 4 shows the effect of seven compounds on the expression of the COL3A1 gene in fibroblasts.
Figure 5 shows the effect of seven compounds on the production of prostaglandin E2 in macrophages.
Figure 6 shows that the presence of seven compounds, daphnin, daphnetin-8- [beta] -glucoside, rutaran, daprenetin, daphnetin, chamaecromone, and isoquercitrin, promotes migration of keratinocytes .
Figure 7 shows that the presence of seven compounds, daphnin, daphnetin-8-? -Glucoside, rutarnins, daprenetin, daphnetin, chamaecromone, and isoquercitrin, The results are confirmed.
FIG. 8 is a graph showing the expression of type-1 procollagen in human UVB-irradiated human fibroblasts, daphnin, daphnetin-8-? -Glucoside, rutalen, daphnetrine, daphnetin, The results are shown in Fig.

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.

Example  1: Identification of wound healing activity of 7 compounds

In this example, in vitro wound healing and inflammation inhibition was performed on seven compounds: daphinine, daphthenin-8-? -Glucoside, rutarncin, daphnoetin, daphne tin, chamaecromone and isoquercitrin Efficacy was confirmed. These compounds were purchased commercially and used.

1. Beta - Catechin (β- catenin ) Activation confirmation

HaCaT human keratinocytes were cultured in wells of 24 well plates containing DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5% (v / v)% fetal bovine serum for 24 hours in a 37 ° C and 5% CO ₂ incubator Respectively. 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) were then added to the cultured cells using Fugene 6 (Roche Applied Science, Indianapolis, IN) and incubated for 24 hours under the same conditions. The above seven compounds were added to each concentration and incubated for 48 hours under the same conditions. Cells were lysed and luciferin was added to measure luminescence. As a control, the same amount of solvent, i.e., dimethylsulfoxide (DMSO), was used.

The results are shown in Table 1 and Fig.

sample Concentration (uM) Luciferase activity
(Multiple of control) Control group DMSO One Daphne Norretin 10 1.2 Daphne Norretin 20 2.2 Isoquercitrin 10 1.2 Isoquercitrin 20 1.6 Chamaone Crotonne 10 1.2 Chamaone Crotonne 20 1.4 Daphne Tin 10 1.1 Daphne Tin 20 1.4 Daphne 10 1.7 Daphne 20 1.8 Daphnetin-8- [beta] -glucoside 10 1.6 Daphnetin-8- [beta] -glucoside 20 1.9 Rutthalensine 10 1.2 Rutthalensine 20 1.5

Figure 1 shows the presence of seven compounds: daphnin, daphnetin-8- [beta] -glucoside, rutarnine, daphnoetin, daphnetin, chamaecromone, and isoquercitrin, Fig. In FIG. 1, A, B, C, D, E, F, and G are respectively daphnin, daphnetin-8-? -Glucoside, rutalen, daprenetin, daphnetin, Quercitrin.

As shown in Table 1 and Fig. 1, the presence of daphnin, daphnetin-8-? -Glucoside, rutarnins, daphnoetin, daphenetin, chamaecromone, and isoquercitrin, Significantly increased transcriptional activity. These results indicate that these seven compounds are effective in wound healing.

2. In fibroblasts COL1A1  And COL3A1  Effect on gene expression

The seven compounds, daphnin, daphnetin-8-? -Glucoside, rutalenin, daprenetin, daphnetin, chamaecromone, and isoquercitrin, promote the expression of collagen genes in human fibroblasts I checked whether the following.

Human fibroblast Hs68 was added to the wells of a 6-well microtiter plate containing 2 ml of DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 2.5% (v / v) 2 x 10 < ⁶ > cells / well, and cultured under the same conditions as above until the confluency reached 80%.

The cells were cultured in the same conditions as those 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 from the cells using TRIzol reagent (Invitrogen, Carlsbad, Calif., USA) and reverse transcribed, 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. The relative mRNA expression level of each gene was normalized to the beta -actin value.

Fig. 2 is an electrophoresis image showing the effect of seven compounds on the expression of COL1A1 and COL3A1 genes in fibroblasts.

Figure 3 shows the effect of seven compounds on the expression of the COL1A1 gene in fibroblasts. The results of FIGS. 4 and 5 are the results of analysis from the electrophoresis photograph of FIG.

As shown in FIG. 4, it was found that the effect of activating the expression of the COL1A1 gene, which is a collagen synthesis-related factor, in chamaese, daphnetin, daphnin, and daphnetin-8-? -Glucoside was remarkable.

Figure 5 shows the effect of seven compounds on the expression of the COL3A1 gene in fibroblasts. As shown in FIG. 5, the expression of the COL3A1 gene, which is a factor related to collagen synthesis, is expressed by the expression of daphinine, daphnetin-8-? -Glucoside, rutalenin, daphnoetin, daphnetin, chamaecromone and isoquercitrin And the activation effect was remarkable.

beta -actin COL1A1 COL3A1 Forward primer SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 5 Reverse primer SEQ ID NO: 2 SEQ ID NO: 4 SEQ ID NO: 6

3. In macrophages, prostaglandins E _{2 (} prostaglandin E ₂ : PGE ₂ ) On the production

The effect of macrophages on the production of prostaglandin E ₂ , an inflammation-promoting factor, was confirmed in the presence of the seven compounds.

RAW 264.7 (ATCC TIB-71) was used as a macrophage. These cells are made from Abelson murine leukemia virus-induced tumors of mice and have the characteristics of adhering to and growing with monocytes and macrophages. RAW 264.7 cells were placed in wells of a 24-well microtiter plate containing DMEM (Dulbecco's Modified Eagle's Media: Hyclone) medium containing 10 (v / v)% fetal bovine serum for 24 hours And cultured under the same conditions as described above. Each of the seven compounds was then added to the medium at the indicated concentration (uM) and incubated for 24 hours under the same conditions. Thereafter, the culture medium in which the cells were cultured was obtained and the degree of production of PGE ₂ secreted into the medium was measured using the PGE2 ELISA assay kit. Negative controls used the same amount of DMSO and positive controls used lipopolysaccharide (LPS).

Figure 9 shows the effect of seven compounds on the production of prostaglandin E2 in macrophages. 9, the vertical axis represents the amount of prostaglandin E ₂ released into the culture medium. As shown in Fig. 9, the seven compounds significantly inhibited the production of prostaglandin E2 compared to the control group. Among them, the inhibitory effect of daphnoretin, chamaecromone, and daphenetin was particularly excellent.

4. Influence on migration of human keratinocytes

These 7 compounds, namely daphinin, daphnetin-8-? -Glucoside, rutalenin, daprenetin, daphnetin, chamaecromone and isoquercitrin, migrate in the human keratinocytes The following table shows the results.

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, HaCaT human keratinocytes 2 x 10 < ⁵ > cells / well. Next, the keratinocytes were cultured in an incubator at 37 ° C and 5% CO2 until the confluency reached 80%, and a single cell layer in the medium was scratched with a p10 pipette tip to induce "scratch-damage" .

Next, on the scraped cell monolayer, 200 μl of serum-free DMEM was incubated with the above seven compounds: daphinine, daphthenin-8-? -Glucoside, rutalenin, daphnoretin, daphenetin, , And isoquercitrin in a concentration of 20 [mu] M medium. Thereafter, human keratinocytes were cultured for 24 hours under the same conditions as the culture conditions described above. As a negative control, DMSO used to dissolve the extract was treated in the same amount. After that, the cell layer was stained with a crystal violet reagent, and the extent to which the scratch scar was recovered was confirmed.

Figure 6 shows that the presence of seven compounds, daphnin, daphnetin-8- [beta] -glucoside, rutaran, daprenetin, daphnetin, chamaecromone, and isoquercitrin, promotes migration of keratinocytes .

As shown in Figure 6, the scratch damage of cells treated with negative control remained relatively unhealed after 24 hours, whereas daphinine, isoquercitrin, and daphthenin-8- [beta] -glucoside were treated In the cells, scratch-damage was healed due to cell proliferation and migration at the wound edge and the area of scratching was reduced after 24 hours.

5. Inhibition of collagenase-1 secretion by fibroblasts induced by UV irradiation

The above seven compounds, namely, Matrix Metalloproteinase-1 (DOPA), daphnin, daphnetin-8-? -Glucoside, rutalin, daprenetin, daphnetin, chamaecromone and isoquercitrin, MMP-1).

First, human fibroblasts were added to a 24-well plate incubator containing DMEM medium containing 2.5% fetal bovine serum at a concentration of 1 × 10 ⁵ cells / well, and incubated at 90% confluency Lt; / RTI > Then, the seven compounds dissolved in serum-free DMEM medium were added to each well at a concentration of 10 μM for 24 hours, and UVB or 15 mJ was irradiated with UV light. The 7 compounds dissolved in serum-free DMEM medium were then added at a concentration of 10 [mu] M each. After 24 hours, the cell culture was collected, centrifuged and the supernatant was harvested.

Thereafter, the level of MMP-1 in the supernatant was measured using the collagen MMP-1 ELISA kit (QIA55, Merck & Co., USA) from the supernatant collected. First, the supernatant was added to a well of a 96-well plate in which a mouse anti-MMP-1 antibody was uniformly coated with a primary antibody, followed by incubation at room temperature for 2 hours to form an antigen-antibody complex . Two hours later, anti-MMP-1 antibody conjugated with horseradish peroxidase as a chromophore was added to the wells of a 96-well plate and incubated for 1 hour. After 1 hour, tetramethylbenzidine, a substrate, was added and incubated at room temperature for 30 minutes to induce color development. After the reaction was stopped by adding a final buffer, the color of the reaction solution was yellow. Respectively. The absorbance of the wells of the yellow-colored 96-well plate was measured at 450/540 nm using a spectrophotometer.

Figure 7 shows that the presence of seven compounds, daphnin, daphnetin-8-? -Glucoside, rutarnins, daprenetin, daphnetin, chamaecromone, and isoquercitrin, The results are confirmed. As a result, as shown in Fig. 7, it was confirmed that daphnin, daphnetin-8-? -Glucoside, rutalenin, daphnoetin, daphenetin, chamaecromone and isoquercitrin were excellent in the inhibitory effect on MMP- The results are summarized as follows. Especially, isoquercitrin was found to be the most effective. Epigallocatechin gallate (EGCG) was used as a positive control.

6. Experiments to stimulate collagen expression in fibroblasts induced by UV

Type-1 procollagen is a compound that inhibits the expression of the seven compounds: daphinine, daphnetin-8-? -Glucoside, rutalin, daprenetin, daphenetin, chamaecromone and isoquercitrin. Of the cells.

First, human fibroblasts were placed in a 24-well plate incubator containing DMEM (Dulbecco's Modified Eagle's Media) medium containing 2.5% fetal bovine serum at a concentration of 1 × 10 ⁵ cells / well, Lt; / RTI > until grown to fluency. Thereafter, the seven compounds dissolved in serum-free DMEM medium were added at a concentration of 10 μM for 24 hours, and then irradiated with UVB, that is, 15 mJ, using a UV irradiator. Then, the above seven compounds dissolved in serum-free DMEM medium were further added at a concentration of 10 [mu] M and cultured in the same manner. Pre-addition of UV group is pretreatment and addition after UV irradiation is added twice to confirm the efficacy of the sample for posttreatment. After 24 hours, the cell culture was collected, centrifuged, and only the supernatant was harvested. Procollagen Type I C-Peptide (EIA Kit) (Cat. # MK101, Takara Bio Ink. , Japan) was used to measure the level of procollagen type-I C-peptide (PIP) in the supernatant harvested. PIP is a peptide that is cleaved from the process of converting collagen into collagen in the course of synthesizing collagen in vivo. As the level of PIP is proportional to the level of collagen, it is used as an index to identify collagen level in the art.

Specifically, a mouse anti-PIP monoclonal antibody ("antibody-POD conjugate ") conjugated with horseradish peroxidase (POD) to the well of a 96-well plate uniformly coated with a mouse anti-PIP monoclonal antibody Quot;) was added, and then the cell culture solution and standard solution were added, respectively, and the mixture was allowed to stand at 37 DEG C for 3 hours. The wells were then washed and a substrate solution containing hydrogen peroxide and tetramethylbenzidine was added and incubated at room temperature for 15 minutes. After that, stop solution was added to the well to stop the reaction and the absorbance was measured at 450 nm using a plate reader.

FIG. 8 is a graph showing the expression of type-1 procollagen in human UVB-irradiated human fibroblasts, daphnin, daphnetin-8-? -Glucoside, rutalen, daphnetrine, daphnetin, The results are shown in Fig. As a result, as shown in Fig. 8, seven compounds promoted type-1 procollagen expression, and in particular, daphinine, isoquercitrin, daphnetin-8-? -Glucoside, Promoting collagen expression. Daphenetin-8- [beta] -glucoside promoted type-1 procollagen expression most significantly.

According to the above example, seven compounds, namely daphinine, daphnetin-8-? -Glucoside, rutalin, daprenetin, daphnetin, chamaecromone and isoquercitrin, The inhibitory effect was excellent.

In addition, according to the above-described embodiment, it was confirmed that seven compounds, namely, daphinine, daphnetin-8-? -Glucoside, rutalen, daprenetin, daphenetin, Quercitrin has been shown to reduce the expression of MMP-1 from human fibroblasts and promote collagen expression. That is, the seven compounds promoted the expression of collagen while reducing the expression of collagenase in the condition of irradiation with ultraviolet light in human fibroblasts. Thus, the seven compounds can promote collagen synthesis in the skin, for example, in the skin, including human fibroblasts, to maintain and promote the elasticity of the tissue. Thus, the seven compounds are effective in improving the texture of human fibroblasts, for example, improving skin wrinkles or preventing skin aging.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Composition for treating wound daphnin and use thereof <130> PN113419 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 agccatgtac gtagccatcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 ctctcagctg tggtggtgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 tgagcggacg ctaaccccct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 cagacgggac agcactcgcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 agccatgtac gtagccatcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 tacggggcaa aaccgccagc 20

Claims (11)

Daphnin, a pharmaceutically acceptable salt, or solvate thereof; Daphnetin-8- [beta] -glucoside, a pharmaceutically acceptable salt, or solvate thereof; Daphnoretin, a pharmaceutically acceptable salt, or solvate thereof; And a pharmaceutical composition for the treatment of wounds comprising daphnetine, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient. 2. The composition of claim 1, wherein the compound increases the transcriptional activity of &bgr; -catenin. The composition according to claim 1, wherein the compound increases the expression of the collagen gene. A method of treating a wound in an individual comprising administering to the individual a composition of any one of claims 1 to 3 effective to treat a wound, wherein said individual is a non-human mammal. delete delete delete delete delete delete delete

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