KR101434163B1 - A pharmaceutical with anti oxidant, anti inflammatory and skin whitening effect comprising the extract from aerial part of pueraria thunbergiana - Google Patents

Abstract

The present invention relates to a composition having anti-oxidant, anti-inflammatory, and skin whitening effects and containing a kudzu aerial part extract as an active ingredient. More specifically, the present invention relates to a composition which can promote anti-oxidant, anti-inflammatory, and skin whitening effects without side effects using natural materials. The composition which can promote an anti-oxidant effect according to the present invention concentration-dependently exhibits an anti-oxidant effect as a result of a DPPH experiment. Moreover, the composition which can promote an anti-inflammatory effect according to the present invention can control the generation of NO and the expression of iNOS and COX-2. In addition, the composition which can promote a skin whitening effect according to the present invention can prevent melanin synthesis in cells, prevent melanin synthesis caused by α-MSH, inhibit melanin accumulation, control the expression of melanin synthesis enzymes and factors related to the synthesis, and improve skin whitening effect even at an in vivo level.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition having antioxidant, anti-inflammatory and whitening effect, which contains an aerial part extract as an active ingredient. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antioxidant,

The present invention relates to a composition having antioxidant, antiinflammatory and whitening effect, which comprises an extract of the above-ground part as an active ingredient, and more particularly to a composition using natural materials to promote antioxidative, antiinflammatory and skin whitening efficacy without side effects To the composition.

The composition capable of enhancing the antioxidant efficacy according to the present invention has a technical feature that shows the antioxidant effect in a concentration-dependent manner as a result of the DPPH test.

In addition, the composition capable of enhancing the anti-inflammatory effect according to the present invention has a technical feature capable of inhibiting NO production, iNOS and COX-2 expression.

In addition, the composition capable of enhancing skin whitening effect according to the present invention not only inhibits intracellular melanin synthesis but also inhibits melanin synthesis caused by? -MSH, inhibits melanin accumulation, and inhibits melanin synthesis and synthesis Inhibits the expression of related factors, and has technical characteristics that can enhance skin whitening even at in vivo level.

In general, reactive oxygen species, known as noxious oxygen, are known as antioxidant enzymes (superoxide dismutase, catalase, clutathione peroxidase, glutathione reductase) and non-enzymatic antioxidants (tocopherol, ascorbic acid, carotenoid, flavonoid , glutathione), the normal cellular function of the human body is maintained.

However, oxidative stress caused by excessive production of active oxygen and defective function of the defense system may be caused by skin antioxidant breakdown, initiation of lipid peroxidation reaction, protein oxidation, DNA oxidation, chain breaks such as collagen, hyaluronic acid, Creation of wrinkles by cross-linking, melanin generation, etc., accelerate skin aging.

Enzymatic substances are self-produced in the body and include antioxidant enzymes such as superoxide dismutase (SOD), catalase, and peroxidase.

Non-enzymatic substances are nutrients that have to be absorbed from the outside of the body. They are in the form of low-molecular compounds and help the action of polymeric antioxidants (enzymes) in the body or remove active oxygen by themselves. Beta-carotene, vitamin A, vitamin C (ascorbic acid), vitamin E (tocopherol) and flavonoids are representative antioxidants.

The synthetic antioxidants developed so far include butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT) and nordihydro-guaiaretic acid (NDGA). Natural antioxidants include tocopherol, gossypol, sesamol, lecitthin, cephalin, ether extract components of red pepper, extracts from wheat germ, soybean, pepper, and tomato, and extracts of mainly florboloid pigments.

However, synthetic antioxidants are toxic in vivo and are likely to cause allergies and tumors. Moreover, they are weak against temperature and are easily destroyed by heating once.

On the other hand, melanin is a brown or black polymer pigment widely distributed in animal and plant systems, and determines human skin color. It controls ultraviolet light, cytokines, growth factors and hormones. Melanocyte stimulating hormone (α-MSH) plays a major role in controlling melanin production.

In summary, α-MSH activates adenylyl cyclase by binding to MC1R (melanocortin 1 receptor), a membrane receptor that is secreted in the midgut of the pituitary gland and expressed only in melanocytes, and activates PKA (protein kinase A) And activates intracellular cAMP response element binding protein (CREB), thereby increasing the expression of MITF (microphthalmia-associated transcription factor), a specific transcription factor in melanocytes.

This MITF stimulates the expression of melanin producing enzyme tyrosinase and related protein (tyrosinase related protein), thereby controlling the melanin production of melanocytes.

Thus, α-MSH promotes tyrosinase activity through activation of adenylyl cyclase, PKA, CREB, and MITF, thereby increasing the proliferation and pigmentation of melanocytes.

Inflammation is a mechanism to regenerate damaged areas that have been altered by stimulation in the body, but persistent inflammation can damage mucous membranes and cause pain, swelling, redness, and fever.

It is known that macrophages are involved in various host reactions and are involved in the maintenance of homeostasis. During inflammatory reaction, nitric oxide (NO) produced by iNOS (inducible nitric oxide synthase) and PGE2 (cyclooxygenase 2) prostaglandin E2) and the like. These iNOS and COX-2 have been shown to increase expression by growth factors, cytokines, and the like, leading to inflammation-related diseases.

Therefore, substances that inhibit the activity of inflammatory mediators such as NO and PGE2 are highly likely to be developed as therapeutic agents for the treatment and improvement of various inflammatory diseases such as acne, inflammatory dermatitis, and contact dermatitis.

The present applicant intends to find a composition capable of enhancing antioxidant efficacy without adverse effect by using a natural-derived material. In addition, it is intended to find a composition which not only inhibits intracellular melanin synthesis but also prevents melanin synthesis caused by alpha-MSH, which is the most important and most important signal in the skin coloring mechanism, thereby enhancing skin whitening. Also, it is intended to find a composition capable of enhancing the anti-inflammatory effect by inhibiting the production of NO, iNOS and COX2.

Meanwhile, Korean Patent Registration No. 10-0628650 discloses a cosmetic composition for skin whitening comprising an extract of Angelica keiskei koidz. And Puerariae Radix extract as an active ingredient, .

This document describes a melanin inhibitory effect experiment using B16F1 pigmented cells.

Second, the skin-related bioactivity of the garlic extract and fractions (Korean Journal of Hairdressing, Vol. 18, No. 4, 2012, pp.858-864) discloses that the tyrosinase inhibitory activity of gallstones is measured .

Third, the antioxidative and whitening effect of Puerariae radix, Bacillus thuringiensis, and P. thunbergii (Journal of the Korean Chemical Society, vol.25, no.2, 2008.06, 219-225) .

As a whole, the prior art does not disclose the use of the above-ground parts (stems and leaves), and in addition, the tyrosinase inhibitory activity is mainly measured for the browns and crabs, and the melanin staining caused by the α-MSH It is not mentioned about prevention.

It is an object of the present invention to provide a composition which is capable of enhancing antioxidant, anti-inflammatory and skin whitening efficacy without using side effects by using a natural-derived material.

It is also an object of the present invention to provide a composition exhibiting an antioxidative effect as a result of DPPH test.

It is also an object of the present invention to provide a composition capable of enhancing anti-inflammatory efficacy by inhibiting NO production, iNOS and COX-2 expression.

It is also an object of the present invention to prevent melanin synthesis caused by α-MSH corresponding to the highest signal in the skin development mechanism, to inhibit melanin accumulation, and to inhibit melanin accumulation, Inhibiting the expression of an involved factor and enhancing skin whitening even at an in vivo level.

The present invention is to provide a composition having antioxidant, anti-inflammatory and whitening effect, a health functional food, and a cosmetic composition containing the ground top extract as an active ingredient.

The present invention provides an extract obtained by extracting with a high purity lower alcohol or water.

The present invention provides a variety of solvent fraction extracts by extracting a ground part with a lower alcohol to obtain an alcohol extract, diluting the alcohol extract with distilled water, and repeatedly fractionating with an organic solvent.

The composition according to the present invention may be provided as a pharmaceutical composition for antioxidant, anti-inflammation and skin whitening, and each composition may be prepared as a health functional food using a foodstuff acceptable processing agent. The health functional food using the composition according to the present invention may be provided in various forms such as functional beverage, health supplement, tea, confectionery, and the like.

In order to use the present invention as a pharmaceutical composition, it may be prepared by a known method in the pharmaceutical field, and may be mixed with the carrier itself, a pharmaceutically acceptable carrier, excipient, diluent or the like to prepare a powder, granule, Or injections, and the like. They may also be administered orally or parenterally.

The effective dose of the composition according to the present invention can be appropriately selected according to the degree of absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, severity of the disease to be treated,

The composition according to the invention may be used in cosmetics or feed for livestock and may in each case be prepared using treatments or additives which are acceptable in cosmetics or feeds.

Formulations such as pills, granules, beverages, tablets, capsules and the like may be prepared using the composition according to the present invention, and in this case, a treatment agent may be added to prepare each formulation.

In the case of cosmetic compositions using the composition according to the present invention, the content is 0.0001 to 10.0% by weight, preferably 0.0005 to 10.0% by weight, and most preferably 0.005 to 5.0% by weight based on the total cosmetic composition. If the content is less than 0.0001% by weight, a clear skin whitening improvement effect can not be expected. If the content is more than 10.0% by weight, no marked increase of the effect is observed by increasing the content.

The ingredients contained in the cosmetic composition of the present invention include the ingredients conventionally used in cosmetic compositions and include customary adjuvants such as, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition according to the present invention can be prepared into any formulation conventionally produced in the art. For example, they may be formulated as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, But is not limited thereto.

More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

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

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

When the formulation of the present invention is an interfacial active agent-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 sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

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

The cosmetic of the present invention includes a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, sphingolipids, polymeric polysaccharides, and polymer peptides.

Examples of the water-soluble vitamin include vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C and vitamin H And their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (sodium ascorbic acid-2-phosphate, magnesium ascorbate-2-phosphate etc.) can also be added to water-soluble vitamins . The water-soluble vitamin can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzymatic method, or a chemical synthesis method.

Examples of useful vitamins include vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (DL-alpha tocopherol, D-alpha tocopherol, D-alpha tocopherol and the like) , Derivatives thereof (such as palmitic acid ascorbin, stearic acid ascorbic acid, dipalmitic acid ascorbin, acetic acid DL-alpha tocopherol, nicotinic acid DL-alpha tocopherol vitamin E. DL-pantothenyl alcohol, D-pantothenyl alcohol, Ether, etc.) are also included in the usable vitamins used in the present invention. Usability Vitamins can be obtained by a conventional method such as a microorganism conversion method, a purification method from a culture of a microorganism, an enzyme or a chemical synthesis method.

Sphingo lipids may be any as long as they can be compounded in cosmetics, and preferable examples thereof include ceramides, phytosphingosine and sphingoglycolipids. Sphingoid lipids can be purified from ordinary mammals, fish, shellfish, yeast or plants by conventional methods or can be obtained by chemical synthesis.

The polymer polysaccharide may be any compound as long as it can be compounded in cosmetics, and examples thereof include hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.). For example, chondroitin sulfate or a salt thereof can be usually purified from mammals or fishes.

The polymeric peptide may be any compound as long as it can be compounded in the cosmetic, and preferably collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin and the like. The polymeric peptide can be obtained by a conventional method such as purification from a culture broth of a microorganism, an enzymatic method, or a chemical synthesis method, or it can be purified from natural products such as ducks such as pigs and cows and silk fiber of silkworms.

The cosmetic of the present invention may be blended with other essential ingredients, if necessary, in combination with the essential ingredients. Examples of the compounding ingredients which may be treated include organic solvents such as a preservative component, a moisturizer, an emollient, a surfactant, an organic and inorganic pigment, an organic powder, an ultraviolet absorbent, an antiseptic, a bactericide, an antioxidant, a plant extract, a pH adjuster, A blood circulation promoter, a cold agent, a restriction agent, and purified water.

The above components may be mixed within a range not to impair the objects and effects of the present invention, but are preferably 0.01 to 5% by weight, more preferably 0.01 to 5% by weight based on the total weight of the composition 0.01 to 3% by weight.

The composition according to the present invention has an effect of using antioxidant, anti-inflammation and skin whitening efficacy without side effects by using a natural-derived material.

In addition, the composition according to the present invention has an antioxidative effect as a result of DPPH experiment.

In addition, the composition according to the present invention has an effect of enhancing the anti-inflammatory effect by inhibiting NO production, iNOS and COX-2 expression.

In addition, the composition according to the present invention not only inhibits intracellular melanin synthesis but also prevents melanin synthesis caused by alpha-MSH corresponding to the highest signal in the skin pigmentation mechanism, inhibits melanin accumulation, Inhibits the expression of factors involved in the inflammation , and has the effect of enhancing skin whitening even in the in vivo level.

Fig. 1 shows the result of measuring the antioxidant activity of the above-ground extract.
Fig. 2 shows the results of measuring the inhibitory effect on melanin synthesis in the submerged phase of the extract.
FIG. 3 shows the results of measuring the inhibitory effect on the melanin synthesis of the above-ground EtOAc fraction extract.
FIG. 4 shows the results of measuring the inhibitory effect of α-MSH-induced melanin synthesis on the ground-based extract.
FIG. 5 shows the results of measuring the inhibitory effect of α-MSH induced melanin synthesis of the above-ground EtOAc fraction extract.
FIG. 6 shows the results of measurement of the inhibitory effect on the NO production of the ground-based extract.
FIG. 7 shows the results of measurement of the inhibitory effect of the EtOAC fraction extract from the ground-based part on NO production.
Fig. 8 shows the result of measuring the NO production inhibitory effect of the ground surface element component.
FIG. 9 shows the results of measuring inhibitory effect of iNOS expression on the extracts of EtOAC fraction derived from the ground-top extract and the ground-top extract.
FIG. 10 shows the results of measurement of COX-2 expression inhibitory effect of EtOAC fraction extracts from ground-top extract and ground-top extract.
FIG. 11 shows the results of measuring the inhibitory effect of the extract of EtOAc from the ground-based extract on the inhibition of melanin accumulation.
FIG. 12 shows the results of measuring the inhibitory effect of tyrosinase activity of the EtOAc fraction extract from the ground-based extract.
FIG. 13 shows the results of measuring the inhibitory effect of tyrosinase glycosylation on the extract of EtOAc fraction from the ground-based extract.
Fig. 14 shows the results of measurement of the mRNA-inhibiting effect of the melanin synthesis-related enzyme in the EtOAc fraction extract from the ground-based extract.
FIG. 15 shows the results of measuring the inhibitory effect of MITF on the EtOAc fraction extract from the ground-based extract.
16 is a view showing a sample application and a UV irradiation process in an experimental animal.
FIG. 17 is a graph showing the effect of L * value on the EtOAc fraction extract from the ground-top extract in a mouse animal model in which pigmentation was induced by UVB irradiation.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the embodiments described in the present specification, the reference examples, and the drawings are merely the most preferred examples of the present invention, and not all of the technical ideas of the present invention are described. Therefore, It should be understood that various equivalents and modifications may be present.

Manufacturing example  1. Preparation of 칡 aerial part extract

Distilled water and ethanol were mixed in proportions to extract the above ground (stem and leaf) extracts, and the respective extracts were fractionated with ethyl acetate (EtOAc) to obtain an EtOAc fraction extract.

1) Extraction of distilled water from above ground

1) Distilled water was added to 2,000 g of the above-ground part, heated at 80 to 100 ° C for 3 hours, filtered, and the filtrate was vacuum-dried to obtain 439.5 g of a dried product.

② The procedure of ① was vacuum-dried, and the extract was dissolved in 3,900 mL of distilled water and the same amount of EtOAc was added to the reaction mixture. The EtOAc fraction extract and the distilled water fraction were obtained twice.

2) 30 30% ethanol extract from above ground

1) 30 g of 30% ethanol was added to 2,000 g of the ground part, heated at 80 to 100 ° C for 3 hours, filtered, and the filtrate was vacuum dried to obtain 409.2 g of a dried product.

② The process of vacuum drying in step ①, dissolving the extract in 3,600 mL of distilled water and partitioning by adding the same amount of EtOAc was repeated twice. 17.4 g of EtOAc extract and 317 g of distilled water fraction were obtained.

3) 70 70% Ethanol Extraction of Above Ground

1) 70 g of ethanol was added to 2,000 g of the above-ground part, and the mixture was heated at 80 to 100 ° C for 3 hours, filtered, and the filtrate was vacuum-dried to obtain 436.7 g of a dried product.

② The procedure of ① was vacuum-dried, and the extract was dissolved in 3,800 mL of distilled water. The same amount of EtOAc was added to the mixture to separate the fractions twice. 31.5 g of EtOAc extract and 342.6 g of distilled water fraction were obtained.

4) 95 95% Ethanol Extraction of Above Ground

(1) 10 g of 95% ethanol was added to 2,000 g of the above-ground part, and the mixture was heated at 80 to 100 ° C for 3 hours, filtered and vacuum dried to obtain 284.8 g of a dried product.

② The procedure of ① was vacuum-dried, and the extract was dissolved in 2,300 mL of distilled water and the same amount of EtOAc was added to the reaction mixture twice. The EtOAc extract (35.3 g) and distilled water fraction (177.8 g) were obtained.

The list and yield of the extracts obtained as a result of the extraction are summarized in Table 1 below. The samples of [Table 1] were used in the following experimental examples.

Sample number List yield One 칡 Distilled water extract 21.98% 2 칡 30% Ethanol extract 20.46% 3 칡 Above ground 70% ethanol extract 22.86% 4 칡 Aboveground portion 95% ethanol extract 14.24% 5 칡 Ground-water distilled water extract EtOAc fraction 3.09% 6 칡 30% Ethanol extract in the above-ground portion EtOAc fraction 4.83% 7 칡 Above-ground 70% ethanol extract EtOAc fraction 9.07% 8 칡 Above-ground 95% ethanol extract EtOAc fraction 10.77%

Manufacturing example  2. Preparation of ground-based extracts

To the water or ethanol extract of Preparation Example 1, distilled water may be added, and then hexane may be added to separate into a water layer and a hexane layer. Ethyl acetate may be further added to the water layer to separate the ethyl acetate layer and the water layer, and the butanol layer and the water layer may be finally added to the water layer.

In addition, distilled water may be added to the methanol extract of the above-ground part, and then hexane may be added to separate the water layer and the hexane layer. Ethyl acetate may be further added to the water layer to separate the ethyl acetate layer and the water layer, and the butanol layer and the water layer may be finally added to the water layer.

In addition, distilled water may be added to the C ₁ -C ₅ aliphatic alcohol extract of the ground portion, and then hexane may be added to separate the water layer and the hexane layer. Ethyl acetate may be further added to the water layer to separate the ethyl acetate layer and the water layer, and the butanol layer and the water layer may be finally added to the water layer.

Manufacturing example  3. Composition with antioxidant, anti-inflammatory and whitening efficacy

Antioxidant, anti-inflammatory and whitening effect is prepared so as to contain as an active ingredient the ground-based extract prepared using Preparation Example 1 or Production Example 2.

Depending on the design conditions, it may also be formulated so as to include a part of the dried powder and / or garlic extract as an active ingredient, which may be used as a pharmaceutical composition, a health functional food composition or a cosmetic composition.

Experimental Example  1. Antioxidant effect of 칡 aerial part extract

1-1. Experimental course

The DPPH assay is one of the most common experiments to measure antioxidant efficacy. The principle of DPPH assay is that the deep purple Diphenylpicrylhydrazyl is reduced to Diphenylpicrylhydrazine by the antioxidant and turns into a pale yellow. The stronger the antioxidant power of the antioxidant, the closer it is to the pale yellow.

Also, when measuring the absorbance, a dark purple color is detected but a light yellow color is not detected. Therefore, the smaller the absorbance value is, the higher the antioxidant power is. The experimental procedure is as follows.

① 18 groups of 0.1 mM DPPH solution (Diphenylpicrylhydrazyl) were prepared. Group 1 was set as negative control and no manipulation was performed. Group 1 was set as positive control group and treated with ascorbic acid 50 μM. The remaining 16 groups were divided into three groups: 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. The other four groups contained 10 μg / mL of the extract of the topsoil ethyl acetate fraction, 50 μg / mL, and 100 μg / mL, respectively.

② Using a spectrophotometer at a wavelength of 520 nm, we observed how the color of sample (1) changes.

1-2. Experiment result

FIG. 1 is a graph showing the antioxidative effect of the above-ground top extract, wherein FIG. 1 (A) is a graph showing the results of the water fraction extract and FIG. 1 (B) is a graph showing the results of the ethyl acetate fraction extract (INHIBITION RATIO = {1- (experiment / control)} x100)

The water extract and EtOAc fraction extracts showed higher antioxidative effect than the negative control. All of them also showed antioxidant effect in a concentration dependent manner.

In particular, the EtOAc fraction extract showed higher antioxidant activity than the positive control at high concentration.

Experimental Example  2. Inhibitory effect on the intracellular melanin synthesis of 칡 aerial part extract

2-1. Experimental course

(1) B16F10 cells were inoculated into 6 wells of two wells so as to be in total 13 groups, and cultured for 24 hours for stabilization.

② Group 1 was set as a negative control group and treated with 10 μM, 1 mM each of kojic acid, 10 μM and 1 mM each of arbutin.

③ The remaining 8 groups were set as the experimental group. The ethanol extracts from the top of the ground, the 30% ethanol extract from the top of the ground, the 70% ethanol extract from the top of the ground, the 95% ethanol extract from the top of the ground, the EtOAc fraction from the top of distilled water, , 70% of the above-ground part, EtOAc fraction of ethanol extract, and EtOAc fraction extract of 95% ethanol extract of the top part were all treated at 100 μg / mL.

After 2 days, the culture was removed and the cells were recovered by treatment with trypsin. The cells were centrifuged at 10,000 to 13,000 rpm for 15 minutes. The obtained pellet was washed with alcohol, and then washed with 1 N NaOH solution containing 10% DMSO The incubation time was 1 hour in a thermostat bath to dissolve the melanin, and the absorbance was measured at 405 nm.

2-2. Experiment result

2 shows the result of measuring the inhibitory effect on melanin synthesis of the ground-based extract (* p <0.05, ** p <0.01, ** p <0.001)

The results showed that kojic acid and arbutin inhibited melanin synthesis in a dose - dependent manner. Arbutin inhibited melanin synthesis up to 41.72% at 1 mM compared with control.

Four extracts of EtOAc fraction showed significant effect. In particular, the EtOAc fraction extract showed a higher inhibitory effect on melanin formation than the positive control, and the EtOAc fraction of the 30% ethanol extract inhibited melanin production up to 9.76%.

Experimental Example  3. Effect of concentration-dependent inhibition of melanin synthesis on the EtOAc fraction

3-1. Experimental course

1) B16F10 cells were cultured in a total of 21 groups in which 6 wells were grouped into 2 wells.

② Group 1 was set as a negative control group and treated with 10 μM, 1 mM each of kojic acid, 10 μM and 1 mM each of arbutin.

The remaining 16 groups were divided into 5 μg / mL, 10 μg / mL, 50 μg / mL and 100 μg / mL each of the EtOAc fraction of the top of distilled water , 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively, of the 70% ethanol extract of the above-ground portion of the soil at a concentration of 10 μg / mL, 50 μg / mL, % Ethanol extract EtOAc fractions were treated with 5 μg / mL, 10 μg / mL, 50 μg / mL and 100 μg / mL, respectively.

After 2 days, the culture was removed and the cells were recovered by treatment with trypsin. The cells were centrifuged at 10,000 to 13,000 rpm for 15 minutes. The obtained pellet was washed with alcohol, and then washed with 1 N NaOH solution containing 10% DMSO The incubation time was 1 hour in a thermostat bath to dissolve the melanin, and the absorbance was measured at 405 nm.

3-2. Experiment result

FIG. 3 shows the results of measuring the inhibitory effect on the melanin synthesis of the EtOAc fraction of the ground-portion extract.

As a result, all of the EtOAc fraction extracts inhibited melanin production in a concentration - dependent manner. Especially, at a concentration of 100 ug / ml of the ethyl acetate fraction of the water extract and 50 ug / ml of the ethyl acetate fraction of the ethanol extract, the melanin synthesis inhibitory effect was higher than that of the positive control arbutin 1 mM.

Experimental Example  4. Inhibitory effect of α-MSH on melanin synthesis

4-1. Experimental course

① B16F10 cells were cultured in 12 wells of 6-well plates.

② Group 1 was set as a negative control group and treated with α-MSH at 100 nM for 24 hours to induce melanin synthesis. 2 group was pretreated with α-MSH for 24 hours and treated with kojic acid at 10 μM and 1 mM.

③ The remaining 8 groups were set as the experimental group. The ethanol extracts from the top of the ground, the 30% ethanol extract from the top of the ground, the 70% ethanol extract from the top of the ground, the 95% ethanol extract from the top of the ground, the EtOAc fraction from the top of distilled water, , 70% of the above-ground part, EtOAc fraction of ethanol extract, and EtOAc fraction extract of 95% ethanol extract of the top part were all treated at 100 μg / mL.

After 2 days, the culture was removed and the cells were recovered by treatment with trypsin. The cells were centrifuged at 10,000 to 13,000 rpm for 15 minutes. The obtained pellet was washed with alcohol, and then washed with 1 N NaOH solution containing 10% DMSO The incubation time was 1 hour in a thermostat bath to dissolve the melanin, and the absorbance was measured at 405 nm.

4-2. Experiment result

4 is a graph showing the effect of suppressing melanin synthesis caused by? -MSH.

As a result of measurement, melanin synthesis by α-MSH treatment suppressed melanin synthesis in a concentration-dependent manner by the positive control kojic acid.

칡 The inhibition effect of the EtOAc fraction of the ground part was significantly inhibited. Especially, all of the extracts of EtOAc fraction inhibited melanin synthesis more than kojic acid 1 mM. In particular, in the ethyl acetate fraction extract, melanin production was reduced to the level before the α-MSH treatment in the case of 30% ethanol (6 times) and 70% (7 times).

Experimental Example  5. Concentration-dependent inhibitory effect of EtOAc fraction on melanin synthesis induced by α-MSH

5-1. Experimental course

① 40 plates were prepared, B16F10 cells were cultured on each plate, and 10 groups were separated into 4 groups.

(2) In the 10 plates, two plates were set as the negative control group, one plate was treated without any treatment, and the other plate was treated with 100 nM of? -MSH alone. 10 μg / ml, 50 μg / ml, 100 μg / ml, and 10 μM / ml of kojic acid were added to each well of 10 μM, 1 mM each of the positive control arbutin / mL.

③ The other 10 plates were treated with 5 ㎍ / mL, 10 ㎍ / mL, 50 ㎍ / mL and 100 痢 / mL of EtOAc fraction of 30% ethanol extract at the upper part in the same manner as in ② in negative control and positive control.

④ The other 10 plates were treated with 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL of the EtOAc fraction of the 70% ethanol extract at the upper part in the same manner as in the control of the negative control group and the positive control group .

⑤ The remaining 10 plates were treated with 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL of EtOAc fraction of 95% ethanol extracts at the upper part in the same manner as in ② in the negative control and positive control groups.

After 2 days, the culture was removed and the cells were recovered by treatment with trypsin. The cells were centrifuged at 10,000 to 13,000 rpm for 15 minutes. The obtained pellet was washed with alcohol, and the cells were washed with 1 N NaOH solution containing 10% DMSO The incubation time was 1 hour in a thermostat bath to dissolve the melanin, and the absorbance was measured at 405 nm.

5-2. Experiment result

5 is a graph showing the concentration-dependent inhibitory effect of the EtOAc fraction on melanin synthesis induced by? -MSH.

As a result, all of the EtOAc fraction extracts inhibited the melanin production induced by α-MSH in a concentration-dependent manner, and the 30% ethanol extract had the highest effect among the EtOAc fraction extracts.

Experimental Example  6. Inhibitory effect of 칡 aerial part extract on NO production

6-1. Preparation for experiment

1) Reagents and equipment

DMEM medium, trypsin-ethylenediaminetetraacetic acid (EDTA) and fetal bovine serum (FBS) were purchased from Gibco Laboratories. Lipopolysaccharide (LPS) was purchased from Sigma. 96-Well tissue culture plates and other tissue culture dishes were purchased from Falcon. Absorbance was measured using a BioRad Microplate Reader.

2) Cell culture

RAW 264.7 macrophages were purchased frozen at ATCC. RAW 264.7 macrophages were cultured in DMEM medium containing FBS (10%), penicillin (100 U / ml) and streptomycin sulfate (100 g / ml) in a humid 37, 5% CO ₂ incubator.

6-2. Experimental course

1) Nitrite Assay

The cultured cells were cultured at a density of 5 × 10 ⁵ cells / well in a 96-well plate (100 μL), cultured for 24 hours, and after 24 hours, the medium was removed. After each sample was diluted with DMEM, LPS / ml), the amount of NO released from the cells in the medium after 24 hours was measured using Griess reagent (0.1% (w / v) N- (1-naphathyl) -ethylenediamine and 1% (w / v) sulfanilamide in % (v / v) phosphoric acid). The reaction was then measured at 540 nm using an ELISA microplate reader (Bio Rad Laboratories Inc., California, USA, Model 550).

2) Western Blot

Analysis RAW 264.7 macrophages were cultured in a 60 mm dish at a density of 3 × 10 ⁵ cells / well for 24 hours. RIPA buffer was added to RAW 264.7 macrophages, followed by centrifugation at 14,000 xg for 25 minutes at 4 ° C, and the supernatant was transferred to a tube. Protein quantitation was performed using a BSA protein kit. Each sample was run on a 12% Spolyacrylamide gel and transferred to nitrocellulose membrane (NC membrane). The transferred NC membrane was blocked with fresh blocking buffer (0.1% Tween 20 in Tris-buggered saline) containing 5% nonfat milk, and then iNOS and COX-2 antibody were diluted 1: 1000 and reacted for 1 hour. After incubation for 1 hour with 2: 1 anti-mouse IgG (anti-mouse IgG), ECL solution was added to the NC membrane at 1: 1 and light was emitted. Respectively. In the same manner, actin is measured using actin antibody.

6-3. Experiment result

FIG. 6 shows the results of measurement of the inhibitory effect on the NO production of the ground-based extract.

NO is overexpressed by iNOS (inducible nitric oxide synthase) expressed by inflammation inducers such as IL-1β, TNF-α, and LPS and cancer-causing factors, and persistent overexpression of NO produced promotes mucosal injury, As a result, some of them induce diseases such as cancer. Therefore, we investigated the inhibitory effect of LPS on the NO production in the RAW 264.7 cells of H ₂ O, 30% EtOH, 70% EtOH, and 95% EtOH extracts. RAW 264.7 cells were pretreated with 25, 50 and 100 μg / ml of extracts for 3 hours, and LPS (1 μg / ml) was treated for 24 hours.

As a result, the inhibition of NO production was observed with increasing concentrations of the above-ground H ₂ O, 30% EtOH, 70% EtOH, and 95% EtOH extracts. Among them, 70% EtOH extract showed the most excellent NO production inhibitory effect.

Experimental Example  7. EtOAc from ground-based extract Of fractions  NO generation inhibitory effect

7-1. Preparation for experiment

Was carried out in the same manner as in Experimental Example 6-1.

7-2. Experimental course

Was carried out in the same manner as in Experimental Example 6-2.

7-3. Experiment result

FIG. 7 shows the results of measuring the inhibitory effect of the EtOAC fraction extract derived from the ground-based extract on NO production.

Inhibition of NO production by LPS - induced inflammation in RAW 264.7 cells of EtOAC fractions from ground - based extracts. RAW 264.7 cells were pretreated with EtOAC fractions at 25, 50, and 100 μg / ml for 3 hours, and LPS (1 μg / ml) was treated for 24 hours.

As a result, the EtOAC fractions from the ground-based extract showed inhibition of NO production with increasing concentrations. Among them, EtOAC fraction of 70% alcohol extract showed the most superior NO production inhibitory effect.

Experimental Example  8. 칡 Inhibitory effect of NO on the surface indicator

8-1. Preparation for experiment

Was carried out in the same manner as in Experimental Example 6-1.

8-2. Experimental course

Was carried out in the same manner as in Experimental Example 6-2.

8-3. Experiment result

Fig. 8 shows the result of measuring the NO production inhibitory effect of the ground surface element component.

Inhibition of NO production by LPS induced inflammation in RAW 264.7 cells of Genistein, Formononetin, Daidzin, and Daidzein, the surface components of the ground, . RAW 264.7 cells were pretreated with 25, 50 and 100 μg / ml of genistein, pomonotin, didezin, and daidzein for 3 hours, treated with LPS (1 μg / ml) for 24 hours, Production inhibitory effect was measured.

As a result, except for daidzein, the concentrations of genistein, pomonotenine, and daidzein increased, indicating inhibition of NO production. Among them, daidzein showed the most excellent inhibitory effect on NO production.

Experimental Example  9. 칡 Ground-top extract and 칡 Ground-top extract-derived EtOAc Of fractions  Inhibitory effect of iNOS protein expression

9-1. Preparation for experiment

Was carried out in the same manner as in Experimental Example 6-1.

9-2. Experimental course

Was carried out in the same manner as in Experimental Example 6-2.

9-3. Experiment result

FIG. 9 shows the results of measuring inhibitory effect of iNOS on the extracts of EtOAc fraction derived from the ground-top extract and the ground-top extract.

Inhibition of iNOS protein expression by LPS-induced inflammation in RAW 264.7 cells of the H ₂ O, 30% EtOH, 70% EtOH, and 95% EtOH extracts and EtOAc fractions from the ground-root extract were investigated.

RAW 264.7 cells were pretreated with 40 μg / ml of each extract at a concentration of 40 μg / ml for 3 hours and treated with LPS (1 μg / ml) for 24 hours. Western blot analysis was performed to measure the inhibitory effect of iNOS protein.

As a result, the inhibition of iNOS expression was superior in the EtOAc fractions derived from the ground-based extracts, compared with the ground-based H ₂ O, 30% EtOH, 70% EtOH and 95% EtOH extracts. Among them, 70% EtOH-derived EtOAc fraction and 95% EtOH-derived EtOAc fraction showed the best inhibitory effect on iNOS expression.

Experimental Example  10. Ground-top extracts and EtOAc from ground-top extracts Of fractions  Inhibitory effect of COX-2 protein expression

10-1. Preparation for experiment

Was carried out in the same manner as in Experimental Example 6-1.

10-2. Experimental course

Was carried out in the same manner as in Experimental Example 6-2.

10-3. Experiment result

FIG. 10 shows the results of measuring inhibitory effect of COX-2 on the extracts of EtOAc fraction derived from the ground-top extract and the ground-top extract.

The inhibition of COX-2 protein expression by LPS-induced inflammation in RAW 264.7 cells was investigated in the EtoAc fractions obtained from the above-ground H ₂ O, 30% EtOH, 70% EtOH and 95% EtOH extracts. RAW 264.7 cells were pretreated with 40 μg / ml of each extract at a concentration of 40 μg / ml for 3 hours, and treated with LPS (1 μg / ml) for 24 hours. Western blot analysis was performed to determine COX-2 protein expression inhibition.

As a result, the inhibition of COX-2 expression was superior in the EtOAc fractions obtained from the ground-based extracts, compared with the H ₂ O, 30% EtOH, 70% EtOH and 95% EtOH extracts. Among them, 30% EtOH-derived EtOAc fraction, 70% EtOH-derived EtOAc fraction and 95% EtOH-derived EtOAc fraction showed the best COX-2 inhibitory effect.

Experimental Example  11. From the above-ground extract, EtOAc Of fractions  Melanin accumulation inhibitory effect

11-1. Experimental course

Fontana-Masson staining was performed to observe the degree of pigmentation by staining melanin with black. Using the B16F10 melanoma cell line, we directly observed the whitening effect of EtOAc fractions from the ground-top extract. The experimental procedure is as follows.

(1) B16F10 cells were plated on a chamber slide at a density of 7 × 10 ³ cells per well and adhered for 24 hours, followed by treatment with α-MSH (100 nM).

② After 24 hours of incubation, samples and α-MSH (100 nM) were treated for 48 hours.

After removing all of the culture medium, it was fixed in 4% formalin solution for 20 minutes, washed twice with 0.1% Triton X-100 PBS, and then reacted with FM Silver Nitrate Solution preheated with 56 ° C water bath for 2 hours.

④ After washing 3 times, it was stained with Gold Chloride solution for 1 minute, washed again 3 times, and reacted for 5 minutes in 5% sodium thiosulfate solution for 1 minute.

After washing three times, the cells were stained for 5 minutes with Nuclear Fast Red Solution, washed three times with 0.1% Triton X-100 PBS, and observed with an optical microscope.

11-2. Experiment result

11 is a photograph of B16F10 cells stained with Fontana-Masson and observed under an optical microscope.

As a result, it was confirmed that melanin staining was less than that of the control group treated with α-MSH (100 nM) at a high concentration, but the concentration of arbutin and kojic acid used in the positive control group did not show a significant effect. The concentration-dependent inhibition of melanin pigmentation at 10, 50 and 100 g / ml concentrations was confirmed.

In addition, the EtoAc fraction of the 30% EtOH extract showed a similar effect to that of the control group not treated with α-MSH (100 nM).

Experimental Example  12. From the ground-top extract-derived EtOAc Of fractions  Enzyme Inhibitory Effect on Melanin Synthesis

12-1. Measurement of intracellular tyrosinase activity

i) Experimental process

In order to investigate the effect of the above-ground extract on tyrosinase activity, an enzyme that regulates melanin synthesis in cells, α-MSH (100 nM) was added to increase melanin synthesis and tyrosinase was extracted from the sample-treated B16F10 cell And the activity thereof was evaluated. The experimental procedure is as follows.

1) B16F10 melanoma cells were treated with α-MSH (100 nM) for 24 hours and α-MSH (100 nM) at various concentrations.

After incubation for 48 hours with the sample, the cells were harvested with trypsin, and the culture broth of pellet was completely removed with PBS. Then, 5 mM EDTA and 1% (V / V) triton X-100 and 0.1% The cells were lysed on ice for 30 minutes with 0.1 M sodium phosphate buffer (SPB pH 6.8).

③ Tyrosinase was obtained from the supernatant by centrifugation at 15,000 rpm for 30 minutes at 4 ° C, and the amount of protein was calculated using Bradford reagent.

④ The same amount of protein diluted in 0.1 M SPB (pH 6.8) and 0.1% (V / V) L-DOPA were reacted at 37 ° C for 1 hour and absorbance was measured at 475 nm.

ii) Experimental results

FIG. 12 is a graph showing the activity of tyrosinase in cells after treatment with EtOAc fraction extracts from ground-top extract.

Both of the EtOAc fraction extracts from aerial part extracts inhibited tyrosinase activity in a concentration-dependent manner compared with α-MSH (100 nM) treatment. Especially No. 6, the inhibitory effect was excellent even at low concentrations and the activity was lower than the tyrosinase activity of the control group without α-MSH (100 nM). No. 7 also showed tyrosinase inhibitory effect at a similar level to the control group not treated with α-MSH (100 nM) at a high concentration.

12-2. Measurement of activity of enzymes related to melanin synthesis

i) Experimental process

In order to investigate the effect of the aerial part extract on tyrosinase expression in cells, B16F10 cells were treated with α-MSH (100 nM) and western blot analysis. The experimental procedure is as follows.

1) B16F10 melanoma cells were inoculated in a 10-cm culture dish at 3 × 10 ⁵ cells for 24 hours and then treated with α-MSH (100 nM).

② After 24 hours, samples were treated with α-MSH (100 nM) at various concentrations and cultured for 48 hours. All cells were collected with trypsin.

③ After completely removing the pellet culture solution with PBS, it was dissolved in ice buffer for 20-30 minutes with lysis buffer containing proteinase inhibitors.

④ Lysate was centrifuged at 13,000 rpm for 20 minutes and the supernatant was collected using Bradford reagent. Twenty-five μg of protein and 5 samples of buffer (0.225 M Tris-HCl, 50% glycerol, 5% SDS, 0.05% bromophenol blue, and 0.25 M β-mercaptoethanol) were inactivated by heating at 95 ° C for 5 minutes and then electrophoresed on SDS polyacrylamide gel.

⑤ Protein of electrophoresed gel was transferred to PVDF membrane and washed with 5% skim milk or 5% BSA for 1 hour.

⑥ The primary antibody was diluted in the blocked membrane, reacted at 4 ° C for 24 hours, washed with PBST, diluted with primary-specific HRP conjugate secondary antibody, and reacted at room temperature for 1 hour.

⑦ After washing with PBST, the band was photographed with ChemiDoc and analyzed with image J.

ii) Experimental results

13 is a Western blotting result of the melanin synthesis-related enzyme after treatment with the EtOAc fraction extract from the ground-root extract.

As a result, two lines of bands reacting to tyrosinase antibody were observed. Tyrosinase is a protein that is activated by N-glycosylation in cells. If it is not glycosylated properly, it does not have melanin synthesis ability. In the Western blot results, tyrosinase, which is glycosylated in the upper bands with a larger protein size, and tyrosinase, which is not glycosylated in the lower bands, has the effect of inhibiting glycosylation of tyrosinase by the bark extract.

12-3. Enzymes related to melanin synthesis mRNA  Confirmation of expression

i) Experimental process

In order to investigate whether the whitening effect of the over-the-ground extract was due to the regulation of mRNA expression of melanin synthesis-related enzymes, M16F10 cells producing melanin were treated with α-MSH (100 nM) to increase melanin synthesis, And RT (realtime) -PCR was performed. The experimental procedure is as follows.

(1) α-MSH (100 nM) was treated with B16F10 melanoma cells for 24 hours, and treated with α-MSH (100 nM) for 24 hours.

② After incubation for 48 hours with the sample, the cells were harvested with trypsin and the culture of the pellet was completely removed with PBS.

③ mRNA was extracted with Eszy-Blue Total RNA extraction kit (iNtRON Biotechnology, Korea) and mRNA content was measured by Nano Drop.

④ Mix the extract with primer using TaqMan RNA-to-Ct 1-Step Kit and mix with the extract on the StepOne machine for 15 minutes at 48 ° C, 10 minutes at 95 ° C, 15 seconds at 95 ° C, 1 minute at 60 ° C The threshold cycle value was measured by repeating ~ 60 times.

ii) Experimental results

FIG. 14 is a Western blotting result of the melanin synthesis-related enzyme after treating the extract of EtOAc fraction derived from the ground-based extract.

As a result, it was confirmed that both of the EtOAc fraction extracts from the ground-based extract reduced tyrosinase mRNA expression in a concentration-dependent manner and that mRNA expression was suppressed at a lower level than that of the control group treated with α-MSH (100 nM) . In addition, the mRNA expression of tyrosinase-related protein 1 (TRP1) was also inhibited in a concentration-dependent manner.

Experimental Example  13. From the above-ground extract, EtOAc Of fractions  Inhibitory effect on melanin synthesis-related factors

13-1. Experimental course

Was carried out in the same manner as i) of Experimental Example 12-2.

13-2. Experiment result

FIG. 15 is a graph showing changes in MITF expression by Western blotting after treatment with the above-ground extracts.

In order to investigate the effect of the above-ground extract on the MITF regulating tyrosinase expression, the expression of MITF was examined by western blotting of the extract-treated cells treated with α-MSH to promote melanin synthesis and treated with No.6 , There was a concentration-dependent inhibitory effect on MITF expression. Also inhibited the expression of MITF at high concentrations.

Experimental Example  14. in vivo  Gt; &lt; RTI ID = 0.0 &gt; EtOAc &lt; / RTI & Of fractions  Whitening effect

14-1. Preparation for experiment

In vivo experiments for inhibiting pigmentation were carried out according to the approval and management of Wonkwang University Animal Experimental Ethics Committee. 6-week-old HRM2 hairless mice were purchased from the central experimental animal, and were adapted for 1 week in the SPF animal breeding room. Water and feed were supplied freely.

14-2. Experimental course

(1) UV-irradiated control group (UV + base cream applied), positive control group (UV + kojic acid 1% cream applied) cream), No. 6 3% application group (UV + No. 6 3% cream application), and divided into 5 groups by 3 animals. The drug was applied 3 days before UV irradiation until the end of the experiment.

② For animal control, anesthetized with ketamine (i.p. 2 ml / kg), 100 mg of cream was applied to the entire back, and then absorbed for 30 minutes.

③ UV irradiation was carried out using a lamp that emits UVB at 306 nm after removing the remaining cream after 30 minutes of application.

④ The amount of ultraviolet light was measured with a UV-meter, and the irradiation amount was gradually increased at 100 mJ / cm, 150 mJ / cm, and 200 mJ / cm, and was irradiated once a day for 5 days each.

⑤ The degree of pigmentation according to the coating material was measured at the L value center of the same site using a colorimeter, and it was performed 1 day before the application of the sample and once a day until the end of the experiment. During the sample application period, measurements were made after pre-application anesthesia.

16 is a view showing a sample application and a UV irradiation process in an experimental animal.

The L * value (lightness value) measured by colorimetric method was compared by using a control cream without a UVB, a base cream, and a UVB group as a control.

14-3. Experiment result

FIG. 17 is a graph showing the L * value measured after treatment with the ground-based extract in a mouse animal model in which pigmentation was induced by UVB irradiation.

The results showed that L * value was lowered due to pigmentation during UVB irradiation, and when kojic acid 1% cream was used as a positive control, it showed significant difference from 12 days after UVB treatment.

In the case of the cream containing 1% of the sample, there was a significant difference from the 9th day after the positive control and the L * value was higher than the kojic acid group.

In particular, when applying cream containing 3% of sample, a remarkable whitening effect was confirmed. From the 6th day of experiment, L * value similar to that of CON group was measured as well as a significant difference as compared with UVB group. CON group.

For more clear comparison, the change of the L * value before and after the experiment inducing the pigmentation by the equation (1) was graphed and it was confirmed that the CON group was increased while the UVB group was decreased.

Kojic acid group showed less decrease than UVB group and L * value was increased in the group treated with Kojic acid.

In Experimental Examples 1 to 14, it was confirmed that both of the water-phase water fraction extract and EtOAc fraction extract showed antioxidative effects in a concentration-dependent manner as a result of the DPPH test, and inhibited intracellular melanin synthesis and inhibited melanin And inhibited the expression of NO, iNOS and COX-2.

It was also confirmed by Fontana-Masson staining that the inhibition of melanin synthesis induced by α-MSH was inhibited by the EtOAc fraction at the upper part of the ground. The tyrosinase activity of melanin synthesis enzyme tyrosinase was inhibited and inhibited by tyrosinase glycosylation. And inhibit the expression of MITF, thereby confirming the mechanism by which mRNA transcription of tyrosinase and tyrosinase-related protein 1 is reduced.

In addition, 1% cream showed better whitening effect than kojic acid 1% cream, which was used as a positive control, and 3% cream , The lightness value was higher than that of the normal control without UVB irradiation.

Claims (10)

delete delete delete delete delete delete delete delete delete 화장 Cosmetic composition with antioxidant, anti-inflammatory and whitening effect, containing an extract of the above-ground part (Leaf and stem)
The above ground-top extract is extracted with ethanol or water, further fractionated with ethyl acetate,
Treated with B16F10 melanoma cell line or 50-100 占 퐂 / ml on RAW 264.7 cell line, 0.1-10% by weight on the basis of cream applied to the skin of HRM2 hairless mice,
Inhibition of melanin synthesis, melanin accumulation, tyrosinase, tyrosinase related protein-1 and microphthalmia-associated transcription factor (MITF), and whitening effect in HRM2 hairless mice induced by UV-induced pigmentation . &Lt; / RTI &gt;
A cosmetic composition for skin whitening which improves pigmentation caused by? -MSH or ultraviolet rays.

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