Method for obtaining collagen peptides from sea star, elastic liposome comprising collagen peptides from sea star, and cosmetic composition comprising the same

Abstract

The present invention relates to a method for obtaining collagen peptides from sea stars, elastic liposomes containing collagen peptides from sea stars, and cosmetic compositions containing the same. According to the present invention, since collagen peptides having excellent skin absorbability, antioxidant and wrinkle improvement activity are prepared using sea stars, which cause adverse effects on marine ecosystems and are difficult to handle, the existing animal collagen can be replaced, and collagen having high extraction efficiency can be provided. In addition, since a method for loading collagen peptides into elastic liposomes is provided, the limitations of low skin absorbability of animal collagen and marine collagen can be overcome, the transdermal absorption rate can be greatly improved, and using the same, a cosmetic composition effective against oxidation and skin wrinkle improvement can be provided.

Description

Technical Field

[0001] This invention relates to a method for obtaining collagen peptides from starfish, an elastic liposome containing collagen peptides from starfish, and a cosmetic composition containing the same. More specifically, it relates to a method for obtaining low molecular weight collagen peptides with antioxidant and wrinkle-improving effects from starfish, an elastic liposome loaded with collagen peptides from said starfish, and a cosmetic composition for improving wrinkles by including the liposome, which has excellent skin absorption and antioxidant effects. Background Technology

[0002] Collagen is a fibrous protein found in most animals, especially mammals, and is a substance that makes up all connective tissues in the body, such as skin and cartilage. Collagen is formed by three polypeptide molecules twisted together in a triple helix.

[0003] As is well known, collagen is related to skin moisture content; therefore, consuming foods rich in collagen can prevent skin aging, joint weakness, and blood vessel damage. However, during actual ingestion and oral administration, collagen is broken down into amino acids such as glycine and proline through protein hydrolysis before being absorbed. Therefore, to supplement a lack of collagen through intake, it is necessary to consume additional vitamins such as vitamin A or vitamin C, iron, and other essential nutrients for collagen synthesis.

[0004] In addition, there are products on the market that contain collagen molecules or fibers themselves, but since proteins are high molecules, they cannot penetrate the skin. Therefore, they are unlikely to have much effect. Even in low molecular weight form, they cannot penetrate the stratum corneum except for the pores and sweat glands that make up less than 0.1% of the skin.

[0005] To date, the raw materials for collagen production have mainly been supplied by livestock such as cattle and pigs. However, there have been recent harmful issues caused by the outbreak of mad cow disease. Furthermore, due to the fact that animal collagen cannot enter the halal market for religious reasons, research is being actively conducted to use marine organisms as raw materials.

[0006] For example, Korean Patent Publication No. 10-1071338 discloses a method for obtaining collagen hydrolysate from the shells or scales of marine organisms such as pufferfish or sea bream, and Korean Patent Publication No. 10-2006-0091350 discloses a polymeric scaffold for tissue engineering made using collagen extracted from marine organisms.

[0007] However, marine collagen obtained from marine organisms has limitations in terms of extraction quantity and efficiency compared to animal collagen, due to the limited availability of such organisms.

[0008] On the other hand, starfish, which inhabit nearshore waters, represent a significant amount of marine debris, estimated to cost 400-500 million won annually to manage. Despite their high reproductive and regenerative capacity, they negatively impact marine ecosystems, leading to reduced yields in aquaculture farms and posing a challenge for fishermen. Therefore, research is underway to explore ways to utilize them as an alternative resource. Currently, some starfish are dried and spread on farmland to increase yields or used to produce calcium carbonate fertilizer. Recently, their use in de-icing agents has also been proposed.

[0009] Therefore, if collagen peptides with excellent skin absorption rates are prepared using readily available starfish, not only can environmental problems be solved, but cosmetic compositions that are effective in improving the skin can also be provided. Summary of the Invention

[0010] In order to solve the problems of the prior art, the purpose of this invention is to provide a method for preparing collagen peptides from starfish.

[0011] Another object of the present invention is to provide an elastoliposome comprising collagen peptides derived from starfish.

[0012] Another object of the present invention is to provide an antioxidant cosmetic composition comprising collagen peptides derived from starfish.

[0013] Another object of the present invention is to provide a cosmetic composition comprising collagen peptides derived from starfish for improving skin wrinkles.

[0014] Another object of the present invention is to provide an antioxidant cosmetic composition comprising elastin liposomes containing collagen peptides derived from starfish.

[0015] Another object of the present invention is to provide a cosmetic composition comprising an elastin liposome containing collagen peptides derived from starfish for improving skin wrinkles.

[0016] To achieve the above objectives, the present invention provides a method for preparing collagen peptides from starfish, comprising: (a) treating the starfish with an alkaline solution to remove non-collagenous substances; (b) adding the starfish with the non-collagenous substances removed to an acidic solution containing one or more acidic compounds selected from tartaric acid, ascorbic acid, and citric acid to extract collagen; (c) adding a protease to the solution from which the collagen is extracted for hydrolysis; and (d) separating the collagen peptides from the solution.

[0017] In this invention, the acid solution may contain 0.05 to 0.5% by weight of an acid compound.

[0018] In this invention, the enzyme may be one or more of subtilisin, pepsin, collagenase, and trypsin.

[0019] In this invention, the molecular weight of the collagen peptide can be from 1550 to 1700 Da.

[0020] The present invention provides an elastic liposome comprising: a phospholipid layer containing phospholipids and a surfactant; and a collagen peptide derived from starfish loaded within the phospholipid layer.

[0021] In this invention, the collagen peptides derived from starfish may contain more than 30% hydrophilic amino acids.

[0022] In this invention, the surfactant can be a glycoside, sucrose, or glycerol surfactant.

[0023] In this invention, the particle size of the elastic liposomes can be 50 to 600 nm.

[0024] Furthermore, the present invention provides an antioxidant cosmetic composition comprising collagen peptides derived from starfish prepared by the above method.

[0025] Furthermore, the present invention provides a cosmetic composition for improving skin wrinkles, comprising collagen peptides derived from starfish prepared by the above method.

[0026] Furthermore, the present invention provides an antioxidant cosmetic composition comprising elastin liposomes, which contains collagen peptides derived from starfish prepared by the above method.

[0027] Furthermore, the present invention provides a cosmetic composition comprising elastin liposomes for improving skin wrinkles, which comprises collagen peptides derived from starfish prepared by the above method.

[0028] Invention Effects

[0029] According to the present invention, collagen peptides with excellent skin absorption and antioxidant and wrinkle-improving activities are prepared by utilizing starfish, which have adverse effects on marine ecosystems and are difficult to manage. Therefore, they can replace existing animal collagen and provide collagen with high extraction efficiency. Furthermore, since a method for loading collagen peptides onto elastin liposomes is provided, the limitations of low skin absorption rates of animal and marine collagen can be overcome, significantly improving transdermal absorption. Using this, cosmetic compositions effective in anti-oxidation and wrinkle improvement can be provided. Attached Figure Description

[0030] Figure 1 The illustration shows live / dead cell images according to the type of enzyme in an experimental example according to the present invention. Figure 1 In the images, (a) is a live image of Bacillus subtilis protease, (b) is a live image of pepsin, (c) is a live image of C-0130, (d) is a live image of trypsin, (e) is a dead image of Bacillus subtilis protease, (f) is a dead image of pepsin, (g) is a dead image of C-0130, and (h) is a dead image of trypsin. Detailed Implementation

[0031] The specific embodiments of the present invention will now be described in more detail. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used in this specification consists of names that are well-known and commonly used in the art.

[0032] This invention relates to a method for preparing collagen peptides from starfish, an elastic liposome comprising the collagen peptides from starfish prepared by said method, and a cosmetic composition comprising the elastic liposome for anti-oxidation and improvement of skin wrinkles.

[0033] Starfish muscle tissue has a variety of physiological functions, such as the elasticity for catching mollusks 1.5 times the size of their own arms, and the tissue regeneration ability for regenerating damaged arms. Furthermore, these properties are believed to be closely related to collagen.

[0034] However, the body wall of starfish is complexly composed of bone plates (calcium carbonate), proteins, pigments, and odor components, differing significantly from the collagen extraction materials from terrestrial animals. Therefore, it is difficult to effectively extract collagen by directly applying known extraction methods such as acetic acid extraction and pepsin extraction.

[0035] Starfish contain a large amount (20-30% by weight) of bone fragments (calcium carbonate) in their body walls. Therefore, it is necessary to define the conditions for removing non-collagenous substances. When extracting collagen using known methods such as acetic acid extraction and acidic protease extraction, the calcium carbonate in the body wall reacts with acetic acid, causing a neutralization reaction. As a result, it is difficult to maintain optimal extraction conditions. Furthermore, when excessive acid is used to adjust the pH, the large amount of calcium acetate produced as a result of the neutralization reaction increases the ionic strength of the solution, causing collagen to precipitate and become incorporated into the enzyme reaction residue. Therefore, there are problems of significant collagen loss and reduced economic efficiency.

[0036] In addition, the collagen of warm-blooded animals denatures at a temperature of about 35 to 40°C, while the collagen of starfish denatures at a temperature of 25°C, which is relatively low. Therefore, in order to avoid thermal deformation, it must be processed at a low temperature.

[0037] The method for preparing collagen peptides from starfish according to the present invention includes: (a) treating the starfish with an alkaline solution to remove non-collagenous substances; (b) extracting collagen by adding the starfish from which the non-collagenous substances have been removed to an acidic solution; (c) hydrolyzing the solution by adding a protease to the solution from which the collagen is extracted; and (d) separating the collagen peptides from the solution.

[0038] The starfish that can be used in this invention are any starfish belonging to the class Asteridae of the phylum Echinodermata, such as *Asterias amurensis*, *Ophioplocus japonucus*, *Asterinapectinifera*, *Certonardoa semiregularis*, *Ophiothrix exigua*, *Solaster*, *Culcita novaeguineae*, *Ophioplocus japonucus*, *Acanthaster planci*, *Ophiactis savignyi*, *Astropecten polyacanthus*, *Coscinasterias acutispina*, *Astropecten scoparius*, *Protoreaster nodosus*, *Ophiotheladanae*, and *Astropecten scoparius Valenciennes*.

[0039] In the method of the present invention, the starfish is first sliced ​​and then treated with an alkaline solution to remove non-collagenous substances, thereby obtaining starfish bone slices.

[0040] In addition to collagen, the body wall of starfish contains many non-effective components such as proteins, subcutaneous fat, odor-causing components (amines, fatty acids, carbonyl compounds, sulfur compounds, etc.), and inorganic substances (calcium carbonate). Therefore, non-collagenous substances can be removed by treating with an alkaline solution.

[0041] The alkaline solution can be any mixed solution with a pH sufficient to separate the non-effective components from the starfish. For example, it can be a mixed solution containing an alkaline compound and a solvent with a pH in the range of 9 to 14.

[0042] The alkaline compound can be any alkali metal salt capable of adjusting the pH of the solution, such as any one or more selected from sodium hydroxide, calcium hydroxide, potassium hydroxide, etc., with sodium hydroxide being the most preferred. The solvent is not limited; for example, water can be used.

[0043] The alkaline solution may contain 1 to 20% by weight of an alkaline compound.

[0044] To treat the starfish with alkali, the starfish slices can be immersed in an alkaline solution and left for 12 to 48 hours.

[0045] After alkali treatment, bone fragments with collagen attached to the starfish body wall are obtained. The yield of the obtained starfish bone fragments is preferably about 10 to 30% by weight of the initial starfish weight.

[0046] The obtained starfish bone fragments were added to an acidic solution to extract collagen.

[0047] The acid can be a pH-adjusting acid compound, such as tartaric acid, ascorbic acid, or citric acid. In a preferred embodiment of the invention, the acid compound can be a mixture of tartaric acid and ascorbic acid in a weight ratio of 10:1 to 1:10.

[0048] The acid solution may contain 0.05 to 0.5% by weight of an acid compound, more preferably 0.1 to 0.4% by weight. When the concentration of the acid solution is too low, the collagen extraction efficiency is too low. As the concentration of the acid solution increases, the extraction efficiency increases, but when the concentration exceeds about 0.25% by weight, the extraction efficiency decreases again. Therefore, in terms of extraction efficiency, it is preferable to use an acid solution in the range of no more than 0.5% by weight.

[0049] In this invention, it is preferable to add the starfish bone fragments to an acidic solution followed by ultrasonic treatment to promote collagen extraction. The ultrasonic treatment can be performed at 10 to 100 kHz for 20 to 200 minutes, more preferably at 30 to 50 kHz for 40 to 80 minutes.

[0050] After ultrasonic treatment, the mixture can be left to stand for 5 to 15 hours until the acid-base reaction is complete.

[0051] Next, protease is added to hydrolyze the extracted collagen into low molecular weight collagen peptides.

[0052] The collagen peptides from starfish prepared by the method of this invention have a molecular weight of approximately 1550 to 1700 Da, depending on the type of enzyme used. This is lower than the approximately 1900 Da value of fish skin collagen (marine collagen) or the approximately 2400 Da value of porcine skin collagen. Therefore, it is expected to be more beneficial for skin penetration.

[0053] The enzymes used can include subtilisin, pepsin, collagenase, trypsin, etc. Subtilisin can produce collagen peptides with the lowest molecular weight and is also the preferred choice in terms of improving wrinkles.

[0054] In one embodiment of the present invention, it was confirmed that when Bacillus subtilis protease is used as an enzyme to decompose collagen peptides, collagen peptides with the best anti-wrinkle effect compared with other enzymes can be prepared.

[0055] Based on the weight of starfish bone fragments, the enzyme is preferably added at 0.01 to 1% by weight, more preferably 0.05 to 0.4% by weight.

[0056] The enzyme treatment temperature and time should be such that the protease can fully hydrolyze the starfish. For example, the hydrolysis temperature and time can be 10 to 65°C and 1 to 10 hours, respectively. The hydrolysis temperature can be a temperature at which the protease has high activity, which is known, and therefore can be appropriately adjusted depending on the type of enzyme. For example, the hydrolysis temperature of trypsin can be in the range of 35 to 40°C.

[0057] After enzyme treatment, collagen peptides can be separated. The separation of collagen peptides can be achieved, for example, by centrifugation to remove salt and separating the supernatant, followed by freeze-drying the supernatant to obtain collagen peptides in powder form.

[0058] The prepared starfish collagen peptides have a particle size of approximately 1 μm in solvent, exhibit no cytotoxicity, and possess antioxidant activity. This contrasts with porcine and fish skin collagen, which lack antioxidant activity.

[0059] Furthermore, the collagen peptides derived from starfish of the present invention possess anti-wrinkle activity. In one embodiment of the present invention, it was confirmed that when comparing the anti-wrinkle activity using the inhibition rate of MMP-1 expression in cells, the collagen derived from starfish showed a 2 to 3-fold higher MMP-1 expression inhibition rate than fish skin collagen and porcine skin collagen.

[0060] The collagen peptides derived from starfish of the present invention can be used alone in cosmetic compositions for anti-oxidation and for improving skin wrinkles, or they can be loaded into elastin liposomes for use.

[0061] The elastosomes of collagen peptides loaded with starfish according to the present invention solve the problem that collagen peptides are difficult to pass through the intercellular lipids of the stratum corneum, thereby overcoming the limitation of skin absorption rate and ensuring optimal collagen peptide performance.

[0062] In particular, this invention has found that the efficiency of loading collagen peptides isolated from starfish onto elastin liposomes can be significantly improved compared to porcine or fish skin collagen commonly used in the prior art. This is believed to be because the collagen peptides from starfish contain a large number of hydrophilic amino acids compared to porcine or fish skin collagen. As shown in Table 1 below, the hydrophilic amino acid ratio of the collagen peptides from starfish is approximately 40%, which is more than 1.5 times higher than that of porcine or fish skin collagen, which contains approximately 25% hydrophilic amino acids.

[0063] Table 1

[0064] starfish Tilapia pigskin alanine 105.0 124.0 115.0 Arginine 94.0 51.0 48.0 Aspartic acid 78.0 46.0 44.0 Cysteine - 3.0 - glutamic acid 106.0 76.0 72.0 glycine 232.0 333.0 341.0 Histidine - 8.0 5.0 Isoleucine 19.0 10.0 10.0 Leucine 16.0 23.0 22.0 Lysine 18.0 23.0 25.0 Methionine - 2.0 6.0 Phenylalanine 4.0 14.0 1.0 proline 108.0 119.0 123.0 serine 40.0 35.0 33.0 threonine 34.0 23.0 16.0 Tyrosine 7.0 3.0 1.0 Valine 28.0 19.0 22.0 L-proline 111.0 86.0 97.0 Total 1000 998 981 Total hydrophilicity 377.0 265.0 244.0 hydrophilicity % 38％ 27％ 25％

[0065] Based on the above, the starfish-derived collagen peptides of the present invention can contain more than 30% hydrophilic amino acids, preferably more than 35%, and particularly more than 38%. In one embodiment of the present invention, it was confirmed that starfish-derived collagen peptides containing about 40% hydrophilic amino acids exhibit significantly superior elastosome loading efficiency compared to collagen peptides from pig skin or fish skin.

[0066] Elastic liposomes were proposed to overcome various shortcomings of existing liposomes, such as low capture efficiency, instability in formulations, low solubility of active ingredients, and the possibility of lipid oxidation and hydrolysis. Furthermore, they can be prepared by adding surfactants that impart elasticity to phospholipids.

[0067] The elastic liposomes according to the present invention consist of a phospholipid layer containing phospholipids and surfactants, and a collagen peptide derived from starfish as a loaded body inside the phospholipid layer. These components not only contain phospholipids with a structure similar to skin cells, but also possess excellent deformability due to increased elasticity, thereby enabling efficient penetration and movement between keratinocytes, resulting in excellent transdermal absorption efficiency.

[0068] The phospholipids act as intercellular lipids, preventing the skin's active ingredients from escaping, while also functioning as a semi-permeable membrane to absorb external moisture.

[0069] In this invention, the phospholipid component may be a commonly used phospholipid in the art, such as a fatty acid chain having 12 to 24 carbon atoms, and may contain one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, but is not limited thereto. In this invention, the phospholipid component is preferably phosphatidylcholine.

[0070] The purpose of including the surfactant is to improve transdermal absorption by imparting elasticity to the interface of the liposome phospholipid layer. The surfactant may be a glucosinolate surfactant, a sucrose surfactant, or a glycerol surfactant, with glucosinolate surfactants being the most preferred.

[0071] The glucoside surfactant may be cetearyl glucoside, decyl glucoside, coco glucoside, behenyl alcohol, arachidyl alcohol, arachidyl glucoside, C10-20 alkyl glucoside, etc., with cetearyl glucoside being the most preferred.

[0072] The sucrose surfactants may include sucrose monostearate, sucrose distearate, and sucrose tristearate.

[0073] In addition, the glycerol surfactants mentioned can be polyglyceryl-6 caprylate, polyglyceryl-4 caprate, polyglyceryl-3 methylglucose distearate, etc.

[0074] In one embodiment of the present invention, it was confirmed that when cetearyl glucoside, a glucoside surfactant, was used as a surfactant to prepare elastic liposomes, the skin absorption rate was 3 to 5 times better than that of other surfactants.

[0075] The phospholipids and surfactants can be mixed in a weight ratio of 3:1 to 20:1, more preferably in a weight ratio of 7:1 to 12:1.

[0076] Furthermore, the collagen peptides derived from starfish of the present invention may comprise 1 to 100% by weight relative to the weight of the phospholipid layer mixed with phospholipids and surfactants, without particular limitation. In experimental examples of the present invention, it has been shown that the range of collagen peptides with the best skin absorption rate varies depending on the content of the phospholipid layer, and the best skin absorption rate is observed when the collagen peptides comprise 1% by weight of the phospholipid layer and 0.1% by weight of the collagen peptides relative to the weight of the solvent.

[0077] The elastosomes loaded with collagen peptides from starfish have a particle size of 50 to 600 nm, with most particles having a size of 100 to 200 nm. This is much smaller than collagen peptides in solvent, which are approximately 1 μm in size. In the experimental examples of the present invention, a trend of increasing particle size was observed with increasing phospholipid content, which was determined to be an increase in the thickness of the elastosome membrane when a predetermined amount or more of phospholipid was added.

[0078] Collagen peptides with a particle size of about 1 μm in solvents are hardly absorbed by the skin in the stratum corneum. However, elastin liposomes, due to their smaller particle size and elasticity, can exhibit excellent skin absorption.

[0079] The collagen peptides derived from starfish and the elastin liposomes containing them of the present invention have excellent antioxidant activity and skin wrinkle improvement effects, and therefore can be used in cosmetic compositions.

[0080] The content of the elastin liposomes may be from 0.1% to 50% by weight relative to the total weight of the cosmetic composition.

[0081] The cosmetic compositions of the present invention can be prepared into any dosage form conventionally prepared in this field, such as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing facial cleansers, oils, foundations, emulsion foundations, wax-based foundations, sprays, masks, etc., but are not limited thereto.

[0082] Furthermore, the cosmetic composition of the present invention, depending on the type of cosmetic, such as facial cleansing cosmetics, basic cosmetics, makeup, hair care cosmetics, functional cosmetics, etc., may include cosmetics containing various additives with different ingredients.

[0083] Example

[0084] The present invention will now be described in more detail through embodiments. These embodiments are for illustrative purposes only, and it will be understood by those skilled in the art that the scope of the invention should not be construed as being limited to these embodiments.

[0085] Preparation Example 1: Preparation of Collagen Peptides from Starfish

[0086] Immerse 1,000g of starfish in 1L of 5% sodium hydroxide solution for 24 hours to remove non-collagenous substances and ensure 200g of bone fragments with attached collagen.

[0087] An acid compound, prepared by mixing tartaric acid and ascorbic acid in a 1:1 ratio with approximately 2 g of undried starfish bone slices, was added to 50 mL of distilled water at concentrations of 0.05, 0.25, 0.5, 1.0, and 2.5% by weight. The mixture was then sonicated at 38 kHz for 1 hour and left to stand for at least 10 hours to complete the acid-base reaction.

[0088] Based on the weight of starfish bone slices, 0.1% by weight of enzymes were added to degrade collagen peptides to a low molecular weight form using Bacillus subtilis protease, pepsin, collagenase (C-0130), collagenase (C-0130) buffer solution, trypsin, and trypsin buffer solution.

[0089] The salts produced during the acid / base reaction in the lower layer are removed by centrifugation, and the supernatant is separated. The separated supernatant is freeze-dried to obtain collagen peptides in powder form.

[0090] Experimental Example 1: Confirming the physical properties based on the collagen peptide extraction process

[0091] 1-1. Extraction efficiency based on acid addition amount

[0092] Table 2 summarizes the collagen peptide extraction efficiency based on the amount of acid added.

[0093] Table 2

[0094]

[0095] Calcium ascorbate, formed by the reaction of ascorbic acid and calcium carbonate (a component of bone fragments), is water-soluble. Therefore, it is included in the extraction efficiency when the supernatant is freeze-dried after centrifugation. Thus, after confirming the yield excluding the calcium ascorbate that might be produced assuming 100% reaction with the added ascorbic acid, it was confirmed that the highest yield was obtained when 0.25% by weight of acid was added, and the yield gradually decreased with increasing acid content.

[0096] This is because calcium tartrate, the salt formed by the reaction of tartaric acid and calcium carbonate, is removed from the lower layer during centrifugation. However, calcium tartrate is preferentially formed over calcium ascorbate.

[0097] 1-2. Extraction efficiency based on the type of enzyme

[0098] To understand the extraction efficiency based on the type of enzyme, the results of adding six enzymes to 0.05% by weight of acid-treated samples are shown in Table 3 below.

[0099] Table 3

[0100]

[0101] Extraction efficiency was calculated based on the dried mass of the collagen extract after alkali treatment, relative to the mass of the bone fragments with attached collagen. The buffer solution used was EDTA dissolved in TESCA when it was C-0130, and dissolved in PBS when it was trypsin.

[0102] The extraction efficiency of all enzymes except collagenase C-0130 showed almost the same trend, confirming that there was no significant difference even in the case of buffer solution.

[0103] 1-3. Based on the molecular weight of collagen peptides according to the type of enzyme

[0104] The molecular weights of collagen peptides based on enzyme type were confirmed by gel permeation chromatography (GPC) and are shown in Table 4 below.

[0105] Table 4

[0106] Example 3 Example 4 Example 5 Example 6 Comparative Example 3 enzymes Subtilisin pepsin C-0130 trypsin X Molecular weight (Da) 1587 1650 1681 1699 2981

[0107] The table above confirms that regardless of the type of enzyme, collagen extract exists as a low molecular weight peptide of approximately 1700 Da due to the influence of the enzyme. Furthermore, in the case of Bacillus subtilis protease, collagen peptides with a minimum molecular weight of less than 1600 Da can be prepared.

[0108] 1-4. Cytotoxicity assays based on the type of enzyme (MTT assay)

[0109] To measure cell viability, human fibroblasts (HDF) were cultured for 24 hours in DMEM, 10% FBS, and 1% penicillin-streptomycin medium. Then, samples of each enzyme from Examples 3 to 6 were added to the medium at a concentration of 0.2 to 1.0 mg / mL, the medium was changed, and the cells were cultured for another 24 hours. MTT solution was then added, and the cells were cultured for an additional 4 hours.

[0110] Table 5 below shows the absorbance measured at 560 nm after the culture medium was removed and DMSO was added.

[0111] Table 5

[0112]

[0113] In survival tests conducted at various concentrations based on the type of enzyme, all concentrations showed a survival rate of over 85%, and in the case of subtilisin, a survival rate of over 92% was observed with almost no cytotoxicity.

[0114] 1-5. Perform cytotoxicity assays (Live / Dead imaging) based on the type of enzyme.

[0115] As described in Examples 1-4, after culturing for another 24 hours at a concentration of 0.2 mg / mL, calcein-AM (live) and ethidium homodimer (dead) were added to the PBS bulk solution. After 30 minutes, the confocal imaging results were displayed. Figure 1 middle.

[0116] exist Figure 1 In the images, (a) is a live image of Bacillus subtilis protease, (b) is a live image of pepsin, (c) is a live image of C-0130, (d) is a live image of trypsin, (e) is a dead image of Bacillus subtilis protease, (f) is a dead image of pepsin, (g) is a dead image of C-0130, and (h) is a dead image of trypsin.

[0117] It was confirmed that there was no cytotoxicity in any of the experimental groups.

[0118] 1-6. Antioxidant activity of collagen peptides based on the type of enzyme.

[0119] The antioxidant properties of collagen peptides based on the enzyme were confirmed by a DPPH free radical scavenging capacity test.

[0120] After reacting the DPPH solution with the sample solutions from Examples 3 to 6 according to their concentrations, the results of confirming the free radical scavenging ability by measuring absorbance at 517 nm are shown in Table 6 below. Antioxidant activity was confirmed using vitamin C as a 100% control.

[0121] Table 6

[0122]

[0123] It exhibits excellent antioxidant activity of over 90% against all enzymes, and shows the best antioxidant activity at 0.2 mg / mL.

[0124] 1-7. Comparison based on the anti-wrinkle activity of enzymes and collagen peptides

[0125] Human fibroblasts (CCD-986sk) were cultured for 24 hours in DMEM, 10% FBS, and 1% penicillin-streptomycin medium. Samples of each enzyme from Examples 3 to 6 were added to the medium at a concentration of 1 mg / mL. After changing the medium, the cells were irradiated with UVB for 20 minutes and cultured for 24 hours. The culture supernatant was incubated with coating buffer, followed by treatment with washing buffer and blocking buffer. Then, after treatment with primary and secondary antibodies at various dilutions, the culture supernatant was removed, and the cells were treated with washing buffer. Finally, the absorbance measured at 405 nm after incubation with pnPP (substrate solution) in the dark for 1 hour was converted into the MMP-1 expression inhibition rate, as shown in Table 7.

[0126] Table 7

[0127]

[0128] The table above confirms that the Bacillus subtilis protease-treated samples showed the best MMP-1 expression inhibition rate compared to other enzymes.

[0129] Experimental Example 2: Comparison of collagen peptides from starfish, porcine skin, and fish skin.

[0130] Experiments were conducted to compare the collagen peptides from starfish extracted using the extraction process determined in Experiment Example 1 with collagen peptides from pig skin and fish skin.

[0131] The porcine skin collagen peptides and fish skin collagen peptides used in the experiment are shown in Table 8 below.

[0132] Table 8

[0133] Manufacturer raw material Protein content Pig skin collagen peptides Xiamen Huaxuan Gelatin pig ≥90％ Fish skin collagen peptides Xiamen Huaxuan Gelatin Tilapia ≥90％

[0134] 2-1. Comparison of cytotoxicity of collagen peptides

[0135] The MTT assay for cell viability was performed in the same manner as in Examples 1-4. The starfish collagen used was the collagen from Example 3. The MTT cell viability results are shown in Table 9 below.

[0136] Table 9

[0137]

[0138] In the table above, porcine skin collagen and fish skin collagen showed cell growth rates of less than 80% at concentrations above 0.4 mg / mL. However, overall, none of the three collagen samples showed significant cytotoxicity.

[0139] 2-2. Comparison of the molecular weights of collagen peptides

[0140] The molecular weights of the three collagen peptides were confirmed by gel permeation chromatography (GPC) and are shown in Table 10 below.

[0141] Table 10

[0142] enzymes Starfish Collagen Peptides Pig skin collagen peptides Fish skin collagen peptides Molecular weight (Da) 1587 2406 1901

[0143] It was confirmed that the molecular weight of collagen from starfish is much lower than that of collagen from pig skin and fish skin.

[0144] 2-3. Comparison of the antioxidant activities of collagen peptides

[0145] The antioxidant activity of DPPH was analyzed in the same manner as in Experiments 1-6, and is shown in Table 11 below.

[0146] Table 11

[0147]

[0148] Starfish collagen showed excellent antioxidant activity, while neither porcine skin collagen nor fish skin collagen showed antioxidant activity.

[0149] 2-4. Comparison of the anti-wrinkle activity of collagen peptides

[0150] The MMP-1 expression inhibition rate was analyzed in the same manner as in Experiments 1-7 and is shown in Table 12 below.

[0151] Table 12

[0152]

[0153] By comparing the MMP-1 expression inhibition rate of cells exposed to ultraviolet light to determine anti-wrinkle activity, it was confirmed that the collagen peptides from starfish were about 3 times higher than those from fish skin, and showed a higher MMP-1 expression inhibition rate than those from pig skin, thus demonstrating significantly superior anti-wrinkle activity.

[0154] Preparation Example 2: Preparation of Elastoliposomes Loaded with Collagen Peptides

[0155] Following the proportions in Table 13, phospholipids, surfactants, and collagen peptides were placed in a 50 mL round-bottom flask and thoroughly dissolved in 20 mL of ethanol. After completely removing the solvent using a rotary evaporator, 20 mL of distilled water was added to fully dissolve the solvent. To homogenize the elastoliposome particles, the mixture was ultrasonically treated at 30 kHz for 15 minutes to prepare the elastoliposomes.

[0156] Table 13

[0157]

[0158] Experimental Example 3: Confirmation of the physical properties of elastic liposomes

[0159] 3-1. Loading efficiency of elastic liposomes

[0160] The phospholipid was phosphatidylcholine, and the surfactants were polyglyceryl-6 caprylate and polyglyceryl-4 caprate (TEGO SOLVE 90, Evonik Corporation). The loading efficiency of each component ratio was measured.

[0161] After preparing elastic liposomes, unloaded collagen peptides were separated by filtration using a 450 nm syringe filter. The collagen peptides loaded onto the purified elastic liposomes were then quantified using a BCA assay. Loading efficiency was calculated by measuring the ratio of the total collagen peptides to the BCA assay quantification value before loading, and the results are shown in Table 14 below.

[0162] Table 14

[0163] Sample Name Load efficiency (%) EL1 / 0.1 88.3 EL3 / 0.1 63.9 EL5 / 0.1 27.9 EL10 / 0.1 42.1 EL1 / 0.5 57.3 EL3 / 0.5 61.5 EL5 / 0.5 55.1 EL10 / 0.5 44.0 EL1 / 1 31.7 EL3 / 1 65.1 EL5 / 1 53.2 EL10 / 1 47.1

[0164] 3-2. Comparison of particle size of elastic liposomes

[0165] The particle size of collagen peptides and elastin liposomes from starfish was measured and is shown in Table 15 below.

[0166] Table 15

[0167] Sample Name Particle size (nm) Collagen peptides from starfish 1022 EL1 / 0.1 109 EL3 / 0.1 121 EL5 / 0.1 129 EL10 / 0.1 194 EL1 / 0.5 200 EL3 / 0.5 565 EL5 / 0.5 388 EL10 / 0.5 177 EL1 / 1 145 EL3 / 1 190 EL5 / 1 204 EL10 / 1 176

[0168] In the case of collagen peptides derived from starfish, the particle size in the solvent was approximately 1 μm, confirming that the elastin liposome particle size does not exceed 1 μm in nm.

[0169] Overall, the particle size also showed an increasing trend with the increase of phospholipid content. This is thought to be because the thickness of the elastosome membrane increases when a certain amount of phospholipid is added.

[0170] 3-3. Comparison of skin absorption rates of elastin liposomes

[0171] To compare skin absorption rates, the skin layer of the acceptor plate coated with artificial skin was hydrated, and the corresponding sample was filled into each well of the donor plate along with buffer solution. The hydrated acceptor plate was then filled with buffer solution and incubated on the donor plate. The absorbance of each plate was then analyzed using a microplate reader, and the results of the skin permeability measurements are shown in Table 16 below.

[0172] Table 16

[0173] Sample Name <![CDATA[Skin absorption rate (mg / cm 2 / h)]]> Collagen peptides from starfish 0 EL1 / 0.1 2392 EL3 / 0.1 1219 EL5 / 0.1 642 EL10 / 0.1 624 EL1 / 0.5 1645 EL3 / 0.5 968 EL5 / 0.5 141 EL10 / 0.5 1657 EL1 / 1 740 EL3 / 1 490 EL5 / 1 0 EL10 / 1 768

[0174] In the table above, in the case of collagen extracts with a particle size of approximately 1 μm in solvent, no skin absorption rate was measured because they are hardly absorbed by the skin in the stratum corneum.

[0175] On the other hand, in the case of samples prepared with elastoliposomes, different skin absorption rates were observed based on the ratio, which is somewhat similar to the trend of particle size.

[0176] Therefore, considering the economics of large-scale production, it is preferable to use EL1 / 0.1, which has an appropriate ratio of collagen extract loading efficiency and particle size, to prepare elastin liposomes, exhibiting the best skin absorption rate.

[0177] 3-4. Analysis based on surfactant loading efficiency

[0178] Based on an EL1 / 0.1 preparation ratio, elastic liposomes were prepared using the following candidate surfactants, and their loading efficiency, particle size, and skin absorption rate were compared. The prepared elastic liposome samples were named according to the type of surfactant as follows.

[0179] Table 17

[0180]

[0181] The loading efficiency of the elastoliposomes according to the type of surfactant was measured in the same manner as in Experimental Example 3-1 and is shown in Table 18 below.

[0182] Table 18

[0183] Sample Name Load efficiency (%) EL1 / 0.1-SF1 82.0 EL1 / 0.1-SF2 79.9 EL1 / 0.1-SF3 88.3 EL1 / 0.1-SF4 80.3

[0184] The loading efficiency of EL1 / 01-SF3 elastoliposomes using cetearyl glucoside surfactant was found to be the highest.

[0185] 3-5. Based on the particle size analysis of surfactants

[0186] The particle size of the elastosomes according to the type of surface activity was measured and is shown in Table 19 below.

[0187] Table 19

[0188] Sample Name Particle size (nm) EL1 / 0.1-SF1 109 EL1 / 0.1-SF2 165 EL1 / 0.1-SF3 110 EL1 / 0.1-SF4 151

[0189] The table above confirms that, depending on the type of surfactant, the particle size ranges from 100 nm without significant deviation.

[0190] 3-6. Analysis based on the skin absorption rate of surfactants

[0191] The skin absorption rate of elastin liposomes was measured according to the type of surfactant and is shown in Table 20 below.

[0192] Table 20

[0193] Sample Name <![CDATA[Skin absorption rate (mg / cm 2 / h)]]> EL1 / 0.1-SF1 2392 EL1 / 0.1-SF2 1333 EL1 / 0.1-SF3 6455 EL1 / 0.1-SF4 2070

[0194] The table above shows approximately 2000 mg / cm³. 2 The skin absorption rate was / h, however, the skin absorption rate of EL1 / 01-SF3 elastin liposomes using cetearyl glycoside surfactant was 6455 mg / cm³. 2 / h, which is a very high value, about 3 to 5 times higher than that of samples using other surfactants.

[0195] Experiment Example 4: Comparison of elastoliposomes loaded with collagen peptides from starfish, porcine skin, and fish skin.

[0196] Using the same composition and surfactant as EL1 / 0.1-SF3 in Experimental Example 3, elastin liposomes loaded with porcine skin collagen peptides and fish skin collagen peptides were prepared, respectively. The sample names are shown in Table 21 below.

[0197] Table 21

[0198] Sample Name Loaded collagen peptides EL-St Collagen peptides from starfish EL-Po Pig skin collagen peptides EL-Fi Fish skin collagen peptides

[0199] 4-1. Based on the loading efficiency of collagen peptides on elastin liposomes

[0200] The loading efficiency of elastin liposomes according to the type of collagen peptide was measured in the same manner as in Experiment 3-1, and is shown in Table 22 below.

[0201] Table 22

[0202] Sample Name Load efficiency (%) EL-St 88.3 EL-Po 0 EL-Fi 17.0

[0203] Collagen peptides derived from starfish were found to have a loading efficiency more than 6 times higher. This is believed to be because the proportion of hydrophilic groups in the amino acid sequence of starfish collagen peptides is approximately 40%, higher than that of collagen peptides from pig and fish skin, making it easier to form elastin liposomes.

[0204] 4-2. Based on the particle size of the elastin liposomes of collagen peptides

[0205] The particle size of elastin liposomes was measured according to the type of collagen peptide and is shown in Table 23 below.

[0206] Table 23

[0207] Sample Name Particle size (nm) EL-St 110 EL-Po 98 EL-Fi 94

[0208] Based on the type of collagen peptide, the particle size of the elastoliposomes was approximately 100 nm, with no significant deviation, but the measured particle sizes of EL-Po and EL-Fi were smaller. Judging from the loading efficiency data, the elastoliposomes were prepared without any loading material, and the particle size was reduced.

[0209] 4-3. Based on the skin absorption rate of collagen peptides and elastin liposomes

[0210] The skin absorption rate of elastin liposomes according to the type of collagen peptide was measured in the same manner as in Experiment 3-3, and is shown in Table 24 below.

[0211] Table 24

[0212] Sample Name <![CDATA[Skin absorption rate (mg / cm 2 / h)]]> EL-St 6455 EL-Po 4466 EL-Fi 5428

[0213] It has been confirmed that the skin absorption rate of collagen peptides from starfish is much higher than that of collagen peptides from pigskin and fish skin.

[0214] 4-4. Based on the antioxidant activity of collagen peptides in elastin liposomes

[0215] The DPPH antioxidant activity of elastin liposomes was measured according to the type of collagen peptide and is shown in Table 25 below.

[0216] Table 25

[0217]

[0218] Experimental results showed that elastin liposomes loaded with collagen peptides from starfish exhibited excellent antioxidant activity, while elastin liposomes loaded with collagen from pig skin and fish skin did not show antioxidant activity.

[0219] 4-5. Based on the skin wrinkle-inhibiting activity of collagen peptides in elastin liposomes.

[0220] The skin wrinkle-inhibiting activity of elastin liposomes was measured according to the type of collagen peptides and is shown in Table 26 below.

[0221] Table 26

[0222]

[0223] Experimental results showed that elastin liposomes loaded with collagen peptides from starfish exhibited excellent skin wrinkle-inhibiting activity, while elastin liposomes loaded with collagen from pig skin and fish skin had no or weak skin wrinkle-inhibiting activity.

[0224] Experiment Example 5: Confirming the activity of collagen peptides from starfish based on enzyme type.

[0225] In Experiment 1, the loading efficiency and skin permeability of elastin liposomes of collagen peptides from starfish obtained using the same methods as in Experiments 3 and 4 were evaluated using subtilisin, pepsin, C-0130 and trypsin as enzymes. The results were compared with those of porcine skin collagen peptides and fish skin collagen peptides and are shown in Table 27 below.

[0226] Table 27

[0227]

[0228] The table above confirms that when using Bacillus subtilis protease as the enzyme, it exhibits the lowest molecular weight, best cell viability, superior anti-wrinkle activity, high loading efficiency, and excellent skin permeability. Furthermore, it was confirmed that collagen peptides extracted from starfish using pepsin, collagenase, and trypsin still possess significantly superior antioxidant properties, loading efficiency, and skin permeability compared to collagen peptides from pig skin and tilapia (fish skin).

[0229] In particular, C-0130, which had the lowest loading efficiency among the four enzymes, also showed a loading efficiency of more than 3.8 times that of fish skin collagen peptides and exhibited superior skin permeability.

[0230] This difference is thought to be due not only to the molecular weight of the type of degrading enzyme, but also to the fact that collagen peptides from starfish contain a large number of hydrophilic amino acids compared to collagen peptides from pig or fish skin.

[0231] Based on the above description, those skilled in the art should understand that the present invention can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the above embodiments are exemplary in all respects and not restrictive, and the scope of the invention should be determined by the meaning and scope of the claims, not by the details of the invention, and all modifications or variations derived from the concept of equivalents are included within the scope of the invention.

Claims

1. An elastic liposome, characterized in that, include: The phospholipid layer contains phospholipids and surfactants; as well as Collagen peptides from starfish loaded within the phospholipid layer. The surfactant mentioned above is a glycoside surfactant. The elastic liposomes have a particle size of 50 to 600 nm. The collagen peptides derived from starfish are prepared by a method comprising the following steps: (a) Treating starfish with an alkaline solution to remove non-collagenous substances; (b) Adding starfish from which the non-collagenous substances have been removed to an acidic solution containing 0.1 to 0.4% by weight of a mixture of tartaric acid and ascorbic acid to extract collagen, wherein the weight ratio of tartaric acid to ascorbic acid is 10:1 to 1:

10. (c) Adding subtilisin to the extracted collagen solution to hydrolyze the extracted collagen; and (d) Separate the collagen peptides from the solution.

2. The elastic liposome according to claim 1, characterized in that, The collagen peptides derived from starfish contain more than 30% hydrophilic amino acids.

3. An antioxidant cosmetic composition, characterized in that, It contains the elastic liposomes according to claim 1 or 2.

4. A cosmetic composition for improving skin wrinkles, characterized in that, It contains the elastic liposomes according to claim 1 or 2.

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