KR102494244B1 - Composition for skin regeneration and wound treatment comprising an absolute type essential oil derived from a Smilax china Flower as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for skin regeneration and wound healing comprising absolute essential oil derived from Smilax china Flower as an active ingredient, and specifically, to extracting absolute type essential oil from Smilax china Flower A method is provided, and the essential oil prepared through the method has confirmed excellent skin regeneration, skin elasticity enhancement and wound healing effects through cell proliferation and migration and collagen synthesis induction effects at the skin cell level, so this is a cosmetic composition or a composition for external application for skin. can be used as

Description

Composition for skin regeneration and wound treatment comprising an absolute type essential oil derived from a Smilax china Flower as an active ingredient}

The present invention relates to a composition for skin regeneration and wound healing comprising, as an active ingredient, absolute type essential oil derived from Smilax china Flower, and more specifically, absolute type from Smilax china Flower It relates to a method for extracting essential oil and a cosmetic composition or composition for external application for skin containing the essential oil prepared therefrom.

Skin is a barrier that minimizes the loss of water in the body and protects the human body from inflow of harmful substances (pathogens and allergens) from the external environment and physical stress, and consists of the epidermis, dermis, and subcutaneous fat. ) consists of three layers. Skin is a thin layer composed of squamous epithelium, and keratinocytes account for 95% of the skin epidermis. Keratinocytes of the epidermis protect the skin by forming keratin through proliferation, migration, and differentiation from the basal layer, and are involved in inflammatory and immune responses by producing various cytokines. In addition, by inducing collagen synthesis, the elasticity of the skin and the formation of the basement membrane are improved to control the adhesion and proliferation of skin cells, improve the barrier function to selectively penetrate harmful substances, and interact with the epidermis and dermis. provides Type I, III, IV, V, VI and VII collagen are distributed in human skin. Type I collagen constitutes more than 85% of the dermal layer of the skin, and type IV collagen is the main structural protein of the basement membrane. Synthesis of type I and IV collagen enhances skin elasticity and improves basement membrane formation, thereby regulating the adhesion and proliferation of skin cells. Due to this, migration and proliferation of skin epidermal keratinocytes and induction of collagen synthesis promote skin regeneration and wound healing as well as promote skin elasticity and help maintain healthy skin.

"Essential oil" derived from natural products is a general term for aromatic oils (fragrant and volatile substances obtained from plant leaves, stems, fruits, flowers, roots, etc.), and is classified in various ways according to extracts, extraction methods, uses and purposes. Essential oils derived from natural products are classified into essential oils, absolutes, and concretes according to extraction methods. The aromatic components of essential oils derived from natural products have effects such as soothing and relieving tension through the skin, circulatory system, and olfactory system, and have therapeutic effects such as antibacterial action, immunity enhancement, and anti-allergic action. It is also used as a raw material for food flavoring and cosmetics.

On the other hand, Smilax China is a creeping shrub belonging to the Liliaceae family (Smilacaceae). It is a thorny vine that is common in various places in the south of Hwanghae-do in Korea, and its roots are thick and winding. The leaves are alternate, round, glossy green, and the edges are flat. Flowers bloom yellow on trees picked by males and females in late spring. Smilax vine grows to a length of 2 m and flowers bloom in May. Its medicinal properties are flat and low, and it has the effects of bloating, diuresis, and detoxification, so it is used as a treatment for joint pain, hemp, species, inflammation, dysentery, lymphadenitis, syphilis, and carcinoma.

However, research on skin physiological activity using Smilax chinensis is insufficient to date, and in particular, there is no study using essential oil contained in Smilax chinensis in large quantities. While studying the in vitro skin restoring effect by extracting essential oil from Smilax chinensis flowers, the present inventors found that the essential oil derived from Smilax chinensis flower has excellent skin regeneration, skin elasticity enhancement and wound healing through cell proliferation and migration and collagen synthesis inducing effects. It was confirmed that the effect was shown and the present invention was completed.

KR10-2014-0072126

In order to solve the above problems, the present invention has completed the present invention by extracting essential oil from Smilax chinensis flowers, identifying its components, and confirming skin elasticity enhancement, skin regeneration and wound healing effects. In addition, the present invention is to provide a manufacturing method of Smilax china L. flower essential oil.

In order to solve the above problems, the present invention provides a cosmetic composition for skin regeneration or wrinkle improvement containing essential oil extracted from Smilax China flowers as an active ingredient.

In one embodiment of the present invention, the essential oil may be characterized in that it is extracted by an organic solvent extraction method using a volatile solvent, but is not limited thereto.

In one embodiment of the present invention, the flowers of Smilax China may not be dried.

In one embodiment of the present invention, the essential oil may be an absolute obtained by extracting concrete extracted by an organic solvent extraction method with alcohol to remove insoluble wax, but is not limited thereto.

In one embodiment of the present invention, the composition is nonanal, tetradecanal, nonadecane, n-hexadecanoic acid, ethyl palmitate ), n-Eicosan, 1-Nonadecene, 1-Heptadecene, n-Heneicosane, 9,12,15-octa Decatrinoic acid, methyl ester (9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)), Docosane, Muscalure, Tricosane, tetracosane ( Tetracosane）, 1-Docosane, Pentacosane, Hexacosane, 1-Octadecanol, Heptacosane and β-Sitosterol ), but may be characterized in that it includes a volatile organic compound of, but is not limited thereto.

In one embodiment of the present invention, the essential oil may be characterized in that it is included in the range of 1 ~ 250 μg / mL, but is not limited thereto.

In one embodiment of the present invention, the composition may induce proliferation and migration of epidermal keratinocytes, but is not limited thereto.

In one embodiment of the present invention, the composition induces the phosphorylation process and signal transduction process of serine/threonine protein kinase (Akt) or extra-epidermal keratinocyte signal-regulating kinase (Erk1/2) in epidermal keratinocytes It can be characterized, but is not limited thereto.

In one embodiment of the present invention, the composition may be characterized in that it induces the synthesis of epidermal keratinocyte collagen type I or collagen type IV, but is not limited thereto.

In one embodiment of the present invention, the composition may be characterized in that it induces an out growth promoting effect of epidermal keratinocyte collagen type I or collagen type IV sprouting, but is not limited thereto. .

In addition, the present invention is a composition for external application for skin for wound treatment comprising essential oil extracted from flowers of Smilax China as an active ingredient, wherein the essential oil is extracted with alcohol from concrete extracted by an organic solvent extraction method to obtain insoluble wax. Characterized in that it is an absolute removed, characterized in that the essential oil is included in the range of 1 to 250 μg / mL, characterized in that the composition induces the proliferation and migration of epidermal keratinocytes, and the composition is characterized in that the epidermis It is characterized by inducing the phosphorylation process or signal transduction process of serine/threonine protein kinase (Akt) or epidermal keratinocyte extracellular signal-regulating kinase (Erk1/2) in keratinocytes, and the composition is epidermal keratinocyte collagen type It is characterized by inducing the synthesis of collagen type I or collagen type IV, and the composition induces an out growth promoting effect of epidermal keratinocyte collagen type I or collagen type IV sprouting. Provided is a composition for external application for skin for treatment.

In addition, the present invention includes a first step of washing the buds of Smilax chinensis just before it blooms; a second step of precipitating the Smilax chinensis flower buds in n-hexane; A third step of obtaining solid concrete by evaporating and concentrating the precipitate in a rotary evaporation; A fourth step of dissolving the concrete in ethyl alcohol, storing it at minus 20 ° C, and then removing insoluble wax through a vacuum cooling filtration; and 5 steps of preparing Smilax china L. flower essential oil by concentrating, separating, and purifying using a rotary evaporation.

The present invention identifies the components of Absolute type essential oil extracted from Smilax chinensis flowers, and proves its skin elasticity enhancement, skin regeneration and wound healing effects, and induces cell proliferation and migration at the skin cell level for skin regeneration and wound healing has an excellent effect on In addition, the essential oil extracted from Smilax china L. flower according to the present invention exhibits excellent skin regeneration and wound healing effects, and thus can be applied in various ways such as cosmetics or external skin preparations.

1 is a flow chart showing a process of extracting absolute type essential oil from Smilax china L. flowers in one embodiment of the present invention.
2 is a graph showing the toxicity of Absolute type essential oil extracted from Smilax china L. flower to epidermal keratinocytes in one embodiment of the present invention.
Figure 3 is for the proliferation and migration effect of epidermal keratinocytes of Smilax china L. flower absolute type essential oil. (A) is a graph showing the effect of Smilax china L. flower Absolute type essential oil on the proliferation of epidermal keratinocytes in one embodiment of the present invention, and (B) is Smilax china L. flower Absolute type in one embodiment of the present invention It is a graph showing the effect of essential oil on migration of epidermal keratinocytes.
4 is a diagram showing the effect of Smilax china L. flower absolute type essential oil on collagen synthesis. (A) is a graph showing the quantification of the effect of Smilax china L. flower absolute type essential oil on the synthesis of collagen type I in one embodiment of the present invention, and (B) is Smilax china L. flower Absolute type in one embodiment of the present invention It is a graph showing the quantification of the effect of essential oil on collagen type IV synthesis.
Figure 5a is an image showing the effect of Smilax china L. flower absolute type essential oil on collagen sprouting out growth in epidermal keratinocytes according to an embodiment of the present invention.
5B is a graph showing the quantification of the effect of Smilax china L. flower absolute type essential oil on collagen sprouting out growth in epidermal keratinocytes according to an embodiment of the present invention.
Figure 6a is an image showing the effect of Smilax china L. flower absolute type essential oil on phosphorylation of serine/threonine protein kinase (Akt) and extracellular signal-regulating kinase (Erk1/2) in epidermal keratinocytes in one embodiment.
6B is a graph showing the quantification of the effect of Smilax china L. flower absolute type essential oil on the phosphorylation of serine/threonine protein kinase (Akt) in epidermal keratinocytes according to one example.
6C is a graph showing the quantification of the effect of Smilax china L. flower absolute type essential oil on the phosphorylation of epidermal keratinocyte extracellular signal-regulating kinase (Erk1/2) according to one embodiment.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following implementation examples are presented as examples of the present invention, and if it is determined that a detailed description of a well-known technology or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the description of the claims described later and equivalent scopes interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express a preferred embodiment of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the contents throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

Throughout this specification, '%' used to indicate the concentration of a particular substance is solid/solid (w/w) %, solid/liquid (w/v) %, and Liquid/liquid is (v/v) %.

In one aspect, it relates to a cosmetic composition for skin regeneration or wrinkle improvement comprising essential oil extracted from Smilax China flowers as an active ingredient.

In one embodiment, the essential oil can be prepared by extracting Smilax China flowers by an organic solvent extraction method to obtain concrete, dissolving the same in an organic solvent and filtering to remove insoluble wax to obtain an absolute product.

In one embodiment, a first step of washing the flower buds of Smilax China of the present invention just before blooming; a second step of precipitating the Smilax chinensis flower buds in n-hexane; A third step of obtaining solid concrete by evaporating and concentrating the precipitate in a rotary evaporation; A fourth step of dissolving the concrete in ethyl alcohol, storing it at minus 20 ° C, and then removing insoluble wax through a vacuum cooling filtration; and 5 steps of preparing Smilax china L. flower essential oil by concentrating, separating, and purifying using a rotary evaporation, whereby Smilax china L. flower essential oil having a skin regeneration or wrinkle improvement effect can be prepared.

The organic solvent extraction method and the method of separating the absolute from the extract may include all methods that can be modified by a person skilled in the art.

In one embodiment, the essential oil extracted from Smilax China flowers is nonanal, tetradecanal, nonadecane, n-hexadecanoic acid, ethyl palmylic acid. Ethyl palmitate, n-Eicosan, 1-Nonadecene, 1-Heptadecene, n-Heneicosane, 9,12 ,15-Octadecatrienoic acid, methyl ester (9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)), Docosane, Muscalure, Tricosane , Tetracosane, 1-Docosane, Pentacosane, Hexacosane, 1-Octadecanol, Heptacosane and β-Sitosterol (β-Sitosterol) may contain volatile organic compound components.

In one embodiment, Smilax China flowers may be buds just before blooming and may be undried flowers.

In one embodiment, the cosmetic composition of the present invention may use essential oil extracted from Smilax China flowers at a concentration of 1 to 250 μg/mL, and it is best to include essential oil at a concentration of 50 to 250 μg/mL. It is more preferable because it has regenerative and wound healing effects.

The term "essential oil" used in the present invention is a general term for aromatic oils (volatile substances) extracted and separated from plants (plant leaves, stems, fruits, flowers, roots, etc.), extracts, extraction methods, uses and purposes Essential oils derived from natural products are classified into essential oils, absolutes, concretes, etc., depending on the extraction method.

As used herein, the term "extract" means an active ingredient isolated from a natural product, that is, a material showing a desired activity. The extract may be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes any form formulated using the dry powder or the extract thereof. In addition, the extract includes a fractionated extract that has undergone the extraction process. The extraction method of the extract is not particularly limited, and may be extracted by, for example, stirring extraction, shaking extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction. As the extraction solvent, a polar solvent such as water or a C ₁ -C ₄ lower alcohol, a non-polar solvent such as hexane, chloroform, dichloromethane or ethyl acetate, or a mixture of two or more thereof may be used.

The cosmetic composition of the present invention may be prepared by including a cosmetically effective amount of the essential oil extracted from Smilax China flower of the present invention described above and a cosmetically acceptable carrier.

As used herein, the term "cosmetically effective amount" means an amount sufficient to achieve the skin regeneration effect through the proliferation of epidermal keratinocytes of the composition of the present invention described above.

The appearance of the cosmetic composition contains a cosmetic or dermatologically acceptable medium or base. These are all formulations suitable for topical application, e.g. solutions, gels, solids, anhydrous pasty products, emulsions obtained by dispersing an oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and It may be provided in the form of a non-ionic follicular dispersant, or in the form of a cream, toner, lotion, powder, ointment, spray or conceal stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition according to an embodiment of the present invention is not particularly limited in its dosage form, for example, softening lotion, astringent lotion, nutrient lotion, nutrient cream, massage cream, essence, eye cream, eye essence, cleansing It can be formulated into cosmetics such as cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil and body essence.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fibers, vegetable fibers, wax, paraffin, starch, tracanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. this can be used

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro propellants such as carbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate , fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives, or ethoxylated glycerol fatty acid esters.

The cosmetic composition of the present invention can be applied to skin, lotion, cream, essence, pack, foundation, color cosmetics, sunscreen, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, eyebrow pencil , It can be applied to cosmetics such as lotion and detergents such as shampoo and soap.

The cosmetic composition according to an embodiment of the present invention may further include functional additives and ingredients included in general cosmetic compositions in addition to the essential oil extracted from the Smilax China flower. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts.

In addition to the above functional additives, the cosmetic composition of the present invention may further contain components included in general cosmetic compositions as needed. Ingredients other than those included include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation accelerators, cooling agents, antiperspirants, purified water and the like.

In one aspect, the present invention is a composition for external application for skin for wound treatment comprising, as an active ingredient, an essential oil extracted from Smilax China flowers, wherein the essential oil is insoluble by extracting concrete extracted by an organic solvent extraction method with alcohol. It is characterized in that it is an absolute wax from which wax is removed, and the essential oil is included in the range of 1 to 250 μg/mL, and the composition induces proliferation and migration of epidermal keratinocytes, wherein the composition is characterized in that it induces the phosphorylation process or signal transduction process of serine/threonine protein kinase (Akt) or signal-regulating kinase (Erk1/2) outside epidermal keratinocytes in epidermal keratinocytes, and the composition is characterized in that epidermal keratinocytes Characterized in that the synthesis of collagen type I or collagen type IV is induced, and the composition induces an out growth promoting effect of epidermal keratinocyte collagen type I or collagen type IV sprouting. , It relates to a composition for external application for skin for wound treatment.

In one embodiment, the composition for external application for skin of the present invention may be used for epidermal keratinocyte proliferation, regeneration, and collagen formation, which is used for skin elasticity, regeneration, and/or Or it may be useful for wound healing.

In one embodiment, the composition for external application for skin of the present invention may use essential oil extracted from Smilax China flowers at a concentration of 1 to 250 μg/mL, and the concentration of 50 to 250 μg/mL is most excellent. It is more preferable because it has skin regeneration and wound healing effects.

In one aspect, the present invention comprises a first step of washing the buds of Smilax china L. flowers just before they bloom; a second step of precipitating the Smilax chinensis flower buds in n-hexane; A third step of obtaining solid concrete by evaporating and concentrating the precipitate in a rotary evaporation; A fourth step of dissolving the concrete in ethyl alcohol, storing it at minus 20 ° C, and then removing insoluble wax through a vacuum cooling filtration; and 5 steps of preparing Smilax china L. flower essential oil by concentrating, separating, and purifying using a rotary evaporation.

Advantages and characteristics of the present invention, and methods for achieving them will become clear with reference to examples or experimental examples described later in detail. Hereinafter, the present invention will be described in detail by examples or experimental examples. However, these examples or experimental examples are intended to specifically explain the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of Smilax chinensis flower absolute type essential oil

1 is a flowchart showing a process of extracting essential oil from Smilax china L. flowers according to a preferred embodiment of the present invention, and Smilax china L. flower essential oil was prepared by the method shown here. In this embodiment, Smilax chinensis flowers, which grow wild at Hoseo University (Asan, Korea), were collected in May and used. The whole flower buds of Smilax chinensis were collected just before blooming, washed, and then extracted using organic solvent extraction method by 3 kg of raw grass. That is, non-dried Smilax chinensis flowers were precipitated in n-hexane, and the precipitate thus obtained was evaporated and concentrated in a rotary evaporation to obtain solid concrete. Then, the concrete was dissolved by adding ethyl alcohol, stored at minus 20 ° C, passed through a vacuum cooling filtration to remove insoluble wax, and concentrated using a rotary evaporator again and separation/purification to prepare Smilax chinensis flower essential oil. In addition, the prepared Smilax china L. flower essential oil was stored in a freezer at minus 80 ° C.

Example 2. Component analysis of Absolute type essential oil from Smilax chinensis flower

The component analysis of Smilax china L. flower essential oil was requested to KBSI (Korea Basic Science Institute, Seoul Center). A gas chromatography/mass spectrometer (6890N GC/5975i MS, Agilent, USA) was used, and a DB5-MS (30 m × 50 μm, 0.25 μm) was used as a column of GC/MS, and a carrier gas ( carrier gas) was analyzed by flowing helium at 1 mL/min. The temperature of the oven was maintained at 40℃-2℃/min-230℃-5℃/min-300℃ (5 minutes), and the injection temperature was analyzed at 280℃. In addition, the temperature of the interface was analyzed at 300℃, the temperature of the ion source was 230℃, and the temperature of the analyzer was analyzed at 150℃, and the mass range was analyzed at 40~800 m/z.

Identification of Smilax china L. flower essential oil components was confirmed by matching the retention indices (RI) of volatile components with the retention times of standard substances or by comparing the mass spectrum of volatile substances with the mass spectrum of the Wiley 7Nist 05 library. RI was determined under the same conditions as the sample using C6 ~ C24 (nalkane). As a result, it was confirmed that it was composed of a total of 20 components as shown in Table 1 below.

Experimental Example 1. Measurement of toxicity (cell viability) of Smilax chinensis Absolute type essential oil on keratinocytes

The toxicity (cell viability) on keratinocytes was investigated using Smilax china L. essential oil obtained in Example 1 above. The keratinocytes were put in a 96 well-plate coated with 0.1 mg/mL of collagen (collagen, type I) at a concentration of 5 × 10 ³ cells/100 μL/well, and then incubated in a 37°C, 5% CO ₂ incubator for 12 hours. cultured. After 12 hours of culture, Smilax chinensis essential oil was treated at a concentration of 1 to 250 μg/mL and cultured for 24 hours. After treatment with EZ-cytox, absorbance was measured at 450 nm, and the results are shown in FIG. 2 .

As shown in FIG. 2, the cytotoxicity of keratinocytes was not observed in Smilax china L. essential oil at a concentration of 1 to 250 μg/mL, and in particular, at a concentration of 50 to 150 μg/mL, cell viability increased. . Therefore, it was confirmed that the concentration of the essential oil component of Smilax chinensis used in the present invention was preferably 250 μg/mL or less, and was applied to subsequent experiments.

Experimental Example 2. Effect of Absolute type essential oil of Smilax china L. flower on the proliferation of epidermal keratinocytes

In order to confirm the effect of Smilax china L. flower essential oil obtained in Example 1 on the proliferation of skin epidermal keratinocytes, the experiment was conducted as follows.

Human skin-derived keratinocytes (HaCat) were cultured in a DMEM medium containing 10% fetal bovine serum and used in the experimental example of the present invention. Epidermal keratinocytes were injected into a 96-well microtiter plate so that the number of cells per well was 3 X 10 ³ cells/100 μL/well, and cultured in a 37°C, 5% CO ₂ incubator.

Each well was treated with 100 μL at a concentration of 1 to 250 μg/mL essential oil of Smilax chinensis, and incubated for 48 hours. EZ-cytox and DMEM were mixed at a ratio of 1:2, treated with 30 μL per well, and reacted in an incubator for 30 minutes. After measuring the absorbance at 450 nm using an ELISA plate reader (Synerge2, BioTek, USA), the result of skin epidermal keratinocyte proliferation of Smilax china L. flower essential oil was obtained by converting it into percentage.

3(A) is a graph showing the proliferation effect of epidermal keratinocytes of Smilax china L. flower essential oil extracted in Example 1 of the present invention. Here, the negative control group did not use Smilax china L. flower essential oil, and the positive control group used epidermal growth factor (EGF 50 ng/mL) conventionally used for wound healing and skin regeneration.

As a result, as shown in FIG. 3(A), it was found that the Smilax china L. flower essential oil according to the present invention induces the proliferation of epidermal keratinocytes in a concentration-dependent manner.

That is, the proliferation of epidermal keratinocytes by epidermal growth factor (50 ng/mL) was induced by 137.08±10.99%, and compared to the positive control, 144.91±9.38% of proliferation was induced at the concentration of 100 μg/mL of Smilax china L. flower essential oil. In particular, at a concentration of 150 μg/mL, a proliferation induction rate of 183.34±9.60% was shown, effectively inducing the proliferation of keratinocytes.

Experimental Example 3. Effect of Absolute type essential oil on migration of epidermal keratinocytes

In order to confirm the effect of Smilax china L. flower essential oil obtained in Example 1 on migration of skin epidermal keratinocytes, the experiment was conducted as follows.

Human skin-derived epidermal keratinocytes (HaCat) were cultured in a DMEM medium containing 10% fetal bovine serum and used in the experimental example of the present invention. The migration rate of epidermal keratinocytes was investigated using a 48-well Boyden microchemotaxis chamber. First, Smilax china L. flower essential oil was treated in the lower chamber at a concentration of 1 to 250 μg/mL, respectively, and type I collagen-coated 8 μm pores A polycarbonate membrane was placed on top. Thereafter, epidermal keratinocytes were treated in the upper chamber at 1 × 10 ⁶ cells/50 μL per well. After incubating the chamber in a 37°C incubator for 3 hours and 30 minutes, the membrane was fixed and stained with Diff-Quick solution.

The number of cells that migrated through the Membrane was counted and converted into percentage to secure the result of migration of epidermal keratinocytes of Smilax china L. flower essential oil.

3(B) is a graph showing the migrating effect of skin keratinocytes of Smilax china L. flower essential oil extracted according to Example 1 of the present invention. In the negative control group, Smilax chinensis flower essential oil was not used, and in the positive control group, epidermal growth factor (EGF 1 ng/mL), which is conventionally used for wound healing and skin regeneration, was used.

As a result, as shown in FIG. 3(B), Smilax china L. flower essential oil according to Example 1 of the present invention induced migration of epidermal keratinocytes in a concentration-dependent manner. In particular, 100 μg/mL of Smilax china L. flower essential oil Concentration showed a migration induction rate of 208.57 ± 11.92%, and a maximum migration rate of 257.30 ± 8.93% at a concentration of 150 μg/mL, indicating that Smilax china L. flower essential oil effectively induces migration of keratinocytes.

Experimental Example 4. Effect of Absolute type essential oil from Smilax china L. flower on the synthesis of collagen type I and collagen type IV in epidermal keratinocytes

In order to measure the effect of Smilax china L. flower essential oil obtained in Example 1 on the synthesis of collagen type I and IV, which are ECM constituents necessary for the proliferation and migration of epidermal keratinocytes, the following experiment was conducted.

After injecting monoclonal anti-collagen I or IV Anti-body (2 μg/well/PBS) into a 96-well microtiter plate, the plate was coated at room temperature for 12 hours. After blocking for 1 hour at room temperature with blocking buffer, samples were treated at 100 μL per well and reacted at room temperature for 90 minutes. Samples were culture medium (CM) of epidermal keratinocytes not treated with Smilax china L. flower essential oil and epidermal keratinization treated with Smilax china L. flower essential oil at concentrations of 50 μg/mL, 100 μg/mL, 150 μg/mL, and 200 μg/mL. The culture medium (CM) of the cells was treated. Then, polyclonal anti-collagen I or IV Anti-body was treated in each well and reacted at room temperature for 90 minutes, and then avidin-HRP was treated in each well and reacted at room temperature for 60 minutes. Supersignal ELISA pico chemiluminescent substrate was injected at 100 μL per well and reacted for 10 minutes, and then the degree of luminescence was measured using an ELISA plate reader. The results for secretion are shown in FIG. 4 . In particular, as shown in FIG. 4(A), the synthesis of collagen type I was induced by 169.47±1.89% at the concentration of 200 μg/mL essential oil of Smilax china L. flower, and as shown in FIG. 4(B), 150 μg of Smilax china L. flower essential oil 163.56±1.52% of the synthesis of collagen type IV was induced at /mL concentration.

Experimental Example 5. Effect of Absolute type essential oil of Smilax chinensis on the out growth of epidermal keratinocyte collagen sprouting

In order to measure the effect of Smilax china L. flower essential oil obtained in Example 1 on the out growth of epidermal keratinocyte collagen sprouting, an experiment was conducted as follows.

Type I collagen and epidermal keratinocytes were mixed to a concentration of 2.5 × 10 ⁷ cells/mL, neutralized by treatment with NaOH (1 N) solution, and then spotted by 5 μL on a 24-well plate and dried at room temperature for 10 minutes. An artificial skin tissue was formed. Samples were treated for each concentration and cultured in an incubator at 37° C. and 5% CO ₂ for 48 hours. The culture medium was removed, cells and tissues were fixed and stained using Diff-Quik solution, and the results were obtained by photographing the stained cells and tissues.

5 is a result showing the effect of Smilax china L. flower essential oil extracted in Example 1 of the present invention on the outgrowth of collagen sprouting of epidermal keratinocytes, and the negative control group was without using Smilax china L. flower essential oil, and the positive control group is using epidermal growth factor (EGF 50 ng/mL), which is used for conventional wound healing and skin regeneration.

As a result, as shown in FIG. 5a, Smilax chinensis flower essential oil was found to induce the outgrowth of epidermal keratinocyte collagen sprouting in a concentration-dependent manner. That is, compared to the positive control group, it was found that 150 μg/mL of Smilax chinensis flower essential oil effectively induced outgrowth of keratinocyte collagen sprouting. In particular, as shown in FIG. 5B, 215.40 ± 14.37% of Smilax chinensis flower essential oil was induced at a concentration of 100 µg/mL, and in particular, 265.03 ± 14.37% was induced at a concentration of 150 µg/mL.

Experimental Example 6. Investigation of the effect of Smilax chinensis flower absolute type essential oil on the signal transduction process in epidermal keratinocytes

Phosphorylation process of intracellular serine/threonine protein kinase (Akt) and signal-regulating kinase (Erk1/2), which are the main signal transduction processes in which Smilax china L. flower essential oil obtained in Example 1 regulates the proliferation and migration of epidermal keratinocytes. In order to measure the effect on Smilax chinensis, the concentration of Smilax china L. flower essential oil presented in Example 3 was treated, and immunoblot was performed using each specific antibody (P-Akt, Akt, P-Erk1/2, Erk1/2). (Immuno Blot) method was used.

Figure 6a shows the effect of Smilax china L. flower essential oil extracted in Example 1 of the present invention on the phosphorylation process of serine/threonine protein kinase (Akt) and extracellular signal-regulating kinase (Erk1/2) in epidermal keratinocytes. In the negative control group, Smilax chinensis flower essential oil was not used, and in the positive control group, epidermal growth factor (EGF 50 ng/mL) used for wound healing and skin regeneration was used.

As a result, as shown in FIGS. 6b and 6c, Smilax china L. flower essential oil induced phosphorylation of serine/threonine protein kinase (Akt) and extracellular signal-regulating kinase (Erk1/2) in epidermal cells in a concentration-dependent manner, particularly , Phosphorylation of intracellular serine/threonine protein kinase (Akt) was induced by 141.76±7.51%, and phosphorylation of extracellular signal-regulating kinase (Erk1/2) was induced by 163.09±8.41% at the concentration of 150 μg/mL essential oil of Smilax chinensis flower. appeared to be

So far, the present invention has been looked at mainly with its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from a descriptive point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (11)

In a composition for external application for skin for wound treatment comprising essential oil extracted from Smilax China flowers as an active ingredient,
The essential oil is an absolute composition obtained by dissolving concrete extracted by hexane in alcohol to remove insoluble wax. delete delete According to claim 1,
The essential oils are Nonanal, Tetradecanal, Nonadecane, n-Hexadecanoic acid, Ethyl palmitate, n-Eicosan Eicosan), 1-Nonadecene, 1-Heptadecene, n-Heneicosane, 9,12,15-Octadecatrinoate, methyl ester (9, 12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)), Docosane, Muscalure, Tricosane, Tetracosane, 1-Docosane (1 Containing volatile organic compounds of -Docosane, Pentacosane, Hexacosane, 1-Octadecanol, Heptacosane and β-Sitosterol phosphorus, composition. According to claim 1,
The essential oil is contained in the range of 1 ~ 250 μg / mL, the composition. According to claim 1,
The composition, wherein the essential oil induces the proliferation and migration of epidermal keratinocytes. According to claim 1,
Wherein the essential oil induces the phosphorylation process and signal transduction process of serine/threonine protein kinase (Akt) or epidermal keratinocyte signal-regulating kinase (Erk1/2) in epidermal keratinocytes. According to claim 1,
The essential oil is to induce the synthesis of epidermal keratinocyte collagen type I or collagen type IV, composition. According to claim 1,
The composition, wherein the essential oil induces an out growth promoting effect of epidermal keratinocyte collagen type I or collagen type IV sprouting. delete delete

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