KR102558352B1 - Cosmetic composition for skin improvement comprising hemp extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for improving skin comprising a cannabis extract as an active ingredient.
In addition, the present invention relates to a composition for improving skin comprising a cannabis extract and exosomes as active ingredients.
The hemp extract of the present invention alone strengthens the dense coupling of keratinocytes without toxicity, increases collagen synthesis, and at the same time inhibits the expression of collagenase (MMP-1), so it can improve skin and treat inflammatory skin diseases. When treating hemp extract and exosomes at the same time, it can show a significantly better skin improvement effect than when treated alone.

Description

Cosmetic composition for skin improvement comprising hemp extract as an active ingredient}

The present invention relates to a cosmetic composition for skin improvement comprising hemp extract as an active ingredient.

Human skin undergoes changes due to various internal and external factors as we age. Internally, the secretion of various hormones that regulate metabolism is reduced, and the function of immune cells and the activity of cells are lowered, resulting in a decrease in the biosynthesis of constituent proteins necessary for the body. Externally, due to the destruction of the ozone layer, the content of ultraviolet rays reaching the surface of the sun increases and environmental pollution is further intensified, resulting in an increase in free radicals and active free radicals, thereby reducing the thickness of the skin, increasing wrinkles, and reducing elasticity. In addition, various changes such as frequent skin troubles occur.

As skin aging progresses, reduction in division of keratinocytes and fibroblasts, reduction in collagen synthesis, increase in MMP production, and increase in signal transmission of melanin production occur, which causes wrinkles to increase and skin elasticity to decrease, and spots, freckles, and age spots to increase. In particular, the balance of hormones is broken around the age of 40, and the cell regeneration ability, collagen synthesis ability, and the moisture content of the stratum corneum, which is important for skin moisturizing, are significantly reduced. It is suggested that the cause of this is a decrease in the lipid components of the stratum corneum and a decrease in natural moisturizing factors. As a result, in aging skin, the amount of moisture supplied from the lower epidermis to the stratum corneum is insufficient, resulting in severe skin dryness, resulting in inflammation and pruritus. On the other hand, wrinkles in the skin are caused by an imbalance between the synthesis and degradation of collagen. In normal skin, the synthesis of type I collagen and its degradation enzyme, MMP-1, are in balance. However, in photoaged skin, the synthesis of type I and III collagen is reduced, and the activity of MMP-1 is increased. These MMPs (matrix metalloproteinases) are proteolytic enzymes that degrade the extracellular matrix and are known to be involved in wound healing or tissue regeneration in a normal state.

Due to these changes, interest in materials capable of suppressing skin aging and social demand for cosmetics containing them are increasing, and developing materials that are biocompatible and have excellent skin penetration rate has emerged as an important task. Recently, various methods have been applied, and among them, various plant resources and peptides have been studied for the recovery of skin damage.

Under this background, the present inventors confirmed that the hemp stem extract strengthens the dense coupling of keratinocytes without toxicity, increases collagen synthesis, and at the same time inhibits the expression of collagenase (MMP-1) to improve the skin. When treated with exosomes, it was confirmed that they showed excellent skin barrier strengthening and collagen synthesis promoting effects compared to when treated alone, and completed the present invention.

Korean Patent Publication No. 10-2022-0091957

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

One exemplary object of the present invention is to provide a cosmetic composition for skin improvement comprising a cannabis extract as an active ingredient.

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

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 cosmetic composition for skin improvement comprising a cannabis extract as an active ingredient.

Hemp (Cannabis sativa L.) refers to an annual plant of the genus Cannabaceae, which has been widely cultivated in tropical and temperate regions around 12,000 years ago, mainly in Central Asia. indica and var. As kafiristanica, it may be Cannabis sativa subspecies sativa, Cannabis sativa subspecies indica, or Cannabis sativa subspecies ruderalis, In addition, it may be a generic name for cannabis plants' genetic crossing, self-crossing, or hybridization plants thereof, but is limited thereto it is not going to be

In the present invention, 'hemp' may be used interchangeably with hemp, which means all cannabis used for other purposes than hallucinogenic drugs.

In the present invention, "extract" refers to an active ingredient separated from a natural product, that is, a substance showing a desired activity, and the extract can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes a dry powder of the extract or any form formulated using the same. In addition, the extract includes fractionated extracts subjected to the extraction process.

In the present invention, the extraction method is not particularly limited as long as it is a method capable of efficiently extracting the active ingredient of hemp, and may be extracted by, for example, alcohol extraction, cold needle extraction, ultrasonic extraction, reflux extraction, hot water extraction, etc., but is not limited thereto.

The type of extraction solvent is not particularly limited as long as it can efficiently extract the active ingredient from hemp. For example, water, an organic solvent, or a mixed solvent thereof may be used, and the liquid extracted using the extraction method may be used directly or concentrated and / or dried.

The organic solvent may be methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof. The extract may be prepared by warming. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient contained in the plant may vary, so an organic solvent capable of efficiently extracting the active ingredient of hemp should be selected.

In the present invention, the extract may be used after being concentrated or diluted, and a distillate of the extract may be used.

The extraction time is not particularly limited as long as the active ingredient of hemp can be efficiently extracted, and may be, for example, 30 minutes to 60 hours, 1 hour to 48 hours, 2 hours to 36 hours, 3 hours to 24 hours, 4 hours to 24 hours, 6 hours to 60 hours, 10 hours to 60 hours, 24 hours to 48 hours, or 36 hours to 48 hours. It is not.

As an example of the present invention, the cannabis stem extract may be extracted twice for 48 hours at room temperature by an ethanol organic solvent.

In the present invention, 'active ingredient' refers to a component that exhibits the desired activity alone or that can exhibit activity together with a carrier having no activity itself.

In the present invention, "skin improvement" refers to all activities that improve or benefit the skin by using the cannabis extract of the present invention.

As an example, the skin improvement of the present invention may mean skin inflammation relief, skin epidermal improvement, wrinkle improvement, anti-aging and/or skin moisturizing, but is not limited thereto.

In the present invention, “wrinkle improvement” means maintaining or enhancing wrinkles and elasticity of the skin.

In the present invention, "anti-aging" means the prevention of skin aging, and includes both chronological aging (endogenous aging), which is natural aging caused by physiological changes in the body over time, and photoaging occurring in areas exposed to sunlight (photogenic aging) that prevents or delays.

In the present invention, "skin moisturizing" means to increase the feeling of moisture in the skin and to maintain 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 skin improvement of the present invention may be an effect realized by strengthening the skin barrier by increasing the expression of dense binding markers ZO-1, occludin, and claudin-1.

The skin improvement of the present invention may be an effect implemented by enhancing skin elasticity by promoting collagen synthesis and suppressing the expression of collagen degrading enzymes at the same time.

In the examples of the present invention, it was confirmed that the cannabis stem extract exhibited a skin improvement effect without cytotoxicity. Specifically, it was confirmed that the hemp stem extract exhibits skin improvement effects by promoting cell compaction, increasing collagen synthesis, and suppressing the expression of collagen degrading enzymes.

In addition, the cosmetic composition for skin improvement of the present invention may further include exosomes derived from cannabis stems.

The exosome is one of extracellular vesicles, and refers to biological nanoparticles with a size of 30 to 300 nm generated from intracellular multivesicles. Exosomes are particles that are attracting attention as a next-generation drug delivery system by playing a major role in intercellular signal transmission, and contain various active ingredients depending on their origin. Exosomes themselves are known to play an important role in pathology of diseases such as cancer and degenerative diseases as well as physiological activities such as immune response, nerve function, and stem cell maintenance function.

In the present invention, the exosome refers to exosomes extracted (separated) from cannabis stems, and the extraction method and conditions are not limited as long as it can include a physiologically active ingredient capable of exhibiting a skin improvement effect. For example, ultracentrifuge, density centrifuge, use of a column, PEG precipitation (PEG precipitation), chromatography (chromatography), immuno-magnetic separation (immuno-magnetic separation (IMS) and acoustic purification) It can be extracted by methods such as (acoustic separation, acoustic purification), but is not limited thereto.

In the cosmetic composition of the present invention, the cannabis stem extract and the cannabis stem-derived exosomes may be in the form of a mixed agent, or the cannabis stem extract and the cannabis stem-derived exosomes may be formulated and treated simultaneously or sequentially. In the case of sequential processing, the order is not limited. For example, the cannabis stem extract or cannabis stem-derived exosomes may be firstly treated, and then the cannabis stem-derived exosomes or hemp stem extracts may be treated.

In the present invention, the hemp extract and the hemp stem-derived exosomes may be contained in an amount of 0.1 to 100% by weight based on the total weight of the cosmetic composition, and the hemp stem-derived exosomes may be included in a weight ratio of 10 ^-4 to 5x10 ^-3 relative to the weight ratio of the cannabis stem extract.

As an example, when the cannabis stem extract is treated at 25 to 100 ug/ml and treated simultaneously with exosomes, that is, when co-administered, it may be treated at 0.001 to 1 ug/ml, which is a concentration level of exosomes that does not exhibit cytotoxicity.

In the examples of the present invention, it was confirmed that the simultaneous treatment of cannabis stem extract (25ug/ml) and hemp stem-derived exosomes (0.001 or 0.01ug/ml) showed excellent skin improvement effects without cytotoxicity. Specifically, it was confirmed that simultaneous treatment showed a synergistic effect on strengthening the skin barrier and promoting collagen production, compared to the case of single treatment of cannabis stem extract or exosome, respectively.

In addition to the hemp extract, the cosmetic composition according to the present invention may be composed of various ingredients commonly used in cosmetic compositions, for example, water-soluble ingredients, powder ingredients, oil, surfactants, moisturizers, viscosity modifiers, preservatives, antioxidants, fragrances, pigments, etc.

Non-limiting examples of the usable surfactant include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants. More specifically, the anionic surfactants include alkylbenzene sulfonates, polyoxyalkylene alkyl sulfate ester salts, alkyl sulfate ester salts, olefin sulfonates, alkyl phosphates, polyoxyalkylene alkyl ether phosphates, dialkyl sulfosuccinates, fatty acid salts, and the like. Oil derivatives, fatty acid diethanolamide, etc. are mentioned. Examples of cationic surfactants include tertiary aliphatic amine salts, alkyltrimethylammonium halides, and dialkyldimethylammonium halides, and examples of amphoteric surfactants include amide betaine type, imidazolinium betaine type, and sulfobetaine type.

Examples of the moisturizing agent include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, and sorbitol. Benzoic acid, dehydroacetic acid, paraoxybenzoic acid ester (methyl paraoxybenzoate, butyl paraoxybenzoate, etc.), phenoxyethanol etc. are mentioned as said preservative. In addition, as the antioxidant, ascorbic acid, BHA, and the like may be mentioned, and in addition, an ultraviolet absorber, an anti-inflammatory agent, and a cooling agent may be added.

The cosmetic composition of the present invention may be formulated into a formulation selected from the group consisting of solutions, external ointments, creams, foams, nourishing lotions, softening toners, packs, softening waters, emulsions, makeup bases, essences, soaps, liquid cleansers, bath additives, sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays. It is not limited.

The cosmetic composition of the present invention may further include one or more acceptable carriers in cosmetic formulations, and as usual ingredients, for example, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservatives, fragrances, etc. may be appropriately mixed, but is not limited thereto. Here, "acceptable carrier in cosmetic formulations" is a compound or composition that is already known and used or can be included in a cosmetic formulation, or a compound or composition to be developed in the future, which has no toxicity, instability or irritation that is more adaptable to the human body when in contact with the skin. The carrier may be included in the composition of the present invention in an amount of about 1 wt % to about 99.99 wt %, preferably about 90 wt % to about 99.99 wt %, based on the total weight of the composition. However, since the ratio varies depending on the formulation in which the composition of the present invention is prepared and its specific application area (face, neck, etc.) or its preferred application amount, etc., the ratio should not be construed as limiting the scope of the present invention in any aspect.

Acceptable carriers in cosmetic preparations included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component. In particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether may be additionally included, but is not limited thereto, and these may be used alone or in combination of two or more.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc. may be used. In particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fat Group esters, polyethylene glycol or sorbitan fatty acid esters may be used, but are not limited thereto, and these may be used alone or in combination of two or more.

When the formulation of the present invention is a suspension, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tracanth, etc. may be used as carrier components, but are not limited thereto, and these may be used alone or in combination of two or more.

When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto, and these may be used alone or in combination of two or more.

Another aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating inflammatory skin disease comprising a cannabis extract as an active ingredient.

The 'hemp extract' and 'active ingredient' are as described above.

In the present invention, "inflammatory skin disease" refers to all skin diseases caused by inflammation, and specifically refers to skin diseases accompanied by inflammatory reactions such as itching (pruritus), edema, erythema, peeling, etc. due to various stimulating factors. Specifically, there are allergic dermatitis, acne, atopy, urticaria, contact dermatitis, seborrheic dermatitis, neurodermatitis, xeroderma, erythema and psoriasis due to various causes, but are not limited thereto.

In addition, the pharmaceutical composition for preventing or treating inflammatory skin diseases of the present invention may further include exosomes derived from hemp stem.

The 'exosome', 'exosome derived from cannabis stem', and 'exosome extraction method' are as described above.

In the pharmaceutical composition of the present invention, for combined administration of the cannabis stem extract and the cannabis stem-derived exosomes, the cannabis stem extract and the cannabis stem-derived exosomes may be mixed, or the cannabis stem extract and the cannabis stem-derived exosomes may be formulated and administered simultaneously or sequentially. In the case of sequential administration, the cannabis stem extract may be administered first, then the cannabis stem-derived exosome may be administered, or the reverse order may be administered.

When the cannabis stem extract and the hemp stem-derived exosomes are co-administered, the administration concentration ratio of the cannabis stem extract and the hemp stem-derived exosomes may be 1: 10 ^-4 to 1: 10 ^-2 , specifically 1:10 ^-4 to 1: 5x10 ^-3 _, 1:10 ^-4 to 1: 10 ^-3 or 1:10 ^-4 to 1: 5x10 ^-4 , Any concentration ratio effective for inflammatory skin diseases is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, for example, a carrier for oral administration or a carrier for parenteral administration may be further included. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like.

In addition, carriers for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like. In addition, a stabilizer and a preservative may be further included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration.

The pharmaceutical composition of the present invention may be formulated into a formulation for oral administration or parenteral administration according to the administration route as described above. When formulated, one or more buffers (eg, saline or phosphate buffered saline (PBS)), antioxidants, bacteriostats, chelating agents (eg, EDTA or glutathione), fillers, bulking agents, binders, adjuvants (eg, aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations contain the pharmaceutical composition of the present invention and at least one excipient, for example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, It may be prepared by mixing sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethyl cellulose, and hydroxypropylmethyl-cellulose or gelatin. Tablets or dragees may be obtained, for example, by combining the active ingredient with a solid excipient which is then milled and processed into a mixture of granules after adding suitable auxiliaries.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water or liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics or preservatives may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and may further include an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.

In the case of parenteral administration, the pharmaceutical composition of the present invention may be formulated according to a method known in the art in the form of injection, transdermal administration, and nasal inhalation with a suitable parenteral carrier. In the case of the injection, it must be sterilized and must be protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof, and/or solvents or dispersion media containing vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, PBS containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, and thimerosal may be further included. Also, in most cases, the injection may further include an isotonic agent such as sugar or sodium chloride.

Transdermal preparations include ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. Here, 'transdermal administration' means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.

For administration by inhalation, the compositions to be used according to the present invention may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or another suitable gas. In the case of pressurized aerosols, dosage units may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in all pharmaceutical chemistry generally known prescriptions, Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour.

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, and the effective dose level may be determined according to the patient's state of health, type of disease, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, combination or simultaneous use of drugs, 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.

In the present invention, 'active ingredient' refers to a component that exhibits the desired activity alone or that can exhibit activity together with a carrier having no activity itself.

In the present invention, "prevention" means any action that suppresses or delays the symptoms of an inflammatory skin disease by administration of the composition of the present invention, and "improvement" in the present invention means that the composition of the present invention is applied to relieve symptoms of inflammatory skin disease, such as erythema, bleeding, scars, dry skin, edema, hematoma, soreness, epidermal thickening, and itching. In the present invention, "treatment" refers to all activities in which symptoms of inflammatory skin disease are improved or beneficially changed by administration of the composition of the present invention.

For example, the pharmaceutical composition of the present invention promotes cell tight coupling by increasing the expression of genes related to cell tight coupling, thereby exhibiting effects of preventing, improving or treating inflammatory skin diseases. Genes associated with cell tight coupling may be ZO-1, occludin, or claudin-1, but are not limited thereto.

For example, the pharmaceutical composition of the present invention can improve, alleviate symptoms related to skin inflammation and exhibit efficacy in preventing, ameliorating or treating inflammatory skin diseases by increasing the expression of the above-described genes related to cell tight coupling.

The hemp extract of the present invention not only strengthens the dense coupling of keratinocytes without toxicity, increases collagen synthesis, but also inhibits the expression of collagenase (MMP-1) at the same time, so it can improve skin and treat inflammatory skin diseases.

1 is a result of evaluating toxicity in normal cells HEK293, HFE145, and HS68 cells according to treatment with cannabis stem extract.
Figure 2 shows the result of confirming the expression levels of dense binding marker proteins (ZO-1, occludin, claudin-1) after treating the cannabis stem extract to keratinocytes (HaCaT cells).
Figure 3 shows the results confirming by MTT assay that treatment of the cannabis stem extract to keratinocytes (HaCaT cells) promotes cell growth in a concentration-dependent manner.
5 shows the characteristics of cannabis stem-derived exosomes extracted (separated) by each of the membrane filter + UC (untra centrifugation method), membrane filter + PEG (polyethylene glycol), and membrane filter + exosome kit methods. Nanoparticle Tracking Analysis (NTA) The result was confirmed.
Figure 6 is a result of confirming the cytotoxicity in skin keratinocytes (HaCaT cells) according to the combined administration of exosomes (extracellular vesicles) and hemp stem extract for each extraction (separation) method.
Figure 7 confirms the cytotoxicity in skin keratinocytes (HaCaT cells) when 0.01 ug/ml exosomes and 25, 50, and 100 ug/ml hemp stem extracts are administered individually or in combination, and dense connective tissue markers (ZO-1, occludin) when administered in combination with stem extracts at each concentration (25, 50, 100 ug/ml) and exosomes at 0.01 ug/ml concentration This is the result of confirming the skin barrier improvement effect on the expression of occludin and claudin-1.
8 is a result of confirming the effect on expression changes of dense connective tissue markers (ZO-1, occludin, and claudin-1) when the cannabis stem extract (25ug/ml) and exosomes (0.001ug/ml) were individually or simultaneously treated on skin keratinocytes.
9 is a result of confirming the cytotoxicity in fibroblasts (HS68 cells) according to individual or combined administration of exosomes (extracellular vesicles) and hemp stem extract for each extraction (separation) method.
10 is a result confirming that cytotoxicity and collagen production are promoted when fibroblasts are individually or simultaneously treated with hemp stem extract and exosomes.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited only to these examples.

Example 1. Preparation of hemp stem extract

The hemp (cheongsam, hemp) stem used in the present invention was provided and used by the Andong Agricultural Cooperative Association located in Andong, Gyeongsangbuk-do, Korea.

After cutting 3.4 kg of hemp stems to 3-4 cm, they were extracted twice for 48 hours at room temperature with 17 L of ethanol. After removing the solvent from the extracted extract using a rotary evaporator, 76 g of cannabis stem extract was prepared.

Example 2. Confirmation of skin improvement effect of hemp stem extract

2.1. Evaluation of normal cytotoxicity of cannabis stem extracts

In order to evaluate the toxicity of the cannabis stem extract to normal cells, HEK293, HFE145, and HS68 cells were seeded in 2x10 ⁴ cells in a 96-well plate, and after 24 hours, the cannabis stem extract was simultaneously treated at concentrations of 25, 50, and 100 μg / ml, and further cultured for 24 hours. Then, after treating the cells with MTT dye (5 mg/ml), additional incubation was performed for 4 hours, and the formazan crystal remaining in the dish was dissolved in DMSO, and the OD value was measured at a wavelength of 550 nm.

As a result, there was no difference in the survival rate of normal cell lines HEK293, HFE145, and HS68 cell lines at all concentrations (25, 50, or 100 μg/ml) treated with hemp stem extract. That is, no cytotoxicity was observed (FIG. 1).

2.2. Confirmation of effect on skin barrier activity

The dense binding of keratinocytes can strengthen the skin barrier to exhibit various effects such as preventing foreign antigen invasion, preventing immune response (itching, etc.), preventing internal moisture loss, and improving skin texture.

Specifically, human-derived keratinocyte lines (HaCaT cell lines) were seeded in 1.2x10 ⁶ cells in a 6-well plate, cultured for 24 hours, and then treated with 10 ng/ml of TNF-α and IFN-γ respectively to inhibit dense binding. Simultaneously, hemp stem extract was treated at concentrations of 25, 50, and 100 μg/ml and further cultured for 24 hours. Thereafter, all the cells were harvested, and the expression levels of ZO-1, occludin, and claudin-1, which are dense binding markers, were confirmed by western blotting. For Western blotting, proteins were extracted from the harvested cells using a passive lysis buffer, followed by electrophoresis on a 4-12% Bis-Tris gel, and transfer to a membrane using a semi-dry transfer system. After blocking with 5% BSA, the membrane was reacted with primary antibodies against ZO-1, occludin, and claudin-1 at 4°C overnight. After reacting the secondary antibody for 1 hour at room temperature, an image was obtained using a LAS4000 mini camera, and the band for each protein was quantified using Image J software.

As a result, when the keratinocyte HaCaT cell line was treated with the cannabis stem extract, it was confirmed that the expression of intercellular tight coupling markers (ZO-1, occludin, and claudin-1), which had been decreased by TNF-α and IFN-γ treatment, increased in a concentration-dependent manner at the protein level (FIG. 2). That is, it was confirmed that the cannabis stem extract prevented damage to dense bonds caused by TNF-α and IFN-γ and rather strengthened dense bonds at high concentrations.

2.3. Confirmation of effect on keratinocyte proliferation

In order to evaluate the growth promoting effect of keratinocytes according to the cannabis stem extract, MTT assay was performed. Specifically, 10 ⁴ human-derived keratinocyte lines (HaCaT) were seeded in a 96-well plate and cultured for 24 hours, and the experimental group was treated with hemp stem extract at concentrations of 25, 50, and 100 ug/ml, and then cultured for 24 hours and 48 hours, respectively, and MTT dye (5 mg/ml) was added. Thereafter, the mixture was further reacted for 4 hours, the medium was removed and dimethyl sulfoxide was added to each well to dissolve the dye for 20 minutes, and the degree of cell growth was analyzed by measuring absorbance at a wavelength of 490 nm.

As a result, it was confirmed that the hemp stem extract promoted the growth of keratinocytes and promoted the growth in a concentration-dependent manner (FIG. 3). That is, it was confirmed that the hemp stem extract exhibited the wound-healing effect of keratinocytes.

2.4. Confirmation of effects on expression of collagen synthesis and degrading enzymes

The human-derived fibroblast cell line (HS68 cells) was seeded in 1.2x10 ⁶ cells in a 6-well plate, and after 24 hours, hemp stem extract was treated with concentrations of 25, 50, and 100 μg/ml and further cultured for 24 hours. Thereafter, all the cells were harvested, RNA and protein were extracted, and expression levels of COL1A1 and MMP1 were analyzed. For mRNA expression, cDNA was synthesized from RNA extracted using M-MLV Reverse Transcriptase, and qRT-PCT was performed using SYBR Premix Ex Taq II to analyze the expression levels of COL1A1 and MMP1 genes. Marker expression was quantified by comparison with GAPDH gene mRNA expression. The protein expression levels of COL1A1 and MMP1 were analyzed by the Western blotting method described above.

As a result, when fibroblast Hs68 cells were treated with hemp stem extract, the expression of mRNA and protein of collagen-1 (COL1A1) was increased, and at the same time, the expression of MMP-1 enzyme that decomposed collagen was decreased in a concentration-dependent manner. The dual function of maximizing the content of skin collagen was confirmed. In particular, it showed the best collagen synthesis efficacy at a concentration of 50 ug/ml (FIG. 4).

Example 3. Confirmation of skin improvement effect of hemp stem extract and exosomes

3.1 Exosome extraction method and characterization

(1) Membrane filter + ultracentrifugation (Membrane filter +UC)

Hemp stem-derived exosomes were extracted using a membrane filter and an ultracentrifuge. Specifically, after washing the cannabis stem, PBS buffer (pH 7.4) was put into a blender at a ratio of 1:2 (w/w) and sufficiently pulverized. Then, the supernatant is collected after centrifugation at 3,000 xg for 20 minutes, and the supernatant is collected after centrifugation at 10,000 xg for 60 minutes. The collected supernatant was filtered with a 0.8 um membrane to collect the filtered solution, and the filtered solution was again filtered with a 0.2 um membrane to collect the filtered solution. After centrifugation of the solution filtered by membrane filtering at 100,000 xg for 120 minutes, the pellet was dissolved in PBS buffer (pH 7.4) and centrifuged once more at 100,000 xg for 120 minutes. The obtained pellet was dissolved in PBS buffer (pH 7.4) and stored frozen at -70 ° C or less until use in experiments.

(2) Membrane filter + PEG

Hemp stem-derived exosomes were extracted using a membrane filter and polyethylene glycol (PEG). Specifically, after washing the cannabis stem, PBS buffer (pH 7.4) was put into a blender at a ratio of 1:2 (w/w) and sufficiently pulverized. Thereafter, the supernatant is collected after centrifugation at 3,000 xg for 20 minutes, and centrifugation is performed again at 10,000 xg for 60 minutes to collect the supernatant. After the collected supernatant was filtered with a 0.8 um membrane, the filtered solution was additionally filtered with a 0.2 um membrane filter to obtain a solution. After membrane filtering, PEG 10% (w / v) was added to the filtered solution, completely dissolved, incubated at 4 ° C for 12 hours, and centrifuged at 10,000 rpm for 60 minutes.

(3) Membrane filter + exosome kit

Hemp stem-derived exosomes were extracted using a membrane filter and an exosome kit. Specifically, after washing the cannabis stem, PBS buffer (pH 7.4) was put into a blender at a ratio of 1:2 (w/w) and sufficiently pulverized. Thereafter, the supernatant collected after centrifugation at 3,000 xg for 20 minutes is centrifuged again at 10,000 xg for 60 minutes to collect the supernatant. The collected supernatant was filtered with a 0.8 um membrane, and the filtered solution was additionally filtered with a 0.2 um membrane filter to collect the filtered solution. This solution was extracted using an exosome kit (Qiagen, miRCURY Exosome Cell/Urine/CSF Kit) and stored frozen at -70°C or less until used in experiments.

(4) Evaluation of characteristics of exosomes

In order to evaluate the properties of the cannabis-derived exosomes extracted by the methods (1) to (3), nanoparticle tracking analysis (NTA) was performed. As a result of checking the number of particles per unit volume of exosomes extracted by each method, it was confirmed that 1x10 ¹⁰ to 1x10 ¹² exosomes exist per unit volume of 1 ml (FIG. 5).

3.2. Confirmation of effect on skin barrier activity of keratinocyte lines

(1) Toxicity evaluation of hemp stem extract and exosomes in keratinocytes

In order to evaluate the toxicity of exosomes (extracellular vesicles) alone or in combination with cannabis stem extract, HaCaT cells were treated with exosomes isolated from cannabis (hemp) stems by the above three methods according to concentration, and 25 ug / ml of cannabis stem extract. Specifically, each cell line was seeded in ⁴ pieces of 1.5x10 per well in a 96-well plate, cultured for 24 hours, then the culture medium was removed, and the exosomes (0.001, 0.01, 0.1, 1, 10ug/ml) or hemp stem extract (25ug/ml) were added to the culture medium for an additional 24 hours, followed by the method described in 2.1. MTT assay was performed by

Additionally, the cytotoxicity of the combined treatment of exosomes and hemp stem extract

It was performed in the same manner as in the above-described method, and the treatment materials and concentrations were i) 25ug/ml of cannabis stem extract alone, ii) alone (0.001ug/ml or 0.01ug/ml) of exosomes isolated from cannabis stems using PEG, iii) treatment of exosomes (0.001ug/ml or 0.01ug/ml) and hemp stem extract (25ug/ml), respectively, mixing, and then incubation at room temperature for 30 minutes. After incubation, each was treated and cultured for 24 hours.

As a result, it was confirmed that there was no significant difference in the toxicity of the exosomes depending on the extraction method, and even when the hemp stem extract and the exosomes were treated in combination, they did not show cytotoxicity, but rather showed the efficacy of slightly promoting cell growth (FIG. 6).

(2) Check the skin barrier strengthening effect

In order to evaluate the skin barrier strengthening effect of exosomes alone or co-treated with hemp stem extract in human-derived keratinocytes (HaCaT cells), 1.2x10 per well in 6 wells.⁶After seeding and culturing for 24 hours, i) 25, 50, 100ug/ml of hemp stem extract by concentration alone, ii) alone (0.01ug/ml) of exosome isolated from hemp stem using PEG, iii) mixing of exosomes (0.01ug/ml) and hemp stem extract (25, 50, 100ug/ml) by concentration, followed by mixing at room temperature for 30 minutes. After incubation for 24 hours, each was treated for 24 hours, and the expression level of epithelial cell dense connective tissue markers (ZO-1, occludin, and claudin-1) was analyzed by Western blotting and real-time qPCR methods.

As a result, the simultaneous treatment of cannabis stem extract (25 ug/ml) and exosomes (0.01 ug/ml) among several combinations significantly increased the expression of ZO-1 and occludin, and did not affect the expression of claudin-1, so it was confirmed that there was a combined effect in strengthening the skin barrier without side effects at the corresponding concentration (FIG. 7). That is, it was confirmed that the combined administration of hemp stem extract and exosomes had a synergistic effect on strengthening the skin barrier.

Based on these results, the cannabis stem extract was fixed at a concentration of 25ug/ml, and it was confirmed whether there was a combined effect in strengthening the skin barrier even at a lower exosome concentration (0.001ug/ml). Similar to the concentration of 0.01ug/ml exosomes, it was confirmed that when the cannabis stem extract and the exosomes were simultaneously treated, a synergistic effect was observed in strengthening the skin barrier compared to the treatment of each alone (FIG. 8).

3.3. Confirmation of effects on collagen synthesis and expression of degrading enzymes in fibroblast cell lines

(1) Toxicity evaluation of hemp stem extract and exosomes in fibroblasts

In order to evaluate the toxicity of exosomes (extracellular vesicles) alone or in combination with cannabis stem extract, HaCaT cells were treated with exosomes and/or cannabis stem extracts isolated from hemp (hemp) stems by the above three methods at each concentration and the survival rate was confirmed. Specifically, each cell line was seeded in ⁴ wells of 1.5x10 per well in a 96-well plate, cultured for 24 hours, then the culture medium was removed, and exosomes (0.1, 1, 10, 100ug/ml) or hemp stem extract (25ug/ml) were added thereto, followed by further culturing for 24 hours, and the MTT assay was performed by the method described in 2.1. .

In addition, the cytotoxicity of low-concentration exosomes and hemp stem extracts individually or in combination was performed in the same manner as described above, and the treatment materials and concentrations were i) hemp stem extract 25ug/ml alone, ii) exosome isolated from hemp stem using a kit (0.001ug/ml or 0.01ug/ml), iii) exosomes (0.001ug/ml or 0.01ug/ml) and cannabis stem extract (25ug/ml) were respectively treated, mixed, and incubated at room temperature for 30 minutes, and then each was treated and cultured for 24 hours.

As a result, in fibroblasts (HS68 cells), exosomes exhibited cytotoxicity regardless of the extraction method at a concentration of 0.1 ug/ml or more, but in the case of exosomes extracted through the PEG method, cytotoxicity was not observed at a concentration of 0.01 ug/ml or less (FIG. 9). However, it was confirmed that the simultaneous treatment of hemp stem extract and exosomes rather improved the survival of fibroblasts, unlike the case of treatment with exosomes alone (FIG. 9).

(2) Check the effect of promoting collagen synthesis

In order to confirm the effect of promoting collagen production according to the combined administration of exosomes and hemp stem extract, 1.2x10 ⁶ fibroblast cell lines (HS68 cells) were seeded in a 6-well plate and cultured for 24 hours. Then, i) 25 or 50ug/ml of cannabis stem extract alone, ii) exosome alone (0.01ug/ml), iii) exosome (0.01ug/ml) and hemp stem extract (25 or 50ug/ml) were mixed and incubated at room temperature for 30 minutes, and then each was treated and cultured for 24 hours. Thereafter, the degree of cell viability was analyzed by performing the MTT assay as described above, and the expression levels of collagen degrading enzyme (MMP-1) and collagen protein gene (COL1A1) were confirmed by Western blotting.

As a result, all treatment conditions did not show toxicity in Hs68 fibroblasts. In addition, the expression level of collagen protein increased when the cannabis stem extract and exosomes were treated in combination with the cannabis stem extract, compared to the case where the cannabis stem extract and exosome were treated alone. Based on these results, the hemp stem extract was fixed at a concentration of 25ug/ml, and it was confirmed whether there was a combined effect on strengthening the skin barrier even at a low exosome concentration (0.001ug/ml). As a result, it was confirmed that the expression of collagen protein (COL1A1) was increased and the expression level of MMP-1 was further decreased when the cannabis stem extract and exosomes were treated in combination, compared to the case of treatment with the cannabis stem extract or exosome alone, and the exosome concentrations of 0.01 and 0.001ug/ml were both confirmed to synergistically increase the expression of collagen protein when administered in combination with the cannabis stem extract (25ug/ml) (FIG. 10).

Claims (17)

A cosmetic composition for skin improvement comprising hemp stem extract as an active ingredient, wherein the hemp stem extract is contained in a concentration of 10 to 200 μg/ml, a cosmetic composition.
delete According to claim 1,
The skin improvement is characterized by strengthening the skin barrier, relieving itching, maintaining skin moisture, increasing collagen synthesis or inhibiting collagen degradation, a cosmetic composition.
According to claim 1,
The composition is a cosmetic composition characterized in that it increases the expression of the dense binding markers ZO-1, occludin, and claudin-1.
According to claim 1,
A cosmetic composition further comprising hemp stem-derived exosomes.
According to claim 5,
The cosmetic composition is in the form of a mixture in which cannabis stem extract and hemp stem-derived exosomes are mixed, or cannabis stem extract and hemp stem-derived exosomes are formulated and treated simultaneously or sequentially.
According to claim 5,
Characterized in that the hemp stem-derived exosomes are contained in 0.0001 to 10 ug / ml, cosmetic composition.
According to claim 5,
The hemp stem-derived exosomes are contained in a weight ratio of 10 ^-4 to 5x10 ^-3 relative to the weight ratio of the hemp stem extract, cosmetic composition.
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