KR102671105B1 - Cosmetic composition for improving skin barrier comprising hemp exosome extract and plant-derived extract - Google Patents

Abstract

The present invention relates to a cosmetic composition for moisturizing skin and improving the skin barrier, comprising hemp exosome extract, hydrolyzed pea protein, and hemp stem extract. , by mixing collagen extract and/or phytosterol at a specific ratio and proceeding with the mixture aging process, it can exhibit excellent skin moisturizing and skin barrier improvement effects, as well as skin whitening and skin wrinkle improvement effects.

Description

Cosmetic composition for improving skin barrier comprising hemp exosome extract and plant-derived extract {Cosmetic composition for improving skin barrier comprising hemp exosome extract and plant-derived extract}

The present invention relates to a cosmetic composition for moisturizing skin and improving the skin barrier, comprising hemp exosome extract, hydrolyzed pea protein, and hemp stem extract.

The skin is the largest organ in the human body and plays a variety of roles, including a barrier function that protects other important organs of the human body from the outside and prevents finite components from escaping, a temperature control function, an excretion function, and a respiratory function. It is a very important institution. Aging of such skin occurs due to various internal and external factors, and aging is largely divided into intrinsic aging caused by genetic causes and extrinsic aging caused by environmental causes. In particular, ultraviolet rays reduce the amount of hyaluronic acid, thereby reducing the moisture content in the skin and increasing transepidermal moisture loss, causing damage to the skin barrier, which causes the skin to become rough, its elasticity to decrease, and wrinkles to form.

The moisture balance of the skin is regulated by the epidermis. The epidermis is made up of dead cells, but it plays a very important role in maintaining moisture in the skin. The epidermis contains keratinocytes, which mainly produce lipids and do not synthesize substantially collagen. The epidermis, located in the outermost layer of the skin, performs a protective function to defend against various external physical, chemical and mechanical stimuli and prevent excessive dissipation of body moisture through the skin. This protective function is possible through the normal formation and maintenance of the stratum corneum, which is composed of keratinocytes.

Keratinocytes are cells formed through gradual changes in form and function as basal cells that continuously proliferate in the lowest layer of the epidermis (stratum basale) move to the stratum corneum, forming old keratin after a certain period of time. Cells are shed from the skin and new keratinocytes rising from the lowest layer of the epidermis take over their function, repeating the process of epidermis differentiation or keratinization. During this keratinization process, keratinocytes produce Natural Moisturizing Factor (NMF) and intercellular lipids such as ceramide, cholesterol, and fatty acids, so that the stratum corneum acts as a barrier to the outside, acting as a skin barrier. It has functions.

However, recently, harmful factors for the skin have been increasing, and changes in eating habits have slowed down the formation and shedding of the stratum corneum, and the decrease in the function of keratinocytes has reduced the amount of moisturizing factors and lipids in the stratum corneum, causing the stratum corneum to return to normal. The number of people with skin that does not function as a skin barrier is increasing. This breakdown in skin barrier function causes various skin diseases such as dry skin, atopic dermatitis, contact dermatitis, and psoriasis. In order to maintain appropriate skin moisture, various studies have been conducted to supply moisture from outside, prevent moisture loss from the body, and develop moisturizers. However, these diseases are not symptomatic with only existing moisturizers with a normal moisture retention function. Although relief can be expected, fundamental cure is difficult.

Meanwhile, filaggrin (Filament aggregating protein; filaggrin) is a protein that plays an important role in the formation of the stratum corneum. It is mainly involved in the formation of filaments by combining with keratin, and in the process of creating the stratum corneum, it is processed into forms such as profilaggrin, filaggrin, and natural moisturizing factor (NMF). Ultimately, it forms a natural moisturizing factor, which makes up 20-30% (dry weight basis) of the stratum corneum, and has excellent moisture absorption properties, so it plays the role of a humectant that regulates moisture in the stratum corneum.

Involucrin is a type of cytoplasmic protein synthesized in human squamous epithelial cells. In normal epidermis, involucrin begins to appear in the upper spinous layer and granular layer, and after being synthesized, it is located near the cell membrane and is destroyed by transglutamidase. It is a major structural protein of the cornified cell envelope that binds to membrane proteins and acts as a support.

Aquaporins are a transmembrane protein (major intrinsic protein family) that allows only water molecules to selectively pass through and exists in all cell membranes, and has been studied as a moisturizing agent through a mechanism that increases moisture absorption in the skin. Among these, aquaporin-3 is expressed in keratinocytes of the skin, and the absorption and movement of water and glycerol through aquaporin-3 prevents moisture loss from cells and increases skin elasticity.

Against this background, the present inventors studied a complex of natural ingredients that can moisturize the skin and restore the barrier function of the skin epidermis. As a result, hemp exosome extract, hemp stem extract, and hydrolyzed pea protein were used in a specific ratio. The present invention was completed by developing a composition that improves the skin barrier and has excellent skin moisturizing effects by increasing the expression of filaggrin, involucrin, and aquaporin by using a natural extract complex prepared through a mixing and maturation process as an active ingredient.

Republic of Korea Patent Publication No. 10-2023-0164611

The purpose of the present invention is to provide a cosmetic composition for moisturizing the skin and improving the skin barrier containing a natural extract as an active ingredient.

In order to achieve the above object, the present invention provides a cosmetic composition for moisturizing skin and improving the skin barrier, comprising hemp exosome extract, hydrolyzed pea protein, and hemp stem extract.

In this specification, “active ingredient” refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

In this specification, “skin barrier” refers to the stratum corneum, which is the upper layer of the epidermis, the outermost layer of the skin, and is composed of keratinocytes. It is the most important first line of defense against toxic substances, microorganisms, mechanical stimulation, and ultraviolet rays, and its function is to provide an environment in which the skin can perform its normal functions by suppressing electrolyte and moisture loss through the skin.

As used herein, “skin barrier improvement” refers to improving or strengthening the barrier function of the stratum corneum located in the outermost layer of the skin, by increasing the expression of involucrin and/or filaggrin. It means improving or strengthening a function.

In this specification, “extract” refers to an extract obtained by extraction treatment of the natural material, a filtrate of the extract, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or It may include extracts of all formulations that can be formed using the extract itself and the extract, such as mixtures thereof. The extract according to the present invention can be obtained by separating and obtaining the extract using extraction and separation methods known in the art, and the extract defined in the present invention is an active ingredient prepared by the above method using an appropriate solvent. The raw materials can be extracted as is or by pulverizing them. At this time, the appropriate solvent that can be used to obtain the extract may be any solvent acceptable in the art, and the extract may be extracted with any solvent selected from the group consisting of water, organic solvents, or mixed solvents thereof. You can. The extraction method can be any method such as cold immersion, reflux, heating, ultrasonic radiation, or supercritical extraction, considering the polarity, extraction degree, and preservation degree of the active substance.

The cannabis exosome extract of the present invention refers to 'cannabis-derived exosomes' obtained from green young stems and roots from which the leaves of Cannabis sativa L. have been removed. According to one embodiment of the present invention, the hemp-derived exosomes (cannabis exosome extract) may be obtained by grinding the stems and roots of hemp using a grinder, followed by sonication and filtration. The ultrasonic treatment is preferably set to a low power of 50 to 60 Hz and performed 3 to 5 times on/off for 15 to 30 seconds.

According to one embodiment of the present invention, the hemp-derived exosomes may be obtained by sonication and filtration, followed by separation and purification using tangential flow filtration (TFF). The tangential-flow filtration (TFF) method removes other impurity particles smaller than the pores of the filter using the surface of a multi-filter with a cut-off value (Molecular Weight Cut Off, MWCO) of 100 to 500 kDa, then 1 It is preferable to concentrate and purify to /4~1/20 volume.

According to one embodiment of the present invention, the hemp-derived exosomes are separated and purified using tangential-flow filtration (TFF), and then purified at 100,000 to 200,000 using ultracentrifugation (UC). Those obtained by centrifugation at ×g are preferable in terms of obtaining high purity exosomes.

According to one embodiment of the present invention, the hemp-derived exosomes include grinding the stems and roots of hemp using a grinder; Sonicating and filtering the ground hemp; Separating and purifying the sonicated cannabis grind liquid using tangential-flow filtration (TFF); Centrifuging the separated and purified cannabis-derived exosomes using ultracentrifugation (UC) method; And it may be prepared by a method comprising filtering the centrifuged cannabis-derived exosomes with a filter.

According to one embodiment of the present invention, the hemp-derived exosomes may be extracellular vesicles with an average particle size of 50 to 250 nm, and the number of particles is 1.0 × 10 ⁷ to 1.0 per unit volume of 1 ml. It may be ×10 ¹⁰ particles.

Hydrolyzed Pea Protein of the present invention refers to a raw material composed of peptides with a molecular weight of 3,000 or less (average 2,000) extracted from peas, and these peptides are located in the skin stem located in the lower part of the epidermis, that is, the epidermal layer of the skin. It is known to act as a stem cell activator that promotes collagen synthesis by activating cells. The hydrolyzed pea protein of the present invention may be made by hydrolyzing peas ( Pisum sativum ) using acid, enzymes, or other methods, and can be obtained by purchasing commercially available hydrolyzed pea protein. It may be used.

The hemp stem extract of the present invention means extracted from the mature stems of hemp ( Cannabis sativa L.). According to one embodiment of the present invention, the hemp stem extract is prepared by pulverizing the hemp ( Cannabis sativa L.) stems and powdering them, and then extracting the hemp (Cannabis sativa L.) stems from 28 to 36 degrees Celsius at 15 to 25°C using water (purified water or distilled water) as an extraction solvent. It may be prepared by maturing the extracted extract for 5 to 10 days at a temperature of 5 to 15 ℃, preferably, the extract extracted for 30 to 34 hours at 18 to 22 ℃ for 6 to 8 days at a temperature of 8 to 12 ℃. It may be manufactured by aging, and may further include filtration and/or sterilization before and after aging. It is preferable to include the process of maturing the extract when producing the ginseng stem extract in that the effects of improving the skin barrier, skin moisturizing, whitening, and improving skin wrinkles can be enhanced compared to the non-ripened case.

The hemp exosome extract, hydrolyzed pea protein, and hemp stem extract of the present invention may be mixed at a weight ratio of 10:1.5 to 2.5:3.5 to 4.5, preferably 10:1.9 to 2.1:3.9 to 4.1. It may be mixed at a weight ratio of , and when mixed at that ratio, the effects of improving skin barrier, skin moisturizing, skin whitening, and improving skin wrinkles can be significantly better than when mixed at other ratios.

According to one embodiment of the present invention, the mixture of hydrolyzed pea protein and hemp stem extract may be aged for 8 to 15 days at a temperature of 9 to 15 ° C. and then mixed with hemp exosome extract, , Preferably, the mixture of hydrolyzed pea protein and hemp stem extract may be aged for 9 to 11 days at a temperature of 10 to 14 ℃ and then mixed with hemp exosome extract, and this maturation process Through this, it is possible to ensure that each ingredient harmonizes well and at the same time, further improve the synergistic effect achieved by the combination of ingredients. According to one embodiment of the present invention, when the aging process is performed, skin barrier improvement, skin moisturizing, and skin Effects such as whitening and skin wrinkle improvement can be significantly improved.

In addition, the cosmetic composition of the present invention may further contain one or more active ingredients selected from collagen extract and phytosterol, and may preferably contain both collagen extract and phytosterol.

The collagen extract refers to extracting oil-soluble components from collagen. The collagen extract of the present invention may use oil-soluble components extracted from plant- or fish-derived collagen, or purchase a commercially available collagen extract. It may be used as such.

The phytosterols refer to a mixture of sterols obtained from higher plants. The phytosterols of the present invention can be sterols derived from plants or sterols extracted/isolated from plants, and commercially available phytosterols can be purchased and used. It may be.

The hemp exosome extract, hydrolyzed pea protein, hemp stem extract, collagen extract and phytosterol of the present invention may be mixed at a weight ratio of 10:1.5 to 2.5:3.5 to 4.5:2.5 to 3.5:0.5 to 1.5. , Preferably, it may be mixed at a weight ratio of 10:1.9~2.1:3.9~4.1:2.9~3.1:0.9~1.1, and when mixed at that ratio, it improves the skin barrier and moisturizes the skin more than when mixed at other ratios. , the effects of skin whitening and skin wrinkle improvement can be significantly improved.

According to one embodiment of the present invention, the mixture of hydrolyzed pea protein, hemp stem extract, collagen extract, and phytosterol is aged at a temperature of 9 to 15°C for 8 to 15 days, and then aged for 8 to 15 days with hemp exosome extract and It may be mixed, and preferably, the mixture of hydrolyzed pea protein, hemp stem extract, collagen extract, and phytosterol is aged at a temperature of 10 to 14°C for 9 to 11 days, and then mixed with hemp exosome extract. It may be mixing, and through this aging process, each ingredient can be well harmonized and at the same time, the synergistic effect achieved by the combination of ingredients can be further improved. According to one embodiment of the present invention, the skin barrier is improved when the aging process is performed compared to when the hemp exosome extract, hydrolyzed pea protein, hemp stem extract, collagen extract, and phytosterol are mixed as is without going through the aging process. , the effects of skin moisturizing, skin whitening, and skin wrinkle improvement can be significantly improved.

In the specification of the present invention, a mixture of the hemp exosome extract, hydrolyzed pea protein, and hemp stem extract; A mixture of hemp exosome extract, hydrolyzed pea protein, hemp stem extract, collagen extract and phytosterols; A mixture of hemp exosome extract, hydrolyzed pea protein, hemp stem extract, collagen extract, or phytosterol; was designated as 'natural extract complex'.

Additionally, in the specification of the present invention, a mixture of hydrolyzed pea protein and hemp stem extract; A mixture of hydrolyzed pea protein, hemp stem extract, collagen extract and phytosterols; A mixture of hydrolyzed pea protein, hemp stem extract, collagen extract, or phytosterol; was designated as 'natural extract mixture'.

The cosmetic composition of the present invention may strengthen the function of the skin barrier by increasing the expression of one or more proteins or genes selected from the group consisting of involucrin, filaggrin, and aquaporin-3, and may additionally cause skin whitening and skin wrinkles. This may indicate an improvement effect.

The natural extract complex is preferably contained in an amount of 0.001 to 10.0% by weight based on the total weight of the cosmetic composition. If it is less than 0.001% by weight, the effect of improving the skin barrier and moisturizing the skin is minimal, and if it exceeds 10.0% by weight, the effect of improving the skin barrier, moisturizing the skin, whitening the skin, and improving wrinkles is only slightly increased due to the increase in content, and economic efficiency is low. You can.

In addition to the above-mentioned active ingredients, the cosmetic composition of the present invention can be prepared by including skin moisturizing ingredients, skin whitening ingredients, skin wrinkle improving ingredients, skin protection ingredients, etc. known in the art to reinforce and add skin-related activities. , For the above ingredients, refer to the Functional Cosmetics Code according to the Cosmetics Act (Ministry of Food and Drug Safety Notification “Functional Cosmetics Standards and Test Methods”) and the Health Functional Food Code of the Health Functional Foods Act (Ministry of Food and Drug Safety Notification “Health Functional Food Standards and Specifications”). can do. One or more of these ingredients may be included in the cosmetic composition of the present invention along with the active ingredients.

According to one embodiment of the present invention, in addition to the above-mentioned active ingredients, the cosmetic composition of the present invention contains Lactobacillus fermentation product and Camellia flower extract to further improve the effects of skin moisturizing, skin whitening, skin wrinkle improvement, and skin barrier improvement. , medicinal buckwheat extract, sophora root extract, forsythia fruit extract, bellflower root extract, lactobacillus fermentation filtrate, centella asiatica extract, bifida fermentation lysate, lotus seed extract, burdock seed extract, honeysuckle flower extract, grown root extract, syringa root Any selected from the group consisting of extract, propolis extract, yeast fermentation filtrate, Bacillus/Lactobacillus/ginseng root fermentation extract, pear extract, damask rose flower water, melon extract, vetiver root oil, and American pine leaf/stem extract. It may additionally contain one or more ingredients.

The cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing, oil, powder. It can be formulated as foundation, emulsion foundation, wax foundation, pack, massage cream, spray, etc., but is not limited to this. More specifically, it includes serum, lotion, nutritional lotion, nutritional cream, massage cream, essence, paste, mask pack, patch, gel, cream, lotion, powder, foundation, makeup base, soap, cleanser, oil, wax and spray. It may be one or more formulations selected from the group consisting of, and may preferably be used in serum form. Ingredients other than the above-mentioned essential ingredients can be appropriately selected and mixed by a person skilled in the art according to other formulations or purposes of use, etc.

The cosmetic composition of the present invention preferably contains a cosmetically acceptable medium or base. These are all formulations suitable for topical application, such as solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspension microemulsions, microcapsules, microgranules or ionic forms (liposomes) and/or It may be provided in the form of a warm vesicular dispersion, or in the form of a cream, toner, lotion, powder, ointment, spray or conceal stick. It can also be manufactured in the form of foam or an aerosol composition further containing compressed propellant. In addition, the cosmetic composition of the present invention can be prepared in the form of an adjuvant for topical or systemic application commonly used in the field of dermatology by containing a dermatologically acceptable medium or base, and these compositions are commonly used in the field. It can be manufactured according to the method.

In addition, the cosmetic composition includes, in addition to the active ingredients of the present invention, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, Commonly used in water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or in cosmetics. It may contain auxiliaries commonly used in the cosmetic field, such as any other ingredients. These auxiliaries are introduced in amounts commonly used in the field of cosmetology.

The composite of hemp exosome extract, hydrolyzed pea protein, and hemp stem extract of the present invention and the composite of hemp exosome extract, hydrolyzed pea protein, hemp stem extract, collagen extract, and phytosterol have each component As the ripening process is carried out in the process of mixing ginseng stem extract and natural extract mixture in a specific ratio, the expression of involucrin, filaggrin, and aquaporin-3, which are proteins related to skin moisturization and skin barrier, is significantly increased. , It shows an excellent effect of recovering damaged skin barrier and at the same time shows skin whitening and skin wrinkle improvement effect, so it can be useful as a cosmetic composition for moisturizing skin and improving skin barrier, especially as a cosmetic composition in the form of a serum.

Hereinafter, the present invention will be described with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

Preparation Example 1. Preparation of cannabis exosome extract

The young stems and roots from which the cannabis leaves were removed were thoroughly washed, then purified water was added to the washed cannabis at a weight ratio of 10 to 20 times (wt%) and ground at a speed of 30 to 50 rpm. The pulverized liquid was sonicated for 30 minutes using a low-speed ultrasonication machine, and suspended matter in the pulverized liquid was sequentially removed through 300 mesh and 100 mesh. Each recovered pulverized liquid was used as a sample for exosome isolation.

To produce cannabis-derived exosomes, tangential-flow filtration (TFF) and ultracentrifugation (UC) methods were combined. After centrifugation at 4,000 × g for 20 minutes, the pellet layer was discarded, the supernatant was collected, and exosomes were separated using the TFF method. Specifically, other impurity particles smaller than the pores of the filter were removed using the surface of a multi-filter with a cutoff value of 100 to 500 kDa in the TFF system, and the solution containing hemp-derived exosomes was concentrated. Exosomes concentrated to about 1/10 times the starting volume were subjected to ultracentrifugation as a second process. After the first ultracentrifugation at 100,000 × g for 70 minutes, the second ultracentrifugation was performed at 150,000 × g for 70 minutes. All centrifugation processes were performed at 4°C. The exosomes extracted in this way were filtered through a 0.22 μm filter to remove impurities such as cell debris, waste, and large particles, and finally, hemp-derived exosomes (cannabis exosome extract, Preparation Example 1) were obtained.

Example 1. Preparation of hemp stem extract

The stems from which the leaves of hemp (hemp) were removed were prepared, washed, cut into pieces, dried, ground in a blender, and pulverized to prepare dried hemp stem powder. 10 g of dried hemp stem powder was added to 100 ml of purified water, extracted at a temperature of 20°C for 32 hours, and then filtered through a 300 mesh filter to obtain an extract. After the filtered hemp stem extract was aged at a temperature of 10°C for 7 days, the aged hemp stem extract was sterilized through a 0.2 ㎛ filter, and the solvent was removed using a rotary evaporator to obtain the hemp stem extract (Example 1). Manufactured.

Comparative Example 1. Preparation of unripened hemp stem extract

10 g of dried hemp stem powder prepared in the same manner as in Example 1 was added to 100 ml of purified water, extracted at a temperature of 20°C for 32 hours, and then the extract was filtered through a 300 mesh filter and sterilized through a 0.2 ㎛ filter without an aging step. Next, the solvent was removed using a rotary evaporator to prepare an unripened hemp stem extract (Comparative Example 1) that did not undergo the ripening step.

Example 2 and Comparative Example 2. Preparation of natural extract complex

Hemp stem extract, hydrolyzed pea protein, collagen extract, and/or phytosterol prepared by the method of Example 1 or Comparative Example 1 were mixed in the weight ratio shown in Table 1 below, and aged at a temperature of 12°C for 10 days. , the aged product was freeze-dried to prepare a natural extract mixture.

The natural extract mixture was mixed with the cannabis exosome extract prepared by the method of Preparation Example 1 at the weight ratio shown in Table 1 below to produce a natural extract complex (Examples 2-1 to 2-7 and Comparative Examples 2-1 to 2- 6) was prepared.

Hemp Exosome Extract
(Production Example 1) Natural extract mixture (maturation stage after mixing) Hydrolyzed Pea Protein hemp stem extract
(Example 1) Unripened hemp stem extract
(Comparative Example 1) Collagen Extract phytosterol Example 2-1 10 2 4 - - - Example 2-2 10 3 3 - - - Example 2-3 10 4 2 - - - Example 2-4 10 5 5 - - - Example 2-5 10 2 4 - 3 One Example 2-6 10 2 4 - 2 2 Example 2-7 10 2 4 - One 3 Example 2-8 10 2 4 - 4 - Example 2-9 10 2 4 - - 4 Comparative Example 2-1 10 2 - 4 - - Comparative Example 2-2 10 2 - 4 3 One

Comparative Example 3. Preparation of natural extract complex excluding mixing and maturation step

Hemp stem extract, hydrolyzed pea protein, collagen extract and/or phytosterol prepared by the method of Example 1 or Comparative Example 1, hemp exosome extract prepared by the method of Preparation Example 1 without the aging step, and the table below By mixing to a weight ratio of 2, natural extract complexes (Comparative Examples 3-1 to 3-4) excluding the mixing and aging step were prepared.

Hemp Exosome Extract
(Production Example 1) Natural extract mixture (excluding the maturation step after mixing) Hydrolyzed Pea Protein hemp stem extract
(Example 1) Unripened hemp stem extract
(Comparative Example 1) Collagen Extract phytosterol Comparative Example 3-1 10 2 4 - - - Comparative Example 3-2 10 2 4 - 3 One Comparative Example 3-3 10 2 - 4 - - Comparative Example 3-4 10 2 - 4 3 One

Experimental Example 1. Evaluation of skin barrier improvement and skin moisturizing efficacy of natural extract complex

To confirm the effect of increasing the synthesis of involucrin and filaggrin, which play an important role in the barrier and moisturizing functions of skin cells, and aquaporins-3, which play an important role in water movement in skin cells. To this end, the cells were treated with the natural extract complexes of the examples and comparative examples, respectively, and then the protein expression levels of involucrin, filaggrin, and aquaporin-3 were confirmed through Western blotting.

First, human keratinocytes HaCaT were inoculated and the natural extract composite samples prepared in Example 2 and Comparative Examples 2 to 3 were added, respectively, and cultured in DMEM medium, 37°C, 5% CO ₂ . After culturing for 24 hours, the cells were recovered, disrupted, and only the protein was extracted and stored at -40°C. The extracted protein was quantified using BCA saasy. The protein amount of each sample was diluted to 30 g, electrophoresed on a 12% SDS-gel, and then transferred to a PVDF membrane. After reacting with the primary antibodies, aquaporin antibody (rabbit anti-aquaporins-3 antibody), involucrin antibody (rabbit anti-involucrin antibody), and filaggrin antibody (rabbit polyclonal), the remaining antibodies were washed and removed, and the secondary antibody was added. (rabbit HRP-coupled anti-IgG antibody). After removing the remaining antibody, a reagent (peroxidase substrate and chromogen solution) that reacts with the antibody to cause a color reaction was added. The developed band was measured through image analysis (BioRad, Universal Hood, USA) and quantified based on the control group (100%) treated with DMSO only, and the results are shown in Table 3.

Expression level of involucrin (%) Expression level of filaggrin (%) Expression level of aquaporin-3 (%) Example 2-1 159.2 158.1 155.0 Example 2-2 136.4 138.5 132.7 Example 2-3 135.6 136.3 138.6 Example 2-4 139.5 135.2 134.1 Example 2-5 168.3 164.1 165.9 Example 2-6 145.2 147.3 148.7 Example 2-7 146.1 149.0 147.2 Example 2-8 140.7 141.4 143.5 Example 2-9 141.8 142.7 144.6 Comparative Example 2-1 120.4 118.5 117.8 Comparative Example 2-2 121.9 120.9 118.4 Comparative Example 3-1 118.0 119.6 121.3 Comparative Example 3-2 119.3 122.8 120.2 Comparative Example 3-3 109.8 110.4 112.1 Comparative Example 3-4 110.6 113.2 111.5 control group 100.0 100.0 100.0

As shown in Table 4, the natural extract complexes corresponding to Comparative Examples 2-1 to 2-1 and 3-1 to 3-4 were not subjected to any aging process during the production of hemp stem extract and/or natural extract mixture. In this case, almost no effect was shown compared to the control group, and in the case of the natural extract complex corresponding to Examples 2-1 to 2-9, where the aging process was performed when preparing the hemp stem extract and natural extract mixture according to the present invention, more than 30% It showed effect. In particular, when HaCaT keratinocytes were treated with the natural extract complexes corresponding to Examples 2-1 and 2-5, the expression of involucrin, filaggrin, and aquaporin-3, which play an important role in skin barrier function and moisturization, all increased. was found to be the highest. Meanwhile, in the case of the natural extract complex corresponding to Examples 2-6 to 2-9, hemp exosome extract, hemp stem extract, and hydrolyzed pea protein were mixed in the same ratio as Example 2-5, but collagen As the mixing ratio of extract and/or phytosterol was different, the expression level was lower than that of Example 2-1 containing only three components.

Therefore, when preparing the hemp stem extract and the natural extract mixture, the maturation process is carried out, and at the same time, hemp exosome extract, hydrolyzed pea protein, and hemp stem extract are included in a weight ratio of 10:2:4. Example 2- 1 and the natural extract complex corresponding to Examples 2-5 containing hemp exosome extract, hydrolyzed pea protein, hemp stem extract, collagen extract, and phytosterol in a weight ratio of 10:2:4:3:1. It was confirmed that it was manufactured under optimal conditions to achieve excellent skin barrier improvement and moisturizing effects.

Experimental Example 2. Measurement of tyrosinase activity inhibition effect

Tyrosinase plays a very important role in the production of skin melanin. It acts as a tyrosine hydroxylase, which oxidizes tyrosine within the melanosome to create DOPA, and as a DOPA oxidase, which oxidizes DOPA to create DOPA quinone, thereby synthesizing melanin polymer. Since it acts as an important enzyme, the possibility of a whitening effect can be inferred by evaluating the inhibitory activity of tyrosinase. Therefore, in order to evaluate the whitening effect of different natural extract complexes in Examples and Comparative Examples of the present invention, tyrosinase inhibitory activity was measured.

Tyrosinase was isolated and purified from mushrooms and purchased from Sigma (USA). The substrate, tyrosine, was dissolved in 0.05 M sodium phosphate buffer solution (pH 6.8) and used at a concentration of 0.1 mg/ml. The natural extract complexes prepared in Example 2 and Comparative Examples 2 to 3 were dissolved in a high concentration of 1,3-butylene glycol aqueous solution, and then diluted in sodium phosphate buffer solution to a concentration of 300 ㎍/㎖ to prepare the sample solution. It was used as. Put 0.5 ml of the tyrosine solution in a test tube, add 0.5 ml of the sample solution, and leave it in a thermostat at 37°C for 10 minutes. Then, 0.5 ml of the 250 U/ml tyrosinase solution was added and reacted again at 37°C for 10 minutes. In the case of the control group (group without sample), 0.5 ml of buffer solution was added instead of each sample solution and the same reaction was performed. Arbutin was used as a positive control. After the reaction was completed, the test tube was quickly cooled by placing it on ice to completely stop the enzyme reaction, and the absorbance at a wavelength of 475 nm was measured using a spectrophotometer. The effect of inhibiting tyrosinase activity was calculated using Equation 1 below to evaluate whitening efficacy, and the results are shown in Table 4 below.

[Equation 1]

Tyrosinase activity inhibition effect (%) = [1-(absorbance of group with sample added/absorbance of group without sample)] × 100

sample Tyrosinase activity inhibition effect (%) Example 2-1 69.1±0.6 Example 2-2 54.9±2.2 Example 2-3 58.7±1.4 Example 2-4 52.6±3.7 Example 2-5 73.8±1.9 Example 2-6 64.0±2.1 Example 2-7 63.5±2.2 Example 2-8 60.1±3.0 Example 2-9 62.2±1.3 Comparative Example 2-1 47.3±0.8 Comparative Example 2-2 46.0±1.5 Comparative Example 3-1 42.5±0.8 Comparative Example 3-2 44.4±2.7 Comparative Example 3-3 36.7±0.4 Comparative Example 3-4 37.9±1.5 Positive control group (arbutin) 65.3±1.6

As shown in Table 5, the natural extract complexes of Examples 2-1 to 2-9, which were subjected to the aging process during the production of hemp stem extract and natural extract mixture, had a tyrosinase inhibitory effect at a level similar to or superior to that of the positive control group. appeared high, but the natural extract complexes of Comparative Examples 2-1 to 2-2 and Comparative Examples 3-1 to 3-4 did not show a significant difference compared to the control group. In particular, Examples 2-1 and 2-5 It was confirmed that the natural extract complex had a higher tyrosinase inhibition effect than arbutin, a positive control, and had excellent whitening activity.

Experimental Example 3. Measurement of elastase activity inhibition effect

When elastase, an enzyme that decomposes elastin, is active, elastin, an elastic fiber, is broken down and skin wrinkle formation is accelerated. Therefore, the wrinkle improvement effect can be inferred by evaluating the effect of inhibiting elastase activity. You can. Therefore, in order to evaluate the wrinkle-improving effect of the natural extract complexes according to the Examples and Comparative Examples of the present invention, the elastase activity inhibition ability was measured.

Elastase derived from human white blood cells was used, and the synthetic substrate MeOSuc-Ala-Ala-Pro-Val-pNA was used as the elastase substrate. A 100mM Tris (pH 7.5) solution was used as the buffer solution. Elastase was used in a final amount of 0.2mU using a buffer solution. In addition, the synthetic substrate of elastase was made into a 100mM solution using DMSO and then diluted with a buffer solution so that the final concentration was 05mM. At this time, the positive control was set to contain quercetin, known as an elastase inhibitor, at a concentration of 100 ppm. The natural extract composite samples prepared in Example 2 and Comparative Examples 2 to 3 were also added to 100 ppm. The reaction was carried out in a 96-well plate and reacted at room temperature for 20 minutes. Absorbance was measured at 405 nm at 1-minute intervals using a spectrophotometer, and the slope of absorbance versus time was determined to be the enzyme activity. The elastase inhibition rate was calculated using Equation 2 below, and the results are shown in Table 5.

[Equation 2]

Elastase inhibition rate (%) = [(control slope - sample slope)/sample slope)] × 100

sample Elastase inhibition rate (%) Example 2-1 79.62 Example 2-2 50.84 Example 2-3 51.26 Example 2-4 53.70 Example 2-5 87.18 Example 2-6 66.39 Example 2-7 67.45 Example 2-8 65.16 Example 2-9 63.72 Comparative Example 2-1 40.19 Comparative Example 2-2 41.97 Comparative Example 3-1 39.18 Comparative Example 3-2 36.61 Comparative Example 3-3 31.03 Comparative Example 3-4 32.59 Positive control group (quercetin) 53.21

As can be seen from the results in Table 5, it was confirmed that the natural extract complex according to Example 2 of the present invention had excellent elastase inhibition ability. The natural extract complexes according to Comparative Examples 2 and 3 also showed a certain degree of effect, but it was less effective than the natural extract complexes corresponding to Examples 2-1 to 2-9, in which the aging process was performed both when preparing the hemp stem extract and when preparing the natural extract mixture. It was insufficient, and a significantly superior effect was confirmed, especially in the natural extract complexes corresponding to Examples 2-1 and 2-5.

Experimental Example 4. Measurement of recovery after skin barrier damage

In order to confirm whether the natural extract complex of the present invention enhances lipid barrier recovery after skin barrier damage in actual human skin, the effect of improving transepidermal water loss was measured. For this purpose, 5% by weight of each natural extract complex prepared in Examples 2-1 to 2-7 and Comparative Examples 2-1 to 2-2 and 3-1 to 3-4 relative to the total composition is included, and a skin softener A serum-type composition was prepared according to a conventional method by mixing 8% by weight, 10% by weight of thickener, 7% by weight of solubilizer, 8% by weight of other additives, and the remaining amount of purified water, and used as a test product.

Fifty-two volunteers in good health and with normal skin without any dermatological disorders were selected as subjects. For the test, the skin was allowed to rest for 30 minutes after washing the test area, and then the transepidermal water loss (TEWL) of the test area was measured using Tewameter TM300 (Courage+Khazaka electronic GmbH, Germany). After measurement, a chamber impregnated with 1% (w/v) SLS (Sodium Lauryl Sulfate) was attached to the test area for 24 hours. After 24 hours, the chamber attached to the test area was removed and the transepidermal water loss (TEWL) of the test area was measured using a Tewameter. After completing the measurement, the subjects applied a serum-type composition containing the natural extract complexes of Examples 2-1 to 2-7 and Comparative Examples 2-1 to 2-2 and 3-1 to 3-4 on the skin twice a day. It was applied and used. One and two weeks after the start of the test, transepidermal water loss (TEWL) was measured for subjects who used the test product to evaluate the effect of improving the skin barrier. The transepidermal water loss (TEWL) improvement rate was calculated using Equation 3 below and is shown in Table 6 below.

[Equation 3]

TEWL improvement rate (%) = [(TEWL measurement result after 0 week damage - TEWL measurement result 1 or 2 weeks after serum application)/(TEWL measurement result after 0 week damage - TEWL measurement result before 0 day damage)] × 100

Transepidermal water loss improvement rate (%) Test product (serum-type composition) 1 week later 2 weeks later Example 2-1 79.51 80.67 Example 2-2 58.92 65.16 Example 2-3 57.47 60.53 Example 2-4 59.44 63.01 Example 2-5 81.09 89.42 Example 2-6 75.15 77.26 Example 2-7 74.38 78.95 Comparative Example 2-1 41.22 48.09 Comparative Example 2-2 45.26 49.74 Comparative Example 3-1 39.63 42.80 Comparative Example 3-2 38.70 46.18 Comparative Example 3-3 27.81 34.34 Comparative Example 3-4 29.54 35.97

As a result, as shown in Table 6 above, the transepidermal water loss (TEWL) improvement rate was more than 50% at the site of use of the serum-type composition containing the natural extract complex of Examples 2-1 to 2-7 after 1 week of product use. It was found to be more than 60% after 2 weeks, showing a higher rate of improvement in transepidermal water loss than when using a serum-type composition containing the natural extract complex of the comparative example. In particular, when the serum-type composition containing the natural extract complex of Examples 2-1 and 2-5 was used, the improvement rate of transepidermal water loss after 2 weeks was shown to be as high as 80% or more, which shows that the present invention It was confirmed that the natural extract complexes of Examples 2-1 and 2-5 were excellent in skin moisturizing and recovery effects of damaged skin barrier.

So far, the present invention has been examined focusing on its embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims, not the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (10)

In a cosmetic composition for moisturizing skin and improving the skin barrier, comprising hemp exosome extract, hydrolyzed pea protein, and hemp stem extract,
The hemp exosome extract, the hydrolyzed pea protein, and the hemp stem extract are mixed in a weight ratio of 10:2:4,
The hemp stem extract is obtained by first maturing the extract of Cannabis sativa L. stems for 30 to 34 hours at 18 to 22°C using water as an extraction solvent at a temperature of 8 to 12°C for 6 to 8 days,
The composition is made by secondary maturing a mixture of the hydrolyzed pea protein and the hemp stem extract for 9 to 11 days at a temperature of 10 to 14 ° C. and then mixing it with the hemp exosome extract,
Cosmetic composition for moisturizing skin and improving skin barrier. delete delete delete According to paragraph 1,
The cosmetic composition further includes collagen extract and phytosterol. According to clause 5,
The composition is a cosmetic composition in which hemp exosome extract, hydrolyzed pea protein, hemp stem extract, collagen extract, and phytosterol are mixed in a weight ratio of 10:2:4:3:1. According to clause 5,
The composition is a mixture of hydrolyzed pea protein, hemp stem extract, collagen extract, and phytosterol, secondarily aged at a temperature of 10 to 14°C for 9 to 11 days, and then mixed with hemp exosome extract, Cosmetic composition. According to paragraph 1,
The cosmetic composition is a cosmetic composition that strengthens the function of the skin barrier by increasing the expression of one or more proteins or genes selected from the group consisting of involucrin, filaggrin, and aquaporin-3. According to paragraph 1,
The cosmetic composition exhibits skin whitening and skin wrinkle improvement effects. According to paragraph 1,
The cosmetic composition consists of serum, lotion, nutritional lotion, nutritional cream, massage cream, essence, paste, mask pack, patch, gel, cream, lotion, powder, foundation, makeup base, soap, cleanser, oil, wax, and spray. A cosmetic composition, which is one or more formulations selected from the group.