KR101346779B1 - Composition for inhibiting growth or metastasis of keloids - Google Patents

Abstract

The present invention provides a pharmaceutical composition and a cosmetic composition for inhibiting growth or metastasis of keloids, wherein the pharmaceutical composition and the cosmetic composition comprise an extract of Aneilema keisak as an active ingredient. The extract inhibits the expression of Smad2, which is a signaling material in the TGF-β signaling system, thereby effectively inhibiting growth and/or metastasis of keloids.

Description

Composition for inhibiting growth or metastasis of keloids

The present invention relates to a composition for inhibiting growth or metastasis of keloids, and more particularly, aneilema cassac ( Aneilema) keisak ) relates to a pharmaceutical composition and / or cosmetic composition for inhibiting growth or metastasis of a keloid comprising the extract as an active ingredient.

Keloids are caused by abnormal accumulation of extracellular matrix (ECM) such as excessive secretion of collagen, also called 'benign dermal fibrous tumor'. Keloids, unlike regular scars, are harder, stick out over the skin, have a reddish, irregular surface.

Keloid fibroblasts (KFs) isolated from keloids are pathologically associated with transforming growth factor-beta (TGF-β) dependent signaling systems in terms of regulating extracellular matrix production and fibroblast overgrowth. It is closely related (J Biol Chem. 1986: 261: 4337-4345. Plast Reconstr Surg. 1996: 98: 827-833). TGF-β signaling promotes collagen biosynthesis and keloid cell growth, and inhibition of TGF-β signaling through reduction of TGF-β-neutralizing antibodies and RNA of Smad2 reduces scarring and collagen biosynthesis. Decreased (Int J Surg. 2007: 5: 278-285).

Therefore, it is expected that materials that inhibit TGF-β signaling system may be usefully applied to pharmaceutical compositions or functional cosmetic compositions for the growth and / or metastasis of keloids.

The present inventors searched for various natural products to develop materials derived from natural products having the growth and / or transfer activity of keloids. Surprisingly, we found that Aneilema keisak ) extract was found to effectively inhibit the growth and / or metastasis of keloids by inhibiting the expression of Smad2 (Receptor regulated smad 2), a signaling agent of the TGF-β signaling system in keloid fibroblasts.

Therefore, the present invention is Aneilema Caesak ( Aneilema) It is an object of the present invention to provide a pharmaceutical composition for inhibiting growth and / or metastasis of keloids comprising keisak extract as an active ingredient.

In addition, the present invention is Aneilema Caesak ( Aneilema) It is an object of the present invention to provide a cosmetic composition for inhibiting growth and / or metastasis of keloids containing keisak extract as an active ingredient.

In accordance with an aspect of the present invention, Aneilema Caesak Keisak ) Provided is a pharmaceutical composition for inhibiting growth or metastasis of keloids comprising the extract as an active ingredient. In the pharmaceutical composition according to the present invention, the dosage form of the composition may be an external dosage form selected from the group consisting of a solution, a suspension, an emulsion, an ointment, a pasta, and a patch.

According to another aspect of the invention, Aneilema Caesak Keisak ) Provided is a cosmetic composition for inhibiting growth or metastasis of keloids comprising the extract as an active ingredient.

In the pharmaceutical composition or the cosmetic composition according to the present invention, the extract is an extraction method comprising extracting Aneilema keisak (C ₁ ~ C ₄ alcohol or C _{1 ~} C ₄ alcohol and a mixed solvent of water) Can be obtained by The extraction is to dry the aerial part of Aneilema keisak , and the resulting dried starch is extracted with a solvent of C ₁ -C ₄ alcohol or C ₁ -C ₄ alcohol and water, preferably By ultrasonic extraction.

According to the present invention, Aneilema Caesak keisak ) extracts have been shown to effectively inhibit the growth and / or metastasis of keloids by inhibiting the expression of Smad2 (Receptor regulated smad 2), a signaling agent of the TGF-β signaling system in keloid fibroblasts. Thus, Aneilema Caesak keisak ) extract can be usefully used in pharmaceutical compositions or cosmetic compositions for inhibiting growth or metastasis of keloids.

Figure 1 shows the results of evaluating the effect of Aneilema keisak extract in human keloid fibroblasts (hKFs).
1A measures the growth rate of cells when human dermal fibroblasts (hDFs) and human keloid fibroblasts (hKFs) were dispensed and cultured at 37 ° C. and 5% CO ₂ conditions at three different conditions for 24 hours. One result. In FIG. 1A, the control group (Cont) indicates the culture in DMEM medium supplemented with 10% FBS and 0.1% gentamycin, and the SD indicates the culture in DMEM medium supplemented with 0.1% FBS and 0.1% gentamycin. SD + TGF shows cultured in DMEM medium supplemented with 0.1% FBS, 10 ng / ml TGF-β ligand and 0.1% gentamycin.
Figure 1 B is the result of analyzing the translocation into the nucleus of Smad4 by immunofluorescence (Immunofluorescence) when treated with various natural product extracts to hKFs activated by the TGF-β ligand-activated signaling system.
FIG. 1C shows anilema cassock ( Aneilema ) in hKFs treated with TGF-β ligand to activate signaling system. keisak ) When the extract was treated, the protein expression level of Smad2 in the cells was analyzed by immunoblotting.
FIG. 1D shows the chemotherapeutic bleomycin (BLM; Bleomycin) and anileema cassac ( Aneilema ) when treated with TGF-β ligands. keisak ) The results were analyzed by immunoblotting of the amount of Smad2 protein and the amount of phosphorylated protein of the activated form Smad2 when each extract was treated.
FIG. 2 shows Aneilema on hKFs keisak ) When treated with extract, the results of evaluation of the pathological response are shown.
FIG. 2A shows the same number of cells (1 × 10 ⁴ cells) of hKFs, and then treated with Aneilema keisak extract, after 1, 2, 3 and 4 days. This is the result of measuring the number.
2B is Aneilema Caesak ( Aneilema) keisak ) The cell cycle was analyzed by flow cytometry for hKFs cells treated with different concentrations.
Figure 2 C is the result of measuring the amount of mRNA of Col3A1 when treated with the concentration of Aneilema keisak extract ( Aneilema keisak ).
2D mechanically scrapes identically divided hKFs cells with a sterile tip at 90% density using Aneilema keisak ) is the result of measuring the cell migration (migration) by treating the extract by concentration.
FIG. 2E shows ethanol (CON, control), bleomycin (BLM) 10 μg / ml, and aneilema cassac ( Aneilema ) in hKFs cells keisak ) 10, 20, 40 ㎍ / ml of the extract was treated by immunofluorescence to determine whether the generation of gamma-H2AX foci using the result, it was confirmed whether the DNA damage.

The present invention is Aneilema Caesak ( Aneilema) Keisak ) provides a pharmaceutical composition for inhibiting growth or metastasis of keloids comprising the extract as an active ingredient.

The invention also provides aneilema kasak ( Aneilema) keisak ) Provides a cosmetic composition for inhibiting growth or metastasis of keloids comprising the extract as an active ingredient.

In the pharmaceutical composition or the cosmetic composition according to the present invention, the extract is an extraction method comprising extracting Aneilema keisak (C ₁ ~ C ₄ alcohol or C _{1 ~} C ₄ alcohol and a mixed solvent of water) Can be obtained by Preferably, the extraction comprises drying the aerial part of Aneilema keisak , and extracting the dried starch obtained with a C _{1 ~} C ₄ alcohol or a mixed solvent of C _{1 ~} C ₄ alcohol and water This can be done by.

The amount of the extractant used is not particularly limited and, for example, in a ratio of about 1 to 10 times the volume, preferably about 1 to 5 times the volume, based on 1 weight of the dried outpost of Aneilema keisak . Can be used. The extraction may be carried out by an extraction method such as cold extraction, hot water extraction, ultrasonic extraction, and reflux cooling extraction for about 1.5 to 48 hours, preferably about 2 to 24 hours. Preferably, the extraction can be performed by ultrasonic extraction at room temperature (about 25 ° C), and the extraction process can be performed once or plural times, preferably about 3 times. The obtained extract can be obtained in a liquid or powder form, if necessary, by a conventional method, for example, at a temperature of about 30 to 70 캜, by concentration under reduced pressure or drying under reduced pressure.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier and may be formulated in various formulations according to conventional methods. Preferably, the pharmaceutical composition according to the present invention may be formulated in an external dosage form selected from the group consisting of solutions, suspensions, emulsions, ointments, pastas, and patches, but is not limited thereto.

The pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose , Hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose 2910, polyethylene glycol 6000, polyvinyl pyrrolidone, methyl hydroxybenzoate, propyl hydroxy benzoate and the like. Also included are diluents or excipients, such as fillers, extenders, wetting agents, surfactants and the like. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, suppositories, and the like. Non-aqueous solvents and suspending agents include vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, and ethyl oleate. Such as injectable esters and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Aneilema Caesak contained in the pharmaceutical composition of the present invention The dosage of the keisak ) extract depends on the condition of the patient with the geloid , the severity of the disease, the form of the formulation, the route of administration and the duration, and may be appropriately selected by those skilled in the art. For example, the aneilema kasak ( Aneilema keisak ) extract may be topically administered to the keloid site at a dose of 0.1-100 mg, preferably 1-10 mg per day. Therefore, the pharmaceutical composition of the present invention is suitable for topical administration at a dose of 0.1 to 10 mg / day, preferably 1 to 10 mg / day, Aneilema Cassak ( Aneilema) keisak ) extracts. Each unit dosage form may be formulated to be suitable for administration once or several times a day.

The cosmetic composition according to the present invention can be prepared in various forms according to a method conventionally used in the field of cosmetic composition. For example, the cosmetic composition of the present invention may be in the form of a conventional cosmetic composition such as a cream, a pack, a lotion, an essence, a lotion, a foundation and a makeup base. Can be prepared according to a conventional method. The Aneilema Caesak The content of keisak ) extract may, for example, range from 0.001 to 20% by weight, preferably 0.01 to 15% by weight, most preferably 0.1 to 10% by weight, per unit cosmetic. Of course, the content may be different depending on the degree of inhibition or growth of the desired keloid.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of extract

Aneilema keisak outposts were harvested and dried for about 7 days. 700 ml of anhydrous methanol was added to 279.7 g of dried outposts, and ultrasonic extraction was performed for 2 hours. The ultrasound extraction procedure was performed twice more. The obtained extract was dried under reduced pressure to obtain an extract of 23.24 g of extract.

Test example. Anti-keloid activity assessment

1. Materials and Test Methods

(1) reagent

The primary antibodies, anti-smad4 (cat # SC-7966), anti-β-actin (cat # SC-47778), and anti-alpha-tubulin (cat # SC-8035) antibodies, are Santa cruz Biotechnology, Inc (Santa). cruz, CA, USA). Anti-cleaved PARP (cat # 9546), anti-phosphorylated Rb (pRb, ser807 / 811: cat # 9308), anti-phosphorylated Akt (pAkt, ser473: cat # 4060X), anti Phosphorylated H2AX (pH2AX, ser139: cat # 9718), anti-phosphorylated Smad2 (pSmad2, ser465 / 467: cat # 3101), anti-Smad2 (cat # 3103) antibodies are described in Cell Signaling Technology (Danvers, MA, USA ). Anti-mouse, anti-rabbit antibodies bound to a secondary antibody, peroxidase, were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA). Anti-mouse IgG bound to secondary antibody Alexa 594 was purchased from Molecular Probes (Eugene, OR, USA). Cy2 bound anti-rabbit IgG was purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA). Bleomycin (BLM: Bleomycin, cat # 203408) was purchased from Calbiochem (San Diego, CA, USA).

(2) Cell culture

Human dermal fibroblasts (hDF) and human keloid fibroblasts (hKFs) were obtained from skin samples from patients donated from Gangbuk Samsung Hospital (IRB # KBC-10126). That is, the human dermis skin was separated by treatment with 0.25% Dipase (Gibco) at 4 DEG C for 16 hours. The dermis skin containing the fibroblasts was excised and placed in a Dulbecco's modified Eagle's medium supplemented with 100 U / ml penicillin, 100 ug / ml streptomycin and 10% fetal bovine serum (FBS) modified Eagle's medium (DMEM). Each cell group was maintained until confluence and treated with trypsin. Cells with 2-3 passages were used for the test. The obtained human dermal fibroblasts and keloid fibroblasts were maintained under 37 ° C. and 5% CO ₂ conditions in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamycin (Chemicon).

(3) RNA isolation and quantitative real-time PCR analysis

Total RNA was extracted from the cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions, and then treated with DNase I. Trizol was removed by adding chloroform, and mRNA was precipitated using isopropanol. The precipitated mRNA was washed with 75% ethanol. The light density was measured using a UV spectrometer at 260 nm to 280 nm to determine the amount and purity of the RNA, and agarose gel electrophoresis was performed to confirm the integrity of the RNA. The gene-specific primers used are shown in Table 1 below. GAPDH was used as a housekeeping gene.

gene SEQ ID NO: order Smad2
One Forward 5'-AAACCCTTACCACTATCAGA-3 ' 2 Reverse 5'-TTTGATTCAACTGTTGGTCA-3 ' Col1A2
3 Forward 5'-GGTGGTGGTTATGACTTTGG-3 ' 4 Reverse 5'-TCTGGGTGGCTGAGTCTCAA-3 ' Col3A1
5 Forward 5'-GCTCTGCTTCATCCCACTATTA-3 ' 6 Reverse 5'-AACATTCTCCAAATGGAATT-3 ' GAPDH
7 Forward 5'-AAGGGTCATCATCTCTGCCC-3 ' 8 Reverse Gt;

All amplification procedures were performed under the following conditions in a final reaction mixture (20 μl) containing a final concentrate of 1 × SYBR supermix, 500 nmol / l of gene specific primer, and 2 μl of template: Initial denaturation at 95 ° C. For 45 minutes, followed by 45 cycles at 95 ° C for 45 seconds, 58 ° C for 45 seconds, 72 ° C for 45 seconds, and final elongation at 72 ° C for 5 minutes. After amplification, standard and threshold values of each reaction were determined using a software package (Light cycler480II Roche). To confirm the validity of the PCR, the amplified product was separated into 2% agarose gels and visualized by staining with ethidium bromide.

(4) Immunoblotting

Cells were washed twice with cold phosphate buffered saline (PBS), lysed in 300 μl of tissue lysis buffer and centrifuged at 14,000 rpm for 10 minutes. About 20 μg of total protein was isolated by SDS-PAGE. Proteins were transferred to PVDF membranes and blocked for 1 hour with 5% skim milk powder in Tris-buffered saline (TBS-T) containing 0.1% Tween-20. Incubated with primary antibody in TBS containing 1% BSA solution for 1 hour at room temperature or 4 hours at 4 ℃. The membrane was washed with TBS-T solution and reacted with HRP-bound secondary antibody (0.1 μg / ml; Jackson Immuno Research Laboratories, West Grove, PA) for 45 minutes. Immune responses were detected using an enhanced chemiliminescence detection system (Amersham Biosciences, Piscataway, NJ).

(5) Immunofluorescence

Cells grown on round glass coverslips (# 1, VWR) were fixed for 4 minutes with 4% paraformaldehyde and then infiltrated with PBS containing 0.1% Triton X-100 for 2 minutes. The immobilized cells were further incubated for 1 hour in TBS containing 3% BSA to block and then incubated for 1 hour with specific primary antibodies. The cells were washed three times with TBS-T solution and then incubated with Cy2-binding anti-rabbit secondary antibody (Jacson ImmunoResearch Laboratories) for 45 minutes. Cell DNA was counterstained with 4 ', 6-diimidino-2-phenylindole (DAPI, 0.2 μg / ml in PBS).

(6) FACs analysis

Cells grown in a culture medium containing ethanol as a control and the Aneilema keisak extract obtained in Example 1 were removed from the culture plate using trypsin, washed twice with PBS, and then washed at -20 ° C. The cells were fixed at 4 ° C. for 16 hours using 3 ml of 70% ethanol stored. The immobilized cells were reacted with PBS containing 100 μg / mL of RNase A (Invitrogen) for 30 minutes, and then again with PBS containing 50 μg / mL of propidium iodide (Sigma) for 30 minutes. I was. DNA content measurement was performed using a flow cytometer (FACScanAnalyzer, BD Biosciences), the analysis of the results using a Sync Wizard Model, ModFit LT software package (BD Biosciences). 2N and 4N peaks from cells that have stopped the cell cycle with G1 / S to identify the percent of the first phase (G1 phase), the DNA synthesizer (S phase), and the second phase / cell divider (G2 / M phase). Was set.

(7) Migration assay

After dispensing cells at a density of 90%, scrape the same amount with a micropipette tip, wash twice with PBS, and then ethanol as a control and analeema casa obtained in Example 1 ( Aneilema) keisak ) was incubated for 24 hours in a cell culture medium containing each extract. After that, the degree of metastasis of the cells was photographed, and then Image Pro. Analyzed using the program.

(8) Statistical analysis

Graphical data are presented as mean ± standard deviation (meas ± SD). Statistical significance between the three groups and between groups was analyzed by one way ANOVA, Bonferroni post-test, and Student t-test. Significance was assumed at p <0.05 (*) and p <0.01 (**). Statistical analysis was analyzed by SAS Statistical Analysis v.9.13.

2. Results and discussion

Human dermal fibroblasts (hDFs) and human keloid fibroblasts (hKFs) were dispensed and cultured at 37 ° C. and 5% CO ₂ conditions at three different conditions for 24 hours to measure the growth rate of the cells. Namely, first condition (Cont): culture using medium containing 10% FBS (normal cell cycle state). Second Condition (SD): Culture using medium containing 0.1% FBS (collected into resting cell cycle). In the third condition (SD + TGF-β: cultured with medium containing 0.1% FBS and 10 ng / ml TGF-β ligand. Both hDFs and hKFs were treated with 0.1% fetal bovine serum, which was performed to induce The growth of cells was inhibited in the culture medium containing TGF-β, but the inhibition of hKFs growth was observed in the resting phase induced with TGF-β ligand (A in Fig. 1). It has been reported to promote the biosynthesis of (ECM: Extracellular matrix) constituents elastin, fibronectin, collagen types I and III (Int J Mol Med. 2009: 24: 283-293) .TGF-β signaling system Is intended for the development of keloid growth inhibitors caused by abnormal accumulation of ECM components. Can serve as a target.

The present inventors conducted tests on various kinds of natural product extracts in order to find a substance having an inhibitory activity on the TGF-β signaling system. TGF-β in hKFs was treated with hKFs treated with TGF-β ligands at 10 ng / ml to activate the signaling system. The activity of β signaling system was measured. TGF-β signaling system binds to Smad4, a signaling mediator through phosphorylation of Smad2 / Smad3 proteins, translocates into the nucleus to regulate transcription of various genes (Nature 2003: 425: 577-584). In order to evaluate the effect of TGF-β signaling on the activity of Smad4, the translocation of Smad4 into the nucleus was evaluated by immunofluorescence. Significantly inhibited translocation into the nucleus (FIG. 1B), and it was revealed through immunoblotting that the inhibition of TGF-β signaling system by this extract was due to the inhibition of Smad2 protein expression (FIG. 1). C). As a result of confirming the extract c, Aneilema Caesak ( Aneilema keisak ) extract.

In addition, in the presence of TGF-β ligands, Bleomycin (BLM) and Aneilema Cassak ( Aneilema ), which are chemotherapeutic agents for the treatment of keloids keisak ) When the extract was dissolved and treated in ethanol, respectively, the amount of protein of Smad2 and the amount of phosphorylated protein of Smad2, which is an activated form, were analyzed by immunoblotting. As can be seen from the result of D of FIG. 1, Aneilema Caesak keisak ) extracts inhibited both Smad2 and phosphorylated Smad2 protein levels, whereas bleomycin (BLM) did not affect the amount of Smad2 protein and phosphorylated Smad2 protein. Thus, Aneilema Caesak keisak ) extract has a keloid therapeutic activity by a different mechanism of action than chemotherapeutic bleomycin.

Aneilema Caesak prepared in Example 1 keisak ) extract inhibited the proliferation of hKFs, stopped the cell cycle during the second resting period, and increased the degree of cell aging (A and B of FIG. 2). That is, after dispensing hKFs into 1 x 10 ⁴ cells, the extract prepared in Example 1 was treated by dissolving in ethanol (control was treated only with ethanol), and after 1, 2, 3, 4 days When the number was analyzed, the growth rate of hKFs decreased in a concentration dependent manner (A of FIG. 2). In addition, the cell cycle was analyzed by flow cytometry for the hKFs cells treated with the extracts prepared in Example 1 at concentrations of 0, 10, 20, and 40 ㎍ / mL, respectively. 4N peak, indicating (B of Figure 2, upper panel) was increased. In addition, beta-galactosidase staining (β-galactosidase staining) was performed to measure cell aging. Aneilema kasak ( Aneilema) keisak ) hKFs treated with the extract can be seen to increase the cell aging compared to the control (Fig. 2 B, subpanel).

Fibrosis caused by excessive accumulation of collagen types I and III in ECM components promotes cell proliferation of keloid fibroblasts, which is known to be increased by TGF-β signaling. (Biochem J 1987: 247: 597-604, FASEB J. 2004: 18: 816-827). The extract obtained in Example 1 was dissolved in ethanol and treated separately, followed by real-time PCR. As a result, it was confirmed that the amount of mRNA of Col3A1 encoding collagen type III protein decreased in a concentration-dependent manner. (C in FIG. 2). In addition, aneilema cassac ( Aneilema) obtained in Example 1 by mechanically scraping hKFs cells equally divided at a density of 90% using a sterile tip keisak ) extracts were dissolved in ethanol and treated at different concentrations. Migration decreased concentration dependently (D in FIG. 2).

Chemotherapeutic agents, such as bleomycin, mitomycin C, 5-fluorouracil, doxorubicin, are known to cause DNA damage to form g-H2AX nuclear foci (Cancer Res. 2010: 70: 4112-4122). However, ethanol (CON, control), bleomycin (BLM) 10 μg / ml, and extracts 10, 20, 40 μg / ml prepared in Example 1 were treated in hKFs cells in ethanol and treated by immunofluorescence. Nuclear staining with phosphorylated H2AX and DAPI, one of the indicators of DNA damage, revealed no signs of DNA damage in the cells treated with the extract (FIG. 2E).

Thus, Aneilema Caesak keisak ) extracts are expected to be useful as pharmaceutical compositions and / or functional cosmetics for inhibiting keloid growth and metastasis by effectively inhibiting cell overgrowth, collagen secretion, and cell metastasis in human keloid fibroblasts. do. In particular, the extract does not cause DNA damage, unlike bleomycin used as a keloid chemotherapeutic agent, it can be seen that it has excellent stability.

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Claims (9)

Aneilema Caesak keisak ) Pharmaceutical composition for inhibiting growth or metastasis of keloids comprising the extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the formulation of the composition is an external formulation selected from the group consisting of a solution, a suspension, an emulsion, an ointment, a pasta, and a patch. The method according to claim 1 or 2, wherein the extract comprises extracting anilema keisak with C ₁ -C ₄ alcohol or a mixed solvent of C ₁ -C ₄ alcohol and water. A pharmaceutical composition, characterized in that obtained. The method of claim 3, wherein the extraction is dried anerial part of Aneilema keisak , the dried starch obtained is a C _{1 ~} C ₄ alcohol or a mixed solvent of C _{1 ~} C ₄ alcohol and water Pharmaceutical composition, characterized in that performed by extraction. The pharmaceutical composition according to claim 3, wherein the extraction is performed by ultrasonic extraction. Aneilema Caesak keisak ) Cosmetic composition for inhibiting growth or metastasis of keloids comprising the extract as an active ingredient. The method according to claim 6, characterized in that the extract is obtained by an extraction method comprising extracting Aneilema keisak with C ₁ -C ₄ alcohol or a mixed solvent of C ₁ -C ₄ alcohol and water. Cosmetic composition. 8. The method according to claim 7, wherein the extraction is to dry the aerial part of Aneilema keisak , the dried starch obtained is a C _{1 ~} C ₄ alcohol or a mixed solvent of C _{1 ~} C ₄ alcohol and water Cosmetic composition, characterized in that it is carried out by extraction. The cosmetic composition according to claim 7, wherein the extraction is performed by ultrasonic extraction.

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