KR102321532B1 - Liposome composition comprising skin physiologically active substances and method of preparing the same - Google Patents

Abstract

It relates to a liposome composition containing a skin bioactive substance and a method for preparing the same, and according to the liposome composition containing a skin bioactive substance and a method for preparing the same according to an aspect, it is physically and chemically stable and can enhance skin absorption of active ingredients. By delivering a skin bioactive substance to the skin using a liposome that can

Description

Liposome composition containing skin bioactive substances and manufacturing method thereof

It relates to a liposome composition containing a skin bioactive substance and a method for preparing the same.

The skin is histologically composed of the epidermis, dermis, and subcutaneous fat, and the thickness varies according to the site, age, and sex. Various skin conditions exist, but each of these skins performs a role such as a barrier function from the outside and other physiological functions. The stratum corneum is a layer of keratinized dead cells, in which the insoluble protein keratin, which has relatively high hydrophobicity, is accumulated at a high density with an intercellular lipid bilayer, which is a thin layer of lipids, stacked therebetween. As a structure, the unique hierarchical structure of a matrix rich in lipids and insoluble proteins makes it impermeable to chemical factors, including various compounds from the external environment. In particular, it is very difficult for hydrophilic physiologically active substances to permeate due to such a hydrophobic multi-structure of the stratum corneum.

Therefore, in general, the transport of substances through the keratin in healthy skin, especially the skin permeation of the hydrophilic physiologically active material, is not easily achieved due to various protective actions of the skin, and even if it is permeated, only a very low concentration is permeated. However, in the case of cosmetics, the physiologically active substances contained in the cosmetics must pass through the stratum corneum, the outermost layer of the skin, in order to exhibit their efficacy. , there is a problem that the physiologically active substances contained in cosmetics do not sufficiently exhibit their functions.

A representative example of a lipid material-based vesicle for transdermal penetration is liposomes. Liposomes are spherical vesicles with a structure in which a phospholipid bilayer contains water. In addition to promoting skin permeation, stabilizing unstable substances that are easily oxidized, and controlling the release rate of captured substances. and has been widely used in cosmetics.

Because it has both hydrophilic and hydrophobic regions of liposomes, both poorly soluble drugs and water-soluble drugs can be included in its structure, and biocompatibility and safety are high by using lipids or cholesterol having biocompatibility. On the other hand, the physical stability of the liposome membrane itself is low, the collection efficiency is limited and the material to be collected is low, and there are many cases where it does not penetrate deep into the skin and remains in the stratum corneum.

Since the lipid layer spacing between keratinocytes in the stratum corneum of the skin is approximately tens of nanometers, vesicles having a size of several hundred nanometers or more, such as liposomes, do not easily pass through the lipid layer. Therefore, in order for the lipid-based vesicle to easily pass through the lipid layer, the vesicle itself must be more flexible and elastically deformable. was developed

Ethosome containing ethanol in the liposome to increase the transdermal permeation of the active ingredient, ethanol approaches the lipid layer of keratinocytes and lowers the Tm of the lipid, increases the fluidity of the lipid layer, and loosens the barrier membrane with a strong structure. Although it increases the skin penetration efficacy of cicle, it may cause skin irritation by ethanol. The transfersome is a structure made by mixing a single-chain surfactant called an edge activator with a phospholipid when preparing a vesicle using lecithin. The edge activator together with the phospholipids constitute the curved surface of the spherical vesicle membrane, and the edge activator having a different radius of curvature than the phospholipids weakens the lipid bilayer membrane of the construct, thereby inducing easy deformation of the membrane of the construct. A lot of technical research is being conducted on a transfersome, a lipid material-based vesicle for percutaneous penetration, which improves physical and chemical stability and improves skin permeability by increasing the flexibility of the structure.

Accordingly, the present inventors prepared a transfersome-type external preparation for skin containing a hydrophilic physiologically active ingredient using a transfersome capable of enhancing skin delivery by supporting a hydrophilic physiologically active ingredient, and confirmed the antioxidant and anti-aging effects of the composition. invention was completed.

M. M. Elsayed, O. Y. Abdallah, V. F. Naggar, and N. M. Khalafallah, Lipid vesicles for skin delivery of drugs: Reviewing three decades of research, Int J. Pharm., 332, 1-16 (2007)

One aspect is a liposome comprising a phospholipid and an edge activator; And to provide a cosmetic composition comprising a hydrophilic physiologically active material supported on the liposome.

Another aspect is a liposome comprising a phospholipid and an edge activator; And to provide an external preparation for skin comprising a hydrophilic physiologically active material supported on the liposome.

Another aspect is a liposome comprising a phospholipid and an edge activator; And to provide a pharmaceutical composition comprising a hydrophilic physiologically active material supported on the liposome.

Another aspect is to provide a method for preparing the composition.

One aspect is a liposome comprising a phospholipid and an edge activator; And it provides a composition comprising a hydrophilic physiologically active material supported on the liposome.

The composition may be a composition for improving skin beauty, preventing, improving, or treating skin condition, skin damage, or skin aging.

The term "skin beauty" refers to returning the damaged skin due to skin damage or skin aging to the state before it was damaged in such a way as to improve skin wrinkles, enhance volume, improve elasticity, or regenerate skin. The skin includes all skin parts of the body including the face, hands, arms, legs, feet, chest, belly, back, buttocks, and scalp.

The term "skin damage" refers to external physical damage, penetration of chemicals, bacteria, fungi, viruses, etc., exposure to ultraviolet rays, moisture loss of the skin, wrinkles due to aging, oxidation by free radicals (active oxygen), etc., skin pigmentation Includes damage from a clinical and cosmetic point of view of skin tissue or cells, including deposition, skin inflammation, seborrheic dermatitis, redness (redness, erythema), edema, lichenification, eczema, pruritus (itchiness), and atopic dermatitis. .

The term "skin aging" includes intrinsic aging over time and extrinsic aging caused by external environments. The skin aging may include skin wrinkles, blemishes, blemishes, and the like. The skin wrinkles may be fine wrinkles caused by skin aging. Skin wrinkles may be due to photoaging, age, facial expression, lack of moisture, or a combination thereof. The photoaging may be skin aging caused by exposure to ultraviolet rays (including UVA, UVB, and UVC). The skin wrinkle improvement may be to suppress or inhibit the generation of wrinkles on the skin, or to alleviate wrinkles already generated.

The liposome may refer to a spherical vesicle of a lipid bilayer containing a phospholipid and an edge activator.

The phospholipids include phosphatidylcholine, soybean lecithin, sphingomyelin, cholesterol, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, cardiolipin, plasma. Dicetylphosphate, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphatidylethanolamine and ele It may be at least one selected from the group consisting of osteoylphosphatidylserine. In addition, the phospholipid is 0.05 wt% to 20 wt%, 0.1 wt% to 15 wt%, 0.1 wt% to 10 wt%, 0.1 wt% to 8 wt%, 0.2 wt% to 15 wt%, 0.2 wt% with respect to the total composition wt% to 10 wt%, 0.2 wt% to 8 wt%, 0.1 wt% to 4 wt%, 0.2 wt% to 4 wt%, 0.2 wt% to 2 wt%, 0.8 wt% to 15 wt%, 0.8 wt% to 8 wt%, 2 wt% to 15 wt%, 2 wt% to 10 wt%, or 2 wt% to 8 wt%.

The edge activator may refer to a material that provides flexibility to the lipid bilayer. In general, edge activators are selected to increase fluidity in the lipid bilayer, allowing them to provide smaller or more elastic vesicles to be highly deformable, for penetrating skin pores with small size. The edge activator may include a silicone-containing edge activator, a cationic edge activator, an anionic edge activator, a nonionic edge activator, a zwitterionic edge activator. More specifically, the edge activator is sodium deoxycholate (Sodium deoxycholate), sodium lauryl sulfate (sodium lauryl sulfate), sodium lauryl ether sulfate (sodium lauryl ether sulfate), disodium lauryl sulfosuccinate ( disodium lauryl sulfosuccinate), lysolecithin, polyglycerin-10 laurate, lauryl PEG/PPG-18/18 methicone, PEG-12 dimethicone, PEG/PPG-15/15 dimethicone, cetyl trimethyl ammonium Chloride (cetyl trimethyl ammonium chloride), alkyl dimethyl benzyl ammonium chloride (alkyl dimethyl benzyl ammonium chloride), cocoamidopropyl betaine (cocoamidopropyl betaine), sodium cocoamphoacetate (sodium cocoamphoacetate), cocamide MEA (cocamide MEA), It may be at least one selected from the group consisting of polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-80, sorbitan monocetearate and steareth-2. . In addition, the edge activator is based on the total composition, 0.005 to 10% by weight, 0.01 to 10% by weight, 0.01 to 5% by weight, 0.01 to 3% by weight, 0.01 to 2% by weight, 0.01 to 2% by weight 1 wt%, 0.02 wt% to 2 wt%, 0.02 wt% to 1 wt%, or 0.02 wt% to 0.8 wt% may be included.

The cosmetic composition may further include water (purified water) and polyhydric alcohol. The water and polyhydric alcohol may be added to form a continuous phase. The polyhydric alcohol may be at least one selected from the group consisting of glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, ethylene glycol, and polyethylene glycol. The polyhydric alcohol is 2 wt% to 30 wt%, 2 wt% to 25 wt%, 2 wt% to 15 wt%, 5 wt% to 30 wt%, 5 wt% to 25 wt%, 5 wt% with respect to the total composition % to 20% by weight, or 5% to 15% by weight.

In one embodiment, the diameter of the liposome is 5 to 1,500 nm, 5 to 1,000 nm, 10 to 1,500 nm, 10 to 1,000 nm, 10 to 800 nm, 10 to 500 nm, 5 to 500 nm, 5 to 300 nm , 10 to 300 nm, 5 to 200 nm, 5 to 100 nm, or 10 to 100 nm.

In the present specification, the term “physiologically active substances” may refer to substances capable of imparting useful activity to an individual, for example, skin regeneration, skin anti-aging, skin wrinkles, skin whitening, and the like. Non-limiting examples of the bioactive material may include caviar extract, proteoglycan or fucoidan.

In one embodiment, the physiologically active material may include all of caviar extract, proteoglycan and fucoidan.

In the present specification, the term "caviar" is used to include not only salted sturgeon eggs, but also natural sturgeon eggs and processed products thereof. Thus, "caviar" may be used interchangeably with "sturgeon roe." On the other hand, sturgeon amniotic fluid can be obtained when using the method of squeezing eggs without cutting the belly of the sturgeon to collect eggs from the sturgeon. The sturgeon eggs and sturgeon amniotic fluid can be easily obtained by directly collecting from the sturgeon or by purchasing commercially available ones.

The caviar extract may be a nucleic acid mixture extracted from caviar.

As used herein, the term “nucleic acid mixture” may refer to an unspecified form in which a nucleic acid fragment having a reduced molecular weight of a nucleic acid, which is a biopolymer, is mixed. The nucleic acid may be DNA. The mixture of the nucleic acid fragments is a nucleic acid polymer, and since it is a biocomponent, there is no allergic reaction or rejection reaction in the body, and the main component is made of DNA or RNA having a negative charge, so it has hydrophilicity and is very easy to apply to pharmaceutical compositions and cosmetics. can be used in the composition.

In one embodiment, the nucleic acid may be DNA.

The nucleic acid fragment may be about 10 kDa to 2000 kDa, about 10 kDa to 1900 kDa, about 10 kDa to 1800 kDa, about 10 kDa to 1700 kDa, about 15 kDa to 1700 kDa, about 15 kDa to 1650 kDa, about 15.5 to 1650 kDa. kDa, it may have a molecular weight of about 16 to 1650 kDa.

The nucleic acid mixture is treated with saline, an organic solvent or a surfactant such as SDS, and then heated to an aqueous solution obtained, centrifuged to obtain a supernatant, and then subjected to several steps such as lowering the pH again to precipitate and separate the nucleic acid complex material. It can be separated according to a general method known in the art, such as a method.

Specifically, the nucleic acid mixture is prepared by adding purified water to the caviar raw material, and homogenizing it to produce a homogenate; adding and dissolving a surfactant and deep sea water extract minerals to the homogenate produced in the above step to produce a lysate; adjusting the pH of the lysate produced in the above step; obtaining a precipitate by adding alcohol to the pH-adjusted lysate obtained in the above step; Purifying the precipitate obtained in the above step to obtain a purified product containing low molecular weight DNA; and/or drying the purified product obtained in the above step to obtain low molecular weight DNA; It may be prepared by a method comprising a.

As used herein, the term "proteoglycan" is an acidic mucopolysaccharide complexed with a protein, the sugar molecule contained therein has nitrogen, and glycosaminoglycan ( glycosaminoglycan (GAG) contains a structure to which sugar chains are bound. Proteoglycan has the water-moisturizing power of hyaluronic acid, fills the gap between collagen fibers, and can play a role in enhancing the moisturizing properties and elasticity of the dermal layer.

As used herein, "fucoidan" may include a sulfated polysaccharide in which fucose extracted from seaweed and a sulfate group are bonded. The fucoidan is synthesized in the intracellular Golgi apparatus and may exist as an intercellular material or a cell wall constituent material of brown algae, and has high viscosity due to water solubility, and fucose, galactose, mannose, and xylose. , uronic acid, etc., have a function as a dietary fiber.

Desalting and deodorizing the prepared brown algae in the fucoidan; grinding the desalted and deodorized brown algae; Enzyme treatment on the pulverized brown algae; obtaining crude fucoidan from the enzyme-treated brown algae; and/or purifying the crude fucoidan to obtain fucoidan.

In one embodiment, the bioactive material is 0.005 wt% to 50 wt%, 0.005 wt% to 40 wt%, 0.01 wt% to 10 wt%, 0.005 wt% to 10 wt%, 0.001 wt% with respect to the total composition to 2 wt%, 0.04 wt% to 10 wt%, or 0.004 wt% to 1.5 wt%.

The component ratio of the proteoglycan: fucoidan: nucleic acid mixture extracted from caviar is 1:0.5 to 2: 3 to 8, 1:0.8 to 1.5: 3 to 8, 1:0.5 to 2: 4 to 6, or 1:0.8 to 1.5: may be 4 to 6, and in one embodiment, may be 1:1:5.

The composition may be a cosmetic composition. The cosmetic composition may be prepared in various forms, and in one embodiment, the composition is a lotion (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream , moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack or sheet, and aerosol It may be prepared in any one formulation selected from the group. The composition of such a formulation can be prepared according to a method conventional in the art. The cosmetic composition may further include preservatives, stabilizers, surfactants, solubilizers, moisturizers, emollients, UV absorbers, preservatives, bactericides, antioxidants, pH adjusters, organic and inorganic pigments, fragrances, cooling agents or limiting agents, etc. have. The blending amount of the additional ingredients such as the moisturizing agent can be easily selected by those skilled in the art within the range that does not impair the purpose and effect of the present invention, and the blending amount is 0.001 to 5% by weight, specifically 0.01 to 3% by weight based on the total weight of the composition % by weight.

In addition, the composition may be a composition for external application to the skin.

In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component normally used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extract, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

In another embodiment, the composition may be a pharmaceutical composition.

The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection.

Another aspect provides a method of making the composition.

The method comprises the steps of preparing a mixture by mixing a phospholipid and an edge activator; dissolving a hydrophilic physiologically active substance in an aqueous phase; preparing a mixed homogenate containing liposomes by mixing and homogenizing the mixture in an aqueous phase in which the physiologically active material is dissolved; and loading the physiologically active material in the mixed homogenate into the liposome (eg, using a microfluidizer).

It relates to a liposome composition containing a skin bioactive substance and a method for preparing the same, and according to the liposome composition containing a skin bioactive substance and a method for preparing the same according to an aspect, it is physically and chemically stable and can enhance skin absorption of active ingredients. By delivering a skin bioactive substance to the skin using a liposome that can

1 is a schematic view showing a liposome containing a physiologically active ingredient according to an embodiment.
2 is a photograph showing a liposome containing a physiologically active ingredient according to an embodiment.
3 is a graph showing the results of particle size analysis of liposomes containing a physiologically active ingredient according to an embodiment.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. These Examples and Experimental Examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these Examples.

Examples 1 to 5: Preparation of liposomes containing bioactive substances

As shown in the composition of Table 1 below, phospholipid lecithin (lipoid company, lipoid P75-3) and edge activator polysorbate-80 or polyglycerin-10 laurate were mixed with polyalcohols glycerin and butylene glycol at 60 to 70 ° C. It was dissolved by heating. Next, caviar DNA, which is a physiologically active ingredient to be loaded on the liposome, was prepared. Caviar DNA was prepared as described in Korean Application No. 10-2019-0105223. Specifically, after adding SDS and sodium chloride extracted from deep sea water so that the final concentration of sodium dodecyl sulfate (SDS) in the pulverized caviar is about 1% by weight and the final concentration of sodium chloride extracted from deep sea water is about 1.25% by weight, 10 Slowly inverting about 30 to 30 times so that the pulverized product (homogenate), SDS, and sodium chloride extracted from deep sea water are evenly mixed, and then stirred at room temperature (for example, 18 to 25° C.) for about 1 hour to dissolve seafood was obtained. Sodium hydroxide was added to the lysate to make the pH 9.5 to 10.5, and then stirred at 60 to 75° C. for 1 to 2 hours. Thereafter, citric acid was added to adjust the final pH to pH 6.7 to 7.3, and then centrifuged at about 10,000 rpm using a centrifuge to obtain a pH-adjusted lysate (supernatant). Ethanol was added to the pH-adjusted lysate by volume so that lysate: ethanol = 25 to 35: 65 to 75, and then precipitated overnight under conditions of 0 to 4 ° C. A precipitate (crude extract) was obtained by centrifugation at 7,000 rpm, which was dried to obtain low molecular weight DNA (yield: 4%).

Fucoidan was prepared as described in Korean Application No. 10-2019-0105016. Specifically, 100 times (by weight) purified water was added to the pulverized brown algae and stirred, and then the enzyme (a mixture of two enzymes selected from Pectinex, Flavozyme, Alcalase, Celluclast and Biscozyme) was added. and extracted. After the extraction was completed, the enzyme was left at 100° C. for more than 20 minutes to stop the enzymatic action. After centrifugation at 8000 rpm for 15 minutes to separate the brown algae raw material and the supernatant, 1M HCl was added to adjust the pH to 2.0, and then calcium chloride (CaCl2) was added to remove alginic acid. Then, the supernatant was separated by centrifugation at 8000 rpm for 15 minutes, and the pH of the separated supernatant was adjusted to pH 7 using 1M NaOH. Then, the supernatant was concentrated using a rotary evaporator, and after concentration was adjusted to 7-10 brix, the same amount of ethanol as the concentration was added to obtain crude fucoidan as a precipitate. The precipitate was dissolved in purified water, and again ethanol was added to obtain fucoidan through a re-refining process, which was finally freeze-dried.

Proteoglycan was purchased from Korea COSPAC and used.

Next, the three physiologically active ingredients prepared above were added to distilled water and dissolved by heating to 30~70°C. The heated and dissolved phospholipid and edge activator mixture solution was slowly added to an aqueous solution containing physiologically active ingredients at 30 to 70 ° C., and homogenized at 30 to 70 ° C. at 1,000 to 10,000 rpm using a homogenizer. The homogenized mixture was treated three times at 30-70° C. at 100-3,000 bar pressure using a microfluidizer to prepare liposomes containing physiologically active ingredients.

Raw material name Example 1 Example 2 Example 3 Example 4 Example 5 Lecithin (P75-3) 0.5 1.0 5.0 0.5 0.5 Polysorbate-80 0.05 0.1 0.5 0.05 0 Polyglycerin-10 Laurate 0 0 0 0 0.05 butylene glycol 10.0 10.0 20.0 0 10.0 glycerin 0 0 0 10.0 0 caviar DNA 0.05 0.1 0.5 0.05 0.05 proteoglycan 0.01 0.02 0.1 0.01 0.01 fucoidan 0.01 0.02 0.1 0.01 0.01 Distilled water To 100 To 100 To 100 To 100 To 100

1 is a schematic view showing a liposome containing a physiologically active ingredient according to an embodiment.

2 is a photograph of a liposome containing a physiologically active ingredient according to an embodiment.

In addition, particle size analysis was performed on the liposome containing the physiologically active ingredient of Example 1 prepared above. For particle size analysis, the particle size and particle size distribution were analyzed using Malvern's Zetasizer Nano ZS90, which can measure the particle size at a scattering angle of 90 degrees using dynamic light scattering, and the results are shown in FIG. 3 .

3 is a graph showing the results of particle size analysis of liposomes containing a physiologically active ingredient according to an embodiment.

As shown in Figure 3, it was found that the liposome according to one embodiment has a diameter of 10 to 1000 nm.

Comparative Examples 1 and 2: Preparation of transfersomes

A transfersome was prepared in the same manner as in Example 1, except for the composition shown in Table 2, except that the composition was different from that of Example 1.

Raw material name Comparative Example 1 Comparative Example 2 Comparative Example 3 Lecithin (P75-3) 0.5 0.5 0 Polysorbate-80 0.05 0 0 butylene glycol 10.0 10.0 0 caviar DNA 0 0.05 0.05 proteoglycan 0 0.01 0.01 fucoidan 0 0.01 0.01 Distilled water To 100 To 100 To 100

Experimental Example 1. Antioxidant activity analysis

A free radical scavenging test and a free radical scavenging test were performed to confirm the antioxidant effect as a skin aging improvement effect assay test for Example 1, Comparative Example 1 and Comparative Example 3. The free radical scavenging test uses that the absorbance of stable DPPH exhibits the maximum absorbance at 540 nm. Specifically, as the free radical DPPH is cleared by the sample and becomes a transparent color from purple, that is, as the free radical scavenging rate increases, the 540 nm This is a method that uses the decrease in absorbance at wavelengths.

First, in 1 mL of 0.1 mM 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH, Sigma) solution, dilute the sample of the above example to an appropriate concentration in methanol, mix 1 mL, and leave at 37°C for 15 minutes After that, the absorbance was measured at a wavelength of 540 nm using a microplate reader (Thermo max, USA). In the free radical scavenging test, the control was measured by adding 1 ml of DPPH and 1 ml of methanol, and 1 ml of methanol and 1 ml of the sample were added to obtain respective color correction values for the sample and the control. Positive control As L-ascorbic acid was used, the free radical scavenging rate was calculated using Equation 1 below, and the results are shown in Table 3 below. In Table 3, IC ₅₀ is the concentration of the sample required to remove 50% of free radicals, and the smaller the value, the higher the antioxidant activity.

[Equation 1]

Free radical scavenging rate (%) = 100 - ((absorbance of sample / absorbance of control) x 100)

sample Free radical scavenging rate, IC ₅₀ (ppm, μg/ml) Example 1 23.50 Comparative Example 1 N.D. Comparative Example 3 25.75 L-Ascorbic Acid 22.50

As a result of Table 3, Example 1 and Comparative Example 3 showed similar antioxidant ability compared to L-ascorbic acid, a strong synthetic antioxidant, and it was confirmed that 50% of the free radical scavenging rate showed antioxidant efficacy at a trace concentration. This means that the composition according to one embodiment not only improves the delivery of the physiologically active material by liposome loading, but also mixes other components for preparing the liposome, and the antioxidant activity of the physiologically active component is not reduced.

Experimental Example 2. Analysis of skin wrinkle inhibition and improvement activity

An elastase inhibition activity test was performed to test the skin wrinkle suppression and improvement effects of Example 1, Comparative Example 1 and Comparative Example 3. Elastin is a component constituting the matrix layer in the dermis of the skin, and as the skin ages, elastin is decomposed, the matrix layer in the dermis collapses, and wrinkles occur. Using these principles of N- Ella stay's substrate by the method of measuring the enzyme activity of Ella's stay for decomposing elastin (elastin) succinyl - (Ala) ₃ p- nitroaniline (N-succinyl- (Ala) ₃ p-nitroaniline, Sigma) can be used to measure the elastase activity by measuring the absorbance at a wavelength of 405 nm for the color change caused by the decomposition of p-nitroaniline.

Specifically, the buffer solution is pH 8.0, 0.267 M Trizma-HCl (Sigma), the substrate solution is 8.8 mM N-Succinyl-(Ala)3 p-nitroaniline, and the enzyme solution is 10 μg/mL of pig pancreatic elastase (from Sigma). prepared) was used. After mixing 60 μL of buffer, 20 μL of substrate solution, and 100 μL of each sample diluted in tertiary distilled water for each concentration, add 20 μL of enzyme solution and react for 15 minutes in a water bath at 25°C to determine the amount of p-nitroaniline produced. It was measured at a wavelength of 405 nm using a microplate reader (UVT-06685, Thermo max, USA). The control group for measuring the elastase activity was measured in the same manner by adding third distilled water instead of the sample, and the case where the color correction value for each was obtained by adding third distilled water instead of the enzyme solution was set. In order to determine the elastase activity inhibitory effect of Example 1, Comparative Example 1 and Comparative Example 3, the sample used for the test was oleanolic acid (Sigma-Aldrich), which is currently known to have excellent effect as a wrinkle-improving functional raw material. ) was set as a positive control. Elastase inhibition rate was calculated numerically using Equation 2 below, and the results are shown in Table 4 below. In Table 4, IC ₅₀ is the concentration of the sample required to inhibit the elastase activity by 50%, and the smaller the value, the higher the inhibition rate.

[Equation 2]

Elastase activity inhibition rate (%) = 100 - ((absorbance of sample / absorbance of control) x 100)

sample Elastase activity inhibition rate, IC ₅₀ (mg/mL) Example 1 0.23 Comparative Example 1 N.D. Comparative Example 3 0.25 oleonolic acid 0.21

As shown in Table 4, the elastase activity inhibition rates of Example 1 and Comparative Example 3 were 0.23 and 0.25 mg/mL, respectively, and it was confirmed that they had anti-wrinkle and anti-aging effects similar to those of oleonolic acid. . This means that the composition according to one embodiment not only improves the delivery of the physiologically active material by liposome loading, but also mixes other components for preparing the liposome, the anti-wrinkle activity of the physiologically active component is not reduced. .

Experimental Example 3. MMP expression inhibition activity analysis

After UV irradiation and sample addition of Example 1, Comparative Example 1 and Comparative Example 3, an enzyme immunoassay (ELISA) was performed to measure the concentration of MMP-1.

Specifically, using a UV chamber, UVA was irradiated to human dermal fibroblasts with an energy of 5 J/cm 2 . The UV irradiation amount and incubation time established the conditions for maximizing MMP expression in fibroblasts through preliminary experiments. The negative control group was wrapped in silver foil and kept in the UVA environment for the same time. UVA emission was measured using a UV radiometer. The medium was not changed during UVA irradiation, and after UV irradiation was completed, the medium containing the sample was exchanged. Thereafter, the cells were further cultured for 24 hours, and the medium was recovered and added to 96-wells. Thereafter, the wells were treated with a primary antibody (MMP-1 (Ab-5) monoclonal antibody) and reacted at 37° C. for 60 minutes. In the well, the secondary antibody, whole mouse, alkaline phosphatase conjugated (anti-mouse IgG), was reacted again for about 60 minutes, and then an alkaline phosphatase substrate solution (1 mg/mlρ-nitrophenyl phosphate in diethanolamine buffer) was reacted at room temperature for 30 minutes. and the absorbance was measured at 405 nm with a microplate reader. As a control, a sample to which no sample was added was used, and as a positive control, oleonolic acid was used, and the results are shown in Table 5 below.

test group Treatment concentration (%) MMP-1 expression inhibition rate (%) Example 1 10 25 Comparative Example 1 10 0 Comparative Example 3 10 17 Oleanolic acid 0.05 20

As shown in Table 5, it was confirmed that the composition of Example 1 showed an inhibitory rate of 25% or more with respect to MMP-1, which was induced during UV irradiation, compared to the control group that was not treated with the sample.

In addition, Comparative Example 3 was directly treated without supporting the skin bioactive material in the liposome, and it was found that the liposome composition according to one embodiment had better MMP-1 inhibitory activity than Comparative Example 3. This means that the composition according to one embodiment not only improves the delivery of the physiologically active material by liposome loading, but also mixes other components for preparing the liposome, the MMP-1 inhibitory activity of the physiologically active component is not reduced. it means.

As a result of the above, it was found that the liposome composition carrying skin physiological activity according to one embodiment has an effect of improving skin condition, for example, antioxidant, anti-aging, anti-wrinkle, or skin barrier improvement.

Claims (8)

liposomes comprising 0.1 wt% to 10 wt% of phospholipid and 0.01 wt% to 5 wt% of an edge activator with respect to the total composition; and
It contains 0.01 to 10% by weight of a hydrophilic skin bioactive material with respect to the total composition, supported on the liposome,
The hydrophilic skin bioactive material will include a low molecular weight DNA fragment having a molecular weight of 10 to 2000 kDa extracted from caviar, proteoglycan and fucoidan,
A cosmetic composition for improving skin beauty. The cosmetic composition of claim 1, wherein the liposome has a diameter of 10 to 10,000 nm. The method according to claim 1, wherein the phospholipid is phosphatidylcholine, soybean lecithin, sphingomyelin, cholesterol, phosphatidic acid, phosphatidylserine, phosphatidyl glycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, cardiolipin , Plasmalogen, dicetylphosphate, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoyl A cosmetic composition that is at least one selected from the group consisting of phosphatidylethanolamine and eleostearoylphosphatidylserine. The method according to claim 1, wherein the edge activator is sodium deoxycholate (Sodium deoxycholate), sodium lauryl sulfate (sodium lauryl sulfate), sodium lauryl ether sulfate (sodium lauryl ether sulfate), disodium lauryl sulfosuccinate (disodium lauryl sulfosuccinate), lysolecithin, polyglycerin-10 laurate, lauryl PEG/PPG-18/18 methicone, PEG-12 dimethicone, PEG/PPG-15/15 dimethicone, cetyl trimethyl ammonium cetyl trimethyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride, cocoamidopropyl betaine, sodium cocoamphoacetate, cocamide MEA , at least one selected from the group consisting of polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-80, sorbitan monocetearate and steareth-2 phosphorus cosmetic composition. The method according to claim 1, wherein the low molecular weight DNA fragment extracted from the caviar,
adding purified water to the caviar raw material and homogenizing to produce a homogenate;
adding and dissolving a surfactant and deep sea water extract minerals to the homogenate produced in the above step to produce a lysate;
adjusting the pH of the lysate produced in the above step;
obtaining a precipitate by adding alcohol to the pH-adjusted lysate obtained in the above step;
Purifying the precipitate obtained in the above step to obtain a purified product containing low molecular weight DNA; and
A cosmetic composition prepared by a method comprising the step of drying the purified product obtained in the above step to obtain low molecular weight DNA. The method according to claim 1, wherein the fucoidan comprises the steps of desalting and deodorizing prepared brown algae; grinding the desalted and deodorized brown algae; Enzyme treatment on the pulverized brown algae; obtaining crude fucoidan from the enzyme-treated brown algae; and purifying the crude fucoidan to obtain fucoidan. The cosmetic composition according to claim 1, wherein the skin cosmetic improvement is anti-wrinkle, antioxidant, or anti-aging. 0.1% to 10% by weight of phospholipids based on the total composition, and
liposomes comprising an edge activator in an amount of 0.01% to 5% by weight based on the total composition; and
It contains 0.01 to 10% by weight of a hydrophilic skin bioactive material with respect to the total composition, supported on the liposome,
The hydrophilic skin bioactive material comprises a low molecular weight DNA fragment having a molecular weight of 10 to 2000 kDa extracted from caviar, proteoglycan and fucoidan, a composition for external application for skin for improving skin condition.

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