KR102088439B1 - Nrf2 active complex, Nrf2 induction revitalizing agent, Revitalizing cosmetics containing the same and Manufacturing method thereof - Google Patents

Abstract

The present invention relates to an Nrf2 activating complex having high active oxygen scavenging activity as well as having NO activity suppression and skin whitening effects, a cosmetic product comprising the same, a serum type cosmetic product (including a mask pack), and a method for manufacturing the same. More specifically, provided is an invention which introduces different two types of materials in addition to an Inula japonica extract as an Nrf2 activator, and manufactures an Nrf2 induction revitalizing formulation using the same to support activation of Nrf2 distributed in the skin epidermal layer and highly improve active oxygen scavenging performance in the skin, thereby providing cosmetic products having excellent skin elasticity improvement and antioxidant effects and having excellent skin inflammation prevention and skin whitening effects.

Description

Nrf2 active complex, Nrf2 induction revitalizing agent containing the same, cosmetic containing the same and method for manufacturing the same {Nrf2 active complex, Nrf2 induction revitalizing agent, Revitalizing cosmetics containing the same and Manufacturing method thereof}

The present invention has an active oxygen scavenging activity while having a skin whitening effect Nrf2 active complex, a formulation comprising the Nrf2 active complex that effectively restores stressed and tired skin, cosmetics using it, serum type cosmetics (including mask packs), and It relates to a method of manufacturing it.

As the root stem, the roots are discoid, columnar, etc., and the shape is uneven. The discoid is 3 ~ 10cm in diameter, 0.5 ~ 3cm in thickness, and the thin one is almost columnar, 1 ~ 3cm in diameter, and 5 ~ 8cm in length. Some are split vertically. The cross-section is gray-brown to yellowish brown, and the brown layer is clear in the vicinity of the formation layer. And, the side is dark brown, there are transverse wrinkles and the skin is yellowish brown, the diameter of the neck is about 3 to 4 times the skin, and the outer surface is very hard but not fibrous. In addition, it has a peculiar smell and taste is bitter and bitter. When chewing it, it is like chewing fine sand. Diabetes prevention and treatment composition using the extract of bokbokhwa (Korea Publication No. 2005-0003669), plant disease control activator (Korea Publication No. 2010-0048351), constipation treatment (Korea Registration No. 1286467) Although the technology has been known, there is no known technology for a skin improving agent using a bokbok extract.

Free radicals are substances that are constantly made in the normal metabolic process of the human body, and usually 2 to 5% of the oxygen we breathe is converted into free radicals. The reason why these free radicals are dangerous is because of their strong oxidation. Excessive harmful free radicals in the body damage cells, proteins, and DNA, causing abnormalities in cell structures, functions, and signaling systems, which eventually lead to inflammation or aging in the body. One of the causes of the generation of free radicals, the sun's ultraviolet rays cause reactive oxygen species (ROS), causing diseases such as erythema, tree species, ultraviolet burns, photoaging and skin cancer.

Peroxide anion ^{_{(Superoxide anion, O 2 - ·}} ) ROS _{containing, H 2 O 2 (hydrogen peroxide} ), and · OH (hydroxy radical), etc. are being continuously generated through respiration and metabolism, in vivo SOD ( Enzymes such as superoxide dismutase), Gpxes (glutathione peroxidase), and catalase are used to prevent ROS damage. However, excessive ROS generation by the sun's rays exceeds the antioxidant defense capacity in vivo, resulting in oxidative stress. That is, it reacts with DNA, cell membranes, and proteins to cause damage to cells or tissues, which are known to promote various inflammatory diseases, Parkinson's disease, photoaging, and cancer.

Therefore, Nrf2 distributed in the epidermal layer of the skin is a transcription factor that acts on the removal of free radicals generated from inside and outside the human body. When activated, it can prevent skin aging, skin cells, and damage to skin tissue.

Republic of Korea Registered Patent No. 10-1857526 (announcement date 2018.05.14.)

As a result of continuous research, the present inventors are able to protect and improve the skin by promoting the activity of Nrf2, which removes free radicals that cause skin aging, and fermentation of tobacco leaf aroma extract together with the above-mentioned bokbok extract. When the mixture of water and a specific compound is used, the effect of promoting Nrf2 activity is greatly increased, and it is found that there is also an effect of inhibiting NO production and skin whitening, thereby completing the present invention. That is, the present invention is to provide a Nrf2 activation complex agent containing three types of substances such as sunbokhwa extract as an active ingredient, an Nrf2 induction revitalizing agent containing the same, a cosmetic product using the same, a serum (including a mask pack), and a method for manufacturing the same .

The present invention for solving the above problems is to provide an activating complex of Nrf2 comprising a bokbok extract, a desmarestia tabacoides extract and a compound represented by Formula 1 below.

[Formula 1]

A in Formula 1 is an alkylene group of C ₂ to C ₄ , and each of R ¹ and R ² is independently a hydrogen atom, -OH, -COOH or -COH.

In addition, the present invention is to provide an Nrf2 induction revitalizing agent comprising the Nrf2 activation complex.

In addition, the present invention is to provide a cosmetic comprising Nrf2 induction revitalizing agent, preferably a serum-type cosmetic (including a mask pack).

In addition, the present invention is to provide a method for preparing the Nrf2 induction revitalizing agent.

The Nrf2 induction revitalizing agent of the present invention helps the activity of Nrf2 distributed in the skin epidermal layer and greatly improves the free radical scavenging performance in the skin, thereby improving skin elasticity and excellent antioxidant effect, regaining the revitalization of rough and dull skin It can give subtle gloss and shine to the skin, and also suppresses NO activity in skin cells and has excellent skin whitening effect, and the cosmetics introduced with the Nrf2 induction revitalizing agent of the present invention can be applied to the skin at any time regardless of use time. Do.

1 is a result of measuring the luciferase activity of Experimental Example 1.
2 is a result of Western blot measurement of Experimental Example 2.
Figure 3 is a result of the measurement of oxidative stress defense against Raw264.7 cells of tobacco leaf agar extract.
Figure 4 is a graph of RFU change for each positive control Trolox concentration carried out in Experimental Example 7.
5 is a netAUC standard curve graph for each positive control Trolox concentration in Experimental Example 7.

Hereinafter, the present invention will be described in more detail.

The activating complex of Nrf2 of the present invention includes a sunbok extract, which is prepared by mixing 4 to 10 parts by weight of sunbokhwa with respect to 100 parts by weight of purified water; Obtaining the extract by extracting the raw material by performing hot water extraction for 4 to 8 hours at 90 to 100 ° C; Preparing a primary filtrate obtained by first filtering the extract; After adding and stirring 1,2-hexanediol and ethylhexylglycerin to the first filtrate, filtering the second step to prepare a sunbok extract; may be used by performing the process of.

In the preparation of the bokbok extract, the primary filtering may be performed with a filter membrane having an average pore size of 20 to 30 μm, preferably filtering with a filter layer having an average pore size of 23 to 27 μm.

In addition, the secondary filtering may be performed with an average pore size of 8 to 15 μm filter film, preferably 8 to 12 μm filter film.

The luciferase activity effect, free radical scavenging activity effect, and oxidative stress defense effect of the sunbok extract are disclosed in Patent No. 10-1857526.

Activation complex of Nrf2 of the present invention relates to a complex agent that provides NO activity suppression and skin whitening effect while increasing the Nrf2 activation effect of the Nrf2 activator using the bokbok extract of Republic of Korea Patent No. 10-1857526 as an active ingredient, It further includes two active ingredients.

The activating complex of Nrf2 of the present invention includes a tobacco leaf aquatic extract, and enhances the activation of Nrf2 in a sun-dried extract, and serves to impart an inhibitory effect on NO in the skin. Tobacco leaf aquatic algae, scientific name Desmarestia tabacoides, is also known as Tabakogusa. It is distributed in Japan, domestic defensive teams, Chuja-do, and Jeju-do. Its morphological features are very large blades, short stems, and broad ovate blades. The leaves are generally slanted, monocotyledonous, and have a midrib. Yeolpyeon becomes a large oval shape when it is opened and similar to tobacco leaves. The middle ear is slightly raised in the lower part, but gradually fades toward the upper part and there is a side vein that coexists. The side vein is almost a number of small vein at that point. The color of tobacco leaf trail is brown, and it has the characteristic of changing to turquoise in the air. The seaweeds belonging to Desmarestia contain acid, and the seaweed extracts belonging to Desmarestia are known to contain nitric acid, sulfate citric acid, etc., and it is known that these acids have high acidity.

In the present invention, the tobacco leaf aroma extract is 20 to 30 parts by weight of the tobacco leaf aroma, preferably 22 to 30 parts by weight, more preferably 22 to 28 parts by weight, even more preferably 24 to 100 parts by weight of purified water Preparing a raw material by mixing 26.5 parts by weight; Extracting the raw material by performing distillation extraction for 4 to 6 hours at 110 ° C. to obtain an extract; Preparing a primary filtrate obtained by first filtering the extract; After adding and stirring 1,2-hexanediol to the first filtrate, filtering the second step to prepare a tobacco leaf aquatic acid extract; may be used by performing the process of.

When producing the tobacco leaf aroma extract, the primary filtering may be performed by filtering with an average pore size of 20 ~ 30㎛ filter film, preferably filtering with an average pore size of 23 ~ 27㎛ filter film.

In addition, the secondary filtering may be performed with an average pore size of 8 to 15 μm filter film, preferably 8 to 12 μm filter film.

Tobacco leaf aroma extract of the present invention prepared in this way, when the concentration is 30% by weight or less of an aqueous solution, RAW264.7 cell viability may be 100% or more, preferably 20 to 30% by weight of the tobacco leaf aquatic extract When included, RAW264.7 cell viability may be 99.8-105%.

In addition, when the tobacco leaf aroma extract of the present invention is contained in 200ppm or less, the RAW264.7 cell viability may be 100% or more.

In addition, when the concentration of the tobacco leaf aroma extract of the present invention is 10 to 200ppm, when measured according to the following Equation 1, the inhibition rate of nitrogen monoxide production in RAW264.7 cells may be 10 to 45%, preferably The inhibition rate of nitrogen monoxide production in RAW264.7 cells may be 25-45%.

[Equation 1]

NO production inhibition rate (%) = (A-B) / A × 100%

In Equation 1, A is the amount of NO produced in RAW264.7 cells (100%) when the concentration of the tobacco leaf aroma extract in the NO activity inhibitor is 0 ppm, and B is RAW264 at a specific concentration of the tobacco leaf aroma extract in the NO activity inhibitor. .7 This is a measure of the amount of NO produced in cells (%).

The Nrf2 activating complex of the present invention comprising tobacco leaf aroma extract inhibits the activity of iNOS (inductive NO synthas), COX-2 (cyclooxygenase-2), and / or interleukin receptor IL2, IL6, thereby inhibiting NO activity And / or have an anti-inflammatory effect.

The activating complex of Nrf2 of the present invention includes a compound represented by the following Chemical Formula 1 in order to enhance the effect of Nrf2 activity of the sunbok extract with imparting skin whitening effect.

[Formula 1]

A of Formula 1 is a C ₂ ~ C ₄ alkylene group, preferably a C ₂ ~ C ₄ alkylene group, more preferably an ethylene group. Further, each of R ¹ and R ^{2 in} Formula 1 is independently a hydrogen atom, -OH, -COOH or -COH, preferably each of R ¹ and R ² is independently -OH, or -COOH, more preferably R ¹ and R ² are -OH.

The compound represented by Chemical Formula 1 is mixed with 5 to 10 parts by weight of elongated roots, with respect to 100 parts by weight of an ethanol aqueous solution having a concentration of 90 to 95% by volume, and then 20 to 35 ° C for 5 to 7 days, preferably 6 to 7 After performing reflux cooling extraction at 20 ~ 30 ℃ for a day, the cooling extract was concentrated under reduced pressure and dried to prepare a crude product; With respect to 100 parts by weight of water, after mixing 3 to 10 parts by weight of the crude extract, preferably 4 to 8 parts by weight, to prepare a mixed solution, 80 to 120 parts by weight of methylene chloride with respect to 100 parts by weight of the water in the mixed solution , Preferably after adding 90 ~ 105 parts by weight, followed by shaking extraction and fractionation into a water layer and an organic solvent layer, removing the organic solvent layer to obtain a fractional solution of the water layer; 3 steps of mixing the fractional solution and ethyl acetate in a volume ratio of 1: 8 ~ 10, shaking extracting, fractionating, and concentrating the organic solvent layer under reduced pressure to obtain an ethyl acetate extract; Four steps to obtain the five fractions obtained by fractionating the ethyl acetate extract with a mixed solvent containing chloroform and methanol in a 5 to 20: 1 volume ratio, preferably 15 to 16: 1 volume ratio, on a column filled with silica gel. ; And semi-prep separation of the third fraction of step 4 using a column to obtain 5 fractions, followed by 5 steps of recrystallization of methanol;

The fraction obtained in step 4 can be semi-prep separated from the third fraction of 5 fractions using rum in step 5, and methanol recrystallization in step 5 is 5 fractions obtained by semi-preparation The second fraction may be recrystallized from methanol.

The Nrf2 activating complex of the present invention may include a bokbok extract, a tobacco leaf aquatic extract, and a compound represented by Formula 1 in a weight ratio of 1: 0.8 to 2.0: 0.05 to 0.2, preferably 1: 0.8 to 1.5 : Inclusion in a weight ratio of 0.08 to 0.15 is advantageous in terms of properly exhibiting Nrf2 activation, NO activity suppression, and whitening effect in the skin.

Next, the Nrf2 induction revitalizing agent of the present invention uses the Nrf2 activating complex comprising the above-mentioned bokbok extract, the tobacco leaf aroma extract and the compound represented by Formula 1, an aqueous solution; Emulsions; The Nrf2 activating complex; Spices; And purified water. At this time, the Nrf2 activating complex is a mixture of the above-mentioned bokbok extract, the tobacco leaf aroma extract and the compound represented by the formula (1) in purified water and 35 to 50: 50 to 15% by volume, preferably 40 ~ 50: 50 to 60% by volume.

In addition, the Nrf2 induction revitalizing agent of the present invention comprises 15 to 20% by weight of the aqueous solution, 18 to 25% by weight of the oily solution, 1 to 10% by weight of the activated complex of Nrf2, 0.05 to 1% by weight of the fragrance, and the balance Contains purified water, preferably, 16 to 19.5% by weight of the aqueous solution, 18.5 to 23% by weight of the oily solution, 6.0 to 9.0% by weight of the activated complex of Nrf2, 0.1 to 0.5% by weight of the fragrance, and the remaining amount of purified water do.

In addition, if the oil solution is less than 18% by weight, there may be a problem in maintaining skin moisturization due to insufficient oil, and if it exceeds 25% by weight, excessive oil components may be contained, which may cause problems such as obstruction of skin breathing or oily skin's porosity. have. In addition, if the active complex of Nrf2 is less than 1% by weight, the content thereof is too small to prevent free radical removal, NO activity suppression and whitening effect, and if it exceeds 10% by weight, it is compatible with other ingredients enhanced by excessive use. Due to the poor sex, the quality of the product may rather deteriorate, and it is uneconomical because the effect of suppressing NO activity due to overuse is almost insignificant.

Among the components of the Nrf2 induction revitalizing agent, the fragrance may be a general one used in the art, and specific examples include Perfume 85147, Perfume 25399, and the like.

[Award amount]

Among the components of the Nrf2 induction revitalizing agent of the present invention, the aqueous solution includes a skin conditioning agent, a metal ion blocker, a thickener, a moisturizer, a viscosity reducing agent, a sterilizing preservative, and a solvent.

The skin conditioning agent among the aqueous solution components may be used alone or in combination of two or more selected from sodium hyaluronate, hyaluronic acid, licorice powder and hydrolite, preferably hyaluronic acid, Two or more selected from licorice powder and hydrolite may be used by mixing hyaluronic acid, licorice powder and hydrolite. And, when using a mixture of hyaluronic acid, licorice powder and hydrolite, hyaluronic acid, licorice powder and hydrolite in a weight ratio of 1: 0.01 to 0.2: 1 to 3, preferably hydrolite: 1: 0.02 to 0.15: It is recommended to mix and use in a weight ratio of 1.2 to 2.7.

The metal ion blocking agent in the aqueous solution component serves to prevent deterioration by metal ions that may be contained in the formulation, and the amount thereof is used in an amount of 0.1 to 2 parts by weight, preferably 100 parts by weight of the skin conditioning agent. 0.2 to 1.5 parts by weight can be used, wherein the amount of metal ion blocking agent used is less than 0.1 part by weight, the metal ion blocking effect may decrease, and when it is used in excess of 2 parts by weight, there is no effect of increasing metal ion blocking, so it is an uneconomical problem. It may be possible to use within the above range. The metal ion blocker may be used in general in the art, preferably disodium EDTA (disodium EDTA), dipotassium EDTA (dipotassium EDTA), trisodium LED, tetrasodium ID It can be used alone or in combination of two or more selected from A and rice phytate (rice phytate).

Next, the thickener in the aqueous solution component serves to increase skin adhesion by imparting appropriate viscosity and stickiness to the formulation, and the amount of the thickener used is 0.5 to 2 parts by weight, preferably 100 parts by weight of the skin conditioning agent. 0.5 to 1.7 parts by weight may be used, and if the amount of xanthan gum used is less than 0.5 parts by weight, the amount of use is too small, so the effect of using it is insufficient, and when it is used in excess of 2 parts by weight, the viscosity of the preparation becomes too high, and It is recommended to use within the above range, since there may be a problem that the stickiness is too high and the productability is poor. As a thickener, a general one used in the art may be used, and preferably, one or two or more selected from xantham gum, guarhydroxy trimonium chloride and hydroxy ethyl cellulose may be used.

Next, the moisturizing agent in the aqueous solution component serves to increase skin moisturizing together with the water vapor barrier of the emulsion, the amount of which is used in an amount of 40 to 80 parts by weight, preferably 42 to 100 parts by weight of the skin conditioning agent. ~ 70 parts by weight can be used, at this time, if the amount of the moisturizer is less than 40 parts by weight, the skin moisturizing effect may be deteriorated, and even if it is used in excess of 80 parts by weight, there is no moisturizing effect, so within the above range It is good to use. In addition, as the moisturizing agent, one type selected from methylpropanediol and glycerin may be used alone or in combination of two or more types.

Next, the viscosity-reducing agent in the aqueous solution component is used to increase the adhesive force with the skin and the absorbency of the skin by maintaining an appropriate viscosity, the amount of which is used in an amount of 45 to 90 parts by weight with respect to 100 parts by weight of the skin conditioning agent, Preferably, 50 to 85 parts by weight, and more preferably 52 to 80 parts by weight, may be used. At this time, if the amount of the viscosity reducing agent is less than 45 parts by weight or more than 90 parts by weight, the viscosity of the formulation is too high or lowered, and thus the skin There may be a problem in that the adhesive strength is too low or the stickiness is too high and the feeling of use is deteriorated. In addition, a general viscosity reducing agent used in the art may be used as the viscosity reducing agent, preferably di (C2 to C5 alkylene) glycol, and more preferably dipropylene glycol. .

Next, the sterilizing preservative in the aqueous solution component is used to prevent the Nrf2 induction revitalizing agent from being contaminated by microorganisms, and can be used without particular limitation as long as it is a sterilizing preservative that is not prohibited as a cosmetic material. Clofenesin and the like can be used. And, the amount of its use may be 2 to 10 parts by weight, preferably 4 to 8 parts by weight, based on 100 parts by weight of the skin conditioning agent, and if the amount is less than 2 parts by weight, the effect of preventing contamination by microorganisms may be insufficient. In addition, use in excess of 10 parts by weight may cause skin trouble as excessive use, so it is preferable to use within the above range.

Next, the solvent in the aqueous solution component may be used alone or in combination of two or more selected from hexanediol, propylene glycol, butylene glycol, and pentylene glycol, preferably hexanediol and It may be used by mixing butylene glycol, or more preferably, by mixing 1,2-hexanediol and 1,3-butylene glycol. In addition, the amount of the solvent used may be 30 to 60 parts by weight, preferably 35 to 50 parts by weight relative to 100 parts by weight of the skin conditioning agent, and when used in less than 30 parts by weight, dissolution and mixing between the aqueous phase components may not be good. And it is uneconomical to use more than 60 parts by weight.

[Payment]

Among the components of the Nrf2 induction revitalizing formulation of the present invention, the emulsion includes a moisture evaporation blocking agent, tocopheryl acetate, a skin conditioning agent, an emulsifying agent, a skin softening agent, a nonionic surfactant, and a film forming agent.

Moisture evaporation blocking agent in the emulsion component serves to maintain the proper moisture content and maintain the proper moisture content together with the moisturizer when maintaining the proper moisture during preparation of the Nrf2 induction revitalizing formulation and applying the prepared formulation to the skin. May be used, preferably one or two selected from caprylic / capric triglyceride oil, MCT oil and squalane, and more preferably, MCT oil and squalane It is good to mix and use in 5-10: 1 weight ratio.

Next, the tocopheryl acetate in the emulsion component serves to prevent the formulation from oxidizing and deteriorating over time, the amount of which is used in an amount of 0.1 to 5 parts by weight, preferably 100 parts by weight with respect to 100 parts by weight of a water vapor blocking agent. 0.4 to 2 parts by weight can be used. At this time, if the amount of tocopheryl acetate used is less than 0.1 parts by weight, the amount may be too small to prevent the antioxidant effect, and if the content exceeds 5 parts by weight, there may be a problem that causes irritation to the skin. It is good to use.

Next, the skin conditioning agent in the emulsion component serves to supply nutrients to the skin, and can be used by mixing one or two selected from shea butter and coconut oil, macadamia nut oil, and jojoba oil. , Preferably, shea fat and coconut oil may be used by mixing in a weight ratio of 1: 0.4 to 0.8. The skin conditioning agent is used in an amount of 10 to 35 parts by weight, preferably 13 to 30 parts by weight, with respect to 100 parts by weight of the water evaporation blocking agent. There may be a problem that may be insignificant, and if the content exceeds 35 parts by weight, the amount is too excessive, and it may be a problem that a portion of the skin remains after the skin is absorbed, so it is preferable to use within the above range.

Next, the emulsifier in the emulsion component is intended to increase the compatibility and emulsifying power between the emulsion composition, can be used in general used in the art, preferably cetearyl glucoside-cetearyl alcohol (cetearyl glucoside and cetearyl alcohol), cetearyl alcohol, glyceryl stearate, PEG-6 sorbitan stearate, PEG-20 stearate, PEG-40 stearate, and PEG-100 stearate alone or Two or more kinds may be used in combination, and more preferably, a species selected from cetearyl glucoside-cetearyl alcohol and cetearyl alcohol may be used alone or in combination of two or more species. In addition, the amount of the emulsifier used may be 30 to 60 parts by weight, preferably 40 to 55 parts by weight, based on 100 parts by weight of the skin conditioning agent. It may not work well, and it is uneconomical to use more than 60 parts by weight.

Next, the skin softener in the emulsion component serves to soften rough skin, the amount of which is used, based on 100 parts by weight of the skin conditioning agent, 2 to 10 parts by weight, preferably 3 to 7.5 parts by weight Good, and at this time, if the amount of the skin softener is less than 2 parts by weight, there may be a problem that the effect may be insignificant, and if the content exceeds 10 parts by weight, the amount of use of the skin softener may be excessive and stickiness may increase. Therefore, it is recommended to use within the above range. In addition, the skin softener may include one or two or more selected from an oily agent containing at least one medium chain monoglyceride, glyceryl caprate, polyglyceryl-2-laurate and polyglyceryl-10-laurate. It can be used in combination, and preferably, one or more selected from an oil agent containing at least one medium chain monoglyceride and polyglyceryl-10-laurate, or a mixture of two or more. And, as a specific example of the oil agent containing one or more intermediate chain monoglycerides, alcohol line MCM (AKOLINE MCM).

Next, the nonionic surfactant in the emulsion component serves as a dissolution aid for the compositions together with the emulsifier, and general nonionic surfactants used in the art may be used, and specific examples include Tween 20 and Tween 40 And it may be used alone or in combination of two or more selected from Tween 60. In addition, the amount of the nonionic surfactant used may be 7 to 20 parts by weight, preferably 10 to 15 parts by weight, based on 100 parts by weight of the skin conditioning agent.

Next, the film-forming agent in the emulsion component serves to protect the skin from unnecessary contaminants, etc. after the formulation is applied to the skin, and a general one used in the art may be used, and as a specific example, sepi Plus 400 (sepiplus 400) can be used. In addition, the amount of the film forming agent used may be 1.5 to 8 parts by weight of the film forming agent, preferably 2.5 to 7 parts by weight based on 100 parts by weight of the skin conditioning agent. The effect of using it is small, so it is not possible to use it, and if it is used in an amount exceeding 8 parts by weight, solid content in the emulsion component may be generated.

In addition, the emulsion may further include a thickening stabilizer. The amount of use may be 1 to 10 parts by weight, preferably 2 to 8 parts by weight, with respect to 100 parts by weight of the skin conditioning agent, and when it exceeds 10 parts by weight, the amount of use is too excessive to exceed the viscosity to be achieved. Since there may be a problem, it is recommended to use within the above range. In addition, the thickening stabilizer is one or two selected from sodium polyacrylate, polyacrylate, polyisobutene, carbomer, polysorbate 20, polysorbate 50, polysorbate 60 and polysorbate 80. It can be used by mixing the above, preferably, one or two or more selected from polyacrylate, polyisobutene and polysorbate 80 can be used by mixing.

Hereinafter, a method of manufacturing the Nrf2 induction revitalizing agent of the present invention described above will be described.

The Nrf2 induction revitalizing formulation of the present invention is prepared by preparing an emulsion and an aqueous solution, respectively, and emulsifying them under a specific condition temperature, respectively, to prepare an aqueous solution and an emulsion; Step 2 to prepare a mixture by adding the aqueous solution to the emulsion and then stirred; It can be prepared by performing a process comprising; three steps of mixing the mixture under 40 ~ 50 ℃, Nrf2 activated complex, flavoring and purified water and stirring at a stirring speed of 2,000 ~ 3,000rpm.

And, the oil phase of the first step is a skin conditioning agent, tocopheryl acetate, emulsifier, skin softening agent, water evaporation blocking agent and nonionic surfactant, after mixing, 60 ~ 75 ℃, preferably 65 ~ 72 ℃ temperature It can be prepared by heating and stirring to prepare a dissolved solution, and then dispersing and stirring the film forming agent in the solution.

In addition, the aqueous solution of step 1 can be prepared by mixing the skin conditioning agent, metal ion blocker, thickener, moisturizer, viscosity reducing agent, preservative, and solvent, followed by heating and stirring to 50 to 60 ° C.

Ingredients used in the oil and water phase and the amount of use thereof are the same as described above.

Step 2 can be prepared by adding the aqueous solution to the oil phase, and then performing emulsification for 1 to 4 minutes at a stirring speed of 2,000 to 3,000 rpm under 70 to 80 ° C.

And, the three-stage bokbok extract may be prepared by the method described above.

The Nrf2 induction revitalizing agent of the present invention thus prepared may have a pH of 6.0 to 7.5, preferably pH of 6.5 to 7.2, more preferably pH of 6.7 to 7.1. In this case, if the pH is less than 6.0, the effect of inhibiting NO activity by the tobacco leaf aroma extract may be insufficient, and if the pH exceeds 7.5, there may be a problem that the effect of activating Nrf2 by the pre-enriched extract may be somewhat reduced. It is good to have a pH.

Various types of cosmetics may be manufactured using the Nrf2 induction revitalizing agent of the present invention described above, and for example, cosmetics such as a cream type, a serum type (including a mask pack) may be manufactured.

Hereinafter, the present invention will be described in more detail based on Examples. However, it should not be construed as limiting the scope of the present invention by the following examples.

[Example]

Preparation Example 1: Preparation of Sunbokhwa extract

5 parts by weight of pre-incubation was prepared with respect to 100 parts by weight of purified water to prepare raw materials.

Next, the raw material was extracted from hot water at 90 to 100 for 4 to 8 hours to obtain an extract.

Next, the extract was filtered with a filter membrane having an average pore size of 25 μm to obtain a primary filtrate.

Next, after adding 5 ml of 1,2-hexanediol and 0.1 ml of ethylhexylglycerol to 94.9 ml of the filtrate, stir sufficiently, and then performing secondary filtering with a filter membrane having an average pore size of 10 µm. A bokbok extract was prepared.

The luciferase activity effect, free radical scavenging activity effect, and oxidative stress defense effect of the sunbok extract are disclosed in Patent No. 10-1857526, and the present invention cites this.

Preparation Example 2: Preparation of tobacco leaf aquatic extract

Prepared to prepare raw materials by mixing 20 to 30 parts by weight of dried and powdered tobacco leaf powder with respect to 100 parts by weight of purified water. Next, the raw material was distilled and extracted at 110 ° C. for 4 hours to obtain an extract. Next, the extract was filtered with a filter membrane having an average pore size of 25 μm to obtain a primary filtrate. Next, 2 ml of 1,2-hexanediol was added to 94.9 ml of the filtrate, followed by sufficient stirring, followed by secondary filtering of the stirred liquid with a filter membrane having an average pore size of 10 µm to prepare tobacco leaf aquatic acid extract.

Experimental Example 1: Measurement of luciferase activity of tobacco leaf aroma extract

(1) Tobacco leaf aquatic extract of Preparation Example 2 was mixed with distilled water, 20 ppm (pH 1.7), 200 ppm (pH 1.7), 2,000 ppm (pH 1.7), 20 ppm (pH 4.8), 200 ppm (pH 4.8), 2,000 ppm (pH 4.8), 20ppm (pH 6), 200ppm (pH 6), 2,000ppm (pH 6), 20ppm (pH 7), 200ppm (pH 7), 2,000ppm (pH 7) tobacco leaf agar extracts are prepared respectively Did. At this time, the pH was adjusted by dropwise addition of 1M NaOH.

In addition, 10 ppm, 20 ppm, 50 ppm, 100 ppm, 200 ppm, 1,000 ppm, and 2,000 ppm were prepared by mixing the tobacco leaf aquatic extract of Preparation Example 1 with an 80% aqueous ethanol solution.

(2) HaCaT reporter cell (HaCaT reporter cell, HaCaT-ARE-GFP-luciferase cell) was prepared by inserting luciferase as an antioxidant response element (ARE) into the human skin cell line HaCaT cell, and this cell line was used to generate lucifera. Azase activity was measured. This cell line is a measure that can rapidly detect the activity of Nrf2 because GFP or luciferase is expressed when ARE is activated by Nrf2.

Measurement of luciferase activity is RPMI1640 media 0ug / ml (control) in HaCaT reporter cells (HaCaT-ARE-GFP-luciferase cells), 20ppm (pH 1.7), 200ppm (pH 1.7), 2,000 ppm (pH 1.7), 20ppm (pH 4.8), 200ppm (pH 4.8), 2,000ppm (pH 4.8), distillation 20 ~ 2,000ppm, 20 ~ 2,000ppm of 80% ethanol aqueous solution and sulfoaphane (Solforaphane, SFN, Positive) of broccoli extract Control) and then incubated for 24 hours after treatment at a concentration of 10 Um, and then the data was measured by measuring the value of protein lysate (Lysate) and the value of luciferase activity. .

As a result of the measurement, luciferase activity was smaller than that of sulforaphane (SFN), but it was confirmed that it exhibited an antioxidant reaction (see Table 1 below). Specifically, the activity at 200 ppm of pH 7 was the best antioxidant reaction, and at 2,000 of pH 7, there was no significant increase effect compared to 200 ppm of pH 7. However, in the case of 2000 ppm of pH 1.7, it is thought that the luciferase value splattered due to the low acidity, and there is an ambiguous problem to presume that 2,000 ppm (pH 1.7) has an antioxidant reaction. The values in Table 1 below are numerical values based on the control value 1. That is, it is the magnification for the control group.

division Fold induction of Luciferase activity Control 1.00 Sulforaphane (SFN) 5.22 pH 1.7-20ppm 0.68 pH 1.7-200ppm 0.78 pH 1.7-2000ppm 1.63 pH 4.8-20ppm 0.72 pH 4.8-200ppm 0.79 pH 4.8-2000ppm 0.96 pH 6.0-20ppm 1.21 pH 6.0-200ppm 1.25 pH 6.0-2000ppm 1.35 pH 7.0-20ppm 1.63 pH 7.0-200ppm 3.09 pH 7.0-2000ppm 3.11

(3) The luciferase activity measurement was performed in the same manner as the luciferase activity measurement. It is shown in.

In FIG. 1, luciferase activity was measured by dissolving and dividing tobacco leaf extract in six concentrations in an 80% aqueous ethanol solution, and the best antioxidant reaction was confirmed at 80% ethanol-200 ppm.

Experimental Example 2: Western blot measurement

HaCaT cells were treated with a control group (LPS, lipopolysaccharide), tobacco leaf aquatic extract, 80% ethanol aqueous solution 20ppm, 80% ethanol aqueous solution 200ppm, and then treated with 2 μg / ml LPS to induce an inflammatory reaction after 1 hour. Next, after incubation for 24 hours, the amount of protein was quantified using Western blot, and the results are shown in A and B of FIG. 2. At this time, the extract of tobacco leaf agar is 20ppm (pH 1.7), 200ppm (pH 1.7), 20ppm (pH 4.8), 200ppm (pH 4.8), 20ppm (pH 6), 200ppm in the same manner as in Experimental Example 2 (1). (pH 6), 20ppm (pH 7), 200ppm (pH 7) tobacco leaf agar extract was prepared and measured using this.

In FIG. 2A, Western blot was measured using COX-2 and cJun antibodies, which are inflammation markers, in order to confirm whether they also respond to inflammatory reactions among those showing an antioxidant reaction through luciferase activity, pH 1.7-200ppm There was an antioxidant reaction, but as it was thought to be due to acidity, cJun activity showed no effect on inflammation, pH7 200ppm reduced both COX-2 and cJun, and 80% ethanol aqueous solution-200ppm reacted in cJun, resulting in pH We decided that 7-200ppm is the most suitable.

Next, B of FIG. 2 is a result confirming that the 20xm (pH 7) and 200ppm (pH 7) COX-2 as well as an iNOS inflammation marker show a response to inflammation.

Experimental Example 3: Measurement of cell viability for tobacco leaf extract

In order to confirm the cytotoxicity of the tobacco leaf agar extract, the mouse macrophage cell line RAW 264.7 cells were measured by an inflammatory model.

(1) Cell culture

The mouse macrophage cell line, RAW 264.7 cells, contains 10% fetal bovine serum (SH30406.02 from Thermo) and 1% penicillin / streptomycin (SV30010 from Thermo), DMEM medium (high glucose, Using Thermo Thermo SH30243.01), the cells were cultured in a 5% CO ₂ incubator using a 100 mm cell culture dish. Cells that reached confluence were maintained by subculture using a scraper (90030 from SPL).

(2) Cell viability experiment

The dehydrogenase present in the mitochondria in living cells produces a colorant called formazan in Tetrazolium Salt (WST). Therefore, the number of living cells can be measured by measuring this. A 96 well cell culture plate (3595 from Corning, Inc.) was added 1 × 10 ⁴ cells / well of RAW 264.7 cells as a DMEM culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin / stereptomycin. Incubate for 24 hours. After that, the sample was replaced with DMEM medium contained by the concentration of the tobacco leaf aroma extract (10ppm, 20ppm, 50ppm, 100ppm, 200ppm, 1,000ppm) and incubated for 48 hours. Using the EZ-CYTOX reagent (EZ-1000 from Daeilab) on the cultured cells and measuring the absorbance at 570 nm, cell viability by concentration was measured according to Equation 2 below, and the results are shown in Table 2 below. .

In addition, the control (control) was measured using raw 264.7 cells not treated with tobacco leaf streaks.

[Equation 2]

Cell viability = (absorbance of sample-added group / absorbance of untreated group) × 100%

division Cell viability (% of control group) Control (0% by weight) 100 10ppm 120 20ppm 120 50ppm 105 100ppm 100 200ppm 97 1000ppm 95 3000ppm 53

Looking at the experimental results in Table 2, the test group treated with tobacco leaf extract at a concentration of 200 ppm or less did not show a statistically significant difference in cell viability compared to the control group, but at 2,000 ppm, the viable viability was rapidly decreased. Was confirmed. That is, it was confirmed that at a concentration of 1000 ppm or less, it did not show cytotoxicity to RAW264.7 cells.

Experimental Example 4: Real time RT PCR measurement

Interleukin receptors IL2, IL6, inflammatory markers iNOS and COX-2 were measured by real-time polymerase chain reaction (Real-time RT PCR) to confirm that mRNA levels were not reduced.

Tobacco leaf extract of Preparation Example 2 was prepared 20ppm (pH 7), 200ppm (pH 7) and 1000ppm (pH 7). In addition, as a control (control), Raw264.7 cells were prepared and LPS (lipopolysaccharide) was prepared.

Raw 264.7 cells were treated with a control and tobacco leaf extract, respectively, and after 1 hour, 2 μg / ml LPS was treated (incubated for 18 hours) to induce an inflammatory reaction, RNA was extracted, and extracted RNA was extracted. After synthesis with cDNA again, mRNA levels were measured by real-time polymerase chain reaction, and the results are shown in Table 3 (IL2, iL6, iNOS, COX-2), respectively.

Classification (Relative mRNA Level) IL2 IL6 iNOS  COX-2 Control One One One One LPS 2.67 9.65 3.77 4.84 pH 7- Tobacco leaf extract 20 ppm 1.28 3.32 2.52 3.14 pH 7- Tobacco leaf extract 200 ppm 0.76 1.80 1.63 0.94 pH 7- Tobacco leaf extract 1000 ppm 0.68 1.51 1.38 0.90

Looking at Table 3, when the tobacco leaf aroma extract treatment, it was confirmed that the phase II mRNA levels of IL2, IL6, iNOS and COX-2 decrease. On the other hand, when only LPS was treated, it showed a result of increasing Phase II mRNA level. Through this, it was confirmed that the tobacco leaf aquatic extract reduced iNOS, COX-2, IL2 and IL6 factors.

Experimental Example 5: Measurement of oxidative stress defense test of tobacco leaf extract

(1) Oxidative stress defense test against Raw264.7 cells

Raw264.7 cells (control), Raw264.7 cells treated with only 2 μg / ml lipopolysaccharide (LPS) at concentrations of 26 μg / ml, and LPS (2 μg / ml) and tobacco leaf aroma extract (200 ppm) in RAW 264.7 cells. In the case of processing together, Raw264.7 intracellular defense was measured, and the results are shown in FIG. 3.

In the control of FIG. 3, DNA staining is performed on RAW 264.7 cells to determine whether DNA damage occurs due to oxidative stress, and DAPI, 8-OH-dG (8-hydroxy deoxyguanosine) and MERGE levels in the cells. It is measured.

Referring to FIG. 3, it can be seen that the treatment with LPS alone and the treatment with tobacco leaf aquatic extract together with LPS provide oxidative stress protection.

Experimental Example 6: Measurement of NO production suppression of tobacco leaf extract

(1) Test substance treatment

RAW 264.7 cells were dispensed into 96-well plates at 4 × 10 ⁴ cells / dishdensity and attached to the plates for 24 hours. 1 hour after each treatment with the tobacco leaf aroma extract, in order to induce an inflammatory reaction, 2 μg / ml concentration of LPS (lipopolysaccharide) was treated and cultured for 18 hours.

(2) Measurement of nitrogen monoxide (NO) production

The NO level generated in RAW 264.7 cells and present in the culture was measured using a NO detection kit based on the Greases reaction (21021 from Intron). After incubating 100 µl of the culture solution presumed to have NO in a 96 well plate, add 50 µl of N1 buffer (sulfanilamide) and react at room temperature for 10 minutes. Subsequently, 50 μl of N2 buffer (naphthylethylenediamine) was added and reacted at room temperature for 10 minutes, and absorbance was measured at 540 nm, and each NO production amount was calculated using a standard calibration curve obtained using a nitrite standard. As a positive control (Positive control) L-NMMA 50μM (N ^G -Methyl-L-arginineacetatesalt, Sigma M7033) was used.

Each result confirms the statistical significance of the test results using the t-test (two-sided verification) function of Microsoft's Office Excel 2007, and the results are shown in Table 4 below. In addition, the NO production inhibition rate in Table 4 was calculated based on Equation 1 below.

[Equation 1]

NO production inhibition rate (%) = (A-B) / A × 100%

In Equation 1, A is NO production amount in RAW264.7 cells (100%) when the concentration of the tobacco leaf aroma extract in the NO activity inhibitor is 0%, and B is RAW264 at a specific concentration of the tobacco leaf aroma extract in the NO activity inhibitor. .7 This is a measure of the amount of NO produced in cells (%).

division NO production inhibition rate (% of control group) Control (0% by weight) 100 Positive control group 45.2% pH7-10ppm 10.3% pH7-100ppm 18.5% pH7-200ppm 35.2% pH7-1500ppm 43.8% pH7-3000ppm 40.5%

Looking at the measurement results in Table 4, the positive control L-NMMA inhibited the production of NO in RAW264.7 cells by about 45% in the 50 μM concentration range. And, it can be seen that the tobacco leaf aroma extract inhibits the production of Nitirc oxide (NO) in RAW264.7 cells at a statistically significant level in a concentration range of 10 ppm, 100 ppm, 200 ppm, and 1500 ppm. When treated at 200 ppm or less, it was confirmed that about 10 to 44% of NO was inhibited, and at 3000 ppm, the NO production inhibition rate was rather lowered.

Preparation Example 3: Preparation of a compound represented by Formula 1

With respect to 100 parts by weight of an aqueous solution of ethanol at a concentration of 92% by volume, 15 parts by weight of dried walnut roots were added, and reflux cooling extraction was performed at 25 ° C for 7 days to obtain a cooling extract, which was concentrated under reduced pressure with a concentrated concentrator to obtain crude extract It was prepared.

Next, after mixing 10 parts by weight of the crude extract with respect to 100 parts by weight of distilled water to prepare a mixed solution, 100 parts by weight of methylene chloride with respect to 100 parts by weight of the mixed solution, followed by shaking extraction, water layer and organic solvent layer After fractionating with and removing the organic solvent layer, a fractional solution of the water layer was obtained.

Next, after mixing the fractional solution and ethyl acetate in a ratio of 1: 5 by weight, this was shake-extracted to fractionate the organic solvent layer, and then the solution of the organic solvent layer was concentrated under reduced pressure with a reduced pressure concentrator to obtain an ethyl acetate extract.

Next, the ethyl acetate extract was fractionated with a mixed solvent containing loroform and methanol in a volume ratio of 15: 1 in a column filled with silica gel to obtain 5 fractions, and the 3rd fraction was semi- using a capsule capsule column. Separated by semi-prep to give 5 fractions again. At this time, acetonitrile was used as the developing solvent.

The second fraction of this was recrystallized with methanol to obtain a final product represented by the following Chemical Formula 1-1.

[Formula 1-1]

A in Formula 1-1 is an ethylene group, and R ¹ and R ² are independently -OH.

¹ H-NMR (500MHz, Acetone-d6): δppm 6.14 (t, 1H, H-4), 6.43 (d, 2H, H-2, 6), 6.75 (d, 2H, H-3 ′, 5 ′ ), 6.79 (d, 1H, H-α), 6.94 (d, 1H, H-β), 7.33 (d, 2H, H-2 ', 6');

¹³ C-NMR (125MHz, Acetone-d6): δppm 102.64 (C-4), 105.76 (C-2), 108.20 (C-6), 115.84 (C-3 ′), 116.48 (C-5 ′), 127.02 (C-α), 128.79 (C-6 ′), 129.38 (C-2 ′), 130.42 (C-β), 131.40 (C-1 ′), 141.31 (C-1), 158.37 (C-4) ′), 159.37 (C-3, 5)

Example 1: Preparation of Nrf2 activated complex

After preparing a mixture of the bokbok extract of Preparation Example 1, the tobacco leaf aroma extract of Preparation Example 2 and the compound represented by Chemical Formula 1-1 of Preparation Example 3 in a weight ratio of 1: 1 to 0.1, and then mixing with water , pH was adjusted to 7.

Examples 2 to 3 and Comparative Examples 1 to 4

Prepared in the same manner as in Example 1, the bokbok extract, tobacco leaf aroma extract and the compound represented by the formula 1-1 to a weight ratio as shown in Table 5 to prepare an Nrf2 activated complex of pH7.

Classification (weight ratio) Sunbokhwa Extract Tobacco leaf extract Compound represented by Formula 1-1 Example 1 One One 0.10 Example 2 One 1.5 0.12 Example 3 One One 0.18 Comparative Example 1 One 0.5 0.10 Comparative Example 2 One 2.2 0.10 Comparative Example 3 One One 0 Comparative Example 4 One One 0.25

Experimental Example 7: Measurement of antioxidant and oxygen radical scavenging activity

For the antioxidant activity (ORAC-FL assay, Oxygen Radical Antioxidant Capacity) test method, fluorescence intensity was measured by reacting fluorescein, a test substance, and AAPH, a peroxyl radical (Excitation: 485nm, Emmision: 525 nm). Specifically, the sample, control group 1, bokbok extract, each of the complexes of Examples 1 to 3 and Comparative Examples 1 to 3, was diluted to a concentration of 0.1% by volume, and Trolox (6-hydroxy-2,5, as a positive control). 7,8-tetramethylchroman-2-carboxylic acid) was used.

Then, 25 μl of each test substance was dispensed into a black 96-well plate, and then 150 μl of Fluoresein was added and reacted at 37 ° C. for 30 minutes. Next, 25 µl of AAPH (2,2'-Azobis (2-amidinopropane) dihydrochloride) was dispensed into each well, and fluorescence was measured under Excitation 485 and Emission 528 nm conditions (60 minutes, interval 3 minutes).

The result was calculated by subtracting the blank fluorescence from the sum of the fluorescence over time, and expressed as net AUC corresponding to the area between the blank and the sample graph. Then, the Trolox equivalent (mM TE / g) was calculated using the Trolox standard curve to calculate the Trolox equivalent (net AUC value) of the corresponding concentration.

For the positive control Trolox, net area under the curve (AUC) was measured in a concentration range of 5 to 50 uM (see FIG. 4), and a net AUC standard curve for each concentration was prepared (see FIG. 5).

And, the results of the oxygen radical scavenging activity of the bokbok extract, Examples 1 to 3 and Comparative Examples 1 to 4 are shown in Table 6 below. Table 6 below shows the average values measured three times.

Concentration of bokbokhwa extract (%) Treatment concentration (% by volume) Trolox equivalent (mM TE / g) Control group (pre-purified extract) 0.1 65.43 Example 1 83.85 Example 2 89.93 Example 3 85.36 Comparative Example 1 70.98 Comparative Example 2 86.02 Comparative Example 3 76.63 Comparative Example 4 85.40

Looking at the measurement results in Table 6, when the 0.1% concentration treatment with only bokbok extract shows 65.43 mM TE / g equivalent of Trolox, it is carried out using the tobacco leaf aroma extract and the compound represented by Formula 1-1 at the same treatment concentration It was confirmed that Examples 1 to 3 have better oxygen radical scavenging activity.

And, in the case of Comparative Example 1 in which the tobacco leaf aroma extract was used in an amount of less than 0.8 weight ratio, there was a problem in that the Trolox equivalent value was significantly lower when compared with Example 1, and also, a comparison in which the compound represented by Formula 1-1 was not used Example 3 also had a problem that the Trolox equivalent level was significantly low.

In addition, in the case of Comparative Example 2 in which the tobacco leaf aroma extract was used in excess of 2.0 weight ratio, when compared with Example 2 in which the tobacco leaf aroma extract was used in a 1.5 weight ratio, there was a problem that the Trolox equivalent value was lowered, expressed by Chemical Formula 1-1. In the case of Comparative Example 4 in which the compound to be used was in excess of 0.2 weight ratio, when compared with Example 3, there was no increase in the Trolox equivalent value.

Experimental Example 8: Real time RT PCR measurement

It is known that when free radicals are generated, enzymes related to the Phase II metabolic system react to the free radicals and their levels increase rapidly. At this time, the increase of the protein related to the regulation of Phase II metabolism is regulated in the transcription step, and the transcription factors that regulate it are Nrf2, HO-1, and NQO-1.

Accordingly, it was measured by real-time polymerase chain reaction (Real-time RT PCR) to confirm that the mRNA level is increased.

HaCaT cells were treated with 20 ug / ml of each of the Nrf2 activating complexes of Preparation Example 1, Control Examples 1 to 3 and Comparative Examples 1 to 4, which are controls, to extract RNA, and the extracted RNA was again synthesized by cDNA. After that, the mRNA level was measured by Real Time RT PCR, and the results are shown in Table 7 (Nrf2, HO-1, NQ01). Table 7 shows the phase II mRNA levels of Nrf2, HO-1, and NQO-1 after 4 hours.

division
(Relative mRNA level) Nrf2 HO-1 NQ01 0 hours 4 hours 0 hours 4 hours 0 hours 4 hours Control group (pre-purified extract) 1.0 1.08 1.0 2.22 1.0 1.37 Example 1 1.0 1.24 1.0 2.58 1.0 1.69 Example 2 1.0 1.39 1.0 2.75 1.0 1.79 Example 3 1.0 1.35 1.0 2.64 1.0 1.85 Comparative Example 1 1.0 1.11 1.0 2.34 1.0 1.40 Comparative Example 2 1.0 1.30 1.0 2.80 1.0 1.75 Comparative Example 3 1.0 1.15 1.0 2.46 1.0 1.42 Comparative Example 4 1.0 1.36 1.0 2.66 1.0 1.84

In the case of Comparative Example 1, Phase II mRNA levels of Nrf2, HO-1, and NQO1 were better than those of the control group, but showed significantly lower results when compared to Example 1. And, in the case of Comparative Example 2, compared with Example 2, the HO-1 level was higher, but the NQ01 and Nrf2 levels were slightly lowered. And, in the case of Comparative Example 3, compared to Example 1, Nrf2, HO-1 levels were low, in particular, NQ01 levels showed significantly lower results. And, in the case of Comparative Example 4, compared with Example 3, there was no significant difference.

Through the above experimental examples, the Nrf2 activating complex of the present invention uses a combination of a sunbok extract and a tobacco leaf aroma extract and a compound represented by Formula 1-1 rather than a sunbok extract alone, and has an antioxidant activity when used in an appropriate ratio. Excellent was confirmed.

Experimental Example 9: Melanin biosynthesis inhibitory effect on skin melanocytes

After collecting the human skin tissue, cut the skin tissue into fine sections, and wash it with a phosphate buffer solution (PhosphateBufferedSaline) containing antibiotics (50mg / ml gentamicin, 50mg / ml amphotericin, 50U / ml penicillin, 50µg / streptomycin) And treated with 0.25% Trypsin / EDTA for 5 minutes, incubated for 15 days in an incubator with 5% CO ₂ and 37 ° C. in M2 medium (Promocell), a melanocyte selection medium, for 15 days, and then the melanocyte cells were again 0.25% Trypsin / The cells were removed with EDTA, counted with a hemocytometer, inoculated with 1 × 10 ⁴ per well into 24-wells containing M2 medium (Promocell), and cultured at 37 ° C until 80% or more of the cells adhered to the well duct.

After incubation for 1 day, dopa, a precursor of the melanin pigment, was added to 0.08 mM, and then Examples 1 to 3 and Comparative Examples 3 to 4 were added at a test concentration of 0.1% by weight, respectively, and cultured for 3 days to add saline to the control group. The cells from which the medium has been removed are washed with a phosphate buffer (PBS, Phosphate Buffered Saline), and treated with trypsin to recover the cells. The recovered cells were counted using a hematocytometer, centrifuged for 10 minutes, and then the supernatant was removed to obtain a precipitate.

The cell precipitate was dried at 60 ° C., and then 100 μl of 1M sodium hydroxide solution containing 10% was added to obtain intracellular melanin in a 60 ° C. bath. With this solution, the absorbance was measured at 490 nm with a microplate to compare the melanin content per cell with the Nrf2-activated complex and the control group (Examples 1 to 3 and Comparative Examples 3 to 4), and compared to the control group 100 It was expressed as% based on%, and the results are shown in Table 8 below.

division Melanin content per cell Control 100% Example 1 66.3% Example 2 55.2% Example 3 46.8% Comparative Example 1 68.5% Comparative Example 2 64.4% Comparative Example 3 98.2% Comparative Example 4 40.6%

Looking at the measurement results of Table 8, compared to the control group, Examples 1 to 3 showed a result in which the melanin content was significantly reduced, and in particular, when comparing Examples 1 to 3, the compounds represented by Chemical Formula 1-1 As it increased, the melanin content tended to decrease. Particularly, in the case of Comparative Example 3 in which the compound represented by Chemical Formula 1-1 was not used, the melanin reduction effect was insufficient. In addition, when comparing Example 1 and Comparative Example 2, the increase in the content of tobacco leaf aroma extract tended to slightly increase the melanin reduction effect.

Through the above experiment, it was confirmed that the Nrf2 activation complex of the present invention has a whitening effect through melanin reduction.

Preparation Example 1: Preparation of Nrf2 induction revitalizing formulation

(1) Preparation of aqueous solution

After completely dispersing the components of Table 9 in a solvent, purified water was added, and then heated and stirred at 56 to 57 ° C to completely dissolve to prepare a water phase solution.

Award amount division Kinds Parts by weight Skin conditioning Hyaluronic acid (1% concentration) 39.2 Licorice powder 2 Hydrolite-5 58.8 Metal ion blocker EDTA-2NA 0.39 Thickener Xanthan gum (Keltrol F) 0.98 Moisturizer glycerin 49.06 Viscosity reducing agent Dipropylene glycol 58.83 solvent 1,2-hexanediol 19.61 1,3-butylene glycol 19.61 Antiseptic preservative Clofenesin 5.88

(2) Preparation of oily liquid

After mixing the components of Table 10, stirring and stirring with an agitator at 68 ° C., followed by dispersing and mixing evenly by introducing a film-forming agent to prepare an emulsion.

Emulsion division Kinds Usage (parts by weight) Skin evaporation blocker MCT oil 87.5 Squalane 12.5 Tocopheryl Acetate Vit-E Acetate 0.625 Skin conditioning Shea butter 12.50 Coconut oil 6.25 Emulsifier Cetearyl glucoside and
Cetearyl alcohol (Emulgade PL 68/50) 18.75 Cetearyl alcohol (KALCOL 6850) 25 Skin softener One or more medium chain monoglycerides
Included payment (AKOLINE MCM) 5 Nonionic surfactant Tween 60 12.5 Film-forming agent SEPIPLUS 400 3.75

(3) After the aqueous solution was slowly added to the oil solution prepared above, the mixture was stirred for 2 minutes at a stirring speed of 2,500 rpm under 74 to 75 ° C using a homomixer, and then the Nrf2 activated complex at 45 ° C ( The Nrf2 activation complex of Example 1: purified water = 45.5: 64.5% by volume) and fragrance (Perfume 85147) were added and slowly stirred to prepare an Nrf2 induction revitalizing formulation, and the prepared formulation was of serum type (including mask pack).

In addition, the prepared formulation was prepared to include 17.90% by weight of the aqueous solution, 20.70% by weight of the oily solution, 6.54% by weight of the Nrf2 activated complex, 0.28% by weight of the fragrance, and the balance of water.

Claims (11)

delete delete delete delete delete 15-20% by weight of the aqueous solution; Oil solution 18 to 25% by weight; Nrf2 activated complex 1 to 10% by weight; Fragrance 0.05 to 1% by weight; And the remaining amount of purified water;
The aqueous solution is 0.1 to 2 parts by weight of a metal ion blocking agent, 0.5 to 2 parts by weight of a thickener, 40 to 80 parts by weight of a moisturizer, 45 to 90 parts by weight of a viscosity reducing agent, and 2 to 2 parts of a sterilizing preservative, based on 100 parts by weight of a skin conditioning agent 10 parts by weight and 30 to 60 parts by weight of solvent,
The emulsion is based on 100 parts by weight of the water vapor barrier, 0.1 to 5 parts by weight of tocopheryl acetate, 10 to 35 parts by weight of the skin conditioning agent, 30 to 60 parts by weight of the emulsifier, 2 to 10 parts by weight of the skin softener, and nonionic interface Contains 7 to 20 parts by weight of active agent and 1.5 to 8 parts by weight of film forming agent,
The Nrf2 activating complex includes a bokbok extract, a tobacco leaf aroma extract and a compound represented by the following formula 1 in a weight ratio of 1: 0.8 to 2.0: 0.05 to 0.2,
The tobacco leaf aroma extract is a cosmetic product characterized in that the concentration of inhibition of nitrogen monoxide production in RAW264.7 cells is 10 to 45% when measured according to Equation 1 below when the concentration of the tobacco leaf aroma extract is 10 to 200 ppm. Nrf2 induction revitalizing agent for;
[Formula 1]

A in Formula 1 is a C ₂ ~ C ₄ alkylene group, each of R ¹ and R ² is independently a hydrogen atom, -OH, -COOH or -COH,
[Equation 1]
NO production inhibition rate (%) = (AB) / A × 100%
In Equation 1, A is the amount of NO produced in RAW264.7 cells (100%) when the concentration of the tobacco leaf aroma extract in the NO activity inhibitor is 0 ppm, and B is RAW264 at a specific concentration of the tobacco leaf aroma extract in the NO activity inhibitor. .7 This is a measure of the amount of NO produced in cells (%). A cosmetic comprising the Nrf2 induction revitalizing agent of claim 6. delete delete delete Step 1 for preparing each of the aqueous solution and the oil phase;
Step 2 to prepare a mixture by adding the aqueous solution to the emulsion and then stirred;
Performing the process comprising; 3 steps of mixing the mixture under 40 ~ 50 ℃, Nrf2 activated complex, fragrance and purified water and stirring at a stirring speed of 2,000 ~ 3,000rpm,
The tobacco leaf acid extract,
1 step of preparing raw materials by mixing 20 to 30 parts by weight of tobacco leaf acid extract with respect to 100 parts by weight of purified water; 2 steps to obtain the extract by extracting the raw material by performing distillation extraction for 4 to 6 hours under 100 ~ 110 ℃; 3 steps for preparing a primary filtrate obtained by primary filtering the extract; And adding and stirring 1,2-hexanediol to the primary filtrate, followed by secondary filtering to prepare tobacco leaf aquatic extract; prepared by performing a process comprising
The compound represented by Formula 1,
With respect to 100 parts by weight of an ethanol aqueous solution having a concentration of 90 to 95% by volume, 5 to 20 parts by weight of walnut roots are mixed, reflux cooling extraction is performed at 20 to 35 ° C for 5 to 7 days, and the cooling extract is concentrated under reduced pressure and Step 1 to prepare a crude extract by drying; With respect to 100 parts by weight of water, 3 to 10 parts by weight of the crude extract was mixed to prepare a mixed solution, and then 80 to 120 parts by weight of methylene chloride was added to the mixed solution, followed by shaking extraction and fractionation into a water layer and an organic solvent layer, Step 2 to remove the organic solvent layer to obtain a fractional solution of the water layer; After the fractional solution and ethyl acetate are mixed, the mixture is shaken and fractionated to concentrate the organic solvent layer under reduced pressure to obtain an ethyl acetate extract; 4 steps to obtain the 5 fractions obtained by fractionating the ethyl acetate extract with a mixed solvent containing chloroform and methanol in a volume ratio of 5 to 20: 1 in a column filled with silica gel; And 5 steps of semi-prep separation of the fraction of step 4 using a column to obtain 5 fractions, followed by 5 steps of recrystallization of methanol.
The Nrf2 activating complex includes a bokbok extract, a tobacco leaf aroma extract and a compound represented by the following formula 1 in a weight ratio of 1: 0.8 to 2.0: 0.05 to 0.2,
When the concentration of the tobacco leaf aroma extract is 10 to 200 ppm, when measured according to Equation 1 below, Nrf2 characterized in that the inhibition rate of nitrogen monoxide production in RAW264.7 cells is 10 to 45%. A method for preparing an induction revitalizing agent;
[Formula 1]

A in Formula 1 is a C ₂ ~ C ₄ alkylene group, each of R ¹ and R ² is independently a hydrogen atom, -OH, -COOH or -COH,
[Equation 1]
NO production inhibition rate (%) = (AB) / Aⅹ100%
In Equation 1, A is the amount of NO produced in RAW264.7 cells (100%) when the concentration of the tobacco leaf aroma extract in the NO activity inhibitor is 0 ppm, and B is RAW264 at a specific concentration of the tobacco leaf aroma extract in the NO activity inhibitor. .7 This is a measure of the amount of NO produced in cells (%).

Images