KR101587233B1 - Skin topical composition for treating wound, recovering wound or preventing forming scar - Google Patents

Abstract

The present invention relates to a composition to treat wounds, to heal wounds, or to suppress formation of scars, which comprises naphthochalcone derivative represented by chemical formula 1 as an active ingredient. The composition can be used as a pharmaceutical composition for external application to the skin and a cosmetic composition to effectively treat wounds, to heal wounds or to suppress formation of scars.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for external application for skin for inhibiting wound healing, wound healing, or scar formation, and a wound treatment method using the same. BACKGROUND ART [0002]

The present invention relates to a composition for external application for skin for wound healing, wound healing or scar formation inhibition, and more particularly to a composition for external application for skin for wound healing, wound healing or scar formation inhibition comprising a novel naphthocalhodextrin derivative as an active ingredient .

Skin is the outermost organ that functions as a barrier to the body. In the epidermis of the skin, keratinocytes constitute more than 90% of all cells. When skin damage occurs, keratinocytes in the wound area undergo morphological changes and migrate from sedentary cells to migrating cells. The phenotypic phenotype of the migrating cells changes during cell re-epithelialization with cell-cell attachment and cell-substrate attachment. These phenotypes resemble the epithelial-mesenchymal transition (EMT) process in part, which plays an important role in cancer metastasis. Cells that undergo epithelial-mesenchymal transition (EMT) loss during re-epithelialization and cancer metastasis lose contact with each other and with the extracellular matrix (ECM). The extracellular matrix is degraded by the substrate metalloproteinase (hereinafter referred to as MMP), which is a kind of zinc-containing endopeptidase. The relationship between epithelial-mesenchymal transition (EMT) and metalloproteinases (MMPs) has been revealed, and the expression of metalloproteinase is one of the key features of epithelial cells after they have undergone epithelial-mesenchymal transition lost. Metalloproteinase-1, 2, 9, 13, etc. are associated with tissue remodeling and cell migration processes. Membrane 1-metalloproteinase is spatially and temporally regulated during the MCF10A cell migration, which is mediated by the degradation of periplasmic proteins in the laminin-5 (Ln-5) g2 chain. The epithelial-mesenchymal transition is affected by a wide range of regulatory factors. PI3K / AKT / mTOR signaling pathway and MAPK (ERK) have been reported to be involved in the regulation of epithelial-mesenchymal transition. Increased phosphorylation of AKT is associated with induction of epithelial-mesenchymal transition and cell migration. Moreover, the transcription factors Snail and Slug play an essential role in cell migration. Slug expression is observed to be upregulated in wound areas of the in vivo, ex vivo, and in vivo environments. Re-epithelialization was reduced in Slug-null mice compared to wild-type mice.

 Chalcone is a substance abundant in edible plants, known as a precursor of flavonoids or isoflavonoids, and chalcone derivatives are found in many species of Coreopsis. Representative examples of calf's include 2 ', 6'-dihydroxy-4'-methoxycalone, and carthamine, which are contained in plants such as cinnamon, safflower, pepper and the like. Chalcone has a biologically interesting function such as antioxidant, cytotoxic, anticancer, and antimicrobial action. Naphthocalocone is a precursor of various drugs. Naphthocalocone derivatives can be isolated from nature or biochemically synthesized and have a variety of pharmacological actions. Several newly synthesized naphtalocalcone derivatives are known to have antimicrobial action, treatment of endometriosis, and anti-inflammatory action. Considering flavonoid structural analogs, alpha-naphtho flavone is known to promote pro-collagen production and anti-aging in dermal fibroblasts.

Prior art related to the present invention is Korean Patent Application No. 10-2011-0101715, which discloses a composition for improving wrinkles containing a chalcone derivative as an active ingredient. The chalcone derivative of the present invention inhibits collagen synthesis and collagenase activity Wherein the wrinkle-improving composition is a wrinkle-improving composition.

SUMMARY OF THE INVENTION The present invention has been accomplished on the basis of the above problems, and it is an object of the present invention to solve the above-mentioned problems and to solve the above problems, And to provide an effective external wound healing composition, wound healing, or scar formation inhibitory composition containing a tococalcon derivative (TDPN) as an effective ingredient.

Disclosure of Invention Technical Problem [6] The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a 2- (5- (2,4,6-trimethoxyphenyl) -4,5-dihydro- -pyrazol-3-yl) naphthalen-1-ol, hereinafter referred to as TDPN) as an active ingredient.

In one embodiment of the present invention, the pharmaceutical composition preferably induces epithelial-mesenchymal transition, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition preferably promotes cell migration in epidermal keratinocytes, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition preferably promotes phosphorylation of extracellular signal regulatory phosphorylase (ERK) and protein kinase B, but is not limited thereto.

The present invention also provides a pharmaceutical composition for inhibiting scar formation, which comprises the naphthocalkon derivative represented by the general formula (1) as an active ingredient.

In one embodiment of the present invention, the pharmaceutical composition preferably induces epithelial-mesenchymal transition, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition preferably promotes cell migration in epidermal keratinocytes, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition preferably promotes phosphorylation of extracellular signal regulatory phosphorylase (ERK) and protein kinase B, but is not limited thereto.

The pharmaceutical composition containing the naphtalocalone derivative according to the present invention can be administered orally or parenterally in the form of cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma Thereby providing a pharmaceutical composition. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium, stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, a weighting agent, a binder, a wetting agent, a disintegrant, a surfactant, and the like is usually used.

Compositions for parenteral use may also be presented in unit dosage form (e.g., as a single dose of ampoule) or as a small bottle containing several doses and in the form of an appropriate excipient (as shown below). The composition may be in the form of a solution, suspension, emulsion, infusion device or delivery device for insertion, or may be present in the form of a dry powder to be reconstituted with water or another suitable solution prior to use. Apart from the active protein of the invention, the composition may also contain parenterally suitable carriers and / or excipients. The compositions of the present invention may be incorporated into microspheres, microcapsules, nanoparticles, liposomes or the like for controlled release. Furthermore, the compositions may also contain suspending, dissolving, stabilizing or pH adjusting substances, tonicity adjusting substances, and / or dispersing substances.

The composition containing the naphtalocalone derivative of the present invention can be used as a preparation for topical application to the skin which can be applied to the skin, such as cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma The composition may be used as a pharmaceutical composition in the form of an external preparation, but is not limited thereto.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at a daily dose of 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg. The administration may be carried out once a day or several times. The dose is not intended to limit the scope of the invention in any way.

The present invention also provides a cosmetic composition for improving scarring comprising the naphthocalkon derivative represented by Formula 1 as an active ingredient.

In one embodiment of the present invention, the cosmetic composition preferably induces epithelial-mesenchymal transition, but is not limited thereto.

In another embodiment of the present invention, the cosmetic composition preferably promotes cell migration in epidermal keratinocytes, but is not limited thereto.

In another embodiment of the present invention, the cosmetic composition preferably promotes phosphorylation of extracellular signal regulatory phosphorylase (ERK) and protein kinase B, but is not limited thereto.

The cosmetic composition may further comprise ingredients that can ordinarily be contained in the cosmetic composition and may include one or more selected from the group consisting of, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances and carriers However, the present invention is not limited thereto.

The cosmetic composition may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, surfactant-free cleansing, oil, powder foundation, emulsion foundation, wax foundation or spray , But is not limited thereto. More specifically, the cosmetic composition is preferably a formulation such as a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder. It is not.

Hereinafter, the present invention will be described in detail.

TDPN Effect of dermal wound treatment

Whole-thickness incisions were made on the backs of mice and the naphtalocalophan derivatives or vaseline of the present invention were applied daily to wound areas. TDPN treated mice exhibited wound anastomosis about 9 days faster than the vaseline treated animals (Figure 1a). The mean length of open wound area was measured by image program and the percentage of TDPN treated group was untreated (Fig. 1B). Tissue samples were obtained on days 9, 12, and 15, and H & E staining was performed. On day 9, the wound treated with TDPN had an epidermal anastomosis complete and dermal anastomosis was not sufficient and an open area (yellow arrow) was seen, and inflammation and necrosis still remained in the external skin (Fig. 2a). Vaseline treatment In the wound, necrosis and inflammation were involved in the wound area and the leading edge of the dermal layer was observed (black arrow), whereas epidermal anastomosis was not complete. The granular tissue formation in the dermis was incomplete and dermal anastomosis was observed in progress (Fig. 2a). At day 12, in the wound treated with TDPN, epidermis and dermal anastomosis were complete and hair follicles started to show, but the differentiation did not start yet. Contraction of the granular tissue squeezes the hair follicle and pushes it toward the wound. (Fig. 2a) Negative control In the treated wound, granular tissue formation was progressing and dermal anastomosis was complete, but epidermal proliferation was insufficient and epidermal anastomosis was incomplete (Fig. 2a). On day 15, in TDPN- The epidermis and dermis anastomosis were complete and glandular differentiation was ongoing through the wound area (Fig. 2a). There is still no sign of nodules resembling early sweat or sebaceous glands in wound treated with negative control on day 15 (Fig. 2a). The collagen accumulation was stained by Marson tricolor (Fig. 2b). In the TDPN treated laboratory animals, collagen accumulation continued until day 12 and the distribution of collagen fibers was not flat. (Fig. 2b). On the 15th day, however, collagen density was high and flattened like normal dermis. (Fig. 2b). In negative control treated wounds, collagen accumulation was not significantly different from the TDPN treated group (Fig. 2b), suggesting that wound anastomosis promotion may be affected by faster dermal differentiation.

Keratinocytes  Through activation TDPN Of epidermal growth promoting effect

We performed Ki-67 antibody staining to determine cell proliferation (Figure 3). In both cases, we showed the highest diffusion index at day 9. On day 12 in the TDPN group and on the 15th day in the negative control treatment group, cells located in the bilayer of the epidermis (yellow arrow) showed a strong response as seen in normal epidermis (Fig. 3) (Fig. 3). These results suggest that TDPN mainly activates epidermal keratinocytes rather than dermal fibroblasts.

TDPN On by Of keratinocytes  Induction of migration

Re-epithelialization involves migration and proliferation of keratinocytes. The inventors of the present invention have tested these two processes through MTT analysis and wound healing assay for keratinocytes (HaCaT cells), in which TDPN was treated at different concentrations for 24 hours. In the migration assay, TDPN promoted HaCaT cell migration in a dose-dependent manner up to 20 μM. However, there was little difference between 10 μM and 20 μM (FIG. 4A). TGF-β is known to induce epithelial-mesenchymal transition (EMT) and migration of various cell types including keratinocytes, (Fig. 4B) and Transwell infiltration assay (Fig. 4C) in order to compare the ability of TGF- These results show that TDPN induces migration and infiltration comparable to TGF-β at the same time interval of treatment.

We have tested cell growth since migration rates can be affected by growth rate. And keratinocyte proliferation was not affected by TDPN, but was confirmed by three independent analyzes including MTT analysis, blood count calculation analysis, and Hofstede staining analysis (FIG. 4d). These results showed no significant change between treated and untreated groups. To confirm these results, proteins associated with the cell cycle were examined in the treatment groups. Levels of cyclin-dependent kinase E, D1, p53, p21 were not significantly changed in the TDPN treated group (Fig. 4e). These data suggest that TDPN induces keratinocyte migration but not proliferation.

ERK (Extracellular signal regulated phosphorylase) and AKT  Phosphorylation and TDPN of Keratinocytes  Relevance to migration effect

We have examined the expression levels of regulator proteins associated with cell migration such as AKT and ERK (extracellular signal regulatory phosphorylase). The keratinocytes were treated with TDPN for different times and protein lysates were analyzed for phosphorylation of AKT and ERK (extracellular signal regulatory phosphorylase). We used a TDPN stock containing about 0.05% DMSO. At each time point, cells treated with TDPN dissolved in DMSO or DMSO were collected and analyzed by western blotting. The relative density between the TDPN treated group and the control group at each particular time point was calculated (Figure 5a). Within two hours, ERK (extracellular signal regulatory phosphorylase) and AKT signal were activated. These signals increased in a dose-dependent manner at a time point of 2 hours (Figure 5b) and treatment with an inhibitor specific for ERK (extracellular signal regulatory phosphorylase) and AKT weakened TDPN-induced phosphorylation (Figure 5c ) In order to confirm that TDPN-induced keratinocyte migration is mediated by ERK (extracellular signal regulated phosphorylation enzyme) and AKT signal, we used the PIK3 inhibitor (LY49002, 50) and the MEK inhibitor (PD98059, 50) Wound healing analysis was performed using pretreated, TDPN treated or untreated cells. The results showed that the inhibition of AKT and ERK (extracellular signal regulated phosphorylase) mechanisms eliminated the effects of TDPN on migration.

TDPN Of the epithelial-mesenchymal transition (EMT)

The inventors of the present invention have examined whether TDPN promotes re-epithelization through epithelial-mesenchymal transition (EMT). The present inventors treated TGF-β1 (10 ng / mL) and TDPN for 24 hours and observed morphological and biochemical changes. Under the above conditions, morphological changes were not significant (Fig. 7)

The morphological changes of keratinocytes by TGF-β in vitro vary depending on the cell type. HaCaT cells treated with TGF-β1 for 24 hours formed only actin stress fibers, but not morphologically. At the cellular level, however, the major epithelial-mesenchymal transition (EMT) markers such as the epithelial marker proteins Zo-1 and E-cadherin, the hepatic markers, and the transcription factor Slug have changed. Western blotting results confirmed that TDPN treatment increased Slug expression in a time-dependent manner (Fig. 6A), dose-dependent (Fig. 6B) and decreased E-cadherin and Zo-1. This suggests that TDPN treatment causes loss of cell-cell adhesion. In addition, the level of hepatic markers fibronectin slightly increased (Figure 6d)

In epithelial-mesenchymal transition (EMT) tumors and keratinocytes migrating, degeneration and remodeling of the ECM is necessary. The epithelial-mesenchymal transition (EMT) mechanism has been reported to involve MMP expression. In the present invention, it was revealed that the levels of MMP-2 and MMP-9 were not altered by TDPN treatment (Fig. 8). Western blot analysis showed that MMP-1 protein levels in TDPN- (FIG. 6C), and the transcriptional expression patterns showed similar results (FIG. 6D).

As can be seen from the present invention, the present invention relates to a dermatological composition for accelerating wound healing or wound healing, which comprises a chalcone derivative represented by the general formula (1), wherein the composition comprises an effective amount of a dermatological drug composition for the treatment of wound healing, ≪ / RTI >

FIG. 1 is a view showing the degree of wound anastomosis after induction of a full-thickness incision wound of the dermis. a) represents the degree of wound closure taken every 3 days in the TDPN treated (upper) and negative control (lower). b) represents the average of the open wound area measured by the image program and the relative percentage to the original wound.
FIG. 2 is a histological view of dermal wound tissue during treatment. FIG. a) shows the result of H & E staining. b) indicates the result of marzone tricolor staining.
FIG. 3 is a graph showing Ki-67 staining results showing the proliferation of dermally wound tissues during the course of treatment. a) shows the results in the TDPN treated group, and b) shows the results in the control group.
Figure 4 shows the results for the effect of TDPN on migration and proliferation of HaCaT cell lines. a) represents the length of the wound measured after one month of TDPN treatment. b) shows the results of migration analysis for cells. c) shows the results of analysis of the infiltration of TDPN-treated cells using the Transwell chamber method. d) shows the results of MTT analysis, Hoechst staining and trypan blue staining analysis for the proliferation of HaCaT cell lines. e) represents the Western blot analysis of the proteins involved in the cell cycle.
Figure 5 shows ERK (extracellular signal regulatory kinase) and AK phosphorylation associated with the promotion of keratinocyte migration by TDPN. a) shows the results of Western blotting at each time point after treatment with DMSO or 10 μM TDPN and the relative ratio to the control. b) shows the Western blot results obtained after 2 hours of treatment with DMSO or 10 μM TDPN. c) shows Western blot results when pretreated with PI3K inhibitor, MEK inhibitor and treated with TDPN. d) shows the results of wound healing analysis when PI3K inhibitor or MEK inhibitor and TDPN were simultaneously treated.
Figure 6 shows the results for TDPN-induced epithelial-mesenchymal transition (EMT) -like variation. a) shows the results of western blot analysis according to the TDPN treatment time and the relative ratio to the control group according to the repeated experiment. b) shows the western blot analysis result according to the TDPN treatment concentration and the ratio to the GAPDH according to the repeated experiment. c) shows Western blot analysis results for MMP1, collagen I, III, and fibronectin at 24 hours after treatment with 10 μM TDPN. d) shows the relative level of mRNA expression for GAPDH in HaCaT cell line treated with TDPN.
Figure 7 shows morphological changes of HaCaT cells when TDPN was treated for 24 hours. The control group used TGF-β1.
FIG. 8 is a graph showing the results of gelatinization of the medium obtained from HaCaT cells treated with 24-hour TDPN.

The present invention will be described in more detail with reference to the following non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

The naphthocalkon derivative used in the examples of the present invention is a naphtalocalone derivative of the formula (1), which is 2- (5- (2,4,6-trimethoxyphenyl) -4,5-dihydro-1H- pyrazol- ) naphthalen-1-ol (TDPN). This derivative was synthesized by Professor Lim, Yunho (Division of Bioscience and Biotechnology, Konkuk University, Seoul, Korea), Konkuk University. The TDPN stock solution was stored at -20 ° C as a partial sample and diluted to final concentration before use.

Example  1. Cell culture

Human keratinocyte cell line HaCaT was cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin / streptomycin (PAA). The cells were cultured in a humid environment at 37 ° C and 5% CO ₂ . RPMI Medium is a medium developed by the Roswell Park Memorial Institute, a medium used for cell culture. This medium contains a large amount of phosphoric acid and is optimized for use in a 5% CO ₂ environment.

Example  2. scratch  Wound healing analysis

Because the doubling time of HaCaT is approximately 24 hours, the cells were seeded at 50% confluency in the incubator 24 hours prior to wound healing scratch analysis. In the present invention, cells were performed until the formation of a monolayer. Scratches were made by drawing across the culture dish surface with a sterile p-200 pipette tip into the cell monolayer. After the scratches were made, the culture medium was supplemented with the naphthococalone derivatives of the present invention. (Hereinafter referred to as DMSO, Amresco) was used as a control. At the 0 and 24 hours after treatment, a 4x magnification cell tomography was obtained using an Olympus IX70 microscope equipped with a digital camera. The scratches were measured using Image J software and the difference in the widths of scratches between the first and last scratches was calculated.

Example  3. MTT  analysis

Cells were seeded in 96-well plates at a density of 3xlO < ⁴ > cells / mL in 200 [mu] l cells in each well. MTT solution (10 μl of 5 mg / ml solution; Amresco) was added to each well and incubated for 4 hours at 37 ° C. Subsequently, the medium was weakly removed, replaced with 150 μl DMSO and incubated for 30 minutes with shaking to dissolve the precipitate. Samples were measured at 570 nm with a spectrophotometer.

Example  4. Western Blat  analysis

Cells were treated with the naphtalocalophan derivatives of the present invention or demethylsulfoxide as a control in a time-dependent and dose-dependent manner. Cells were harvested by scraping and lysed with RIPA buffer solution. The BCA method (Thermo Scientific) was used for protein concentration determination. Biochemical method refers to bicinchoninic acid method. It is a method in which protein is reduced to copper monovalent ion under alkaline condition. Copper monovalent ion reacts with green biocide to form pink .

The extracts were analyzed by SDS-PAGE and then Western blotted with the appropriate antibodies.

The following antibodies were used for Western blotting: p21 (catalog no. 2947), AKT (9272) phosphorylated AKT (9271S), ERK (extracellular signal regulatory phosphorylase) (4695), Slug (9585S), and GAPDH (2118); E-cadherin (610181) and Zo-1 (610966) were derived from BD Science Transduction; Cyclin E (sc-247), Cyclin D1 (sc-246), p53 (sc-126), phosphorylated ERK (extracellular signal regulated kinase) (sc-7383), collagen I (sc-28888), fibronectin (sc-9068) from Santa Cruz Biotechnology; Metalloproteinase-1 (444209) is from Calbiochem. Secondary antibodies used in Western blots were anti-mouse (PI-2000; Vector Laboratories), anti-rabbit (PI-1000; Vector Laboratories), and anti-goups (AP-107P; Millipore). The blots were analyzed using Image J software and the relative change in the ratio of target protein to dimethicyl sulfoxide control was determined.

Example  5. RT - PCR

The cells were seeded in a 60 mm culture dish and treated with the naphtalocalone derivative of the present invention. The RNA of the cells treated with the naphthocalphone derivative of the present invention was extracted using Trizol reagent (Invitrogen). Subsequently, 2 μg of total RNA was used to synthesize cDNA using a reverse transcriptase kit (Promega). The resultant cDNA was used to perform RT-PCR using the G-Taq kit (Cosmo Genetech, Seoul, Korea) according to the manufacturer's instructions.

RT-PCR used the gene-specific primers in Table 1 below (forward and reverse primers, respectively), which were selected using the Blast Primer program.

Forward primer Reverse primer GADPH 5'-GAAGGTGAAGGTCGGAGTC-3 ' 5'-GAAGATGGTGATGGATTTC-3 ' p53 5'-ACACGCTTCCCTGGATTGG-3 ' 5'-CTGGCATTCTGGGAGCTTCA-3 ' p21 5'-GTCAGTTCCTTGAGCCG-3 ' 5'-GAAGGTAGAGCTTGGGCAGG-3 ' MMP-1 5'-AGGGGAGATCATCGGGAC-3 ' 5'-GGCTGGACAGGATTTTGG-3 ' MMP-2 5'-AACACCTTCTATGGTGCCC-3 ' 5'-ACGAGCAAAGGCATCACCA-3 ' MMP-7 5'-TACAGTGGGAACAGGCTAGG-3 ' 5'-GGCACTCCACATCTGGGC-3 '

Table 1 shows the sequence of the gene-specific primers.

The results of RT-PCR were analyzed using Image J software and the relative change in the ratio of target protein to dimethicyl sulfoxide control was determined.

Example  6. MMP Zymography

MMP < / RTI > zymography was performed according to the method described in Gogly et al, Anal Biochem, 255, pp 211-216, 1998; 8% sodium dodecyl sulfate (SDS) gel (0.01 mg / ml) containing gelatin was used. SDS in the gel was removed by incubation twice with 200 ml of 2.5% Triton X-100 (30 minutes each) at 4 ° C. Thereafter, the gel slabs were incubated in culture buffer overnight at 37 ° C. The gel was then fixed and stained with 0.05% Coomassie blue R-250 for 1 hour. Molecular weight protein markers were immediately visualized in the stained band against the blue color of the dyed gelatin background. The gelatinase activity was clearly seen as clear zones of lysis against a blue background. The gel was scanned to make a permanent record of the results.

Example  7. Transwell  Infiltration analysis

Transwell infiltration assays were performed using commercially available transwell plates (Corning). Cells were harvested by trypsin treatment and 7xlO ⁴ cells / well in 100 μl of medium seeded into the insertion wells. Harvest wells were set with medium containing fetal bovine serum as a chemoattractant factor. After 24 hours, the medium of the insertion wells was replaced with 100 μl of free serum medium containing the naphthocalonone derivative of the present invention, TGF-β1, dimethysulfoxide. Invasive cells of recipient wells were harvested by trypsin treatment and counted by a hemocytometer.

Example  8. Hoechst ( Hoechst ) Dye growth assay

The swollen staining proliferation assay was performed as described in Richards et al., Exp Cell Res, 159, pp. 235-246, 1985; In the 96-well plate wells are of 200㎕ cells were seeded at a density of 3x10 ⁴ cells / ㎖. The cells were treated with the naphthocalon cone derivatives or dimethysulfoxide of the present invention for 24 hours. A solution of Floust 33342 (1 μl of 10 mM stock solution) was added to each well and incubated for 30 minutes at 37 ° C. Cell proliferation was estimated by directly measuring changes in cell fluorescence using a spectrophotometer (SPECTRAFLUOR, Tecan). The machine was equipped with a 365nm broadband filter for emission of the beam, and a 450nm narrow band interference filter was installed for the introduction of the UV filter into the light.

Example  9. Electrical Cell-Substrate Impedance Sensing ( ECIS ) Migration Analysis

HaCaT cells were seeded into the ECIS array at a density of 1 x 10 ⁵ cells / cm 2, and the impedance (ratio of voltage to current at the AC circuit) was measured using an ECIS instrument. A 2400 mA wounding pulse was processed at 6400 kHz for 20 seconds. The media was then replaced with media containing the naphtalcocalon derivatives of the present invention, TGF-? 1, or dimethysulfoxide. The results were output to the ECIS program.

Example  10. Tree plate blue  Cell count analysis

5xlO < ⁴ > cells / well were seeded in 24-well plates. After 24 hours of incubation, the cells were treated at various concentrations with the naphtalcocalon derivatives or dimethysulfoxide of the present invention. All cells were collected by trypsin treatment in each well. Survival cells were identified by trypan blue staining and counted. This experiment was repeated three times.

Example  11. Selection and breeding of experimental animals

BALB / C mice at 4 weeks to 6 weeks of age were placed in conditions free of pathogen for 2 weeks in our laboratory prior to conducting the experiment. Experimental animals were kept under controlled environmental conditions (light / dark cycle, temperature and humidity) and during the experiment, mice were allowed free access to food and water. All animal experimental procedures were carried out in accordance with the guidelines of the IACUC of Cheju National University.

Example  12. Wound recovery experiment and analysis

Mice were anesthetized using intraperitoneal injection of ketamine (100 mg / kg). The back surface was shaved by a laser and a 8 mm diameter biopsy punch was used to create a circular full thickness incision wound of 8 mm diameter on the back surface of the mouse. Mice were divided into 6 groups according to the reagents applied to the wound site (n = 6)

The following reagents were used in wound areas. Vaseline (Unilever Korea), TDPN (Supplied from Konkuk University). The drug was treated daily with the same amount, and the same amount of compound was prepared by mixing with vaseline according to the following ratio: TDPN 200 [mu] M and the same volume of DMSO as negative control. The mixture was applied directly to the wound with a plastic stick (about 50 ug / mouse / day). The digital pictures about the wounds were taken every day with a digital camera (Sony). The wound images were analyzed with an image analysis program (Image J, National Institutes of Health, Bethesda, MD, USA).

Example  13. Immunohistochemical analysis

Mice were euthanized on days 9, 12, 15, and 18 for histological analysis of wound sites. After that, the wound area was cut out, divided into two, and fixed with 10% formalin. The samples were subjected to H & E (hematoxylin and eosin) staining, Masson trichrome staining and normal histological processing of Ki 67.

Claims (12)

1. A pharmaceutical composition for wound healing or wound healing, comprising a naphthocalonide derivative represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition induces epithelial-mesenchymal transition. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition promotes cell migration in epidermal keratinocytes. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition promotes phosphorylation of extracellular signal regulatory phosphorylase (ERK) and protein kinase B. 1. A pharmaceutical composition for inhibiting scar formation, comprising a naphthocalon cone derivative represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition induces epithelial-mesenchymal transition. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition promotes cell migration in epidermal keratinocytes. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition promotes phosphorylation of extracellular signal regulatory phosphorylase (ERK) and protein kinase B. A cosmetic composition for improving scarring comprising a naphthocalon cone derivative represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

The cosmetic composition for improving scarring according to claim 9, wherein the cosmetic composition induces epithelial-mesenchymal transition. The cosmetic composition for improving scarring according to claim 9, wherein the cosmetic composition promotes cell migration in epidermal keratinocytes. The cosmetic composition for improving scarring according to claim 9, wherein the cosmetic composition promotes phosphorylation of extracellular signal regulated phosphorylase (ERK) and protein kinase B.

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