KR100758960B1 - Cosmetic composition containing diospyros kaki fruit extract stabilized with nanoliposome - Google Patents

Abstract

A cosmetic composition containing Diospyros kaki fruit(persimmon) extract stabilized with nanoliposome is provided to whiten the skin by inhibiting tyrosinase activity, melanin synthesis and collagenase(MMP-1; matrix metalloproteinase-1) activity, reduce the skin irritation, and inhibit discoloration and separation of Diospyros kaki fruit(persimmon) extract by stabilizing with nanoliposome. A cosmetic composition for skin whitening contains 0.1-30.0 wt.%, preferably 0.01-60 wt.% of a Diospyros kaki fruit(persimmon) extract stabilized with nanoliposome which is prepared by passing the liposome containing phospholipid, glutathione, 95% modified ethanol, glycerin and polysorbate 20 through a high pressure emulsifying device, and is formulated as lotion, gel, water-soluble liquid, cream, essence, make up base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color or eyebrow pencil.

Description

Cosmetic composition containing Diospyros kaki fruit extract stabilized with nanoliposome

The present invention relates to a cosmetic composition containing a persimmon extract stabilized with nano-liposomes, more specifically, a whitening effect, wrinkle improvement effect, and stimulation-releasing effect containing 0.1 to 30.0% by weight of persimmon extract stabilized with nanoliposomes It relates to a cosmetic composition.

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 melanocytes 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.

That can only been known to be involved synthesis of expression and melanin of tyrosinase, in regulators of cytokine (cytokine), copper, zinc, metal ions and interferon, prostaglandins, histamine, etc., such as iron cells which participate in the transmission (baksunam, for 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 due to insufficient whitening effects, safety problems for the skin, formulation and stability when formulated in cosmetics, etc. It is limited.

On the other hand, in addition to these existing whitening efficacy substances, research is continuously conducted to find whitening active ingredients among natural products, among which is Sangbaekpi (Japanese Patent Publication No. 55-44375, Bovine 64-26507, Bovine ( -) 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 and discoloration, 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, 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 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 The expression of collagenase (hereinafter referred to as 'MMP-1') and gelatinase (gelatinase (hereinafter referred to as 'MMP-2')) is accelerated to decrease the elasticity of the skin and form 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 inhibit or improve skin aging around the world. 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 became aware of the senses. Persimmon Tree Persimmon Tree Persimmon Tree kaki is the fruit of Thunberg). The persimmon tree is a deciduous arbor tree, about 15m high, the bark is black gray, split and has brown hair on the body. Leaves are regenerated, intact, elliptical ovate, long ovate or obovate, pointed, broad, basal or basal, 7 ~ 17㎝ long, 4 wide ~ 10㎝ with no sawtooth, and leaf disease (葉 ~) is 5 ~ 15㎜ in length with hairs. Flowers bloom in May-June, flowers are yellowish white, bisexual or unisexual, hang on the axillary leaves, with small hairs on the surface of calyx and corolla, 18mm long, 15mm diameter, calyx 10mm long, 12 wide Mm. The fruit matures in October, and the berries ripen yellowish red. Persimmon fruit ( Disopyros kaki ) is an alkaline fruit that is rich in nutrition and contains about 11 to 15% of glucose and fructose, and vitamin C contains 20 to 50 mg, which is about 5 to 12 times the amount of apples per 100 g of edible portion. Of course, it is rich in vitamin A and rich in tannic acid. In particular, tannic acid is known to play a role in lowering blood pressure without altering ECG as well as a pharmacological action that stops diarrhea or improves upset stomach. Kim, Tae-Jung, Resources Plant of Korea, Seoul National University Press, 1996). However, the efficacy of such whitening and inhibiting the aging effect is not unknown, persimmon (Disopyros kaki ) has not been applied to cosmetics.

An object of the present invention is to provide a cosmetic composition containing a persimmon extract stabilized with a nanoliposome, devised to solve the above problems.

It is another object of the present invention to provide a cosmetic composition containing a persimmon extract stabilized with nanoliposomes, characterized in that it contains 0.1 to 30% by weight of the persimmon extract stabilized with nanoliposomes based on the total weight of the cosmetic composition. .

Another object of the present invention is to provide a cosmetic composition containing persimmon extract stabilized with nano liposomes that can be applied to skin cosmetics and inhibits the production of melanin and the activity of enzymes related thereto when contained in cosmetics and exhibits excellent whitening effect. will be.

Another object of the present invention is applicable to skin cosmetics and stabilized with nano-liposomes exhibiting excellent wrinkle improvement effect such as anti-wrinkle effect by acting on the antioxidant effect, collagen synthesis effect, substrate metalloproteinase when contained in cosmetics It is to provide a cosmetic composition containing the extract.

It is another object of the present invention to provide a cosmetic composition containing persimmon extract stabilized with nanoliposomes that are applicable to skin cosmetics and exhibit an irritating effect of reducing skin irritation that may occur with cosmetic bases when contained in cosmetics. .

In order to achieve the above object, the present inventors select persimmon among various natural products, and develop a cosmetic composition containing persimmon extract stabilized with nanoliposomes, thereby increasing the selection range of various cosmetic raw materials, and without skin irritation It provides a method for producing a cosmetic having excellent qualitative and functional effect.

The present invention relates to a cosmetic composition containing a persimmon fruit extract and a cosmetic composition containing a persimmon extract stabilized with nanoliposomes. The persimmon extract used in the present invention is contained in the leachate obtained by leaching and transferring from persimmon fruit, or the concentrate obtained by partially or totally concentrating the leachate, or the extract extract and extract prepared by drying the concentrate. It contains the chemicals that exert their main effects.

Nanoliposomes to stabilize the persimmon extract is preferably obtained by passing a liposome containing phospholipid, glutathione, 95% modified ethanol, glycerin, polysorbate 20 or more times through a high-pressure emulsifier.

In addition, the present invention is characterized in that the persimmon extract stabilized with the nanoliposomes containing 0.1 to 30.0% by weight based on the total weight of the total cosmetic composition. In addition, the cosmetic composition according to the present invention may contain, in addition to the above-mentioned extract, other components that can give a synergistic effect to the main effect, preferably within the range of not impairing the main effect of the present invention.

In addition, the persimmon extract stabilized with the nanoliposome according to the present invention is used as the extraction solvent at least one selected from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane Extracted at 70 to 85 ℃, and the resulting suspension is characterized in that the liquid obtained by filtration or concentrated under reduced pressure or freeze-dried at 50 ℃ or less.

In addition, the present invention, the cosmetic composition is a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) or water-in-oil (W / O) type base cosmetic formulation and oil-in-water or water-in-oil makeup base , Foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, teak color and eyebrow pencils are characterized in that it is selected from the color cosmetic formulations. The composition may be somewhat fluid and may have the appearance of a white or colored cream, ointment, milk lotion, serum, essence, paste or mousse. It may optionally be applied to the skin 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 composition 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. have. 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.

The subject of the present invention is also the use of a cosmetic composition containing a persimmon extract stabilized with nanoliposomes used as skin whitening agent, anti-aging agent, skin irritant.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative description of the present invention, the scope of the present invention is not limited to these Examples.

< Example  One: With nanoliposomes  Preparation of Stabilized Persimmon Extract>

The persimmons were chopped, refluxed with 70% aqueous ethanol solution for 3 hours, chilled, and filtered through a Whatman # 10 filter paper. The filtered extract was concentrated under reduced pressure and lyophilized at 50 ° C or lower. Persimmon extract was prepared by using one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol so as to contain 0.001 to 60.0% by weight of the vacuum concentrate and the lyophilisate.

Next, nanoliposomes were obtained as follows.

Nanoliposomes are small in size by passing liposomes containing phospholipid, glutathione, polysorbate 20, and ethanol five times through a high pressure emulsifier. Phospholipids, glutathione, 95% modified ethanol, glycerin, and polysorbate 20 were emulsified under high pressure to form a closed bilayer nanoliposome. The phase A of Table 1 was heated up to 95 degreeC, and it mixed and dissolved uniformly. It was cooled to 40 ° C and phase B was mixed. This was passed through a high pressure emulsifier (Microfluidizer M210EH, USA) five times under conditions of a pressure 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 4 Glutathione 0.1 95% modified ethanol 20 glycerin 30 Polysorbate 20 4 B phase Persimmon Extract 20 Purified water Remaining amount Sum 100

Note) Unit: weight%

The persimmon extract is added 0.01 to 60% by weight based on the total weight of nanoliposomes. When added in less than 0.01% by weight it is difficult to expect the effect, even when added in excess of 60% by weight does not show a clear increase in efficacy.

Nanoliposomes stabilized persimmon extract is contained in 0.1 to 30% by weight based on the total weight of the cosmetic composition. If it is less than 0.1% by weight, it is difficult to expect an effective effect, and if it exceeds 30% by weight, there is no apparent increase in efficacy. Therefore, 0.1-30 weight% is preferable.

< Example  2: Mushroom Tyrosinase  Used Tyrosinase  Activity Inhibition Effect Measurement>

In order to confirm the whitening effect of the persimmon extract obtained in Example 1 it was determined by looking at the degree of inhibition of the function of the enzyme tyrosinase (tyrosinase).

Tyrosinase is an enzyme that accelerates the oxidation of tyrosine in vivo and helps melanin production. In this example, the 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) is applied. The whitening effect was determined.

The inhibitory effect of tyrosinase activity was investigated using kojic acid, arbutin, lettuce extract, oil-soluble licorice extract and persimmon extract prepared in Example 1 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. The activity inhibition rate (%) for tyrosinase was calculated by Equation 1, and the IC ₅₀ value is the concentration of a substance that inhibits tyrosinase enzyme activity by 50%.

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

A: Absorbance before reaction of the well in which the sample is placed

B: Absorbance after reaction of the well in which the sample was placed

C: Absorbance before reaction of the well without sample

D: absorbance after reaction of the well without sample

As a result of testing the inhibitory effect of tyrosinase activity, the IC ₅₀ value of the persimmon extract was 0.01%, which was superior to the conventional whitening agents known as kojic acid, arbutin, and lettuce extract (Table 2). .

 Sample Name Inhibitory effect of tyrosinase (IC ₅₀ )  Kojisan 0.02%  Arbutin 0.40%  Persimmon Extract 0.01%  Lettuce extract 0.7%  Soluble Licorice Extract 0.03%

< Example  3: B16F1 Melanosite  Intracellular Use Tyrosinase  Determination of Enzyme Inhibitory Effect>

This Example is to determine the degree of inhibition of tyrosinase enzyme activity in B16F1 melanocytes in order to determine the whitening effect of the persimmon extract obtained in Example 1 by measuring the tyrosinase enzyme activity in the cell.

The B16F1 melanocytes used in this example are cell strains derived from mice and 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, the cell number was measured, and the cells were recovered by centrifugation. 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. The inhibition rate of tyrosinase enzyme activity (%) of B16F1 melanocytes was calculated by the equation (2), IC ₅₀ value is the concentration of a substance that inhibits the activity of the intracellular tyrosinase enzyme 50%.

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

A: Absorbance of Wells Without Samples

B: Absorbance of Wells Added Samples

As a result of measuring the inhibitory effect of tyrosinase enzyme activity of B16F1 melanocytes, the IC ₅₀ value of the persimmon extract was 0.01%. It was shown to inhibit (Table 3). As a result, it can be seen that the persimmon extract of the present invention exhibits a whitening effect by inhibiting the activity of tyrosinase enzyme in the cell.

 Sample Name Inhibitory effect of tyrosinase activity (IC ₅₀ )  Kojisan  0.03%  Arbutin  0.2%  Persimmon Extract  0.01%  Lettuce extract  0.65%  Soluble Licorice Extract  0.02%

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

In this example, in order to confirm the whitening effect of the persimmon extract obtained in Example 1, the whitening effect was determined according to the degree of inhibition of melanin production for B16F1 melanocytes.

B16F1 melanocytes used in this example 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 melanocytes used in this example were 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 R. Lotan and D. Lotan, Cancer Res , 40 : 3345-3350, 1980) was modified with a slight modification. 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. Melanin inhibition rate (%) of B16F1 melanocytes was calculated by the equation (3), IC ₅₀ value is the concentration of the substance inhibits 50% melanin production.

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

A: Melanin amount in wells without sample

B: Melanin amount in the wells to which the sample was added

As a result of testing the melanin production inhibitory effect of B16F1 melanocytes, the IC ₅₀ value of the persimmon extract was 0.005%, which was superior to the conventional whitening agents hydroquinone, arbutin, oil-soluble licorice extract, and extract of lettuce extract (Table 4).

 Sample Name Melanin synthesis inhibitory effect (IC ₅₀ )  Hydroquinone  0.02%  Arbutin  0.4%  Persimmon Extract  0.003%  Lettuce extract  4.5%  Soluble Licorice Extract  0.03%

< Example  5: Antioxidant Effect Measurement>

In this Example, the active oxygen radical scavenging effect was measured to confirm the antioxidant effect of the persimmon extract obtained in Example 1.

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. The result of the active oxygen scavenging effect was calculated by Equation 4, and the results are shown in Table 5.

Reactive oxygen scavenging effect (%) = [1- (St-So) / (Bt-Bo)] X 100

St: absorbance at 560 nm after enzyme reaction of sample solution

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before enzyme reaction of enzyme-free sample solution

Bo: absorbance at 560 nm before enzyme reaction of enzyme-free blank test solution

Sample Name Antioxidative effect(%) Persimmon Extract 0.001% 40 0.005% 80 0.01% 96 Green tea extract 98 Vitamin E 91

< Example  6: Collagen Synthesis Effect Measurement Experiment>

In order to observe the collagen synthesis effect of the persimmon extract obtained in Example 1 was tested by treating directly human fibroblasts obtained from human or commercially purchased.

DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% fetal bovine serum and human fibroblasts (5,000 cells / well) were put into a 96-hole plate incubator and cultured until 70-80% growth. The persimmon extract was then treated with 0.01% and 0.001% concentrations, respectively, and cell culture fluid was collected and collagen synthesized 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. At this time, the reaction absorbance of the cell culture without the extract was used as a control. Collagen synthesis effect was calculated by the equation (5), the results are shown in Table 6.

Collagen Synthesis Effect (%) = (Reaction Absorbance of Cell Culture Treated with Persimmon Extract / Reaction Absorbance of Control) X 100

As shown in Table 6 it can be seen that the persimmon extract has an excellent collagen production promoting effect.

Sample Name Collagen Synthesis Effect (%) Persimmon Extract 0.01% 90 0.001% 60 Control 0

< Example  7: temperament Metalloprotein  Degrading enzymes ( MMP -1) Determination of Activity Inhibition Effect>

Measurement of the inhibitory effect of the substrate metalloproteinase (MMP-1) activity of the persimmon extract prepared in Example 1 was carried out 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.

The results showed that the persimmon extract according to the present invention inhibits collagenase activity in a concentration-dependent manner as shown in Table 7. 1,10-phenanthroline used as a positive control showed a 70% activity inhibitory effect at 2mM. As a result, it can be seen that the persimmon extract has an excellent collagenase activity inhibitory effect.

sample MMP-1 activity inhibitory effect (%) Persimmon Extract 0.002% 50 0.005% 70 0.01% 90 Positive control (2 mM, 10-phenanthroline) 70

< Example  8: Persimmon extract after ultraviolet irradiation MMP -1 expression inhibition evaluation>

In this Example, ELISA (Enzyme Linked ImmunoSorbent Assay) was performed to measure the expression level of MMP-1 after UV irradiation and sample addition of the persimmon extract obtained in Example 1 (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 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 the absorbance was measured at 405 nm using an absorbance spectrometer. At this time, the reaction absorbance of the cell culture solution without the extract was used as a control. MMP-1 expression inhibition effect was calculated by the equation (6), the results are shown in Table 8.

% Inhibition of MMP-1 expression = [(A-B) / A] X 100

A: Absorbance of Wells Without Samples

B: Absorbance of Wells Added Samples

As shown in Table 8, the persimmon extract according to the present invention showed an MMP-1 expression inhibitory effect in a concentration-dependent manner. Retinoic acid used as a positive control showed an inhibitory effect of 45% expression at 3.5uM. As a result, it can be seen that the persimmon extract has an excellent inhibitory effect on MMP-1 expression.

Sample Name % Inhibition of MMP-1 expression Persimmon Extract 0.001% 30 0.005% 60 0.01% 90 Retinol (3.5 μM) 45

< Example  9: inflammation-related enzymes ( hyaluronidase ) Activity Inhibition Effect Measurement>

This example is to determine the anti-inflammatory effect of the persimmon extract obtained in Example 1 by determining the enzyme activity of the inflammation-related enzyme hyaluroniadiaze enzyme.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and has a function of causing inflammation. In this example, the method of inhibiting the activity of the enzyme to measure the anti-inflammatory effect (Y. Kakegawa, et al . , Japanese J of Inflammation , 4 : 437-438, 1984) was used to determine the anti-inflammatory effect.

Inhibitory effect of hyaluronia diase was investigated using comfrey, seesaw, triticale, oil-soluble licorice extract, and persimmon extract as samples. Inhibitory activity of hyaluronidiases of each sample was measured as follows.

100 μl of sample and 50 μl of hyaluronidiase solution (type IV-S, Sigma, 400U / mL) were added and reacted at 37 ° C. for 20 minutes, followed by enzyme activation solution (compound 48/80 CaCl ₂ ㆍ 2H ₂ O, Sigma). , 0.1 mg / ml) was added to 100 µl and reacted again at 37 ° C. for 20 minutes. 250 µl of a hyaluronic acid solution (0.4 mg / ml) was added thereto and reacted at 37 ° C. for 40 minutes, and 100 µl of 0.4N NaOH was added to terminate the reaction. 100 μl of potassium borate solution was added thereto, reacted at 95 ° C. for 3 minutes, cooled, and then, 3 ml of ρ -dimethylaminobenzaldehyde solution was added and reacted at 37 ° C. for 20 minutes, followed by color development. Absorbance was measured at 565 nm to determine the inhibition rate for hyaluronidiases. Inhibition rate (%) for hyaluronidiases was calculated by the equation (7), IC ₅₀ value is the concentration of a substance that inhibits the hyaluronidiase enzyme activity by 50%.

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

A: Enzyme activity of wells without sample

B: Enzyme Activity of Well Added Samples

As a result of testing the inhibitory effect of hyaluronidiaze enzyme activity, the IC ₅₀ value of the persimmon extract was 0.02%, which is a useful anti-inflammatory licorice extract, comfrey extract, tritical herb extract, and seesaw extract, It showed a superior effect (Table 9).

 sample Inhibitory effect of hyaluronidiases (IC ₅₀ )  Comfrey Extract  0.3%  Seesaw extract  0.4%  Soluble Licorice Extract  0.05%  Triticale extract  0.3%  Persimmon Extract  0.02%

Example 10 Evaluation of Stimulation Relaxation Effect Using Cell Culture Technology

This Example is a cell by Sodium Lauryl Sulfate (hereinafter referred to as "SLS") which is a substance causing skin irritation using cell culture technology to evaluate the stimulating relaxation effect of the persimmon extract obtained in Example 1 The stimulating effect was evaluated to the extent of inhibiting death.

SLS is a substance that is used as a criterion for evaluation of skin irritation based on cosmetics, and is a substance that binds to cell membranes to inhibit cell metabolism and destroys cell membranes to cause skin irritation.

This example evaluates the effect of reducing apoptosis by SLS when SLS and persimmon extracts are added to human fibroblasts at the same time.

The Hs68 fibroblasts used in this example were used as a human dermal cells in the ATCC (American Type Culture Collection, Accession No .: 1635).

Stimulation relaxation test using the cell culture technology was carried out as follows.

Human-derived fibroblasts were dispensed at a concentration of 1 X 10 ⁵ per well in a 96-well plate incubator, incubated for 24 hours, treated with 0.01% SLS alone, 0.01% SLS and persimmon extract (0.01%) simultaneously for 24 hours. Further incubation. After incubation, MTT (Sigma) reagent was added and incubated for 4 hours, the culture medium was removed, 1N NaOH / isopropanol solution was added and stirred for 20 minutes, and the absorbance was measured at 565 nm with an absorbance spectrometer. The effect of inhibiting apoptosis was measured.

Experimental results have found that persimmon extract is an irritant that reduces skin irritation that can occur when applied with a cosmetic substrate by inhibiting apoptosis caused by SLS (Table 10).

 Sample Name  Fibroblast survival rate (%)  0.01% SLS  25  0.01% SLS + 0.01% Persimmon Extract  80

< Experimental Example  1>

In this Experimental Example, the cosmetics containing the nanoliposomes stabilized persimmon extract obtained in Example 1 were prepared and the skin whitening effect of humans was evaluated by comparative experiments with Comparative Example 1.

To prepare an emulsion according to the conventional method for preparing a cosmetic using the raw material of Table 11. Comparative Example 1 was prepared in the same manner as in Preparation Examples 1 to 3 except that the liposomes stabilized persimmon extract obtained in Example 1 was not added.

Raw material (% by weight) Production Example Comparative Example 1 One 2 3 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 Nanoliposome Stabilized Persimmon Extract 10 One 0.1 - incense Quantity Quantity Quantity Quantity antiseptic Quantity Quantity Quantity Quantity Purified water Remaining amount Remaining amount Remaining amount Remaining amount Total (% by weight) 100 100 100 100

Note) Unit: Weight%

For 20 experimenters (20-35 year old female), the emulsion prepared in Preparation Examples 1 to 3 on the right side of the face, and the emulsion prepared in Comparative Example 1 on the left side of the face for 2 consecutive days each Applied. 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. Table 12 compares the facial skin color of the experimenter using the emulsion prepared in Preparation Example 3 with the facial skin color of the experimenter using the emulsion made in Comparative Example 1.

As shown in Table 12, it can be seen that the whitening effect is excellent in the facial skin of the experimenter applying the emulsion containing nanoliposomes stabilized persimmon extract.

 Product used  Preparation Example 3  Comparative Example 1  Experiment number  Right skin color  Left skin color  One  1.5  2.5  2  2.0  3.0  3  1.5  3.0  4  2.0  3.0  5  2.5  3.0  6  1.5  2.5  7  2.5  3.5  8  2.0  3.5  9  2.0  3.0  10  1.5  2.0  11  2.0  3.0  12  1.5  2.5  13  1.5  2.0  14  2.0  2.0  15  2.0  3.0  16  1.5  2.5  17  2.0  3.0  18  2.5  3.0  19  1.0  2.5  20  1.5  3.5

< Experimental Example  2>

In this Experimental Example, the emulsion prepared in Preparation Examples 1 to 3 and Comparative Example 1 was applied to humans to evaluate the effect of improving skin wrinkles.

For 20 experimenters (women aged 20-35 years), the emulsion prepared in Preparation Examples 1 to 3 was applied to the right side of the face, and the emulsion prepared in Comparative Example 1 was applied to the left side of the face twice a day for 2 consecutive months. It was.

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.

Table 13 compares the average eye wrinkle depth and wrinkle reduction rate of the experimenter using the emulsion prepared in Preparation Example 2 with the experimenter using the emulsion of Comparative Example 1. As shown in Table 13, it can be seen that the skin fine wrinkle improvement effect is excellent in the skin around the facial eye of the experimenter applying the emulsion containing nanoliposomes stabilized persimmon extract.

Preparation Example 2 Comparative Example 1 Before use 2 months after use Before use 2 months after use Average fine wrinkle depth (AU) 0.201 0.150 0.199 0.195 Wrinkle Reduction (%) 25.4% 2.0%

n = 20, p <0.01

< Experimental Example  3>

This experimental example evaluated the irritation-reducing effect of the cosmetic containing nanoliposomes stabilized persimmon extract obtained in Example 1 by human patch experiment. This evaluation is a result of evaluation obtained by directly applying the result obtained in Example 10 to the human body.

In general, SLS that causes irritation in cosmetic formulations and the product prepared in Preparation Examples 1 to 3 were mixed and patched for 24 hours, 48 hours, and 72 hours to evaluate the irritation-releasing effect based on the stimulation induction index.

FINN CHAMBER (FINLAND) was used to coat 0.2 mg of each product on the upper arm of 50 healthy men and women aged 20 to 50 years, and the acute irritation index was evaluated after 24 hours. After evaluation, the same amount of product was applied to the same site again, and the delayed stimulation index after 48 and 72 hours was evaluated.

As a result of the test, the product containing the nanoliposome stabilized persimmon extract was patched and no skin redness occurred after 24 hours, 48 hours, and 72 hours.

The results of this evaluation indicate that the nanoliposomes stabilized persimmon extract have a significant effect of reducing skin irritation by substrates (surfactants, fragrances, alcohols) that cause irritation when used in cosmetics.

< Experimental Example  4: Stability Experiment

The stability of the formulation for the cosmetic containing nanoliposomes stabilized persimmon extract according to the present invention was measured by the following method.

Preparation Example 1 and Comparative Preparation Example 1 {Instead of using a nanoliposome stabilized persimmon extract of Example 1, 10% by weight persimmon extract of Example 1 (that is, persimmon extract not stabilized with nanoliposomes) was added Except that, except that the emulsion prepared in the same manner as in Preparation Example 1 was stored in an opaque vitreous container in a constant temperature bath at 45 ° C. for 12 weeks, and kept in a completely shaded refrigerator at 4 ° C. The separation, discoloration, and precipitation of the samples stored in an opaque glass container for 12 weeks and stored for 12 weeks in a circulation chamber (once / day) circulating at -5 ° C to 45 ° C were measured. . The results of the evaluation of the product separation and the degree of discoloration classified into the following six grades are shown in Table 14 below.

Product discoloration evaluation criteria:

0: no change, 1: very little separation (discoloration), 2: little separation (discoloration), 3: little severe separation (discoloration), 4: severely separation (discoloration), 5: very severe separation (discoloration)

 Temperature  Preparation Example 1  Comparative Preparation Example 1  45 ℃  0  3  4 ℃  0  2  cycle  0  3

As can be seen in Table 14, Preparation Example 1 stabilized through nanoliposomes was stable without discoloration or separation symptoms at 4 ° C., 45 ° C., and circulation test, but was used directly in formulations without stabilization (ie, stabilization with nanoliposomes). In the case of Comparative Production Example 1 (not using a raw material), separation (discoloration) occurs and it is understood that it is unstable.

In addition, the results obtained by classifying the product precipitation degree into the following six grades are shown in Table 15 below.

Product sedimentation evaluation criteria:

0: no change, 1: very little sedimentation, 2: little sedimentation, 3: little sedimentation

4: severely settling, 5: extremely severely settling

 Temperature  Preparation Example 1  Comparative Preparation Example 1  45 ℃  0  3  4 ℃  0  2  cycle  0  2

As can be seen in Table 15, the cosmetics stabilized with the nanoliposomes according to the present invention do not cause precipitation, and it can be seen that they have been stabilized.

As such, when the persimmon extract is added without stabilization, it readily reacts with other weak ionic polymers in the formulation, causing precipitation and discoloration. However, when the persimmon extract is stabilized with nanoliposomes and applied to the formulation as in the present invention, it can be seen that precipitation and discoloration reaction are minimized even when the persimmon extract is added in excess.

< Experimental Example  5: size of nanoparticles Transdermal  Absorption Measurements>

The degree of transdermal absorption of the examples in which the nanoliposomes were obtained was measured. Nanoparticles having sizes of 50 nm, 80 nm, 120 nm, and 500 nm were prepared using PMMA polymers to which fluorescent substances were added by chemical bonding, and then applied to the skin of female hairless guinea pigs. After elapsed, the cross section of the skin was cut to measure the fluorescence emitted from the percutaneously absorbed nanoparticles.

Preparation of fluorescent PMMA polymer nanoparticles covalently bonded fluorescent molecules to the carboxyl group of the methacrylic acid of polymethylmethacrylate-co-methacrylic acid (molar ratio of methylmethacrylate and methacrylic acid is 84:16). The preparation method is briefly described as follows. 34 g of polymethylmethacrylate-co-methacrylic acid was dissolved in 150 ml of anhydrous methylene chloride, and then 132 mg of dicyclohexylcarbide and 74 mg of N-hydroxysuccinimide were added to the carboxyl group. Activated. After stirring for 1 hour, 222 mg of fluoresceamine was added to react the primary amine of the fluorescein amine with the activated carboxyl group to form an amide bond, thereby allowing the fluorescent molecules to be covalently bonded to the polymer chain. The reaction proceeded for 5 hours at room temperature and dark conditions. Dicyclohexylurea, a by-product formed after the reaction, is removed through a 0.45 μm nylon filter, and then precipitated in diethyl ether to first remove the reaction reagents and unreacted residues, and to stand in an excess of tertiary distilled water for one day. The waters were removed and dried using a vacuum oven at 40 ° C. As a result, PMMA nanoparticles having a size of 50 nm, 80 nm, 120 nm and 500 nm to which fluorescent materials were added were prepared.

Eight weeks of female hairless guinea pigs (strain IAF / HA-hrBR) were used for percutaneous absorption testing of nanoparticles by size. The skin of the abdominal region of the guinea pig was cut out and mounted in Franz-type diffusion cells (Lab Fine Instruments, South Korea). 50 mM phosphate buffer (pH 7.4, 0.1 M NaCl) was added to the receiving vessel (5 ml) of the Franz-type diffusion cell. The diffusion cell was mixed and dispersed at 600 rpm while maintaining 32 ° C, and 50 µl of a 10% (w / v) solution of fluorescent PMMA nanoparticles prepared for each size was placed in each donor container. Absorption and diffusion were performed according to a predetermined time (average 12 hours), and the area of skin where absorption and diffusion occurred was 0.64 cm 2. After the diffusion of nanoparticles is completed, 10 ml of dried kimwipes or ethanol is used to wash away the unabsorbed nanoparticles remaining on the skin, and the cross section of the skin is cut to absorb the fluorescent PMMA nanoparticles. The distribution of was measured. The measurement results in the skin distribution of the nanoparticles are shown in Table 16 below.

 Average size of nanoparticles  Percutaneous absorption area  Remarks  50 nm  35 μm  80 nm  17 μm  120 nm  12㎛  500 nm  7㎛

From the experiments it can be seen that the percutaneous absorption of the nanoparticles largely depends on the average particle diameter. It was confirmed that the nanoparticles having an average diameter of about 50 nm pass through the epidermis and are absorbed and diffused to the upper end of the dermal layer. When the particle size exceeded 500 nm, the nanoparticles stayed on the skin surface and no longer absorbed into the skin. Skin absorption of nanoparticles with an average diameter of about 50 nm is diffused into the intercellular lipids of the skin, and the hydrophobicity of the polymer is thought to have an effect of promoting such transdermal absorption. In addition, when the active ingredient having poor percutaneous absorption or instability is trapped in the nanoparticles having an average particle diameter of about 50 nm, transdermal absorption and delivery of the active ingredient may be possible through delivery into the skin by a polymer.

That is, the cosmetic composition containing the persimmon extract of the present invention and nanoliposomes stabilized persimmon extract showed an excellent effect on skin improvement such as the whitening effect, irritation relief effect, wrinkle improvement effect as described above.

As described above, the persimmon extract according to the present invention is a whitening effect and free radical scavenging effect, such as tyrosinase activity inhibitory effect, melanin synthesis inhibitory effect, to prevent melanin production, which is a substance causing blemishes, freckles and skin pigmentation, Wrinkle improvement effects such as activity of collagenase (MMP-1) and expression inhibition are excellent, skin stimulation effect such as skin cell death suppression effect that reduces skin irritation caused by cosmetic substrate, and enzyme activity inhibitory effect related to inflammation induction Excellent mitigation effect

Furthermore, the persimmon extract was stabilized with nanoliposomes and showed little discoloration in stability and temperature change, and no separation occurred, indicating that the persimmon extract was very stable.

Therefore, the present invention is to prepare a cosmetic composition, such as a lotion, cream, milky lotion, pack containing a nano liposome stabilized persimmon extract having the above-mentioned effect of skin whitening effect wrinkle improvement effect, irritation relief functional cosmetic composition Can provide.

In the above description of the preferred embodiment of the present invention, those skilled in the art may make various modifications without departing from the gist of the present invention, and such modifications belong to the protection scope of the present invention. The protection scope of the invention should be construed as described in the claims.

Claims (9)

A cosmetic composition containing Diospyros kaki extract stabilized with nanoliposomes as a skin improving component. delete The method of claim 1, Cosmetic composition containing a persimmon extract stabilized with nano-liposomes, characterized in that the persimmon extract stabilized with the nano-liposomes contained 0.1 to 30.0% by weight based on the entire cosmetic composition. The method of claim 1, The persimmon extract is a cosmetic composition containing a persimmon extract stabilized with nano-liposomes, characterized in that it contains 0.01 to 60% by weight based on the total weight of the stabilized nano-liposomes. The method according to any one of claims 1, 3 and 4, The nanoliposomes are obtained by passing a liposome comprising a phospholipid, glutathione, 95% modified ethanol, glycerin, polysorbate 20 through a high pressure emulsifier, cosmetic composition containing a persimmon extract stabilized with nanoliposomes. The method according to any one of claims 1, 3 and 4, Formulation of the cosmetic composition is a base cosmetic formulation consisting of a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) and water-in-oil (W / O) type and oil-in-water and water-in-oil makeup base, foundation Cosmetic composition, characterized in that any one selected from the skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, teak color and eyebrow pencil formulations. The method according to any one of claims 1, 3 and 4, Persimmon extract stabilized with the nanoliposome is a cosmetic composition, characterized in that it is used for skin whitening. The method according to any one of claims 1, 3 and 4, Persimmon extract stabilized with the nano liposome is a cosmetic composition, characterized in that used for wrinkle improvement. The method according to any one of claims 1, 3 and 4, Persimmon extract stabilized with the nano liposome is a cosmetic composition, characterized in that it is used for skin irritation relief.