Method for obtaining collagen peptides from starfish, elastic liposomes containing starfish-derived collagen peptides, and cosmetic compositions containing the same

Abstract

To provide an elastic liposome containing a starfish-derived collagen peptide.SOLUTION: The present invention provides an elastic liposome comprising: a phospholipid layer comprising phospholipids and a surfactant; and a starfish-derived collagen peptide carried inside the phospholipid layer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [1] The present invention relates to a method for obtaining collagen peptides from starfish, a method for obtaining collagen peptides derived from starfish, More specifically, the present invention relates to a method for obtaining low molecular weight collagen peptides from starfish that have antioxidant and anti-wrinkle effects, elastic liposomes carrying the starfish-derived collagen peptides, and cosmetic compositions for anti-wrinkle treatment that contain the same and have excellent skin absorption and antioxidant effects. [Background technology]

[0002] [2] Collagen is a fibrous substance found in most animals, especially mammals. Collagen is a protein that makes up the majority of all connective tissues in the body, including skin and cartilage. Collagen is made up of three polypeptide molecules twisted around each other in a triple helix, forming a rope-like structure.

[0003] [3] Collagen is responsible for skin moisture, so consuming collagen-rich foods can help improve skin It is well known that collagen can prevent skin aging, weakened joints, and blood vessel damage. However, when it is actually ingested or orally administered, it is broken down into amino acids such as glycine and proline through the protein breakdown process before being absorbed. Therefore, in order to replenish missing collagen through intake, it is necessary to take additional vitamins A, C, and iron, which are necessary for collagen synthesis.

[0004] [4] There are also products on the market that contain collagen molecules or fibers that are applied to the skin. However, because proteins are high molecular weight substances, they cannot penetrate the skin, and this is unlikely to have a significant effect. Even in the form of low molecular weight substances, they can penetrate pores, which make up less than 0.1% of the skin. Penetration of the stratum corneum, excluding the sweat glands, is impossible.

[0005] [5] Until now, collagen production materials have mainly come from livestock such as cows and pigs. Recently, the harmfulness of animal collagen has come to light due to the BSE (bovine spongiform encephalopathy) scare. Due to issues such as the inability to enter the halal market for religious reasons, research into using marine organisms as materials is actively being conducted.

[0006] [6] For example, Korean Patent Publication No. 10-1071338 discloses the use of marine organisms such as pufferfish and sea bream. A method for obtaining collagen hydrolysate from shells and scales is described, and Korean Patent Publication No. 10-2006-0091350 describes a tissue engineering method using collagen extracted from marine organisms. describes polymeric supports for use in

[0007] [7] However, marine collagen obtained from marine organisms is limited in the marine organisms from which it can be extracted. However, compared to animal collagen, there is a problem that the amount of collagen extracted is limited and the extraction efficiency is low.

[0008] [8] Meanwhile, starfish that live in coastal waters are a marine waste that requires a budget of 400-500 million won per year to dispose of. While starfish are discarded and have a high reproductive and regenerative capacity, they have a negative impact on the marine ecosystem, reducing the yield of fish farms and causing problems for fishermen, so research is being conducted to find ways to utilize them as a resource. Currently, these starfish are used in some areas by drying them and scattering them on agricultural soil to increase yields, and in other areas to produce calcium carbonate fertilizer, and recently, it has been suggested that they could be used as a snow removal agent.

[0009] [9] Therefore, we developed a collagen peptide with excellent skin absorption rate using readily available starfish. If we could produce a chido, we would be able to provide a cosmetic composition that is effective in improving skin while solving environmental problems. Summary of the Invention

[0010] Detailed Description of the Invention Technical challenges

[10] To solve these problems of the prior art, the object of the present invention is to provide a method for producing collagen peptides from starfish.

[0011]

[11] Another object of the present invention is to provide elastic liposomes containing starfish-derived collagen peptides.

[0012]

[12] Another object of the present invention is to provide an antioxidant cosmetic composition containing starfish-derived collagen peptides.

[0013]

[13] Another object of the present invention is to provide a cosmetic composition for improving skin wrinkles, which contains a starfish-derived collagen peptide.

[0014]

[14] Another object of the present invention is to provide an antioxidant cosmetic composition comprising elastic liposomes containing starfish-derived collagen peptides.

[0015]

[15] Another object of the present invention is to provide a cosmetic composition for improving skin wrinkles, which comprises elastic liposomes containing starfish-derived collagen peptides. [Problem to be solved by the invention]

[0016] Problem solving means

[16] To achieve the above object, the present invention provides a method for producing a starfish, comprising the steps of (a) treating a starfish with an alkaline solution to remove non-collagenous substances, (b) adding the starfish from which the non-collagenous substances have been removed to an acid solution containing one or more acid compounds selected from the group consisting of tartaric acid, ascorbic acid, and citric acid to extract collagen, and (c) (d) adding a protease to the solution into which the collagen has been extracted to hydrolyze it; and (e) isolating the collagen peptide from the solution.

[0017]

[17] In the present invention, the acid solution may contain 0.05 to 0.5 wt % of an acid compound.

[0018]

[18] In the present invention, the enzyme is selected from the group consisting of subtilisin, pepsin, It can be one or more of collagenase and trypsin. Cut.

[0019]

[19] In the present invention, the collagen peptide may have a molecular weight of 1550 to 1700 Da.

[0020]

[20]

[21] The present invention also provides a phospholipid layer comprising phospholipids and a surfactant; and an elastic liposome containing a starfish-derived collagen peptide carried inside the phospholipid layer.

[0021]

[22] In the present invention, the starfish-derived collagen peptide may contain 30% or more hydrophilic amino acids.

[0022]

[23] In the present invention, the surfactant may be a glucoside-based, sucrose-based, or glyceryl-based surfactant.

[0023]

[24] In the present invention, the particle size of the elastic liposome may be 50 to 600 nm.

[0024] [twenty five]

[26] The present invention also provides an antioxidant cosmetic composition containing the starfish-derived collagen peptide produced by the above-mentioned method.

[0025]

[27] The present invention also provides a cosmetic composition for improving skin wrinkles, which contains the starfish-derived collagen peptide produced by the above-mentioned method.

[0026]

[28] The present invention also provides an antioxidant cosmetic composition comprising elastic liposomes containing starfish-derived collagen peptides produced by the above-mentioned method.

[0027]

[29] The present invention also provides a cosmetic composition for improving skin wrinkles, which comprises elastic liposomes containing starfish-derived collagen peptides produced by the above-mentioned method. [Effects of the Invention]

[0028] Effect of the invention

[30] According to the present invention, collagen peptides with excellent transdermal absorption and antioxidant and anti-wrinkle activity are produced using starfish, which are difficult to process and have a negative impact on the marine ecosystem, making it possible to provide collagen with high extraction efficiency as an alternative to existing animal collagen. Furthermore, a method for utilizing collagen peptides by encapsulating them in elastic liposomes overcomes the limited transdermal absorption of animal collagen and marine collagen, significantly improving transdermal absorption, and using the resulting collagen, it is possible to provide cosmetic compositions that are effective in antioxidant and anti-wrinkle properties. [Brief explanation of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[31] Figure 1 shows live / dead images of cells according to the type of enzyme in an experimental example of the present invention. Figure 1(a) shows a subtilisin / live image, (b) shows a pepsin / live image, (c) shows a C-0130 / live image, (d) shows a trypsin / live image, (e) shows a subtilisin / dead image, (f) shows a pepsin / dead image, (g) shows a C-0130 / dead image, and (h) shows a trypsin / dead image. DETAILED DESCRIPTION OF THE INVENTION

[0030] MODE FOR CARRYING OUT THE INVENTION

[32] Specific embodiments of the present invention are described in more detail below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0031]

[33]

[34] The present invention relates to a method for producing collagen peptides from starfish and the collagen peptides produced by the method. The present invention relates to elastic liposomes containing starfish-derived collagen peptides, and cosmetic compositions containing the same for antioxidant and wrinkle-reducing effects.

[0032]

[35] Starfish musculature is elastic enough to catch shellfish up to 1.5 times the size of their arms. It has various physiological functions, such as the ability to regenerate tissue and grow damaged limbs, and it is believed that these properties are closely related to collagen.

[0033]

[36] However, the starfish body wall is made up of a complex mixture of bone fragments (calcium carbonate), proteins, pigments, and odor components, which differs in many ways from collagen extraction materials from land animals. Therefore, it is difficult to effectively extract it using known extraction methods such as acetic acid extraction and pepsin extraction.

[0034]

[37] In other words, starfish body walls contain a large amount (20-30% by weight) of bone fragments (calcium carbonate), and it is necessary to investigate the conditions for removing non-collagenous substances. When collagen is extracted using the already known acetic acid extraction method or acid protease extraction method, the calcium carbonate in the body wall reacts with acetic acid to cause a neutralization reaction, making it difficult to maintain optimal extraction conditions. When excess 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 mixed into the enzyme reaction residue, resulting in significant collagen loss and reduced economic viability.

[0035]

[38] Furthermore, the thermal denaturation temperature of collagen in starfish collagen is relatively low at 25°C, compared to about 35–40°C in warm-blooded animals, so it must be processed at low temperatures to avoid thermal denaturation.

[0036]

[39] The method for producing collagen peptides from starfish of the present invention includes the steps of (a) treating the starfish with an alkaline solution to remove non-collagenous substances, (b) adding the starfish from which the non-collagenous substances have been removed to an acid solution to extract collagen, (c) adding a proteolytic enzyme to the solution from which the collagen has been extracted to hydrolyze it, and (d) separating the collagen peptides from the solution.

[0037]

[40] The starfish that can be used in the present invention may be any that belong to the Echinodermata phylum and the Starfish class, such as the Amur starfish, brittle star, starfish, red starfish, spiny brittle star, common starfish, mangrove starfish, brittle star, crown-of-thorns starfish, little brittle star, spiny maple shell, columbine starfish, maple shell, lump starfish, and brittle star.

[0038]

[41] In the method of the present invention, first, the starfish is cut into small pieces and then treated with an alkaline solution to remove non-collagenous substances, thereby obtaining starfish bone fragments.

[0039]

[42] The body wall of starfish contains a considerable amount of non-active ingredients, such as proteins other than collagen, subcutaneous fat, odor-causing substances (amines, fatty acids, carbonyl compounds, sulfide compounds, etc.), and inorganic substances (calcium carbonate). Therefore, non-collagenous substances can be removed through alkaline solution treatment.

[0040]

[43] The alkaline solution may be a mixed solution having a pH that allows non-active ingredients to be separated from the starfish, or may be a mixed solution containing an alkaline compound and a solvent and having a pH in the range of 9 to 14.

[0041]

[44] The alkaline compound may be any alkaline salt capable of adjusting the pH of the solution. For example, the aqueous solution may contain one or more selected from sodium hydroxide, calcium hydroxide, potassium hydroxide, etc., with sodium hydroxide being most preferred. The solvent is not limited, and for example, water can be used.

[0042]

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

[0043]

[46] To treat the starfish with alkali, the starfish can be cut into small pieces and immersed in an alkali solution for 12 to 48 hours.

[0044]

[47] Once the alkali treatment is complete, bone fragments with collagen attached to the body wall of the starfish can be harvested. The yield of the harvested bone fragments is preferably about 10 to 30% by weight of the original weight of the starfish.

[0045]

[48] ​​The harvested starfish bone fragments are added to an acid solution to extract collagen.

[0046]

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

[0047]

[50] The acid solution may contain 0.05 to 0.5 wt. % of the acid compound, more preferably 0.1 to 0.4 wt. %. If the concentration of the acid solution is too low, the collagen extraction efficiency will be low. As the concentration of the acid solution increases, the extraction efficiency increases, but above about 0.25 wt. %. Therefore, the acid solution should not exceed 0.5% by weight. It is preferable to use the above in terms of extraction efficiency.

[0048]

[51] In the present invention, after adding the starfish bone chips to the acid solution, it is preferable to perform ultrasonic treatment to accelerate collagen extraction. The ultrasonic treatment can be performed at 10 to 100 kHz for 20 to 200 minutes, and more preferably at 30 to 50 kHz for 40 to 80 minutes.

[0049]

[52] Once sonication is complete, the solution should be left for 5 to 15 hours to allow the acid-base reaction to complete. can.

[0050]

[53] Next, proteolytic enzymes are added to hydrolyze the extracted collagen into low molecular weight collagen peptides.

[0051]

[54] The starfish-derived collagen peptides produced by the method of the present invention have a molecular weight of about 1550 to 1700 Da depending on the type of enzyme, which is lower than that of fish collagen (marine collagen) at about 1900 Da and pigskin collagen at about 2400 Da. Therefore, it is expected to be more advantageous for skin penetration.

[0052]

[55] The enzyme may be subtilisin, pepsin, collagenase, trypsin, etc., and subtilisin is capable of producing collagen peptides with the lowest molecular weight and is also the most preferred in terms of wrinkle improvement performance.

[0053]

[56] In one embodiment of the present invention, it was confirmed that when collagen peptides are decomposed using subtilisin as an enzyme, collagen peptides having the most excellent wrinkle-improving effect can be produced compared to other enzymes.

[0054]

[57] The enzyme is preferably added in an amount of 0.01 to 1% by weight based on the weight of the starfish bone fragments. The content is preferably 0.05 to 0.4% by weight, and more preferably 0.05 to 0.4% by weight.

[0055]

[58] The temperature and time of the enzyme treatment may be any temperature that allows the protease to sufficiently hydrolyze the starfish. For example, the hydrolysis temperature and time may be in the range of 10 to 65°C and 1 to 10 hours, respectively. The hydrolysis temperature is determined by the protease. This is a temperature at which trypsin has high activity, and this temperature can be adjusted appropriately depending on the type of enzyme. For example, the hydrolysis temperature for trypsin can be in the range of 35 to 40°C.

[0056]

[59] Once the enzymatic treatment is complete, the collagen peptides can be isolated, for example by centrifugation to remove salts and separating the supernatant, which can then be freeze-dried to obtain powdered collagen peptides.

[0057]

[60] The starfish-derived collagen peptides produced have a particle size of approximately 1 μm in solvent, are completely non-cytotoxic, and have antioxidant activity, in contrast to both pig skin collagen and fish collagen, which have no antioxidant activity.

[0058]

[61] Furthermore, the starfish-derived collagen peptide of the present invention has anti-wrinkle activity. In one example of the present invention, when the anti-wrinkle activity was compared based on the rate of inhibition of MMP-1 expression in cells, it was confirmed that starfish-derived collagen exhibited a 2- to 3-fold higher MMP-1 expression inhibition rate than fish collagen and pigskin collagen.

[0059]

[62] The starfish-derived collagen peptide of the present invention can be used by itself in cosmetic compositions for antioxidant and anti-wrinkle purposes, or can be used after being supported in elastic liposomes.

[0060]

[63] The elastic liposomes carrying starfish-derived collagen peptides according to the present invention solve the problem of collagen peptides having difficulty passing through the intercellular lipids of the stratum corneum, overcome the limitations of skin absorption rate, and ensure optimal collagen peptide performance.

[0061]

[64] In particular, the present invention has found that collagen peptides isolated from starfish can be loaded into elastic liposomes with significantly higher efficiency than the commonly used pigskin collagen or fish collagen. This is thought to be because starfish-derived collagen peptides contain a larger amount of hydrophilic amino acids than pigskin or fish collagen. As shown in Table 1 below, starfish-derived collagen peptides contain approximately 40% hydrophilic amino acids, which is more than 1.5 times higher than the approximately 25% hydrophilic amino acids contained in pigskin or fish collagen.

[0062]

[65] [Table 1]

[66] [Table 1]

[0063]

[67]

[68] From this perspective, the starfish-derived collagen peptide of the present invention can contain 30% or more, preferably 35% or more, particularly 38% or more hydrophilic amino acids. In one embodiment of the present invention, a starfish-derived collagen peptide containing about 40% hydrophilic amino acids is It was confirmed that the collagen peptide showed significantly superior elastic liposome loading efficiency compared to pigskin or fish collagen peptide.

[0064]

[69] Elastic liposomes overcome the low entrapment efficiency, instability in the formulation, and inactivity of existing liposomes. It has been proposed to compensate for some of the drawbacks, such as the low solubility of the phospholipid components and the possibility of lipid oxidation and hydrolysis, and can be prepared by adding a surfactant that imparts elasticity to the phospholipids.

[0065]

[70] The elastic liposomes according to the present invention are prepared by mixing phospholipids and surfactants. It is composed of a phospholipid layer containing starfish-derived collagen peptides as a carrier inside the phospholipid layer. The ingredients not only contain phospholipids that have a similar structure to skin cells, but also have increased elasticity and excellent deformability, allowing them to effectively penetrate and move between the stratum corneum, resulting in excellent transdermal absorption efficiency.

[0066]

[71] The phospholipids play the role of intercellular lipids. It acts as a semipermeable membrane that prevents skin-beneficial ingredients from escaping from the skin and also functions as a permeable membrane that draws in moisture from the outside.

[0067]

[72] In the present invention, the phospholipid component may be a phospholipid commonly used in the art, for example, a phospholipid having a fatty acid chain of 12 to 24 carbon atoms, including, but not limited to, one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol. In the present invention, the phospholipid component is preferably phosphatidylcholine.

[0068]

[73] The surfactant is included to impart elasticity to the interface of the liposome phospholipid layer and improve transdermal absorption. Examples of surfactants that can be used include glucoside-based, sucrose-based, and glyceryl-based surfactants, with glucoside-based surfactants being most preferred.

[0069]

[74] The glucoside surfactants include cetearyl glucoside, decyl glucoside, coco glucoside, and behenyl Alcohol (behenyl alcohol), arachidyl alcohol, arachidyl glucoside, C10-20 alkyl glucoside ) can be used, with cetearyl glucoside being the most preferred.

[0070]

[75] The sucrose-based surfactants that can be used include sucrose monostearate, sucrose distearate, and sucrose tristearate.

[0071]

[76] Examples of the glyceryl surfactant include polyglyceryl-6 caprylate, polyglyceryl-4 caprate, Polyglyceryl-3 Methylglucose Distearate and the like can be used.

[0072]

[77] In one example of the present invention, it was confirmed that when elastic liposomes were prepared using cetearyl glucoside, a glucoside surfactant, as a surfactant, they exhibited a skin absorption rate that was 3 to 5 times higher than that of other surfactants.

[0073]

[78] The phospholipid and surfactant may be mixed in a weight ratio of 3:1 to 20:1, with a weight ratio of 7:1 to 12:1 being more preferred.

[0074]

[79] Furthermore, the starfish-derived collagen peptide of the present invention may be contained in an amount of 1 to 100 wt% of the weight of the phospholipid layer containing the phospholipid and surfactant, but is not particularly limited thereto. In the experimental examples of the present invention, it was found that the range of collagen peptide with the best skin absorption rate varies depending on the content of the phospholipid layer, and the best skin absorption rate was achieved by containing 1 wt% of the phospholipid layer and 0.1 wt% of the collagen peptide relative to the weight of the solvent. showed.

[0075]

[80] Elastic liposomes loaded with starfish-derived collagen peptides have a particle size of 50 to 600 nm, with most of them being on the order of 100 to 200 nm. This is significantly smaller than collagen peptides, which are approximately 1 μm in size in solvent. In the experimental examples of the present invention, the particle size tends to increase as the phospholipid content increases. This is believed to be because the thickness of the elastic liposome membrane increases when a certain amount of phospholipid is added.

[0076]

[81] Collagen peptides with a particle size of about 1 μm in a solvent have almost no skin absorption in the stratum corneum, but elastic liposomes can exhibit excellent skin absorption due to their smaller particle size and elasticity.

[0077]

[82] The starfish-derived collagen peptide of the present invention and elastic liposomes containing it have excellent antioxidant activity and anti-wrinkle effects on the skin, and can be used in cosmetic compositions.

[0078]

[83] The elastic liposomes are contained in an amount of 0.1 to 50% by weight based on the total weight of the cosmetic composition. It's okay to be surrounded.

[0079]

[84] The cosmetic compositions of the present invention may be prepared in any dosage form commonly used in the art, including, but not limited to, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, sprays, mask packs, and the like.

[0080]

[85] In addition, the cosmetic composition of the present invention may include cosmetics containing various additives with different ingredients depending on the type of cosmetic, such as facial cleansing cosmetics, basic cosmetics, color cosmetics, hair cosmetics, and functional cosmetics. [Example]

[0081]

[86]

[87] Example

[88]

[89] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0082]

[90]

[91] Production Example 1: Production of starfish-derived collagen peptides

[92]

[0083]

[93] After immersing 1,000 g of starfish in 1 L of 5% sodium hydroxide solution for 24 hours, The bone fragments were left to stand for a while, and non-collagenous substances were removed, leaving 200 g of bone fragments with collagen adhered thereto.

[0084]

[94] Approximately 2 g of fresh starfish bone fragments were added to 50 mL of distilled water with a 1:1 mixture of tartaric acid and ascorbic acid at concentrations of 0.05, 0.25, 0.5, 1.0, and 2.5 wt%. After ultrasonic treatment at 38 kHz for 1 hour, The mixture was left standing for 10 hours or more to complete the acid-base reaction.

[0085]

[95] Subtilisin, pepsin, collagenase (C-0130), collagenase (C-0130) buffer solution, trypsin, and trypsin buffer solution were added at 0.1 wt% of the enzymes based on the weight of the starfish bone fragments to decompose collagen peptides into low molecular weight forms.

[0086]

[96] The salts formed by the acid / base reaction in the lower layer were removed using a centrifuge, and the supernatant was separated. The separated supernatant was then freeze-dried to obtain powdered collagen peptides.

[0087]

[97]

[98] Experimental Example 1: Confirmation of physical properties by collagen peptide extraction process

[99]

[0088]

[0100] 1-1. Extraction efficiency depending on the amount of acid added

[0101] The collagen peptide extraction efficiency depending on the amount of acid added is summarized in Table 2 below.

[0102]

[0089] [Table 2]

[0103] [Table 2]

[0090]

[0104]

[0105] Ascorbic acid reacts with calcium carbonate, a component of bone spicules, to form a In the case of calcium ascorbate, since it is water-soluble, after centrifugation The extraction efficiency is included when the supernatant is freeze-dried. Therefore, the yield excluding calcium ascorbate that can be produced under the assumption that 100% of the added ascorbic acid reacted was confirmed. The highest yield was observed when 0.25 wt% of acid was added. It was confirmed that the yield gradually decreased with increasing dosage.

[0091]

[0106] This is because calcium tartrate, a salt formed by the reaction of tartaric acid and calcium carbonate, It is removed from the lower layer during centrifugation, which is thought to be due to the preferential formation of calcium tartrate over calcium ascorbate.

[0107]

[0092]

[0108] 1-2. Extraction efficiency depending on the type of enzyme

[0109] To examine the extraction efficiency depending on the type of enzyme, six types of enzymes were added to 0.05 wt % acid-treated sample. The results are shown in Table 3 below.

[0110]

[0093] [Table 3]

[0111] [Table 3]

[0094]

[0112]

[0113] The extraction efficiency was calculated by comparing the mass of bone fragments to which collagen was attached after alkaline treatment with the mass of collagen. The calculation is based on the dry mass of the freeze-dried extract of TESCA and Triglyceride. In the case of apsidal leukemia, a buffer solution of EDTA dissolved in PBS was used.

[0095]

[0114] The extraction efficiency showed almost the same tendency for each enzyme except for C-0130, which is collagenase. It was also confirmed that there was no significant difference in the case of buffer solutions.

[0096]

[0115]

[0116] 1-3. Collagen peptide molecular weight by enzyme type

[0097]

[0117] The molecular weight of the collagen peptide depending on the type of enzyme was confirmed by GPC (Gel Permeation Chromatograph) and is shown in Table 4 below.

[0118]

[0098] [Table 4]

[0119] [Table 4]

[0099]

[0120]

[0121] In the table above, regardless of the type of enzyme, the collagen extract It has been confirmed that collagen exists in the form of a low molecular weight peptide of approximately 1700 Da, and in the case of subtilisin, it has been confirmed that collagen peptides with the lowest molecular weight of 1600 Da or less can be produced.

[0100]

[0122]

[0123] 1-4. Cytotoxicity test by enzyme type (MTT assay)

[0101]

[0124] To measure cell viability, human fibroblasts (HDFs) were cultured in DMEM, 10% FBS, and 1% penicillin-streptomycin medium for 24 hours. Thereafter, each enzyme sample from Examples 3 to 6 was added to the medium at a concentration of 0.2 to 1.0 mg / mL, and after changing the medium, the medium was further cultured for 24 hours, and then an MTT solution was added and the medium was further cultured for 4 hours.

[0102]

[0125] After removing the medium, DMSO was added and the absorbance was measured at 560 nm. The results are shown in Table 5 below. shown in.

[0126]

[0103] [Table 5]

[0127] [Table 5]

[0104]

[0128]

[0129] In survival rate tests according to the type and concentration of enzyme, all showed a survival rate of 85% or more. In the case of subtilisin, the survival rate was over 92%, confirming that there was almost no cytotoxicity.

[0105]

[0130]

[0131] 1-5. Cytotoxicity test by enzyme type (Live / Dead imaging)

[0132] As in Experimental Example 1-4, after 24 hours of additional incubation at a concentration of 0.2 mg / mL, calcein AM (live) and ethidium homodimer (dead) were added to the PBS solution, and after 30 minutes, confocal imaging was performed. The results are shown in Figure 1.

[0106]

[0133] In Figure 1, (a) is a subtilisin / live image, (b) is a pepsin / live image, (c) is a C-0130 / live image, (d) is a trypsin / live image, and (e) is a subtilisin / dead image. (f) shows the pepsin / dead image, (g) shows the C-0130 / dead image, and (h) shows the trypsin / dead image.

[0134] No cytotoxicity was observed in any of the experimental groups.

[0107]

[0135]

[0136] 1-6. Antioxidant activity of collagen peptides depending on the type of enzyme

[0108]

[0137] The antioxidant properties of collagen peptides were confirmed through the DPPH radical scavenging test. I acknowledged it.

[0109]

[0138] After reacting the DPPH solution with the sample solutions of Examples 3 to 6 at different concentrations, The radical scavenging ability was confirmed by measuring absorbance at 100 nm, and the results are shown in Table 6 below. The antioxidant activity was confirmed using vitamin C as a 100% control.

[0139]

[0110] [Table 6]

[0140] [Table 6]

[0111]

[0141]

[0142] It showed excellent antioxidant activity of approximately 90% or more against all enzymes, and showed the highest antioxidant activity at 0.2 mg / mL.

[0112]

[0143]

[0144] 1-7. Comparison of anti-wrinkle activity of collagen peptides with enzymes

[0145]

[0113]

[0146] Human fibroblasts CCD-986sk were cultured for 24 hours in a medium containing DMEM, 10% FBS, and 1% penicillin-streptomycin. Each enzyme sample from Examples 3 to 6 was added to the medium at a concentration of 1 mg / mL, and after changing the medium, the cells were irradiated with UVB for 20 minutes and cultured for 24 hours. The culture supernatant was incubated with a coating buffer, and then treated with a washing buffer and a blocking buffer. After treatment with diluted primary and secondary antibodies at different concentrations, the culture supernatant was removed and treated with washing buffer. Finally, the cells were incubated in pnPP (substrate solution) in the dark for 1 hour, and the absorbance at 405 nm was measured. The results were converted into MMP-1 expression inhibition rates and are shown in Table 7 below.

[0147]

[0114] [Table 7]

[0148] [Table 7]

[0115]

[0149]

[0150] In the table above, the subtilisin-treated sample had the best MM compared to the other enzymes. It was confirmed that the expression of P-1 was suppressed.

[0116]

[0151]

[0152] Experimental Example 2: Starfish-derived collagen peptide, pigskin collagen peptide, and Comparison of Fish Collagen Peptides

[0117]

[0153]

[0154] Starfish-derived collagen extracted by the extraction process determined through Experimental Example 1 Experiments were carried out to compare the peptides with pig skin collagen peptides and fish collagen peptides.

[0118]

[0155] The pig skin collagen peptide and fish collagen peptide used in the experiment were , respectively, as shown in Table 8 below.

[0156]

[0119] [Table 8]

[0157] [Table 8]

[0120]

[0158]

[0159] 2-1. Comparison of cytotoxicity of collagen peptides

[0121]

[0160] The MTT assay cell viability test was carried out in the same manner as in Experimental Examples 1 to 4. The starfish collagen used was the collagen of Example 3. The results of the MTT cell viability were as follows: Shown in Table 9.

[0161]

[0122] [Table 9]

[0162] [Table 9]

[0123]

[0163]

[0164] In the table, pig skin collagen and fish collagen showed cell growth rates of 80% or less at concentrations of 0.4 mg / mL or higher, but overall, no significant cytotoxicity was observed in any of the three collagen samples.

[0124]

[0165]

[0166] 2-2. Comparison of molecular weights of collagen peptides

[0125]

[0167] The molecular weights of the three collagen peptides were confirmed by GPC (Gel Permeation Chromatograph) and are shown in Table 10 below.

[0168]

[0126] [Table 10]

[0169] [Table 10]

[0127]

[0170]

[0171] Collagen derived from starfish is much lower than pigskin and fish collagen. It was confirmed that the polymer had a low molecular weight.

[0128]

[0172]

[0173] 2-3. Comparison of the antioxidant activity of collagen peptides

[0129]

[0174] The DPPH antioxidant activity was analyzed in the same manner as in Experimental Examples 1 to 6, and the results are shown in Table 11 below. showed.

[0175]

[0130] [Table 11]

[0176] [Table 11]

[0131]

[0177]

[0178] Starfish collagen showed excellent antioxidant activity, while pigskin collagen and fish collagen showed poor antioxidant activity. None of the collagen samples exhibited antioxidant properties.

[0132]

[0179]

[0180] 2-4. Comparison of collagen peptides' anti-wrinkle activity

[0133]

[0181] The MMP-1 expression inhibition rate was analyzed in the same manner as in Experimental Example 1-7, and the results are shown in Table 12 below. did.

[0182]

[0134] [Table 12]

[0183] [Table 12]

[0135]

[0184]

[0185] When comparing the wrinkle-preventing activity based on the rate of UV-induced inhibition of MMP-1 expression in cells, starfish collagen peptide was found to be approximately three times more effective than fish collagen peptide, and pigskin collagen peptide. It has been shown to have a higher MMP-1 expression inhibition rate than methicone and has a significantly superior anti-wrinkle activity. You can confirm it.

[0136]

[0186]

[0187] Production Example 2: Production of elastic liposomes carrying collagen peptides

[0188]

[0137]

[0189] Add phospholipids and surfactants to a 50 mL round flask according to the manufacturing ratios in Table 13 below. The agent and collagen peptide were added and thoroughly dissolved in 20 mL of ethanol. After completely removing the solvent using a rotary evaporator, 20 mL of distilled water was added and the mixture was thoroughly dissolved. To homogenize the elastic liposome particles, the mixture was sonicated at 30 kHz for 15 minutes. Elastic liposomes were prepared using the above method.

[0190]

[0138] [Table 13]

[0191] [Table 13]

[0139]

[0192]

[0193] Experimental Example 3: Confirmation of the physical properties of elastic liposomes

[0194]

[0140]

[0195] 3-1. Loading efficiency of elastic liposomes

[0141]

[0196] The phospholipid is phosphatidylcholine, and the surfactant is polyglyceryl caprylate. The carriers used were Polyglyceryl-6 Caprylate (Polyglyceryl-6 Caprylate) and Polyglyceryl-4 Caprate (TEGO SOLVE 90, EVONIK), and the carrier efficiency at each composition ratio was measured.

[0142]

[0197] After preparing the elastic liposomes, they are filtered through a 450 nm syringe filter and loaded. The purified elastic liposomes were disrupted by ultracentrifugation. The collagen peptides loaded afterwards were quantified by BCAA assay. The loading efficiency was calculated by calculating the ratio of the BCA Assay quantification value of the total collagen peptides before loading to the BCA Assay quantification value, and the results were calculated as follows: Shown in Table 14.

[0198]

[0143] [Table 14]

[0199] [Table 14]

[0144]

[0200]

[0201] 3-2. Comparison of particle size of elastic liposomes

[0145]

[0202] The particle sizes of starfish-derived collagen peptides and elastic liposomes were measured and shown in the table below. 15.

[0203]

[0146] [Table 15]

[0204] [Table 15]

[0147]

[0205]

[0206] In the case of starfish-derived collagen peptide, the particle size is about 1 μm in the solvent, and the elasticity is It was confirmed that the particle size of the liposomes was in the nm range and did not exceed 1 μm.

[0148]

[0207] Generally, as the phospholipid content increases, the particle size also tends to increase, which is thought to be due to the increase in the thickness of the elastic liposome membrane when a certain amount of phospholipid is added.

[0149]

[0208]

[0209] 3-3. Comparison of skin absorption rates of elastic liposomes

[0150]

[0210] To compare the skin absorption rate, the acceptor plate coated with artificial skin was After hydrating the skin layer of the donor plate, the corresponding sample was filled into each well of the donor plate together with the buffer solution. After hydration, the acceptor plate was filled with the buffer solution and placed on the donor plate and incubated. The absorbance of each plate was then analyzed using a microplate reader to measure the skin permeation rate, and the results are shown in Table 16 below.

[0211]

[0151] [Table 16]

[0212] [Table 16]

[0152]

[0213]

[0214] In the above table, in the case of a collagen extract having a particle size of about 1 μm in the solvent, There was almost no skin absorption in the stratum corneum, and the skin absorption rate was not measured.

[0153]

[0215] On the other hand, samples made with elastic liposomes showed various skin absorption rates depending on the ratio. This was somewhat similar to the particle size trend.

[0154]

[0216] Therefore, taking into consideration the economics of the process during oxidation, the collagen extract should be prepared in an appropriate ratio. It has the highest carrying efficiency and particle size, and is an elastic liposome with EL1 / 0.1, which shows the best skin absorption rate. It is preferable to manufacture a sphere.

[0155]

[0217]

[0218] 3-4. Analysis of carrier efficiency by surfactant

[0156]

[0219] Elastic liposomes were prepared using the following candidate surfactants at a production ratio of EL1 / 0.1, and the loading efficiency, particle size, and skin absorption rate were compared. The prepared elastic liposome samples are named as follows according to the type of surfactant:

[0220]

[0157] [Table 17]

[0221] [Table 17]

[0158]

[0222]

[0223] The loading efficiency of elastic liposomes depending on the type of surfactant was evaluated in the same manner as in Experimental Example 3-1. The results are shown in Table 18 below.

[0224]

[0159] [Table 18]

[0225] [Table 18]

[0160]

[0226]

[0227] EL1 / 01-SF3 Elastic Liposomes with Cetearyl Glucoside Surfactant It was confirmed that the loading efficiency of the SiO2 was the highest.

[0161]

[0228]

[0229] 3-5. Particle size analysis using surfactants

[0162]

[0230] The particle size of the elastic liposomes was measured depending on the type of surfactant, and the results are shown in Table 19 below. did.

[0231]

[0163] [Table 19]

[0232] [Table 19]

[0164]

[0233]

[0234] In the table above, particles in the 100 nm range are obtained without significant deviation depending on the type of surfactant. It was confirmed that the diameter was

[0165]

[0235]

[0236] 3-6. Analysis of skin absorption rate by surfactants

[0166]

[0237] The skin absorption rate of elastic liposomes depending on the type of surfactant was measured, and the results are shown in Table 2 below. 0 shown.

[0238]

[0167] [Table 20]

[0239] [Table 20]

[0168]

[0240]

[0241] In the table above, it is approximately 2000 mg / cm 2 The skin absorption rate was about / h, but cetearyl EL1 / 01-SF3 elastic liposomes using glucoside surfactants exhibited a density of 6455 mg / cm 2The skin absorption rate of 1000mg / h was extremely high, about 3 to 5 times higher than that of samples using other surfactants.

[0169]

[0242]

[0243] Experimental Example 4: Starfish-derived collagen peptide, pigskin collagen peptide and fish Comparison of elastic liposomes loaded with collagen peptides

[0244]

[0170]

[0245] Elastic liposomes loaded with pig skin collagen peptide and fish collagen peptide were prepared using the same composition and surfactant as EL1 / 0.1-SF3 in Experimental Example 3. The names of the samples are listed in Table 21 below.

[0246]

[0171] [Table 21]

[0247] [Table 21]

[0172]

[0248]

[0249] 4-1. Loading efficiency of elastic liposomes using collagen peptides

[0173]

[0250] The loading efficiency of elastic liposomes depending on the type of collagen peptide was examined in the same manner as in Experimental Example 3-1. The results were measured in the same manner and are shown in Table 22 below.

[0251]

[0174] [Table 22]

[0252] [Table 22]

[0175]

[0253]

[0254] The loading efficiency of starfish-derived collagen peptide was confirmed to be more than six times higher. This is thought to be because the hydrophilic group ratio in the amino acid sequence of starfish collagen peptide is about 40%, which is higher than that of pigskin and fish collagen peptide, making it easier to form elastic liposomes.

[0176]

[0255]

[0256] 4-2. Particle size of elastic liposomes made from collagen peptides

[0177]

[0257] The particle size of elastic liposomes was measured depending on the type of collagen peptide, and the results are shown in Table 23 below. shown in.

[0258]

[0178] [Table 23]

[0259] [Table 23]

[0179]

[0260]

[0261] The particle size of the elastic liposomes was 100 nm without any significant deviation depending on the type of collagen peptide, but the particle size of EL-Po and EL-Fi was measured to be smaller. Considering the loading efficiency data, this suggests that elastic liposomes were produced without loading substances, resulting in a reduced particle size.

[0180]

[0262]

[0263] 4-3. Skin absorption rate of elastic liposomes using collagen peptides

[0181]

[0264] The skin absorption rate of elastic liposomes depending on the type of collagen peptide is shown in Experimental Example 3-3. Measurements were carried out in the same manner and are shown in Table 24 below.

[0265]

[0182] [Table 24]

[0266] [Table 24]

[0183]

[0267]

[0268] The skin absorption rate of starfish-derived collagen peptides is higher than that of pigskin and fish collagen. It was confirmed that this was significantly higher than that of peptides.

[0184]

[0269]

[0270] 4-4. Antioxidant activity of elastic liposomes with collagen peptides

[0185]

[0271] The DPPH antioxidant activity of elastic liposomes was measured according to the type of collagen peptide. Shown in Table 25 below.

[0272]

[0186] [Table 25]

[0273] [Table 25]

[0187]

[0274]

[0275] As a result of the experiment, elastic liposomes carrying starfish-derived collagen peptides were found to have excellent The elastic liposomes loaded with pigskin and fish collagen showed no antioxidant activity.

[0188]

[0276]

[0277] 4-5. Anti-wrinkle activity of elastic liposomes containing collagen peptides

[0189]

[0278] The skin wrinkle-inhibiting activity of elastic liposomes was measured according to the type of collagen peptide. The results are shown in Table 26 below.

[0279]

[0190] [Table 26]

[0280] [Table 26]

[0191]

[0281]

[0282] As a result of the experiment, elastic liposomes carrying starfish-derived collagen peptides were found to have excellent It was confirmed that elastic liposomes loaded with pigskin and fish collagen had no or weak skin wrinkle inhibitory activity.

[0192]

[0283]

[0284] Experimental Example 5: Confirmation of the activity of starfish-derived collagen peptides depending on the type of enzyme

[0285]

[0193]

[0286] In Experimental Example 1, subtilisin, pepsin, C-0130, and trypsin were used. The elastic liposome loading efficiency and skin permeability of the starfish-derived collagen peptide obtained as an enzyme were measured in the same manner as in Experiments 3 and 4, and the pigskin collagen peptide and fish collagen peptide were also measured. The results were compared with those for the antigen peptide and are shown in Table 27 below.

[0287]

[0194] [Table 27]

[0288] [Table 27]

[0195]

[0289]

[0290] From the above table, when subtilisin is used as the enzyme, the lowest molecular weight and excellent fineness are obtained. It was confirmed that the collagen peptides extracted from starfish using pepsin, collagenase, and trypsin still exhibited significantly superior antioxidant properties, binding efficiency, and skin permeability compared to the collagen peptides from pigskin and tilapia (fish).

[0196]

[0291] In particular, C-0130, which had the lowest binding efficiency among the four enzymes, showed a binding efficiency of more than 3.8 times that of fish collagen peptide, and also showed superior skin permeability.

[0197]

[0292] These differences are not simply due to the molecular weight of the enzymes involved, but are due to the This is believed to be because the collagen peptides derived from pig skin contain a larger amount of hydrophilic amino acids than collagen peptides from pig skin or fish.

[0198]

[0293]

[0294] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention is based on the technical idea It will be understood that the present invention may be embodied in other specific forms without changing the essential features or characteristics. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims that follow, and equivalent concepts thereof, rather than the above detailed description.

Claims

1. a phospholipid layer comprising a phospholipid and a surfactant; and An elastic liposome comprising a starfish-derived collagen peptide carried inside the phospholipid layer.

2. The elastic liposome according to claim 1, wherein the starfish-derived collagen peptide contains 30% or more hydrophilic amino acids.

3. The elastic liposome according to claim 1 , wherein the surfactant is a glucoside-based, sucrose-based, or glyceryl-based surfactant.

4. The elastic liposome according to claim 1, wherein the particle diameter of the elastic liposome is 50 to 600 nm.

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