WO2023224274A1 - Cosmetic composition comprising supercritical extract of fusidium coccineum strain cells - Google Patents

Abstract

A composition comprising a supercritical extract of Fusidium sp. strain cells is provided. The composition inhibits the expression of MMP-1 increased by UV rays, and increases the expression of collagen and elastin, and thus has excellent effects on skin condition improvement such as skin aging reduction, skin wrinkle reduction and skin elasticity improvement.

Description

Cosmetic composition comprising supercritical extract of Fucidium cocineum strain cells

It relates to a cosmetic composition containing a strain extract.

Fusidium coccineum is a fungus that produces fusidic acid, a steroid antibiotic. Fucidic acid is effective against Gram-positive bacteria, especially staphylococcal infections, and is also used as an internal or external medicine. Additionally, fusidic acid is used in drugs in the form of sodium fusidate. In the cosmetics field, an indicator substance called beta-glucogallin is secured and utilized in the fermented product of the above strain to improve skin.

On the other hand, when a substance that becomes a gas or liquid at normal temperature and pressure exceeds a certain high temperature and pressure limit called the supercritical point, the evaporation process does not occur, so it enters a state in which gas and liquid cannot be distinguished, that is, a critical state. , The substance in this state is called a supercritical fluid. Supercritical fluids are characterized by large changes in molecular density. The molecular density is close to that of a liquid, but the viscosity is low and close to that of a gas. In addition, it spreads quickly and has a thermal conductivity as high as water. This characteristic is very useful in chemical reactions, and when used as a solvent, it exhibits the unique property of extremely increasing the solvent concentration around the solute (dissolved molecule). Supercritical fluids can continuously change their density from a state close to an ideal gas to a state close to a liquid density by changing the pressure and temperature, and the ease of controlling these physical properties can be used to separate substances by using them in reaction and separation processes. It is called supercritical extraction. Supercritical extraction can change the solubility of the supercritical fluid by controlling conditions such as temperature and pressure during the extraction stage, allowing selective extraction of specific components.

No research has been conducted on the supercritical extract of the strain itself, rather than the culture medium of the Fucidium cocineum strain or the fermented product of the strain, so additional research is needed.

One aspect is to provide a cosmetic composition for improving skin condition containing a supercritical extract of Fusidium sp. strain cells as an active ingredient.

Another aspect is to provide a composition for external skin application for improving skin condition, which contains a supercritical extract of Fusidium sp. strain cells as an active ingredient.

Another aspect is to provide a health functional food composition for improving skin condition containing a supercritical extract of Fusidium sp. strain cells as an active ingredient.

Another aspect includes treating or administering to a subject in need thereof an effective amount of a supercritical extract of Fusidium sp. strain cells or a composition comprising the supercritical extract. It provides methods for prevention, improvement, or treatment.

Another aspect provides the use of a supercritical extract of cells of a strain of Fusidium sp. for use in preparing a composition for improving skin condition.

One aspect provides a composition for improving skin condition containing a supercritical extract of Fusidium sp. strain cells as an active ingredient.

The type of the Fucidium genus strain is not limited as long as it is a strain belonging to the Fucidium genus. For example, the Fusidium genus strain may be Fusidium coccineum , Acremonium fusidioides , or Ramularia coccinea . The strains of the Fucidium genus may be known or directly isolated. In one embodiment, the Fusidium genus strain may be a Fusidium coccineum strain (Accession Number: KCCM 121013P).

As used herein, the term “cell” can be obtained by pure isolation and culturing of a strain. In one embodiment, the cells are grown at an appropriate temperature in a medium (e.g., Potato Dextrose Agar (PDA) or Potato Dextrose Broth (PDB) medium) that supplies nutrients so that Fucidium strains can grow and survive in vitro. It may be obtained after culturing at (e.g., 10 to 40°C) for an appropriate time (e.g., 4 to 500 hours). Additionally, the cells may be obtained by filtering and centrifuging the culture medium of the strain to remove the supernatant. Additionally, the cells may be obtained by freeze-drying, then crushing or sterilizing.

As used herein, the term “supercritical extract” may refer to cells of a Fucidium genus strain extracted by a supercritical extraction method. The supercritical extract may be obtained by supercritically extracting cell lysate of a Fucidium genus strain or a freeze-dried product thereof with carbon dioxide (CO ₂ ) at a temperature of 40 to 80° C. and a pressure of 150 to 500 bar. The supercritical extract is a cell lysate of a Fucidium genus strain or a freeze-dried product thereof extracted 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. It could be. In addition, the supercritical extract may be a cell lysate of a Fucidium genus strain or a freeze-dried product thereof extracted at a pressure of 150 to 500 bar, 150 to 450 bar, 150 to 350 bar, 200 to 350 bar, or 250 to 350 bar. . At this time, if the temperature and pressure conditions of the supercritical extract are below or above the above range, there is a problem that the effect of improving skin condition cannot be sufficiently exerted.

The supercritical extract may include the extract itself, its dilution, concentrate, or lyophilisate.

The 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 seed oil, shea butter oil, etc. .

In one embodiment, the composition may further include cells of the Fucidium genus strain.

In another embodiment, the extract may be included in an amount of 0.00000001 to 50% by weight based on the total weight of the composition. The extract is, for example, 0.00000001 to 50% by weight, 0.0000001 to 50% by weight, 0.000001 to 50% by weight, 0.00001 to 50% by weight, 0.0001 to 50% by weight, 0.001 to 50% by weight, 0.01 to 0.01% by weight. It may be included at 50% by weight, 0.1 to 50% by weight, or 1 to 50% by weight. At this time, if the content of the extract is less than the above range, there is a problem that the skin condition improvement effect cannot be sufficiently exerted, and if it exceeds the above range, there is a problem that the unit price of the raw materials increases.

The term “included as an active ingredient” means that the supercritical extract is added to an extent that can produce the effects mentioned in this specification, and various ingredients are added as sub-ingredients for drug delivery and stabilization to formulate it in various forms. It means becoming.

The composition may be in a liquid state or a dry state. In one embodiment, the composition may be in dry powder form.

The drying method for preparing the composition in a dry state may be a method generally used in the art and is not particularly limited. Non-limiting examples of the drying method include air drying method, natural drying method, spray drying method, freeze drying method, etc. These methods can be used alone or at least two methods can be used together.

In one embodiment, the supercritical extract of Fucidium cocineum strain cells reduces the expression of MMP-1 increased by ultraviolet rays compared to the strain culture medium, and significantly increases the expression of collagen and elastin regardless of the presence or absence of ultraviolet irradiation. confirmed.

Therefore, the composition according to one embodiment may improve skin condition or have a skin regenerative effect.

The skin condition improvement may include suppressing or improving skin aging, improving skin wrinkles, improving skin elasticity, strengthening the skin barrier, or moisturizing the skin. The improvement in skin condition may include suppressing or improving skin aging, improving skin wrinkles, or improving skin elasticity.

As used herein, the term "skin aging" refers to both tangible and intangible changes that occur in the skin as one ages, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to a decrease in wound recovery, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical removal ability, immune response, sensory function, and temperature regulation.

The composition may be used to improve skin aging caused by extrinsic or intrinsic factors. The exogenous factors refer to various external factors, such as ultraviolet rays (light), and the endogenous factors are also referred to as chronological factors and mainly refer to factors that occur due to the passage of time. In other words, the skin aging is specifically not only a symptom of premature aging induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemicals, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age. It is a concept that includes wrinkles, loss of elasticity, skin sagging, and dryness. In addition, wrinkles include stimulation caused by changes in internal and external factors that change the components that make up skin tissue, causing wrinkles. The aging may be photoaging. The term “photoaging” is a phenomenon caused by external environmental factors, the most representative factor being ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cutting of matrix proteins, and abnormal cross-linking, and repetition of this mechanism causes skin aging that is evident in appearance.

As used herein, the term “wrinkle” refers to a state in which the skin loses its elasticity and becomes loose, for example, the skin may be folded. The term “prevention or improvement of skin wrinkles” may refer to any action that prevents or improves wrinkles by suppressing the expression of factors related to wrinkles, or increases the total amount of collagen.

The term “improvement of skin elasticity” may refer to any action that prevents or improves the loss of skin elasticity and loosening, or increases the total amount of collagen and elastin in the skin.

The “skin barrier strengthening” may refer to any action that improves the function of the skin barrier, which is located on the outermost layer of the skin and prevents moisture and nutrition loss. Impaired skin barrier function can refer to any changes that appear on the skin due to decreased or damaged skin barrier function. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, etc.

The term “skin moisturizing” may refer to any action that maintains skin moisture or prevents moisture loss.

The term “skin regeneration” may refer to any action in which new skin is created on damaged or old skin areas.

The term “prevention” includes suppressing the occurrence of a disease. The term “treatment” includes inhibiting, alleviating, or eliminating the development of a disease. The term “improvement” may refer to any action that results in at least reducing the severity of a parameter associated with the alleviation or treatment of a condition, such as symptoms.

The composition may be a cosmetic composition.

The cosmetic composition may have, for example, an softening lotion, a nourishing lotion, 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 cosmetic formulation.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the supercritical extract as an active ingredient, for example, stabilizers, solubilizers, vitamins, pigments, and flavoring agents. and a carrier.

Additionally, the composition may be a composition for external skin application.

The external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The skin external preparations include ingredients commonly used in external skin preparations such as cosmetics and medicines, such as aqueous ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or a combination thereof may be appropriately mixed according to need. The skin external preparations include metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, tocopherol acetate, glytylitinic acid, tranexamic acid, and the like. Drugs such as derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, and saccharides such as trehalose can also be appropriately mixed.

Additionally, the composition may be a pharmaceutical composition.

The pharmaceutical composition may additionally include 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, disintegrant, binder, lubricant, 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 monohydrogen phosphate anhydride, 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 syrup, or combinations thereof. Parenteral dosage forms may be injections, ointments, creams, etc.

Additionally, the composition may be a health functional food composition.

The health functional food composition can be used alone or in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw materials. There are no particular restrictions on the types of health functional foods. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The health food composition also contains nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. It may contain the carbonating agent used, or a combination thereof. The health functional food composition may also contain pulp for the production of natural fruit juice, fruit juice beverage, vegetable beverage, or a combination thereof.

Another aspect includes treating or administering to a subject in need thereof an effective amount of a supercritical extract of Fusidium sp. strain cells or a composition comprising the supercritical extract, thereby preventing the condition in the subject. , improving, or treating methods are provided.

The condition of the subject may be a skin-related condition. The skin-related condition may be skin aging, skin wrinkles, skin elasticity, etc.

As used herein, the term “effective amount” means an amount effective enough to produce the above-mentioned effects.

As used herein, the terms “administering,” “introducing,” and “implanting” are used interchangeably and are used interchangeably to introduce a composition into an individual by a method or route that results in at least partial localization to the desired site according to one embodiment. It may refer to the arrangement of the composition according to one embodiment of.

Administration can be done by methods known in the art. Administration may be administered directly to the subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. The administration may be administered systemically or locally. The treatment or administration may include application to the skin.

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, for example, moisturizing the skin, strengthening the skin barrier, treating skin wounds, improving skin wrinkles, improving skin elasticity, or having an anti-aging effect.

The administration of the composition according to one embodiment is 0.1 mg to 1,000 mg, 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 mg per day. 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 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 formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The frequency of administration can be once a day or more than twice within the range of clinically acceptable side effects, and can be administered at one or two or more locations, daily or at intervals of 2 to 5 days. The number of days of administration can be from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage per kg is the same as for humans, or the above dosage is converted into, for example, the volume ratio (e.g., average value) of the organs (heart, etc.) between the target animal and human. One dose can be administered.

Another aspect provides the use of a supercritical extract of cells of a strain of Fusidium sp. for use in preparing a composition for improving skin condition.

Details regarding the composition for improving skin condition, Fucidium genus strain, and supercritical extract are as described above.

The cosmetic composition according to one aspect has excellent effects on improving skin aging, skin wrinkles, and skin elasticity by suppressing the expression of MMP-1 increased by ultraviolet rays and increasing the expression of collagen and elastin.

Figure 1 is a graph showing the effect of an extract according to one aspect on the expression of MMP-1.

Figure 2 is a graph showing the effect of an extract according to one aspect on the expression of COL1A1.

Figure 3 is a graph showing the effect of an extract according to one aspect on the expression of ELN.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Preparation example]

Preparation Example 1. Obtaining Fucidium cocineum strain cells and preparing cell freeze-dried material

Fucidium cocineum strain cells were obtained. Specifically, the Fusidium coccineum strain (KCCM12013P) was received and enriched 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. Afterwards, the culture medium of the strain was filtered using a 0.45 ㎛ filter to recover cells. In order to disrupt and sterilize the recovered cells, they were freeze-dried for 72 hours at about -70°C and about 20 mTorr. Finally, cell freeze-dried material of the Fucidium cocineum strain was obtained.

[Example]

Example 1. Preparation of supercritical extract of Fucidium cocineum strain cells

A supercritical extract of Fucidium cocineum strain cells was prepared. Specifically, the freeze-dried product obtained in the above preparation example was subjected to supercritical extraction for 3 hours at about 60°C and 300 bar using CO ₂ gas and sunflower seed oil (carrier oil).

[Comparative example]

Comparative Example 1. Preparation of Fucidium cocineum strain culture medium

A culture medium of Fucidium cocineum strain was prepared. Specifically, the Fusidium coccineum strain (KCCM12013P) was received, suspended in PDB medium at a speed of 100 rpm/min, and enriched and cultured at 25°C for 300 hours. Afterwards, the culture medium was passed through a 0.2 ㎛ filter, sterilized through the filter, and a culture medium of the Fucidium cocineum strain was prepared.

[Experimental example]

Experimental Example 1. Confirmation of anti-aging and anti-wrinkle activity

The anti-aging and anti-wrinkle activities of the supercritical extract of Fucidium cocineum strain cells were confirmed.

Specifically, the human fibroblast cell line (Human dermal fibroblast, Hs68) was distributed at 3.5x10 ⁵ in a 6-well plate and then cultured in an incubator at 37°C and 5% CO ₂ conditions for 24 hours. Afterwards, the medium was removed, DPBS (Dulbecco's Phosphate-Buffered Saline) was added, and UVB of 20 mJ/cm ² was irradiated or not. Immediately after UVB irradiation, DPBS was removed and the medium was replaced with a medium without FBS (Fetal Bovine Serum), then treated with Example 1 or Comparative Example 1 and further cultured for 24 hours. As a negative control, a group untreated with UV light and strain culture was used, and as a positive control, a group treated with 1 μM of EGCG (epigallocatechin-3 gallate) was used. Afterwards, RNA was isolated from the cells of each sample using Trizol (RNA iso, DAKARA, Japan), RNA was quantified at 260 nm with nanodrop, and cDNA was synthesized in an amplifier using 2 μg of RNA each. (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, Cyber Green (SYBR Green supermix, Applied Biosystems, USA) was used for the target genes MMP-1 (Matrix metalloproteinase-1), COL1A1 (Collagen, type 1, alpha 1), and ELN (elstin). Real-time polymerase chain reaction was performed in a real-time PCR machine (Step One Plus, AppliedBiosystems, USA) by adding primers and cDNA. The expression level of the gene was finally analyzed through correction for the β-actin gene.

Figure 1 is a graph showing the effect of supercritical extract on the expression of MMP-1 according to one aspect.

Figure 2 is a graph showing the effect of supercritical extract on the expression of COL1A1 according to one aspect.

Figure 3 is a graph showing the effect of supercritical extract on the expression of ELN according to one aspect.

As a result, as shown in Figure 1, it was confirmed that MMP-1 expression increased due to ultraviolet irradiation, and the increased expression of MMP-1 was significantly reduced by the treatment in Comparative Example 1 and Example 1. Specifically, it was confirmed that the treatment of Example 1 had an excellent effect in reducing MMP-1 expression compared to Comparative Example 1.

In addition, as shown in Figures 2 and 3, Example 1 was confirmed to increase the expression of COL1A1 and ELA in fibroblasts regardless of the presence or absence of ultraviolet irradiation. Specifically, Example 1 was confirmed to significantly increase the expression of COL1A1 and ELA compared to the positive control group (ECGC). Meanwhile, Comparative Example 1 increased the expression of COL1A1 and ELA when not irradiated with ultraviolet rays, but it was confirmed that the expression of COL1A1 decreased after irradiation with ultraviolet rays.

That is, the supercritical extract of Fucidium cocineum cells according to one aspect inhibits the expression of MMP-1, which is increased by ultraviolet rays, and collagen (COL1A1) and elastin (ELA) compared to the Fucidium cocineum strain culture medium. It significantly increases the expression of , and is effective in anti-aging, improving skin wrinkles, improving skin elasticity, improving skin barrier, and improving skin moisturization.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (8)

1. A cosmetic composition for improving skin condition comprising a supercritical extract of Fusidium sp. strain cells as an active ingredient.
2. The cosmetic composition according to claim 1, wherein the strain is selected from the group consisting of Fusidium coccineum , Acremonium fusidioides , and Ramularia coccinea .
3. The cosmetic composition according to claim 1, wherein the improvement of skin condition includes improvement of skin aging, improvement of skin wrinkles, or improvement of skin elasticity.
4. The cosmetic composition according to claim 1, which inhibits the expression of MMP-1 increased by ultraviolet rays.
5. The cosmetic composition according to claim 1, which increases the expression of one or more genes among COL1A1 and ELA (Elastin).
6. A composition for external application for skin for improving skin condition, comprising a supercritical extract of Fusidium sp. strain cells as an active ingredient.
7. A health functional food composition for improving skin condition containing supercritical extract of Fusidium sp. strain cells as an active ingredient.
8. Use of a supercritical extract of Fusidium sp. strain cells for use in preparing a composition for improving skin condition.

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