阅读说明：本技术 一种源自酵母细胞外囊泡和溶解物的化妆品组合物 (Cosmetic composition derived from extracellular vesicles and lysates of yeast ) 是由 黄淳锡 赵一志 齐秋月 魏茂林 金荣熙 金延埈 于 2021-09-27 设计创作，主要内容包括：本发明提供一种化妆品组合物,其以源自酿酒酵母的外泌体及其溶解物为有效成分,具有抗老化、皮肤再生、改善皮肤弹性或改善皮肤皱纹功能。另外,培养液中含有增殖所需成分和细胞排出的代谢产物等多种成分,其既含有对皮肤有益的成分,还含有对皮肤有害的成分,因此,本发明的目的在于,从酵母培养液中只提取对皮肤实际有用的成分,提供一种具有优异效果的化妆品组合物。(The present invention provides a cosmetic composition having anti-aging, skin-regenerating, skin elasticity-improving or skin wrinkle-improving effects, which comprises an exosome derived from saccharomyces cerevisiae and a lysate thereof as active ingredients. Further, since the culture solution contains various components such as components necessary for growth and metabolites discharged from cells, and contains components beneficial to the skin and components harmful to the skin, it is an object of the present invention to provide a cosmetic composition having an excellent effect by extracting only components actually useful to the skin from a yeast culture solution.)

1. A cosmetic composition comprises an effective ingredient separated from one or more substances derived from extracellular vesicles of Saccharomyces cerevisiae and a lysate thereof.

2. The cosmetic composition of claim 1, wherein: the yeast is Saccharomyces cerevisiae RD-03RI SC (C) (accession number: KCTCSD 1359).

3. The cosmetic composition of claim 1, wherein: the cosmetic raw material composition is used for resisting aging, regenerating skin, improving skin elasticity or improving skin wrinkles.

4. The cosmetic composition according to claim 1 or 2, characterized in that: the extracellular vesicles or their lysates are isolated by ultracentrifugation or ultrafiltration membranes.

5. The cosmetic composition of claim 1, wherein: the extracellular vesicles, or lysate thereof, may be isolated using ultrafiltration membranes.

6. The cosmetic composition of claim 1, wherein: the size of the extracellular vesicles is 30-200 nm.

7. The cosmetic composition of claim 1, wherein: the cosmetic composition further comprises one or more additives selected from butanediol, hexanediol, ethylhexylglycerin and phenoxyethanol.

8. The cosmetic composition of claim 1, wherein: the cosmetic composition is selected from astringent, nourishing cream, massage cream, essence, facial mask, gel, skin care lotion, ointment, patch or spray.

9. The cosmetic composition of claim 1, wherein: the cosmetic composition further comprises an extracellular vesicle transporter for delivering the active ingredient of the extracellular vesicle or a lysate thereof to the skin or cells or into the extracellular vesicles.

10. An encapsulation method comprising encapsulating the active ingredient naturally by introducing the active ingredient into an extracellular vesicle using the delivery material according to claim 9 in the case of culturing a yeast, or artificially encapsulating the active ingredient into an extracellular vesicle by ultrasonic treatment after introducing the active ingredient into the extracellular vesicle in the case of culturing a yeast.

11. Use of a cosmetic composition according to any one of claims 1 to 3, 5 to 9 for improving the condition of mammalian skin.

12. Use of the cosmetic composition according to any one of claims 1 to 3 and 5 to 9 for the preparation of a medicament for skin rejuvenation or for improving skin elasticity or for improving skin wrinkles.

Technical Field

The present invention relates to a cosmetic composition containing an active ingredient separated from one or more substances derived from extracellular vesicles of Saccharomyces cerevisiae and their lysates.

Background

It is known that, due to aging, ultraviolet light, etc., the synthesis of Prostaglandin E2(Prostaglandin E2) increases and the production of inflammation-inducing factors, etc. increases as COX-2, an enzyme that produces inflammatory cytokines, etc., increases in cells. The increased production and synthesis of MMPs in various inflammatory reactions trigger collagen degradation, which in turn reduces skin elasticity and forms skin wrinkles. In particular, when sunlight and ultraviolet rays are irradiated to the skin, a large amount of radicals are generated, and the antioxidant defense function of the skin is destroyed by the radicals, which causes an increase in wrinkles, skin sagging, and the like, and accelerates skin aging. Therefore, in order to reduce skin wrinkles and maintain skin elasticity, the production of reactive oxygen species and free radicals is suppressed, inflammatory reactions are suppressed, and skin regeneration of wounds is promoted, thereby protecting the skin.

As is known, substances effective in improving skin wrinkles include adenosine, retinoic acid (retinoids), etc., but adenosine has a less significant clinical effect, and retinoic acid cannot be used in women of childbearing age and has side effects such as erythema, so that anti-aging functional cosmetics using active ingredients of plants or natural substances are currently receiving wide attention. However, in a functional cosmetic containing a plant extract as an active ingredient, there is a problem that a foreign body sensation may be generated during evaporation of a solution after the application to the skin, and the duration is short.

Many membrane (membrane) vesicles are discharged from cells to the extracellular environment, and these vesicles are generally called Extracellular Vesicles (EV) and Extracellular Vesicles (EV), and are classified into exosomes (exosomes), Microvesicles (microviscles) and Outer membrane vesicles (Outer membrane vesicles) according to their origin, size and production process, but they are not very different in appearance and have a common property of being secreted to the outside of cells, and thus they are collectively called "extracellular vesicles".

Exosomes are vesicles of several tens to several hundreds of nanometers in size, each composed of a double lipid membrane having the same structure as a cell membrane, and contain a protein, nucleic acid (mRNA, miRNA, etc.), and the like, which are called exosome substances, inside the vesicles. Exosomes also include a wide range of signaling factors (signalingfactors) that are specific to the cell type and can be regulated differently depending on the environment of the different secreting cells.

Exosomes are intercellular signal transmission media secreted by cells, and various cell signals transmitted by the exosomes can regulate cell actions such as activation, growth, movement, differentiation, dedifferentiation, apoptosis (apoptosis), necrosis (necrosis) and the like of target cells. The exosomes contain different special genetic materials and bioactive factors according to the properties and states of cells from different sources. If derived from proliferating stem cell exosomes, they can regulate cell migration, proliferation and differentiation, reflecting the stem cell characteristics associated with tissue regeneration (Nature Review Immunology 2002(2) 569-. Due to such characteristics, studies have been made to use extracellular vesicles for the purpose of diagnosis, treatment, and the like (patent document 2).

Exosomes are also called cytograms, and they are excreted not only from stem cells and cancer cells, but also from various biological cells such as plants, bacteria (bacteria), fungi (fungi), algae (algae), and the like. For example, exosomes can be isolated from mesenchymal stem cell culture fluid, fibroblast culture fluid, or yeast culture fluid.

In recent years, fermented cosmetics have been favored, and the types of yeast used for cosmetics have been increasing day by day. The yeast used in cosmetics is about hundreds of kinds, and among the raw materials for fermenting cosmetics, the most reverberant for consumers is galactoenzyme fermentation filter (galactomyces fermentfiltrater). The galactase was inspired by the fact that the skin of the hands of the brewer is elastic and clean compared to its age, the yeast found during the study on various yeasts.

The galactonase fermentation filtrate is rich in vitamins, minerals, amino acids, organic acids, ferment, yeast peptide, etc. However, people who are sensitive to the fermentation components of the galactonase fermentation filtrate may have side effects such as skin allergy, reddening, or dermatitis (patent document 1).

At present, the change of the components of the fermentation of the above-mentioned fermented products and the effects on the skin are studied relatively much, but studies on the influence of a single pure yeast culture solution on the skin are lacking.

Documents of the prior art

Patent document

(patent document 0001) korean laid-open publication No.: no. 10-2020-0028290.

(patent document 0002) korean laid-open publication no: no. 10-2019-0050286.

Disclosure of Invention

The present invention aims to provide a cosmetic composition having anti-aging, skin-regenerating, skin elasticity-improving or skin wrinkle-improving effects, which contains as an active ingredient exosomes derived from saccharomyces cerevisiae and its lysates.

As described above, since the culture solution contains various components such as components necessary for growth and metabolites discharged from cells, and contains components beneficial to the skin and components harmful to the skin, the present invention aims to extract only components actually useful to the skin from the yeast culture solution, thereby providing a cosmetic raw material composition having an excellent effect.

The cosmetic composition according to the present invention comprises an effective ingredient separated from one or more substances among extracellular vesicles derived from saccharomyces cerevisiae and a lysate thereof.

Preferably, the yeast is Saccharomyces cerevisiae RD-03RI SC (C) (accession No.: KCTC SD 1359). The strain was deposited at 2021, 11 months at the Korean Collection of type strains (KCTC) located in New City, Jingyi, N.C. 181, Robei, Korea, with the deposit number SD1359, and classified as Saccharomyces cerevisiae RD-03, by the subsidiary research institute of Ruidi' an, Inc.

Preferably, the cosmetic composition is used for anti-aging, skin regeneration, skin elasticity improvement or skin wrinkle improvement.

Preferably, the extracellular vesicles or lysate thereof are isolated by ultracentrifugation or ultrafiltration membrane.

More preferably, the extracellular vesicles or lysate thereof may be isolated using ultrafiltration membranes.

Preferably, the size of the extracellular vesicles is 30-200 nm.

Preferably, the cosmetic composition further comprises one or more additives selected from butylene glycol, hexylene glycol, ethylhexylglycerin and phenoxyethanol.

Preferably, the cosmetic composition is selected from the group consisting of lotions, nourishing creams, massage creams, essences, facial masks, gels, skin lotions, ointments, patches, sprays, and the like.

Preferably, as another embodiment of the present invention, the extracellular vesicles may be used for a delivery of an active ingredient of the extracellular vesicles or a lysate thereof to skin or cells or a delivery substance in the extracellular vesicles.

Preferably, as another embodiment of the present invention, the effective ingredient is put into the extracellular vesicles by using the extracellular vesicle transfer material during the culture of yeast, and the effective ingredient is encapsulated naturally, or the effective ingredient is put into the extracellular membrane vesicles during the culture of yeast, and then encapsulated artificially in the extracellular vesicles by ultrasonic treatment.

The cosmetic composition containing the active ingredient separated from one or more substances selected from the group consisting of extracellular vesicles derived from saccharomyces cerevisiae and a lysate thereof according to the present invention can be used for a cosmetic method for improving the skin condition of mammals, in addition to therapeutic uses.

Preferably, the cosmetic method comprises the steps of:

step a): applying the cosmetic composition directly to the skin of a mammal;

step b): contacting or applying a patch, mask or mask paper coated or impregnated with said cosmetic composition to the skin of a mammal, or sequentially performing steps (a) and (b) above.

In addition, the method also comprises the step (c): after step (b), removing said patch, mask or mask paper from the skin of the mammal and applying said cosmetic composition to the skin of the mammal.

In another aspect of the present invention, there is also provided the use of the above cosmetic composition for improving the condition of mammalian skin.

In still another aspect of the present invention, there is provided a use of the above cosmetic composition for preparing a medicament for skin regeneration or improving skin elasticity or improving skin wrinkles.

The present invention provides a cosmetic composition which extracts only actually useful active ingredients from extracellular vesicles derived from saccharomyces cerevisiae and a lysate thereof, and has the effects of anti-aging, skin regeneration, skin elasticity improvement, skin wrinkle improvement, and the like.

Drawings

FIG. 1 shows an image of a transmission electron microscope in example 2 of the present invention.

FIG. 2 roughly shows the results of test example 2.

Fig. 3a and 3b roughly show the results of test example 3.

FIG. 4 roughly shows the results of test example 4.

FIG. 5 roughly shows the results of test example 5.

Fig. 6a and 6b roughly show the results of test example 6.

Fig. 7a and 7b roughly show the results of test example 7.

Detailed Description

The present invention will be described in further detail with reference to specific examples so that those skilled in the art can easily practice the present invention. The embodiments of the present invention are provided to more fully explain the present invention to those having ordinary skill in the art. Therefore, the embodiments of the present invention may be variously modified, and the scope of the present invention is not limited to the following embodiments.

Throughout the present specification, the word "comprise" or "comprises" a part of a constituent element means that the constituent element is not excluded and other constituent elements are included when the constituent element is not described in a specific exclusive meaning.

Throughout the present specification, when a step is said to be "on" or "before" a step, it includes not only the case where the step has a direct time sequence relationship with another step, but also the case where the time sequence order of two steps is interchangeable in the sequence as in the step of mixing the steps thereafter, and there is an indirect time sequence relationship, which has the same right.

The terms "about" and "substantially" and the like as used throughout the specification are used in the sense of being inclusive of manufacturing tolerances and materials, and are used in a sense of being or close to values in order to facilitate understanding of the invention and to prevent unauthorized infringement by illegal use of the exact value or disclosure of the exact value. Throughout this specification, the use of the terms "… step" or "step of …" does not represent a "step for …".

The present invention provides a cosmetic raw material composition containing an active ingredient separated from one or more substances selected from extracellular vesicles derived from Saccharomyces cerevisiae and lysates thereof.

Preferably, the composition in the cosmetic raw material has the functions of anti-aging, skin regeneration, skin elasticity improvement or skin wrinkle improvement.

In the present invention, the yeast culture fluid is separated in two different ways, ensuring extracellular vesicles derived from yeast and their lysates. Human fibroblasts are used for treating extracellular vesicles and lysates thereof, so as to obtain culture conditions and an extracellular vesicle separation method with the best collagen synthesis capacity.

The manufacturing method of the extracellular vesicles adopts an ultra-speed centrifugal separation method, but considering mass production, the ultrafiltration membrane adopted by the invention is better, and if a process for setting the cut-off molecular weight of the ultrafiltration membrane in a segmented manner is established, the extracellular vesicles with various particle sizes can be obtained, and the product can be refined.

The size of the extracellular vesicles separated by the method is equivalent to 30-200 nm, and the extracellular vesicles derived from the yeast can be regarded as natural nanostructures. The yeast-derived extracellular vesicles themselves are recognized to have the effect of preventing and improving skin wrinkles, and thus can be applied to products.

In addition, the nanometer structure body has the characteristic of being easy to combine with the wrinkle-removing and anti-aging functional physiological active substance. These nanostructures were used in cosmetics as a delivery material, and nano cosmetics were developed, which are easily absorbed into the skin because their size is far larger than the skin cell interval. In other words, the nanostructure can function as a delivery agent for easily delivering ingredients of cosmetics to the inside of the skin or to the inside of extracellular vesicles.

By using these nanostructures as a mediator, more products can be derived by adding the active ingredient to the yeast-derived extracellular vesicles. For example, in the case of culturing yeast, a method of naturally encapsulating an active ingredient by adding the active ingredient to a medium or a method of artificially encapsulating an active ingredient by instantaneously inducing a structural change of extracellular vesicles by ultrasonic treatment can be employed.

The present invention will be described in more detail below with reference to examples and test examples.

Comparative examples 1 to 8: yeast culture filtrate

Yeast (Saccharomyces cerevisiae RD-03RI-SC (C)) (KCTCSD1359)) was inoculated onto YMBroth, cultured with stirring at 27 ℃ and 37 ℃ for 8, 16, 24, and 48 hours, centrifuged at 4 ℃ and 4000rpm for 20 minutes, and the supernatant was filtered with 0.22 μm PES to prepare comparative examples 1 to 8.

Comparative examples 9 to 16: yeast-derived extracellular vesicle solution using separation reagent

After the yeast culture filtrate of comparative examples 1 to 8 was reacted with a total exosome separating agent (Totalexosome isolationreagent), the supernatant was removed by centrifugation at 10,000rpm for 1hr at 4 ℃ to secure extracellular vesicle particles. Comparative examples 9 to 16 were prepared by resuspending in phosphate buffer.

Comparative examples 17 to 24: yeast-derived extracellular vesicle lysate using separation reagent

The yeast culture filtrate of comparative examples 1 to 8 was reacted with the total exosome-separating agent, and then centrifuged at 4 ℃ and 10,000rpm for 1hr to remove the supernatant, thereby securing extracellular vesicle particles. Resuspend with distilled water and perform ultrasonic (Sonication) treatment 3 times at 5-minute intervals to produce comparative examples 17 to 24.

The culture conditions of comparative examples 1 to 24 were as shown in Table 1 below.

TABLE 1

Example 1: yeast-derived extracellular vesicle solution using ultrafiltration membrane

Yeast (Saccharomyces cerevisiae RD-03RI-SC (KCTCSD1359)) was inoculated onto YMBroth, cultured at 27 ℃ under stirring for 48 hours, centrifuged at 4 ℃ and 4000rpm for 20 minutes, and the supernatant was filtered with 0.22 μm PES to give a yeast culture filtrate. Filtering with 40nm ultrafiltration membrane device to remove residual liquid. Example 1 was produced by recovering EV filtered through a 40nm ultrafiltration membrane apparatus using 1/10 distilled water for the yeast culture filtrate and filtering the EV with 0.22 μm PES.

Example 2 Yeast-derived extracellular vesicle lysate Using Ultrafiltration Membrane

The extracellular vesicle solution obtained in example 1 was subjected to ultrasonic treatment 3 times at intervals of 5 minutes, to prepare example 2. A transmission electron micrograph of these extracellular vesicle lysates is shown in FIG. 1.

Test example 1: evaluation of protein content (comparative examples 1 to 24)

In order to compare the total protein content under different yeast culture conditions, BCA assay was performed using comparative examples 1-24 as reagents.

As shown in Table 2 below, the protein content was the highest in comparative examples 1 to 8 in the form of the yeast culture filtrate, and the tendency of the protein content to decrease was more pronounced with increasing culture time, which was found to be an influence of the medium components. The protein content of comparative examples 9-24 was less than 100. mu.g/ml, indicating that most of the protein containing media components had been removed when the extracellular vesicles were isolated.

TABLE 2

Comparative example	Total protein content (μ g/ml)
		1	2170.56
2	1995.65
		3	1840.28
4	1795.11
		5	2143.35
6	1794.31
		7	1786.45
8	1789.47
		9	44.74
10	39.84
		11	87.89
12	95.30
		13	65.11
14	99.52
		15	72.54
16	117.74
		17	32.16
18	57.56
		19	52.99
20	68.75
		21	49.03
22	33.00
		23	43.04
24	61.30

Test example 2: evaluation of fibroblast proliferation potency (comparative examples 9 to 24)

As aging progresses, the number and function of fibroblasts responsible for the expression of collagen and elastin decrease. Fibroblasts (HumanDermal Fibroplast) were cultured on a plate for 24 hours, and then comparative examples 9 to 24 were treated with reagents and cultured for 24 hours. The proliferation potency of the agent-treated fibroblasts relative to the sterile-treated group was calculated by MTT reagent treatment.

As shown in FIG. 2, the extracellular vesicles isolated from the yeast culture solution at 27 ℃ for 48 hours, that is, the cell growth ability of the treated group of comparative example 20, was the strongest, and the concentration thereof showed the dependency results in the 0.01 to 0.5% treated group.

Test example 3: evaluation of collagen Synthesis ability (comparative examples 12 and 20, examples 1 and 2)

As aging progresses, the number and function of fibroblasts responsible for the expression of collagen and elastin decrease. Cortical absorption of large molecular weight collagen is nearly impossible and biosynthesis is most efficient in skin cells. Collagen is synthesized inside cells as procollagen (procollagen), and then secreted outside the cells and neutralized into collagen fibers. PIP (ProcollagenTypeIC-tertiary peptide) secreted to the outside of the cell was confirmed by ELISA (Enzyme-Linked Dimmosbetssay), and the ability of collagen synthesis by agent treatment was evaluated in human fibroblasts.

As shown in FIGS. 3a and 3b, comparative examples 12 and 20 are higher in synthesis ability than example 2.

Test example 4: ergosterol confirmation Test (TLC)

Ergosterol, also known as previtamin D, is one of the components of fungi such as yeast and mushrooms, and can be used as EVmembraemarker according to 0087. The presence of Ergosterol was confirmed by thin layer chromatography (Thinlayerchromatograph). Example 2 was lyophilized, the solvent was removed, the suspension was suspended in methanol, and 100. mu.l of the supernatant was titrated after centrifugation. For comparison, spotting was performed by dissolving the Ergosterol standard with methanol, and after developing the developing solution, it was confirmed whether or not a spot having the same Rf value as that of the standard was formed under irradiation of UV254 nm.

As shown in the TLC (thin layer chromatography) results of fig. 4, example 2 forms spots with the same Rf value as the Rf of the Ergosterol standard, thus confirming the presence of Ergosterol.

Test example 5: protein profiling (SDS-PAGE/Gel stabilizing)

SDS-PAGE and Brilliant Blue Gel Staining were performed to confirm the protein profiles that make up the extracellular vesicles. After freeze-drying example 2, suspending in distilled water, loading on gel (gel), and analyzing Protein map by using two Protein markers (Protein Marker) of 10-180 kDa and 40-300 kDa.

As shown in FIG. 5, example 2 consists of 60-75 kDA and 100-180 kDa proteins.

Test example 6: particle distribution and concentration Analysis (Nanotracking Analysis)

In order to confirm the particle distribution and concentration of the extracellular vesicles present in examples 1 and 2, analysis was performed using a nanoparticle tracking analyzer (NS 300).

As shown in FIGS. 6a and 6b, the average particle diameter of examples 1 and 2 was 150 to 220nm, and the particle concentration was about 1X10⁸～1x10¹⁰particles/ml。

Test example 7: ZETA potential Analysis (Zetapotential Analysis)

In order to confirm the dispersion stability of the extracellular vesicle particles present in examples 1 and 2, analysis was performed using a ZETA potential analyzer.

As shown in FIGS. 7a and 7b, the ZETA potentials of examples 1 and 2 were-19.7 mV to-21 mV, and the degree of dispersion was considered to be stable.

The cosmetic raw material composition may further comprise one or more additives selected from butanediol, hexanediol, ethylhexyl glycerin and phenoxyethanol. The above additives are necessary for forming the subsequent dosage form, but are not limited thereto.

The cosmetic composition is selected from the group consisting of smoothing toner, nourishing cream, massage cream, essence, pack, gel, skin lotion, ointment, patch, and spray, but is not limited thereto.

In addition, the cosmetic raw material composition containing the active ingredient separated from one or more substances among the saccharomyces cerevisiae-derived extracellular vesicles and the lysate thereof of the present invention can be used for a cosmetic method for improving the skin condition of mammals, in addition to therapeutic use.

Preferably, the cosmetic method comprises the following steps:

step a): applying the cosmetic composition directly to the skin of a mammal;

step b): contacting or applying a patch, mask or mask paper coated or impregnated with said cosmetic composition to the skin of a mammal, or sequentially performing steps (a) and (b) above.

In addition, the method also comprises the step (c): after step (b), removing said patch, mask or mask paper from the skin of the mammal, and applying said cosmetic composition to the skin of the mammal.

Although the embodiments of the present invention have been described in detail, the scope of the present invention is not limited thereto, and those skilled in the art will appreciate that various modifications and variations are possible without departing from the technical spirit of the present invention as defined in the appended claims.