KR102125895B1 - Inhibitor for NO activator having extracts derived from natural plant, Anit-inflammation agent containing of the same, Revitalizing cosmetics containing the same and Manufacturing method thereof - Google Patents

Abstract

The present invention relates to an inhibitor of NO activity using natural resources, and an anti-inflammatory agent containing the same. More specifically, a complex extract including two or three extracts is used as an inhibitor of NO activity to be used in anti-inflammatory drugs, cosmetics using the same, external preparations for skin such as ointments, and health supplements.

Description

Inhibitor for NO activator having extracts derived from natural plant, Anit-inflammation agent containing of the same, Revitalizing cosmetics containing the same and Manufacturing method thereof}

The present invention is an anti-inflammatory agent derived from a natural plant extract containing the NO activity inhibitor that suppresses NO active ingredients that cause inflammation in the skin, etc., and effectively restores stressed and tired skin, cosmetics using the same, and methods for manufacturing the same It is about.

Inflammatory reactions are caused by the human body's defense mechanism against external stimuli to maintain homeostasis by mediating external stimuli such as LPS (lipopolysaccharide) derived from microorganisms and internal stimuli such as arachidonic acid.

LPS is a substance present in the outermost layer of the cell wall of Gram negative bacteria and is known to play a variety of roles in the interaction between pathogenic bacteria and eukaryotes. In particular, LPS is recognized as a foreign substance in the human body and activates the cell defense system, thereby activating the inflammatory pathway, one of the immune defense systems.

When cells are stimulated by external stimuli such as LPS, the expression of iNOS (inducible NO synthas) and COX-2 (cyclooxygenase-2) is induced, which is the intracellular signal transduction substance that mediates the inflammatory response. It releases nitric oxide (NO) and prostaglandin E2 (PGE ₂ ). The excessive release of NO and PGE ₂ causes an inflammatory reaction in the human body and tissues.

In general, NO is a modulator that regulates normal physiological activities such as physiological phenomena such as blood pressure regulation and neurotransmission mediator, and also plays a pivotal modulator in the immune response. However, NO overexpressed by iNOS causes an inflammatory response and abnormal immune system. In addition, COX-2 is an enzyme that converts arachidonic acid to prostaglandin (PGE ₂ ), and when COX-2 is overexpressed, PGE ₂ is excessively generated, which serves as another inflammatory pathway.

Non-steroidal anti-inflammatory drugs (NSAIDS), a widely used agent for the treatment of most inflammatory diseases, are prostaglandin from arachidonic acid called cyclooxygenase (COX). By inhibiting the enzyme activity involved in biosynthesis, it exhibits anti-inflammatory action, which causes serious side effects such as gastrointestinal disorders, liver disorders, and renal impairment in addition to the main therapeutic action, which is a limitation in long-term use (Rajakariar R et al 2006).

Therefore, there is almost no side effect, and it is widely used for a long period of time, and it is widely required to develop a new anti-inflammatory analgesic that is excellent in anti-inflammatory efficacy, which is also a reason why research on material development through verification of efficacy from natural resources has been activated recently. The popularity of skin external preparations and cosmetics with anti-inflammatory effect using natural resources is increasing, and research on natural cosmetic materials and products without these side effects is increasing.

Republic of Korea Registration No. 10-1164083 (announcement date 2012.07.16.) Republic of Korea Registration No. 10-1964809 (announcement date 2019.03.27.)

As a result of continuously researching the NO activity-inhibiting component derived from the natural plant components developed and patented by the present inventors, mixing and using an extract derived from a specific natural plant can increase the NO-inhibition efficacy while minimizing or minimizing side effects. By knowing, the present invention was completed. That is, the present invention is an NO-activator containing a complex extract using a herbal extract (and/or a fraction thereof) and a tarragon leaf and stem (leaf/stem) extract, an anti-inflammatory agent comprising the same, a cosmetic product using the same and preparation thereof I want to provide a method.

In order to solve the above problems, the NO activity inhibitor of the present invention is to provide an NO activity inhibitor comprising an herbal Robert extract (and/or a fraction thereof) and a tarragon leaf and stem (leaf/stem) extract.

In addition, the present invention is to provide an anti-inflammatory agent comprising the NO activity inhibitor.

In addition, the present invention is to provide a cosmetic comprising the anti-inflammatory agent.

In addition, the present invention is to provide a method for preparing the anti-inflammatory agent.

The NO activity inhibitor of the present invention has an excellent anti-inflammatory effect by suppressing the production of NO by LPS without cytotoxicity, and inhibiting the intracellular activity of the inflammatory markers iNOS, COX-2, TNFα, and IL6. Therefore, the NO activity inhibitor of the present invention is suitable for use as an anti-inflammatory agent, and further, it can be applied to cosmetics and the like to provide a natural external preparation for skin that has an anti-inflammatory effect due to skin troubles and acne, preferably natural cosmetics.

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

The herb robert (herbs robert) is a kind of geranium (Geranium robertianum), which is wild in Europe, and has small flowers of reddish purple in summer.

Tarragon (Scientific name: Artemisia dracunculus, Tarragon) is a plant that is used as a spice with a sweet aroma and a spicy, bitter taste that French people regard as the queen of spices, and mainly uses leaves for cooking. Taragon is a tonic, which is good for the head, heart, and liver, helping to recover from fatigue, and in the Middle Ages, it was considered a plant that promotes stamina, and its leaves are rich in iodine, minerals, salt, and vitamin C, which have also been used to treat vitamin deficiency. The tarragon stem grows about 50 to 90 cm, and the leaves are shiny, but are narrow, long, and green. And, there is a feature that a small bud is formed, but the fruit is not formed because the flower does not bloom.

NO active inhibitor of the present invention includes a herbal extract and a tarragon leaf and stem (hereinafter referred to as "leaves / stems") of the complex extract comprising an extract, the complex extract is herbal Robert extract and tarragon leaf / stem extract 1: 1.5 to 3.0 in a weight ratio, preferably, Herb Robert extract and tarragon leaf / stem extract may include a 1: 1.8 to 2.5 weight ratio. When the amount of the tarragon leaf/stem extract used is less than 1.5 weight ratio, the synergistic effect of inhibiting NO activity may be insufficient, and when it is used in excess of 3.0 weight ratio, the synergistic effect of inhibiting NO activity decreases, and the cell viability of RAW 264.7 cells decreases somewhat. Since there may be a problem, it is better to mix and use within the above range.

In addition, the NO activity inhibitor of the present invention may further include the safflower seed extract as a composite extract material for increasing the NO activity suppression effect and skin whitening effect while slightly reducing the toxicity of the tarragon leaf/stem extract. The city complex extract comprises herbal robert extract, tarragon leaf/stem extract and safflower seed extract in a ratio of 1: 1.5 to 3.0: 0.2 to 0.5, preferably herbal robert extract, tarragon leaf / stem extract and safflower seed extract 1: 1.8 ~ 2.5: It is good to include 0.2 to 0.4 by weight. At this time, if the amount of safflower seed extract is less than 0.2% by weight, the amount of use is too small, so the improvement effect due to the introduction thereof may be inadequate, and even if it is used in excess of 0.5% by weight, there is no further increase in whitening effect, Herb Robert extract and/or tarragon Since the leaf/stem usage is relatively small, the NO activity inhibiting effect may be reduced, so it is preferable to use within the above range.

The herbal Robert extract is prepared by mixing 15 to 35 parts by weight of Herb Robert, preferably 20 to 30 parts by weight, more preferably 20 to 25 parts by weight based on 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 70 to 80°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 herbal robert extract; may be used by performing the process of.

When manufacturing the herbal robert 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.

In the present invention, the tarragon leaf/stem extract is prepared by mixing 10 to 30 parts by weight of the tarragon leaf/stem extract with respect to 100 parts by weight of purified water, preferably 12 to 25 parts by weight, and more preferably 12 to 18 parts by weight. To do; Extracting the raw material by performing hot water extraction for 4 to 8 hours at 90 to 100° C. to obtain an extract; 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 tarragon leaf/stem extract extract; may be used by performing the process of.

In the present invention, the safflower seed extract comprises: extracting safflower seed powder with n-hexane at 15 ~ 30°C, and then removing the fat from the extract; A second step of extracting and filtering the fat-removed extract at 15 to 30° C. with an ethanol aqueous solution having a concentration of 60 to 75% by volume, preferably 65 to 75%; Concentrating and filtering the ethanol extract under reduced pressure to obtain a reduced pressure concentrate; And filtering the concentrated product under reduced pressure to obtain a safflower seed extract as a filtrate; a product prepared by performing a process including a can be used.

When preparing the safflower seed extract, the first step is repeated 2 to 5 times, preferably 3 to 4 times, to prepare an extract from which fat is removed, and then, in step 2, a re-extraction process with an aqueous ethanol solution is performed. Can.

The reduced-pressure concentration of the above step 3 can be performed through a general method used in the art, and is performed for removing solvent and impurities.

In addition, the reduced pressure concentration in step 3 and the filtering in step 4 may be performed through a general reduced pressure concentration and filtering method used in the art, for example, the filtering is preferably an average pore size of 8 ~ 15㎛ filter membrane, preferably Filtering can be performed with an 8-12 μm filter film.

Herbal robert extract and tarragon leaf/stem extract; Or herbal Robert extract, tarragon leaf / stem extract and safflower seed extract; NO inhibitors of the present invention incorporating the complex extract containing; when the composite extract containing less than 2.0% by weight, RAW264.7 cell viability is 96.00 % Or more, preferably, when the complex extract is included in an amount of 0.125 to 2.0% by weight, the cell viability of RAW264.7 may be 98.00 to 103.00%, more preferably 99.0 to 103%.

In addition, the NO activity inhibitor of the present invention, when the concentration of the complex extract is 0.1 to 1.0% by weight, measured according to the following equation (1), the inhibition rate of nitrogen monoxide (NO) production in RAW264.7 cells is 25 to 43 %, preferably 26 to 40%, more preferably NO production inhibition rate may be 28.2 to 36.5%, preferably when the concentration of the complex extract is 0.1 to 0.5% by weight, based on the following equation (1) When measured, the rate of inhibition of nitrogen monoxide production in RAW264.7 cells may be 23.4 to 33.5%, preferably 25.0 to 30.5%.

[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 complex extract in the NO activity inhibitor is 0%, and B is in RAW264.7 cells at a specific concentration of the complex extract in the NO activity inhibitor. NO is the measured value (%).

The NO activity inhibitor of the present invention inhibits the activity of iNOS (inductive NO synthas), COX-2 (cyclooxygenase-2), tumor necrosis factor TNF-α (tumor necrosis factor α) and/or interleukin receptor IL6, It may have an NO inhibitory effect and/or an anti-inflammatory effect.

The anti-inflammatory agent of the present invention includes the herbal robert extract and tarragon leaf/stem extract; Or Herb Robert extract, tarragon leaf / stem extract and safflower seed extract; using a NO-activator containing a complex extract containing; Emulsions; The activator; Spices; And purified water.

In addition, the anti-inflammatory agent of the present invention contains 20 to 30 parts by weight of the oily liquid, 1 to 30 parts by weight of the NO activity inhibitor, 0.05 to 2 parts by weight of the fragrance, and the purified water of the residual amount with respect to 100 parts by weight of the aqueous solution. Preferably, with respect to 100 parts by weight of the aqueous solution, 22 to 28% by weight of the emulsion, 5 to 30 parts by weight of the NO activity inhibitor, 0.1 to 1 parts by weight of the perfume and the balance of purified water. At this time, if the oil solution is less than 20 parts by weight, there may be a problem in maintaining skin moisturization due to insufficient oil, and if it exceeds 35 parts by weight, excessive oil components may be contained, which may cause problems such as obstruction of skin breathing or oily skin, etc. . In addition, if the NO activity inhibitor is less than 1 part by weight, the content may be too small to show the NO activity inhibitory effect, and if it exceeds 30 parts by weight, the quality of the product is rather reduced due to poor compatibility with other components due to excessive use. It may fall off, and it is uneconomical because the effect of inhibitors of NO activity due to overuse is almost insignificant.

Among the anti-inflammatory agent components, the fragrance may be a general one used in the art, and specific examples include Perfume 85147, Perfume 25399, and the like.

[Awards]

Among the components of the anti-inflammatory agent of the present invention, the aqueous solution includes a skin conditioning agent, a metal ion blocking agent, a thickener, a moisturizing agent, a viscosity reducing agent, a sterilizing preservative and a solvent.

The skin conditioning agent in the aqueous solution component 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 kinds of 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 the 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 is less than 0.5 parts by weight, the amount of the xanthan gum is too small, and thus the effect of using it is insufficient. It is recommended to use within the above range since the stickiness may increase so that there may be a problem of poor marketability. As a thickener, a general one used in the art can be used, and preferably, one or more of one selected from xantham gum, guarhydroxy trimonium chloride and hydroxy ethyl cellulose can be used in combination.

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 a moisturizing agent, one type selected from methyl propanediol 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 adhesion to 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 based on 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, and when 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 may be too high or low, resulting in a skin contact with 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. Further, as the viscosity reducing agent, a general viscosity reducing agent used in the art may be used, preferably di(C2 ~ C5 alkylene) glycol, and more preferably dipropylene glycol. .

Next, the sterilizing preservative in the aqueous solution component is used to prevent the anti-inflammatory 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. Phenesin 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 Butylene glycol may be used by mixing, or more preferably, 1,2-hexanediol and 1,3-butylene glycol may be used by mixing. 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 components of the aqueous solution may not be good. It is uneconomical to use more than 60 parts by weight.

[Payment]

Among the components of the anti-inflammatory agent of the present invention, the oily liquid 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.

The moisture evaporation blocking agent in the emulsion component serves to maintain the proper moisture content during the manufacture of the anti-inflammatory agent and to maintain the proper moisture content together with the moisturizing agent when applying the prepared formulation to the skin. , Preferably, one or two selected from caprylic/capric triglyceride oil, MCT oil, and squalane can be used in combination, and more preferably, 5 to 5 MCT oil and squalane are used. It is good to mix and use in a 10:1 weight ratio.

Next, the tocopheryl acetate in the emulsion component serves to prevent the formulation from being oxidized and deteriorated 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. It 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 and is shiny, so it is preferable to use within the above range.

Next, the emulsifier in the emulsion component is intended to improve 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 the rough skin, the amount of use is 2 to 10 parts by weight, preferably 3 to 7.5 parts by weight based on 100 parts by weight of the skin conditioning agent 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, there may be a problem that the use amount is too high and stickiness may be severe when used. It is recommended to use within the above range. In addition, the skin softening agent may be used alone or in combination of two or more medium chain monoglyceride-containing oil agents, glyceryl caprate, polyglyceryl-2-laurate, and polyglyceryl-10-laurate. It can be used by mixing, 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 at least one medium chain monoglyceride is 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 external contaminants, etc. after the formulation is applied to the skin, and a general one used in the art can be used. 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 if 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. And, as the thickening agent, 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 for preparing the anti-inflammatory agent of the present invention described above will be described.

The anti-inflammatory preparations of the present invention are prepared by emulsifying the oily liquid and the aqueous solution, respectively, and emulsifying them under a specific condition temperature. Step 2 to prepare a mixture by adding the aqueous solution to the emulsion and then stirring; It can be prepared by performing a process comprising; three steps of mixing and stirring the mixture, herbal Robert extract (and/or fractions thereof), flavor and purified water at 40 ~ 50 ℃ and stirring speed of 2,000 ~ 3,000rpm.

Then, the oil phase of the first step is a skin conditioning agent, tocopheryl acetate, an emulsifier, a skin softener, a moisture evaporation blocking agent and a nonionic surfactant, and then a temperature of 60 to 75°C, preferably 65 to 72°C. 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 the first stage can be prepared by mixing a skin conditioning agent, a metal ion blocker, a thickener, a moisturizer, a viscosity reducing agent, a sterilizing preservative, and a solvent, followed by heating and stirring to 50 to 60°C.

The components used in the oil and water solutions and the amounts used are the same as described above.

In the second step, a mixture can be prepared by adding an aqueous solution to an oily phase and then emulsifying at a stirring speed of 2,000 to 3,000 rpm at 70 to 80° C. for 1 to 4 minutes.

In addition, the herbal extract of step 3 (and/or a fraction thereof) may be prepared by the method described above.

The anti-inflammatory preparation of the present invention thus prepared may be pH 5.4 to 6.2, preferably pH 5.8 to 6.0, more preferably pH 5.85 to 5.95.

Various types of cosmetics may be manufactured using the anti-inflammatory agent of the present invention described above, and for example, cosmetics such as a cream type and a serum type 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 Herb Robert Extract

The raw material was prepared by mixing 23 parts by weight of the dried herb robert with respect to 100 parts by weight of purified water.

Next, the raw material was extracted by hot water extraction at about 78 to 80° C. for 6 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 ethylhexylglycerin 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. Herb Robert extract was prepared.

Preparation Example 2: Preparation of tarragon leaf/stem extract

Raw materials were prepared by mixing 16 parts by weight of dried tarragon leaves and stems with respect to 100 parts by weight of purified water.

Next, the raw material was extracted by hot water extraction at 95 to 98° C. for 6 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 ethylhexylglycerin 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 tarragon leaf/stem extract was prepared.

Preparation Example 3: Preparation of safflower seed extract

The dried safflower seeds were crushed to prepare safflower seed powder. Next, after mixing the safflower seed powder and n-hexane at a concentration of 99.99% by volume, which is a non-polar solvent, in a weight ratio of 1:22, and extracting at 24 to 25°C, fat was removed. The extraction and fat removal were repeated three times to obtain an extract from which fat was removed. Next, the fat ratio-extracted extract and an aqueous ethanol solution having a concentration of 72 to 73% by volume were mixed at a weight ratio of 1: 10, and then extracted at 24 to 25°C to obtain an ethanol extract. Next, the ethanol extract was concentrated under reduced pressure, and then filtered to obtain a reduced pressure concentrate. Next, the ethanol extract was concentrated under reduced pressure, and then filtered to obtain a reduced pressure concentrate. Next, safflower seed extract was prepared by filtering the reduced pressure concentrate with a filter membrane having an average pore size of 10 μm.

Example 1: Preparation of complex extract (NO activity inhibitor)

The herbal extract of Preparation Example 1 and the tarragon leaf/stem extract of Preparation Example 2 were mixed at a weight ratio of 1:2.2 to prepare a composite extract.

Example 2: Preparation of complex extract (NO activity inhibitor)

The herbal extract of Preparation Example 1 and the tarragon leaf/stem extract of Preparation Example 2 were mixed at a weight ratio of 1:1.6 to prepare a composite extract.

Example 3: Preparation of complex extract (NO activity inhibitor)

The herbal extract of Preparation Example 1 and the tarragon leaf/stem extract of Preparation Example 2 were mixed at a weight ratio of 1:2.7 to prepare a composite extract.

Example 4

The herbal extract of Preparation Example 1, the tarragon leaf/stem extract of Preparation Example 2, and the safflower seed extract of Preparation Example 3 were mixed at a weight ratio of 1:2.2:0.3 to prepare a composite extract.

Comparative Example 1

Herb Robert extract alone of Preparation Example 1 was prepared as a NO activity inhibitor.

Comparative Example 2

The tarragon leaf/stem extract alone of Preparation Example 2 was prepared as an NO activity inhibitor.

Comparative Examples 3 to 4

A composite extract (NO activity inhibitor) was prepared in the same manner as in Example 1, but a composite extract having the composition shown in Table 1 below was prepared, respectively, to prepare a NO activity inhibitor.

Classification (weight ratio) Herb Robert Extract Tarragon leaf/stem extract Safflower Seed Extract Example 1 One 2.2 - Example 2 One 1.6 - Example 3 One 2.7 - Example 4 One 2.2 0.30 Comparative Example 1 One - - Comparative Example 2 - One - Comparative Example 3 One 1.2 - Comparative Example 4 One 3.3 -

Experimental Example 1: Measurement of cell viability for complex extract (NO activity inhibitor)

In order to confirm the cytotoxicity of the extract, RAW 264.7 cells, a mouse macrophage cell line, 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) from DMEM medium (high glucose, The culture was cultured in a 5% CO ₂ incubator using a 100 mm cell culture dish using SH30243.01 from Thermo. Cells that reached confluence were maintained by subculture using a scraper (90030 from SPL).

(2) Cell viability experiment

The dehydrogenase present in mitochondria in living cells produces a chromosome called formazan in Tetrazolium Salt (WST). Therefore, it is possible to measure the number of living cells by measuring this. A 96 well cell culture plate (3595 from Corning, Inc.) was used as a DMEM culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin/stereptomycin in RAW 264.7 cells at a concentration of 1×10 ⁴ cells/well. Incubate for 24 hours. Subsequently, the sample is replaced with DMEM medium contained in each concentration of the complex extract (0%, 0.125 wt%, 0.25 wt%, 0.5 wt%, 1 wt%, 2 wt%, 4 wt%) and cultured for 48 hours. Using the EZ-CYTOX reagent (EZ-1000 of Daeilab) on the cultured cells and measuring the absorbance at 450 nm, cell viability by concentration was measured according to Equation 2 below, and the results are shown in Table 2 below. . The measurement results in Table 1 below show the average values of three repeated experiments, and are measured relative to the control group (0% by weight of extract).

[Equation 2]

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

division 0 wt% 0.125% by weight 0.25 wt% 0.5 wt% 1 wt% 2 wt% 4 wt% Control 100% - - - - - - Comparative Example 1 - 100.03 100.26 101.17 102.68 103.81 102.23 Comparative Example 2 - 99.98 98.06 97.02 94.32 91.22 89.75 Comparative Example 4 - 99.82 99.40 98.24 95.61 93.22 90.17 Example 1 - 100.47 100.52 100.85 102.57 100.66 97.04 Example 2 - 100.51 100.96 101.58 102.98 100.98 98.66 Example 3 - 99.87 99.58 99.89 98.84 96.08 95.95 Example 4 - 100.55 100.67 101.38 102.89 101.42 98.09

Looking at the experimental results in Table 2, the test group treated with Herb Robert extract of Comparative Example 1 at a concentration of 2.0% or less did not show a statistically significant difference in cell viability compared to the control group, so RAW264 at a concentration of 2.0% or less .7 did not show cytotoxicity to cells. In addition, in the case of Comparative Example 2, in which the tarragon leaf/stem extract was used alone, when compared to the control and Comparative Example 1, the tarragon leaf/stem extract was somewhat toxic and showed a relatively low cell survival rate. On the other hand, in the case of Examples 1 to 3, compared to Comparative Example 1, the cell viability was somewhat low, but when compared with Comparative Example 2, it showed very good cell survival rate, and Example 4 in which safflower seed extract was introduced In the case, it showed a relatively high cell viability than Examples 1 to 3, and it is believed that the safflower seed extract is due to the mitigating effect of the toxicity of the tarragon leaf/stem extract.

In addition, in the case of Comparative Example 4 in which the tarragon leaf/stem extract was used in excess of 3.0 weight ratio with respect to 1 weight ratio of Herb Robert extract, it was confirmed that there was a problem in that cell viability was significantly lowered when compared to Example 3.

Experimental Example 2: Measurement of NO production inhibition of complex extract (NO activity inhibitor)

(1) Test substance treatment

RAW 264.7 cells were dispensed onto a 60 mm plate at a 2×10 ⁶ cells/dish density and attached to the plate for 24 hours. In order to induce an inflammatory reaction, a new medium containing LPS (lipopolysaccharide) at a concentration of 1 µg/ml and a test substance was treated and cultured for 24 hours.

(2) Measurement of nitrogen monoxide (NO) production

The NO level produced in RAW 264.7 cells and present in the culture was measured using a NO detection kit based on the Greases reaction (detection kit, 21021 from intron). After incubating 100 µl of the culture medium presumed to have NO in 96 well plates, add 50 µl of N1 buffer (sulfanilamide) and react at room temperature for 10 minutes. Subsequently, 50 µl of N2 buffer (naphthylethylenediamine) was added, 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-arginine acetate salt, Sigma M7033) was used.

Each result confirms the statistical significance of the test result by using the t-test (two-sided verification) function of Microsoft's Office Excel 2007, and the results are shown in Table 3 below. And, the NO production inhibition rate in Table 3 was calculated based on the following equation (1).

[Equation 1]

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

In Equation 1, A is NO production in RAW264.7 cells when the extract concentration in the NO activity inhibitor is 0% (100%), and B is NO in RAW264.7 cells at a specific concentration of the extract in the NO activity inhibitor. It is a measured value (%) of production. The measurement results in Table 2 below show the average values of three repeated experiments, and are the values measured relative to the control group (0% by weight of the extract).

NO production inhibition rate (%) 0 wt% 0.125% by weight 0.25 wt% 0.5 wt% 1 wt% 2 wt% Control 0% - - - - - Positive control
(L-NMMA 50μM) 61.05% - - - - - Comparative Example 1 - 16.62% 19.98% 22.07% 24.76% 22.54% Comparative Example 2 - 1.24% 2.35% 3.88% 3.96% 3.98% Comparative Example 3 - 18.95% 21.83% 26.14% 26.92% 27.54% Comparative Example 4 - 27.67% 30.81% 33.22% 35.04% 35.72% Example 1 - 25.34% 26.52% 31.97% 33.25% 31.17% Example 2 - 25.03% 25.80% 28.31% 30.72% 29.38% Example 3 - 27.08% 28.64% 33.78% 36.33% 36.30% Example 4 - 26.05% 27.14% 32.76% 35.18% 32.24%

Looking at the measurement results in Table 3, the positive control L-NMMA inhibited the production of NO in RAW264.7 cells by about 61% in the 50 μM concentration range. In addition, it can be confirmed that the herbal robert extract of Comparative Example 1 inhibits the production of Nitirc oxide (NO) of RAW264.7 cells at a statistically significant level in a concentration range of 0.125 to 2.0%. When the treatment was 0.125% by weight to 1% by weight, it was confirmed that about 10 to 25% of NO was inhibited, and at 2% by weight, there was a problem that the inhibition rate of NO production was lowered. And, in the case of the tarragon leaf/stem extract of Comparative Example 2, the effect of inhibiting NO production was insufficient.

On the other hand, in the case of Example 1 using a mixture of herb robert extract and tarragon leaf/stem extract, when compared with Comparative Example 1, it showed excellent NO production inhibition rate overall, and showed high NO production inhibition rate even at 0.5 to 1% by weight.

And, in the case of Example 4 in which safflower seed extract was further introduced, it showed a relatively better NO production suppression effect than Example 1. In addition, with respect to the Herb Robert extract 1 weight ratio, in the case of Comparative Example 3 in which the tarragon leaf/stem extract was used in a weight ratio of less than 1.5, when compared with Examples 1 and 2, the effect of inhibiting NO production was significantly reduced.

In addition, with respect to the Herb Robert extract 1 weight ratio, in the case of Comparative Example 4 in which the tarragon leaf/stem extract was used in excess of 3.0 weight ratio, when compared to Example 3, Comparative Example 4 showed excellent results in NO production at low concentration. From the concentration exceeding 0.5% by weight, rather than Example 3, the results showed a lower NO production inhibition rate.

Preparation Example 1: Preparation of anti-inflammatory preparations and cosmetics

(1) Preparation of aqueous solution

After completely dispersing the components of Table 4 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 5, stirring and stirring with an agitator at 68°C, and then 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) 100 parts by weight of the prepared aqueous solution was added to the main emulsification tank, 25 parts by weight of the oil phase was put in a separate oil phase tank, and each was heated to 80° C., and then mixed and emulsified while gradually adding the oil phase to the main tank. Ordered. Next, an oil phase was added, followed by emulsification at a rate of 2,500 rpm for the homer mixer and 40 rpm for the agitator for about 3 minutes. After the emulsification is complete, the contents of the azimixer are cooled at a rate of 12 rpm, and then 22 parts by weight of the complex extract of Example 1 and 0.12 parts by weight of perfume (Perfume 85147) are added to the contents at 45° C., and then the stirring is stopped at 38° C. and 25° C. It was aged in to prepare a creamy soft cream type cosmetic (milky white creamy).

Preparation Example 2: Preparation of anti-inflammatory agents and cosmetics

In the same manner as in Preparation Example 1, a soft cream type cosmetic (milky white creamy phase) was prepared, but instead of 22 parts by weight of the complex extract of Example 1, 27 parts by weight of the complex extract of Example 2 was prepared.

Through the above Examples and Experimental Examples, it was confirmed that the complex extract has an excellent anti-inflammatory effect, and has an oxidative stress defense effect. It is believed that the present invention is capable of developing NO active inhibitors and anti-inflammatory agents containing a complex extract, and providing various applied products such as cosmetic preparations, ointments using ointments, and health supplements using the same.

Claims (12)

delete Herbal robert extract; And tarragon leaf and stem extract; contains a composite extract containing 1: 1.6 to 2.7 weight ratio,
When the Herb Robert extract is contained in an amount of 2% by weight or less, the cell survival rate of RAW264.7 is 96.00% or more,
When the concentration of Herb Robert extract is 0.1 to 0.5% by weight, the NO activity inhibitor characterized in that the inhibition rate of nitrogen monoxide (NO) production in RAW264.7 cells is 25 to 43%, as measured according to the following equation (1) ;
[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 complex extract in the NO activity inhibitor is 0%, and B is in RAW264.7 cells at a specific concentration of the complex extract in the NO activity inhibitor. It is a measured value of NO production (%). The NO activity inhibitor according to claim 2, wherein the complex extract further comprises safflower seed extract. The NO activity inhibitor according to claim 3, wherein the herbal extract, tarragon leaf/stem extract and safflower seed extract are contained in a weight ratio of 1:1.6 to 2.7:0.2 to 0.5. delete delete Awards; Emulsions; The NO activity inhibitor of claim 2; Spices; And purified water;
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 emulsion includes a moisture evaporation blocking agent, tocopheryl acetate, skin conditioning agent, emulsifying agent, skin softening agent, nonionic surfactant and film forming agent,
An anti-inflammatory formulation comprising 100 to 30 parts by weight of the aqueous solution, 20 to 30 parts by weight of the emulsion, 1 to 30 parts by weight of the NO activity inhibitor, 0.05 to 2 parts by weight of the perfume, and the balance of purified water. delete The anti-inflammatory agent according to claim 7, characterized in that the pH is 5.8 to 6.0. Cosmetic comprising the anti-inflammatory agent of claim 9. The cosmetic product according to claim 10, which is non-irritating to acne skin. 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 stirring;
Performing a process comprising; 3 steps of mixing the mixture, the complex extract, fragrance and purified water under 40 ~ 50 ℃ and stirring at a stirring speed of 2,000 ~ 3,000rpm,
The complex extract includes herbal robert extract and tarragon leaf and stem extract, or herbal robert extract, tarragon leaf and stem extract and safflower seed extract ,
The complex extract is a method for producing an anti-inflammatory agent, characterized in that it comprises a herbal extract and tarragon leaf and stem extract in a weight ratio of 1: 1.6 to 2.7.