CN118338892A - Novel composition comprising exosomes derived from schizonepeta as active ingredient - Google Patents
Novel composition comprising exosomes derived from schizonepeta as active ingredient Download PDFInfo
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- CN118338892A CN118338892A CN202380014846.8A CN202380014846A CN118338892A CN 118338892 A CN118338892 A CN 118338892A CN 202380014846 A CN202380014846 A CN 202380014846A CN 118338892 A CN118338892 A CN 118338892A
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- schizonepeta
- skin
- exosomes
- cosmetic
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Abstract
The present invention provides a composition for improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing or promoting wound healing, which comprises an exosome derived from schizonepeta as an active ingredient. The composition of the present invention has a lower possibility of containing residual solvents, other impurities, etc. than conventional hot water extracts of nepeta, solvent extracts of nepeta and filters of these extracts, and is very effective in improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing or promoting wound healing.
Description
Technical Field
The invention relates to a preparation method of a Chinese medicinal composition containing schizonepeta herbNEPETA CARTARIA) as active ingredient, and more particularly to a composition for improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing or accelerating wound healing, which comprises exosomes derived from schizonepeta as active ingredient.
Furthermore, the present invention relates to a cosmetic composition for improving skin elasticity, reducing skin wrinkles or regenerating skin comprising the composition, and to a pharmaceutical composition for anti-inflammatory, wound healing or accelerating wound healing comprising the composition.
Background
Skin aging is known to result in reduced skin elasticity and increased skin wrinkles, and it is known that reduced skin elasticity and formation of skin wrinkles occur due to reduced synthesis of collagen and stimulated expression of collagenase matrix metalloproteinases (matrix metalloproteinase, MMPs).
In addition, it is known that in skin cells, the enzyme COX-2 that produces inflammatory cytokines increases due to aging progression or Ultraviolet (UV) rays, resulting in an increase in the synthesis of prostaglandin E2 (prostaglandin E2) and an increase in the production of inflammatory inducers. Due to the inflammatory response, MMP biosynthesis is increased, leading to collagen degradation and to reduced skin elasticity and skin wrinkles formation. In particular, when sunlight and ultraviolet rays are directly irradiated on the skin, a large amount of radicals are generated, and these radicals may damage the antioxidant defense system of the skin, thereby increasing wrinkles, relaxing the skin, and accelerating skin aging. Substances known to be effective in reducing skin wrinkles include adenosine and retinoic acid. However, adenosine has little efficacy in clinical practice, and retinoic acid cannot be used in pregnant women and has side effects such as erythema.
Inflammation is the defensive response of the body against physical or chemical injury, bacterial, fungal or viral infection, or pathological conditions caused by a variety of allergens, and the like. The inflammatory response appears to be part of the innate immune response. Various substances and physiological and chemical phenomena are involved in the inflammatory response, and recent studies have shown that various inflammatory cytokines play an important role in the inflammatory response. The major cytokines involved in the inflammatory response include IL-1. Beta., TNF-. Alpha., IL-6, IL-8, IL-12, IFN-. Beta.and the like. The increased expression and secretion of these cytokines and their activation are associated with a complex array of physiological responses including secretion of inflammatory mediators, immune cell infiltration, cell migration and tissue destruction, and symptoms such as erythema, edema, fever and pain.
In general, if the infectious agent is removed from the body and damaged tissue is regenerated, the inflammatory response does not become a significant problem and the affected area returns to its normal state. However, acute or chronic inflammatory diseases occur if the infectious agent is not removed from the body or the inflammatory response is excessive or sustained due to internal substances of the body. Non-steroidal anti-inflammatory drugs, neuropeptide antagonists, COX inhibitors, antihistamines and immunosuppressant drugs such as cyclosporin a are used to reduce or treat the inflammatory response or inflammatory disease caused thereby, but have such problems: they cause adverse effects such as skin atrophy, vasodilation, depigmentation, hypersensitivity, tolerance, neutropenia, etc. In addition, there are such limitations: the above drugs only help to control the symptoms associated with inflammation to a certain level, not to treat it at all.
Recently, it has been reported that the cell secretory group (secretome) contains a variety of bioactive molecules that regulate cell behavior. In particular, the cell secretory group contains "exosomes" (exosomes) having an intercellular signaling function, and thus components and functions thereof have been actively studied.
Cells shed a variety of membrane vesicles into their extracellular environment, and these released vesicles are commonly referred to as extracellular vesicles (extracellular vesicle, EV). EV is also known as a cell membrane-derived vesicle, exonuclear granules (ectosome), shedding vesicles, microparticles, exosomes, etc., and is also used in some cases differently from exosomes.
Exosomes are vesicles of tens to hundreds of nanometers in size, comprising phospholipid bilayer membranes having the same structure as cell membranes. Such exosomes include proteins, nucleic acids (mRNA, miRNA, etc.), etc., referred to as exosome carriers (cargo). Exosome cargo is known to include a wide range of signaling factors, and these signaling factors are specific to cell types and are regulated differently depending on the environment of the secreting cells. Exosomes are known to be intercellular signaling mediators secreted by cells and through which various cellular signals are transmitted to regulate cellular behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis and necrosis of target cells. Exosomes contain specific genetic material and bioactive factors, depending on the nature and status of the cells from which the exosomes are derived. Exosomes derived from proliferating stem cells regulate cell behavior such as cell migration, proliferation, and differentiation, and summarize the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569-579).
That is, exosomes called "avatars" of cells contain bioactive factors, such as growth factors, similar to cells, and act as carriers for transporting bioactive factors between cells, i.e., for mediating communication between cells. Exosomes are known to be released not only from animal cells such as stem cells, immune cells, fibroblasts and cancer cells, but also from cells of various organisms (e.g. plants, bacteria, fungi and algae). For example, the exosomes may be isolated from a culture medium of plant cells or plant stem cells, as well as a culture medium of cancer cells, immune cells, mesenchymal stem cells, and the like.
However, studies on the isolation, purification and characterization of exosomes derived from plant cells or plant stem cells remain insufficient, and most of these studies are only used for market purposes, in which case the extracellular vesicles mixed in the plant juice filtrate are simply referred to as exosomes. Therefore, more detailed characterization and functional studies of plant cell-derived exosomes are needed.
Catmint (catnip), a scientific name of schizonepeta, is a perennial herb belonging to the genus schizonepeta in the family Labiatae and order cheiliformes. Herba Schizonepetae is the favorite cat and is commonly referred to as catmint. Herba Schizonepetae is a herb that has a peppermint flavor after it is dried and is also known as catmint. Herba Schizonepetae is native to asia and europe, and is distributed in korea, japan, china, zhongya, africa, europe and north america. It is known for its pain relief and antipyretic properties. However, the current technology remains with the use of leaf, stem or whole plants of nepeta cataria by hot water extraction or solvent extractionThe obtained extract can be used as food or cosmetic ingredient level. However, the solvent extract has a problem of toxicity to the human body due to the residual extraction solvent.
The present inventors have found that exosomes derived from nepeta cataria are effective in improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing or accelerating wound healing, and have developed cosmetic compositions for improving skin elasticity, reducing skin wrinkles or accelerating skin regeneration, and pharmaceutical compositions for anti-inflammatory, wound healing or accelerating wound healing, comprising exosomes derived from nepeta cataria as an active ingredient.
Meanwhile, it should be understood that what is described as background art is only for aiding in understanding the background of the present invention and is not to be considered as prior art for the present invention.
Disclosure of Invention
Technical challenges
It is an object of the present invention to provide a composition for improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing or accelerating wound healing, which comprises exosomes derived from schizonepeta as an active ingredient.
It is another object of the present invention to provide a cosmetic composition for improving skin elasticity, reducing skin wrinkles or regenerating skin comprising the same, and a pharmaceutical composition for anti-inflammatory, wound healing or accelerating wound healing comprising the same.
However, the invention as described above is intended to be illustrative, and the scope of the invention is not limited thereto. Further objects and advantages of the present invention will be apparent from the following description, appended claims and drawings.
Solution scheme
The present invention provides a composition for improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing or accelerating wound healing, comprising exosomes derived from schizonepeta as an active ingredient.
The term "catmint" as used herein refers to perennial herbs belonging to the genus schizonepeta in the family Labiatae and the order chebulales, and its scientific name is schizonepeta. Herba Schizonepetae is a herb that has a peppermint flavor after drying and is also known as catmint. Herba Schizonepetae is native to asia and europe, and is distributed in korea, japan, china, zhongya, africa, europe and north america.
The term "exosomes" as used herein refers to nano-sized vesicles secreted or released from plant cells into the extracellular space and having a membrane structure, and is also referred to as exosome-like vesicles or exosome-like particles.
The term "skin elasticity" as used herein refers to a feature in which deformed skin caused by an external force is easily restored to its original shape when the external force is removed. The term "skin wrinkles" refers to fine lines caused by skin aging. Skin wrinkles may be caused by genetic factors, reduction of collagen and elastin present in the dermis of the skin, external environmental factors, and the like. Thus, the term "skin wrinkle reduction or improvement" as used herein refers to inhibiting or preventing the formation of wrinkles on the skin, or reducing wrinkles that have formed.
The term "anti-inflammatory" as used herein means the prevention, inhibition, alleviation, amelioration or treatment of inflammation. By way of example and not limitation of the invention, some examples of inflammatory diseases include dermatitis, atopic dermatitis, eczema, inflammation caused by bacterial, viral or fungal infections, burns, inflammation caused by burns, wounds, inflammation caused by wounds, and the like.
The term "wound" as used herein means a condition in which a portion or all of the body is damaged, and is intended to encompass pathological conditions in which tissue (e.g., skin, muscle, nerve tissue, bone, soft tissue, internal organs, or vascular tissue) that constitutes the internal or external surface of the body is damaged or destroyed. By way of example and not limitation of the present invention, some examples of wounds include bruises, lacerations, stabs, cuts, avulsions, bedsores, tissue damage caused by irradiation, penetrations, gunshot wounds, burns, frostbite, surgical wounds, suture wounds after plastic surgery, wounds caused by chemicals, and the like, and may include any injury to any part of an individual.
The term "iontophoresis (iontophoresis)" as used herein refers to such a method: a microcurrent is caused to flow through the skin to which the active ingredient has been applied, thereby creating a potential difference and changing the electrical environment of the skin, and thus causing the ionized active ingredient to penetrate the skin by an electrical repulsive force. Some examples of iontophoresis used in one embodiment of the present invention include: a method of introducing a microcurrent into skin by flowing the microcurrent from an external power source to an electrode patch on the skin, the microcurrent being generated by the external power source; a method of introducing a microcurrent into the skin, the microcurrent being generated by a battery disposed in an electrode patch on the skin; and a method of introducing a microcurrent into skin through a patch provided with a reverse electrodialysis device on the skin, the microcurrent resulting from a concentration difference between a high concentration electrolyte solution and a low concentration electrolyte solution in the reverse electrodialysis device. However, the present invention is not limited thereto, and of course, various types of iontophoresis may be used.
The term "exosomes derived from nepeta as used herein is intended to include all exosomes isolated from, for example, a culture medium of nepeta plant cells, a culture medium of nepeta callus, a culture medium of nepeta plant stem cells, nepeta juice, or a nepeta biological solution equivalent thereto, or all exosomes derived (e.g., secreted and/or released from) from plant cells or plant stem cells of nepeta.
In a composition according to one embodiment of the invention, the exosomes may be isolated and purified from the nepeta juice.
The composition according to an embodiment of the present invention, which comprises an exosome derived from schizonepeta as an active ingredient, may exhibit at least one of an effect of improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing, or accelerating wound healing.
The composition according to the present invention comprising an exosome derived from schizonepeta as an active ingredient may be a cosmetic composition or a pharmaceutical composition.
The present invention provides a cosmetic composition for improving skin elasticity, reducing skin wrinkles or regenerating skin, which comprises exosomes derived from schizonepeta as an active ingredient. For example, the cosmetic composition may be a shampooSoapCleaning agentSurfactant-containing cleaning agentCreamEmulsionOintmentSkin-activating lotionRepairing agentConditioning agentSuspensionEmulsionPasteGel agentOiling agentWax agentSpray agentAerosol formulationMist agentOr powderAnd is preferably an emulsion or cream.
The present invention also provides a pharmaceutical composition for anti-inflammatory, wound healing or accelerating wound healing, comprising exosomes derived from schizonepeta as active ingredients.
By way of example and not limitation, a pharmaceutical composition according to one embodiment of the present invention may be administered or treated by injection, microneedle, iontophoresis, coating, or a combination thereof. For example, the pharmaceutical composition may be an injectable formulation, an infusible formulation, a spray formulation, a liquid formulation or a patch formulation.
In one embodiment of the present invention, when the composition is used as a pharmaceutical composition, it may comprise a pharmaceutically acceptable carrier, excipient or diluent. Carriers, excipients, and diluents include, but are not limited to: lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginic acid, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, an effective amount of a pharmaceutical composition according to an embodiment of the present invention means an amount that needs to be administered in order to achieve an anti-inflammatory, wound healing or wound healing acceleration effect.
The content of the pharmaceutical composition according to one embodiment in the formulation may be appropriately selected according to the kind, amount, form, etc. of the additional components as described above. For example, the pharmaceutical composition of the present invention may be included in an amount of about 0.1wt% to 99wt%, preferably about 10wt% to 90wt%, based on the total weight of the injectable formulation. Furthermore, the appropriate dosage of the pharmaceutical composition according to an embodiment of the present invention may be adjusted according to the following: the severity of the disease, the type of formulation, the method of formulation, the age, sex, weight, health condition, diet, rate of excretion, the time of administration and the regimen of administration of the patient. For example, when a pharmaceutical composition according to one embodiment of the present invention is administered to an adult, it may be administered at a dose of 0.001mg/kg to 100mg/kg once to several times per day.
Meanwhile, in preparing the composition according to one embodiment of the present invention into a cosmetic composition, components commonly used in cosmetic products, for example, moisturizers, antioxidants, oily components, UV absorbers, emulsifiers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, and the like may be appropriately contained as needed within a range that does not impair the effects of the present invention.
In addition, the cosmetic composition according to an embodiment of the present invention may contain an agent and/or moisturizer for improving skin conditions, which have been used in the prior art, in addition to the exosomes derived from nepeta, within a range that does not impair effects (e.g., improvement of skin conditions, improvement of skin elasticity, reduction of skin wrinkles, skin regeneration, etc.).
The cosmetic composition according to one embodiment of the present invention may be used in various forms, for example, a patchSheet-mounted facial maskFacial mask towelCream, lotion, ointment, suspension, emulsion, paste, emulsion, gel, oil, and peel-off facial maskSpray, aerosol, fog agent and foundationPowder and oiled paper
The cosmetic composition according to one embodiment of the present invention may be used for the purpose of skin elasticity improvement, wrinkle improvement, skin regeneration, etc., and may be prepared into any preparation generally prepared in the art. For example, it may be formulated as: patch, sheet pack, facial mask towel and skin softenerNutritional productAstringent emulsionNourishing creamMassage creamEye creamCleaning creamEssenceEye essenceCleaning emulsionCleaning foamCleaning waterSun creamLipstickSoap, shampoo, surfactant-containing cleanser, and bath preparationBody washBody creamBody oilBody essenceBody cleanerHair dyeHair tonicEtc., but is not limited thereto.
The cosmetic composition according to one embodiment of the present invention may comprise components commonly used in cosmetic products. For example, the cosmetic composition may comprise conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. In addition, depending on the type or intended use of the cosmetic composition, one skilled in the art can appropriately select other components in each formulation of the cosmetic composition without difficulty.
Another embodiment of the present invention provides a cosmetic method for non-therapeutic purposes for regulating a mammalian skin condition using the cosmetic composition. In the cosmetic method of the present invention, the expression "regulating the skin condition" means improving the skin condition and/or prophylactically regulating the skin condition, and the expression "improving the skin condition" means visually and/or tactilely perceivable positive changes in the appearance and feel of the skin. For example, the expression "improving skin condition" may include reduction of skin wrinkles, improvement of skin elasticity or skin regeneration.
The cosmetic method according to one embodiment of the present invention includes: (a) Applying the cosmetic composition directly to mammalian skin; or (b) contacting or adhering a patch, sheet pack or facial mask towel to mammalian skin, said patch, sheet pack or facial mask towel having a cosmetic composition applied thereto or immersed therein; or (a) and (b) may be performed sequentially. In step (a), the cosmetic composition may be an emulsion or a cream.
Or the cosmetic method according to an embodiment of the present invention may further comprise (c): removing the patch, sheet pack or facial mask towel from the mammalian skin after step (b), and applying the cosmetic composition to the mammalian skin. In step (c), the cosmetic composition may be an emulsion or a cream.
By way of example and not limitation, the cosmetic composition may be applied to the skin by microneedle, iontophoresis, direct application, or a combination thereof. For example, the cosmetic composition may be a spray formulation, a liquid formulation, or a patch formulation.
In the cosmetic method according to an embodiment of the present invention, the mammal may be a cat, a human, a dog, a rodent, a horse, a cow, a monkey, or a pig.
In addition, the present invention provides methods for treating inflammation, healing wounds, or accelerating wound healing comprising administering a therapeutically effective amount of a pharmaceutical composition to a mammal, or applying the pharmaceutical composition to skin, an inflammatory region, or an injured region.
By way of example, and not limitation, the pharmaceutical compositions may be administered by injection, microneedle, iontophoresis, or a combination thereof. For example, the pharmaceutical composition may be an injectable formulation, an infusible formulation, a spray formulation, a liquid formulation or a patch formulation.
The mammal may be a cat, human, dog, rodent, horse, cow, monkey or pig.
Advantageous effects of the invention
The composition according to the present invention has a lower possibility of containing impurities such as residual solvents and the like, and has excellent effects on improving skin elasticity, reducing skin wrinkles, regenerating skin, anti-inflammatory, wound healing or accelerating wound healing, as compared with conventional hot water extracts of schizonepeta, solvent extracts of schizonepeta, filters of such extracts, and the like.
It should be understood that the scope of the present invention is not limited to the above effects.
Drawings
FIG. 1 is a graph showing particle size distribution and particle number obtained by Nanoparticle Tracer Analysis (NTA) of the schizonepeta-derived exosomes of the present invention.
FIG. 2 shows fluorescence microscopy images showing fluorescence stained nepeta-derived exosomes delivered to cells in human dermal fibroblasts (green: exosomes delivered to cells; and blue: nuclei).
FIG. 3 is a graph showing the increase in the relative amount of collagen in a concentration-dependent manner after treatment of human dermal fibroblasts with schizonepeta-derived exosomes.
Fig. 4 is a graph showing the increase in migration of human dermal fibroblasts after treatment of scratch-wounds with schizonepeta-derived exosomes.
Fig. 5 shows fluorescence microscopy images showing fluorescence-stained nepeta-derived exosomes delivered to cells in RAW 264.7 cells (green: exosomes delivered to cells; and blue: nuclei).
FIG. 6 is a graph showing the reduction of IL-6 induced by LPS when RAW 264.7 cells were treated with exosomes derived from nepeta cataria.
Detailed Description
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention and are not intended to limit or restrict the scope of the present invention. Those readily inferred from the detailed description and examples of the invention by those skilled in the art are to be construed as falling within the scope of the invention. References mentioned in this invention are incorporated herein by reference.
It should be understood that throughout this specification, when any portion is referred to as "comprising" any component, it does not exclude other components, but may also comprise other components, unless otherwise indicated.
Examples
Example 1: preparation of herba Schizonepetae juice
Leaves and stems of schizonepeta are first washed under running water to remove pesticides. Next, the leaves and stems were cut into pieces of not more than 5cm, and juice was extracted with a low-speed screw at a stirring speed of 40rpm to obtain juice, which was then filtered through a 400-mesh screen to remove large suspended substances. The filtered juice was centrifuged at 10,000Xg for 10 minutes at 4℃to remove proteins and cell walls. The supernatant obtained by the above operation was filtered through a filter membrane having 0.22 μm pores to obtain a filtrate, which was stored at-80 ℃ until purification.
Example 2: purification of nepeta-derived exosomes
The juice prepared as described in example 1 was subjected to step centrifugation and ultracentrifugation to remove a large amount of impurities and separate the exosomes from the schizonepeta source. The thawed nepeta juice was centrifuged at 2,000Xg for 10 minutes and filtered through a filter membrane having 0.22 μm pores to obtain a filtrate. The filtrate was subjected to a first centrifugation at 10,000Xg and a second centrifugation at 20,000Xg, and then the supernatant was recovered. The supernatant was ultracentrifuged at 100,000Xg for 70 minutes at 4℃to obtain a precipitate. The obtained precipitate was suspended in Phosphate-Buffered Saline (PBS) to finally obtain an exosome derived from nepeta cataria. The size and concentration of nepeta-derived exosomes isolated by the above method were analyzed by Nanoparticle Tracer Analysis (NTA) using NS300 (purchased from MALVERN PANALYTICAL) (fig. 1).
Example 3: evaluation of ability of schizonepeta-derived exosomes to be delivered into dermal fibroblasts
To examine whether an exosome of schizonepeta origin would be delivered to human dermal fibroblasts (purchased from ATCC), the following analysis was performed. To perform fluorescent staining of the membranes of schizonepeta-derived exosomes prepared in example 2, exosomes were reacted with PKH67 fluorescent dye (purchased from Sigma-Aldrich). After the reaction, the reaction solution was fractionated with MiniTrap-25 columns (available from Cytiva) to remove free PHK67 that was not stained in the exosome film. Negative controls were prepared by reacting PKH67 fluorescent dye with buffer solution and fractionating the reaction product using MiniTrap-25 columns. The PKH67 stained exosomes were incubated with pre-cultured human dermal fibroblasts and then observed using fluorescence microscopy for the delivery of exosomes into the cells over time. Nuclei were stained with Hoechst fluorochrome (available from Thermo Fisher) and cytoplasm was stained with CellMask orange fluorochrome (available from Thermo Fisher). As a result of detecting whether exosomes would be delivered into cells, it was determined that fluorescent-stained exosomes were delivered into cells and green fluorescence accumulated in cells over time (fig. 2).
Example 4: evaluation of collagen production-stimulating Activity
Human dermal fibroblasts (purchased from ATCC) dispersed in DMEM medium containing fetal bovine serum are dispensed into multi-well plates and then cultured for 24 hours. Human dermal fibroblasts were further cultured in serum-free medium for 48 hours. Thereafter, the schizonepeta-derived exosomes prepared in example 2 were diluted in serum-free medium, and then human dermal fibroblasts were treated with each dilution and cultured. To evaluate collagen production using human dermal fibroblasts, the experimental groups were classified as follows:
(1) Negative control group (control): an experimental group treated with serum-free medium alone;
(2) The group treated with low concentration of schizonepeta-derived exosomes (denoted "low" in fig. 3): experimental groups (treatment concentration: 1.0X10 10 particles/mL) treated with an exosome of schizonepeta origin (prepared in example 2) diluted in serum-free medium; and
(3) The group treated with high concentrations of schizonepeta-derived exosomes (denoted "high" in fig. 3): an experimental group (treatment concentration: 4.0X10 10 particles/mL) was treated with an exosome of schizonepeta origin (prepared in example 2) diluted in serum-free medium.
Human dermal fibroblasts were treated and cultured for 24 hours according to each of the above experimental groups. The medium is collected and centrifuged, and the centrifuged medium is then prepared. The amount of collagen synthesized by human dermal fibroblasts and accumulated in the medium was measured using an EIA kit (purchased from Takara) for type I procollagen C-peptide (PIP). The relative amounts of collagen were determined by normalizing the measured amounts of collagen by dividing the measured amounts of collagen by the total number of cells measured using the MTT assay kit (purchased from Sigma-Aldrich).
As a result, it was determined that the schizonepeta-derived exosomes of the present invention significantly improved collagen synthesis in human dermal fibroblasts in a concentration-dependent manner compared to the negative control (fig. 3).
From the above experimental results, it can be seen that the schizonepeta-derived exosomes of the present invention have excellent effects of enhancing collagen synthesis, i.e., improvement of skin elasticity, reduction of wrinkles and/or skin regeneration.
As can be seen from the above results, the schizonepeta-derived exosomes of the present invention have useful functional activity as a functional cosmetic for skin elasticity improvement, wrinkle reduction and/or skin regeneration, i.e., activity to enhance collagen synthesis. Thus, the schizonepeta-derived exosomes of the present invention can be used as active ingredients in cosmetic compositions for skin elasticity improvement, wrinkle reduction and/or skin regeneration.
Example 5: evaluation of skin regeneration Using dermal fibroblasts
To evaluate whether schizonepeta-derived exosomes prepared as described in example 2 promote wound healing of human dermal fibroblasts (purchased from ATCC), a scratch-wound assay was performed. Human dermal fibroblasts dispersed in DMEM containing fetal bovine serum were distributed at a density of 5,000 cells/well into a culture plate (ImageLock plates; purchased from EssenBio) for wound induction and cultured at 37℃under 5% CO 2 for 24 hours. Scratches were made using WoundMaker (available from EssenBio) after the cells reached a confluency of 90% or higher. To evaluate the skin regeneration effect using human dermal fibroblasts, the experimental groups were classified as follows:
(1) Negative control group (n.c.): an experimental group treated with serum-free medium alone;
(2) Positive control group (p.c.): an experimental group treated with a medium comprising 10% fetal bovine serum;
(3) The group treated with low concentration of schizonepeta-derived exosomes (denoted "low" in fig. 4): experimental groups (treatment concentration: 2.0X10 9 particles/mL) treated with an exosome of schizonepeta origin (prepared in example 2) diluted in serum-free medium; and
(4) The group treated with high concentrations of schizonepeta-derived exosomes (denoted "high" in fig. 4): an experimental group (treatment concentration: 1.2X10 10 particles/mL) was treated with an exosome of schizonepeta origin (prepared in example 2) diluted in serum-free medium.
Thereafter, each experimental group was scratched-wound, and human dermal fibroblasts were cultured at 37 ℃ under 5% CO 2 for 12 hours. Wound healing efficacy was measured for each experimental group using Incucyte (purchased from Sartorius).
As a result of measuring the wound healing efficacy, it was determined that the schizonepeta-derived exosomes of the present invention improved migration of human dermal fibroblasts compared to the negative control (fig. 4).
As can be seen from the above experimental results, the schizonepeta-derived exosomes of the present invention have an excellent effect of promoting human dermal fibroblast migration, i.e., an excellent wound healing effect or skin regeneration effect.
Thus, the schizonepeta-derived exosomes of the present invention can be used as active ingredients of cosmetic compositions for improving skin elasticity, reducing wrinkles and/or regenerating skin, as well as active ingredients of pharmaceutical compositions for wound healing or accelerating wound healing.
Example 6: evaluation of ability of schizonepeta-derived exosomes to deliver into macrophages
To examine whether an exosome derived from schizonepeta was delivered to mouse macrophages (RAW 264.7; purchased from ATCC), the following analysis was performed. To perform fluorescent staining of the membranes of schizonepeta-derived exosomes prepared in example 2, exosomes were reacted with PKH67 fluorescent dye (purchased from Sigma-Aldrich). After the reaction, the reaction solution was fractionated with MiniTrap-25 columns (available from Cytiva) to remove free PHK67 that was not stained in the exosome film. Negative controls were prepared by reacting PKH67 fluorescent dye with buffer solution and fractionating the reaction product using MiniTrap-25 columns. The PKH 67-stained exosomes were incubated with pre-cultured mouse macrophages and then observed using a fluorescence microscope for the delivery of exosomes into cells over time. Nuclei were stained with Hoechst fluorochrome (available from Thermo Fisher) and cytoplasm was stained with CellMask orange fluorochrome (available from Thermo Fisher). As a result of detecting whether exosomes would be delivered into cells, it was determined that fluorescent-stained exosomes were delivered into cells and green fluorescence accumulated in cells over time (fig. 5).
Example 7: evaluation of anti-inflammatory Effect on schizonepeta-derived exosomes
To examine whether the schizonepeta-derived exosomes prepared as described in example 2 exhibited anti-inflammatory effects, the effect of schizonepeta-derived exosomes on IL-6 production in mouse macrophage RAW 264.7 cells was evaluated. RAW 264.7 cells suspended in DMEM medium containing 10% FBS were distributed into each well of a multi-well plate to achieve a confluency rate of 80% to 90%. The next day, RAW 264.7 cells were treated with schizonepeta-derived exosomes diluted in fresh medium containing LPS [ DMEM containing 1% FBS and 200nM LPS (lipopolysaccharide) ] and then RAW 264.7 cells were cultured for 24 hours. The experimental groups for evaluating the anti-inflammatory effect were classified as follows.
(1) Negative control group (n.c.): an experimental group in which RAW264.7 cells were treated with a medium containing LPS alone;
(2) Positive control group (p.c.): an experimental group in which RAW 264.7 cells were treated with a medium containing LPS mixed with dexamethasone (final concentration: 200. Mu.M);
(3) The group treated with low concentration of schizonepeta-derived exosomes (denoted "low" in fig. 6): an experimental group in which RAW 264.7 cells were treated with a schizonepeta-derived exosome (prepared in example 2) diluted in a medium containing LPS (treatment concentration: 6.0X10 9 particles/mL); and
(4) The group treated with high concentrations of schizonepeta-derived exosomes (denoted "high" in fig. 6): an experimental group (treatment concentration: 2.0X10 10 particles/mL) in which RAW 264.7 cells were treated with schizonepeta-derived exosomes diluted in LPS-containing medium (prepared in example 2).
After completion of culture of RAW 264.7 cells in each experimental group, culture supernatants were collected, and the production amount of inflammatory cytokine IL-6 present in the culture supernatants was measured using IL-6ELISA kit. The amount of IL-6 (inflammatory cytokine) produced in the group treated with LPS alone and the amount of IL-6 produced in each experimental group treated with LPS-containing medium mixed with dexamethasone and schizonepeta-derived exosomes (low-concentration and high-concentration exosomes) were measured separately using ELISA kits (purchased from R & D Systems) according to manufacturer's manual (FIG. 6).
As a result, it was confirmed that IL-6 production was inhibited in the following experimental groups compared with that in the negative control group: wherein RAW 264.7 cells were treated with LPS plus schizonepeta-derived exosomes (low and high concentrations of exosomes) prepared in example 2 (fig. 6).
As can be seen from the above experimental results, the schizonepeta-derived exosomes of the present invention have excellent anti-inflammatory effects. Thus, the schizonepeta-derived exosomes of the present invention are useful as active ingredients in pharmaceutical compositions for anti-inflammatory, wound healing and/or accelerating wound healing.
While the invention has been described with reference to certain embodiments, the scope of the invention is not limited to these embodiments. It will be understood by those skilled in the art that various modifications and changes may be made without departing from the spirit and scope of the present invention, and that such modifications and changes also fall within the scope of the present invention.
Claims (19)
1. Cosmetic composition for improving skin elasticity, reducing skin wrinkles, and regenerating skin comprises exosomes derived from herba Schizonepetae (NEPETA CARTARIA) as active ingredient.
2. The cosmetic composition of claim 1, wherein the composition is a shampoo, soap, cleanser, surfactant-containing cleanser, cream, emulsion, ointment, lotion, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, mist or powder.
3. The cosmetic composition of claim 1 or 2, wherein the exosomes are isolated from a culture medium of schizonepeta plant cells, a culture medium of schizonepeta callus, a culture medium of schizonepeta plant stem cells, schizonepeta juice, or a schizonepeta biological solution equivalent thereto, or are derived from plant cells or plant stem cells of schizonepeta.
4. The cosmetic composition of claim 3, wherein the exosomes are isolated and purified from nepeta juice.
5. A pharmaceutical composition for anti-inflammatory, wound healing or accelerating wound healing comprising exosomes derived from schizonepeta as active ingredients.
6. The pharmaceutical composition of claim 5, wherein the composition is administered or treated by injection, microneedle, iontophoresis, coating, or a combination thereof.
7. The pharmaceutical composition of claim 5, wherein the composition is an injectable formulation, an infusible formulation, a spray formulation, a liquid formulation, or a patch formulation.
8. The pharmaceutical composition of any one of claims 5 to 7, wherein the exosomes are isolated from the medium of schizonepeta plant cells, the medium of schizonepeta callus, the medium of schizonepeta plant stem cells, schizonepeta juice, or a schizonepeta biological solution equivalent thereto, or the exosomes are derived from plant cells or plant stem cells of schizonepeta.
9. The pharmaceutical composition of claim 8, wherein the exosomes are isolated and purified from nepeta juice.
10. Cosmetic method for non-therapeutic purposes of improvement of skin elasticity, reduction of skin wrinkles or skin regeneration of mammalian skin, using a cosmetic composition comprising exosomes derived from schizonepeta as active ingredient.
11. The cosmetic method of claim 10, wherein the method comprises:
(a) Applying the cosmetic composition directly to the mammalian skin; or (b) contacting or adhering a patch, sheet pack or facial mask towel to or onto the mammalian skin, the patch, sheet pack or facial mask towel having the cosmetic composition applied thereto or immersed therein; or (a) and (b) may be performed sequentially.
12. The cosmetic method of claim 11, wherein in step (a), the cosmetic composition is an emulsion or cream.
13. The cosmetic method of claim 11 or 12, further comprising step (c): removing the patch, the sheet pack or the pack towel from the mammalian skin after step (b), and applying the cosmetic composition to the mammalian skin.
14. The cosmetic method of claim 13, wherein in step (c), the cosmetic composition is an emulsion or cream.
15. The cosmetic method of any one of claims 10 to 12, wherein the mammal is a cat, human, dog, rodent, horse, cow, monkey or pig.
16. A method for treating inflammation, promoting wound healing or accelerating wound healing, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an exosome derived from schizonepeta as an active ingredient to a mammal other than a human, or administering a pharmaceutical composition comprising an exosome derived from schizonepeta as an active ingredient to the skin, inflammatory region or injured region of a mammal other than a human.
17. The method of claim 16, wherein the pharmaceutical composition is administered by injection, microneedle, iontophoresis, or a combination thereof.
18. The method of claim 16, wherein the pharmaceutical composition is an injectable formulation, an infusible formulation, a spray formulation, a liquid formulation, or a patch formulation.
19. The method of any one of claims 16 to 18, wherein the mammal is a cat, dog, rodent, horse, cow, monkey, or pig.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2022-0010217 | 2022-01-24 | ||
| KR10-2023-0006863 | 2023-01-17 |
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| Publication Number | Publication Date |
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| CN118338892A true CN118338892A (en) | 2024-07-12 |
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