KR20220153866A - External Composition Comprising Plant Extracellular vesicle of Rose for Improving Skin - Google Patents

Abstract

The present invention relates to an external preparation composition comprising a rose-derived extracellular vesicle as an active ingredient and a method for preparing an external preparation composition for improving skin. The rose-derived extracellular vesicle according to the present invention provides various skin improvement effects of moisturizing, anti-aging, skin wall strengthening, protecting cells from damaging by anti-stress, skin regenerating, anti-aging by wound healing, skin pore contraction, sebum inhibition, cell energy enhancement and acne inhibition, and skin protection from ultraviolet rays or blue rays. Accordingly, the external preparation composition comprising rose-derived extracellular vesicle as an active ingredient can be effectively used for improving skin.

Description

External Composition Comprising Plant Extracellular Vesicle of Rose for Improving Skin}

The present invention relates to a composition for external application for skin improvement comprising rose-derived extracellular vesicles as an active ingredient; And it relates to a method for preparing the composition for external application for skin improvement.

The skin is the largest organ in the body by weight and consists of three layers: the keratinized outer epidermis, the inner connective tissue rich in blood vessels, the dermis, and the innermost subcutaneous tissue. , nails, sensory receptors, and glands) together form the integumentary system. The skin forms the boundary between the inside and outside of the body to perform major functions of various organs in the body.

The skin is essential for maintaining homeostasis, and in addition to protective functions, it is an important organ that performs various functions such as regulating body temperature, inhibiting water loss, containing sensory receptors, synthesizing various biochemical substances, and excreting small amounts of waste.

However, compared to other organs, the skin has many opportunities to come into direct contact with external stimuli or various pathogens, and in particular, when ultraviolet rays are absorbed into the skin, a photochemical reaction causes skin lesions. In addition, the number of patients with skin diseases caused by factors commonly occurring in modern society, such as mental stress, obesity, alcohol, and tobacco, is rapidly increasing.

Accordingly, development of various skin protectants to inhibit skin damage and protect skin cells is being actively conducted. The downside is that side effects occur.

Under this background, as a result of intensive research efforts to develop natural-derived materials useful for skin improvement, the present inventors have found that extracellular vesicles obtained from rose plant cell cultures or culture solutions exhibit excellent skin improvement effects, and large quantities using plant cell culture technology The present invention was completed by confirming that production was possible.

Republic of Korea Patent No. 10-1998032

The present invention aims to solve the above problems and other problems related thereto.

An exemplary object of the present invention is to provide a composition for external application for skin improvement comprising a rose plant cell culture, or extracellular vesicles derived from the culture medium as an active ingredient.

Another exemplary object of the present invention is to provide a method for preparing the composition for external application.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and another problem not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

As one aspect for achieving the above object, the present invention provides a composition for external application for skin improvement comprising rose plant cell culture or extracellular vesicles derived from the culture medium as an active ingredient.

The term "plant cell culture" of the present invention refers to a culture product including a cell mass produced by culturing a tissue cut from a plant in a medium, and in a broad sense, callus or agro, which is a cell mass that has lost stress that causes normal organ formation or tissue differentiation. It includes cultures of plant tumor tissues caused by infections such as bacteria.

In the present invention, the rose plant cell culture refers to a culture obtained by culturing a part of a rose plant, for example, a leaf, petal, stem, placenta, etc., or a part thereof in an induction medium or subsequent subculture. .

In the present invention, rose plant cell culture is a concept commonly used with callus, which generally means plant tissue culture, and also includes placental cell culture, and plant cell culture means a culture medium obtained by filtering cells from the rose plant cell culture do.

The term “extracellular endoplasmic reticulum” of the present invention is a cell-derived vesicle with a size of 30-150 nm, including exosomes, and cultures eukaryotic cells and body fluids such as blood and urine of multicellular eukaryotic organisms can be observed in a single culture.

In the present invention, "skin improvement" refers to all activities that improve or benefit the skin by using the rose plant cell culture or extracellular vesicles derived from the culture medium of the present invention.

The skin improvement of the present invention is an example, skin moisturizing, skin barrier improvement, skin aging prevention, skin regeneration or wound healing improvement, skin immunity improvement and defense enhancement, skin inflammation relief or improvement, skin soothing, ultraviolet rays or blue light It may include skin protection, antioxidant, skin protection from skin stress, skin cell energy promotion, skin pore reduction, sebum inhibition, or acne improvement, but is not limited thereto.

The term "moisturizing the skin" of the present invention means increasing the feeling of moisture in the skin and maintaining a moist state. If you increase the skin moisturizing effect, it can have a beneficial effect on improving wrinkles and increasing elasticity of the skin.

The term “improvement, strengthening of the skin barrier” of the present invention refers to improving the thickness of the epidermal layer by promoting the differentiation of keratinocytes in the skin barrier composed of keratinocytes.

The term "skin aging" of the present invention is a concept that includes both endogenous and extrinsic aging. Such skin aging also includes improvement, prevention, and prevention of various phenomena arising from skin aging, such as wrinkle improvement, wrinkle prevention, and elasticity improvement.

The term "skin protection from ultraviolet rays or blue light" of the present invention refers to the prevention or improvement of damage or symptoms caused by exposure of the skin to ultraviolet rays, for example, wrinkles caused by ultraviolet rays, reduced skin elasticity, photoaging, or skin keratinization. Blue light, commonly referred to as blue light, is a type of visible light that occurs frequently in electronic devices in daily life and can be perceived by the eyes. , It is known that it can induce skin aging, and it means skin protection from such blue light stimulation.

The term "pore reduction" of the present invention means reducing the size of pores in the skin surface from which hair grows. If the pores are enlarged, waste materials and bacteria can easily penetrate into the body and bacterial infection and acne can easily occur. Therefore, by reducing the pores, it is possible to maintain a clean skin condition and to obtain a protective effect from bacterial infection.

The term "sebum suppression" of the present invention refers to reducing the amount of oil exuded from the skin. Since inflammation and acne occur on the skin surface when sebum is excessively secreted, it is possible to improve the health of the skin and maintain a fresh skin condition by inhibiting sebum secretion.

The term "acne inhibition or improvement" of the present invention means to reduce the occurrence of acne caused by excessive sebum secretion and pore closure, or to subside the occurrence of acne.

The term "skin cell damage protection" of the present invention means protection from cell damage by blocking the cause of skin cell damage, for example, increasing resistance to stress, which is one of the causes of skin cell damage. An increase in gene expression may be included, but is not limited thereto. For example, it means suppressing skin aging or protecting the skin from various environmental stresses, and such environmental stresses may include ultraviolet rays, pollution, wind, or temperature.

The term "contained as an active ingredient" of the present invention means that the composition for external application for skin includes an effective amount capable of exhibiting various skin improvement effects as described above.

Specifically, in the experimental example of the present invention, various skin improvement effects of rose-derived extracellular vesicles were confirmed. Specifically, the expression of water/glycerol transporter AQP3 (Aquaporin 3) and FLG (Filaggrin) known as a natural moisturizing factor increased. , and it was confirmed that the expression level of the TERT (Telomerase Reverse Transcriptase) gene, which is an anti-aging related gene, increased. In addition, in order to confirm the enhancement of skin immunity and defense, it was confirmed that LL37 and hBD-2 expressions were increased, and autophagy was activated through an increase in LC-3 expression. It can be seen that it is effective in suppressing inflammation through the reduction of COX-2 gene expression and iNOS gene expression, and it can be seen that it has excellent skin soothing ability in confirming the skin soothing effect targeting subjects. It was confirmed that the present composition had an excellent skin protection effect from skin cells even when irradiated with ultraviolet rays and blue light. In addition, the cell damage protection effect was confirmed by the increase in the expression level of LEA protein, which increases resistance to stress as the expression level increases, and the effect of wound healing, pore shrinking effect by precipitation of hemoglobin, sebum inhibitory effect, and increase in ATP concentration The cell energy enhancement effect was confirmed. In addition, it was confirmed that the acne improvement effect appeared in the test subjects who applied the lotion containing rose-derived extracellular vesicles. Based on these test examples, the composition for external application of the present invention moisturizes the skin, strengthens the skin barrier, protects cells from damage caused by anti-stress, improves skin immunity, enhances defense, anti-aging by wound healing, heals wounds, improves scars, reduces pores, It was confirmed that it can be usefully used for skin improvement because it has excellent effects such as sebum suppression, cell energy enhancement, and acne suppression.

In the present invention, the composition for external application for skin may be a cosmetic composition or a pharmaceutical composition.

In the cosmetic composition, a carrier acceptable for cosmetic formulations may be included. Here, "acceptable carrier in cosmetic formulation" means a compound or composition that is already known and used that can be included in a cosmetic formulation, or a compound or composition to be developed in the future, which exhibits toxicity, instability, or irritation more than the human body can adapt to when in contact with the skin. say that there is no The carrier may be included in the external composition for skin of the present invention in an amount of about 1% to about 99.99% by weight, preferably about 90% to about 99.99% by weight, based on the total weight of the composition. However, since the ratio varies depending on the formulation of the composition for external application for skin of the present invention, its specific application area (face, neck, etc.) or its preferred application amount, the ratio does not limit the scope of the present invention in any aspect. should not be construed as limiting.

Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, UV scattering agents, UV absorbers, coloring agents, perfumes, and the like may be exemplified as the carrier. Compounds or compositions that can be used as the alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, coloring agent, fragrance, etc. are already known in the art. Therefore, those skilled in the art can select and use an appropriate corresponding material or composition.

The composition for external application for skin according to the present invention can be prepared in various forms, such as lotion, essence, gel, emulsion, lotion, cream (oil-in-water type, water-in-oil type, multi-phase), solution, suspension (anhydrous and aqueous). , anhydrous products (oil and glycol-based), gels, masks, packs, powders, or capsules (soft capsules, hard capsules) with a coating such as gelatin.

In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing an oil phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes), nonionic It may be provided in the form of a vesicular dispersion, cream, toner, lotion, powder, ointment, spray or conceal stick. In addition, it can be prepared in the form of a foam (foam) or the form of an aerosol composition further containing a compressed propellant.

In addition, the composition for external application for skin of the present invention may further include a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant agent, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, and an ion. type or nonionic emulsifier, filler, sequestering and chelating agent, preservative, vitamin, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic or lipophilic active agent, lipid vesicle or commonly used in cosmetics It may contain adjuvants commonly used in cosmetology or dermatology, such as any other ingredient. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

The skin in the present invention is a concept that includes not only the face, but also the scalp and the whole body, and as a composition for external application for skin that can be applied to such a scalp, there are shampoo, rinse, treatment, hair growth agent, etc., and body cleanser that can be applied to the whole body It can be manufactured in various forms for uses such as

Therefore, the composition for external application for skin according to the present invention provides cell protection by ultraviolet rays, skin moisturizing, anti-aging, skin barrier strengthening, itching relief, cell damage protection by anti-stress, anti-wrinkle, anti-aging by wound healing, pore reduction, It includes the form of functional cosmetics with sebum suppression, cell energy enhancement and acne suppression effects.

In the present invention, the composition for external application for skin may function as a pharmaceutical composition for improving skin condition, for example, wound healing, scar improvement, itching relief, and the like.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent according to conventional methods. Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specific reference may be made to the pharmacopoeia of each country including the 'Korean Pharmacopoeia'. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, carriers, excipients and diluents that may be included in the composition of the present invention may be non-natural carriers, but are not limited thereto.

The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. . Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. to the compound. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The specific formulation of the pharmaceutical composition is known in the art, and reference may be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). These documents are considered as part of this specification.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmacologically effective amount means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. It may be determined according to the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. The dosage and frequency do not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, dogs, cats, cows, horses, pigs, and humans through various routes, and may be preferably human. Any mode of administration may be expected, and may be administered by, for example, oral, intravenous, intramuscular or subcutaneous injection, but is not limited thereto.

As another aspect of the present invention, the present invention provides a method for preparing the composition for external application comprising the following steps. Specifically,

(a) culturing plant cells by separating petals, leaves or placentas of rose plants; (b) obtaining extracellular vesicles from the rose plant cell culture or culture medium; And (c) preparing a composition for external application for skin containing the extracellular vesicles; providing a method for producing a composition for external application for skin improvement containing rose plant cell culture or culture medium-derived extracellular vesicles as an active ingredient do.

Here, the plant cell culture in step (a) includes induction culture for inducing early callus and placental cell culture for petals and leaves, and subsequent subcultures thereof.

The plant cell culture in step (a) is obtained by cancer culture in a medium containing zeatin, sucrose, myo-inositol, and agar, , can be characterized by 20 to 30 passages of culture.

In addition, the subculture may be performed by solid culture and / or liquid culture, but liquid culture may be characterized by performing high-frequency treatment as an attractant treatment.

In the present invention, the high-frequency treatment is performed 4 to 6 times/day at 100 or 400 kHz, treatment is performed for 10 to 20 minutes, and repeated for one week to two weeks.

An appropriate medium may be used for the culture of the present invention, and any medium commonly used for plant tissue culture in the technical field of the present invention may be used without limitation.

In particular, in the present invention, by containing the myo-inositol in the medium, a plant cell culture cultured in this medium or extracellular vesicles obtained from the culture medium exhibits a very excellent skin improvement effect.

In the present invention, if necessary, the rose plant cell culture or culture solution may be obtained as an extract through an extraction process. In the present invention, the extraction is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, cold extraction, solvent extraction, steam distillation, dissolution, compression, ultrasonic extraction, filtration, reflux extraction, and the like, which are performed alone or in combination of two or more methods. can be performed by The extract of the present invention is extracted using an appropriate solvent, and may include, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. The type of extraction solvent used to prepare the extract is not particularly limited, and any solvent known in the art may be used.

In the step (b), the process of separating the extracellular vesicles may be appropriately performed through a conventionally known method. For example, it can be obtained by processing VIB (vesicle isolation buffer), and, if necessary, filter the cells using a 25 um filter bag, and filter the remaining culture medium through a 5, 1, or 0.22 um filter to obtain cells containing exosomes. The ectoplasmic reticulum may be separated and used, and if necessary, concentrated to a volume of about 1/10 to 1/50. A composition for external application for skin containing the thus obtained extracellular vesicles can be prepared through a conventionally known method.

Rose-derived extracellular vesicles according to the present invention are rose plant cell cultures or culture medium-derived extracellular vesicles obtained through specific subcultivation, treatment with an attractant, and culture in a myoinositol-containing medium, and these extracellular vesicles are skin moisturizing, anti-aging, Various skin improvement effects such as skin barrier strengthening, cell damage protection by anti-stress, skin regeneration, anti-aging by wound healing, pore shrinking, sebum suppression, cell energy promotion and acne suppression effect, skin protection from ultraviolet rays or blue light Having said that, a composition for external application containing rose-derived extracellular vesicles as an active ingredient can be usefully used for skin improvement.

1 is a step of preparing a rose-derived extracellular vesicles.
2 is an NTA analysis result confirming the size distribution of rose-derived extracellular vesicles obtained in Example 1.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Preparation of a composition according to the present invention

rose plant cell Induction and culture

Rose plant cells were induced and cultured according to the procedure disclosed in FIG. 1 . Specifically, in the case of callus among rose plant cells used in the present invention, rose petals and leaves were immersed in 70% ethanol for 30 seconds, washed with sterile water, and disinfected again with a disinfectant solution (30% bleach + Tween 20) for 20 minutes After washing with sterilized water three times, use a sharp knife to make a cut at once, and 1 mg/L Zeatin, 3% Sucrose, 1 g/L Myo-inositol (W/O) and 8 g/L agar In the basic MS (Murashige and Skoog) medium (Duchefa, Cat No. M0221), early rose callus was induced by dark culture at 25 ℃ and 70% humidity.

In addition, in the case of placenta cells, ovules and ovaries in rose buds were sterilized with 70% ethanol and 0.5% sodium hypochlorite for 5 minutes and washed several times with sterile water. The ovarian wall was carefully peeled off using tweezers, and the placentas were separated and cultured for 2 to 3 weeks in MS medium (Duchefa, Cat# M0256) containing 5% sucrose. In the case of rosette placenta cell culture, the same medium as the medium used for the induction of roseh callus was used as the culture medium.

The culture medium was adjusted to pH 5.8 using 1 N sodium hydroxide (NaOH). Subculture in the solid state was carried out for 3 weeks, and plant cells were divided into passages through sub-culture, and culture was carried out according to the process shown in the figure below. For the present invention, only plant cells between passage 20 and 30 were used.

[Plant cell culture process]

In particular, rose plant cell cultures of passage 20 or more obtained through solid culture were further subjected to liquid culture (liquid mass culture). This liquid phase mass culture is cultured for 5 weeks in a 500 mL, 20 L, or 100 L bioreactor (Samsung Science Co., Ltd.) with an air supply of 0.1 vvm under the conditions of a temperature condition of 23 ± 2 ° C and a humidity of 70%. prepared.

Treatment of rose callus and placenta cell attractant

After culturing in a 100 L bioreactor for the purpose of increasing the content of physiologically active substances when culturing rose callus and placenta cells, additional culture was performed for 5 weeks in a 500 L pilot reactor connected with a high frequency generator, and 100 or 400 kHz was used at 15-minute intervals throughout the day. The treatment was performed 4 times and this was repeated for a week or two. The medium was cultured for one week and recovered to secure the culture medium.

Example 1-2: Isolation of rose callus-derived extracellular vesicles

Callus derived from mass-cultured rose petals and leaves was finely ground in VIB (Vesicle Isolation Buffer) buffer, and only cells were separated using a 25 μm filter bag. The cells were filtered and the remaining culture medium was sequentially filtered using a 5μm, 1μm, 0.22μm fiter, The rose callus culture medium is concentrated using a MWCO 300 kcross flow filter system (Cytiva, akta flux) until the volume is about 1/10 to 1/50. * VIB composition: 20 mM MES, 2 mM CaCl ₂ , 0.1 M NaCl

Example 1-3: Isolation of extracellular vesicles derived from rose callus culture

Only cells are separated from the callus culture medium derived from mass-cultured rose petals and leaves using a 25 μm filter bag. Cells were filtered and the remaining culture medium was sequentially filtered using a 5 μm, 1 μm, 0.22 μm fiter, and the rose callus culture fluid was 1/10 to 1/10 using a MWCO 300 kDa cross flow filter system (Cytiva, akta flux). It is used after concentrating to a volume of about 50.

Example 1-4: Separation of extracellular vesicles derived from rose placenta cell culture

Separate only the cells from the mass-cultured rose placenta cell culture medium using a 25 μm filter bag. Cells were filtered and the remaining culture medium was sequentially filtered using a 5 μm, 1 μm, 0.22 μm fiter, and the rose callus culture fluid was 1/10 to 1/10 using a MWCO 300 kDa cross flow filter system (Cytiva, akta flux). It is used after concentrating to a volume of about 50.

It was confirmed that the extracellular vesicles obtained through the same process as 1-2 to 1-4 were composed of extracellular vesicles containing exosomes of 30 to 150 nm in size (FIG. 2).

Experimental Example 1: Non-irritating effect confirmation test through skin cell culture

In order to determine whether the composition of the present invention causes toxicity in skin cells and causes skin irritation, MTT assay was performed. To this end, HaCaT cells (keratinocytes) and Detroit cells (fibroblasts) were incubated in 96- It was dispensed into a well plate and incubated for 24 hours in an incubator under conditions of 5% CO ₂ and 37 °C. Thereafter, the cells were treated with the control group and the test material composition, to which purified water was added, and further cultured for 24 hours. Subsequently, 4 μL of 5 mg/mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each well, followed by incubation for 4 hours. . After removing the culture medium, 100 μL of DMSO (Dimethyl sulfoxide) solution was added to dissolve the cells, and then absorbance was measured at 540 nm.

division Treatment concentration (%) HaCaT cell viability (%) control group - 100 rose callus
origin
extracellular endoplasmic reticulum One 102 3 107 5 118 Derived from rose callus culture
extracellular endoplasmic reticulum One 105 3 110 5 125 placenta cells
origin
extracellular endoplasmic reticulum One 102 3 108 5 110

As a result, the composition of the present invention, rose callus, rose callus culture medium, and placenta cell-derived extracellular vesicles, did not show intracellular toxicity to keratinocyte (HaCaT) and fibroblast (Detroit) at 1 to 5% treatment concentration, and cell viability was improved. By confirming the increase, it was confirmed that it was a safe substance with low possibility of causing skin irritation because there was no irritation.

Experimental Example 2: Moisturizing and skin barrier improvement confirmation test

In order to investigate the efficacy of the composition according to the present invention for moisturizing and skin barrier improvement, changes in the expression levels of AQP3 (Aquaporin 3), a water/glycerol transporter, and FLG (Filaggrin), known as a natural moisturizing factor, were investigated. To this end, HaCaT cells were seeded in a 96-well plate and cultured for 24 hours under cell culture conditions. Thereafter, the cells were treated with the control group and the test material composition, to which purified water was added, and further cultured for 24 hours. Real-time PCR was performed to confirm the expression of AQP3 and FLG at the gene level, and the test sequence is as follows. For RNA isolation and cDNA synthesis, SuperPrep ^TM cell lysis & RT Kit for qPCR (Toyobo Co., Cat. SCQ-101) was used. The cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of the previously extracted lysate was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To compare and analyze gene expression, the cDNA synthesized above was used as a template and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (AQP3; Cat. QT00212996, FLG; Cat. QT02448138), and the AQP3 and FLG mRNA expression levels of the samples were quantified with GADPH (Cat. QT01192646). Real-time qPCR conditions were first reacted at 95 ° C for 1 minute, followed by 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle, for a total of 40 cycles.

division Treatment concentration (%) AQP3 gene expression rate FLG gene expression rate control group - One One rose callus
origin
extracellular endoplasmic reticulum One 2.2 2.2 3 3.2 2.6 5 3.6 3.7 Derived from rose callus culture
extracellular endoplasmic reticulum One 1.5 1.9 3 2.9 3.2 5 4.3 3.9 placenta cells
origin
extracellular endoplasmic reticulum One 1.2 1.1 3 1.5 2.3 5 2.6 2.6

As a result, as in the present invention, it was confirmed that the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol contributed to skin moisturizing and strengthening of the skin barrier.

Experimental Example 3: Anti-aging effect confirmation test according to the increase in TERT expression

In order to examine the anti-aging efficacy of the composition according to the present invention, changes in the expression level of TERT (Telomerase reverse transcriptase), known as an enzyme that extends the length of telomeres at the ends of DNA strands, were investigated. Changes in the expression level of TERT (Telomerase reverse transcriptase), known as an enzyme that extends the length of telomeres at the ends of DNA strands, were investigated.

HaCaT cells were seeded in a 96-well plate and cultured for 24 hours under cell culture conditions. Thereafter, the cells were treated with the control group and the test substance composition, and further cultured for 24 hours. Real-time PCR was performed to confirm TERT at the gene level, and the test sequence is as follows.

For RNA isolation and cDNA synthesis, SuperPrep ^TM celllysis & RTKitforqPCR (TOYOBO, Cat.SCQ-101) was used. After removing the medium, the cells were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and stop solution was added. 8 μL of the previously extracted lysate was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To compare and analyze gene expression, the cDNA synthesized above was used as a template and real-time PCR analysis was performed using Thunderbird ^TM SYBRqPCRMix (TOYOBO, Cat.QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, TERT; Cat. QT00073409), and the TERT mRNA expression level of the sample was quantified with GADPH. Real-time qPCR conditions were first performed at 95 ° C for 1 minute, followed by 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle, for a total of 40 cycles.

division Treatment concentration (%) TERT gene expression rate control group - 1.0 rose callus
origin
extracellular endoplasmic reticulum One 1.4 3 2.3 5 2.9 Derived from rose callus culture
extracellular endoplasmic reticulum One 2.7 3 3.5 5 4.2 placenta cells
origin
extracellular endoplasmic reticulum One 1.5 3 2.2 5 2.6

As a result, as in the present invention, it was confirmed that the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol were effective in preventing aging by increasing the amount of TERT mRNA expression compared to the control group.

Experimental Example 4: Skin immunity improvement and defense enhancement confirmation test

It was confirmed whether the composition of the present invention enhances skin immunity and defense through the activity of cathelicidin-related antimicrobial peptides (LL37) and human β-defensin-2 (hBD-2), which have antibacterial effects. To this end, HaCaT cells were seeded in a 96-well plate and cultured for 24 hours under cell culture conditions. Thereafter, the cells were treated with the control group and the test substance composition, to which purified water was added, and further cultured for 24 hours. Real-time PCR was performed to confirm LL37 and hBD-2 at the gene level, and the test sequence is as follows. For RNA isolation and cDNA synthesis, SuperPrep ^TM cell lysis & RT Kit for qPCR (Toyobo Co., Cat. SCQ-101) was used. The cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of the previously extracted lysate was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To compare and analyze gene expression, the cDNA synthesized above was used as a template and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (LL37; Cat. QT00010458, hBD-2; Cat. QT00204617), and the LL37 and hBD-2 mRNA expression levels of the samples were quantified with GADPH (Cat. QT01192646). Real-time qPCR conditions were first reacted at 95 ° C for 1 minute, followed by 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle, for a total of 40 cycles.

division Treatment concentration (%) LL37 gene expression rate hBD-2 gene expression rate control group - One One rose callus
origin
extracellular endoplasmic reticulum One 1.5 1.6 3 2.3 2.2 5 2.7 2.8 Derived from rose callus culture
extracellular endoplasmic reticulum One 1.4 1.9 3 2.6 2.5 5 4.3 3.7 placenta cells
origin
extracellular endoplasmic reticulum One 1.5 1.3 3 2.0 2.5 5 2.1 3.0

As a result, as in the present invention, it was confirmed that the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol increased the expression of LL37 and hBD-2 in skin cells, contributing to skin immunity and defense enhancement.

Experimental Example 5: Autophagy activity expression confirmation test

In order to investigate the autophagy activity effect of naturally degrading its own organelles or cellular components that are unnecessary or nonfunctional in the intracellular regulation process of the composition of the present invention, LC-3 (Microtubule-associated protein 1A) involved in autophagy formation Changes in the expression level of /1B-light chain 3) were investigated. To this end, HaCaT cells were seeded in a 96-well plate and cultured for 24 hours under cell culture conditions. Thereafter, the cells were treated with the control group and the test substance composition, to which purified water was added, and further cultured for 24 hours. Real-time PCR was performed to confirm LC-3 at the gene level, and the test sequence is as follows. For RNA isolation and cDNA synthesis, SuperPrep ^TM cell lysis & RT Kit for qPCR (Toyobo Co., Cat. SCQ-101) was used. The cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of the previously extracted lysate was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To compare and analyze gene expression, the cDNA synthesized above was used as a template and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (LC-3; Cat. QT00055069), and the LC-3 mRNA expression level of the sample was quantified with GADPH (Cat. QT01192646). Real-time qPCR conditions were first reacted at 95 ° C for 1 minute, followed by 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle, for a total of 40 cycles.

division Treatment concentration (%) LC-3 gene expression rate control group - 1.0 rose callus
origin
extracellular endoplasmic reticulum One 1.5 3 2.0 5 3.2 Derived from rose callus culture
extracellular endoplasmic reticulum One 1.9 3 2.8 5 3.8 placenta cells
origin
extracellular endoplasmic reticulum One 1.3 3 2.0 5 2.6

As a result, as in the present invention, it was confirmed that the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol contributed to the activation of autophagy by increasing the expression of LC-3 in skin cells.

Experimental example 6: Inflammation inhibitory effect confirmation test

In order to investigate whether the composition of the present invention has the effect of inhibiting skin inflammation, changes in the expression levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), which are involved in inflammatory reactions, were investigated. To this end, HaCaT cells were seeded in a 96-well plate and cultured for 24 hours under cell culture conditions. Thereafter, an inflammatory response was induced through UVB irradiation, and the cells were further cultured for 4 hours by treating the cells with a control and a test substance composition to which purified water was added. Real-time PCR was performed to confirm COX-2 and iNOS at the gene level, and the test sequence is as follows. For RNA isolation and cDNA synthesis, SuperPrep ^TM cell lysis & RT Kit for qPCR (Toyobo Co., Cat. SCQ-101) was used. The cells from which the medium was removed were washed once with PBS, 50 μL cell lysis mixture was added, reacted for 5 minutes, and 10 μL stop solution was added. 8 μL of the previously extracted lysate was added to 32 μL of the RT reaction mixture, and cDNA was synthesized using PCR at 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To compare and analyze gene expression, the cDNA synthesized above was used as a template and real-time PCR analysis was performed using Thunderbird ^TM SYBR qPCR Mix (TOYOBO, Cat. QPS-201). The primers used in the experiment were Qiagen's QuantiTect primer assays (COX-2; Cat. QT00040586, iNOS; Cat. QT01323189), and the expression levels of COX-2 and iNOS mRNA in the sample were quantified with GADPH (Cat. QT01192646). Real-time qPCR conditions were first reacted at 95 ° C for 1 minute, followed by 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds per cycle, for a total of 40 cycles.

division ultraviolet ray Treatment concentration (%) COX-2 gene expression rate iNOS gene expression rate control group - - 0.2 control group + - 1.0 rose callus
origin
extracellular endoplasmic reticulum One 0.97 0.81 3 0.85 0.79 5 0.75 0.70 Derived from rose callus culture
extracellular endoplasmic reticulum One 0.80 0.80 3 0.75 0.70 5 0.64 0.50 placenta cells
origin
extracellular endoplasmic reticulum One 0.90 0.72 3 0.81 0.66 5 0.72 0.62

As a result, as in the present invention, the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol reduce the expression of COX-2 and iNOS in skin cells compared to the control group irradiated with ultraviolet rays, thereby reducing the inflammatory response. confirmed.

Experimental example 7: Skin soothing effect confirmation test

In order to examine the skin soothing effect according to the skin temperature reduction rate of the essence formulation to which the composition according to the present invention was applied, squares (2 cm X 2 cm) were divided into forearms of 25 subjects, and the product was applied in an amount of 50 μL each, and then before the test It was exposed to direct sunlight for 20 minutes. For measurement, body heat was measured at intervals of 0 minutes (before sample application), 5 minutes (after sample application), and 10 minutes (after sample application) using a computer infrared body heat detector (Digital Infrared Thermographic Imaging; DITI), and the temperature decrease rate was measured. Calculated.

division Treatment concentration (%) Heat reduction rate (%) Before sample application 5 minutes later after 20 minutes control group - 9.2 6.6 3.1 rose callus
origin
extracellular endoplasmic reticulum 5 8.7 6.7 6.2 Derived from rose callus culture
extracellular endoplasmic reticulum 5 8.8 7.9 7.0 placenta cells
origin
extracellular endoplasmic reticulum 5 8.4 6.5 5.5

As a result, in the case of the control group, the heat of the skin rapidly decreased over time, but as in the present invention, the heat reduction rate was continuously maintained by the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol. It was confirmed that there is a skin soothing effect.

Experimental example 8: from UV stimulation skin cells Protective effect confirmation test

In order to examine the effect of the composition of the present invention on protecting skin cells from UV stimulation, changes in cell viability were examined. To this end, HaCaT cells were seeded in a 96-well plate and cultured for 24 hours under cell culture conditions. Thereafter, UVB was irradiated, and the cells were treated with the control and the composition as the test substance to which purified water was added, and further cultured for 24 hours. An ultraviolet crosslinker (Ultra-Violet Products Co., Cat No. CL-1000) emitting ultraviolet light with a wavelength of 302 nm was used as a light source for UV irradiation. Subsequently, cell viability was measured using MTT assay to confirm whether skin cell damage was protected by UV induction.

division ultraviolet ray treatment concentration
(%) Cell viability (%) control group - - 100 control group + - 40.3 rose callus
origin
extracellular endoplasmic reticulum One 50 3 62 5 57 Derived from rose callus culture
extracellular endoplasmic reticulum One 48 3 60 5 76 placenta cells
origin
extracellular endoplasmic reticulum One 46 3 53 5 55

As a result, as in the present invention, the cell viability of the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol was increased compared to the control group irradiated with UV light, confirming the cell protection effect by UV irradiation.

Experimental example 9: Blue Light ( blue light )On by skin cells protective effect test

To investigate whether the composition of the present invention protects skin cells from irradiation of blue light, which has the shortest wavelength and strongest energy among visible light that humans can see, Topview 5450 SMD LED emitting 460 nm wavelength (ITSWELL, Cat No. IWS-L5056-VRI-K3) was used to measure cell viability. To this end, HaCaT cells were seeded in a 96 well plate and cultured for 24 hours under cell culture conditions. Thereafter, the 460 nm wavelength was irradiated with 10 mJ/cm², and the cells were treated with a control and a composition as a test substance to which purified water was added, and further cultured for 24 hours. Subsequently, MTT assay was performed to measure the survival rate of skin cells after blue light induction.

division Blue Light treatment concentration
(%) Cell viability (%) control group - - 100 control group + - 74 rose callus
origin
extracellular endoplasmic reticulum One 72 3 75 5 79 Derived from rose callus culture
extracellular endoplasmic reticulum One 80 3 82 5 93 placenta cells
origin
extracellular endoplasmic reticulum One 70 3 78 5 85

As a result, as in the present invention, the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol can specifically reflect and block the light of the blue light range, which is the visible ray range, and can protect the skin. confirmed.

Experimental example 10: H ₂ O ₂ Oxidation Antioxidant effect confirmation test from toxicity

In order to examine the antioxidant effect from the H ₂ O ₂ oxidative toxicity of the composition according to the present invention, the change in cell viability by H ₂ O ₂ was investigated.

To this end, HaCaT cells were seeded in a 48-well plate and cultured for 24 hours under cell culture conditions. Thereafter, 1 mM H ₂ O ₂ was irradiated, and the cells were treated with the composition as a control and a test material, followed by further incubation for 12 to 16 hours. Thereafter, cell viability was measured using the MTT assay to confirm whether the skin cells were protected from damage caused by the induction of H ₂ O ₂ oxidative toxicity.

division H ₂ O ₂ treatment concentration
(%) Cell viability (%) control group - - 100 control group + - 60 rose callus
origin
extracellular endoplasmic reticulum One 67 3 75 5 87 Derived from rose callus culture
extracellular endoplasmic reticulum One 74 3 82 5 85 placenta cells
origin
extracellular endoplasmic reticulum One 65 3 74 5 74

As a result, as in the present invention, the positive control group (H ₂ O ₂ treatment), cell death by H ₂ O ₂ treatment was inhibited, and H ₂ O ₂ It was confirmed that there is an antioxidant effect from oxidative toxicity.

Experimental example 11: antistress Protein activation effect confirmation test

In order to determine whether the composition of the present invention has a protective effect against intracellular stress, the activity level of late embryogenesis abundant (LEA) protein was investigated. LEA is known to protect cells from damage by increasing resistance to stress as the expression level increases. To this end, HaCaT cells were seeded in a 6-well plate and cultured for 24 hours under cell culture conditions. Thereafter, cells were treated with a control and test material composition to which purified water was added, and cells were additionally cultured for 24 hours. In-Cell ELISA Colorimetric Detection kit (Invitrogen, Cat No. 62200) was used to measure LEA protein expression. ) was used. The medium of the cultured cells was removed, and the cells were fixed by culturing at room temperature for 15 minutes with 100 μL fixing solution. After washing with PBS, 100 μL of Permeabilization solution was added and reacted at room temperature for 15 minutes. After washing with PBS, 100 μL quenching solution was added and reacted at room temperature for 20 minutes. After washing with PBS, 100 μL of blocking solution was added and reacted at room temperature for 30 minutes. After removing the solution, 50 μL of the LEA primary antibody was added and reacted at 4° C. for 24 hours. After removing the antibody, it was washed with PBS, and 50 μL of HRP conjugated secondary antibody was added and reacted for 1 hour. After removing the antibody and washing with PBS, 100 μL of TMB substrate solution was added and reacted at room temperature for 15 minutes in a shaded state. Then, 100 μL stop solution (1 NH ₂ SO ₄ ) was added, and absorbance was measured at 450 nm.

division Treatment concentration (%) LEA protein content (%) control group - 100 rose callus
origin
extracellular endoplasmic reticulum One 110 3 121 5 132 Derived from rose callus culture
extracellular endoplasmic reticulum One 116 3 131 5 140 placenta cells
origin
extracellular endoplasmic reticulum One 107 3 119 5 126

As a result, as in the present invention, it was confirmed that the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol increased the expression level of LEA protein to protect cells from damage.

Experimental example 12: Wound healing effect confirmation test

Wound healing assay was conducted to determine whether the composition of the present invention has wound healing ability by promoting cell proliferation and migration. To this end, a culture insert for wound healing assay was added to each well of a 24-well plate, and 70 μL of HaCaT cells were dispensed into the insert at 90% < confluence and cultured for 24 hours. Thereafter, the insert was removed to obtain a micrograph of the cell pattern at time 0, and the cells were treated with a control and a composition as a test substance to which purified water was added. After additional incubation for 16 hours, the medium was removed, 4% PFA (Paraformaldehyde) was added, and reaction was performed at room temperature for 15 minutes. After the fixation process, the cells were washed 3 times with PBS, and after additional culture for 16 hours, cell patterns were measured by micrographs. The increase rate of the healing area was calculated by measuring the area between cell gaps at 0 and 16 hours using a program called Image J.

division Treatment concentration (%) Healing area increase rate (%) control group - 15 rose callus
origin
extracellular endoplasmic reticulum One 20 3 28 5 42 Derived from rose callus culture
extracellular endoplasmic reticulum One 35 3 42 5 51 One 22 placenta cells
origin
extracellular endoplasmic reticulum 3 23 5 35

As a result, as in the present invention, it was confirmed that the treatment of extracellular vesicles obtained by culturing in a medium containing Myo-Inositol increased the healing area ratio and indirectly contributed to wound healing.

Experimental example 13: Test to confirm the energy enhancement effect of cells

In order to determine whether the composition of the present invention has a cell energy enhancing effect, the activity level of ATP released by cells during metabolism was measured. To this end, Detroit cells were seeded in a 12-well plate and cultured for 24 hours under cell culture conditions. Thereafter, the cells were treated with the control group and the test substance composition to which purified water was added, and the cells were additionally cultured for 24 hours. Take 50 μL of the diluted lysate of the cells cultured through the above process and put it in each 96-well plate together with 50 μL of the ATP standard in the ATP assay kit (Abcam, Cat. ab83355) and add 50 μL of ATP reaction mix The solution was also added to each well and reacted at room temperature for 30 minutes in a shaded state. After the reaction was completed, absorbance was measured at 570 nm, and an ATP standard concentration curve was prepared to calculate the amount of ATP in the sample.

division Treatment concentration (%) ATP content (%) control group - 100 rose callus
origin
extracellular endoplasmic reticulum One 110 3 122 5 125 Derived from rose callus culture
extracellular endoplasmic reticulum One 110 3 120 5 141 placenta cells
origin
extracellular endoplasmic reticulum One 115 3 128 5 132

As a result, as in the present invention, it was confirmed that the amount of ATP increased through the treatment of extracellular vesicles obtained by culturing in a medium containing Myo-Inositol, thereby increasing the energy activity.

Experimental Example 14: Pore shrinking effect confirmation test (in vitro)

In order to find out whether the composition of the present invention has a pore-reducing effect, hemoglobin was used as a protein to replace the skin and tested. To this end, after adding 2 mL each of the control group to which purified water was added to 2 mL of 1 mg/mL hemoglobin solution, the composition as the test substance, and the positive control (Tannic acid), mixing with shaking for 30 seconds, centrifugation at 3,500 rpm for 10 minutes did 2 mL of purified water was added to 1 mL of the supernatant, and absorbance was measured at 407 nm.

division treatment concentration
(%) Hemoglobin precipitation (%) control group - 0 positive control
(Tannic acid) 45 rose callus
origin
extracellular endoplasmic reticulum One 13 5 25 Derived from rose callus culture
extracellular endoplasmic reticulum One 19 5 38 placenta cells
origin
extracellular endoplasmic reticulum One 15 5 32

As a result, as in the present invention, the hemoglobin precipitation rate was increased by the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol, thereby indirectly confirming the pore contraction effect.

Experimental example 15: Sebum inhibitory effect confirmation test

In order to confirm the sebum suppression effect of the essence formulation to which the composition according to the present invention was applied, the sebum secretion of the skin of 10 test subjects was measured after 4 weeks using a skin oil meter (Sebum meter SM810). The test was conducted at 22±2 ℃ and 40±5 % humidity. For oily skin, the measured value is 220 μg/cm ² or more, and in the case of normal skin, it is 100 to 220 μg/cm ² .

division Treatment concentration (%) Skin oil content (μg/cm ² ) 0 days (before use) After 4 weeks of use control group - 231 215 rose callus
origin
extracellular endoplasmic reticulum One 230 188 5 230 162 Derived from rose callus culture
extracellular endoplasmic reticulum One 232 184 5 231 155 placenta cells
origin
extracellular endoplasmic reticulum One 230 178 5 231 165

As a result, in the case of the control group, after 4 weeks of use, the change in sebum oil content was insignificant, but, as in the present invention, the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol showed a reduced amount of skin oil content, suppressing sebum It was confirmed that it worked.

Experimental example 16: Acne improvement effect confirmation test

In order to confirm the acne improvement effect of the lotion formulation to which the extracellular vesicles of the present invention were applied, 10 men and women with acne were asked to apply the lotion evenly over their entire face in the morning and evening for 4 weeks. The judgment of the acne improvement effect was evaluated by evaluating the pre-test condition with 1-3 points with reference to the following criteria according to the degree of feeling of the person participating in the experiment, and then the acne healing effect was evaluated, and the results were shown in the table.

<Evaluation of condition before test>

1 = Moderate acne 2 = Moderate acne 3 = Severe acne

<Determination of acne effect>

-: Almost no effect, +: Slight effect, ++: Much relief, +++: Fully healed

division 0 days
(before use) After 2 weeks of use After 4 weeks of use rose callus
origin
extracellular endoplasmic reticulum Subject 01 3 + ++ Subject 02 2 - ++ Subject 03 2 ++ ++ Subject 04 2 + ++ Subject 05 One - + Subject 06 2 - ++ Subject 07 One + + Subject 08 2 - + Subject 09 3 ++ ++ Subject 10 2 + + Derived from rose callus culture
extracellular endoplasmic reticulum Subject 01 3 + ++ Subject 02 2 + +++ Subject 03 2 + ++ Subject 04 2 + +++ Subject 05 One - ++ Subject 06 2 ++ +++ Subject 07 One + ++ Subject 08 2 + + Subject 09 3 ++ ++ Subject 10 2 - + placenta cells
origin
extracellular endoplasmic reticulum Subject 01 3 + ++ Subject 02 2 ++ +++ Subject 03 2 + ++ Subject 04 2 ++ + Subject 05 One - ++ Subject 06 2 + + Subject 07 One + ++ Subject 08 2 ++ ++ Subject 09 3 ++ ++ Subject 10 2 - ++

As a result, after 2 weeks and 4 weeks of use, as in the present invention, the effect of improving acne in most subjects was confirmed by the extracellular vesicles obtained by culturing in a medium containing Myo-Inositol.

Example 2: Preparation of composition for external application for skin

In order to prepare a composition for external application for skin containing rose-derived extracellular vesicles as an active ingredient according to the present invention, lotion, essence, lotion, cream and gel were prepared according to the composition ratios shown in the table below.

lotion manufacturing

Name of raw material Weight % (w/w) glycerin 5.0 dipropylene glycol 3.0 hyaluronic acid 0.5 Polyoxyethylene Hydrogenated Castor Oil 0.1 polyethylene oleylethyl 0.1 ethanol 5.0 antiseptic 0.15 spices Appropriate amount flash Appropriate amount Rose-derived extracellular endoplasmic reticulum 1.0 Purified water to 100

essence manufacturing

Name of raw material Weight % (w/w) Cetostearyl Alcohol 1.0 self-emulsifying monostearic acid 1.0 beeswax 0.5 squalane 5.0 isocetyloctanoate 3.0 Dimethylsiloxane 0.3 Sorbitan Monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 propylene glycol 0.2 carboxypolymer 0.22 triethanolamine 0.25 antiseptic Appropriate amount Spices Appropriate amount flash Appropriate amount Rose-derived extracellular endoplasmic reticulum 1.0 Purified water to 100

lotion manufacturers

raw material name Weight % (w/w) Cetostearyl Alcohol 0.8 self-emulsifying monostearic acid 1.0 beeswax 0.5 stearic acid 0.5 liquid paraffin 7.0 squalane 5.0 macadamia oil 3.0 isocetyloctanoate 2.0 Dimethylsiloxane 0.3 Sorbitan Monostearate 0.5 Polyethylene glycol monostearate 1.2 glycerin 4.0 propylene glycol 4.0 betaine 4.0 carboxypolymer 0.12 triethanolamine 0.15 antiseptic 0.25 Spices Appropriate amount flash Appropriate amount Rose-derived extracellular endoplasmic reticulum 1.0 Purified water to 100

cream maker

Name of raw material Weight % (w/w) Cetostearyl Alcohol 3.0 self-emulsifying monostearic acid 1.5 Type-specific monostearic acid 1.5 beeswax 0.5 liquid paraffin 8.0 squalane 7.0 isocetyloctanoate 4.0 Refined jojoba oil 4.0 Dimethylsiloxane 0.3 Sorbitan Monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 propylene glycol 4.0 betaine 4.0 xanthan gum 0.06 triethanolamine 0.10 antiseptic 0.25 Spices Appropriate amount flash Appropriate amount Rose-derived extracellular endoplasmic reticulum 1.0 Purified water to 100

gel manufacturing

raw material name Weight % (w/w) glycerin 4.0 propylene glycol 4.0 ethanol 10 Polyoxyethylene Hydrogenated Castor Oil 0.1 carboxypolymer 0.30 triethanolamine 0.30 antiseptic Appropriate amount Spices Appropriate amount flash Appropriate amount Rose-derived extracellular endoplasmic reticulum 1.0 Purified water to 100

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (14)

A composition for external application for skin improvement comprising a rose plant cell culture or culture medium-derived extracellular vesicles as an active ingredient. According to claim 1,
The rose plant cell culture or culture medium is a composition for external application for skin improvement, characterized in that the plant cell culture or culture medium derived from rose petals, leaves or placentas. According to claim 1,
The rose plant cell culture solution is a composition for external application for skin improvement, characterized in that obtained by filtering the cells from the plant cell culture. According to claim 1,
The composition is a composition for external application for skin improvement, characterized in that for moisturizing or skin barrier improvement. According to claim 1,
The composition for external application for skin improvement, characterized in that for preventing skin aging, skin regeneration or improving wound healing. According to claim 1,
The composition is a composition for external application for skin improvement, characterized in that for improving skin immunity and enhancing defense. According to claim 1,
The composition for external application for skin improvement, characterized in that for the relief or improvement of skin inflammation, or skin soothing. According to claim 1,
The composition for external application for skin, characterized in that for protecting the skin from ultraviolet rays or blue light. According to claim 1,
The composition for external application for skin improvement, characterized in that it has an antioxidant activity. According to claim 1,
The composition for external application for skin, characterized in that for protecting the skin from skin stress. According to claim 1,
The composition for external application for skin, characterized in that it has a skin cell energy enhancing effect. According to claim 1,
The composition for external application for skin, characterized in that for reducing skin pores or inhibiting sebum or improving acne. (a) culturing plant cells by separating petals, leaves or placentas of rose plants;
(b) obtaining extracellular vesicles from the rose plant cell culture or culture medium; and
(c) preparing a composition for external application for skin containing the extracellular vesicles;
A method for producing a composition for external application for skin improvement comprising a rose plant cell culture or culture medium-derived extracellular vesicles as an active ingredient. According to claim 13,
The plant cell culture in step (a) is obtained by cancer culture in a medium containing zeatin, sucrose, myo-inositol, and agar. , manufacturing method.

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