KR101505294B1 - Compositions for reducing keloid or scar - Google Patents

Abstract

The present invention relates to a novel hatching enzyme separated and purified from starfish hatching liquid extract, more specifically, confirming the enzyme activity of the starfish hatching enzyme and establishing the maximum activity condition of the enzyme, and the enzyme is a keloid or scar It is possible to use the composition as a cosmetic composition for the treatment or prevention of keloid or scar.

Description

Compositions for reducing keloid or scar formation < RTI ID = 0.0 >

The present invention relates to a composition for reducing keloid or scar formation using a hatching liquid extract containing a starfish hatching enzyme.

Hatching enzymes (HE) are special enzymes that can be released from the internal hatching cells in the blastocyst stage in vertebrate or invertebrate animals to break down the extracellular coat of eggs and allow the larva to come out [1 -4]. Since the 1960s, several reports have been made on the purification and definition of HEs from various species such as insects, echinoderms, amphibians, tibial fish and mammals.

HE is very important for marine species because almost all species are modified in seawater. To date, the sea urchin Strongylocentrotus purpuratus and Strongylocentrotus intermedius , the shrimp Penaeus chinensis , aurantine Ciona intestinalis , and the flounder Paralichthys olivaceus , have been characterized as metallo-proteases. On the other hand, Sea Urchin HE is a collagenase-like (EC 3.4.24.12) associated with many physiological or pathological processes including cell migration, tissue regeneration, neovascularization, infection, tumor invasion and metastasis Are classified [5].

 Collagen constitutes about 70% of human skin dry weight, and predominant collagen is type I, which makes up 80-90% of total collagen, and Ⅲ, which makes up 10-15% [6]. Collagenase has been used in the treatment of many collagen-mediated diseases as medicines or cosmetics that break down excessively accumulated collagen in tissues such as keloids and scars [7]. Unfortunately, the use of HEs, except for modifications, is not well known. Only Zonase X, a salmon HE, has been commercialized as a release and moisturizer in cosmetics [8].

Starfish is an invertebrate belonging to the starfish river and diptera, and contains steroids, glycosides, anthraquinones, alkaloids, phospholipids, peptides and fatty acids fatty acids have been reported [21]. As a marine predator, starfish cause loss of mussel, oyster and scallop [9].

Therefore, the inventors of the present invention have isolated and purified the hatching enzyme from starfish, and confirmed its characteristics and enzyme activity against collagen. Therefore, it is possible to provide a therapeutic or prophylactic cosmetic for improving keloids and scars caused by excessive accumulation of collagen It is expected to be available as a composition.

[1] Katagiri, Chiaki. Properties of the hatching enzyme from frog embryos. J Exp Zool 1975; 193: 109-18. [2] Denuc 'JM. How embryos escape from their envelopes: A new look at the (phylogenetically) old problem of hatching. Meded Kon Acad Wet Belgie 1985; 46: 1-30. [3] Fan, TJ. Mechanism of two hatching enzyme molecules on the vitelline envelope during the hatching process of Xenopus laevis. Acta Zoologica Sinica 2000; 46: 308-13. [4] Yamagami K. Mechanism of hatching in fish. Fish Phil 1988; 11: 447-99. [5] Lepage T, Gache C. Early expression of a collagenase-like hatching gene in the sea urchin embryo. EMBO J 1990; 9: 3003-12. [6] Leena Ala-Kokko, Aarne Rintala, Eeva-Riitta Savolainen. Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures. Journal of Investigative Dermatology 1987; 89: 238-4. [7] Sabatino, Gregory L. (Chester Springs, PA, US), Del Tito Jr., Benjamin J. (Doylestown, PA, US), Bassett, Phillip J. (Newcastle-under- Hazel A. (Nr Crewe, GB), Hitchcock, Antony G. (Crewe, GB). (2010). Compositions and methods for treating collagen-mediated diseases. US 007811560B2. http://www.freepatentsonline.com/7811560.html. [8] Norway. Aqua Bio Technology, Norway, July. 2012. [Online]. Available: http://www.aquabiotechnology.com/index.php-id=5. [9] Barkhouse C L, M Niles, L-A Davidson. A literature review of sea star control methods for shellfish cultures. Can. Ind. Rep. Fish. Aquat. Sci 2007; 279: vii + 38.

It is an object of the present invention to provide a pharmaceutical composition for reducing keloid or scar formation comprising starfish ( Asterias amurensis ) hatched liquid extract as an active ingredient.

It is also an object of the invention starfish (Asterias amurensis ) hatching liquid extract as an active ingredient, and to provide a cosmetic composition for reducing keloid or scar formation.

In addition, it is an object of the present invention to provide a method for producing starfish hatching liquid extract.

The present invention provides a pharmaceutical composition for reducing keloid or scar formation comprising starfish ( Asterias amurensis ) hatched extract as an active ingredient

The present invention provides a cosmetic composition for reducing keloid or scar formation comprising starfish ( Asterias amurensis ) hatched extract as an active ingredient.

In addition, the present invention provides a method for producing starfish hatching liquid extract.

The novel starfish hatching enzyme obtained by extracting the starfish hatching liquid of the present invention has a potency for degradation of the membrane and specifically degrades collagen I and V to prevent or treat keloids or scars caused by excessive accumulation of collagen And can be usefully used as a pharmaceutical composition and a cosmetic composition.

Brief Description of the Drawings Figure 1 shows the elution peak and enzyme activity of starfish hatching enzyme (HE) in DEAE-Sepharose FF anion exchange chromatography.
FIG. 2 is a graph showing elution peaks and enzyme titer of starfish hatching enzyme (HE) in Sephacryl S-100 gel chromatography.
3 is a view showing an SDS-PAGE pattern of Starfish HE;
(a) Peak II (purified HE) and Peak I separated by Sephacryl S-100 gel chromatography were electrophoresed and stained with Coomassie gel to confirm the molecular weight;
Lane 1: Molecular weight standard marker;
Lane 2: Peak II (refined HE);
Lane 3: Peak I; And
(b) standard protein molecular weight versus relative mobility.
FIG. 4 is a diagram showing a part of the amino acid sequence of starfish hatching enzyme (HE) using LC / MS / MS.
Figure 5 shows the effect of pH and temperature on HE titer and stability;
(a) the potency and stability of HE according to pH; And
(b) HE activity and stability with temperature.
FIG. 6 is a graph showing the effect of metal ions on inhibition of HE activity by a chelator or inhibitor;
(a) A graph showing the inhibitory effect of Starfish HE by the chelating agents EDTA and EGTA;
(b) the titer of the HE reduced by the chelator is restored by each of the metal ions; And
(c) inhibitory effect of HE on Leupeptin, TLCK, TPCK and PMSF inhibitors, respectively.
FIG. 7 is a graph showing the reaction rate constants and hydrolysis specificity of the purified starfish hatching enzyme.
8 is a composition table of the emulsion prototype, which is a cosmetic composition containing the starfish hatching enzyme of the present invention.
Fig. 9 is a composition table of the prototype of Some, which contains the starfish hatching enzyme of the present invention.
FIG. 10 shows the effect of the cosmetic composition containing the starfish hatching enzyme of the present invention in LPS-induced NO production.
11 is a chart for confirming the stability of the cosmetic composition containing starfish hatching enzyme at 4 캜 according to the present invention.
12 is a view for confirming the fibroblast proliferation inhibiting effect of the cosmetic composition containing the starfish hatching enzyme of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for reducing keloid or scar formation comprising starfish ( Asterias amurensis ) hatched extract as an active ingredient.

The above-mentioned hatched liquid extract is preferably a starfish-derived starfish hatching enzyme (HE) purified through a method for producing starfish hatching liquid extract of the present invention, but is not limited thereto.

The above-mentioned hatching enzyme preferably has a maximum titer at pH 6.0 to pH 7.0 and 25 to 35 ° C, more preferably at maximum pH at 8.0 and 30 ° C, but is not limited thereto. The hatching enzyme is preferably stable at pH 4.0 to 6.0 and 30 to 40 ° C, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention may further comprise Zn < ^{2 +} >.

The pharmaceutical composition of the present invention may be various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.

Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. The salt is not particularly limited as long as it is pharmaceutically acceptable so long as it is pharmaceutically acceptable and includes, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, , Benzenesulfonic acid, toluenesulfonic acid, and naphthalenesulfonic acid.

The pharmaceutical composition according to the present invention may be formulated into oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations such as ointments and creams, suppositories and sterile injection solutions And may be formulated in any form suitable for pharmaceutical preparations.

The invention also starfish (Asterias amurensis ) hatching liquid extract as an active ingredient, and to provide a cosmetic composition for reducing keloid or scar formation.

The above-mentioned hatched liquid extract is preferably a starfish-derived starfish hatching enzyme (HE) purified through a method for producing starfish hatching liquid extract of the present invention, but is not limited thereto.

The above-mentioned hatching enzyme preferably has a maximum titer at pH 6.0 to pH 7.0 and 25 to 35 ° C, more preferably at maximum pH at 8.0 and 30 ° C, but is not limited thereto. The hatching enzyme is preferably stable at pH 4.0 to 6.0 and 30 to 40 ° C, but is not limited thereto.

The cosmetic composition suppresses the formation of fibroblasts and can suppress the induction of keratin due to excessive collagen production.

The stability of starfish HE to the pH and temperature helps skin care due to its acidity (pH 4.4-5.6), and the change in imbalance in skin causes skin to normally peel off dead cells from the surface.

In addition, the cosmetic composition of the present invention may further contain Zn ^{2 +} .

Also, the cosmetic composition of the present invention can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, , Soaps, cleansing foams, cleansing lotions, cleansing creams, lotions and body cleansers. When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component When the formulation of the present invention is a solution or an emulsion, a solvent, a solvent or an emulsifier is used as a carrier component. Examples of the solvent include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate Glycolate, glycerol aliphatic ester, polyethylene glycol, or fatty acid esters of sorbitan. When the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used. When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

The cosmetic composition of the present invention may comprise a composition selected from the group consisting of water-soluble vitamins, useful vitamins, high molecular weight peptides, polymeric polysaccharides and sphingogly lipids. In addition to the above components, Antimicrobial agents, antioxidants, plant extracts, pH adjusters, alcohols, coloring matters, perfumes, blood circulation accelerators, coolants, antiperspirants, antioxidants, emollient agents, surfactants, organic and inorganic pigment organic powders, , Purified water, and the like may be blended. In addition, the cosmetic composition of the present invention can be prepared into any formulation in which the cosmetic composition is usually prepared, and can be, for example, an emulsion, a cream, a lotion, a pack, a foundation, a lotion, a serum, a cleanser and a hair cosmetic.

In addition,

1) collecting egg of starfish and dejellied to obtain a precipitate;

2) obtaining a sperm from a starfish cellulase;

3) stirring the precipitate of the step 1) and the sperm of the step 2) to prepare a hatched liquid;

4) dialyzing the incubation solution of step 3);

5) eluting the dialyzed solution of the step 4) with a column to obtain a fraction; And

6) dialyzing the active fraction in the fraction obtained in step 5).

In a specific embodiment of the present invention, the present inventors isolated and purified the hatching enzyme from the hatching solution of starfish to obtain a hatching liquor extract containing the 110.9 kDa hatching enzyme. The hatching enzyme is mainly a metal-protease with a metal ion (especially a divalent cation), which is chelated by a chelate such as EDTA or EGTA. In the present invention, it was confirmed that the potency of the starfish HE of the present invention was strongly restored by zinc ions, and the zinc starch was classified as zinc metal-protease. In addition, the inhibitors TLCK and TPCK are used to alkylate and inactivate the histidine residues of the trypsin and chymotrypsin tigers, and the PMSF and leupeptin respectively substitute the hydroxyl groups of the serine residues of the tigecycline with sulfonylation and sulfhydryl (sulfydryl) to inhibit the enzyme titer, starfish HE can be classified as trypsin-type serine protease. Thus, the starfish HE isolated and purified in the present invention confirmed the enzyme characteristics and enzyme titer showing enzyme diversity characterized by zinc metal- and trypsin-type serine protease (see FIG. 3 and Table 1). In addition, the maximum potency conditions of the enzyme according to pH, temperature or metal ion were established (refer to FIGS. 4 and 5 and Table 2), and the enzyme epidemiological characteristics were confirmed, and it was confirmed that the enzyme specifically degraded collagen (See Fig. 6 and Table 3). Thus, the starfish HE of the present invention, which has potent and specific hydrolytic potency against type I collagen, is characterized by excessive accumulation of collagen, which is the connective tissue, and keloid and hypertrophic scar treatment and prevention, which are known to increase the production of type I collagen And it has been confirmed that the prototype cosmetic composition containing the starfish hatching enzyme of the present invention is safe without causing inflammation and thus it can be used as a cosmetic composition for reducing keloid and hypertrophic scar formation. In addition, it has been confirmed that collagen-induced keratin is excessively inhibited by fibroblasts, which are generated when a wound is generated, and thus it can be used as a cosmetic composition for exfoliating.

Hereinafter, the present invention will be described in detail with reference to Examples and Preparation Examples. However, the examples and preparations according to the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples and preparations. Embodiments and manufacturing examples of the present invention are provided to enable those skilled in the art to more fully understand the present invention.

< Example  1> Production and identification of starfish-derived hatchery extract

<1-1> Manufacture of Starfish Hatching Liquid Extract

The present inventors isolated and purified a hatching enzyme (HE) from a starfish hatching solution.

Specifically, body length: 15.7-16.3 cm; Arm length: 8.5-8.9 cm; Weight: 96.1-99.9 g adult starfish ( Asterias amurensis ) were collected in Gangneung (Korea) in May 2011. Approximately 400,000 eggs were placed in 1 liter of Kester artificial sea water (KASW, salinity 35.00 ‰, chlorinity 19.00 ‰, pH 7.8), and KASW was dejelled by adjusting to pH 5.5 with 1 N HCl. After 10 minutes, the supernatant was drained and the precipitate was washed three times with the same volume of KASW. The sperm was squeezed out of the volunteers and stored at 4 ° C until fertilization. About 100,000 eggs contained in 250 ml of KASW were added to the sperm at ~0.002% and gently stirred for 30 seconds. After 10 minutes, the suspension was diluted with KASW to 1%, and the incubation solution was stored at 16 DEG C for 16 hours. Ammonium sulfate was then gently added to 100 ml of the incubation solution overnight to a degree of saturation of 70%. The resulting pellet was centrifuged at 7728 xg for 30 minutes and dissolved in 10 ml of buffer A. The pellet was resuspended in buffer A (10 kDa cutoff membrane, Rancho Dominguez, California, USA) using a spectrum dialysis membrane Lt; / RTI &gt; overnight. The dialyzed precipitate was applied to a pre-equilibrated DEAE-sepharose Fast Flow column (Amersham Pharmacia Biotech, Uppsala, Sweden) (2.6 x 30.0 cm). The bound proteins were eluted with a linear concentration gradient of 0-1.0 M NaCl in a buffer A 300 ml at a flow rate of 1.5 ml / min (Fig. 1). The peak II was fractionated into 3 ml of fraction collector (Gilson, Middleton, WI, USA). More than 50% of the maximal titer fractions were isolated and dialyzed against buffer A and then concentrated to 4 ml by ultrafiltration. The concentrated enzyme solution was applied to a Sephacryl S-100 gel-filtration column (Amersham Pharmacia Biotech, Uppsala, Sweden) (2.6 x 90.0 cm) equilibrated with an equilibrium buffer at a flow rate of 1.5 ml / min (3 ml per tube). Fractions with greater than 50% of the maximum activity were isolated and dialyzed against buffer A.

As a result, a peak II predicted with purified starfish hatching enzyme (HE) was obtained (FIG. 2).

<1-2> Confirm protein concentration

The present inventors measured the protein concentration in the process of separating the starfish differentiating enzyme of Example <1-1> by the Bradford method.

Specifically, the relative protein content of each chromatographically separated fraction using bovine serum albumin as the calibration standard was calculated by measuring the absorbance at 595 nm (Table 1).

<1-3> Hatching of starfish hatching liquid extract enzyme Potency  Confirm

Since Starfish HE has a choriolytic titer for specific membrane degradation, it is important to note that the turbidity is changed by the protein fragments generated by the hydrolysis of the egg shell, and the starfish hatching extract purified using the egg shell membrane is the hatching enzyme And were confirmed by the activity analysis.

Specifically, about 5,000 eggs counted by an optical microscope (DM2500, Leica Microsystem GmbH, Wetzlar, Germany) were dejelled and washed with water, removed through a 100 탆 mesh and squeezed with a size-4 injection needle. The collected membranes were washed twice with distilled water and sonicated for 10 seconds at 35 kHz (MSONIC, Mirae Ultrasonic, Seoul, Korea). After 10 minutes of precipitation, the upper layer of the striatum was obtained and then centrifuged at 1932 xg for 15 minutes (5810R, Eppendorf, Hamburg, Germany). The obtained membrane was thoroughly washed with distilled water and resuspended in 10 ml of buffer A (20 mmol L ^- Tris-HCl, pH 7.4) to obtain an egg envelope as a substrate for the hatching enzyme. 100 쨉 l of the egg shell (500 eggs / ml) and the same volume fraction (Peak I and II) were mixed and incubated at 30 째 C for 30 minutes. The enzyme reaction was stopped by the addition of 2.8 ml of 20% cold trichloroacetic acid (TCA). After incubation at 4 ° C for 30 minutes, the mixture was centrifuged at 1932 × g for 30 minutes at 4 ° C. and absorbance was measured at 280 nm with a spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The rate of increase in absorbance at 0.001 / min at 280 nm is defined as the choriolytic constant: 1 U (ΔA 280, 0.001 / min).

As a result, the peak Ⅱ (HE) showed a yield of 14.28% and the purification degree was 7.42 times. The specific activity of the purified HE was 449.62 U / mg, but the peak I had a lower specific activity than the peak Ⅱ , And non-target enzyme without enzyme activity (Table 1).

step Total protein
(mg) Total potency
(? A 280 0.001 / min) Specific potency
(? A 280 0.001 / min mg) yield
(%) refine
(ship) Hatching liquid 27.00 1637.00 60.63 100.00 1.00 (NH) ₂ SO ₄ precipitate 11.00 821.40 74.67 50.18 1.23 DEAE-Sepharose 6.23 617.75 99.16 37.74 1.64 Peak I 4.95 415.25 83.89 25.37 1.38 Peak II 1.79 283.50 158.82 17.32 2.62 Sephacryl S-100 3.01 554.45 184.20 33.87 3.04 Peak I 2.49 320.65 128.72 19.59 2.12 Peak II 0.52 233.80 449.62 14.28 7.42

Data were presented as standard = SD of triplicate experiments, and statistical significance was analyzed using one-way ANOVA. P <0.05 was considered significant.

<1-4> Confirmation of purity and molecular weight of peaks I and II

The present inventors have found that the purity of the starfish hatching enzyme Peak II isolated from the starfish hatching solution extract in Example <1-1> The molecular weight was confirmed by electrophoresis.

Specifically, the glyceraldehyde-3-phosphate dehydrogenase (36 kDa), egg-derived ovalbumin (45 kDa), glutamic acid dehydrogenase (glutamic acid) dehydrogenase (55 kDa), albumin, bovine serum (66 kDa), rabbit Muscle derived phosphorylase b (97 kDa), E. coli (Sigma-Aldrich, St. Louis, Mo., USA), consisting of β-galactosidase (116 kDa) and myosin (205 kDa) After performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with sodium dodecyl sulfate-polyacrylamide gel (12% separating gel and 5% stacking gel), Coomassie blue staining was performed.

As a result, it was confirmed that the molecular weight of the hatching enzyme peak II was 110.9 kDa, but the peak I was different from the peak II in the characteristics of SDS-PAGE (FIG. 3A). In addition, the relative movement of the protein with respect to the molecular weight is shown in FIG. 3B.

For reference, the starfish HE has a molecular weight of 110.9 kDa, which is similar to that of invertebrate HEs; Sea urchin Strongylocentrotus purpuratus (57 kDa), Paracentrotus lividus (51 kDa), Strongylocentrotus intermedius (44 kDa) and Hemicentrotus pulcherrimus (31 kDa): vertebrates; Flounder ( Paralichthys ) olivaceus (34.8 kDa), teleosts Fundulus heteroclitus (15-40 kDa), medaka Oryzias latipes (LCE, 25.5 kDa, HCE, 24 kDa), Salmo gairdneri Rich (10 kDa) and Esox lucius L. (10-15 kDa): frog-like amphibian Rana pirica (56 kDa) and Xenopus laevis (40 kDa): and the largest among rat-like mammals (74 kDa).

<1-5> Identification of Fraction of Starfish Hatching Liquid Extract

The inventors of the present invention compared some amino acid sequences by LC / MS / MS analysis of starfish hatching enzyme Peak II, which was separated from the starfish hatching extract.

Specifically, PNGase was used for the oligosaccharide digestion from glycoprotein and glycopeptide. That is, 50 μl of the incubation enzyme was added to 50 μl of denaturing buffer (0.5% SDS, 1% β-mercaptoethanol), and the mixture was boiled in water at 100 ° C. for 10 minutes. After cooling at room temperature, 10 μl of reaction buffer (0.05 mM phosphate, pH 7.5), 5 μl of 15% Triton X-100 and 5 μl of PNGase F were added and reacted at 37 ° C for 2 hours. After the reaction, the reaction was stopped by boiling in water at 100 ° C. for 10 minutes, and 10 μl of the gel was removed by SDS-PAGE. After lowering the gel, the peptide was extracted from the gel piece of SDS-PAGE using acetonitrile and aqueous solution, mixed with the first supernatant, and lyophilized. The lyophilized samples were then dissolved in 23 μl of 50 mM ammonium bicarbonate and analyzed by LC / MS / MS. Chromatogram analysis was performed using Ultimate LC system (Dionex, UK). The molecular weight of the protein was determined by reverse phase chromatography (75 μm C18 PepMap colum) using 0.1% formic acid / acetonitrile in three steps of solvent gradient Respectively. The solvent gradient was developed at a flow rate of 200 nl / min for 60 min and the eluent was ionized by electrospray ionization using a Mass-Lynx v4.0 program using a Z-spray source coupled to QTof-micro (Waters Corp.) . The analyzer was operated in a data - independent mode and was selected in order of magnitude based on the intensity of the ions by collision mediation for mass sequencing. Data analysis was performed using the impact energy record based on the mass and charge ratio (m / z) and the charge state of the sample (FIG. 4). The results of the analysis were compared with some of the identified sequences in other starfish using the BLAST (Basic Local Alignment Search Tool) on the data base at http://blast.ncbi.nlm.nih.gov/Blast.cgi.

As a result, the value of pic y (7) in the pic obtained by LC / MS / MS analysis of Peak II isolated and purified from the starfish hatching extract of the present invention was evaluated by LC / MS / MS of the membrane protein of Patiria pectinifera and Acanthaster planci Val-Ser-Ala-Val-Tyr-Asp-Asp-Gln-Gly-Arg. Therefore, it was confirmed that the amino acid sequence of the starfish hatching enzyme of the present invention is similar to the amino acid sequence of the egg membrane protein.

< Example  2> Hatching enzyme Potency  And stability verification

<2-1> pH  And temperature-dependent hatching enzyme Potency  And stability

The inventors have confirmed the potency and thus stability of the incubation enzyme with respect to pH or temperature.

Specifically, sodium acetic acetate buffer (pH 4.0-6.0), phosphate buffer (pH 7.0 and 8.0), or glycine (pH 7.0-6.0) were used instead of the buffer A used in Example 1 to confirm the activity of the hatching enzyme according to pH. NaOH buffer (pH 9.0 and 10.0), and buffer A 10 mM as a blank were pretreated with hatching enzyme at 30 ° C for 1 hour and then adjusted to pH 7.4, and the enzyme activity was analyzed by the method of Example <1-3> .

In order to confirm the activity of the hatching enzyme according to the temperature, the enzyme solution was pretreated at a temperature of 20 to 50 ° C for 1 hour in each buffer A of pH 7.4, The enzyme activity was analyzed.

As a result, the hatching enzyme exhibited the maximum enzyme titer at pH 8.0 and was stable at pH 4.0 to 6.0 (FIG. 5A). In addition, the hatching enzyme showed maximum activity at 30 ° C and was stable in the range of 30-40 ° C (Fig. 5b).

<2-2> Hatching enzyme according to inhibitor and metal ion Potency

The present inventors have measured the effect of metal ions on the reduced HE titer by various inhibitors or chelators.

Specifically, the purified HE lice were pretreated for each of ^{Mg 2 +, Ca 2 +,} Cu 2 + or Zn 3 hours at 4 ℃ by ^{2 +} 5 mM cation. HE from 4 ℃ chelator is EDTA, EDTA, EGTA, Leupeptin, TLCK, TPCK , or after, respectively, for 30 minutes with PMSF 10 mM, in an enzyme activity regeneration respective ^{Mg 2 +, Ca 2 +,} Cu 2 + Or Zn ^{2 +} metal ion. The activity of HE without pretreatment of metal ions was used as a control.

As a result, 1 mM, and by EDTA or EGTA in 5 mM the activity of enriched enzyme inhibited more than 50% (Fig. ^{6a) Ca 2 +, Mg 2} + and was slightly restored by the Cu ^{2 +} (Fig. 6b), zinc The potency of ion-depressed HE was restored to the strongest, more than twice as much as the control (Table 2 and FIG. 6b). Other chelators Leupeptin, TLCK, TPCK or PMSF also reduced the activity of the hatching enzyme by more than 50% (Fig. 6C).

process Relative titer /% HE alone 100.00 0.36 HE + EDTA (10 mmol / L) 62.60 + - 0.38 HE + EDTA (10 mmol / L ) + Ca 2 + (5 mmol / L) 26.10 ± 0.26 HE + EDTA (10 mmol / L ) + Mg 2 + (5 mmol / L) 71.54 + - 0.51 HE + EDTA (10 ^{mmol / L) + Cu 2 +} (5 mmol / L) 0 HE + EDTA (10 ^{mmol / L) + Zn 2 +} (5 mmol / L) 94.31 + - 0.31

<2-3> Confirmation of reaction rate constant and specific hydrolysis of hatching enzyme

The present inventors measured the reaction rate constant and hydrolysis specificity of the purified starfish hatching enzyme.

Specifically, starfish HE purified in Example 1 was incubated with 0.2-1.5 mg / L of dimethyl casein, gelatin or Type I and V collagen. Thereafter, Km of the incubation enzyme for dimethyl casein, gelatin, type I and V collagen and Vmax values are line weaver-Burk plot (Lineweaver-Burk plot) was calculated by the method, Kcat (catalytic efficiency), the Km value and Was calculated based on the Vmax value.

As a result, Km , Vmax and Kcat values were 0.31 mg / ml, 0.17 U / ml and 122.70 / s, HE showed lower Km values for gelatin and higher Kcat values for type I collagen (Table 3 and Figure 7).

Therefore, it was confirmed that Starfish HE hydrolyzes Type I collagen more specifically and effectively than dimethyl casein.

Km ^a
(mg / ml) Vmax
(U / ml) Kcat
(s ^-1 ) Dimethyl casein ^b 0.31 0.17 122.70 Gelatin ^c 0.19 0.08 57.07 Collagen I ^d 1.09 0.12 771.98 Collagen V ^e 0.36 0.09 64.46

^a Km is the Michaelis constant, Vmax is the maximum reaction rate, and kcat is the catalyst efficiency, where kcat = Vmax / [E].

^b Dimethyl casein consists of αβ and κ casein, which mainly form micelles, 27.3, 24.1 and 8 kDa, respectively.

^c Gelatin is a partially hydrolyzed collagen with a broad MW range. The average molecular weights of the three gelatin samples are 29, 47 and 62 kDa, respectively.

The average molecular weight of ^{d, e} type I and V collagen is 300 kDa.

< Example  3> Hatching enzyme content Cosmetics  Identify the stability and effectiveness of the composition

<3-1> Hatching enzyme content Cosmetics  Safety of composition

(Anti-inflammatory) effects of LPS, Crude enzyme, hatching enzyme-containing emulsion (Fig. 8), Some prototype (pretreatment product for producing emulsion) (Fig. 9) and positive control anti-keratinase Arazyme and Saimdang cosmetics.

Specifically, 100 μl of the fibroblast culture was added to a 96-well plate and subjected to a preliminary reaction at 37 ° C and 5% CO ₂ for 24 hours. After preliminary reaction, the culture was washed with PBS, 100 μl of the supernatant was added to a new well plate, and an equal amount of Griess reagent was added. The reaction was carried out in the dark for 10 minutes and absorbance was measured at 540 nm. Antiinflammatory activity was measured using NO production as an indicator.

As a result, the amount of nitric oxide produced in the emulsion prototype was much lower than that of the reference LPS. When the amount of nitric oxide produced is higher than that of LPS (lipopolysaccharide), it may cause inflammation in our skin. The cosmetic composition containing the HE of the present invention was found to be a safe cosmetic composition (FIG. 10).

<3-2> Containing Hatching Enzyme Cosmetics  Confirm the stability of the composition

Emulsion prototypes including the hatching enzyme according to the present invention, Some prototypes, and Crude enzyme were stored in Incubator at 4 ° C, respectively, and enzyme inactivity was measured every 2 weeks.

Specifically, 100 μl of the substrate, 100 μl of the emulsion prototypes, Some prototype and Crude enzyme were mixed with 100 μl of each of the casein at a rate of 10 mg / ml every two weeks, and then incubated at 30 ° C. for 30 minutes Lt; / RTI &gt; After incubation, 2.8 ml of 20% TCA (trichloroacetic acid) solution was added as a reaction termination reagent. The supernatant was centrifuged (1,932 g, 30 min, 4 ° C) and absorbance was measured at 280 nm. 10 ^-3 ). In order to measure the protein content required for measuring the specific activity, 1 ml of the Bovine serum blue solution was mixed with 100 μl of the sample diluted 1/10, and the absorbance at 595 nm was measured and substituted into the following equation / ml &lt; / RTI &gt; The calculated protein content was substituted into the following equation (2) to confirm the specific activity.

Y: absorbance value (595 nm)

X: Protein content (mg / ml).

As a result, there was almost no specific activity change at 4 ° C for 6 weeks in the prototype except Crude Enzyme (FIG. 11).

<3-3> Containing Hatching Enzyme Cosmetics  Confirming the cell growth inhibitory effect of the composition

In order to confirm that the collagen induced by excess collagen is formed by the formation of a large amount of fibroblasts when a skin injury occurs, the inventors of the present invention have found that the prototype emulsion containing the hatching enzyme of the present invention for fibroblasts, .

Specifically, Dulbecco's modified Egle's medium ( DMEM) 10% fetal bovine serum (FBS) for mixing and then injecting the cell culture flasks HS 68 (human foreskin fibroblast cell line) the frequency divider and 37 ℃, in 5% CO ₂ Incubator in Lt; / RTI &gt; The cultured cells were treated with 5 mg / ml, 1 mg / ml or 0.5 mg / ml of LPS, Crude enzymes, Emulsion prototypes including hatching enzymes, Some prototypes, Positive control skin exfoliation cosmetic Arazyme and Saimdang cosmetics . The cell proliferation after the treatment of the samples was measured by EL-800 microplate reader (BioTek instruments, Winooski, VT, USA) at 450 nm in WST-plate at 37 ° C in 5% CO ₂ using WST- Growth lowland area is expressed by the following equation.

(As: absorbance value of the additive group, Ac: absorbance value of the control).

As a result, the cell inhibition effect of the emulsion prototype was similar to that of the exfoliants of Arazyme and Saimdang cosmetics, which were positive control groups, and the cell proliferation inhibition effect was increased as the concentration of the sample increased (FIG. 12).

< Manufacturing example  1 > Preparation of oil-soluble (W / O formulation) ointment agent (% by weight)

HE 0.01-10.0%

Stearyl alcohol 7.0%

Stearic acid 2.2%

Squalene 3.4%

Octyl dodecanol 5.0%

Olive oil 6.0%

Glycerin monoauric fatty acid ester 10.0%

Isopropyl adipate 2.0%

Tego 0.3%

Carboxyl vinyl polymer 3.0%

Propylene glycol 5.0%

Kojic acid 0.05%

Alpha-keto glutamic acid 0.2%

Cysteine 0.2%

Parabens 0.3%

Sterile purified water or 0.02 M phosphate or buffer solution balance

After mixing the above components, a fat-soluble ointment was prepared according to a conventional method.

< Manufacturing example  2 > Preparation of hydrophilic (O / W type) ointment agent (% by weight)

HE 0.01-10.0%

White vaseline 25.0%

Stearyl alcohol 20.0%

Propylene glycol 12.0%

Propylene misstatin 0.1%

Sodium glycerin monolaurate 0.2%

Polyoxyethylene hardened castor oil 4.0%

Betaine-containing cationic emulsifier 0.2%

Glycerin monostearate 0.1%

0.1% paraoxybenzoic acid propylate

Purified water balance

After mixing the above components, a hydrophilic ointment was prepared according to a conventional method.

< Manufacturing example  3> Preparation of nutrition lotion (% by weight)

Glycerin 8.0%

Butylene glycol 4.0%

Hyaluronic acid extract 5.0%

Beta-glucan 7.0%

Carbomer 0.1%

HE 0.05%

Capphilic / capric triglyceride 8.0%

Squalane 5.0%

Cetearyl glucoside 1.5%

Sorbitan stearate 0.4%

Cetearyl alcohol 1.0%

Triethanolamine 0.1%

Purified water balance

After mixing the above ingredients, a nutritional lotion was prepared according to a conventional method.

< Manufacturing example  4 > Preparation of nutritional cream (% by weight)

Glycerin 3.0%

Butylene glycol 3.0%

Liquid paraffin 7.0%

Beta-glucan 7.0%

Carbomer 0.1%

HE 3.0%

Cappylic / capric triglyceride 3.0%

Squalane 5.0%

Cetearyl glucoside 1.5%

Sorbitan stearate 0.4%

Polysorbate 60 1.2%

Triethanolamine 0.1%

Purified water balance

After mixing the above ingredients, a nutritional cream was prepared according to a conventional method.

< Manufacturing example  5> Preparation of Massage Cream (% by weight)

Glycerin 8.0%

Butylene glycol 4.0%

Liquid paraffin 45.0%

Beta-glucan 7.0%

Carbomer 0.1%

HE 1.0%

Cappylic / capric triglyceride 3.0%

Waste 4.0%

Cetearyl glucoside 1.5%

0.9% sorbitan sesquioleate

Vaseline 3.0%

Paraffin 1.5%

Purified water balance

After mixing the above components, a massage cream was prepared according to a conventional method.

< Manufacturing example  6 > Preparation of pack (% by weight)

Glycerin 4.0%

Polyvinyl alcohol 15.0%

Hyaluronic acid extract 5.0%

Beta-glucan 7.0%

Allantoin 0.1%

HE 0.5%

Nonyl phenyl ether 0.4%

Polysorbate 60 1.2%

Ethanol 6.0%

Purified water balance

After mixing the above components, packs were prepared according to a conventional method.

Claims (8)

Starfish (Asterias amurensis ) hatching liquid extract as an active ingredient for reducing keloid or scar formation.
The pharmaceutical composition for reducing keloid or scar formation according to claim 1, wherein the hatching liquid extract is a starfish-derived starfish hatching enzyme (HE).
3. The method according to claim 2, wherein the hatching enzyme is most stable at pH 4.0 to 6.0 and 30 to 40 DEG C with a maximum activity at pH 6.0 to pH 7.0 and at 25 to 35 DEG C to reduce keloid or scar formation A pharmaceutical composition.
A pharmaceutical composition for reducing keloid or scar formation further comprising Zn &lt; ^{2 +} &gt; in the composition of claim 1.
A cosmetic composition for exfoliating, comprising asterias amurensis hatching extract as an active ingredient.
A cosmetic composition for exfoliating, which further comprises Zn ²⁺ in the composition of claim 5.
The cosmetic composition according to claim 5 or 6, wherein the cosmetic composition suppresses the formation of fibroblasts and suppresses the induction of exfoliation by excessive collagen production.
1) collecting egg of starfish and dejellied to obtain a precipitate;
2) obtaining a sperm from a starfish cellulase;
3) stirring the precipitate of the step 1) and the sperm of the step 2) to prepare a hatched liquid;
4) dialyzing the incubation solution of step 3);
5) eluting the dialyzed solution of the step 4) with a column to obtain a fraction; And
6) dialyzing the titer of the fraction obtained in step 5).

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