Composition for skin improvement comprising culture of coast apium graveolens

Abstract

The present invention relates to a composition for improving skin, comprising a culture, a culture filtrate, an extract, or an exosome derived therefrom, which comprises a callus tissue of coast apium graveolens, in particular, a callus tissue of coast apium graveolens. The coastal eryngii callus culture filtrate, the coastal eryngii callus extract and the coastal eryngii callus culture solution source exosome have the effects of wrinkle improvement, moisturizing and skin barrier improvement, aging prevention, skin immunity and defense enhancement, autophagy activation and inflammation inhibition, and have the effects of resisting ultraviolet rays, resisting inflammation, resisting inflammation, resisting inflammation, resisting inflammation, resisting inflammation and the like. The composition has a skin-protecting effect against blue light, oxidative toxicity and stress, a wound-healing effect, a cell-energy-increasing effect, a pore-shrinking effect, a skin-calming effect and an acne-skin-improving effect, and can be effectively used for improving various skin problems.

Description

Technical Field

[0001] The present invention relates to a skin-improving composition comprising Eryngium maritima, in particular comprising a culture of its callus, a culture filtrate, an extract or exosomes derived therefrom.

[0002] National research and development undertakings supporting this invention:

[0003] Project ID: 1425180205

[0004] Project number: RS-2023-00303008

[0005] Department Name: Ministry of SMEs and Startups

[0006] Project management (specialized) organization name: Korea Small and Medium Enterprise Technology and Information Promotion Agency

[0007] Research project name: Small and medium-sized enterprise technology innovation and development (R&D) / Small and medium-sized enterprise technology innovation and development project (market response type - foundation)

[0008] Research topic: Development of functional skin materials using exosomes derived from plant cells

[0009] Contribution rate: 1 / 1

[0010] Name of the project implementing organization: Bio FD&C Co., Ltd. Background Art

[0011] The human skin is the largest organ in the human body and is usually divided into the epidermis, dermis and subcutaneous fat layer. The epidermis, the outermost layer of the skin, is composed of stratified squamous epithelium and serves as a protective barrier for the body against the external environment. From the outside to the inside, the epidermis is divided into the stratum corneum, the stratum lucidum, the stratum granulosum, the stratum spinosum and the stratum basale. The dermis is a layer between the epidermis and the subcutaneous tissue, divided into the papillary dermis and the reticular dermis. It contains blood vessels, collagen, elastin fibers, pores, arrector pili muscles, sebaceous glands, sweat glands, various sensory nerves, fibroblasts and macrophages that are not found in the epidermis, and occupies the largest part of the skin. The skin is always directly exposed to the external environment. As the environment changes, it will be exposed to various stimuli, and various skin diseases will appear due to the stress or defense mechanism caused by it. As the outermost organ of the human body, the skin plays the role of the first layer of biological protective membrane, preventing the loss of water in the human body. In addition, it also blocks the penetration of various harmful substances or pollutants that may flow in from the outside, protects the human body from ultraviolet rays, reduces heat loss by shrinking in a cold environment, and dissipates body heat by sweating in a hot environment, thus playing an important role in maintaining the homeostasis of the human body.

[0012] The causes of skin aging are mainly due to factors such as age increase, ultraviolet rays, dust, and stress. Skin aging is divided into intrinsic aging (intrinsic aging) which is natural aging with time dependence and extrinsic aging (extrinsic aging) through external factors. The representative of extrinsic aging is photoaging (phtoaging) through ultraviolet rays. As the cause of intrinsic aging, the increase in age reduces the role of fibroblasts and the number of cells. As a result, the reduction in the amount of fiber component synthesis and the looseness of the structure lead to reduced elasticity. In addition, if the water in the skin cells disappears, it causes changes in the structure of the stratum corneum and becomes the cause of aging. If the skin aging phenomenon proceeds, there will be a decrease in the division of keratinocytes and fibroblasts, a decrease in collagen synthesis, an increase in the generation of matrix metalloproteinases (MMPs), an increase in the signal transduction of melanin generation, etc., which causes an increase in wrinkles and a decrease in skin elasticity, an increase in dark spots, freckles and age spots, etc. In particular, around the age of 40, as the balance of sex hormones is broken, the cell regeneration ability, the synthesis ability of collagen, and the water content of the stratum corneum, which is important in skin moisturizing, are significantly reduced. This is due to the reduction of the components of the stratum corneum base, the reduction of the natural moisturizing factor, the reduction of the amount of glycerol, etc. In the end, in aging skin, the amount of water supplied from the lower part of the epidermis to the stratum corneum is insufficient, which makes the skin dry and aggravated, so it is easy to develop inflammation and pruritus. On the contrary, the wrinkles of the skin are caused by the imbalance of the synthesis and decomposition of collagen. Usually, the synthesis of type I collagen and the matrix metalloproteinase-1 (MMP-1) as its decomposition enzyme form a balance in the skin. However, in photoaged skin, the synthesis of type I and type III collagen decreases, and the activity of matrix metalloproteinase-1 increases. The above-mentioned matrix metalloproteinases (MMPs, matrix metalloproteinases) are protein decomposition enzymes that show the function of decomposing the extracellular matrix (extracellular matrix). It is known that under normal conditions, it is related to wound healing or tissue regeneration processes. Autophagy is a mechanism that generates energy and removes damaged substances by decomposing aged or damaged intracellular substances and organs when the intracellular energy source is exhausted or the intracellular stress factors are excessive (Hearing & Jimenez, Int J Biochem, 1987, 19 (12), 1141-7). Recently, various research reports have shown that with the progress or continuation of aging, the intracellular autophagic activity decreases sharply. When autophagy is inhibited, the cells accumulate excessively old mitochondria or misfolded proteins, etc., increasing intracellular free radicals and oxidative stress, and ultimately generating gene fragments caused by cell apoptosis. The above fragments stimulate melanocytes to induce the maturation of melanosomes and their delivery to keratinocytes.

[0013] With the above changes, the interest and social demand for materials that can improve damaged skin have also increased, and the development of materials that are biocompatible and have excellent skin permeability has emerged as an important issue. Recently, various methods have been applied, among which various plant resources have been used to study the restoration of the above-mentioned skin damage. Summary of the invention

[0014] Technical issues

[0015] An object of the present invention is to provide a skin-improving cosmetic composition.

[0016] Another object of the present invention is to provide a skin protecting cosmetic composition.

[0017] Another object of the present invention is to provide an antioxidant cosmetic composition.

[0018] Another object of the present invention is to provide a cosmetic composition for skin regeneration.

[0019] Furthermore, the present invention aims to provide a pharmaceutical composition for skin wound healing or anti-inflammation.

[0020] Another object of the present invention is to provide a food composition for improving skin.

[0021] At the same time, the object of the present invention is to provide a method for preparing exosomes derived from Eryngium littoralis callus.

[0022] Technical Solution

[0023] In order to solve the above problems, the present invention provides a skin improvement cosmetic composition comprising Eryngium maritima Cultures, extracts, and cultures of tissues, cells, or callus of Eryngium Maritimum The active ingredient is a filtrate of a culture, an extract of a culture, exosomes derived from tissue, cells or callus, exosomes derived from a culture or exosomes derived from a culture filtrate.

[0024] Furthermore, the present invention provides a cosmetic composition for protecting the skin against light, oxidative toxicity or stress, comprising a culture of tissue, cell or callus, an extract, a filtrate of the culture, an extract of the culture, exosomes derived from the tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of Eryngium maritima as an active ingredient.

[0025] Furthermore, the present invention provides an antioxidant cosmetic composition comprising a culture of tissue, cell or callus, an extract, a filtrate of a culture, an extract of a culture, an exosome derived from a tissue, cell or callus, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

[0026] Furthermore, the present invention provides a cosmetic composition for skin regeneration, comprising a culture of tissue, cell or callus, an extract, a filtrate of a culture, an extract of a culture, an exosome derived from a tissue, cell or callus, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

[0027] Furthermore, the present invention provides a pharmaceutical composition for skin wound healing or anti-inflammation, comprising a culture, extract, culture filtrate, culture extract, exosomes derived from tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of Eryngium maritima as an active ingredient.

[0028] Furthermore, the present invention provides a food composition for improving skin, comprising a culture of tissue, cell or callus, an extract, a filtrate of a culture, an extract of a culture, an exosome derived from a tissue, cell or callus, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

[0029] At the same time, the present invention provides a method for preparing exosomes derived from Eryngium littoralis callus.

[0030] Effects of the Invention

[0031] The Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract and Eryngium littoralis callus culture fluid-derived exosomes of the present invention have wrinkle improvement effects, moisturizing and skin barrier improvement effects, anti-aging effects, skin immunity and defense enhancement effects, autophagy activation effects, inflammation inhibition effects, skin protection effects against ultraviolet rays, blue light, oxidative toxicity and stress, wound healing effects, cell energy increase effects, pore contraction effects, skin calming effects and acne skin improvement effects, and can be effectively used to improve various skin problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure shows the process of preparing callus culture solution of Eryngium maritima.

[0033] Figure 2 This is a graph showing the results of analyzing the particle size of exosomes derived from Eryngium maritima callus culture filtrate.

[0034] Figure 3 This is a graph showing the results of analyzing the particle size of exosomes derived from Eryngium maritima callus.

[0035] Figure 4 This is a graph showing the results of analyzing the particle size of exosomes derived from the callus culture fluid of Eryngium maritima. DETAILED DESCRIPTION

[0036] Hereinafter, the present invention will be described in detail by referring to the accompanying drawings through examples of the present invention. However, the following examples are only used to illustrate the present invention, and the present invention is not limited thereto. If it is judged that the specific description of the technology or structure that is clear to the ordinary technicians in the technical field to which the present invention belongs may unnecessarily confuse the gist of the present invention, its detailed description may be omitted. The present invention can be modified and applied in various ways within the scope of the attached invention claims and the equivalent scope interpreted therefrom.

[0037] Throughout this specification, unless otherwise specified, "%" used to express the concentration of a specific substance is (w / w)% for solid / solid, (w / v)% for solid / liquid, and (v / v)% for liquid / liquid.

[0038] In one embodiment, the present invention relates to a skin-improving cosmetic composition comprising a culture of tissue, cell or callus, an extract, a filtrate of a culture, an extract of a culture, an exosome derived from a tissue, cell or callus, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

[0039] In one example, the composition of the present invention may include an extract of callus, a culture, a culture filtrate, an extract of a culture, or a culture-derived exosome of Eryngium maritima.

[0040] In one example, the composition of the present invention may include an extract of callus tissue, culture, culture filtrate or culture-derived exosomes of Eryngium maritima.

[0041] In one example, the extract can be extracted using one or more solvents selected from the group consisting of water, organic solvents, subcritical fluids and supercritical fluids. The organic solvent can be any one selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, hexane (n-hexane), glycerol, propylene glycol, butylene glycol, ethyl acetate, methyl acetate, dichloromethane, chloroform, ethyl acetate, acetone, dichloromethane, cyclohexane, petroleum ether, benzene and mixed solvents thereof, with hot water extract being the most preferred.

[0042] In one example, skin improvement can be one or more selected from the group consisting of skin wrinkle improvement, skin moisturizing or skin barrier improvement, skin aging prevention, pigmentation inhibition, skin cell energy increase, skin immunity or defense enhancement, inflammation inhibition, pore shrinkage, sebum inhibition or acne improvement, or skin calming.

[0043] In one example, the composition of the present invention can increase the expression of aquaporin 3 (AQP3), filaggrin (FLG), telomerase reverse transcriptase (TERT), cathelicidin-related antimicrobial peptides (LL37, CAMP), human β-defensin-2 (hBD-2), microtubule-associated protein 1A / 1B-light chain 3 (LC-3), or late embryogenesis abundant protein (LEA).

[0044] In one embodiment, the composition of the present invention can reduce the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS).

[0045] In one example, the composition of the present invention can improve wrinkles by increasing the biosynthesis of type I procollagen C-terminal peptide (PIP).

[0046] In one example, the composition of the present invention can improve skin moisturizing and skin barrier by increasing the expression of aquaporin 3 or filaggrin.

[0047] In one example, the composition of the present invention can prevent aging by increasing the expression of telomerase reverse transcriptase.

[0048] In one example, the composition of the present invention can activate autophagy by increasing the expression of microtubule-associated protein 1A / 1B-light chain 3, thereby inhibiting aging such as pigmentation.

[0049] In one example, the composition of the present invention can enhance skin immunity and defense by increasing the expression of cathelicidin-related antimicrobial peptides or human β-defensin-2.

[0050] In one example, the composition of the present invention can inhibit inflammation by reducing the expression of cyclooxygenase-2 or inducible nitric oxide synthase.

[0051] In one example, the composition of the present invention can shrink pores by inhibiting sebum production, thereby improving acne skin.

[0052] In one example, the compositions of the present invention can calm the skin when skin damage is caused by direct sunlight.

[0053] In one embodiment, the present invention relates to a cosmetic composition for protecting the skin against light, oxidative toxicity or stress, comprising a culture, an extract, a filtrate of a culture, an extract of a culture, an exosome derived from a tissue, a cell or a callus of Eryngium maritima, a culture-derived exosome or a culture filtrate-derived exosome as an active ingredient.

[0054] In one example, the light can be ultraviolet light or blue light.

[0055] In one example, the compositions of the present invention can protect the skin from stress by increasing the expression of late embryogenesis abundant proteins.

[0056] In one embodiment, the present invention relates to an antioxidant cosmetic composition comprising a culture of tissue, cell or callus, an extract, a filtrate of a culture, an extract of a culture, exosomes derived from tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of Eryngium maritima as an active ingredient.

[0057] In one embodiment, the present invention relates to a cosmetic composition for skin regeneration, comprising a culture of tissue, cell or callus, an extract, a filtrate of a culture, an extract of a culture, an exosome derived from a tissue, cell or callus, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

[0058] The term "plant cell" used in the present invention may be any plant cell. The plant cell may be a cultured cell, a cultured tissue, a cultured organ or a whole plant, preferably, a cultured cell, a cultured tissue or a cultured organ, more preferably, any form of a cultured cell.

[0059] The term "plant tissue" used in the present invention refers to differentiated or undifferentiated plant tissue, for example, plant leaves, petioles, stems, flowers, buds, roots, seeds or placenta, pollen, cancerous tissues and various forms of cells used in culture, but is not limited thereto, that is, including single cells, protoplasts and callus. Plant tissue can be in situ or in organ culture, tissue culture or cell culture.

[0060] The term "extract" used in the present invention refers to an active ingredient separated from a natural object, that is, a substance showing target activity. The above-mentioned extract can be obtained by a process of extraction using water, an organic solvent or a mixed solvent thereof, including a dry powder (powder) of the extract or all forms thereof. In addition, the above-mentioned extract also includes fractions of the extract fractionated through the above-mentioned extraction process. The extraction method of the extract is not particularly limited, for example, it can be extracted by methods such as stirring extraction, oscillation extraction, hot water extraction, cold soaking extraction, reflux cooling extraction or ultrasonic extraction. The extraction solvent can use a dosage form solvent such as water, a C1-C4 lower alcohol, or a non-polar solvent such as hexane, chloroform, dichloromethane or ethyl acetate, or a mixture of two or more thereof.

[0061] The term "expression" used in the present invention generally refers to a cellular process in which a biologically active peptide is generated from a deoxyribonucleic acid (DNA) sequence and exhibits biological activity in a cell. In this sense, expression includes not only transcription and interpretation processes, but also post-transcriptional and post-interpretation processes that can affect the biological activity of a gene or gene product. The above process includes not only ribonucleic acid (RNA) synthesis, processing and transport, but also peptide synthesis, transport and post-interpretation deformation of polypeptides, but is not limited thereto.

[0062] In the present invention, the above expression can be confirmed by measuring the expression level of gene or messenger RNA (mRNA) by using nucleic acid sequence, nucleic acid sequence complementary to the above nucleic acid sequence, primer pair, probe or primer pair and probe that specifically recognize the above nucleic acid sequence and the fragment of complementary sequence through polymerase chain reaction, real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), reverse transcription polymerase chain reaction, competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), nuclease protection analysis (RNase, S1nuclease assay), in situ hybridization, nucleic acid microarray, Noser hybridization or DNA chip method. It can be confirmed by using antibodies, antibody fragments, nucleic acid aptamers, affinity multimers or peptidomimetics that specifically recognize the full length or fragments of the relevant protein through Western blotting, enzyme linked immunosorbent assay (ELISA, enzyme linked immunosorbent assay) The expression level of the protein can be determined by measuring the expression level of the protein using methods such as radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorting (FACS), mass spectrometry or protein microarray.

[0063] The cosmetic composition of the present invention can be prepared by comprising a cosmetically effective amount of the above-mentioned active ingredient of the present invention and a cosmetically acceptable carrier.

[0064] The term "cosmetically effective amount" used in the present invention refers to a sufficient amount for achieving the whitening, wrinkle improvement or moisturizing effects of the composition of the present invention.

[0065] The cosmetic composition has a cosmetic or dermatologically acceptable medium or base. It can be provided in all dosage forms suitable for topical application, for example, solutions, gels, solids, anhydrous pastes, oil-in-water emulsions, suspensions, microemulsions, microparticles, or ionic (liposomes) and nonionic vesicle dispersions, or in the form of creams, lotions, emollients, powders, ointments, sprays, or concealer sticks. These compositions can be prepared according to the usual methods of the technical field of the present invention. The composition of the present invention can also be used in the form of foam or an aerosol composition that also contains a compressed filler.

[0066] The dosage form of the above-mentioned cosmetic composition of one embodiment of the present invention is not particularly limited. For example, it can be formulated into cosmetics such as skin softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, facial mask, makeup powder, body lotion, body cream and body essence.

[0067] When the dosage form of the cosmetic composition of the present invention is a patch, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicon, bentonite, silicon dioxide, talc or zinc oxide can be used as a carrier component.

[0068] When the dosage form of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component. Especially in the case of a spray, fillers such as chlorofluorocarbons, propane / butane or dimethyl ether can also be included.

[0069] When the dosage form of the cosmetic composition of the present invention is a solution or an emulsion, a solvent, a solvating agent or an emulsifier can be used as a carrier component, for example, water, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.

[0070] When the dosage form of the cosmetic composition of the present invention is a suspension, water, a liquid diluent such as ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and suspending agents such as polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite or tragacanth gum can be used as a carrier component.

[0071] When the dosage form of the cosmetic composition of the present invention is a lotion containing a surfactant, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazoline derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic acid derivatives or ethoxylated glyceryl fatty acid esters can be used as carrier components.

[0072] The cosmetic composition of the present invention can be used in cosmetics such as skin care water, emulsion, face cream, essence, facial mask, foundation, makeup, sunscreen, dual-purpose powder, face powder, pressed powder, base makeup, concealer, eye shadow, lipstick, lip gloss, lip repair balm, eyebrow pencil, lotion, and washing products such as shampoo and soap.

[0073] In addition to the above-mentioned active ingredients, the cosmetic composition of one embodiment of the present invention may also include functional additives and ingredients contained in common cosmetic compositions. The functional additives may include ingredients selected from the group consisting of water-soluble microorganisms, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids and seaweed extracts.

[0074] In addition to the above functional additives, the cosmetic composition of the present invention may also be formulated with ingredients contained in ordinary cosmetic compositions as needed. Other ingredients included may be oil components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH regulators, alcohol, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, pure water, etc.

[0075] In one embodiment, the present invention relates to a pharmaceutical composition for skin wound healing or anti-inflammation, comprising a culture, extract, culture filtrate, culture extract, exosomes derived from tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of Eryngium maritima as an active ingredient.

[0076] In one example, the wound can be an unhealed traumatic wound, tissue destruction caused by radiation exposure, an abrasion, bone gangrene, a burn, an avulsion, a penetrating wound, a gunshot wound, a cut, frostbite, a contusion or bruise, a skin ulcer, dry skin, skin keratosis, cracking, bursting, dermatitis, pain caused by dermatophytosis, surgical wounds, vascular disease wounds, corneal wounds and other wounds, bedsores, pits, diabetic skin erosions and other conditions related to diabetes and poor circulation, chronic ulcers, suture sites after cosmetic surgery, spinal injury wounds, gynecological wounds, chemical wounds or acne.

[0077] In one embodiment, the composition can be applied topically to the skin.

[0078] In one embodiment, the composition may contain a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable carrier, and the carrier may be selected from the group consisting of gauze, a bandage, a tape, a film, a contact patch, and a non-contact patch.

[0079] The term "treatment" used in the present invention refers to all actions to improve or change the wound or the inflammation symptoms associated therewith to a cure by administering the pharmaceutical composition of the present invention. A person skilled in the art of the present invention can refer to the materials published by the Korean Medical Association and the like to know the exact criteria of the diseases for which the composition of the present application is effective and to judge the degree of improvement, enhancement and treatment.

[0080] The therapeutically effective amount of the composition of the present invention can vary according to various factors, such as the method of administration, the target site, the patient's condition, etc. Therefore, when used in humans, the dosage should be determined by considering both safety and efficiency. The amount used in humans can also be estimated based on the effective amount determined by animal experiments. Matters to be considered when determining the effective amount are recorded in, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EWMartin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0081] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" used in the present invention refers to a sufficient and side-effect-free amount for treating a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined based on factors including the patient's health status, the type of disease, the severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the metabolic ratio, the treatment period, the drugs used in conjunction or simultaneously, and other factors well known in the medical field. The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered single or multiple times. The key point is that after considering all the above factors, the minimum amount without side effects is administered, which can be easily determined by ordinary technicians.

[0082] The term "administration" used in the present invention refers to providing a prescribed substance to an individual or patient in any appropriate manner, and can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally or locally in an injection form) or orally according to the target method, and the range of the dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, metabolic rate and severity of the disease. The liquid preparations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin commonly used as simple diluents, a variety of excipients can also be included together, for example, wetting agents, sweeteners, aromatics, preservatives, etc. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, suppositories, etc. The pharmaceutical composition of the present invention can also be administered by any device in which the active substance can move to the target cell. Preferred administration methods and preparations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. The injection can be prepared using aqueous solvents such as physiological saline and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerol, etc.) and other non-aqueous solvents, and may contain stabilizers for preventing deterioration (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, butylated hydroxyanisole (BHA), tocopherol, ethylenediaminetetraacetic acid (EDTA)), emulsifiers, buffers for adjusting pH, preservatives for preventing the development of microorganisms (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) and other pharmaceutical carriers.

[0083] The term "subject" used in the present invention refers to all animals including humans, such as monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs, who suffer from the above-mentioned muscle diseases or anticancer drug side effects or may develop the diseases. The above-mentioned diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the individual. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

[0084] The pharmaceutical composition of the present invention may contain a carrier, a diluent, an excipient or a combination of two or more of the carriers commonly used in biological preparations. The term "pharmaceutically acceptable" used in the present invention refers to a property of being non-toxic to normal cells or humans exposed to the above-mentioned composition. The above-mentioned carrier is not particularly limited as long as it can transport the composition into the body. For example, the compounds recorded in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered physiological saline, glucose solution, maltodextrin solution, glycerol, ethanol and a mixture of one or more of the above components can be used, and antioxidants, buffers, antibacterial agents and other common additives can also be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants can also be added to prepare injection dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, the drug can be preferably formulated according to each disease or component using an appropriate method in the technical field to which the present invention pertains or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0085] In addition, the composition of the present invention may include a carrier, a diluent, an excipient or a combination of two or more of the carriers commonly used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they can transport the composition into the body. For example, compounds recorded in Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered physiological saline, glucose solution, maltodextrin solution, glycerol, ethanol and a mixture of one or more of the above components can be used, and antioxidants, buffers, antibacterial agents and other common additives can also be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants can also be added to prepare injection dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, the appropriate method in the technical field to which the present invention belongs or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) can be preferably prepared according to each disease or component.

[0086] The composition of the present invention may also include one or more active ingredients showing the same or similar functions. Relative to the total weight of the composition, the composition of the present invention includes 0.0001 weight percent to 30 weight percent of the above active ingredients, preferably, 0.001 weight percent to 3 weight percent.

[0087] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, in which case, the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium phosphate, lactose, mannitol, starch syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, palm wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, glucose, sorbitol, and talc, etc. Relative to the above composition, preferably, the pharmaceutically acceptable additive of the present invention is included in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0088] In one embodiment, the present invention relates to an external preparation for wound healing or skin tissue regeneration, comprising a culture, an extract, a filtrate of a culture, an extract of a culture, an exosome derived from a tissue, a cell or a callus, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

[0089] In one embodiment, the present invention relates to a cosmetic composition for wound improvement or skin tissue regeneration, comprising a culture, extract, culture filtrate, culture extract, exosomes derived from tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of Eryngium maritima as an active ingredient.

[0090] In one embodiment, the present invention relates to a food composition for improving skin, comprising a culture of tissue, cell or callus, an extract, a filtrate of a culture, an extract of a culture, exosomes derived from tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of Eryngium maritima as an active ingredient.

[0091] In one example, skin improvement can be one or more selected from the group consisting of skin wrinkle improvement, skin moisturizing or skin barrier improvement, skin aging prevention, pigmentation inhibition, skin cell energy increase, skin immunity or defense enhancement, inflammation inhibition, pore shrinkage, sebum inhibition or acne improvement, or skin calming.

[0092] When the composition of the present invention is used as a food composition, the composition can be directly added, or used together with other foods or food ingredients, and can be appropriately used according to a common method. In addition to the active ingredient, the composition can also include an acceptable food auxiliary additive in food science, and the mixing amount of the active ingredient can be determined according to the purpose used (for example, health care or therapeutic treatment).

[0093] The term "food supplementary additive" used in the present invention refers to a structural element that can be supplementally added to food. A person skilled in the art of the present invention can appropriately select and use the food supplementary additive when preparing various dosage forms of health functional foods. Examples of food supplementary additives include various nutrients, microorganisms, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplementary additives of the present invention are not limited to the above examples.

[0094] The food composition of the present invention may include health functional food. The term "health functional food" used in the present invention refers to food prepared and processed into tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients with useful functionality for the human body. Among them, "functionality" refers to the use of the structure and function of the human body to adjust nutrients or physiological effects and other health uses to obtain useful effects. The health functional food of the present invention can be prepared by the methods commonly used in the general technical field. During the above preparation, it can be prepared by adding raw materials and ingredients commonly added in the general technical field. In addition, the dosage form of the above health functional food can be prepared without restriction as long as it is recognized as a dosage form of health functional food. The food composition of the present invention can be prepared in various forms of dosage forms. Unlike conventional medicines, it uses food as a raw material, so it has the advantages of not having side effects that may occur due to long-term use of medicines, and outstanding portability. The health functional food of the present invention can be taken as an adjuvant for promoting the effect of anticancer agents.

[0095] In addition, the types of health food that can use the composition of the present invention are not limited. At the same time, the composition containing the tissue, cell or callus culture, extract, filtrate of the culture, extract of the culture, exosomes derived from tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of the present invention as an effective ingredient can be prepared by selecting and mixing appropriate other auxiliary ingredients and known additives that can be contained in health functional foods according to the ordinary technicians of the technical field to which the present invention belongs. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, biscuits, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, drinking water, tea, drinks, alcoholic beverages and vitamin complexes, etc., which can be prepared by adding the tissue, cell or callus culture, extract, filtrate of the culture, extract of the culture, exosomes derived from tissue, cell or callus, culture-derived exosomes or culture filtrate-derived exosomes of the present invention as the main ingredients to the prepared juice, tea, jelly and juice.

[0096] In one embodiment, the present invention relates to a method for preparing exosomes derived from Eryngium maritima callus, comprising: a step of inducing and culturing callus from Eryngium maritima tissue; a step of filtering the culture of the callus; a step of concentrating; and a step of isolating and purifying extracellular vesicles.

[0097] In one embodiment, the present invention relates to a method for preparing a callus culture filtrate of Eryngium maritima, comprising: the steps of inducing and culturing callus from tissue of Eryngium maritima; and the step of filtering the callus culture.

[0098] In one embodiment, the present invention relates to a method for preparing an extract of Eryngium maritima callus, comprising: the steps of inducing and culturing callus from tissue of Eryngium maritima; the step of isolating callus from the culture of callus; and the step of extracting the callus of Eryngium maritima using a solvent.

[0099] The present invention is described in more detail by the following examples, but the following examples are only used to embody the content of the present invention, and the present invention is not limited thereto.

[0100] Example 1. Inducing callus of Eryngium littoralis and preparing its culture medium

[0101] Seeds of Eryngium maritimum Callus (purchased from the UK) were soaked in 70% ethanol for 30 seconds and then washed with sterile water. Disinfection was performed with a disinfectant (30% sodium hypochlorite + Tween 20) for 20 minutes. After washing with sterile water three times to germinate the seeds, a sharp blade was used to scratch the leaves of the germinated Eryngium maritimum plant once, and the seeds were cultured in a basic MS (Murashige and Skoog) medium (Duchefa, Cat No. M0221) containing 0.2 mg / L Zeatin (Duchefa, Cat No. Z0917, Cas No. 1637-39-4), 3% sucrose and 8 g / L agar under growth conditions of 25°C and 70% humidity to induce initial Eryngium maritimum callus (EMC). In this case, the medium was adjusted to pH 5.8 using 1N sodium hydroxide (NaOH). Subculture was performed in a solid state for 2 weeks, and liquid mass culture was performed in a 100L bioreactor (Samsung Science Co., Ltd.) at a temperature of 23±2°C and a humidity of 70% in a culture room with an air supply of about 0.1 vvm for 5 weeks to prepare a culture fluid of Eryngium maritima. Figure 1 ).

[0102] Example 2. Preparation of Eryngium maritima callus culture filtrate

[0103] The culture solution prepared in Example 1 was filtered once using a 100 μm filter and a 25 μm filter to prepare a culture filtrate.

[0104] Example 3. Preparation of Eryngium maritima callus extract

[0105] The Eryngium littoralis callus was obtained from the Eryngium littoralis callus culture solution prepared in Example 1 and washed three times with water, then the water was fully removed and dried at 60° C. for 2 hours using a dryer. 50 g of the dried Eryngium littoralis callus powder was added with 25 L of pure water and hot water extracted at 90 to 120° C. for 48 hours to prepare an Eryngium littoralis callus extract.

[0106] Example 4. Preparation of exosomes from Eryngium littoralis callus

[0107] 4-1. Preparation of callus-derived exosomes

[0108] The culture solution of Eryngium littoralis callus, the culture filtrate and the callus were filtered using 50 micron and 25 micron mesh filter bags respectively, and then centrifuged at 3000g for 20 minutes, and only the supernatant was taken to remove the cell residues. The supernatant obtained by filtration was removed by 0.45μm and 0.22μm, respectively, to remove impurities such as cell residues, waste and giant particles. The filtered culture solution of Eryngium littoralis callus was concentrated by ultrafiltration with MWCO 100000Da until the volume was 1 / 20 to 1 / 100. Extracellular vesicles including exosomes were separated and purified from the concentrated solution by size exclusion chromatography. Extracellular vesicles including high-purity exosomes were selectively separated and purified according to size, and exosomes derived from Eryngium littoralis callus were prepared by removing non-exosomal proteins, nucleic acids, lipids, etc.

[0109] 4-2. Analysis of particle size distribution of culture medium-derived exosomes

[0110] The results of dynamic light scattering (DLS) analysis of the particle sizes of exosomes derived from Eryngium maritima callus culture filtrate, Eryngium maritima callus extract, and Eryngium maritima callus culture solution showed that the exosomes derived from Eryngium maritima callus culture filtrate (1.98×10 10 particles / mL) with a particle size of 143.5 nm ( Figure 2 ), exosomes from Eryngium maritima callus (1.70×10 10 particles / mL) with a particle size of 149.4 nm ( Figure 3 ), exosomes from callus culture fluid of Eryngium littoralis (1.74×10 10 particles / mL)( Figure 4 ) has a particle size of 163.9 nm.

[0111] Experimental Example 1. Confirmation of skin toxicity

[0112] In order to confirm whether the three compositions of the present invention (Eryngium maritima callus culture filtrate, Eryngium maritima callus extract, and Eryngium maritima callus culture fluid-derived exosomes) are toxic to skin cells and induce skin irritation, MTT analysis was performed. Specifically, HaCaT cells (keratinocyte) and Detroit cells (fibroblast) were dispensed into a 96-well culture plate together with Dubelcco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic, and cultured in a thermostat under the conditions of 5% CO2 and 37°C for 24 hours. Then, the cells were treated with a control group added with pure water and a composition as a test substance at various concentrations, and then cultured for an additional 24 hours. Next, 4 μL of 5 mg / mL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was added to each well. bromide) solution and incubate for 4 hours to react. After removing the culture medium, 100 μL of dimethyl sulfoxide (DMSO) solution was added to each well to lyse the cells, and then the absorbance was measured at a wavelength of 540 nm.

[0113] Table 1

[0114]

[0115]

[0116] As a result, it was confirmed that at a concentration of 0.5 to 2% (w / w), the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, showed no toxicity in HaCaT cells and Detroit cells, but instead increased cell survival rates and produced a regeneration effect due to cell proliferation (Table 1). It can be seen that the three compositions of the present invention have no cytotoxicity and are safe substances with a low possibility of inducing skin irritation.

[0117] Experimental Example 2: Confirmation of wrinkle improvement effect

[0118] In order to confirm the wrinkle improvement effect of the three compositions of the present invention (Callus culture filtrate of Eryngium littoralis, callus extract of Eryngium littoralis, and exosomes derived from callus culture fluid of Eryngium littoralis), the biosynthesis amount of type I procollagen C-terminal peptide (PIP, Procollagen Type I C-Peptide), which is a precursor of collagen synthesis, was measured. Specifically, Detroit551 cells were injected into a 48-well culture plate and cultured for 24 hours under cell culture conditions. Then, the cells were treated with a control group with added pure water and a composition as a test substance, respectively, and then cultured for 48 hours. Next, 20 μL of the supernatant of the culture medium was taken and placed in an antibody coated microtiter plate together with 20 μL of the PIP standard substance in the PIP EIA kit (Kit) (Takara, Cat.MK101) and 100 μL of the antibody solution labeled with peroxidase was placed in each well, and reacted at a temperature of 37°C for 3 hours. After removing the culture medium and washing four times with 200 μL of phosphate buffer solution (PBS), 100 μL of substrate solution was added to each well and reacted at room temperature for 15 minutes in a light-shielded state. Then, 100 μL of stop solution (1N sulfuric acid (H2SO4)) was added and the absorbance was measured at a wavelength of 450 nm.

[0119] Table 2

[0120]

[0121] As a result, at a concentration of 0.5 to 2% (w / w), the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, all showed an increase in the amount of collagen biosynthesis (Table 2), thus confirming that the three compositions of the present invention have a wrinkle improvement effect.

[0122] Experimental Example 3: Confirmation of moisturizing and skin barrier improvement effects

[0123] In order to confirm the moisturizing effect and skin barrier improvement effect of the three compositions of the present invention (Calliphytum littoralis callus culture filtrate, Calliphytum littoralis callus extract and Calliphytum littoralis callus culture fluid derived exosomes), the expression changes of aquaporin 3 as a water / glycerol carrier and filaggrin known as a natural moisturizing factor were analyzed by real-time polymerase chain reaction (Real-time PCR). Specifically, after culturing HaCaT cells in a 96-well culture plate for 24 hours, the cells were treated with pure water (control group) and the three compositions as test substances, respectively, and cultured for an additional 24 hours. After washing the cultured cells with phosphate buffer solution, 50 μL of the cell lysis mixture of SuperPrepTM cell lysis&RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was added to react for 5 minutes, and 10 μL of the stop solution was added to separate the ribonucleic acid. After adding 32 μL of RT reaction mixture to 8 μL of the isolated RNA, polymerase chain reaction (PCR) was performed at 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes to synthesize complementary deoxyribonucleic acid (cDNA). The above-synthesized complementary deoxyribonucleic acid was used as a template and Qiagen's QuantiTect primer assays (aquaporin 3; Cat. QT00212996 and filaggrin; Cat. QT02448138) were used as primers. The cDNA was synthesized by Thunderbird. TM After reacting at 95°C for 1 minute, SYBR qPCR Mix (TOYOBO, Cat. QPS-201) was used for real-time polymerase chain reaction analysis with 40 cycles of 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. Phosphoglycerate dehydrogenase (GADPH) (Cat. QT01192646) was used to quantify the expression levels of aquaporin 3 and filaggrin.

[0124] Table 3

[0125]

[0126] As a result, it was confirmed that the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, increased the expression of aquaporin 3 and filaggrin at all concentrations compared with the control group, thereby contributing to skin moisturizing and skin barrier improvement (Table 3).

[0127] Experimental Example 4: Confirmation of the anti-aging effect

[0128] In order to confirm the anti-aging effect of the three compositions of the present invention (Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract and Eryngium littoralis callus culture fluid-derived exosomes), the expression level change of telomerase reverse transcriptase (Telomerase reverse transcriptase), which is known to extend the telomere length at the end of the deoxyribonucleic acid chain, was confirmed by real-time polymerase chain reaction using QuantiTect primer assays (telomerase reverse transcriptase; Cat. QT00073409) primers in the same method as in Experimental Example 3.

[0129] Table 4

[0130]

[0131] As a result, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, showed concentration-dependent increases in the expression level of messenger RNA of telomerase reverse transcriptase in skin cells compared to the control group (Table 4), confirming that they are effective in preventing aging.

[0132] Experimental Example 5. Confirmation of skin immunity improvement and defense enhancement

[0133] In order to confirm whether the three compositions of the present invention (Eryngium maritima callus culture filtrate, Eryngium maritima callus extract and Eryngium maritima callus culture fluid-derived exosomes) have the effect of improving skin immunity and defense, the same method as the above-mentioned Experimental Example 3 was used to confirm the changes in the expression levels of Cathelicidin-related antimicrobial peptides (Cathelicidin-related antimicrobial peptides, CAMP) and human β-defensin-2 (human β-Defensin-2), which are peptides with antimicrobial efficacy, by real-time polymerase chain reaction using QuantiTect primer assays (Cathelicidin-related antimicrobial peptides; Cat.QT00010458 and human β-Defensin-2; Cat.QT00204617).

[0134] Table 5

[0135]

[0136]

[0137] As a result, compared with the control group, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes and Eryngium maritima callus extract, which are the compositions of the present invention, showed concentration-dependent increases in the expression levels of cathelicidin-related antimicrobial peptides and human β-defensin-2 in skin cells (Table 5), thus confirming that they contribute to the enhancement of skin immunity and defense.

[0138] Experimental Example 6. Confirmation of autophagy activity

[0139] In order to confirm whether the three compositions of the present invention (Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract and Eryngium littoralis callus culture fluid-derived exosomes) have the autophagic activity efficacy of naturally decomposing unnecessary or dysfunctional small organs or cell structure components in the intracellular regulation process, the expression level changes of microtubule-associated protein 1A / 1B-light chain 3 (Microtubule-associated protein 1A / 1B-light chain 3) related to autophagy formation were confirmed by real-time polymerase chain reaction using QuantiTect primer assays (microtubule-associated protein 1A / 1B-light chain 3; Cat.QT00055069) primers in the same way as in Experimental Example 3 above.

[0140] Table 6

[0141]

[0142] As a result, compared with the control group, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, showed increased expression levels of microtubule-associated protein 1A / 1B-light chain 3 in skin cells in a concentration-dependent manner, thereby confirming that they contribute to the activation of autophagy (Table 6).

[0143] Experimental Example 7. Confirmation of Inflammation Suppression Effect

[0144] In order to confirm whether the three compositions of the present invention (Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract and Eryngium littoralis callus culture fluid-derived exosomes) have a skin inflammation inhibitory effect, the expression level changes of cyclooxygenase-2 and inducible nitric oxide synthase (Inducible nitric oxide synthase) related to inflammatory response were confirmed by real-time polymerase chain reaction using QuantiTect primer assays (cyclooxygenase-2; Cat.QT00040586 and inducible nitric oxide synthase; Cat.QT01323189) primers in the same method as in Experimental Example 3.

[0145] Table 7

[0146]

[0147]

[0148] As a result, compared with the control group, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes and Eryngium maritima callus extract, which are the compositions of the present invention, showed concentration-dependent reductions in the expression of cyclooxygenase-2 and inducible nitric oxide synthase in skin cells that were increased by ultraviolet irradiation (Table 7), thereby confirming the effect of alleviating inflammatory responses.

[0149] Experimental Example 8. Effect of protecting skin cells from ultraviolet rays

[0150] In order to confirm whether the three compositions of the present invention (Eryngium maritima callus culture filtrate, Eryngium maritima callus extract and Eryngium maritima callus culture fluid-derived exosomes) can protect skin cells from ultraviolet stimulation, HaCaT cells were dispensed into a 96-well culture plate and cultured for 24 hours. After irradiation with outdoor ultraviolet light (UVB) using an ultraviolet crosslinker (Ultraviolet Products, Cat No. CL-1000) that emits ultraviolet light of 302 nm wavelength, the cells were treated with pure water (control group) and the composition as the test substance, respectively, and then cultured for an additional 24 hours, and the cell survival rate was determined by MTT analysis.

[0151] Table 8

[0152]

[0153] As a result, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, showed concentration-dependent increases in the cell survival rate of the control group that was reduced by ultraviolet irradiation in skin cells (Table 8), thereby confirming that they have a cell protective effect against ultraviolet rays.

[0154] Experimental Example 9. Cell protection effect against blue light

[0155] In order to confirm whether the three compositions of the present invention (Eryngium maritima callus culture filtrate, Eryngium maritima callus extract, and Eryngium maritima callus culture fluid-derived exosomes) protect skin cells in blue light, which has the shortest wavelength and the strongest energy among visible light that humans can see, Topview 5450SMD LED (ITSWELL, Cat No. IWS-L5056-VRI-K3) was used at 10mJ / cm2 HaCaT cells were irradiated with light of 460 nm wavelength, and treated with pure water (control group) and the composition as the test substance, respectively, and then cultured for 24 hours. The cell survival rate was measured by MTT analysis.

[0156] Table 9

[0157]

[0158] As a result, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, showed a concentration-dependent increase in the survival rate of skin cells that were reduced by blue light irradiation (Table 9), thus confirming that the skin is protected by specifically reflecting and blocking light in the blue light region.

[0159] Experimental Example 10. Confirmation of Antioxidant Effect

[0160] In order to confirm the antioxidant effects of the three compositions of the present invention (Eryngium maritima callus culture filtrate, Eryngium maritima callus extract, and Eryngium maritima callus culture fluid-derived exosomes), HaCaT cells were treated with 1 mM hydrogen peroxide (H2O2), and the cells were treated with pure water (control group), 2 mM N-acetylcysteine ​​(NAC) (positive control group), and the composition as a test substance, respectively, and cultured for 12 to 16 hours, and then MTT analysis was performed to measure changes in cell survival rate related to hydrogen peroxide oxidative toxicity.

[0161] Table 10

[0162]

[0163]

[0164] As a result, compared with the positive control group (treated with hydrogen peroxide), the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, reduced cell apoptosis caused by treatment with hydrogen peroxide at a treatment concentration of 0.5 to 2%, thereby confirming that they have an antioxidant effect against the oxidative toxicity of hydrogen peroxide.

[0165] Experimental Example 11: Confirmation of the protective effect against stress

[0166] In order to confirm whether the three compositions of the present invention (Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract and Eryngium littoralis callus culture fluid-derived exosomes) have a protective effect against stress in cells, the activation degree of the anti-stress protein Late embryogenesisabundant, which is known to protect cells from cell damage by increasing the tolerance to stress as the expression level increases, was analyzed by In-Cell ELISA Colorimetric Detection kit (Invitrogen, Cat No. 62200). Specifically, after culturing HaCaT cells in a 6-well culture plate for 24 hours, the cells were treated with pure water (control group) and the composition as a test substance, respectively, and cultured for an additional 24 hours. After removing the cell culture medium, fix the cells by treating with 100 μL of fixing solution at room temperature for 15 minutes, wash with phosphate buffer solution, treat with 100 μL of permeabilization solution and react at room temperature for 15 minutes. After washing with phosphate buffer solution, treat with 100 μL of quenching solution and react at room temperature for 20 minutes. After washing with phosphate buffer solution, treat with 100 μL of blocking solution and react at room temperature for 30 minutes. Then, after removing the solution, add 50 μL of primary antibody against late embryonic abundant protein and react at 4°C for 24 hours, remove the antibody, wash with phosphate buffer solution, add 50 μL of secondary antibody coupled to horseradish peroxidase (HRP) and react for 1 hour. After removing the antibody and washing with phosphate buffer solution, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added and reacted at room temperature for 15 minutes under light-shielding conditions. Then, 100 μL of stop solution (1N sulfuric acid) was added and the absorbance at a wavelength of 450 nm was measured.

[0167] Table 11

[0168]

[0169] As a result, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes and Eryngium maritima callus extract, which are the compositions of the present invention, showed a concentration-dependent increase in the expression level of late embryonic abundant proteins in skin cells (Table 11), thus confirming the effect of protecting cells from cell damage caused by intracellular stress.

[0170] Experimental Example 12: Confirmation of wound healing effect

[0171] In order to confirm whether the three compositions of the present invention (Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract and Eryngium littoralis callus culture fluid-derived exosomes) have the ability to heal wounds by promoting cell proliferation and mobility, a wound healing assay was performed. Specifically, a culture insert for wound healing assay was set in each well of a 24-well culture plate, and 70 μL of HaCaT cells were injected into the insert at a density of less than 90% (90%<) and cultured for 24 hours. Then, after removing the insert, the cell state was photographed using a microscope (0 hour period), and the cells were treated with pure water (control group), positive control group (EGF) and the composition as the test substance, respectively, and then cultured for 16 hours. After culture, the culture medium was removed, and 4% paraformaldehyde (PFA, Paraformaldehyde) was added and reacted at room temperature for 15 minutes. After the fixation process, the cells were washed three times with phosphate buffer solution, and after additional culture for 16 hours, the cell status was photographed using a microscope (16 hours period), and the area between the cell compartments was measured using the Image J program to calculate the increase rate of the healing area.

[0172] Table 12

[0173]

[0174] As a result, the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, showed increased healing area rates in a concentration-dependent manner compared to the control group (Table 12), confirming that they have a wound healing effect.

[0175] Experimental Example 13: Confirmation of Cell Energy Enhancement Effect

[0176] In order to confirm whether the three compositions of the present invention (Cell culture filtrate of Eryngium littoralis, extract of Eryngium littoralis callus and exosomes derived from culture fluid of Eryngium littoralis callus) have a cell energy-enhancing effect, the activity of adenosine triphosphate (ATP) released during metabolism was measured using a cell adenosine triphosphate assay kit (ATP assay kit) (Abcam, Cat. ab83355). Specifically, Detroit cells were cultured for 24 hours after being cultured in a 12-well culture plate for 15 minutes, and the cells were treated with pure water (control group) and the composition as the test substance and then cultured for 24 hours. 50 μL of the diluted cultured cell lysate was placed in a 96-well culture plate together with 50 μL of adenosine triphosphate standard substance, and 50 μL of adenosine triphosphate reaction mixture (reaction mix) solution was added to each well, and then reacted at room temperature for 30 minutes in a light-shielded state. After the reaction is completed, the absorbance is measured at a wavelength of 570 nm, an adenosine triphosphate standard concentration curve is drawn, and the amount of adenosine triphosphate in the sample is calculated.

[0177] Table 13

[0178]

[0179] As a result, the Eryngium maritima callus culture filtrate, Eryngium maritima callus extract, and Eryngium maritima callus culture fluid-derived exosomes, which are the compositions of the present invention, showed an increase in the amount of adenosine triphosphate in skin cells in a concentration-dependent manner compared to the control group (Table 13), thereby confirming an energy activity enhancing effect.

[0180] Experimental Example 14: Confirmation of pore shrinking effect

[0181] In order to confirm whether the essence formulation of the following Preparation Example 1 containing three compositions of the present invention (Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract and Eryngium littoralis callus culture fluid-derived exosomes) has a pore shrinking effect, squares (2cm×2cm) were divided on the faces of 20 subjects and the essence formulation was treated with a bean-sized amount for 4 weeks, and then hemoglobin was used as a protein to replace the skin to conduct an in vitro test. Specifically, pure water (control group) and the composition as the test substance were treated with cells in 2mL of 1mg / mL hemoglobin solution and shaken for 30 seconds, and then centrifuged at 3500rpm for 10 minutes. After adding 2mL of pure water to 1mL of the centrifuged supernatant, the absorbance was measured at a wavelength of 407nm.

[0182] Table 14

[0183]

[0184] As a result, the control group showed little change in the amount of sebum oil after 4 weeks of use, while in the case of the treatment with the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the composition of the present invention, the amount of skin oil decreased depending on the concentration (Table 14), thus confirming that the composition of the present invention has a sebum suppressing effect.

[0185] Experimental Example 15: Confirmation of skin soothing effect

[0186] The skin soothing effect of the essence formulation of Preparation Example 1 below, which contains three compositions of the present invention (Eryngium littoralis callus culture filtrate, Eryngium littoralis callus extract, and Eryngium littoralis callus culture fluid-derived exosomes), was confirmed by the skin temperature reduction rate. Specifically, after the forearms of 25 subjects were divided into squares (2 cm × 2 cm), 50 μL of the essence formulation of Preparation Example 1 was applied to each of the forearms, and then exposed to direct light for 20 minutes. The body temperature at 0 minutes (before sample application), 0 minutes (immediately after sample application), 5 minutes (after sample application), and 20 minutes (after sample application) was measured by computer infrared thermography (Digital Infrared Thermographic Imaging; DITI) and the temperature reduction rate was calculated.

[0187] Table 15

[0188]

[0189] As a result, in the case of the control group, the heat of the skin decreased rapidly over time, while the heat reduction rate was continuously maintained in the Eryngium maritima callus culture filtrate, Eryngium maritima culture fluid-derived exosomes, and Eryngium maritima callus extract, which are the compositions of the present invention, thereby confirming a skin soothing effect.

[0190] Experimental Example 16: Confirmation of acne improvement effect

[0191] In order to confirm the acne improvement effect of the lotion formulation of Preparation Example 2 below, which contains three compositions of the present invention (filtrate of callus culture of Eryngium maritima, extract of callus of Eryngium maritima, and exosomes derived from callus culture fluid of Eryngium maritima), 10 men and women with acne were subjected to uniform application of the lotion formulation of the present invention on the entire face in the morning and evening for 4 weeks. The acne improvement effect was evaluated according to the degree of feeling of the experimenter with reference to the following criteria: State before application of lotion = 1: slight acne, 2: slightly acne, 3: severe acne; Acne improvement effect = -: almost no effect, +: slight effect, ++: greatly relieved, +++: completely cured.

[0192] Table 16

[0193]

[0194]

[0195] As a result, it was confirmed that most of the subjects' acne was improved when the lotion formulations of the compositions of the present invention were used for 2 weeks and 4 weeks (Table 16).

[0196] Preparation Example 1. Preparation of a skin external application composition in the form of an essence liquid

[0197] An essence liquid dosage form containing the Eryngium maritima callus culture filtrate, the Eryngium maritima callus extract and the exosomes derived from the Eryngium maritima callus culture fluid as the composition of the present invention is prepared.

[0198] Table 17

[0199]

[0200]

[0201] Preparation Example 2. Preparation of a lotion-type skin external application composition

[0202] A lotion formulation containing the Eryngium maritima callus culture filtrate, the Eryngium maritima callus extract, and the Eryngium maritima callus culture fluid-derived exosomes as the composition of the present invention was prepared.

[0203] Table 18

[0204] Raw material name Weight percentage (w / w) glycerin 5.0 Dipropylene glycol 3.0 Compositions of the present invention 1.0 Hyaluronic acid 0.5 Polyoxyethylene hydrogenated castor oil 0.1 Polyethylene ethyl oleate 0.1 Ethanol 5.0 preservative 0.15 spices Moderate Coloring material Moderate Pure water Up to 100 .

Claims

1. A skin-improving cosmetic composition, characterized in that: The invention relates to a composition comprising a culture of tissue, cell or callus tissue, an extract, a filtrate of the culture, an extract of the culture, an exosome derived from the tissue, cell or callus tissue, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

2. The skin-improving cosmetic composition according to claim 1, characterized in that: Skin improvement is one or more selected from the group consisting of skin wrinkle improvement, skin moisturizing or skin barrier improvement, skin aging prevention, pigmentation inhibition, skin cell energy increase, skin immunity or defense enhancement, inflammation inhibition, pore shrinkage, sebum inhibition or acne improvement, or skin calming.

3. The skin improving cosmetic composition according to claim 1, characterized in that: Enhance the biosynthesis of type I procollagen C-terminal peptide.

4. The skin-improving cosmetic composition according to claim 1, characterized in that: Increase the expression of aquaporin 3, filaggrin, telomerase reverse transcriptase, cathelicidin-related antimicrobial peptide, human β-defensin-2, microtubule-associated protein 1A / 1B-light chain 3, or late embryonic abundant protein.

5. The skin-improving cosmetic composition according to claim 1, characterized in that: Reduce the expression of cyclooxygenase-2 and inducible nitric oxide synthase.

6. A cosmetic composition for protecting the skin against light, oxidative toxicity or stress, characterized in that: The invention relates to a composition comprising a culture of tissue, cell or callus tissue, an extract, a filtrate of the culture, an extract of the culture, an exosome derived from the tissue, cell or callus tissue, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

7. The cosmetic composition for protecting the skin against light, oxidative toxicity or stress according to claim 6, characterized in that: The light is ultraviolet light or blue light.

8. An antioxidant cosmetic composition, characterized in that: The invention relates to a composition comprising a culture of tissue, cell or callus tissue, an extract, a filtrate of the culture, an extract of the culture, an exosome derived from the tissue, cell or callus tissue, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

9. A cosmetic composition for skin regeneration, characterized in that: The invention relates to a composition comprising a culture of tissue, cell or callus tissue, an extract, a filtrate of the culture, an extract of the culture, an exosome derived from the tissue, cell or callus tissue, a culture-derived exosome or a culture filtrate-derived exosome of Eryngium maritima as an active ingredient.

10. A pharmaceutical composition for skin wound healing or anti-inflammation, characterized in that: The invention contains the culture of tissue, cell or callus of Eryngium maritima as an effective ingredient, an extract, a filtrate of the culture, an extract of the culture, or exosomes derived from the tissue, cell or callus.

11. A food composition for improving skin, characterized in that: The invention contains the culture of tissue, cell or callus of Eryngium maritima as an effective ingredient, an extract, a filtrate of the culture, an extract of the culture, or exosomes derived from the tissue, cell or callus.

12. A method for preparing exosomes derived from Eryngium littoralis callus, characterized in that: include: Step a), inducing and culturing callus tissue from tissue of Eryngium littoralis; Step b), filtering the culture of callus tissue; Step c), concentration; as well as Step d), isolating and purifying extracellular vesicles.

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