KR101764806B1 - Method for manufacturing complex extract of Lycopodium clavatum and Equisetum arvense and cosmetic composition containing the same - Google Patents

Abstract

The present invention relates to a production method of a complex extract of Lycopodium clavatum and Equisetum Arvense L. containing a large amount of wrinkle alleviation and skin whitening active ingredients, and to a cosmetic composition for wrinkle alleviation and skin whitening, which shows effects of promoting collagen production in the skin, inhibiting elastase, and inhibiting expression of a melanin-related factor by containing the complex extract.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a cosmetic composition for extracting Lycopersicon esculentum and Lycopersicon esculentum,

The present invention relates to a method for producing a complex extract of Lycoris spp. And Lycopersicon esculentum and a cosmetic composition containing the same as an active ingredient. More specifically, the present invention relates to a method for producing a complex extract of lycopersicum and Lycopersicum containing a large amount of active ingredients, To a technique for use as a cosmetic composition for use in cosmetics.

The cause of wrinkles is largely divided into the generation of wrinkles by natural aging and the generation of wrinkles by exposure to ultraviolet rays (photoaging). However, the mechanism of wrinkling due to two causes has not been clarified yet. A common phenomenon found in association with skin changes due to natural aging and photoaging is the reduction and modification of collagen and elastin, the main constituent proteins in the dermis, and the reduction of skin elasticity due to the reduction of the hyaluronic acid, the substrate of the dermis. Particularly, according to the results thus far, reduction and modification of collagen and elastin, which are main proteins in the dermis, are directly related to the generation of wrinkles and the reduction of elasticity of skin.

It is the unequaled desire of all men to have white and fine skin. In general, skin color of human skin and eyes are determined by melanin carotene and hemoglobin. The skin color of a person is determined by various factors such as the distribution of active blood vessels of melanocytes that make melanin pigment, the thickness of skin, and the presence or absence of a coloring matter such as carotenoids and bilirubin, among which melanocyte thyrosinase And melanin, which is produced by the action of various enzymes, is most important. The formation of melanin pigment affects physiological factors such as genetic factor hormone secretion and stress, and environmental factors such as ultraviolet irradiation. Melanin is present in the skin and has an important function to protect the body from ultraviolet light. However, it is known that melanin is over-produced to form spots or freckles, promotes skin aging, and plays an important role in skin cancer induction.

The main function of melanin is to remove these harmful radicals to protect the skin from damage caused by it. Thus, a high amount of melanin means that it has an effective countermeasure to protect the skin from physical or chemical toxicants. Factors that promote the production of melanin include sunlight, ultraviolet light, as well as estrogen or prostaglandine. The biosynthesis of melanin is caused by the formation of specific enzymes such as tyrosinase, tyrosinase related protein-1 (TRP-1) and DCT (dopachrome tautomerase) in the melanosome of melanocyte present in the epidermal basal layer Which is the main enzyme of melanin synthesis, is being used in the early stages of development of melanin polymer inhibitors. In addition to the oxidation by tyrosinase, there are a number of factors affecting the production of melanin in the skin, such as advanced glycation end products (AGEs) or non-enzymatic glycation reactions. Glycation is a non-enzymatic reaction of sugars and proteins that reacts with the carbonyl of the reducing sugar and lysine or arginine, the free amino group of the protein, to form the initial glycation product, the schiff base, , And the compounds thus formed are continuously reacted to generate advanced glycation end products (AGEs), which are browning compounds through a series of reactions such as cross-linking.

This is because the free amino group lysine or arginine of the protein reacts with the carbonyl group of the reducing sugar to form the initial glycation product shiff base, and the compounds formed at this time undergo a series of complicated reactions such as condensation, re-dislocation, oxidation, cleavage, It is a reaction to make a yellowish brown compound (melanoidine). When these AGEs are formed and accumulated, melanin pigment formation and accumulation occurs in the skin. This production of melanin is synthesized in the organelles called melanosomes in the basal layer of melanocytes. When AGEs are generated, the receptor receptor for AGE (RAGE) acts on the receptor site. AGE promotes the expression of the enzyme tyrosinase, which plays a major role in melanogenesis, by promoting the microphthalmia associated transcription factor (MITF), an important transcriptional regulator in melanin synthesis, after binding to RAGE. Finally melanin synthesis is produced. The synthesized melanin pigment is transferred to the adjacent keratinocyte through the dendrite of melanocytes, and the transferred melanin pigment is distributed in various parts of the skin through keratinocytes.

In order to treat or alleviate excessive melanin pigmentation caused by skin pigmentation abnormalities such as dyspnea freckles or pigmentation and ultraviolet ray exposure, ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione or derivatives thereof The substances with inhibitory activity of tyrosinase have been used in cosmetics or medicines because they have insufficient whitening effect. The safety of the skin has been limited due to formulation and stability problems in the formulation of cosmetics.

The present inventors selected natural products having skin wrinkles and whitening effect, prepared complexes therefrom, and studied their activity. In the production of complex extracts of Lycopersicon esculentum and Lentinus edodes, a specific solvent was used in combination with hydrolytic enzymes treatment It is possible to produce an extract having excellent wrinkle and skin whitening activity.

Korean Patent Publication No. 2013-0092258 (2013.08.20) Korean Patent Publication No. 2016-0035683 (2014.04.01)

It is an object of the present invention to provide a method for producing a complex extract of Lycopersicon esculentum and Lycopersicon esculentum containing a large amount of skin whitening and wrinkle improving active ingredients. It is another object of the present invention to provide a cosmetic composition for improving wrinkles and skin whitening comprising the combined extract of Lycopersicon esculentum and Aspergillus as an active ingredient.

To achieve the above object, according to the present invention, there is provided a process for the production of (A) a process for the production of (A) a mixture of water and anhydrous or hydrous alcohol, ethyl acetate, acetone, glycerin, ethylene glycol, Butylene glycol in the presence of at least one extraction solvent; (B) preparing an enzyme-treated extract of lysozyme and horse radish by adding a hydrolytic enzyme to the solvent extract prepared in the step And (C) adding 5 to 10 times volume of a shell lysis solution to the enzyme-treated extract of Lycoris spp. And Rhizoma leaves, and heating to 80 to 100 캜 for extraction.

In the step (A), the lycopersic beans are mixed at a weight ratio of 1 to 15: 15 to 1, preferably at the same weight ratio.

In the step (B), the hydrolytic enzyme may be selected from the group consisting of beta-glucanase, preferably Viscozyme.

In the step (C), the shell dissolving solution is obtained by firing the shell at 1000 to 1500 ° C. for 10 to 30 minutes, adding the crushed shell powder into purified water, heating the mixture at 100 to 120 ° C. for 5 to 30 minutes, and then filtering. The shell dissolving solution as the extraction solvent preferably has a pH of 8 to 10.

At this time, the shell may be at least one shell of shellfish selected from the group consisting of oyster, ramie clams, clams, rolled mussels and mussels.

According to the present invention, there is provided a cosmetic composition for improving wrinkles and whitening comprising 0.1 to 30% by weight of the combined extract of Lycopersicon esculentum and Rhododendron japonica, which is produced by the method of the present invention, based on the total weight of the cosmetic composition.

The cosmetic composition containing the combined extract of Lycopersicon esculentum and Lycopersicum extract prepared according to the production method of the present invention contains a natural extract and is safe, exhibits excellent skin wrinkle and skin whitening effect.

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

Lycopodium (Lycopodium clavatum Linn ㅹ) It is composed of apricot, upright and leaf. It has a thin cylindrical shape with a slight bend, a length of about 2 m, a diameter of 0.1 to 0.3 cm, and a yellowish white fringe below it. The upright branch is branched twice. The leaves are tightly arranged on the stem, arranged in a screw shape, curved and curved, and have a line or needle shape. It is 0.3 ~ 0.5cm long and has yellowish green to light yellowish brown. There is no hair, and the ovary is arctic, and it is the leading edge and it breaks easily. The vagina is flexible and the cut surface is light yellow and the woody part is white. There is a peculiar smell and the taste is cool. Chongsong is located in Jeju Island (Halla Mountain) and its mountainous regions in Korea, and is distributed throughout East Asia including China and Japan. It is mainly called Club moss, ground pine, devil's claw, devil ash. Lycopersa is a fern that lives on the earth from the late Carboniferous period (360 million to 280 million years ago) and has a huge forest in high humidity and high temperature, especially spore breeding plant . Also, it is a promising medicinal plant in the future and has been studied so far. It has acetyl cholinesterase inhibitory activity and contains a large amount of alkaloids such as tricterpenoids (Lycopodine), ricopodine, clavatine and clabatoxin . In the private sector, plant spores are used to heal skin wounds, and they are sprayed on dermatitis, such as wet warts, rashes, and ulcers.

Equisetum Arvense L.) is a perennial herb that grows in the mountains of the country. The growing environment grows in sunny or sunny places. The height is 30 ~ 40cm, and the reproductive organs come out in early spring and form sporangium at the end of the stem like an apartment. Nutrition is hollow and grows diagonally, standing straight from the ground and vertically aligned. Stems are used for medicinal purposes. Also known as papilia. It grows in the grass and grows as the stems grow long. Growing early in the spring is a reproductive stem, at the end of which a number of sporangia run. There are no branches, and light brown leaves like scales turn on the nodes. The nutrient stem grows at the time of reproductive stunting. It stands straight and is green with 30 ~ 40cm in height, with nodes and ridgelines, scaly leaves on the nodes, and branches. The sporophyll number is an elliptical shape, with hexagonal sporangia closely attached, like a back of a turtle, with sporangium of about 7 inside each. It means that the cow is tearing, and the cow is eating well. Reproductive stem is edible, and nutritional stem is diuretic. It is widely distributed in the northern hemisphere from north of the hatchery to one.

According to the present invention, there is provided a process for the production of (A) a process for producing (A) a mixture of water and a water-soluble or water-miscible organic solvent having a carbon number of 1 to 4, ethylacetate, acetone, glycerin, ethylene glycol, propylene glycol, dipropylene glycol, and butylene glycol Extracting at least one extraction solvent; (B) preparing an enzyme-treated extract of lysozyme and horse radish by adding a hydrolytic enzyme to the solvent extract prepared in the step And (C) adding 5 to 10 times volume of a shell lysis solution to the enzyme-treated extract of Lycoris spp. And Rhizoma leaves, and heating to 80 to 100 캜 for extraction.

First, extracts are prepared after mixing lycopersicum gruelosus and horse radishes at a certain ratio. According to one embodiment of the present invention, the lycopods and rhizomes are mixed at a weight ratio of 1: 15: 15-1, preferably at the same weight ratio. The extract may be a hot-water extract or a solvent extract of at least one solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, ethyl acetate, glycerin, ethylene glycol, propylene glycol, dipropylene glycol and butylene glycol to be.

Then, hydrolysis enzyme is added to the above extract to perform enzyme treatment. The hydrolytic enzyme may be selected from the group consisting of beta-glucanase, preferably an enzyme-treated extract using Viscozyme.

Then, the enzyme-treated extract is extracted again with a shell-lysing solution as an extraction solvent. The shell dissolving solution is obtained by baking the shell at 1000 to 1500 ° C. for 10 to 30 minutes, adding the crushed shell powder into purified water, heating the mixture at 100 to 120 ° C. for 5 to 30 minutes, and then filtering. According to one embodiment of the present invention, at least one bark of shellfish selected from the group consisting of oyster, foxtail, clams, rolled and mussels is fired at 1000 to 1500 ° C for 10 to 30 minutes, Crush with powder of crushed cakes of mesh. This is obtained by heating the mixture in purified water at 100 to 120 ° C for 5 to 30 minutes and then filtering. The shell dissolving solution as the extraction solvent preferably has a pH of 8 to 10, more preferably a pH of 10.

The lyophilized extract solution is added to the enzyme-treated lycopersicum lanceolate extract and corn root extract in a volume of 5-10 times volume and heated to 80-100 ° C. to boil and steam to produce a complex extract of Lycopersicon esculentum and Lycopersicon esculentum.

As a result of extracting the enzyme-treated extract by using heat extract as a solvent for shell extract, the disaccharides of glycosides occurred, and as the fibrous component changed to monosaccharide, the content of saccharide increased, various amino acid components increased, Improvement and whitening effect.

The cosmetic composition containing 0.1 to 30% by weight of the combined extract of Lycopersicum sativus L. and Rhododendron L. produced by the above-mentioned method is effective for inhibiting the activity of elastase, collagen biosynthesis and melanin synthesis-related factors, It can be used as a cosmetic composition for skin wrinkle improvement and whitening.

 The cosmetic composition of the present invention can be manufactured into any of the commonly used formulations and examples thereof include lotion, cream, essence, cleansing foam, cleansing water, pack, body lotion, body oil, body gel, shampoo, Conditioner, hair gel, foundation, lipstick, mascara, make-up base and the like.

[Example]

Hereinafter, the present invention will be described in detail by way of the following examples and test examples, but the present invention is not limited to these examples.

Production Example 1: Production of Lycopersicon esculentum extract

10 kg of lycopersicin was washed with purified water and dried. The extract was dried in an extractor (Cosmos-660, Gyeongseo Machine) with a cooling condenser and heated to 80 ℃ ~ 100 ℃. .

 The precipitate was filtered through 300 mesh filter paper, and the precipitate was filtered twice using filter paper of Edbentek No. 5 and Wattman GFC 150 mm filter paper. The mixture was concentrated using a vacuum condenser (Coolace CCA-1100, EYELA) at a temperature of 40 ° C to 50 ° C and then filtered through a spray dryer (B-290, BUCHI) , And dried under the conditions of 100% efficiency (35 cm3 / hr), pump efficiency 25% (7.5 ml / min), Nozzle Cleaner 4 and 30 mm (357 liters / hour) To obtain 500 g of an extract powder of the title product.

Production Example 2: Preparation of Rhizome Extract

10 kg of horse mackerel was washed with purified water and dried. The resulting extract was heated to 80 ° C to 100 ° C in an extractor (Cosmos-660, Kyoreya) equipped with a cooling condenser and weighed 5 times with 70% .

The precipitate was filtered through 300 mesh filter paper, and the precipitate was filtered twice using filter paper of Edbentek No. 5 and Wattman GFC 150 mm filter paper. The mixture was concentrated using a vacuum condenser (Coolace CCA-1100, EYELA) at a temperature of 40 ° C to 50 ° C and then filtered through a spray dryer (B-290, BUCHI) , And dried under the conditions of 100% efficiency (35 cm3 / hr), pump efficiency 25% (7.5 ml / min), Nozzle Cleaner 4 and 30 mm (357 liters / hour) To obtain 500 g of an extract powder of the title product.

Preparation Example 3: Preparation of mixed extract of Lycopersicon esculentum

5 kg of each of lycopersicin and lycopene were washed with purified water and then dried. The extract was dissolved in 5% by weight of 70% ethanol and heated at 80 ° C to 100 ° C in an extractor (Cosmos-660, And extracted three times in total for 2 hours.

 The precipitate was filtered through 300 mesh filter paper, and the precipitate was filtered twice using filter paper of Edbentek No. 5 and Wattman GFC 150 mm filter paper. The mixture was concentrated using a vacuum condenser (Coolace CCA-1100, EYELA) at a temperature of 40 ° C to 50 ° C and then filtered through a spray dryer (B-290, BUCHI) , And dried under the conditions of 100% efficiency (35 cm3 / hr), pump efficiency 25% (7.5 ml / min), Nozzle Cleaner 4 and 30 mm (357 liters / hour) To obtain 500 g of an extract powder of the title product.

Production Example 4: Preparation of enzyme-treated Lycopersicon esculentum extract

Purified water was added to the extract powder of the Lycopersicon esculentum extract prepared in Preparation Example 3, and 0.5% of Viscozyme enzyme was added thereto, followed by reaction at 50 ° C for 2 hours. Next, the enzyme reaction was completed by heating at 95 DEG C for 30 minutes, and then the reaction product was concentrated in vacuo. The concentrate was dried at 60 DEG C for 30 hours. The precipitate was filtered through 300 mesh filter paper, and the precipitate was filtered twice using filter paper of Edbentek No. 5 and Wattman GFC 150 mm filter paper. The mixture was concentrated using a vacuum condenser (Coolace CCA-1100, EYELA) at a temperature of 40 ° C to 50 ° C and then filtered through a spray dryer (B-290, BUCHI) (357 liters / hour), 100% efficiency (35 cm3 / hr), pump efficiency 25% (7.5 ml / min), Nozzle Cleaner 4 and flow meter Rotameter) 100 g of the title powder was obtained.

Production Example 5: Production of shell powder and solution

1 kg of oyster shells were baked at 1200 ° C for 30 minutes in direct and indirect heat, and the shell was pulverized to 200 mesh by a pulverizer. The oyster shell powder was placed in a 10-fold volume of purified water and heated at 100 占 폚 for 30 minutes. The solid powder was separated with a filter to obtain a shell dissolving solution.

Example 1: Preparation of steam hydrothermal extract

The lyophilized solution (pH 10) prepared in Preparation Example 5 was added to the enzyme-treated mixed extract powder prepared in Preparation Example 4 in an amount of 10 times the volume, and the extract solution (Cosmos-660, And heated to ~ 100 ° C for 3 hours. The precipitate was filtered through a 300-mesh filter paper, and the precipitate was filtered twice using a filter paper of Edbentek No. 5 and a Wattman GFC 150 mm filter. The mixture was concentrated at a temperature of 40 to 50 ° C using a vacuum condenser (Coolace CCA-1100, EYELA), and then dried at an inlet temperature of 180 ° C., an aspirator efficiency Dried under conditions of 100% (35 cm3 / hr), pump efficiency 25% (7.5 ml / min), Nozzle Cleaner 4 and 30 mm (357 liters / hour) 100 g of the title extract powder was obtained.

Test Example 1: Confirmation of cytotoxicity

To confirm cytotoxicity, fibroblasts were inoculated into IMDM medium supplemented with 10% FBS at a cell concentration of 5 × 10 ⁵ cells and cultured in a 37 ° C. 5% CO ₂ incubator for 24 hours. After the culture, the culture medium was removed, and the extract prepared in the above Preparation Examples and Examples was diluted with dimethyl sulfoxide to give sample concentrations of 50, 100, 500 and 1000 μg / ml. The diluted solution was treated and cultured for 24 hours 100 ml of MTT (3- [4,5-dimethylthiazol-2yl] -2,5-diphenyltetrazoliumboromide, Sigma, USA) solution was added to each well (3 mg / ml) for further 4 hours. After removing the supernatant, 150 μl of dimethylsulfoxide was added, and the resulting formazan was shaken by shaking for 30 minutes. Absorption was measured at 540 nm using a multimicroplate reader (Molecular device Spectra max 190). Cell viability was calculated according to the following equation and the results are shown in Table 1 below.

Cell survival rate (%) = absorbance of sample-added group / absorbance of control group 100

Sample concentration
(占 퐂 / ml) Cell survival rate (%) Example 1 Production Example 1 Production Example 2 Production Example 3 Production Example 4 0 100 100 100 100 100 50 100 100 100 100 100 100 100 100 100 100 100 500 100 100 100 100 100 1000 99.0 96.8 97.0 97.5 98.2

As shown in the results of Table 1, it was confirmed that the cell survival rate was confirmed at the concentration of 500 μg / ml in all of the extract samples of the examples and the production examples, and it was confirmed that the cytotoxicity was not affected. Respectively.

Test Example 2: Confirmation of the effect of inhibiting the formation of the final glycation end product

Glycation inhibitor assay was performed to measure the ability of the extracts prepared in the above Preparation Examples and Examples to inhibit the production of the final glycation end product. Preparation of the reaction was carried out by modifying the method of Mcpherson et al. (Role of fructose in glycation and cross-linking of proteins. Biochemistry 1998, 27, 1901-1907). (BSA, 5 mg / ml), 25 mM ribose, 1 mM diethylenetriaminepentaacetic acid (DTPA) and 0.02% sodium azide were mixed to a final concentration in a 0.1M phosphate buffer (pH 7.0) 180 占 퐇 was dispensed into each well plate (Nunc, Denmark), and then 20 占 퐇 of each sample (10 mg / ml) was added. At this time, a control group (A) in which glycosylation did not occur without mixing the ribose, a glycosylation control group (B) in which glycosylation occurred in the preparation, and a sample treatment group (C) were prepared.

A sample treatment group (D) without treatment with ribose was also prepared to eliminate the interference of fluorescence by each sample. After completion of the reaction, the reaction was carried out for 7 days at 37 ° C in a constant-temperature and constant-humidity incubator to prevent drying during the reaction. After the reaction was completed, fluorescence was measured at ex = 370 nm / em = 440 nm using a spectrophotometer (VICTOR3, Perkin Elmer, Waltham, MA, USA). Each sample was repeated three times.

The inhibitory activity of the final glycation end product was 100% for the control group (A) without saccharification reaction and 0% for the glycosylation control group (B) in which the glycation reaction occurred. (CD), which excluded the fluorescence of each sample in which no saccharification reaction occurred, was used. As a control group, aminoguanidine was used, and samples were measured by adding Example 1 and Preparation Examples 1 to 4. The inhibition rate (%) of the final glycation end product formation was calculated by the following formula, and the results are shown in Table 2 below.

(%) = 100 - [(C-D) / (A-B) x 100]

Sample Concentration (%) % Inhibition of final glycation end product formation Example 1 Production Example 1 Production Example 2 Production Example 3 Production Example 4 Aminoguanidine 0.5 24.40 15.5 16.2 17.8 20.3 30.25 One 30.52 26.30 25.3 28.2 30.5 36.78 2 38.38 29.60 28.6 30.0 35.6 43.56

As shown in the above Table 2, the combined extract of Lycopersicon esculentum and Lycopersicon esculentum of Example 1 was effective in inhibiting the production of the final glycation products and increased in a concentration-dependent manner. In addition, it was confirmed that the extracts of Preparation Examples 1 to 4 were more effective in inhibiting the production of final glycation end products.

Test Example 3: Confirmation of RAGE Expression Inhibitory Effect

THP-1 cells treated with mAillard reaction products (MRP) for 30 min were reacted for 24 hrs, collected, washed with PBS, and lysed in 50 mM Tris-Cl, 150 mM NaCl, 10 μg / ml leupeptin, 1% NP-40) for 30 minutes and then dissolved by pipetting. After centrifugation, the supernatant was obtained, and proteins were quantified using Bradford reagent. The protein was loaded on 2% SDS PAGE gels based on total lysate 30 μg / lane. Proteins from the electrophoresed gels were transferred to a PVDF membrane at 230 mA for 1 hour and blocked with 5% skim milk for 1 hour. Antibodies were incubated with rabbit polyclonal anti-IκB antibody (1: 1000 dilution), mouse monoclonal anti-RAGE antibody (1: 500 dilution), rabbit polyclonal anti-COX- 1: 1000 dilution) was diluted in 3% BSA / PBS overnight at 4 ° C. Anti-goat IgG conjugated with horseradish peroxidase (HRP): 10000 was diluted in 5% skim milk, reacted at room temperature for 1 hour, and the band was detected using ECL.

Inhibition rate (%) = [(A-B) / A] x 100

A: Amount of RAGE expression in wells to which no sample was added

B: RAGE expression level of the experimental group

Sample concentration
(%) RAGE expression inhibition rate (%) Example 1 Production Example 1 Production Example 2 Production Example 3 Production Example 4 0.5 13.5 5.0 6.3 9.5 11.5 One 22.5 11.2 9.2 13.5 15.6 2 31.5 15.6 18.6 20.5 21.2

As shown in Table 3, the combined extract of Lycopersicon esculentum and Rhizome of Example 1 showed a higher RAGE expression inhibitory effect than that of Preparation Examples 1 to 4.

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Test Example 5: Test for promoting collagen biosynthesis

Fibroblasts were inoculated into IMDM medium supplemented with 10% FBS at a cell concentration of 5 × 10 ⁵ cells and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. And the diluted solution prepared by diluting the extracts of the above Examples and Production Examples into dimethyl sulfoxide at a concentration of 50, 100, 500, and 1000 μg / ml was added thereto. After culturing for 18 hours under the same conditions, The cells were stressed by UV irradiation. After that, fibroblasts were recovered using Trizol reagent (Invitrogen, USA), and mRNA was extracted and cDNA was synthesized through a series of procedures. Gene amplification was carried out using 10x taq polymerase buffer, 10 mM dNTP, 10 mM Tris-HCl buffer (pH 7.0) using a thermal cycler (GenePro, Hangzhou Bioer tech., CHINA). The amplified product was amplified by electrophoresis The PCR reaction was carried out by adding 50 μL of distilled water to 95 ° C for 5 min and 1 cycle / 95 ° C for 1 min / 51 ° C. 2 minutes / 72 degrees 1 minute, 28 cycles amplification and 72 degrees for 5 minutes. The expression rate of the generated procollagen was calculated according to the following equation. As a control, normal fibroblasts without UV irradiation were used.

procollagen expression rate (%) = B / A × 100

A: The amount of procollagen expression in the control group

B: The amount of procollagen expressed in the sample treated and UV irradiated fibroblasts

As a result of the test, the results shown in Table 5 were obtained.

Sample concentration
(占 퐂 / ml) procollagen expression rate (%) Example 1 Production Example 1 Production Example 2 Production Example 3 Production Example 4 50 48.45 11.23 15.32 29.55 31.33 100 79.63 15.69 21.68 48.27 58.65 500 115.68 48.56 52.33 67.89 89.77 1000 184.23 59.63 61.57 97.65 152.46

As can be seen from the results of Table 5, the procollagen expression ratio was higher than that of Production Examples 1 to 4 in Example 1, and it was confirmed that the collagen production promoting effect was more excellent.

Test Example 6: Inhibitory effect of intracellular MITF expression

B16F10 melanoma cells were treated with 5 × 10 ⁵ cells / ㎖ of cells and cultured in Petri dishes at 5% CO ₂ and 37 ° C for 24 hours. Cells were grown in Petri dishes at least 80% and the following extracts were added at concentrations that were above 100% cell survival. The extracts were treated by concentration and cultured in DMED / 10 FBS for 24 hours at 5% CO ₂ and 37 ° C. After 24 hours, the RNA isolation from the cells, cDNA synthesis and RT-PCR were performed according to the method described by Komoroski et al. (Drug Metab. Dispos. 32: 512-518, 2004). The expression of the gene was confirmed by RT-PCR by selecting MITF. GAPDH was used as a house keeping gene to identify differences in gene expression. The extracted RNA was amplified with a dT-adapter primer (DEG-adapter primer) and DEPC water to denature 15 μl of the total RNA. After denaturation at 70 ° C for 5 min, reverse transcription of Moloney murine leukemiavirus (M-MLV) Lt; 0 > C for 60 minutes and 94 DEG C for 5 minutes. The synthesized cDNA was amplified by PCR reaction and the total reaction solution was 5 μl. The final concentration of each reagent is a primer 100pmol, 1.0㎛, dNTP mixture 0.2mM, 5x green or colorless GoTaq Reaction buffer 1x1.5 mM, MgCl 2 (PCR buffer) and GoTaq DNA polymerase 1.25 units. The PCR reaction conditions for denaturation, annealing and extension were as follows: 95 ° C 2 min 95 ° C 30 sec 60 ° C 30 sec 72 ° C 1 min 72 ° C 5 min 30-40 cycles . After PCR, 10 mu l of the product was electrophoresed on 2% agarose gel. The amount of expression was confirmed using Gel Documentation System (Korea Raptec Cat. No. DGS-200D).

Inhibition rate (%) = [(A-B) / A] x 100

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

Sample use
density(%) MITF expression inhibition rate (%) Example 1 Production Example 1 Production Example 2 Production Example 3 Production Example 4 Aminoguanidine (200 mM) 0.5 30.26 10.58 15.66 20.32 25.64 49.22 One 35.65 15.36 17.68 22.35 29.65 2 41.22 21.32 23.67 28.98 31.28

As shown in Table 6, the extract of Lycopersicon esculentum and Rhizome of Example 1 showed superior MITF mRNA gene expression inhibitory effect than the extracts of Production Examples 1 to 4.

Test Example 7: Confirmation of reduction of tyrosinase expression

Cells were plated at 1 × 10 ⁵ cells / mL in 24-well plates and cultured for 24 hours. After 24 hours, samples were prepared on the medium without FBS and antibiotics, and the cells were treated for 24 hours. After washing the sample with phosphate buffered saline (PBS) at pH 6.8, PBS containing 1% Triton X-100 was added to the wells, and the cells attached to the wells were removed with a cell scraper. The cells were rapidly frozen at -70 ° C and thawed. Cell membranes were disrupted by repeating this procedure three times. After centrifugation, the supernatant was used as an enzyme source for tyrosinase activity measurement and as a sample for protein measurement. Protein quantification was performed using a Bio-Rad protein kit. To determine tyrosinase activity, 200 μl of 10 mM L-dopa, 500 μl of 0.1 M PBS (pH 6.8), 300 μl of tyrosinase enzyme source (supernatant from cells) After 1 hour of incubation, the absorbance at 475 nm was measured.

Inhibition rate (%) = [(A-B) / A] x 100

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

Sample concentration
(%) Inhibition rate of tyrosinase expression (%) Example 1 Production Example 1 Production Example 2 Production Example 3 Production Example 4 Control Arbutin 0.5 23.25 10.5 10.56 18.66 19.65 26.68 One 30.56 15.69 18.68 22.84 25.6 40.85 2 42.32 20.23 22.36 28.54 31.54 50.36

As shown in Table 7, the combined extract of Lycoris spp. And Lycoris spp. Of Example 1 showed superior tyrosinase expression inhibitory effect as compared with Preparation Examples 1 to 4.

Test Example 8: Confirmation of Reduction of Melanin Synthesis

The whitening effect of B16F1 melanocytes was evaluated based on the degree of inhibition of melanin formation. The 16F1 melanocyte used in this study is a mouse-derived cell line and is a cell that secretes a melanin pigment called melanin. During the artificial culture of these cells, samples were treated to compare the degree of reduction of melanin pigment. The 16F1 melanocyte used in this test was purchased from ATCC (American Type Culture Collection, Accession No. 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocyte was measured as follows. B16F1 melanocytes were plated at a concentration of 2 × 10 ⁶ cells / mL per well in a 6-well plate. The cells were treated with 0.1% (wt / v) ≪ / RTI > Here, PBS was used as a solvent used to adjust the concentration of the sample. Cells were cultured for 72 hours. Cells were treated with 500 μl of trypsin-EDTA to measure cell numbers, and centrifuged to recover the cells. Quantification of intracellular melanin was carried out by modification of the method of Lotan (Cancer Res., 40: 3345-3350, 1980). The cell pellet was washed once with PBS, and 1 ml of a homogenization buffer solution (50 mM sodium phosphite, pH 6.8, 1% Tripton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to destroy the cells. To the cell filtrate obtained by centrifugation (3,000 rpm, 10 min), 120 μl of 1N NaOH (10% DMSO) was added to dissolve the extracted melanin, and the absorbance of melanin was measured at 405 nm with a microplate reader. Melanin was quantified The percent inhibition of melanin formation in the sample was measured. Melanin formation inhibition rate (%) of B16F1 melanocyte was calculated by the following equation.

Inhibition rate (%) = [(A-B) / A] x 100

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

Sample concentration
(%) Melanin inhibition rate (%) Example 1 Production Example 1 Production Example 2 Production Example 3 Production Example 4 Control Arbutin 0.5 25.68 12.37 13.65 15.86 19.45 22.56 One 31.56 14.56 18.95 20.78 25.64 38.78 2 48.9 19.87 24.5 27.45 30.44 51.46

As shown in Table 8, the best melanogenesis inhibitory effect was obtained in Example 1.

Formulation Example 1: Preparation of a serum containing a complex extract of Lycopersicon esculentum

(Example 1) was prepared according to a conventional method with the composition and content shown in Table 9 below.

Raw material Content (% by weight) Lycopersicon esculentum extract (Example 1) 1.0 Wax 1.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Mineral oil 10.0 Sorbitan stearate 0.5 Pro-type glycerin monostearate 1.0 Stearic acid 1.5 Glyceryl stearate /
FAGE-400 stearate 1.0 Propylene glycol 3.0 Carboxy polymer 0.1 Triethanolamine 0.1 Phenoxyethanol Suitable amount Purified water To 100

Formulation Example 2: Preparation of cream containing Lycopersicon esculentum extract

The cream containing the Lycopersicon esculentum extract (Example 1) was prepared according to a conventional method with the composition and content shown in Table 10 below.

Raw material Content (% by weight) Lycopersicon esculentum extract (Example 1) 1.0 Shea Butter 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.8 Mineral oil 10.0 Dimethicone 3.0 Sorbitan stearate 0.5 Pro-type glycerin monostearate 1.0 Cetearyl alcohol 2.0 Glyceryl stearate /
FAGE-400 stearate 1.0 Propylene glycol 3.0 Carboxy polymer 0.25 Triethanolamine 0.25 Phenoxyethanol Suitable amount Purified water To 100

Test Example 9: Confirmation of formulation stability

The formulations prepared in Formulation Examples 1 and 2 were kept in a room temperature (25 ° C.), refrigerated (4 ° C.) and constant temperature (50 ° C.), kept in an opaque glass container for 12 weeks in refrigerators and incubators Observed (discolored, detached and separated), and the stability was confirmed. The results are shown in Table 11.

Temperature condition Stability verification (discoloration, removal and separation) Formulation Example 1 Formulation Example 2 Room temperature (25 캜) 0 0 Refrigerated (4 ℃) 0 0 Constant temperature (50 ° C) 0 0

<Formulation stability level>

0: No change 1: Minute change 2: Change 3: Extreme change

As shown in the above table, all of Formulation Examples 1 and 2 were found to be stable without deterioration and separation phenomenon under the temperature conditions of 25 캜, 4 캜 and 50 캜.

Claims (5)

(A) at least one selected from the group consisting of water, anhydrous or hydroalcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, dipropylene glycol and butylene glycol Extracting with an extraction solvent;
(B) preparing an enzyme-treated extract of lysozyme and horse radish by adding a hydrolytic enzyme to the solvent extract prepared in the step And
(C) adding a 5 to 10-fold volume of a shell dissolving solution to the enzyme-treated extract of the lycopersicum rhizome (Lycopersicon esculentum), and heating the mixture to 80 to 100 캜 for extraction. The method according to claim 1, wherein the step (A) is carried out at a weight ratio of 1: 15 to 15: 1. The method according to claim 1, wherein the hydrolytic enzyme in step (B) is selected from the group consisting of beta-glucanase. The method according to claim 1, wherein in the step (C), the shell is baked at 1000 to 1500 ° C for 10 to 30 minutes, and the crushed shell powder is heated at 100 to 120 ° C for 5 to 30 minutes in purified water And filtering the resultant mixture. A cosmetic composition for improving and whitening wrinkles containing 0.1 to 30% by weight of the combined extract of Lycopersicon esculenta and Lycopersicum prepared according to any one of claims 1 to 4, based on the total weight of the cosmetic composition.