KR100992042B1 - preparation for cosmetic composition containg ginseng extract stabilized by nanoliposome - Google Patents

Abstract

The present invention comprises the steps of extracting wild ginseng by processing for 30 minutes at a pressure of 1000MPa, temperature 70 ℃ using an ultra high pressure processor; The present invention relates to a method for preparing a cosmetic composition containing a ginseng extract stabilized with nanoliposomes, comprising stabilizing the extracted ginseng extract with nanoliposomes, which are substances causing blemishes, freckles, and skin pigmentation. Excellent anti-wrinkle effect such as whitening effect such as tyrosinase activity inhibiting melanin prevention, melanin synthesis inhibitory effect, free radical scavenging effect, collagenase (MMP-1) activity and expression suppression It has excellent stability without discoloration and separation and precipitation.

Nanoliposomes, melanin, wild ginseng, wrinkles, antioxidants, cosmetics

Description

Preparation method of cosmetic composition containing wild ginseng extract stabilized with nanoliposomes {preparation for cosmetic composition containg ginseng extract stabilized by nanoliposome}

The present invention relates to a method for preparing a cosmetic composition containing a wild ginseng extract stabilized with nanoliposomes, and more particularly, to a method for preparing a cosmetic composition containing a stabilized wild ginseng extract by containing nanoliposomes in a wild ginseng extract.

The factors that determine skin color are basically partial or total pigmentation such as blemishes, freckles and tanning due to freckle and UV exposure, and the distribution of acne and scars, keratin distribution, blood circulation, Stress, health status.

Among the above factors, the most important factor for determining skin color is pigmentation. The pigments that affect skin color include melanin, carotene, and hemoglobin, the most important of which is melanin. The most important factors affecting melanin biosynthesis are ultraviolet rays and hormones in the body. The biosynthesis of melanin is brown and black through a series of oxidation processes, beginning with the conversion of tyrosine, an amino acid, from the melanoma of melanocytes to tyrosinase and dihydroxyphenylalanine. (eumelanin) polymer. This biosynthesis process is carried out in a special type of brown intracellular organelles called melanosomes. Melanosomes, including melanin granules, move from the periphery of the nucleus to the dendritic end, and then into the cytoplasm by the phagocytosis of keratinocytes. Accumulate around the nucleus of the site. The synthesis of melanin, the number of melanosomes, and the movement to surrounding keratinocytes are partially affected by hormones and ultraviolet rays, and primarily by genetic influences.

In addition, intracellular regulators such as cytokines, copper, zinc and iron, interferon, prostaglandin and histamine, which are involved in the expression of tyrosinase, synthesis and transfer of melanin, are known to be involved. Society of Cosmetic Scientists, 25: 77-127, 1999). Therefore, the skin whitening effect can be expected by inhibiting the synthesis of melanin, which determines the skin color, and the activity of tyrosinase, an enzyme involved therein.

Conventionally, ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione, or the like in order to treat or alleviate excessive melanin pigmentation caused by abnormal symptoms of skin pigmentation such as blemishes, freckles, pigmentation, and UV exposure. These derivatives and substances having tyrosinase inhibitory activity have been used in cosmetics and pharmaceuticals.

However, they are very unstable and difficult to deliver to the skin, requiring a special stabilization system and delivery system, and their use is limited due to insufficient whitening effects, safety issues with the skin, formulation and stability problems when formulated in cosmetics, etc. It is becoming.

Meanwhile, in addition to these existing whitening efficacy substances, research is being conducted to find whitening active ingredients among natural products. Among them, baekbaekpi (Japanese Patent Application No. 55-44375, cow 64-26507, cow ( -) 64-83009, 1-1-25687, 5-5-139950, Korean Laid-Open Patent Nos. 1992-002109, 1997-021273), Licorice (Japanese Laid-Open Patent Publication 60-214721) , Bovine 63-23809, bovine 64-63506, pyeong 1-49706, Korean Laid-Open Patent No. 1992-002109, 1997-025601) and many other plant extracts act on tyrosinase. Although it has been found that it inhibits melanin production, they also have many problems in use above the effective concentration in cosmetics or pharmaceuticals in terms of safety, stability, discoloration, etc. and do not produce satisfactory effects.

Next, factors affecting the skin include the generation of wrinkles and the decrease in elasticity due to aging of the skin. Human skin is constantly changing with age and skin aging. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletories, 111: 31-37, 1996). While natural aging is influenced by genetic factors, it is difficult to control artificially. External aging is relatively easy due to environmental factors. Representative external aging factors include ultraviolet rays, and the most prominent external aging phenomenon is wrinkle formation (HW Daniel, Ann Intern Med, 75 (6): 873-880, 1971; GL Grove, et al., J Am) Acad Dermatol, 21: 631-637, 1989; CE Griffiths, et al., Arch Dermatol, 128: 347-351, 1992).

One of the photoaging mechanisms caused by ultraviolet light is via free radical pathways (D Harman, J Gerontol, 11 (3): 298-301, 1956). Free radicals are known to cause breakdown of connective tissue formation, such as cutaneous collagen, cell membrane dysfunction, DNA mutation, protein action, intercellular energy transfer, and metabolism-related molecules (AF Kligman and RM Lavker, J Cutan Aging Cosmet Dermatol, 1: 5-12, 1988). The fact that free radicals are involved in aging means that antioxidants or various chemical scavengers that inactivate the free radicals can be used to delay the aging process.

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but its synthesis decreases with aging and matrix metalloproteinase (MMP), an enzyme that breaks down collagen, especially Expression of collagenase (hereinafter referred to as 'MMP-1') and gelatinase (gelatinase (hereinafter referred to as 'MMP-2')) is promoted, thereby reducing the elasticity of the skin and forming wrinkles. Ultraviolet radiation and free radicals also activate these degrading enzymes.

MMP is classified into MMP-1, MMP-2, and MMP-9 according to its substrate specificity. MMP-1 is a fibroblast type collagenase, an enzyme of epilepsy, and collagen type I, II, III, and 기질 as substrates. There are, Ⅷ, Ⅹ and gelatin, cut into 3/4 or 1/4 length of the original collagen length peptide to facilitate the action of non-specific enzyme (gelatinase). MMP-2 belongs to 72 KD of gelatinase (A) and degrades substrates such as gelatin, collagen types IV, V, VIII, VIII, elastin and fibronectin. MMPs of neutrophils and polymorphonuclear leukocytes are primarily activated by oxidation, which first generates hydrogen peroxide in the cell and is converted to hypochlorous acid by myeloperoxidase in the presence of chlorine ions. Activate it.

In order to prevent aging due to ultraviolet rays, the most common method is to apply a product containing sunscreen directly to the skin.However, in 1988, retinoic acid was reported to be effective in reducing skin roughness and fine wrinkles of aged skin (KS Weiss). , et al., JAMA, 259: 527-532, 1988), research is being actively conducted to develop substances that suppress or improve skin aging worldwide. Among them, vitamins A, C, E, derivatives of vitamins and AHA (alpha-hydroxy acid) are representative substances for improving skin aging known to date (Hermitte, Cosmetics & Toiletries, 107: 63-67, 1992; DR Rosenthal, et al., J Invest Dermatol, 95: 510-515, 1990; TD Ditre, et al., J Invest Dermatol, 34: 187-195, 1996). However, they are very unstable in the natural environment, so there is a problem in use, or somewhat insufficient to show a visible effect.

Therefore, the present inventors have tried to find a substance related to skin whitening and skin aging inhibition in natural products, and came to pay attention to wild ginseng.

Ginseng (Panax ginseng) is a perennial herb belonging to the Araliaceae family, and its root is called Ginseng Radix in oriental medicine, and the tonic, Gangjeong, hematopoietic, warmth, Health, health, fatigue, mental stability, sedative effects are known to be effective. The main active ingredients of ginseng include saponins, sapongenins, polyacetylenes, pyrazine derivatives, maltol, and the like, and ginseng saponins are based on the chemical structure of protopanaxadiol and protopanaxatriol. It is divided into system and oleanonane-based saponins. To date, 19, 10 and 1 compounds have been separated and purified, and diol and triol are known to have different pharmacological effects in the body. Ginseng saponins, unlike other plant saponins, are not toxic and do not show hemolytic action below 0.001%. They are reported to have central nervous system suppression, protein synthesis promotion, adrenal cortex hormone secretion, and immune boosting. It is becoming. On the other hand, wild ginseng is a ginseng naturally occurring in the mountains, indications and utilities are similar to ginseng, but its medicinal effects are excellent, and representative effects can be said to maintain homeostasis of body control functions. Anti-fatigue and antistress action, blood pressure control action, anticancer action, arteriosclerosis and hypertension prevention, gastrointestinal function enhancement, immune function enhancement, antiviral action and the like have been reported based on this action.

On the other hand, the raw materials used up to now as a material of cosmetics was mostly to simply inhibit the enzyme activity, there was a problem that can not control the amount of expression of the enzyme. In addition, consumers are increasingly demanding the use of cosmetics with minimal functional side effects such as skin protection, irritation suppression, inflammation suppression, and sun protection while having high quality functional properties according to special composition, and to minimize skin irritation As consumers' response to cosmetics using natural materials instead of using chemical raw materials is increasing, there is a need to develop natural materials as raw materials for cosmetics.

Accordingly, an object of the present invention is to provide a method for preparing a cosmetic composition containing a wild ginseng extract stabilized with nanoliposomes exhibiting a whitening effect, antioxidant effect, anti-wrinkle effect, and collagen synthesis effect.

In order to achieve the above object, the present invention comprises the steps of extracting wild ginseng by treatment for 30 minutes at pressure 1000MPa, temperature 70 ℃ using an ultra-high pressure treatment machine; It provides a method for producing a cosmetic composition containing the wild ginseng extract stabilized with nano-liposomes comprising the step of stabilizing the extracted wild ginseng extract with nano-liposomes.

Hereinafter, the problem solving means of the present invention will be described in detail.

The present invention provides a method for preparing a cosmetic composition containing a stabilized wild ginseng extract by treating the wild ginseng at pressure 1000MPa, temperature 70 ° C. for 30 minutes, and then mixing nanoliposomes with the extracted wild ginseng extract.

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Meanwhile, the wild ginseng used in the present invention is preferably subjected to a pretreatment process for 5 to 60 minutes with steam or water at 60 to 90 ° C.

On the other hand, in the present invention, the extraction of wild ginseng is not limited to a specific pH, but it is preferable that the pH of the water inside the ultra-high pressure during the ultra-high pressure treatment is reduced to 1-6.

On the other hand, the extraction solvent used in the present invention is not limited to a specific kind, but preferably selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, hexane and mixtures thereof good.

Meanwhile, the wild ginseng extract used in the present invention includes a concentrate obtained by partially or fully concentrating the extract, or an extract extracted by drying the extract and a chemical substance having a main effect contained in the extract. do.

Nanoliposomes are in the form of conventional liposomes, which have the bipolar nature of phospholipid molecules, hydrophobic moieties promote association with hydrocarbon chains, and polar hair groups promote association with hydrophilic components. It serves to stabilize the hydrophilic components. The average particle diameter of the nanoliposomes is 1-100 nm, which improves skin penetration and improves formulation stability.

On the other hand, wild ginseng extract is not limited to a specific range, but preferably contains 0.01 to 60% by weight relative to the total weight of the nanoliposomes, but when added to less than 0.01% by weight, the effect is difficult to expect, 60 When added in excess of% by weight, no marked increase in efficacy effect is seen.

On the other hand, the addition amount of the wild ginseng extract stabilized with nanoliposomes is not limited to a specific range, but preferably contains 0.1 to 30% by weight relative to the total weight of the cosmetic composition, if less than 0.1% by weight It is hard to expect an effective effect, and when it exceeds 30% by weight, no marked increase in efficacy is seen. In addition, the cosmetic composition according to the present invention may contain, in addition to the above-mentioned extract, other components which may preferably give a synergistic effect to the main effect within the range of not impairing the main effect of the present invention.

On the other hand, nanoliposomes are not limited to those obtained through a specific process, but preferably those obtained by passing a liposome comprising phospholipid, glutathione, 95% modified ethanol, glycerin, polysorbate 20 through a high pressure emulsifier good.

Meanwhile, the cosmetic composition containing the wild ginseng extract stabilized with the nanoliposomes of the present invention may be used as an example for skin whitening, skin wrinkle improvement, and skin elasticity.

On the other hand, the formulation of the cosmetic composition containing the wild ginseng extract stabilized with nanoliposomes of the present invention is not limited to a specific kind, for example, lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type And cosmetics consisting of water-in-oil (W / O) type, oil-in-water and water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencil It may have any one appearance selected from the color cosmetic formulation consisting of.

It may also be applied to the skin, optionally in the form of an aerosol, or may be in the form of a solid such as a stick. It may be used as a skin care product and / or makeup product.

The compositions according to the invention may also contain conventional auxiliaries such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants and dyes in the cosmetic field. Can be. The amounts of these various adjuvants are amounts conventionally used in the art, such as from 0.01 to 20% by weight relative to the total weight of the composition. In any case, the adjuvant and its proportions will be chosen so as not to adversely affect the desirable properties of the cosmetic composition according to the invention.

As described above, the cosmetic composition of the present invention containing a wild ginseng extract stabilized by mixing nanoliposomes with a wild ginseng extract has an inhibitory effect on tyrosinase activity, which inhibits melanin production, a substance causing blemishes, freckles and skin pigmentation, and melanin. Excellent whitening effect such as synthetic inhibitory effect, free radical scavenging effect, wrinkle improvement effect such as activity and expression inhibition of collagenase (MMP-1), and excellent stability without discoloration, separation and precipitation in response to temperature change Has

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes modifications of equivalent technical spirit.

Example 1 Preparation of Wild Ginseng Extract

After picking and washing wild ginseng as a sample, using a ultra high pressure processor, the temperature was raised to 70 ° C. under a pressure of 1000 MPa, treated for 30 minutes, cooled to room temperature, and dried in a dryer to a moisture content of 12 to 13% to obtain wild ginseng. After that, the obtained wild ginseng was dispersed and dissolved in purified water to prepare a wild ginseng extract, and then filtered to obtain an extract. (Example 1).

< Experimental Example  1> Determination of active ingredient content of wild ginseng

The control was prepared by screening and washing the same wild ginseng as Example 1, raising the temperature to 70 ° C. for 3 hours using steam, stopping the steam, cooling to room temperature, and drying it to a moisture content of about 12 to 13%. In Comparative Example 1, four-year-old ginseng was used.

The total saponin content and ginsenoside content of each sample were analyzed and the results are shown in Table 1 below.

Example 1 Control Comparative Example 1 Crude saponin 77.8 65.3 22.1 Ginsenoside
(Ginsenoside)
Rg1 0.014 0.0124 0.215 Rg2 0.234 0.2092 0.204 Rg3 1.532 1.2855 0.074 Re 0.012 0.0117 0.210 Rb1 0.076 0.0703 0.128 Rb2 0.275 0.2228 0.081 Rb3 0.081 0.077 0.098 Rc 0.077 0.0765 0.137 Rd 0.271 0.265 0.086 Rh1 0.451 0.435 - Rh2 0.136 0.123 -

As a result of measuring the active ingredient content of wild ginseng, it was confirmed that the content of the irradiated ponin and ginsenoside of Example 1 is high.

From the above results, it was confirmed that the wild ginseng extract extracted under the extraction conditions of the present invention had an increased content of irradiated saponin and ginsenosides.

<Example 2> Preparation of nanoliposomes stabilized wild ginseng extract

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The nanoliposomes were emulsified under high pressure with phospholipid, glutathione, 95% modified ethanol, glycerin, polysorbate 20 (phase A) to prepare a closed bilayer nano liposome.

More specifically, the phase A of Table 2 was heated to 95 ° C., uniformly mixed and dissolved, and then cooled to 40 ° C., followed by mixing with phase B. This was passed through a high pressure emulsifier (Microfluidizer M210EH, USA) five times under conditions of 1200 bar and a flow rate of 500 m / s to obtain a stabilized nanoliposome.

The size of the liposome obtained through one pass was about 125 nm, but the size of the fifth pass was about 70 nm.

Raw material name content A phase Phospholipids 5 Glutathione 0.1 95% denatured ethanol 20 glycerin 30 Polysorbate 20 5 B phase Wild ginseng extract 20 Purified water 19.9 Sum 100

< Experimental Example  2> Measurement of the whitening effect

In order to confirm the whitening effect of the wild ginseng extract stabilized with the nanoliposomes obtained in Example 2, the degree of inhibition of an enzyme called tyrosinase was measured.

Tyrosinase is an enzyme that accelerates the oxidation of tyrosine in vivo and helps melanin production. In Experimental Example 2, a method of inhibiting the function of this enzyme to inhibit the oxidation of tyrosine to form a black polymer called melanin (SH Pomerantz, J Biol chem, 241: 161-168, 1966) was applied. The whitening effect was determined.

The inhibitory effect of tyrosinase activity was investigated using kojic acid, arbutin, lettuce extract and wild ginseng extract prepared in Example 2 as samples. The inhibitory activity against tyrosinase of each sample was determined by placing 15 μl of the sample into a 96-well plate incubator, 150 μl of 50 mM phosphate buffer (pH 6.5) and 25 μl of 1.5 mM L-tyrosine solution, followed by mushroom tyrosinase. 10 μl (1,500 units / ml, Sigma) was added and reacted at 37 ° C. for 20 minutes, and then the absorbance was measured at 490 nm using an absorbance photometer (microplate reader, ELx800, USA) to determine the activity inhibition rate against tyrosinase.

IC ₅₀ The value is the concentration of the substance that inhibits tyrosinase enzyme activity by 50%.

Sample Name Tyrosinase activity inhibitory effect (IC ₅₀ ) Example 2 0.01% Kojisan 0.03% Arbutin 0.45% Lettuce extract 0.65%

IC ₅₀ of wild ginseng extract was tested for inhibitory effect of tyrosinase activity The value was 0.01%, which was higher than that of kojic acid, arbutin and lettuce extract, which were known to be excellent in tyrosinase inhibitory effect.

From the above results, the excellent whitening effect of the cosmetic composition containing the wild ginseng extract stabilized with the nanoliposomes of the present invention could be inferred.

< Experimental Example  3> B16F1 Melanosite  Intracellular Use Tyrosinase  Determination of enzyme activity inhibitory effect

In order to confirm the whitening effect of the wild ginseng extract stabilized with the nanoliposomes obtained in Example 2, the degree of inhibition of tyrosinase enzyme activity in B16F1 melanocytes was determined by measuring intracellular tyrosinase enzyme activity.

B16F1 melanocytes used in Experimental Example 3 are cell strains derived from mice and are cells that synthesize an enzyme called tyrosinase. The degree of inhibition of tyrosinase enzyme activity was evaluated by treating the sample during artificial culture of these cells and by separating the tyrosinase from the cells and measuring the enzyme activity. The B16F1 melanocytes used in this example were used after being sold from ATCC (American Type Culture Collection, Accession No .: 6323). The tyrosinase enzyme activity inhibitory effect of B16F1 melanocytes was measured as follows.

B16F1 melanocytes were dispensed at a concentration of 2 X 10 ⁶ per well in a 6-hole plate incubator, and the cells were attached and incubated for 72 hours by treating the sample at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and then the number of cells was measured and centrifuged to recover the cells. Wash the cell pellet once with PBS, add 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF), vortex for 5 minutes, disrupt the cells, and centrifuge (3,000 rpm, 10 minutes) to recover the supernatant. Put 1.5mM L-tyrosine, 0.06mM L-dopa, and cell supernatant into 50mM sodium phosphate buffer (pH 6.5), and incubate at 37 ℃ for 30 minutes and measure the absorbance at 490nm with an absorbance photometer to inhibit tyrosinase enzyme activity. Was measured.

IC ₅₀ value is the concentration of a substance that inhibits the activity of intracellular tyrosinase enzyme by 50%.

Sample Name Inhibitory effect of tyrosinase activity (IC ₅₀ ) Example 2 0.02% Kojisan 0.04% Arbutin 0.25% Lettuce extract 0.70% Soluble Licorice Extract 0.03%

As a result of measuring the inhibitory effect of tyrosinase enzyme activity of B16F1 melanocytes, IC ₅₀ value of wild ginseng extract was 0.02%, and the activity of tyrosinase was lowered at lower concentrations than koji acid, arbutin, oil-soluble licorice extract, and lettuce extract. It was determined to inhibit effectively.

As a result, it was possible to deduce the excellent whitening effect of the cosmetic composition containing the wild ginseng extract stabilized with the nanoliposomes of the present invention.

< Experimental Example  4> B16F1 Melanosite  Measurement of melanin production inhibitory effect

In order to confirm the whitening effect of the wild ginseng extract stabilized with the nanoliposomes obtained in Example 2, the whitening effect was determined according to the degree of inhibition of melanin production for B16F1 melanocytes.

The B16F1 melanocytes used in the four examples of this experiment are cell strains derived from mice, and cells that secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to evaluate the degree of melanin black pigment reduction. The B16F1 melanosite used in this example was used after being sold from ATCC (American Type Culture Collection, Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed at a concentration of 2 X 10 ⁶ per well in a 6-hole plate incubator, and the cells were attached and incubated for 72 hours by treating the sample at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Quantification of intracellular melanin was performed by modifying the method of Rotan (R. Lotan and D. Lotan, Cancer Res, 40: 3345-3350, 1980). The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with an absorbance spectrometer. Melanin production inhibition rate (%) was measured.

IC ₅₀ The value is the concentration of the substance that inhibits melanin production by 50%.

Sample Name Melanin synthesis inhibitory effect (IC ₅₀ ) Example 2 0.004% Hydroquinone 0.02% Arbutin 0.5% Lettuce extract 4.0% Soluble Licorice Extract 0.03%

As a result of testing the melanin production inhibitory effect of B16F1 melanocytes, the IC ₅₀ value of the wild ginseng extract was measured to be 0.004%, showing melanin synthesis inhibitory effect better than the existing whitening agents hydroquinone, arbutin, oil-soluble licorice extract, and lettuce extract.

From the above results, the excellent whitening effect of the cosmetic composition containing the wild ginseng extract stabilized with the nanoliposomes of the present invention could be inferred.

< Experimental Example  5> Antioxidant effect measurement

The active oxygen radical scavenging effect was measured to confirm the antioxidant effect of the wild ginseng extract stabilized with the nanoliposomes obtained in Example 2.

The reactive oxygen radical scavenging activity was generated by the xanthine-xanthine oxidase system of each sample according to the method of Furuno et al., Biol Pharm Bull, 25 (1): 19-23, 2002). The effect of scavenging free radicals was measured. ₃ mM xanthine, 3 mM EDTA, 0.72 mM nitroblue tetrazolium (NBT), 0.15% BSA (bovine serum albumin) solution and sample were added to 0.05 M Na ₂ CO ₃ buffer and mixed at 25 ° C. The reaction was carried out for 10 minutes. Then, xanthine oxidase (0.25 U / ml) solution was added to each test tube, and it reacted at 25 degreeC for 30 minutes, and the absorbance was measured at 565 nm.

Sample Name density(%) Antioxidative effect(%) Example 2 0.001 37 0.005 81 0.01 95 Green tea extract 0.5 98 Vitamin E 0.5 91

As a result of measuring the antioxidant effect, Example 2 shows that the antioxidant effect (%) compared to the concentration of green tea extract and vitamin E, a representative antioxidant, showed an excellent antioxidant effect.

From the above results, it was possible to deduce the excellent antioxidant effect of the cosmetic composition containing wild ginseng extract stabilized with the nanoliposomes of the present invention.

< Experimental Example  6> Collagen synthesis effect measurement

In order to observe the collagen synthesis effect of the wild ginseng extract stabilized with the nanoliposomes obtained in Example 2, experiments were performed directly on human fibroblasts obtained from humans or purchased commercially. The control group was taken as the reaction absorbance of the cell culture without the Example.

DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% fetal bovine serum and human fibroblasts (5 X 10 ³ cells / well) were put into a 96-hole plate incubator and cultured until 70-80% growth. Next, the ginseng extract was treated with 0.01% and 0.001%, respectively, and the cell culture solution was collected for 1 day. The collagen synthesis amount was measured using a collagen protein measuring instrument (Catalog No .: MK 101, Takara Shuzo Co. Ltd, Japan) Measured. In the measurement method, first, the cell culture solution collected in a 96-hole plate incubator uniformly coated with primary collagen antibody was subjected to antigen-antibody reaction for 3 hours. After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-hole plate incubator and allowed to react for 15 minutes. After 15 minutes, the color developing agent was added to develop color at room temperature, and 1M sulfuric acid was added to stop the color development. The color of the reaction color is yellow, and the intensity of the reaction varies depending on the degree of reaction.

A yellowish 96-hole plate incubator was measured at 450 nm using an absorbance spectrometer.

Sample Name Collagen Synthesis Effect (%) Example 2 0.01% 90 0.001% 65 Control 0

As a result of measuring the collagen synthesis effect, Example 2 showed the excellent collagen production | stimulation effect.

From the above results, it was possible to infer an excellent collagen production promoting effect of the cosmetic composition containing the wild ginseng extract stabilized with the nanoliposomes of the present invention.

< Experimental Example  7> MMP -1 activity inhibition effect

The inhibitory effect of the substrate metalloproteinase (MMP-1) activity of the wild ginseng extract stabilized with the nanoliposome prepared in Example 2 was measured by Wang et al. (Y. Wang, et al., J Biol. Chem., 274: 33043-). 33049, 1999). That is, 20 µl of DQ-collagen and 40 µl of sample dissolved in the reaction buffer at 0.25 mg / ml were added to 100 µl of reaction buffer, and 40 µl of collagenase diluted to 0.5 unit was added. After 20 minutes at room temperature, the fluorescence value was measured using a fluorescence spectrophotometer (LS55, PERKIN ELMER, USA) at an absorption wavelength of 495 nm and an emission wavelength of 515 nm. The fluorescence value was measured. The fluorescence value of the sample itself was also measured and corrected in enzymatic activity calculation. As a positive control, 1,10-phenanthroline (phenanthroline), which is known to have an inhibitory effect on MMP-1 activity, was used.

Sample Name MMP-1 activity inhibitory effect (%) Example 2 0.001% 33 0.005% 70 0.01% 91 Positive control (2 mM, 10-phenanthroline) 73

As a result of measuring the inhibitory effect of MMP-1, Example 2 was found to inhibit collagenase activity in a concentration-dependent manner. The 1,10-phenanthroline used as a positive control showed 73% activity inhibition at 2 mM.

As a result, it was possible to deduce the excellent collagenase activity inhibitory effect of the cosmetic composition containing wild ginseng extract stabilized with the nanoliposomes of the present invention.

< Experimental Example  8> After UV irradiation MMP -1 expression inhibition measurement

Enzyme Linked ImmunoSorbent Assay (ELISA) was performed to measure the expression level of MMP-1 after UV irradiation and sample addition of wild ginseng extract stabilized with nanoliposomes obtained in Example 2 (SE Dunsmore, et al., J Biol Chem., 271: 24576-24582, 1996).

Human fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber. UV irradiation dose and culture time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. Negative controls were wrapped in tinfoil and maintained at the same time in the environment of UVA. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact with the previously dispensed medium, and were irradiated with UVA, exchanged with the medium containing the sample, and then cultured for 24 hours, and the medium was recovered and coated in a 96-hole plate incubator. The primary antibody (monoclonal antibody) was treated and reacted at 37 ° C. for 90 minutes. The secondary antibody, anti-mouse immunoglobulin (alkaline phosphatase conjugated anti-mouse IgG) was reacted for another 90 minutes, washed with a buffer solution, and then washed with an alkaline phosphatase substrate solution (1 mg / ml p-nitrophenyl phosphate in diethanolamine buffer). ) Was reacted at room temperature for 30 minutes and absorbance was measured at 405 nm using an absorbance spectrometer.

At this time, the reaction absorbance of the cell culture solution not treated with Example 2 was used as a control.

Sample Name % Inhibition of MMP-1 expression Example 2 0.001% 30 0.005% 64 0.01% 90 Retinol (3.5 μM) 52

Inhibition of MMP-1 expression after UV irradiation As a result, Example 2 showed the effect of inhibiting MMP-1 expression in a concentration-dependent manner. Retinoic acid used as a positive control showed a 52% expression inhibition effect at 3.5uM.

From the above results, it could be inferred from the excellent MMP-1 expression inhibitory effect of the cosmetic composition containing wild ginseng extract stabilized with the nanoliposomes of the present invention.

< Example  3> Cosmetics  Produce

A cosmetic containing a wild ginseng extract stabilized with the nanoliposomes obtained in Example 2 was prepared.

The components of the following cosmetics were as shown in Table 10, and an emulsion was prepared according to a conventional cosmetic preparation method (Example 3, Example 4, Example 5, and Comparative Example 2).

Raw material (% by weight) Example 3 Example 4 Example 5 Comparative Example 2 Polysorbate-60 1.5 1.5 1.5 1.5 Sorbitan stearaytan 0.5 0.5 0.5 0.5 Stearic acid One One One One Squalane 5 5 5 5 Cetostearyl alcohol 2 2 2 2 Cetyl octanoate 5 5 5 5 Carbomer 0.15 0.15 0.15 0.15 Triethanolamine 0.2 0.2 0.2 0.2 glycerin 5 5 5 5 Wild ginseng extract 10 One 0.1 - Purified water 69.65 78.65 79.55 79.65 Total (% by weight) 100 100 100 100

< Experimental Example  9> Measure whitening effect

The whitening effect of Example 3 and Comparative Example 2 prepared above was measured.

20 experimenters (20-35 year old female) were applied to Example 3 on the right side of the face and Comparative Example 2 on the left side of the face, twice a day for 2 consecutive months. After completion of the test, the skins of the application areas on both sides of the face were compared by using an image analyzer and a color difference meter, and the darkest color was set to 5, the middle color to 3, and the brightest color to 1, and the intermediate level was estimated. (n = 20, p <0.01)

Objective evaluation of the skilled person Subjective Evaluation of the Subject Example 3 Comparative Example 2 Example 3 Comparative Example 2 medium 1.9 2.85 1.65 2.7

As a result of measuring the whitening effect of the cosmetics, the whitening effect was excellent in the facial skin of the experimenter applied to Example 3.

From the above results, it was confirmed that the excellent whitening effect of the cosmetic composition containing the wild ginseng extract stabilized with the nanoliposomes of the present invention.

< Experimental Example  10> Measure the improvement effect of skin wrinkles

Example 4 and Comparative Example 2 were applied to humans to evaluate the effect of improving skin wrinkles.

Twenty test subjects (20-35 year old female) were applied to Example 2 on the right side of the face and Comparative Example 2 on the left side of the face, twice a day for 2 consecutive months.

After the experiment was completed, wrinkles around the face of the eyes were collected with a silicone resin copy (replica) before and after using the product for two months, and the skin fine wrinkle device and the skin image analyzer were used to compare the condition of the eye wrinkles.

Example 4 Comparative Example 2 Before use after use Before use after use Average fine wrinkle depth (AU) 0.198 0.148 0.199 0.194 Wrinkle Reduction (%) 25.25% 2.5%

As a result of measuring the improvement effect of the skin wrinkles of the cosmetics, Example 4 was found to have a higher wrinkle reduction rate compared to Comparative Example 2.

From the above results, it was confirmed that the excellent skin wrinkle improvement effect of the cosmetic composition containing the wild ginseng extract stabilized with the nanoliposomes of the present invention.

< Experimental Example  11> Stability Measurement

The stability of the formulation for the cosmetic containing the wild ginseng extract stabilized with the nanoliposomes of the present invention was measured by the following method.

Example 3 and Comparative Example 3 (instead of using the wild ginseng extract stabilized with the nanoliposomes of Example 2, except that 10% by weight of the wild ginseng extract of Example 1 (ie, the wild ginseng extract not stabilized with the nanoliposomes) was added Then, the emulsion prepared in the same manner as in Example 3) was stored in an opaque glass container in a constant temperature bath at 45 ℃ constant stored for 12 weeks, opaque in a completely shaded refrigerator kept at 4 ℃ constant For samples stored in a vial container for 12 weeks and samples stored for 12 weeks in a circulation chamber (once / day) circulating at -5 ° C to 45 ° C, separation, degree of discoloration and precipitation were measured. The degree of separation and discoloration of the product was classified into six grades (0: no change, 1: very slight separation (discoloration), 2: slight separation (discoloration), 3: slight separation (discoloration), 4: severely Separation (discoloration), 5: Extremely severe separation (discoloration).

Temperature Example 3 Comparative Example 3 45 ℃ 0 3 4 ℃ 0 2 cycle 0 3

As a result of measuring the degree of separation and discoloration of the cosmetic emulsion, Example 3 stabilized with nanoliposomes was stable without discoloration or separation symptoms at 4 ° C., 45 ° C. and circulation test, but Comparative Example 3 containing no nano liposome was isolated. Discoloration occurred, indicating instability.

From the above results it was confirmed that the excellent stability of the cosmetic containing the wild ginseng extract stabilized with the nanoliposomes of the present invention.

In addition, the degree of product precipitation was evaluated by classifying into the following six grades (0: no change, 1: very little precipitation, 2: little precipitation, 3: little precipitation, 4: severe precipitation, 5: extremely severe precipitation).

Temperature Example 3 Comparative Example 3 45 ℃ 0 3 4 ℃ 0 2 cycle 0 2

As a result of measuring the degree of precipitation of the cosmetic emulsion, Example 3 stabilized with nanoliposomes did not occur precipitation, Comparative Example 3 did not contain nanoliposomes precipitated.

From the above results, it was confirmed that the addition of nanoliposomes to wild ginseng extract easily reacts with other weak ionic polymers in the formulation, causing precipitation and discoloration, and contains the wild ginseng extract stabilized with the nanoliposomes of the present invention. The stability of the cosmetic composition was also confirmed.

Claims (9)

Processing wild ginseng using an ultra-high pressure processor at a pressure of 1000 MPa and a temperature of 70 ° C. for 30 minutes and cooling to room temperature, followed by drying to obtain a water content of 12 to 13 wt%; Preparing wild ginseng extract by extracting the dried wild ginseng with purified water; The wild ginseng extract is mixed in a liposome containing phospholipid, glutathione, 95% modified ethanol, glycerin and polysorbate 20 to be contained in an amount of 0.01 to 60% by weight based on the total weight of the nanoliposome, and then passed through a high pressure emulsifier to a nanoliposome. Stabilizing; And Method of producing a cosmetic composition for skin whitening comprising a; containing 0.1 ~ 30% by weight relative to the total weight of the cosmetic composition of the wild ginseng extract stabilized with the nanoliposomes. delete delete delete delete delete delete delete delete