KR20200053082A - A composition for antioxidation, anti-inflammation, antiaging or skin soothing comprising herb complex extracts - Google Patents

Abstract

The present invention relates to a composition for improving skin condition using an herbal composite extract as an active ingredient. The composition of the present invention containing the herbal extract composed of Centella asiatica, wormwood, Illicium verum and Scutellaria roots as an active ingredient has anti-oxidant, anti-inflammatory, anti-aging and skin soothing effects, and has no cytotoxicity and side effects on skin, thereby being safely used in cosmetics and pharmaceutical compositions.

Description

A composition for antioxidation, anti-inflammation, antiaging or skin soothing comprising herb complex extracts using herbal extract as an active ingredient

The present invention relates to a composition for improving skin condition using herbal extracts as an active ingredient, and more specifically, antioxidant, anti-inflammatory, anti-aging and skin containing herbal extracts consisting of medicinal herbs, medicinal herbs, octagonal fennel and gold as active ingredients. It relates to a cosmetic composition and a pharmaceutical composition having a soothing effect.

The skin of the human body is an important organ that plays a role in protecting the body from external stimuli, and the surface of the skin consists of a structure in which the sebaceous membrane secreted from the sebaceous and sweat glands covers the stratum corneum, always replenishing insufficient moisture and maintaining a shiny healthy state. In addition, the skin protects the skin from UV rays and plays an important role in the body, such as performing temperature control functions in the body. Compared to other organs, the skin is an organ in which extinction and production occur periodically, and functions to protect the body and prevent water loss in the body. It is ideal when the moisture content of the stratum corneum is 20%, but when moisture is lost, the stratum corneum falls out and is easily exposed to the outside, causing irritation and skin diseases.

Other factors that accelerate aging include lack of moisture and skin irritation. Although 60-70% of the human body is made up of moisture, the skin loses moisture due to external stimulation, and the skin loses moisture, which causes skin aging such as insufficient moisture and reduced skin elasticity. An inflammatory response is a physiological response that protects a living body from factors that are or are potentially harmful to tissues. Cells and connective tissue are damaged by proteolytic enzymes caused by excessive inflammation, and skin elasticity is reduced, which causes wrinkles, which leads to rapid skin aging. Therefore, it is possible to suppress skin aging by protecting the damaged cells by inhibiting proteins and lipid-degrading enzymes produced by hyperinflammatory.

Dry skin has weakened immune function, and it is easy to cause allergies such as skin erythema, itching, and itching as well as atopic dermatitis. Therefore, it is important to prevent skin moisture loss. Cosmetics used to protect the skin and strengthen the barrier and enhance skin have various ingredients. Solubilizers, emulsifiers, preservatives, fragrances, sunscreens, and other functional active ingredients that are not related to skin protection purposes are It is known to cause irritation, inflammation, allergies, erythema, edema, and itching.

Therefore, interest in substances that alleviate the irritation of cosmetics is increasing, and various methods for alleviating skin irritation have been tried. For example, methods have been tried to remove substances that cause skin irritation. However, since the functional raw material itself often acts as a stimulus, there are limitations in such research, and it is required to use a substance having a stimulating effect and reducing side effects while maintaining the effect of the functional raw material.

Accordingly, the present inventors continued to study to develop new natural substances that have anti-oxidant, anti-inflammatory, anti-aging and skin soothing effects and have less human side effects, resulting in herbal extracts consisting of medicinal herbs, wormwood, octagonal fennel, and gold. The present invention was completed by discovering that it exhibits antioxidant, anti-inflammatory, anti-aging, and skin soothing effects without being cytotoxic.

Therefore, the technical problem to be solved in the present invention is to provide a composition having excellent human stability while having antioxidant, anti-inflammatory, anti-aging and skin soothing effects.

In order to solve the above technical problem, the present invention provides a composition for antioxidant, anti-inflammatory, anti-aging and skin soothing, which includes as an active ingredient an herbal extract consisting of yelp, wormwood, octagonal fennel and gold.

Preferably, the composition comprising the herbal complex extract in the present invention may be a cosmetic composition or a pharmaceutical composition.

In the present invention, the "active ingredient" refers to a component that can exhibit the desired activity alone or itself can exhibit activity with an inactive carrier.

In the present invention, anti-aging means skin protection and improvement of skin condition, skin whitening, prevention or improvement of skin aging and wrinkles, shrinking pores, shrinking, converging skin, moisturizing skin, soothing skin, protecting skin and alleviating inflammatory reactions of skin , Immune disease improvement function, or skin barrier function improvement, skin irritation relaxation, skin cell proliferation and regeneration ability, antioxidant ability, collagen synthesis enhancement ability, skin protection against UV rays, skin damage caused by UV rays, antibacterial, acne improvement, etc. It is an all-inclusive concept.

In the present invention, skin soothing means to alleviate skin troubles, for example, in the case of summer or due to stress, when more sebum occurs, skin troubles occur, and areas where skin troubles occur become more and more red. It means to alleviate these skin problems.

In the present invention, the skin is a concept that includes not only the face, but also the scalp and the whole body, as a composition for external application for skin that can be applied to such scalp, there is a shampoo, conditioner, treatment, hair repellent, etc., and a body cleanser that can be applied to the whole body It can be manufactured in various forms for the purpose of, for example.

According to one preferred embodiment of the present invention, the mixed weight ratio of the herbal complex extract may be variously changed, but 1-5: 1-5: 1-5: 1-5 of bottled grass, wormwood, octagonal fennel, and gold It is preferred to mix in a dry weight ratio of, in particular, it is more preferable to mix 2: 1: 1: 1 in a dry weight ratio of centella, wormwood, octagonal and golden.

The bottled grass used in the present invention is a perennial plant of the mountain-flowering parsley family, and the island of Madagascar in Africa is widely indigenous to high-temperature and humid areas of Southwest Asia and Central and South America, including the origin and coastal regions of the Indian Ocean, and is distributed in Jeju Island and southern island regions in Korea. Doing. In addition, bottle grass has been used for a long time as a medicinal product through traditional therapies in Asia and is known to have an effect on the treatment of skin trauma, chronic ulcers, and brain diseases. In particular, the main components of the water-soluble extracts, Asiatosicoside, Madecassoside, Asiatic Acid, and Madekas acid, are substances having regenerative properties of skin tissue, and have been reported to improve the production of extracellular matrix through promoting collagen production. Among them, Asiaticoside is certified and used as an active ingredient for improving skin wrinkles.

The medicinal mugwort used in the present invention is a perennial herb belonging to the family Asteraceae, and dried dried whole leaves are called cotyledon or lover, and stems and leaves are medicinal, young leaves are edible, and ordinary leaves are used as medicinal materials. It has strong fertility that grows throughout Korea. It is estimated that about 300 of the 400 Atremisia plants are native to Korea. In addition to edible wormwood, medicinal mugwort has been reported to have effects such as bleeding and hemostasis in oriental medicine. The fragrance or essential oil component of mugwort is known to have various physiological activities such as insecticidal, antibacterial and anti-tumor, and the main components are cineole, a-thujon, sesquiterpene, and ses Quiterpene alcohol, camper, terpinene-4-ol, coumarin, capillin, borneol, and the like.

The octagonal fennel used in the present invention belongs to the family Illciacease , and has over 40 species in the United States, Mexico, and Southeast Asia, and has been used as a folk and traditional medicine for the treatment of anti-inflammatory, expectorant, and gastrointestinal diseases. Among them, octagonal fennel is a fruit of star shape and anise, and is mainly grown in China and Vietnam. It is used as a spice in Asia and as a tea in Latin America. Octagonal fennel is a fruit, boiled as it is or boiled in boiling water. The components of the octagonal fennel consist of essential oils, sesquiterpenes, phenylpropanoids, liganan, etc., and in particular, essential oil components include trans-anethole, anisealdehyde, Contains limonene, estragole, anisyl acetate, feniculin, linalool, A-trans-bergamotene and trans-anetol (trans-anethole) contains about 86-93%. Sesquiterpenes include veranisations A, B, C, anisatin, and meoanisatin, and phenylpropanoids and lignans are verimols A, B , C, D, E, F, K, etc. have been reported.

The golden (黃 芩) used in the present invention is a perennial herb belonging to the family Lamiaceae, and has a height of 20-60 cm, distributed in Korea, China, Mongolia, and eastern Siberia, growing in the grass of mountainous regions, and is now commonly cultivated. It is a root with the golden bark removed, and it is collected in the fall and dried by removing the fine roots and the outer skin. It has been used as a treatment for allergic inflammatory diseases such as bronchial asthma, atopic dermatitis, hyperlipidemia and arteriosclerosis in oriental medicine. It contains various physiologically active substances such as golden flavonoids, baicalein, wogonin, and wogonoside. Efficacy has various activities such as anti-inflammatory, anti-allergic, anti-bacterial and anti-viral, and other antioxidant activities such as lipid peroxidation inhibition and radical generation inhibition have been reported.

The term "extract" of the present invention refers to a formulation in which a crude drug is squeezed into an appropriate leach solution and concentrated by evaporating the leach solution, but is not limited thereto, but an extract obtained by an extraction treatment, a dilution or concentrate of the extract, or an extract It may be a dried product obtained by drying, a crude product or a purified product thereof.

According to a specific embodiment of the present invention, the herbal complex extract may be mixed with four dry herbal medicines of bottle grass, medicinal mugwort, octagonal fennel, and gold first, and then extracted with an extraction solvent, or each herbal medicine may be first extracted and then mixed. have.

The herbal extract of the present invention can be prepared using general extraction methods, separation and purification methods known in the art. The extraction method may include hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction, but is not limited thereto.

The extraction solvent used in the present invention is water, alcohols having 1 to 4 carbon atoms, ethyl acetate, glycerin, ethylene glycol, propylene glycol, butylene glycol and mixed solvents thereof (for example, hydrous alcohol, hydrous glycerin, hydrous ethylene glycol) , Hydrous propylene glycol, hydrous butylene glycol), and is preferably extracted with water, a solvent selected from anhydrous alcohol having 1 to 4 carbon atoms or hydrous alcohol. Here, the alcohol having 1 to 4 carbon atoms may be methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, and ethanol is preferred. Here, the hydrous alcohol may contain water in an amount of 0.3 to 90 (wt / wt)%. Of these extraction solvents, hydrous ethanol is most preferred, and the amount of water contained is preferably 30 to 90 (wt / wt)%, more preferably 60 (wt / wt)%.

According to a specific embodiment of the present invention, bottled grass, wormwood, octagonal fennel and golden extract are, for example, bottled water, wormwood, octagonal fennel and gold, each having a volume of 1 to 50 times the dry weight of each of water and 1 to 4 carbon atoms. After extracting the active ingredient by heating extraction at 30 to 100 ℃ for 1 to 48 hours, or by immersion at 4 to 30 ℃ for 3 to 20 days by adding an extraction solvent selected from alcohols or hydrous alcohols thereof, the extraction solvent is used as a vacuum concentrator. Concentration can be obtained respectively. In this case, an extractor equipped with a cooling condenser may be used to prevent evaporation of the solvent.

In the present invention, each use part of bottle grass, wormwood, octagonal fennel, and gold can be used for leaves, flowers, roots and seeds.

In the composition of the present invention, bottled grass, wormwood, octagonal fennel, and golden extract may be added to the composition in an amount of 0.001 to 10.0% by weight relative to the total weight of the cosmetic composition, preferably 0.001 to 5.0% by weight Can be added.

The composition containing the herbal complex extract of the present invention exhibits an antioxidant effect, an anti-inflammatory effect, an anti-aging effect and a skin soothing effect, and has little cytotoxicity as a natural substance.

According to one embodiment of the present invention, there is provided a cosmetic composition comprising the herbal complex extract as an active ingredient.

In the cosmetic composition according to one embodiment of the present invention, in addition to the herbal complex extract as an active ingredient, ingredients conventionally added to the cosmetic composition, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and conventional adjuvants, and Additional carriers can be added.

The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleaning , May be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared in the form of a nutrition cream, astringent lotion, soft lotion, lotion, essence, nutrition gel or massage cream.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in the case of a spray, additionally chlorofluorohydrocarbon, propane / Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or trakant gum and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide as a carrier component Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition, in the cosmetic composition of each formulation, other ingredients other than the above mentioned medicinal herb, medicinal mugwort, octagonal fennel, and golden extract may be arbitrarily selected and blended according to other cosmetic formulations or purpose of use. For example, colorants (pigments), flavors (flavors), suspending agents, emulsifiers, solubilizers, stabilizers, isotonic agents, pH adjusting agents, viscosity modifiers, solvents, preservatives, etc., which are commonly used in cosmetic compositions may be included. .

The pharmaceutical composition according to one embodiment of the present invention includes a pharmaceutically acceptable carrier in addition to the herbal complex extract. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto It does not work. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenteral administration, more preferably applied in a topical application mode by application.

Suitable dosages of the pharmaceutical compositions of the invention are variously prescribed by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. Can be. The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. In addition, in the case of an external preparation, it is good to apply for 1 to 5 times a day in an amount of 1.0 to 3.0 ml on an adult basis and continue for at least 1 month. However, the dosage is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention is prepared in a unit dose form or multi-dose by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art. It can be manufactured by incorporating into a container. At this time, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an exir, powder, granule, tablet, or capsule, and may further include a dispersant or stabilizer.

On the other hand, the herbal complex extract of the present invention is a natural substance, which is harmless to the human body and has little toxicity and side effects, so it can be used safely even for long-term use. In particular, it can be safely applied to cosmetics and pharmaceutical compositions as described above.

The composition containing the herbal extract, medicinal mugwort, octagonal fennel, and golden herbal complex extract of the present invention has antioxidant, anti-inflammatory, anti-aging and skin soothing effects, and has no cytotoxicity and skin side effects, so it can be safely used in cosmetics and pharmaceutical compositions Can be.

1 is a graph showing the amount of tumor necrosis factor-α produced by the herbal extract.
Figure 2 is a graph showing the amount of interleukin (IL) -1β production of herbal complex extracts.
Figure 3 is a graph showing the amount of IL-8 production of herbal extracts.
Figure 4 is a graph showing the cytotoxicity of the herbal extract.

Hereinafter, the present invention will be described in more detail through examples. These examples and the like are only for explaining the present invention in more detail, according to the gist of the present invention, the scope of the present invention is not limited by these reference examples and examples, etc. to those skilled in the art. It will be obvious.

<Example 1> Preparation of oriental medicine complex extract of bottled grass, medicinal mugwort, octagonal fennel, and golden extract

After washing the bottle grass, wormwood, octagonal fennel and golden extract with purified water, add 25 kg of dried bottle grass, wormwood 25 g, octagonal fennel 25 g and golden 25 g (weight ratio of 1: 1: 1: 1) with 2 kg of distilled water and cool the condenser. The extractor was warmed at 60 ° C. for 2 hours, extracted 3 times, filtered with a 300 mesh filter paper, and allowed to stand at room temperature for 1 week. The precipitate was filtered twice with Adventec No. 5 filter paper and Whatman GFC 150 mm filter paper. Subsequently, after concentrating at a temperature of 40-50 ° C. using a vacuum concentrator, 12 g of a lyophilized herbal extract powder was obtained.

<Examples 2 to 17>

A herbal extract was prepared according to the extraction method described in Example 1 by mixing bottle grass, wormwood, octagonal fennel and gold in a weight ratio as shown in Table 1 below.

<Comparative Examples 1 to 15>

A herbal extract was prepared according to the extraction method described in Example 1 by mixing bottle grass, wormwood, octagonal fennel and gold in a weight ratio as shown in Table 1 below.

division Mix weight ratio Example 1 1: 1: 1: 1 Example 2 1: 1: 1: 2 Example 3 1: 1: 2: 1 Example 4 1: 2: 1: 1 Example 5 2: 1: 1: 1 Example 6 1: 1: 2: 3 Example 7 1: 2: 3: 1 Example 8 2: 3: 1: 1 Example 9 3: 2: 3: 1 Example 10 3: 1: 1: 4 Example 11 1: 4: 4: 1 Example 12 1: 4: 2: 4 Example 13 4: 1: 1: 1 Example 14 1: 2: 1: 5 Example 15 1: 1: 5: 1 Example 16 1: 5: 1: 2 Example 17 5: 1: 1: 1 Comparative Example 1 One:-:-:- Comparative Example 2 -:One:-:- Comparative Example 3 -:-:One:- Comparative Example 4 -:-:-:One Comparative Example 5 1: 1:-:- Comparative Example 6 1:-: 1:- Comparative Example 7 1:-:-: 1 Comparative Example 8 -: 1: 1:- Comparative Example 9 -: 1:-: 1 Comparative Example 10 -:-: 1: 1 Comparative Example 11 -:-:One:- Comparative Example 12 1: 1: 1:- Comparative Example 13 1: 1:-: 1 Comparative Example 14 1:-: 1: 1 Comparative Example 15 -: 1: 1: 1

<Test Example 1> Measurement of antioxidant effect

(1) DPPH radical scavenging ability measurement

The electron donating ability (EDA) was measured according to Blois' method (1958). 1 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, stirred, left to stand for 30 minutes, and absorbance was measured at 517 nm. DPPH radical scavenging ability was calculated according to the following equation (1).

At this time, OD _Sample is the absorbance value measured by mixing the sample and DPPH solution, and OD _Blind is the absorbance value measured by adding ethanol instead of the sample.

The measurement of DPPH radical scavenging ability is a method of measuring the electron donating ability, and the larger the reducing power is, the stronger the antioxidant becomes. Based on the degree of reduction of DPPH, the reducing power and antioxidant power of the measurement material can be estimated. DPPH electron donating ability to inhibit oxidation by donating electrons of free radicals by participating in the chain reaction of lipid peroxidation was measured by adjusting and adding the herbal complex extract to a concentration of 10-1,000 ug / ml. As a positive control, antioxidants such as dibutyl hydroxy toluene (BHT) and vitamin C were used, and the 50% inhibitory concentration (IC ₅₀ ) is shown in Table 2 below.

division IC ₅₀ Example 1 25.2 Example 2 26.4 Example 3 24.5 Example 4 26.5 Example 5 35.4 Example 6 31.1 Example 7 23.6 Example 8 24.1 Example 9 22.4 Example 10 24.1 Example 11 24.6 Example 12 25.5 Example 13 33.5 Example 14 25.2 Example 15 21.6 Example 16 23.1 Example 17 32.1 Comparative Example 1 8.1 Comparative Example 2 7.3 Comparative Example 3 8.4 Comparative Example 4 8.3 Comparative Example 5 9.1 Comparative Example 6 11.4 Comparative Example 7 12.1 Comparative Example 8 13.4 Comparative Example 9 13.5 Comparative Example 10 12.1 Comparative Example 11 15.5 Comparative Example 12 16.3 Comparative Example 13 14.2 Comparative Example 14 16.5 Comparative Example 15 15.4 BHT 101.4 Vitamin C 24.8

As shown in Table 2, it was confirmed that the herbal complex extract of the present invention has excellent ABTS radical inhibitory activity and thus has an antioxidant effect. In particular, it was confirmed that it exhibits an excellent effect than the positive control vitamin C.

(2) ABTS radical cation scavenging ability measurement

Mix (1: 1, v / v) of 2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) (14.8 mM) and potassium peroxidate (1: 1, v / v) and in a room temperature cow. It reacted for 16 hours. The ABTS solution was diluted with tertiary distilled water and adjusted to absorbance of 0.700 ㅁ 0.05 at 734 nm. After that, the herbal extract (20 μL) and ABTS solution (180 μL) were placed in a 96-well plate and reacted in the cow for 30 minutes. The absorbance of the reaction solution was measured at 723 nm. Distilled water (200 μL) was used for the blank test, and distilled water (20 μL) and ABTS solution (180 μL) were used for the control group. The free radical scavenging ability was calculated according to the following equation (2).

The experimental values were repeated 3 times and measured and then expressed as the average value.

As a positive control, an antioxidant, dibutyl hydroxy toluene (BHT), was used, and the 50% inhibitory concentration (IC ₅₀ ) is shown in Table 3 below.

division IC ₅₀ Example 1 33.5 Example 2 32.6 Example 3 33.2 Example 4 32.6 Example 5 35.2 Example 6 32.5 Example 7 31.4 Example 8 33.6 Example 9 31.2 Example 10 31.5 Example 11 31.7 Example 12 31.1 Example 13 31.5 Example 14 33.4 Example 15 31.3 Example 16 32.3 Example 17 32.2 Comparative Example 1 4.6 Comparative Example 2 6.4 Comparative Example 3 5.1 Comparative Example 4 7.3 Comparative Example 5 8.5 Comparative Example 6 10.1 Comparative Example 7 12.2 Comparative Example 8 11.3 Comparative Example 9 12.4 Comparative Example 10 11.2 Comparative Example 11 14.1 Comparative Example 12 11.3 Comparative Example 13 14.1 Comparative Example 14 14.3 Comparative Example 15 12.3 BHT 34.1

As shown in Table 3 above, it was confirmed that the herbal complex extract of the present invention has excellent ABTS radical inhibitory activity and thus has an antioxidant effect.

< Test example  2> Anti-inflammatory activity measurement

1) Measurement of NO production

RAW264.7 macrophages were dispensed at 2.5 24-10 ⁵ cells / mL per well into 24-well cell culture plates and cultured for 18 hours under 37 ° C and 5% CO ₂ incubator conditions. The cultured cells were replaced with a medium containing 1 μg / mL of lipopolysaccharide (LPS) to induce the production of NO, and then cultured for 24 hours by adding the herbal complex extract prepared in the above example. Then, the amount of NO generated was mixed with 100 μL of the cell culture supernatant and 100 μL of the Griess reagent (0.1% naphthylethylenediamine dihydrochloride and 1% sulfanylamine in 2.5% phosphoric acid, 1: 1) in a 96-well plate. After reacting for a minute, absorbance was measured at 540 nm using an ELISA reader (Xmark microplate absorbance spectrophotometer, Bio-Rad, XMark ^{TM ,} Japan). The amount of NO generated was calculated by comparing with the standard curve for each concentration of sodium nitrate (NaNO ₂ ). The results are shown in Table 4 below.

NO is one of the highly reactive inorganic low-molecular reactive oxygen, L-arginine, which is synthesized as L-citrulline by NOS, kills bacteria or induces apoptosis against cancer cells, signaling, blood clotting and platelet inhibition , It plays an important role in physiological and pathological reactions in the body, such as neurotransmitters involved in blood vessel expansion. However, excessively formed NO can cause inflammation and cause tissue damage, genetic mutation, neurological damage, and immune diseases such as arthritis and bronchitis (Weise & Nathan 1991; Hyun et al., 2015).

division NO production (%) Example 1 63.3 Example 2 65.2 Example 3 63.4 Example 4 64.4 Example 5 60.8 Example 6 64.1 Example 7 63.3 Example 8 63.1 Example 9 66.3 Example 10 65.8 Example 11 66.6 Example 12 63.4 Example 13 61.5 Example 14 63.4 Example 15 65.7 Example 16 64.8 Example 17 60.2 Comparative Example 1 84.3 Comparative Example 2 86.4 Comparative Example 3 85.3 Comparative Example 4 87.5 Comparative Example 5 88.1 Comparative Example 6 90.9 Comparative Example 7 92.1 Comparative Example 8 91.6 Comparative Example 9 85.4 Comparative Example 10 84.1 Comparative Example 11 84.2 Comparative Example 12 81.3 Comparative Example 13 84.5 Comparative Example 14 84.1 Comparative Example 15 82.1 Control 100 Untreated 43

As a result, the LPS-treated group (control group) showed a higher NO expression level than the LPS-untreated group, Examples 1 to 17 showed up to 40% NO production inhibition rate, and the comparative groups 1 to 15 showed up to 18% It showed the NO production inhibition rate.

2) Measurement of cytokine production

LPS (1 μg) to verify the effect of a mixture of herbal extracts on the production of inflammatory cytokines such as TNF-α, IL-1β and IL-8 through Enzyme-linked immunosorbent assay (ELISA) / mL) to stimulate RAW 264.7 cells, and at the same time treat the extract, incubate for 24 hours in a 37 ° C, 5% CO ₂ incubator, and then recover the culture solution to determine the TNF-α, IL-1β, and IL-6 contents of ELISA. It was carried out according to the method suggested by the manufacturer using a cytokine kit (R & D system, Minneapolis, MN, USA).

The antibody-coated 96-well plate was added with standard solution (100 μL) and cell culture solution (100 μL) for 2 hours at room temperature, washed with washing solution, and then added with search antibody (100 μL) to bind for 1 hour. Ordered. After adding 100 μL of avidin-HRP to each well and reacting for 30 minutes, after washing with washing solution, adding 100 μL of TMB, reacting for 20 minutes in the dark, and adding 100 μL of stop solution to react The cytokine secretion was calculated by measuring the absorbance at 450 nm.

Macrophages are cells that maintain the homeostasis of the body by participating in innate and acquired immunity. When an antigen enters the body, the antigen is removed by phagocytosis or the antigen is presented to other immune cells to induce an inflammatory reaction. It plays a role in preventing the growth of (Lee et al., 2007). Macrophages have increased activity by immune-enhancing substances such as interferon (IFN), glycolipid (LPS), and tumor necrosis factor-α (TNF-α), interleukin (IL) -1β, IL-8 that increase the immune response. , Nitric oxide (NO) secretion, etc. (Hibbs et al., 1998). TNF-α is a multifunctional cytokine secreted when mast cells are activated, involved in immune and inflammatory responses, and promotes secretion of other inflammatory cytokines such as IL-1β and IL-8 (Barnes and Adcock, 1993; Butler et al., 1995). IL-8 is a chemotactic factor that promotes mobilization of neutrophils, lymphocytes, and eosinophils.It is known to activate neutrophils to release lysosomal enzymes and induce the production of active corals, and is involved in inflammatory diseases such as arthritis and sepsis (Harada et al., 1996). In addition, IL-1β is a cytokine produced by monocytes and macrophage cells, which is important for cytokine networks, such as activating T cells and enhancing the activity of cytokines such as IL-2 secreted from T cells. Plays a role (Cah et al., 2010).

The amount of cytokine (TNF-α, IL-1β, IL-8) secreted from the culture supernatant of RAW 264.7 macrophages treated with LPS (1 μg / mL) of the herbal extracts prepared in the above Example was measured, respectively. .

1 is a graph showing the amount of tumor necrosis factor-α produced by the herbal extract. As can be seen here, in the case of TNF-α, the LPS-treated group (control) showed a higher TNF-α expression level compared to the LPS-untreated group, and Examples 1 to 17 showed up to 30% inhibition of TNF-α production. Was confirmed.

Figure 2 is a graph showing the amount of interleukin (IL) -1β production of herbal complex extracts. As shown here, in the case of IL-1β, the LPS-treated group (control group) showed higher IL-1β expression compared to the LPS-untreated group, and the experimental groups 1 to 8 showed up to 29% of IL-1β production inhibition rate. Could confirm.

3 is a graph showing the IL-8 production amount of a mixture of herbal extracts. As shown here, in the case of IL-8, the LPS-treated group (control group) showed higher IL-8 expression compared to the LPS-untreated group, and the experimental groups 1 to 8 showed up to 27% inhibition of IL-8 production. Could confirm.

<Test Example 3> Cytotoxicity test of herbal extracts

The cytotoxicity of the herbal extracts was investigated. In this experiment, human keratinocyte cell line HaCaT cells and mouse macrophage cell line RAW264.7 cell culture were used to perform cytotoxicity experiments (MTT). After treating the samples with HaCaT cells and RAW264.7 cells, the cells were cultured in a serum free state to measure the cytotoxic effect of the cells using an MTT assay. MTT (3- (4,5-dimetylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) is a pale yellow substrate that is cleaved by the respiration enzyme in the mitochondria of living cells and produces dark blue formazan. Since dead cells do not react, the amount of formazan produced is used to measure the number of live cells. The results are shown in FIG. 4.

Figure 4 is a graph showing the cytotoxicity of the herbal extract. As shown here, in the case of HaCaT, the herbal complex extract (Example 5) did not show cytotoxicity at a concentration of 200 ppm or less. However, it showed some cytotoxicity at concentrations above 200 ppm. In the case of RAW264.7, the herbal complex extract did not show cytotoxicity at concentrations below 100 ppm, and showed little cytotoxicity at concentrations above 100 ppm.

<Test Example 4> Experiment to confirm the safety of herbal extracts against human skin

In the cytotoxicity test of Test Example 3, since the herbal complex extract was found to have no cytotoxicity, a skin safety verification experiment was conducted to confirm whether it is safe for human skin. Based on squalene, a formulation containing 1, 10 and 100 ppm of herbal extracts was prepared, and using this, 30 herbal healthy extracts were applied to the upper forearm area every other day for a total of nine 24 hours to obtain a herbal extract. Whether or not it irritated the skin was measured. As a method of application, a pin chamber (Finn chanber, Epitest Ltd, Finland) was used, and 15 µl of the external preparation for skin was added dropwise to the chamber, followed by application. The degree of reaction on the skin each time was scored using Equation 3 below, and the results are shown in Table 5 below.

In Equation 3, in the reactivity, ㅁ is given a score of 1 point, + is 2 points, and ++ is 4 points, and it is determined as a safe composition when the average reactivity is less than 3.

Test substance Number of subjects who responded Average
Reactivity 1 week 2 weeks 3 weeks Primary Secondary 3rd Primary Secondary 3rd Primary Secondary 3rd a b c a b c a b c a b c a b c a b c a b c a b c a b c Control
(Squalene) --- --- --- --- --- --- --- --- --- 0.09 Herbal Compound Extract 1ppm --- --- --- --- --- --- --- --- --- 0.00 10ppm --- --- --- --- --- --- --- --- --- 0.00 100ppm --- --- --- --- --- --- --- --- --- 0.00

In Table 5, a: ㅁ, b: +, and c: ++ are shown.

As shown in Table 5, in the case of the control group, the number of persons corresponding to ㅁ, +, and ++ was 1, 0, and 0, respectively, in the second week's second patch, and responded to the skin for the patch for the remaining period. Did not appear. In addition, in all the test groups in which the herbal complex extract was tested, there was no reaction on the skin. As a result of calculating the results of Table 5 according to Equation 3, the average reactivity of the control group was 0.09, and the average reactivity of the test group was 0.00, 0.00, and 0.00 at 1, 10, and 100 ppm, respectively, and was judged as a safe substance. .

<Production Example 1> Preparation of cosmetic

1-1. Manufacture of cream

As shown in Table 6 below, a cream containing an herbal extract (Example 5) as an active ingredient was prepared according to a conventional method.

ingredient Content (% by weight) Herbal complex extract (Example 5) 0.03 or 0.01 Carbo940940 0.12 Trehalose 3.00 Sorbitol 1.00 1,3-butylene glycol 5.00 Bis-page-18 methyl ether dimethyl silane 1.00 Glycerol 4.00 D-panthenol 0.30 Edith 0.02 Polysorbate 60 0.80 Glycerol monostearate 1.20 Glycerol stearate / page-100 stearate 0.90 Sorbitan Sesquiletate 0.30 Lanette-o 1.60 Cetyl ethylhexonoate 3.00 Caprylic / caric triglyceride 1.00 Cyclomethicone 6.50 Triethanolamine 0.13 Fenonib 0.70 Incense, preservative a very small amount Purified water Balance Sum 100

1-2. Toner manufacturing

As shown in Table 7 below, a toner containing the herbal complex extract (Example 5) as an active ingredient was prepared according to a conventional method.

ingredient Content (% by weight) Herbal complex extract (Example 5) 0.03 or 0.01 Disodium ID 0.01 Amino coat 1.00 D-panthenol 0.10 Trehalose 0.10 Glycerol 3.00 Carbopol Itdi 2020 0.07 Alcohol 3.00 Bis-page-18 methyl ether dimethyl silane 0.20 Phenyl trimethicone 0.10 PAGE-40 Hydrogenated Coastal Oil 0.15 Methylparaben 0.05 Triethanolamine 0.13 Fenonib 0.70 Phenoxyethanol 0.50 Fragrance, preservatives, coloring a very small amount Purified water Balance Sum 100

1-3. Preparation of lotion

As shown in Table 8, a lotion containing the oriental medicine complex extract (Example 5) as an active ingredient was prepared according to a conventional method.

ingredient Content (% by weight) Herbal complex extract (Example 5) 0.03 or 0.01 Carbomer941 0.05 Allantoin 3.00 Herb X Hyaluron 1.00 Methylparaben 0.10 Glycerol 1.10 Disodium ID 0.05 Polysorbate 60 1.00 Solitan Sesquioleate 1.00 Stearic Acid 1.00 Glycerol stearate / page-100 stearate 1.50 Cetyl alcohol 1.30 Propyl paraben 0.05 Butylparaben 0.05 Macadamia oil 0.05 Dimethicone 0.50 Caprylic / Capric Triglyceride 1.00 Mineral oil 4.50 Triethanolamine 0.10 Phenoxyethanol 0.50 incense a very small amount Purified water Balance Sum 100

< Test example  4> Herbal complex extract Skin soothing  effect

After dividing the group into 30 persons per evaluation group for applicants with severe skin troubles, the herbal complex extract obtained in Example 5 was paid to each group, and the product was used once daily for 2 weeks, and then the skin trouble after evaluation and 2 weeks after use The skin soothing effect of each test substance was evaluated by calculating the average by evaluating the relief effect and the skin soothing effect.

Skin trouble relief effect Skin soothing effect Control (purified water) 1.0 2.0 Herbal Complex Extract 4.8 4.9

As can be seen from Table 10, it was confirmed that the herbal complex extract according to the present invention improved both the skin trouble relieving effect, the skin soothing effect, and the skin trouble relieving effect than using only purified water.

Claims (3)

Antioxidant, anti-inflammatory, anti-aging, or skin soothing effects that include herbal extracts mixed with a weight ratio of 1-5: 1-5: 1-5: 1-5. Cosmetic composition having a. Antioxidant, anti-inflammatory, anti-aging, or skin soothing effects that include herbal extracts mixed with a weight ratio of 1-5: 1-5: 1-5: 1-5. Pharmaceutical composition having a. The method according to claim 1 or 2,
A composition characterized in that the inflammation is at least one selected from the group consisting of atopic dermatitis, psoriasis, seborrheic dermatitis, contact dermatitis, erythematous lupus and papule hives.

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