Reverse-phase liposome containing culture medium of fusidium corneum and use thereof

Abstract

The present invention provides a reverse-phase liposome containing a culture medium of Fusidium coccineum strain and a supercritical extract of Fusidium coccineum cells. The reverse-phase liposome demonstrates excellent effects of skin permeation, skin elasticity enhancement, skin lifting improvement, skin moisturization, skin self-growth enhancement, and skin barrier reinforcement.

Description

Reverse-phase liposome containing a culture solution of Fusidium cocineum and use thereof

[0001] The present invention relates to a reverse-phase liposome containing a microbial culture medium and a use thereof.

[0002] The skin envelops the entire body and is largely composed of three layers: the epidermis, the dermis, and the subcutaneous fat layer. The outermost layer, the epidermis, contains the stratum corneum, which acts as a barrier to protect the body from various external irritants. The stratum corneum has a "lamellar structure," and this structural characteristic suppresses moisture evaporation and effectively protects the skin from external stress and harmful stimuli. However, it also hinders the absorption of effective cosmetics ingredients. Therefore, technologies that enhance the absorption rate of this stratum corneum are crucial when developing cosmetic formulations.

[0003] Fusidic acid is a type of antibiotic used in ointments to prevent secondary bacterial infections in human skin wounds.

[0004] Meanwhile, even substances that are gases and liquids at normal temperatures and pressures cease to evaporate when they exceed a certain high temperature and high pressure limit called the supercritical point. This state makes them indistinguishable from gas and liquid, a state in which the two phases are indistinguishable. This state is called a supercritical fluid. Supercritical fluids are characterized by a large variation in molecular density. Their molecular density is close to that of a liquid, but their viscosity is low, making them close to that of a gas. Furthermore, they diffusion is rapid, resulting in thermal conductivity as high as that of water. These characteristics make them extremely useful in chemical reactions. When used as a solvent, they exhibit the unique property of dramatically increasing the solvent concentration around the solute (dissolved molecules). Supercritical fluids can be continuously varied in density by varying pressure and temperature, from a state close to that of an ideal gas to a state close to that of a liquid. This ease of property control is utilized in reaction and separation processes, a technique called supercritical extraction. Supercritical extraction alters the solubility of a supercritical fluid by adjusting conditions such as temperature and pressure during the extraction step, enabling the selective extraction of specific components.

[0005] The inventors of the present invention completed the present invention by preparing a reverse-phase liposome using a supercritical extract of a Fucidium cocineum strain and confirming its skin improvement effect.

[0006] One aspect provides a reverse phase liposome comprising a Fusidium coccineum strain culture and a supercritical extract of Fusidium coccineum strain cells.

[0007] Another aspect provides a cosmetic composition for improving skin condition comprising the above-described reverse liposome as an active ingredient.

[0008] Another aspect provides a composition for external application for skin improvement containing the above-mentioned reverse liposome as an active ingredient.

[0009] Another aspect provides a method for preventing, improving or treating a condition of a subject, comprising the step of treating or administering to a subject in need thereof a composition comprising a reverse phase liposome comprising a culture of a strain of Fusidium coccineum and a supercritical extract of Fusidium coccineum strain cells.

[0010] Another aspect provides a use of a reverse phase liposome comprising a Fusidium coccineum strain culture and a supercritical extract of Fusidium coccineum strain cells for use in preparing a composition for improving skin condition.

[0011] One aspect provides a reverse phase liposome comprising a Fusidium coccineum strain culture and a supercritical extract of Fusidium coccineum strain cells.

[0012] The above “Fusidium coccineum” is a type of fungus, which may be isolated from a plant. The plant is not limited to any specific type, but may be, for example, a ginkgo leaf.

[0013] In one specific example, the Fusidicium cocineum may be a strain deposited as KCCM12013P.

[0014] In this specification, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned medium" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the strain of Fusidium cocineum can grow and survive in vitro. In addition, the culture medium may mean a culture medium obtained by removing the bacterial cells from the bacterial culture obtained by culturing the strain. Meanwhile, the liquid from the culture medium from which the bacterial cells have been removed is also called a "supernatant", and the supernatant may be obtained by allowing the culture medium to stand still for a certain period of time and taking only the liquid in the upper layer excluding the portion that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture medium to remove the sediment at the lower layer and taking only the liquid in the upper layer. The culture medium may include the culture medium itself obtained by culturing the strain, a concentrate or a lyophilized product thereof. The above culture medium may include a culture supernatant obtained by removing the strain from the culture medium, a concentrate thereof, or a lyophilized product thereof. The culture medium, culture conditions, and culture time for culturing the Fusidium cocineum strain may be appropriately selected or modified by those skilled in the art. Specifically, the medium for the Fusidium cocineum strain may be, for example, PDA (Potato Dextrose Agar) or PDB (Potato Dextrose Broth) medium.

[0015] In one specific example, the culture medium of the Fusidium cocineum strain may be a fermented culture medium obtained by inoculating and fermenting the strain. More specifically, it may be a fermented culture medium from which the fungal cells have been removed.

[0016] The term “cell” in this specification may refer to a fungus itself obtained after culturing a Fusidia coccineum strain in a medium that provides nutrients to enable the strain to grow and survive in vitro.

[0017] In this specification, the term "bacterial cell" refers to the strain itself, and includes the strain itself selected by separating from a sample or the like, or the strain isolated from the culture medium by culturing the strain. The bacterial cell can be obtained by centrifuging the culture medium and collecting the portion that has settled to the lower layer, or by allowing the bacterial cell to settle to the lower layer of the culture medium by gravity and then removing the upper liquid after leaving it still for a certain period of time.

[0018] The above cells may be obtained through one or more of the processes of crushing, sterilization, and freeze-drying.

[0019] The term “supercritical extract” as used herein may be obtained by extracting Fusidium cocineum strain cells by a supercritical extraction method. The supercritical extract may be obtained by supercritically extracting Fusidium cocineum strain cells with carbon dioxide (CO2) at a temperature of 40 to 80°C and a pressure of 150 to 500 bar. The supercritical extract may be obtained by extracting Fusidium cocineum strain cells at a temperature of 40 to 80°C, 40 to 75°C, 40 to 65°C, 45 to 80°C, 45 to 70°C, or 50 to 65°C. In addition, the supercritical extract may be obtained by extracting Fusidia coccineum strain cells at a pressure of 150 to 500 bar, 150 to 450 bar, 150 to 400 bar, 150 to 350 bar, 200 to 350 bar, or 250 to 350 bar. In this case, if the temperature and pressure conditions of the supercritical extract are below or exceed the above range, there is a problem in that the skin condition improvement effect cannot be sufficiently exhibited.

[0020] The above supercritical extract may be extracted using a carrier oil for supercritical extraction as a solvent. The carrier oil for supercritical extraction may be, for example, sunflower seed oil, olive oil, squalene oil, jojoba seed oil, mineral oil, tea tree oil, peppermint oil, grape seed oil, camellia japonica seed oil, shea butter oil, etc.

[0021] In one specific example, the supercritical extract may be an oil extract of Fusidia coccineum strain cells.

[0022]

[0023] As used herein, the term “reverse liposome” refers to a liposome in which the hydrophobic portion (tail) faces outward and the hydrophilic portion (head) faces inward in a non-polar solvent environment. The reverse liposome can be dispersed into nano-sized particles.

[0024] The above reversed-phase liposome may contain a supercritical extract of Fucidium cocineum strain cells in an amount of 10 to 45 wt%, 10 to 40 wt%, 10 to 35 wt%, 15 to 45 wt%, 15 to 40 wt%, 15 to 35 wt%, 20 to 45 wt%, 20 to 40 wt%, 20 to 35 wt%, 25 to 45 wt%, 25 to 40 wt%, 25 to 35 wt%, 30 to 45 wt%, 30 to 40 wt%, 30 to 35 wt%, 35 to 40 wt%, but is not limited thereto.

[0025] The above reverse phase liposome may further comprise a surfactant or polyol.

[0026] The surfactant may be a natural surfactant or a synthetic surfactant. Specifically, the natural surfactant may be soybean lecithin, egg lecithin, hydrogenated lecithin (hydrogenated soybean lecithin and hydrogenated egg lecithin), sphingosine, ganglioside, or phytosphingosine, but is not limited to any surfactant commonly used in the art to prepare reversed-phase liposomes. In one specific example, the surfactant may be hydrogenated lecithin.

[0027] The above polyol may be any one selected from the group consisting of propylene glycol, dipropylene glycol, butylene glycol, glycerin, methylpropanediol, isoprene glycol, pentylene glycol and any combination thereof, but is not particularly limited thereto.

[0028] The above reverse-phase liposomes can be prepared using a reverse-phase liposome preparation method known in the art. For example, the above reverse-phase liposomes can be prepared by a method including a step of mixing a strain culture medium, a supercritical extract of strain cells, a surfactant, and a polyol. The mixing can be performed using a stirring device such as a stirrer. The stirring speed and stirring time are not particularly limited, but for example, stirring can be performed at a stirring speed of 2,000 rpm to 10,000 rpm and a stirring time of 1 minute to 12 hours. Devices such as a homogenizer and a microfluidizer can be used.

[0029] In one embodiment, the components were mixed, homogenized at 6,000 rpm for 10 minutes, cooled, and then treated with a microfluidizer to prepare reverse phase liposomes.

[0030] In one specific example, the reverse phase liposome may have enhanced skin permeability.

[0031] In one specific example, the reversed-phase liposome may increase the expression of one or more genes selected from the group consisting of elastin (ELN), hyaluronic acid synthase (HAS), claudin (CLD), and filaggrin (FLG). Specifically, the liposome may increase the expression of ELN, HAS, CLD, or FLG to enhance skin elasticity, improve skin lifting, moisturize skin, enhance skin self-healing power, or strengthen skin barrier.

[0032] In one embodiment, a reverse-phase liposome comprising a Fucidium coccineum strain culture and a supercritical extract of the strain cells not only functions as a formulation that enhances skin permeability to allow active ingredients in the liposome to penetrate into the skin more efficiently, but also functions as a composition for improving skin condition by increasing the expression of elastin, hyaluronic acid synthase, claudin, or filaggrin, thereby exhibiting effects such as improving skin elasticity, improving skin lifting, moisturizing the skin, enhancing skin self-regeneration, or strengthening the skin barrier.

[0033]

[0034] Another aspect provides a cosmetic composition for improving skin condition, comprising the reverse-phase liposome as an active ingredient.

[0035] In the above cosmetic composition, the same elements as those already mentioned among the mentioned term elements are as described above.

[0036] In one specific example, the improvement in skin condition may be improvement in skin elasticity, improvement in skin lifting, skin moisturizing, enhancement of skin self-healing power, or enhancement of skin barrier.

[0037] In this specification, the term "skin elasticity enhancement" may be used interchangeably with "skin wrinkle improvement" and may mean alleviating the degree to which the skin sags or sags, or inhibiting or preventing the formation of wrinkles on the skin, or alleviating wrinkles that have already formed.

[0038] In this specification, the term “skin lifting” may refer to any action that lifts up sagging skin due to oxidation of collagen, elastin, etc., which form the skin connective tissue, or pulls up and smooths wrinkled skin.

[0039] As used herein, the term "skin barrier strengthening" can refer to any action that enhances the function of the skin barrier, located on the outermost layer of the skin and preventing moisture and nutrient loss. Damage to skin barrier function can refer to any change that occurs in the skin due to a decline or damage to the skin barrier function. Examples include increased wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, and acne.

[0040] In this specification, the term “strengthening skin’s self-healing power” can mean all actions that strengthen the skin’s own power and restore damaged skin barriers to health.

[0041] As used herein, the term “skin moisturizing” may mean any action that maintains skin moisture or prevents moisture loss.

[0042] As used herein, the term "improvement" may mean any action that at least reduces the severity of a symptom, for example, a parameter associated with alleviating or treating a condition.

[0043] The reversed liposome is present in an amount of 0.001 wt% to 80 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to It may be included in an amount of 20 wt%, 0.1 wt% to 10 wt%, 0.1 wt% to 5 wt%, 0.1 wt% to 3 wt%, or 0.1 wt% to 2 wt%. In this case, if the content of the liposome is less than the above range, there may be a problem in that the skin condition improvement effect cannot be sufficiently exhibited.

[0044] The above cosmetic composition may have, for example, a cosmetic formulation of a softening toner, a nourishing toner, a massage cream, a nourishing cream, an essence, a pack, a patch, a mask sheet, a gel, an ointment, an ampoule, or a skin adhesive type.

[0045] The ingredients included in the above cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the reverse-phase liposome as an active ingredient, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0046]

[0047] Another aspect provides a composition for external application for skin improvement containing the above-mentioned reverse liposome as an active ingredient.

[0048] In the above skin external preparation composition, the same elements among the mentioned terminology as those already mentioned are as described above.

[0049] The above-mentioned 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 above-mentioned external preparation for skin may be appropriately mixed with ingredients commonly used in external preparations for skin such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or a combination thereof, as needed. The above skin external preparation may also appropriately contain metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; drugs such as caffeine, tannin, tocopherol acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0050]

[0051] Additionally, the composition may be a pharmaceutical composition.

[0052] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The glidant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0053] The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may include granules, powders, liquids, tablets, capsules, dry syrups, or combinations thereof. Parenteral dosage forms may include injections, ointments, creams, and the like.

[0054]

[0055] Another aspect provides a method for preventing, improving, or treating a condition of a subject, comprising administering or treating to a subject in need thereof a composition comprising a reverse phase liposome comprising a culture of a strain of Fusidium coccineum and a supercritical extract of Fusidium coccineum strain cells.

[0056] The condition of the above object may be related to improvement of skin condition.

[0057] As used herein, the terms "administering," "introducing," and "implanting" are used interchangeably and may refer to placement of a composition according to an embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site according to an embodiment.

[0058] Administration may be administered by any method known in the art. Administration may be administered directly to the subject by any means, including intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. Administration may be systemic or local. Treatment or administration may include application to the skin.

[0059] The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of improving skin condition, such as an individual in need of increasing skin elasticity, improving skin lifting, moisturizing the skin, enhancing skin self-healing power, and strengthening the skin barrier.

[0060] The above administration is 0.1 mg to 1,000 mg per day per individual of the composition according to one specific example, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to It may be administered at 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg. However, the dosage may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The number of administrations may be once a day or twice or more within the range of clinically acceptable side effects, and the administration site may be administered at one or more sites, and the total number of administration days may be from 1 to 30 days per treatment at intervals of 2 to 5 days daily or every day. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage per kg as for humans may be administered, or the dosage may be converted to the above dosage based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and humans.

[0061]

[0062] Another aspect provides a use of a reverse phase liposome comprising a Fusidium coccineum strain culture and a supercritical extract of Fusidium coccineum strain cells for use in preparing a composition for improving skin condition.

[0063] Details regarding the above Fusidium cocineum strain, supercritical extract, reverse phase liposome, and use for improving skin condition are as described above.

[0064] Reverse-phase liposomes according to the aspect of the invention not only serve as a formulation that enhances skin permeability and allows the active ingredients in the liposomes to penetrate the skin more efficiently, but also exhibits the effects of increasing skin elasticity, improving skin lifting, moisturizing the skin, enhancing skin self-regenerating power, or strengthening the skin barrier, and thus can be utilized as a composition for improving skin condition.

[0065] Figure 1 is a graph evaluating the skin permeability of reverse-phase liposomes containing a supercritical extract of a Fucidium coccineum strain culture and strain cells according to various aspects.

[0066] Figure 2 is a graph showing the effect of reverse-phase liposomes containing a supercritical extract of a Fucidium coccineum strain culture and strain cells on ELN expression according to the daily aspect.

[0067] Figure 3 is a graph showing the effect of reverse-phase liposomes containing a supercritical extract of a Fucidium coccineum strain culture and strain cells according to the daily aspect on FLG expression.

[0068] Figure 4 is a graph showing the effect of reverse-phase liposomes containing a supercritical extract of a Fucidium coccineum strain culture and strain cells according to the daily aspect on CLD expression.

[0069] Figure 5 is a graph showing the effect of reverse-phase liposomes containing a supercritical extract of a Fucidium coccineum strain culture and strain cells according to the daily pattern on HAS3 expression.

[0070] **p<0.01 vs. None

[0071] In Figure 1, Normal liposome (Fusiderm 0) is the basic liposome of Comparative Example 2, and Normal liposome (Fusiderm X) is the basic liposome of Comparative Example 3.

[0072] In FIGS. 1 to 5, Reverse liposome (Fusiderm O) is the reverse liposome of Example 1, and Reverse liposome (Fusiderm X) is the reverse liposome of Comparative Example 1.

[0073] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0074]

[0075] [Manufacturing example]

[0076] Manufacturing Example 1. Manufacturing of supercritical extract of Fusidium coccineum strain

[0077] The Fusidium coccineum strain (KCCM12013P) was provided and cultured by suspending the strain in PDB (Potato dextrose broth) medium at a speed of 100 rpm / min. The culture temperature was maintained at approximately 25°C and cultured for 300 hours. Thereafter, the culture broth of the strain was filtered using a 0.45 μm filter to recover the cells. In order to disrupt and sterilize the recovered cells, the cells were freeze-dried at approximately -70°C and approximately 20 mTorr for 72 hours to obtain a cell lyophilized product of the Fusidium coccineum strain.

[0078] The cell freeze-dried product of the Fusidium cocineum strain obtained above was supercritically extracted using CO2 gas and sunflower seed oil (carrier oil) at approximately 60°C and 300 bar for 3 hours. Finally, a supercritical extract of the Fusidium cocineum strain cells was obtained.

[0079]

[0080] Manufacturing Example 2. Preparation of Fusidium cocineum strain culture solution

[0081] A culture solution of the Fusidium cocineum strain was prepared. Specifically, it was confirmed that Fusidium cocineum (KCCM12013P) exhibited an optimal growth environment on potato starch agar (PDA). 5 g of the potato starch agar medium was mixed with 100 ml of sterilized distilled water and sterilized at 121°C for 15 minutes before use. After sterilization, it was cooled to 25°C, and 1 g of the isolated fungus was inoculated. The inoculated medium was cultured in an incubator at 28°C with stirring at 100 rpm for 300 hours. After 300 hours, the medium was filter-sterilized through a 0.2 μm filter to obtain a culture solution of Fusidium cocineum.

[0082]

[0083] [Example]

[0084] Example 1. Preparation of reverse-phase liposomes containing a supercritical extract of Fusidia coccineum strain culture and strain cells.

[0085] Reverse-phase liposomes were prepared using the supercritical extracts of the Fusidia coccineum strain cultures and strain cells prepared in Manufacturing Examples 1 and 2. Specifically, all of the components in Table 1 below were mixed, dissolved until the temperature reached 80°C, and homogenized at 6,000 rpm for 10 minutes. After stirring, the mixture was cooled to 30°C and then treated twice with a microfludaizer at 1,300 bar. As a result, the raw material recovery rate was greater than 96%.

[0086] Raw material name Example 1 (weight%) (Reverse liposome: Fusiderm O) Fusidium cocineum supercritical extract 36.5% Medium-chain triglyceride oil - polyquaternium - 512% Hydrogenated lecithin 3% Beta-glucan 3% Methylpropanediol 20% Glycerin 30% Fusidium cocineum culture broth 3% Hexanediol 0.5% Purified water 2%

[0087]

[0088] [Comparative example]

[0089] Comparative Example 1. Preparation of reverse-phase liposomes not containing a supercritical extract of Fusidia coccineum strain culture medium and strain cells.

[0090] Compared to Example 1, reverse-phase liposomes were prepared without the supercritical extract of the Fusidia coccineum strain culture and strain cells. Specifically, reverse-phase liposomes were prepared in the same manner as Example 1 by mixing the components in Table 2 below. Compared to Example 1, medium-chain triglycerides (MCT) were used instead of the supercritical extract, and the remaining components and contents were the same as in Example 1.

[0091] Raw material name Comparative example 1 (weight %) (Reverse liposome: Fusiderm X) Fusidium cocineum supercritical extract - medium-chain triglyceride oil 36.5% Polyquaternium - 512% Hydrogenated lecithin 3% Beta-glucan 3% Methylpropanediol 20% Glycerin 30% Fusidium cocineum culture broth - hexanediol 0.5% Purified water 2%

[0092] Comparative Examples 2 and 3. Preparation of Basic Liposomes

[0093] Compared to Example 1, basic liposomes, rather than reverse-phase liposomes, were prepared. Specifically, all components listed in Table 3 below were mixed, dissolved at 60°C, and homogenized at 6,000 rpm for 10 minutes. After stirring, the mixture was cooled to 30°C, and then subjected to a single microfluidizer treatment at 800 bar. As a result, the raw material recovery rate was greater than 96%.

[0094] Raw material name Comparative example 2 (weight %) (Normal liposome: Fusiderm O) Comparative example 3 (weight %) (Normal liposome: Fusiderm X) Fucidium coccineum culture broth 55% - purified water 32.66% 87.66% Hexanediol 2% 2% Butylene glycol 7.5% 7.5% Lecithin 0.75% 0.75% Dipotassium glycyrrhizate 0.04% 0.04% Ceramide NP 0.025% 0.025% Arginine 0.025% 0.025%

[0095]

[0096] [Experimental Example]

[0097] Experimental Example 1. Confirmation of skin permeability

[0098] In order to confirm the skin permeability of the liposomes manufactured in the above examples and comparative examples, purified water (2 wt%) among the components of reversed-phase liposomes (Example 1 and Comparative Example 1) was replaced with adenosine (2 wt%), or adenosine (2 wt%) was added to the basic liposomes (Comparative Examples 2 and 3) to manufacture liposomes in the same manner as the above manufacturing method. Specifically, a skin permeation device (FDC-6T, Logan, USA) was used, and the relative humidity of the device was maintained at 30-50% during the test. The Strat-M membrane (trandermal diffusion test model, USA, filter diameter 25 mm, thickness 300 μm) was washed with PBS (phosphate buffered saline) for 3-5 minutes and fixed to the upper part of the receptor chamber, and then the receptor chamber was filled with PBS and the temperature of the PBS was maintained at 32±1℃. Thereafter, 0.5 mL of the examples or comparative examples was applied to the membrane (applied area 0.64 cm 2) was uniformly applied to the skin, and after 3 hours, 0.3 mL of PBS was collected from the receptor chamber and adenosine was quantitatively analyzed using HPLC. The skin permeation rate was confirmed by calculating the amount of adenosine detected in the receptor chamber compared to the amount of adenosine in the applied test substance.

[0099] Figure 1 is a graph evaluating the skin permeability of reverse-phase liposomes containing supercritical extracts of Fusidium cocineum cultures and strain cells according to various aspects. In Figure 1, reverse liposome (Fusiderm O) is the reverse-phase liposome of Example 1, reverse liposome (Fusiderm X) is the reverse-phase liposome of Comparative Example 1, normal liposome (Fusiderm O) is the basic liposome of Comparative Example 2, and normal liposome (Fusiderm X) is the basic liposome of Comparative Example 3.

[0100] As a result, as shown in Fig. 1, the skin permeation rate of the reverse-phase liposome (Example 1) containing the supercritical extract of the Fusidia coccineum strain culture and the strain cell showed a better skin absorption effect compared to the reverse-phase liposome (Comparative Example 1) not containing the supercritical extract of the Fusidia coccineum strain culture and the strain cell. In addition, compared to the basic liposome (Comparative Examples 2 and 3) that is not the reverse-phase liposome, it showed an absorption amount about twice as large, showing an excellent skin permeation rate result.

[0101]

[0102] Experimental Example 2. Confirmation of skin condition improvement effect

[0103] The skin condition improvement effect of the reversed-phase liposomes manufactured in the above examples and comparative examples was confirmed. Specifically, human keratinocyte HaCaT cells were cultured in DMEM medium (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% FBS (fetal bovin serum). All cultures were performed in a 37°C, 5% CO2 incubator. The cultured cell lines were treated with the examples or comparative examples at 1% of the culture medium and further cultured for 24 hours. After that, RNA was isolated from the cells of each sample using Trizol (RNA iso, DAKARA, Japan), and then RNA was quantified at 260 nm using a nanodrop. Then, 2 μg of RNA was used for cDNA synthesis in an amplifier (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, real-time polymerase chain reaction was performed on a real-time PCR machine (Step One Plus, Applied Biosystems, USA) using CyberGreen (SYBR Green supermix, Applied Biosystems, USA) along with primers and cDNA for the target genes elastin (ELN), hyaluronic acid synthase (HAS3), claudin 1, and filaggrin. The gene expression levels were analyzed through correction for the β-actin gene.

[0104] Figures 2 to 5 are graphs showing the effects of reverse-phase liposomes containing a supercritical extract of a Fusidium cocineum strain culture and strain cells according to various aspects on ELN, FLG, CLD, and HAS3 expression, respectively. In Figures 2 to 5, reverse liposome (Fusiderm O) is the reverse-phase liposome of Example 1, and reverse liposome (Fusiderm X) is the reverse-phase liposome of Comparative Example 1.

[0105] As a result, as shown in Fig. 2, the reverse-phase liposome (Example 1) containing the supercritical extract of the Fusidia cocineum strain culture and the strain cell showed an ELN gene expression effect approximately three times greater than that of the reverse-phase liposome (Comparative Example 1) not containing the Fusidia cocineum culture and the strain cell extract.

[0106] In addition, as shown in FIGS. 3 to 5, the reverse-phase liposome (Example 1) containing the supercritical extract of the Fusidia cocineum strain culture and the strain cells showed superior expression effects on the FLG, CLD, and HAS3 genes compared to the reverse-phase liposome (Comparative Example 1) not containing the supercritical extract of the Fusidia cocineum strain culture and the strain cells.

[0107]

[0108] In summary of the above results, reverse-phase liposomes containing a supercritical extract of the Fucidium coccineum strain culture and strain cells according to the daily pattern have superior skin permeability compared to reverse-phase liposomes that do not have such a culture, and are effective in increasing skin elasticity, improving skin hydration, and improving the skin barrier.

[0109]

[0110] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A reverse-phase liposome comprising a culture medium of a Fusidium coccineum strain and a supercritical extract of Fusidium coccineum strain cells.

2. In claim 1, the reverse-phase liposome has enhanced skin permeability.

3. A reverse-phase liposome according to claim 1, which increases the expression of at least one gene selected from the group consisting of elastin (ELN), hyaluronic acid synthase (HAS), claudin (CLD), and filaggrin (FLG).

4. In claim 1, the reverse-phase liposome, wherein the Fucidium cocineum strain is deposited as KCCM12013P.

5. A cosmetic composition for improving skin condition, comprising the reverse-phase liposome of claim 1 as an active ingredient.

6. A cosmetic composition according to claim 5, wherein the improvement in skin condition is improvement in skin elasticity, improvement in skin lifting, skin moisturizing, enhancement of skin self-regenerating power, or enhancement of skin barrier.

7. A composition for external application of skin for improving skin condition, comprising the reverse-phase liposome of claim 1 as an active ingredient.

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