KR101816816B1 - Compositions for Angiogenesis, Skin Whitening and Anti-inflammatory Containing Lonicerae Flos Extract - Google Patents

Abstract

The present invention relates to a composition for angiogenesis, whitening, and anti-inflammation containing a Lonicerae Flos extract as an active ingredient. The Lonicerae Flos extract provided in the present invention increases the number of capillaries in skin which are reduced in number as skin aging proceeds, excellently inhibits tyrosinase and nitric oxide (NO) production, and can be applied as compositions for angiogenesis, whitening, and anti-inflammation.

Description

{Compositions for Angiogenesis, Skin Whitening and Anti-inflammatory Containing Lonicerae Flos Extract}

The present invention relates to a composition for angiogenesis, whitening and anti-inflammation comprising Lonicerae Flos extract as an active ingredient.

Angiogenesis (Neovascularization) is the process by which new blood vessels are created. It occurs infrequently in normal in vivo conditions, but is a process that accompanies embryogenesis, luteinizing or wound healing processes.

The process by which new blood vessels are formed is generally caused by the stimulation of new blood vessel promoting factors to reconstruct blood vessels by protease-induced degradation of blood vessel basement membrane, vascular endothelial cell migration, proliferation and formation of vascular endothelial cell differentiation, . The process of angiogenesis is known to be tightly controlled by several types of promoters and inhibitors, including growth factors, cytokines, lipid metabolites and potential fragments of hemostatic proteins.

If blood vessel formation, which is an important process in the development and wound healing and organ formation, does not occur properly, serious diseases will occur. For example, if blood vessels are not formed in the developmental stage, placenta may be undeveloped and cause abortion, and the occurrence of tissue ulcers and ischemia may cause dysfunction of the organs, leading to further death. Recent changes in dietary habits, nutritional status, and aging population have led to a variety of cardiovascular diseases, including arteriosclerosis, myocardial infarction and angina, cerebral infarction, and acute limb ischemia, And the development of new therapies and factors promoting or promoting neovascularization to treat such ischemic diseases is attracting attention.

Currently, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used as therapeutic agents for ischemic vascular disease. However, peptide values are very high and unstable. In addition, disadvantages such as unintended vascular leakage phenomenon in use, promotion of vascular inflammation, abnormally excessive proliferation of vascular smooth muscle cells, induction of inadequate proliferation of tissue fibroblasts, and the like are pointed out Has come. Therefore, there is a continuing demand for new factors with low risk of side effects and high efficacy and safety of treatment.

The most closely related skin whitening is generally known as melanin. The skin color of a person is determined by the content and distribution of melanin in the skin. In addition to genetic factors, skin color is affected by environmental or physiological conditions such as sunlight, fatigue, and stress. Melanin is synthesized in the melanocyte in the epidermis of the skin. Melanosome, the melanocyte in the melanocyte, acts on tyrosine, an amino acid, called tyrosinase, It is converted to quinone (Dopaquinone) and is made by nonenzymatic oxidation. When such a synthesis of melanin occurs excessively in the skin, it darkens the skin tone, and causes stains, freckles, senile plaques, and nevi. Thus, inhibiting the activity of tyrosinase in the skin to inhibit the synthesis of melanin pigment not only makes it possible to whiten the skin tone to brighten the skin tone, but also to reduce skin irritation caused by ultraviolet rays, hormones and hereditary causes, It can improve constipation.

Recently, as the development of technology and quality of life have improved the desire and concern for beauty, it has become necessary to develop a whitening product which prevents excessive melanin production. Accordingly, researches on the development of whitening agent and whitening cosmetic product are actively under way. Many whitening agents are used today to treat melanin deposits, but most of them are toxic to melanocytes and cause side effects. For example, hydroquinone, which has excellent whitening effect, is cytotoxic and has potential for kidney toxicity and carcinogenicity. In addition, it is prohibited for use in whitening cosmetics in Europe due to skin damage such as ochonosis. Kojic acid has excellent whitening effect but is cytotoxic and is known to cause contact dermatitis or cancer. On the other hand, arbutin and ascorbic acid, which are less toxic, have a weak inhibitory effect on tyrosinase. Citrus essential oils such as lemons also have a weak inhibitory effect on tyrosinase. They have been limited in their use due to safety concerns with the skin, stability problems within the formulation and insufficient whitening effects. Therefore, it is important to develop a substance that strongly inhibits tyrosinase with little side effects such as cytotoxicity.

Inflammation refers to the pathological condition of abscesses formed by the infiltration of external infectious agents (bacteria, fungi, viruses, various kinds of allergens). In other words, inflammation is one of the in vivo immune reactions that works when the outsider penetrates into the living body. It can be observed when the skin is irritated or scratched or swollen when it is seen in the peripheral part of the purulent acne. It is. Specifically, when external bacteria invade a specific tissue and proliferate, the leukocyte of the living body recognizes it and actively attacks the proliferated foreign germs. In this process, the dead cells of the leukocyte are accumulated in the invaded tissue At the same time, the cell debris of invading microorganisms killed by leukocytes melts into the invading tissues and abscess forms. The treatment of abscess due to inflammation can be promoted through the anti-inflammatory action. Anti-inflammatory action is the action of inhibiting the growth of invading microorganisms by using an antibacterial agent or activating macrophages that digest foreign substances accumulated in the abscess, And enhancing the function of excretory macrophages.

Generally, an inflammatory reaction is a biological defense reaction process for repairing and regenerating damage caused by an invasion that causes a fundamental change in a cell or tissue of a living body. In the reaction process, localized blood vessels, various tissue cells of body fluids, Lt; / RTI > Normally, the inflammatory reaction induced by external invading bacteria is a defense system to protect the living body. However, when abnormally excessive inflammatory reaction is induced, various diseases appear. These diseases are called inflammatory diseases. Inflammatory diseases are various inflammatory mediators secreted from target cells activated by external stimuli, which amplify and sustain inflammation, and are life-threatening diseases of the human body. These diseases include acute inflammation, diseases in joints such as rheumatoid arthritis, And allergic inflammatory diseases such as bronchial asthma and the like.

Among the various immune response factors, macrophage is an immune system cell present in almost all tissues and plays an important role in biological defense as well as innate immune response as well as acquired immune response with mononuclear cells. LPS (Lipo poly saccharide) is known to be an endotoxin that causes a pulmonary hemorrhagic shock when the body is infected with bacteria. Treatment of mouse RAW 264.7 macrophages with LPS increases the synthesis of proinflammatory cytokines such as IL-1β, IL-6, TNF-α and IL-13 and induces iNOS and COX-2 protein expression. And through the activation pathway of NF-kB and MAPK, the expression factors of proinflammatory cytokines and inflammatory mediators by LPS, to produce nitric oxide. Thus, macrophages induce inflammatory responses by LPS and are thus widely used to search for inflammation-improving materials.

As described above, research and development have been actively carried out in various fields having angiogenesis, whitening, and anti-inflammatory effects. In particular, studies using natural substances have continued to prevent toxicity or irritation to the skin.

On the other hand, Lonicerae Flos is a flower of Lonicera japonica Thunb. Belonging to Caprifoliaceae. The characteristic has a peculiar smell, the taste is sweet and the quality is cold. It contains about 1% of luteolin and inositol, and contains saponin, tannin and lonicerin (luteolin rhamnoglucoside). The efficacy has the effects of dermatological necrosis caused by dysentery, fever, mastitis, stomach ulcer, cystitis, sore throat, tonsillitis, bronchitis, conjunctivitis and swelling, Inflammatory action, antipyretic action, increased leukocyte phagocytosis, stimulation of central nervous system, lowering of serum cholesterol, and prevention of ulcer have been reported.

Honeysuckle (Lonicerae Flos) in the cosmetic compositions described using the extract, in the case of domestic Republic of Korea Patent No. 10-1655222 call "washing cleansers, body cleansers, cleansing gel and shampoo comprising the honeysuckle and jjilrekkot extract using deep ocean water" Korean Patent Laid-Open Publication No. 10-2008-0011546 entitled " Composition for hypodermic skin external application containing ginkgo biloba extract ", Korean Patent Laid-Open Publication No. 10-2005-0100473 "Quot;, and Korean Patent Registration No. 10-1546857 entitled " Anti-inflammatory or moisturizing composition containing ginkgo, europaea, and kelp mixture extract ", and the like.

Accordingly, the present inventors have found that Lonicerae Flos extract not only can increase the number of skin capillary blood vessels that are reduced as the skin ages, but also exhibit tyrosinase inhibitory activity and NO (nitric oxide) It is possible to simultaneously impart new-onset, whitening, and anti-inflammatory effects, thereby completing the present invention.

Korean Patent Publication No. 10-1655222 Korean Patent Publication No. 10-2008-0011546 Korean Patent Publication No. 10-2005-0100473 Korean Patent Publication No. 10-1546857

It is an object of the present invention to provide a composition for angiogenesis, whitening and anti-inflammation derived from natural products.

It is another object of the present invention to provide cosmetics, medicines, or foods containing the composition for angiogenesis, whitening and anti-inflammation.

In order to achieve the above object, the present invention provides a composition for angiogenesis, whitening and anti-inflammation comprising Lonicerae Flos extract as an active ingredient.

In the present invention, the Lonicerae Flos extract is extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, propylene glycol, glycerin, and mixed solvents thereof.

In the present invention, the Lonicerae Flos extract is an enzyme extract extracted by enzymatic treatment of a Ganoderma lucidum extract.

In the present invention, the enzyme is at least one selected from the group consisting of arabinoxyhydrolase, fibrinolytic enzyme, glucanolytic enzyme, hemicellulolytic enzyme, xylan acid degrading enzyme and pectin hydrolyzing enzyme.

In the present invention, the Lonicerae Flos extract is selected from the group consisting of chloroform, methyl chloride, normal-hexane, acetic acid and mixtures thereof, and the enzyme extract obtained by enzymatic treatment of Ganoderma lucidum or Ganoderma lucidum extract is selected from the group consisting of And the fraction is an organic solvent.

In the present invention, the Lonicerae Flos extract is contained in an amount of 0.001 to 10% by weight based on the total weight of the composition.

In the present invention, the Lonicerae Flos extract is characterized in that it has an angiogenic effect, a tyrosinase inhibitory effect and an inhibitory effect on NO (nitric oxide) production.

In the present invention, the composition further comprises Astragali Radix extract, Angelicae Gigantis Radix extract and Glycyrrhizae Radix extract.

In the present invention, the composition comprises 3 to 20% by weight of Lonicerae Flos extract, 40 to 60% by weight of Astragali Radix extract, 10 to 40% by weight of Angelicae Gigantis Radix extract, and Glycyrrhizae Radix extract, And 10 to 25% by weight of the extract.

The present invention also provides a cosmetic comprising the composition.

In addition, the present invention provides a pharmaceutical product comprising the above cosmetic composition.

The present invention also provides a food comprising the cosmetic composition.

The Lonicerae Flos extract provided from the present invention not only can increase the number of skin capillary blood vessels that are reduced as the skin ages, but also has excellent tyrosinase inhibitory effect and NO (nitric oxide) , Angiogenesis, whitening, anti-inflammatory compositions.

1 is a graph showing the cytotoxicity evaluation of Examples 1 to 5 and Comparative Examples 1 to 3.
FIG. 2 is an image obtained by microscopic observation of the angiogenesis state of Example 1 and Comparative Examples 1 to 3. FIG.
FIG. 3 is an image obtained by microscopic observation of the angiogenesis state of Examples 2 to 5. FIG.
Fig. 4 is a graph showing tyrosinase inhibitory activity of Example 1 and Comparative Examples 1 to 3. Fig.
Fig. 5 is a graph showing tyrosinase inhibitory activity of Examples 2 to 5. Fig.
6 is a graph showing the inhibitory effect of NO (nitric oxide) production by Examples 1 and 2 and Comparative Examples 1 and 3.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

According to one embodiment of the present invention, the present invention relates to a composition for angiogenesis, whitening and anti-inflammation comprising Lonicerae Flos extract as an active ingredient.

Honeysuckle according to the invention (Lonicerae Flos extracts may be obtained through various extraction methods known in the art and are specifically selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, propylene glycol, glycerin, and mixed solvents thereof Or may be extracted with a solvent.

Specifically, the content of the extraction solvent may be 2 to 100 times, more preferably 5 to 50 times, and most preferably 10 to 20 times the weight of the Lonicerae Flos solids.

In addition, Lonicerae Flos extract can be extracted by room temperature or warming under conditions that the active ingredient of the extract is not destroyed or the destruction is minimized. The extraction method is not particularly limited, and for example, cold extraction, ultrasonic extraction, reflux cooling extraction, room temperature extraction, hot water extraction and the like can be used depending on the kind of the extract and the extraction method.

Filtration is a process of removing suspended solid particles from an extract, and may be performed by filtering particles using a cotton, nylon, paper, or the like, or by ultrafiltration, freezing filtration, centrifugation, and the like.

The step of concentrating and then drying the filtrate includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying and the like. In some cases, a step of pulverizing the finally dried extract may be added.

According to one embodiment of the present invention, the Lonicerae Flos extract may be an enzyme extract extracted by enzymatic treatment of a gingkoceae solvent extract.

It is preferable that the enzyme is at least one selected from the group consisting of arabinoxyhydrolase, fibrinolytic enzyme, glucanolytic enzyme, hemicellulose degrading enzyme, xylan acid degrading enzyme and pectin hydrolyzing enzyme, It is preferable from the viewpoint of excellent solubility.

According to one embodiment of the present invention, the Lonicerae Flos extract may be prepared by treating an enzyme extract obtained by treating a Ganoderma lucidum extract or a Ganoderma lucidum extract with an enzyme, in the presence of chloroform, methyl chloride, normal-hexane, And a fraction obtained by fractionation with an organic solvent selected from the group consisting of When the fraction is used, the content of the active ingredient to be extracted is increased to increase the number of skin capillary blood vessels as the skin ages, and it is preferable from the viewpoint that the whitening and anti-inflammatory effect can be further improved.

According to one embodiment of the present invention, the Lonicerae Flos extract may be processed into various forms such as an extract, a powder obtained by drying the extract, and a concentrate obtained by concentrating and filtering the extract.

According to one embodiment of the present invention, the Lonicerae Flos extract obtained from the above method may be contained in an amount of 0.001 to 10% by weight, more preferably 0.1 to 1% by weight, based on the total weight of the composition. If the amount of the Lonicerae Flos extract is less than 0.001% by weight based on the total weight of the composition, the function as a composition is poor because the angiogenesis, whitening, and anti-inflammatory effects are not sufficiently obtained. It is not economical because it can exhibit sufficient efficacy even if it is not contained, and stability and spreadability of the formulation are lowered, which makes it difficult to use as a cosmetic product.

The Lonicerae Flos extract is characterized in that it has an angiogenic effect, a tyrosinase inhibitory effect and an inhibitory effect on NO (nitric oxide) production. In other words, since the angiogenesis, whitening, and anti-inflammatory effects can be given at the same time, not only skin beauty but also fundamental skin health improvement can be achieved.

According to an embodiment of the present invention, the composition comprising the Lonicerae Flos extract may further comprise at least one of Astragali Radix extract, Angelicae Gigantis Radix extract and Glycyrrhizae Radix extract, It is preferable since it is more excellent in the efficacy against extract use.

The Astragali Radix extract, Angelicae Gigantis Radix extract and Glycyrrhizae Radix extract can be prepared in the same manner as the extraction method of Lonicerae Flos extract as described above.

At this time, the kinds and locations of Lonicerae Flos , Astragali Radix , Angelicae Gigantis Radix and Glycyrrhizae Radix used for producing the mixed extract are not particularly limited. The composition containing the mixed extract also contains 3 to 20% by weight of Lonicerae Flos extract, 40 to 60% by weight of Astragali Radix extract, 10 to 40% by weight of Angelicae Gigantis Radix extract and 10 to 40% by weight of Glycyrrhizae Radix ) Of 10 to 25% by weight of the extract of the present invention is not only capable of increasing the number of skin capillary blood vessels that are reduced as the skin ages, but also exhibits remarkably superior tyrosinase inhibitory effect and NO (nitric oxide) .

As described above, the Lonicerae Flos extract is excellent in angiogenic efficacy, tyrosinase inhibitory effect and NO (nitric oxide) production inhibitory effect, and thus can be used as a composition for angiogenesis, whitening and anti-inflammation.

The composition for angiogenesis, whitening and anti-inflammation may be used as a cosmetic composition, a pharmaceutical composition or a food composition, but is most preferably used as a cosmetic composition.

According to one embodiment of the present invention, when used as the cosmetic composition, it can be used as an external ointment for skin, a cream, a softening longevity, a convergent lotion, a nutritional lotion, a pack, an essence, a hair tonic, a shampoo, a rinse, a hair conditioner, Lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutritional cream, eye cream, moisturizing cream, hand cream, foundation, nutrition essence, sunscreen, soap, skin lotion, But are not limited to, any of the formulations selected from the group consisting of cleansing foams, cleansing lotions, cleansing creams, cleansing waters, powders, body lotions and body cleansers. The composition of each of these formulations may contain various kinds of bases and additives necessary for formulation of the formulation, and the kinds and amounts of these ingredients can be easily selected by those skilled in the art.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, .

In the case where the formulation of the present invention is bar diesel, in addition to the above-mentioned effective ingredients, there may be used carbomers, triethanolamine, menthol, ethanol, glycerin, disodium iodide, methylparaben, Can be used. Herein, the carbomer and triethanolamine function as a gradual agent, the menthol functions as a fragrance component, the ethanol functions to give a cooling sensation, the glycerin serves as a moisturizing agent, and the disodium is dithiene, And methylparaben is added for its function as an antiseptic. In addition, the fragrance may include various fragrances such as herb fragrance in addition to menthol fragrance.

In addition to the above-mentioned specific ingredients, it is also within the scope of the present invention to apply the known substances widely used in the art to achieve the functions of the thickener, refreshing feeling, fragrance ingredient, .

When the formulation of the present invention is a powder or a spray (spray), lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be added as a carrier component, Propellants such as carbon, propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is added as a carrier component. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid esters of sorbitan, and the like.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

According to one embodiment of the present invention, cosmetic compositions for angiogenesis, whitening and anti-inflammation may be prepared from compositions such as bar diesel, body lotion, body cream, and body oil for accelerating fat decomposition by adding ordinary additives, It can also be manufactured as an aerosol type. In this case, it is preferable that the cosmetic composition is used as a transdermal administration method in which it is directly applied or sprayed on the skin.

According to one embodiment of the present invention, the amount of the cosmetic composition for angiogenesis, whitening and anti-inflammation can be appropriately adjusted depending on individual differences, formulations and forms such as the age and condition of skin fat, Can be usefully used as a composition for an effect.

The composition obtained from the above method exhibits an angiogenic effect, tyrosinase inhibitory effect and NO (nitric oxide) inhibitory activity, and thus can provide excellent angiogenesis, whitening, anti-inflammatory cosmetics, medicines and foods.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

≪ Example 1 >

1-1. Enzyme treatment

After finishing the grinding, 700 g of the mixture was added to 14 g of 75% ethanol, and the mixture was extracted at 60 ° C. for 3 hours. After the extraction, the mixture was filtered with a filter paper to remove solid content, and the dried extract was filtered and concentrated through a rotary evaporator to a high concentration. The concentrate was frozen at -70 ° C to completely evaporate the water through a freeze- Was prepared.

100 g of the prepared giitta extract was suspended in 1 L of 10% ethanol, and the pH of the suspension was adjusted to be between 5.0 and 6.0. 10 mL of pectin hydrolyzing enzyme (Pectinex Ultra SP-L, Novozyme) was treated and reacted at 49.5 ° C and 150 rpm for 18 hours in a constant temperature incubator. After the reaction, the enzyme-treated product was heated at 100 ° C for 30 minutes to stop the activity of the enzyme, and after removing the impurities with a vacuum filter, an enzyme-treated gold-eutectic extract was prepared.

The filtered enzyme treated gold europium extract was fractionated with methyl chloride at a ratio of 1: 1, and the obtained methyl chloride fraction was concentrated with a vacuum concentrator to prepare a final gold fraction.

In order to conduct an efficacy experiment as described below, the obtained gold-silver-containing fraction was dissolved in dimethylsulfoxide so as to have a concentration of 500 mg / mL.

1-2. Production of untreated enzyme

After finishing the grinding, 700 g of the mixture was added to 14 g of 75% ethanol, and the mixture was extracted at 60 ° C. for 3 hours. After the extraction, the mixture was filtered with a filter paper to remove solid content, and the dried extract was filtered and concentrated through a rotary evaporator to a high concentration. The concentrate was frozen at -70 ° C to completely evaporate the water through a freeze- Was prepared.

The thus prepared ginger extract was fractionated with methyl chloride at a ratio of 1: 1, and the obtained methyl chloride fraction was concentrated by using a vacuum concentrator to prepare a final germanium fraction.

In order to conduct an efficacy experiment as described below, the obtained gold-silver-containing fraction was dissolved in dimethylsulfoxide so as to have a concentration of 500 mg / mL.

≪ Comparative Example 1 >

The extracts of hwanggi were prepared in the same manner as in Example 1, except that Hwanggi (DaeDang Herb Co., Ltd., Korea) was used in place of Ganoderma lucidum.

≪ Comparative Example 2 >

The Angelica keiskei Radix extract was prepared in the same manner as in Example 1, except that Angelica keiskei Kappa (Kappa Kagaku Co., Ltd., Korea) was used.

<Comparative Example 3> Preparation of licorice extract

The licorice extract was prepared in the same manner as in Example 1, except that licorice instead of gingkojum (Caffe Herbiche, Korea) was used.

&Lt; Examples 2 to 5 >

A complex extract was prepared by enzymatic treatment and fractionation in the same manner as in the enzyme-treated gold europium extract of Example 1, except that a mixed medicine of gold eu, hwanggi, angelica, and licorice was used instead of gold europaea. The mixing ratio of each medicinal substance used in the combined extract is shown in Table 1 below.

Medicinal Mixing ratio of medicines (% by weight) Example 2 Example 3 Example 4 Example 5 Gold silver 15 2 25 2 Hwanggi 45 20 20 65 Angelica 25 70 5 5 licorice 15 8 50 28

<Experimental Example 1> Cytotoxicity test: MTT assay

In order to confirm whether the extracts of Examples 1 to 5 and Comparative Examples 1 to 3 had irritation to cells using keratinocyte, cytotoxicity experiments were carried out.

The keratinocytes were cultured in an ATCC (American Type Culture Collection). Each cell was washed twice with 1 × 10 ⁴ cells / mL in a 24-well culture plate. DMEM (Dubelco's Modified Eagle Medium, GIBCO, USA) containing 1% penicillin-streptomycin (P / S) and 10% FBS was used as the medium. After incubation for 24 hours in DMEM containing 10% FBS and incubated for up to 50% of the culture vessel surface area, the cells were replaced with 10% FBS / DMEM containing the extracts prepared in Examples 1 to 5 and Comparative Examples 1 to 3, Lt; / RTI &gt; After the incubation, 50 μl of a solution (4 mg / ml) of 3- (4,5-dimethylthiazole) -2,5-diphenyltetrazolium bromide (MTT, Sigma M5655, USA) was added and further cultured for 3 hours After removing the supernatant, 200 μL of dimethylsulfoxide (DMSO, Sigma D2650, USA) solution was added to each well. After stirring for 20 minutes, the Formazan crystals formed were dissolved and then 100 μL of each was taken in 96 well. Absorbance was measured. The degree of survival of skin cells was calculated using the following equation (1) based on the absorbance of the control group using purified water.

[Equation 1]

Cytotoxicity (%) = (absorbance of each sample / control absorbance) X100

As a result, as shown in Fig. 1, in the case of cell survival rate against keratinocyte, it was confirmed that neither the gingival extract of Example 1 nor the combined extracts of Examples 2 to 5 showed toxicity to cell survival.

<Experimental Example 2> Angiogenesis test

Enzyme-treated extracts of Examples 1 to 5 and Comparative Examples 1 to 3 were each treated with cord blood HUVECs to determine whether angiogenesis was induced to induce angiogenesis.

First, 200 μL of 5 mg / mL Matrigel from Corning Inc. was added to each well and cured for 30 minutes in a 24-well culture plate. HUVECs cells were then added to EBM-2 medium supplemented with EGM-2 from Lonza to 5 x 10 ⁴ cells / well and diluted to ⁴⁰ μg / mL in Example 1 and Comparative Examples 1 and 3 with 20 and 40 μg / mL Examples 2 to 5 were each treated and cultured for 18 hours at 37 ° C under 5% CO ₂ . After the incubation, the angiogenesis state was magnified 40 times by a microscope (Nikon eclipse TS100, Japan).

As a result, as shown in FIG. 2, it was confirmed that the Euglena extract had a remarkably superior angiogenic potential due to an increase in the number of skin capillary blood vessels compared to the extracts of Hwanggi, Danggui and Licorice. In addition, it was confirmed that the angiogenic effect was further enhanced when the pectin hydrolase was treated.

It was confirmed that the gingival extract was superior in angiogenesis efficacy. However, in order to further enhance the skin capillary blood flow, the extract of Ganoderma lucidum, Angelica gigantosa extract, and Licorice root extract was mixed in various mixing ratios, Respectively.

As a result, as shown in FIG. 3, it was confirmed that the complex extract of Example 2 had a higher skin capillary blood vessel count than the combined extracts of Examples 3 to 5, and the angiogenic effect was remarkably excellent.

As a result, it was confirmed that the gingival exudate alone had an excellent angiogenesis potency, and when the complex extract containing Gingkoff extract was mixed at an optimum mixing ratio, the gingival exudate alone enhanced the angiogenic effect.

<Experimental Example 3> Whitening efficacy test: Tyrosinase inhibitory activity

Tyrosinase inhibitory activity tests involved in melanin synthesis were carried out using the enzyme-treated extracts of Examples 1 to 5 and Comparative Examples 1 to 3.

In each well of a 96-well microplate, the final concentration of 500 μg / mL (final reaction amount: 300 μL) and the dilutions of 20 and 40 μg / mL of Examples 1 to 5 and Comparative Examples 1 and 3 were added, and 0.1 M potassium phosphate buffer ) Was added, and 100 μL of 1.5 mM L-tyrosine, a tyrosinase substrate, was added, followed by reaction at 37 ° C. for 10 minutes. After that, 10 μL of tyrosinase (1500 U / mL) was added, reacted at room temperature for 5 minutes, and absorbance was measured at 475 nm. The absorbance value was normalized based on the absorbance of the composition and the inhibition rate was calculated using the following equation (2).

&Quot; (2) &quot;

(%) = (1-absorbance of each sample / absorbance of control group) X100

As a result, as shown in FIG. 4, the Euglena extract had a superior tyrosinase inhibitory activity against Angelicae gigantis, but the tyrosinase inhibitory activity was low when compared with the Angelica keiskei extract and licorice extract.

In order to further enhance the tyrosinase inhibitory activity of the gingkoff extract, the extract of Ganoderma lucidum, Angelica gigantosa extract, and Licorice root extract were mixed in various mixing ratios to produce tyrosinase inhibitory effect.

As a result, as shown in FIG. 5, the tyrosinase-inhibiting effect was more excellent when the compound extract of Gingko Ehwa extract alone was used, and further, the compound extract of Example 2 showed remarkable efficacy of tyrosinase inhibitory activity against the compound extracts of Examples 3 to 5 , And it was confirmed that the addition of 40 μg / mL concentration was more effective than the whitening ingredient arbutin.

As a result, it was confirmed that tyrosinase inhibitory effect was enhanced even when Ganoderma lucidum extract alone was used. However, it was confirmed that when Ganoderma lucidum extract, Hwanggi extract, Angelica japonica extract and Licorice extract were mixed at an optimal mixing ratio, .

<Experimental Example 4> Anti-inflammatory activity test: NO (nitric oxide) formation inhibitory activity

In order to confirm the skin irritation-improving effect using the enzyme-treated extracts of Examples 1 and 2 and Comparative Examples 1 to 3, oxytocin (RAW 264.7), which produces nitric oxide (NO) The amount of nitrite type nitrite was measured by adding Griess reagent to the resulting culture. First, a macrophage cell line, RAW 246.7 cells, 10% Fetal bovine serum (FBS) , 1% Penicillin / streptomycin (P / S) containing a Dulveccos's modified eagles medium (DMEM) with the medium 37 ℃, 5% CO ₂ conditions Lt; / RTI &gt; incubator. Cells were treated with 6x10 ⁴ cells per well in a 48 well culture plate and cultured for 24 hours. After culturing, the cells treated with Examples 1 and 2 and Comparative Examples 1 and 3 diluted with concentration (5, 100, 200 μg / mL) were treated with 1 μg / mL of Lipo poly saccharide (LPS) And cultured for additional 22 hours. After culturing, 100 μL of cell culture solution and 100 μL of Griess reagent [1% (w / v) sulfanilamide, 0.1% N-1-naphthylethylene diamine in 2.5% (w / v) phosphoric acid] After the reaction, the absorbance was measured at 540 nm. A standard curve was obtained using sodium nitrate and the resulting nitrite value was calculated. The inhibition rate of NO (nitric oxide) production was calculated using the following equation (3).

&Quot; (3) &quot;

Inhibition rate of NO (nitric oxide) production (%) = (absorbance of each sample / absorbance of control) X100.

As a result, as shown in FIG. 6, it was confirmed that the gingival oyster extract had an inhibitory rate of NO (nitric oxide) production.

In order to further enhance the inhibitory rate of nitric oxide formation in the gingkoff extract, the extract of Ganoderma lucidum was mixed with various concentrations of Hwanggi extract, Angelica gigantosa extract and licorice extract to prepare a complex extract to inhibit nitric oxide (NO) production. As a result, it was confirmed that the effect of inhibiting the formation of NO (nitric oxide) was remarkably improved when the extract of Ganoderma lucidum extract alone was used.

As a result, it was found that nitric oxide (NO) production inhibitory effect was inhibited even when the extracts were used alone. However, when the extracts of Ganoderma lucidum, Hwanggi extract, Angelica gigantosa extract and licorice extract were mixed at an optimum mixing ratio, Production inhibitory effect was further enhanced.

Experimental Example 5 Evaluation of skin irritation

To evaluate the skin irritation using the enzyme-treated extracts of Examples 1 to 2 and Comparative Examples 1 to 3, a skin clinical laboratory was commissioned and tested. First, the subjects were kept in a clean and dry state to keep the test conditions of the test subjects equal to 30 persons. The skin stability was maintained at a temperature of 22 ± 2 ℃ and RH 40 ~ 60% for at least 30 minutes. And then proceeded. The test site was wiped with 70% ethanol and dried. Then, 25 占 퐇 of the enzyme-treated extracts of Examples 1 and 2 and Comparative Examples 1 and 3 were dropped in a Finn chamber, and then adhered to the back region of the subject and fixed. The patches were removed after 24 hours. After removing the patches, the test area was marked with a marking pen and the test area was observed after 30, 24 and 48 hours. The skin contact response was judged by the International Association of Contact Dermatological Research Meetings as: - unstimulated, ±: unstimulated (mild erythematosus that can be detected faintly or barely), +: mildly stimulated (mild erythema, edema and papules) The median skin response (mean score) was calculated from the following equation (4): &quot; (4) &quot; The results are shown in Tables 2-6.

&Quot; (4) &quot;

number Subject Example 1 in Propanediol 30 minutes later After 24 hours After 48 hours One YJY - - - 2 JBK - - - 3 KMA - - - 4 KJY-1 - - - 5 SSJ - - - 6 KMR - - - 7 OSE - - - 8 LHS - - - 9 YYA - - - 10 JHK-1 - - - 11 JSY - - - 12 JHJ - - - 13 KBR - - - 14 PKM - - - 15 SKH - - - 16 KKA - - - 17 KJS - - - 18 SHJ - - - 19 KHY - - - 20 BEJ - - - 21 JHK-2 - - - 22 KJY-2 - - - 23 SCS - - - 24 KYR - - - 25 PIJ - - - 26 KNS - - - 27 JEH - - - 28 YMK - - - 29 PMA - - - 30 PCH - - - Reactivity ± 0 0 0 + 0 0 0 ++ 0 0 0 +++ 0 0 0 Mean score: 0.00 Judgment No stimulation

number Subject Comparative Example 1 in Propanediol 30 minutes later After 24 hours After 48 hours One YJY - - - 2 JBK - - - 3 KMA - - - 4 KJY-1 - - - 5 SSJ - - - 6 KMR - - - 7 OSE - - - 8 LHS - - - 9 YYA - - - 10 JHK-1 - - - 11 JSY - - - 12 JHJ - - - 13 KBR - - - 14 PKM - - - 15 SKH - - - 16 KKA - - - 17 KJS - - - 18 SHJ - - - 19 KHY - - - 20 BEJ - - - 21 JHK-2 - - - 22 KJY-2 - - - 23 SCS - - - 24 KYR - - - 25 PIJ - - - 26 KNS - - - 27 JEH - - - 28 YMK - - - 29 PMA - - - 30 PCH - - - Reactivity ± 0 0 0 + 0 0 0 ++ 0 0 0 +++ 0 0 0 Mean score: 0.00 Judgment No stimulation

number Subject Comparative Example 2 in Propanediol 30 minutes later After 24 hours After 48 hours One YJY - - - 2 JBK - - - 3 KMA - - - 4 KJY-1 - - - 5 SSJ - - - 6 KMR - - - 7 OSE - - - 8 LHS - - - 9 YYA - - - 10 JHK-1 - - - 11 JSY - - - 12 JHJ - - - 13 KBR - - - 14 PKM - - - 15 SKH - - - 16 KKA - - - 17 KJS - - - 18 SHJ - - - 19 KHY - - - 20 BEJ - - - 21 JHK-2 - - - 22 KJY-2 - - - 23 SCS - - - 24 KYR - - - 25 PIJ - - - 26 KNS - - - 27 JEH - - - 28 YMK - - - 29 PMA - - - 30 PCH - - - Reactivity ± 0 0 0 + 0 0 0 ++ 0 0 0 +++ 0 0 0 Mean score: 0.00 Judgment No stimulation

number Subject Comparative Example 3 in Propanediol 30 minutes later After 24 hours After 48 hours One YJY - - - 2 JBK - - - 3 KMA - - - 4 KJY-1 - - - 5 SSJ - - - 6 KMR - - - 7 OSE - - - 8 LHS - - - 9 YYA - - - 10 JHK-1 - - - 11 JSY - - - 12 JHJ - - - 13 KBR - - - 14 PKM - - - 15 SKH - - - 16 KKA - - - 17 KJS - - - 18 SHJ - - - 19 KHY - - - 20 BEJ - - - 21 JHK-2 - - - 22 KJY-2 - - - 23 SCS - - - 24 KYR - - - 25 PIJ - - - 26 KNS - - - 27 JEH - - - 28 YMK - - - 29 PMA - - - 30 PCH - - - Reactivity ± 0 0 0 + 0 0 0 ++ 0 0 0 +++ 0 0 0 Mean score: 0.00 Judgment No stimulation

number Subject Example 2 in Propanediol 30 minutes later After 24 hours After 48 hours One J * S - - - 2 M * I - - - 3 J * S - - - 4 J * Y - - - 5 K * H - - - 6 L * M - - - 7 K * J - - - 8 L * J - - - 9 K * W - - - 10 I * S - - - 11 K * B - - - 12 J * J - - - 13 J * S - - - 14 Y * O - - - 15 Y * W - - - 16 J * R - - - 17 H * H - - - 18 P * O - - - 19 K * M - - - 20 P * J - - - 21 C * N - - - 22 K * A - - - 23 K * J-1 - - - 24 Y * T - - - 25 H * S - - - 26 K * J-2 - - - 27 P * Y - - - 28 O * S - - - 29 J * M - - - 30 L * H - - - Reactivity ± 0 0 0 + 0 0 0 ++ 0 0 0 +++ 0 0 0 Mean score: 0.00 Judgment No stimulation

As a result, as shown in Tables 2 to 6, no skin irritation was observed in Examples 1 and 2 and Comparative Examples 1 and 3 after 30 minutes, 24 hours, and 48 hours after patch removal. The average skin reactivity was 0.00 and determined to be unstimulated according to the criteria of the patch test.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

From 3 to 20% by weight of Lonicerae Flos extract, from 40 to 60% by weight of Astragali Radix extract, from 10 to 40% by weight of Angelicae Gigantis Radix extract and from 10 to 25% by weight of Glycyrrhizae Radix extract A composition for angiogenesis, whitening, and anti-inflammation comprising a mixed extract as an active ingredient.
2. The method according to claim 1, wherein the Lonicerae Flos extract is extracted with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, propylene glycol, glycerin and a mixed solvent thereof. Composition for whitening and anti-inflammation.
The method of claim 1, wherein the honeysuckle (Lonicerae Flos extract is an enzyme extract obtained by enzymatic treatment of Ganoderma lucidum extract.
[Claim 4] The method according to claim 3, wherein the enzyme is at least one selected from the group consisting of arabinoxyhydrolase, fibrinolytic enzyme, glucanolytic enzyme, hemicellulose degrading enzyme, xylan acid degrading enzyme and pectin hydrolyzing enzyme Composition for neonatal, whitening and anti-inflammation.
[2] The method according to claim 1, wherein the Lonicerae Flos extract is selected from the group consisting of chloroform, methyl chloride, normal-hexane, acetic acid, and mixtures thereof, wherein the enzyme extract obtained by enzymatic treatment of Ganoderma lucidum or Ganoderma lucidum extract is Wherein the fraction is a fraction obtained by fractionation with an organic solvent to be selected.


delete The composition for angiogenesis, whitening and anti-inflammation according to claim 1, wherein the Lonicerae Flos extract has an angiogenic effect, a tyrosinase inhibitory effect and an inhibitory effect on NO (nitric oxide) production.


delete delete A cosmetic comprising the composition of any one of claims 1, 2, 3, 4, 5 and 7.
A pharmaceutical product comprising the composition of any one of claims 1, 2, 3, 4, 5 and 7.
A food comprising the composition of any one of claims 1, 2, 3, 4, 5 and 7.

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