KR20230079615A - Liposome comprsings barley sprout extract and vitamin tree fruit extract and cosmetic compositions containing the same - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising a sprout barley extract, a fermented vitamin tree fruit extract, and a vitamin tree fruit supercritical extract, comprising: a sprout barley extract; Fermented vitamin tree fruit extract obtained by fermenting vitamin tree fruit with Lactobacillus rhamnosus HK-9 (accession number: KCCM11254P); And vitamin tree fruit supercritical extract; by including, has antioxidant, skin wrinkle improvement and skin whitening effect.

Description

Cosmetic composition containing sprout barley extract, vitamin tree fruit fermented extract, and vitamin tree fruit supercritical extract {Liposome comprsings barley sprout extract and vitamin tree fruit extract and cosmetic compositions containing the same}

The present invention relates to a cosmetic composition comprising sprout barley extract, vitamin tree fruit fermented extract and vitamin tree fruit supercritical extract.

Human skin aging is being studied in two major ways: “Intrinsic aging” that occurs over time due to changes in the function of cells, which are biological processes for everyone, and continuous exposure to ultraviolet rays (light) It is classified as “Besides the aging of the human nature” that occurs due to exposure to external environmental factors such as , stress, and pollution (smoking).

When skin cells and cell membranes are damaged by the above factors, the permeability of cell membranes changes and the ion balance in cells is broken. . If such changes at the cellular level continue, the structure of skin tissue loses regularity, local scars appear in the dermis, and the thickness of the dermis decreases due to a decrease in collagen fibers or elastic fibers. The crosslinking between collagen fibers increases, and the fine structure of elastic fibers is also damaged, resulting in overall skin elasticity deterioration and skin wrinkles. In addition, collagen, an important component constituting the matrix of the dermal layer of the skin, is a major matrix protein produced by fibroblasts of the skin and exists in the extracellular matrix. It is known to perform important functions such as support. It is known that such collagen is reduced by external stimuli such as age and ultraviolet light, which is closely related to the formation of wrinkles in the skin. Aging not only reduces the synthesis of collagen itself, but also increases the activity of MMPs (matrix metalloproteinases) that decompose connective tissue including collagen, which promotes the degradation of collagen in skin tissue, which is the cause of the decrease in collagen content. It is known to be TIMP (Tissue Inhibitors of Matrix Metalloproteinase), an endogenous inhibitor that inhibits these MMPs, exists. When the activities of MMPs and TIMPs are balanced and properly regulated, physiological tissue reorganization and homeostasis are well maintained.

Meanwhile, the color of human skin is closely related to the concentration and distribution of melanin in the skin. In addition to genetic factors, melanin is also affected by environmental or physiological conditions such as solar ultraviolet rays, fatigue, and stress. Melanin is synthesized in melanocytes in the epidermal layer of the skin. An enzyme called tyrosinase acts on tyrosine, a kind of amino acid, in melanosomes, organelles in melanocytes, to produce DOPA and dopaquinone. ) and then produced through a non-enzymatic oxidation reaction. When the synthesis of melanin occurs excessively in the skin, the skin tone is darkened, and spots, freckles, and the like occur. Therefore, if the synthesis of melanin pigment is inhibited by inhibiting the activity of tyrosinase in the skin, skin whitening can be realized by brightening the skin tone, as well as skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones, and genetic causes. It can improve depression.

In the field of cosmetics, studies on cosmetics containing functional ingredients having skin wrinkle improvement and skin whitening effects are being actively conducted, and interest in cosmetics derived from natural products with excellent skin improvement effects while minimizing skin irritation is increasing.

Patent Document 1. Korean Patent Registration No. 10-2320424

An object of the present invention is to provide a cosmetic composition comprising sprout barley extract, vitamin tree fruit fermented extract and vitamin tree fruit supercritical extract.

One aspect of the present invention is sprout barley extract; Fermented vitamin tree fruit extract obtained by fermenting vitamin tree fruit with Lactobacillus rhamnosus HK-9 (accession number: KCCM11254P); It provides a cosmetic composition for antioxidant, skin whitening and skin wrinkle improvement containing as an active ingredient; and supercritical extract of vitamin tree fruit.

The cosmetic composition may be liposomalized with an oily component including the supercritical extract of the vitamin tree fruit and an aqueous component including the barley sprout extract and the fermented extract of the vitamin tree.

The sprout barley extract may be extracted using 70 to 85% by weight of ethanol as a solvent.

The vitamin tree fruit fermented extract may be extracted by fermenting the vitamin tree fruit at 25 to 45 ° C. for 36 to 60 hours.

The vitamin tree fruit supercritical extract may be extracted at a pressure of 300 to 450 bar and a temperature of 40 to 60 ° C. using carbon dioxide as a supercritical fluid.

The cosmetic composition may be a mixture of the sprout barley extract, the vitamin tree fruit fermented extract, and the vitamin tree fruit supercritical extract in a weight ratio of 1: 0.5 to 1.5: 0.5 to 1.5.

The cosmetic composition may inhibit the activity of tyrosinase.

The cosmetic composition may suppress MMP-1 expression and increase the production of type I procollagen protein.

The cosmetic composition of the present invention is composed of natural materials, is safe for the skin, and has excellent skin wrinkle prevention and improvement effects, whitening effects, and antioxidant effects. More specifically, it effectively inhibits the expression of MMP-1 protein, which directly acts on the formation of skin wrinkles, increases the expression of type I procollagen protein, effectively prevents and improves skin wrinkles, and inhibits tyrosinase. It can effectively act on skin whitening by suppressing the production of melamine.

1 schematically shows a method for preparing a liposomalized cosmetic composition according to an embodiment of the present invention.
Figure 2 is prepared in Example 1 of the present invention (a) freeze-dried powder of sprout barley extract, (b) freeze-dried powder of vitamin tree fruit fermented extract and (c) its TLC result, (d) vitamin tree fruit supercritical It shows a photographic image of the extract.

Hereinafter, the present invention will be described in detail.

The present invention sprout barley extract; Fermented vitamin tree fruit extract obtained by fermenting vitamin tree fruit with Lactobacillus rhamnosus HK-9 (accession number: KCCM11254P); It provides a cosmetic composition for antioxidant, skin whitening and skin wrinkle improvement containing as an active ingredient; and supercritical extract of vitamin tree fruit.

The cosmetic composition may be liposomalized with an oily component including the supercritical extract of the vitamin tree fruit and an aqueous component including the barley sprout extract and the fermented extract of the vitamin tree.

The cosmetic composition of the present invention is liposomalized to completely capture the active ingredients of the barley sprout extract and vitamin tree fruit extract to significantly improve efficacy, as well as improve skin absorption and permeability to quickly and deeply absorb active ingredients into the skin. can

sprout barley extract

Sprout barley (young barley leaves) refers to barley that sprouts from seeds and grows to about 15 to 20 cm. Sprout barley has a high content of inorganic components such as calcium, magnesium, and potassium, and antioxidant functional substances such as vitamin C, vitamin B1, and vitamin E. In addition, it is known to contain functional substances such as saponarin, rutonarin, and isovitexin having antioxidant, anti-inflammatory, and anti-cancer functions.

The sprout barley extract may be extracted at 60 to 90 ° C. for 1 to 10 hours, preferably for 1 hour 30 minutes to 8 hours, but is not limited thereto.

The sprout barley extract may be extracted using water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The mixed solvent may be 20 to 90% by weight of methanol, ethanol, butanol or propanol, and most preferably 70 to 85% by weight of ethanol. In particular, it is most preferable in that the antioxidant, skin whitening, and skin wrinkle improvement effects of the cosmetic composition containing the sprout barley extract using 70 to 85% by weight of ethanol are remarkably increased.

Vitamin Tree Fermented Extract

The vitamin tree, also called sea buckthorn, is a tree that produces yellow to orange fruits. The fruit of the vitamin tree has a much higher vitamin content than other fruits and vegetables, and the content of polyphenols and flavonoids that are effective in antioxidants is also very high.

The vitamin tree fruit fermented extract is obtained by fermenting the vitamin tree fruit with Lactobacillus rhamnosus HK-9 (Accession Number: KCCM11254P) to obtain a vitamin tree fruit fermented extract.

Lactobacillus bulgaricus, Lactobacillus acidophilus MG501, Lactobacillus casei MG311, Lactobacillus rhamnosus MG315, Lactobacillus fermentum MG590, Lactobacillus plantarum MG208, and Bifidobacterium longum MG723 are generally used in cosmetic compositions, but these lactic acid bacteria have excellent antioxidant properties and skin wrinkles when fermenting vitamin tree fruits. Since improvement and skin whitening effects are not obtained, it is preferable to use Lactobacillus rhamnosus HK-9 (accession number: KCCM11254P).

In particular, after incubating Lactobacillus rhamnosus HK-9 (accession number: KCCM11254P) at 25 to 45 ° C, preferably 30 to 40 ° C for 36 to 60 hours, preferably 40 to 50 hours, 1X10 ⁸ to 5X10 ⁹ It is obtained at a concentration of cfu / mL, and may be obtained by inoculating the fruit of a vitamin tree. If at least one of the incubation temperature and time is less than the lower limit, fermentation does not occur sufficiently and improvement in antioxidant, skin whitening and skin wrinkle improvement effects cannot be expected, and conversely, if it exceeds the upper limit, antioxidant, skin whitening and skin wrinkle improvement Rather, it may decrease. In addition, when Lactobacillus rhamnosus HK-9 (accession number: KCCM11254P) has a concentration less than the lower limit, it may be difficult to expect skin whitening and skin wrinkle improvement effects, and even if the culture time is increased, the upper limit cannot be exceeded.

The vitamin tree fruit fermented extract of the present invention is obtained by mixing the pulverized vitamin tree fruit with Lactobacillus rhamnosus HK-9 (Accession Number: KCCM11254P) at 25 to 45 ° C. for 36 to 60 hours, preferably 30 to 60 hours. It is obtained by fermentation at 40°C for 40 to 50 hours. If any one of the fermentation temperature and time is less than the lower limit, a sufficient fermentation effect cannot be expected, and conversely, if it exceeds the upper limit, the vitamin tree fruit fermented extract may deteriorate, which is not preferable.

Vitamin Tree Fruit Supercritical Extract

The vitamin tree fruit supercritical extract may be extracted using carbon dioxide, water, ethane, ethylene, and propane as supercritical fluids, and in particular, as a supercritical fluid, the critical point is low and toxicity When carbon dioxide without carbon dioxide is used, the efficiency of extraction and separation of useful components for wrinkle improvement contained in fat-soluble components of vitamin tree fruit can be remarkable.

The vitamin tree fruit supercritical extract may be extracted at a pressure of 300 to 450 bar and a temperature of 40 to 60 ° C, preferably at a pressure of 350 to 400 bar and a temperature of 45 to 50 ° C. The effective ingredient extraction efficiency of the vitamin tree fruit supercritical carbon dioxide extract obtained when the temperature and pressure conditions of the supercritical extraction satisfy the above range can be significantly improved.

The cosmetic composition of the present invention is characterized in that it is a three-type mixture in which the sprout barley extract, the vitamin tree fruit fermented extract and the vitamin tree fruit supercritical extract are mixed, and when the three-type mixture is used, alone or two of the three extracts Antioxidant, skin whitening, and skin wrinkle improvement effects are significantly improved compared to mixtures mixed with species.

In the cosmetic composition, the sprout barley extract, the vitamin tree fruit fermented extract, and the vitamin tree fruit supercritical extract are mixed in a weight ratio of 1: 0.5 to 1.5: 0.5 to 1.5, preferably 1: 0.9 to 1.1: 0.9 to 1.1 weight ratio. If the mixed weight ratio does not satisfy the above range, antioxidant, skin whitening and skin wrinkles are better than when the sprout barley extract, vitamin tree fruit fermented extract and vitamin tree fruit supercritical extract were used alone or in combination with two. It is preferable to satisfy the above weight ratio in that the improvement efficiency is not greatly improved.

The total sum of the sprout barley extract, the vitamin tree fruit fermented extract and the vitamin tree fruit supercritical extract may be included in 0.0001 to 30% by weight based on 100% by weight of the total cosmetic composition.

In this specification, "skin whitening" means to make the skin beautiful and white, and the skin color of a person is determined by the concentration and distribution of melanin inside the skin. In addition to genetic factors, environmental or It is also influenced by physiological conditions. Melanin is synthesized in melanocytes in the epidermal layer of the skin. An enzyme called tyrosinase acts on tyrosine, a kind of amino acid, in melanosomes, organelles in melanocytes, to produce DOPA and dopaquinone. ) and then produced through a non-enzymatic oxidation reaction. When the synthesis of melanin occurs excessively in the skin, the skin tone is darkened and spots, freckles, and the like are generated. Therefore, if the synthesis of melanin pigment is inhibited by inhibiting the activity of tyrosinase in the skin, skin whitening can be realized by brightening the skin tone, as well as skin hyperpigmentation such as spots and freckles caused by ultraviolet rays, hormones, and genetic causes. can improve

The cosmetic composition of the present invention inhibits the activity of tyrosinase to inhibit the synthesis of melanic pigment, thereby having effects of skin whitening and improvement of dermal hyperpigmentation.

In the present specification, “skin wrinkles” means fine lines caused by skin deterioration, and the causes of such skin wrinkles include genes, reduction of collagen, external environment, and the like. In the present specification, "improvement of skin wrinkles" means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already occurred.

The main function of skin fibroblasts is to regenerate damaged skin tissue through extracellular matrix synthesis and proliferation. Extrinsic aging factors such as photoaging or endogenous aging factors such as accumulation of free oxygen damage and cell aging due to telomere disruption They lower the physiological activity of dermal fibroblasts in the dermal layer. Type I collagen, which accounts for more than 95% of skin extracellular matrix and plays a very important part in the physiological activity of fibroblasts through interaction and signal exchange with adhesion molecules and cytoskeleton of cells. When the synthesis is lowered, the amount of collagen in the skin tissue is reduced, and thus the surface tension is reduced, resulting in a decrease in skin elasticity and formation of skin wrinkles, as well as a decrease in physiologically active functions including cell proliferation and regeneration. cause a vicious cycle. Accordingly, collagen of dermal fibroblasts is directly related to skin elasticity, wrinkle formation, and tissue repair or regeneration upon skin damage. That is, when the synthesis of collagen or procollagen in skin fibroblasts is promoted or the synthesis of matrix metallo-proteinase (hereinafter referred to as 'MMP') that decomposes collagen is inhibited, skin elasticity is improved, skin regeneration, and skin Effects such as wrinkle improvement, wound healing, repair and regeneration of damaged skin tissue, and prevention of skin aging can be obtained.

The cosmetic composition of the present invention inhibits MMP-1 expression and increases the production of type I procollagen protein. Accordingly, the liposome of the present invention may have effects such as skin elasticity improvement, skin regeneration, skin wrinkle improvement, wound healing, repair and regeneration of damaged skin tissue, and prevention of skin aging, thereby improving skin wrinkles.

The cosmetic composition of the present invention may include purified water, additional oily ingredients, and ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, conventional adjuvants such as vitamins, pigments, and fragrances, and A carrier may be further included.

In the cosmetic composition of the present invention, in addition to the above essential components, other ingredients commonly used in cosmetics may be mixed as necessary. Specific examples of the ingredients include fats and oils, moisturizers, emollients, ultraviolet absorbers, pH adjusters, fragrances, blood circulation promoters, cooling agents, antiperspirants, and purified water.

The cosmetic composition of the present invention can be used in various ways, such as cosmetics or facial cleansers having antioxidant, skin whitening, and skin wrinkle improvement effects.

Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent lotion, softening lotion, nutrient lotion, various creams, essences, packs, foundations, etc., and cleansing, face wash, soap, treatment, and beauty essences. etc. Specific formulations of the cosmetic composition of the present invention include cosmetics such as skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, essence, and nutrient essence. Cleansing products such as Ryuwa packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, and body cleansers can be mentioned.

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

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component, and in particular, in the case of a spray, chloro propellants such as fluorohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil may be used.

In the case where the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohols, suspending agents such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

Example 1. Preparation of a mixture of sprout barley extract, vitamin tree fruit fermented extract and vitamin tree fruit supercritical extract

sprout barley extract

10 times the weight of 80% by weight ethanol was added to the pulverized powder of sprouted barley, extracted at 70 ° C. for 120 minutes to obtain an 80% ethanol extract of barley sprouted, and then concentrated and lyophilized to powder.

Vitamin tree fruit fermented extract

A powder obtained by pulverizing 100 g of vitamin tree fruit was prepared. In addition, Lactobacillus rhamnosus HK-9 (Accession No.: KCCM11254P) was added to a shaking incubator with a medium prepared by adding 55% MRS medium and 0.5% L-Cysteine hydrochloride, and 70 to 100 rpm , and pre-incubated at 37 °C for 48 hours.

Then, 1 L of lactobacillus culture medium made of 55% MRS medium and 100 g of vitamin tree fruit powder were added to a 2 L flask, and the pre-cultured Lactobacillus rhamnosus HK-9 culture medium at a concentration of 3X10 ⁹ cells / mL was 5 (v / v)% was fermented at 37 ° C. for 48 hours. Then, the supernatant was obtained by centrifuging the fermented product at 3000 rpm for 10 minutes, and then filtered using 0.45 μM filter paper to obtain a vitamin tree fruit fermented extract.

Figure 2 is prepared in Example 1 of the present invention (a) freeze-dried powder of sprout barley extract, (b) freeze-dried powder of vitamin tree fruit fermented extract and (c) its TLC result, (d) vitamin tree fruit supercritical It shows a photographic image of the extract. Referring to FIG. 2 (c), it can be seen that the fermented vitamin tree fruit extract according to the present invention is converted from a high molecule to a low molecule as fermentation proceeds.

Vitamin Tree Fruit Supercritical Extract

In order to selectively obtain fat-soluble components of vitamin tree fruit, 100 g of vitamin tree fruit powder was put into a supercritical extractor and extracted for 2 hours at 380 bar pressure and 47 ℃ temperature using carbon dioxide as a supercritical fluid to obtain vitamin tree fruit supercritical. A critical extract was obtained.

mixture preparation

The sprout barley 80% ethanol extract, vitamin tree fruit fermented extract, and vitamin tree fruit supercritical extract were mixed and prepared in a weight ratio of 1:1:1.

Example 2. Liposome preparation of barley sprout extract, fermented vitamin tree fruit extract and vitamin tree fruit supercritical extract

The aqueous phase was prepared by mixing the 80% ethanol extract of sprout barley of Example 1 and the fermented extract of vitamin tree fruit of Example 1 at a weight ratio of 1:1.

After mixing 10% by weight of the vitamin tree fruit carbon dioxide supercritical extract of Example 1, 80% by weight of lecithin, and 10% by weight of sodium deoxycholate, the oil phase was prepared.

Each of the water phase part and the oil phase part was heated to 80 °C and mixed at 4000 rpm. Then, the water phase and the oil phase were put into a homogenizer and stirred at 5000 to 6000 rpm for 10 minutes to sufficiently disperse the lecithin. Then, after cooling to 30 °C, liposomes were obtained by treatment with an ultrasonic homogenizer at 40 kHz for 3 minutes. At this time, 80% ethanol extract of sprout barley, fermented extract of vitamin tree fruit, and carbon dioxide supercritical extract of vitamin tree fruit were used in the liposome at a weight ratio of 1:1:1.

Comparative Example 1. Preparation of a mixture containing sprout barley extract and vitamin tree fruit fermented extract

The 80% ethanol extract of sprout barley of Example 1 and the fermented extract of vitamin tree fruit of Example 1 were mixed and prepared in a weight ratio of 1:1.

Comparative Example 2. Preparation of a mixture containing sprout barley extract and vitamin tree fruit supercritical extract

The 80% ethanol extract of sprout barley of Example 1 and the supercritical extract of vitamin tree fruit of Example 1 were prepared by mixing at a weight ratio of 1:1.

Comparative Example 3. Vitamin tree fruit fermented extract and Preparation of a mixture containing vitamin tree fruit supercritical extract

The vitamin tree fruit fermented extract of Example 1 and the vitamin tree fruit supercritical extract of Example 1 were prepared by mixing at a weight ratio of 1:1.

Test Example 1. Antioxidant component analysis

(1) Measurement of total polyphenol content

1 ml of 95% Ethanol, 1 ml of each sample, and 0.5 ml of 50% Folin-Ciocalteu reagent were added to the test tube and reacted at room temperature for 5 minutes. Then, 1 ml of 5% Na ₂ CO ₃ was added, reacted again at room temperature for 1 hour, and absorbance was measured at 725 nm. Using gallic acid (Sigma-Aldrich) as a standard material, the same experiment was performed to obtain a standard curve, and using this, the content of total polyphenols eluted from 1 g of dry sample was calculated based on gallic acid, and compared at a concentration of 1000 μg / ml The results are shown in Table 1 below.

Polyphenol content (mg GAE/g) Example 1 3.15 Example 2 2.75 Comparative Example 1 0.07 Comparative Example 2 0.11 Comparative Example 3 0.17

As shown in Table 1, Example 1 of the present invention was confirmed to have a polyphenol content of 3.15 mg GAE / g and Example 2 of 2.75 mg GAE / g compared to the standard material. On the other hand, Comparative Examples 1 to 3 showed significantly low polyphenol content.

(2) Measurement of total flavonoid content

According to the method of Moreno et al., 75 μL of ethanol, 140 μL of distilled water, 5 μL of 1 M potassium acetate, and 5 μL of 10% aluminum nitrate were sequentially added to 25 μL of the sample, mixed, reacted at room temperature for 40 minutes, and absorbance was measured at 415 nm. . Then, using Quercetin (Sigma Co., USA) as a standard material, the total flavonoid content was calculated from a standard calibration curve obtained in the concentration range of 1 to 500 μg/mL, and is shown in Table 2 below.

Flavonoid content (mg QE/g) Example 1 0.90 Example 2 1.21 Comparative Example 1 0.03 Comparative Example 2 0.06 Comparative Example 3 0.05

As shown in Table 2, the flavonoid content of Examples 1 and 2 was significantly improved compared to Comparative Examples 1 to 3.

Through the results of Test Example 1, mixing all three of the two mixtures of sprout barley extract, vitamin tree fruit fermented extract, and vitamin tree fruit supercritical extract were mixed to improve the total polyphenol and flavonoid content. know how important it is.

Test Example 2. Evaluation of antioxidant activity

Examples 1 to 2 and Comparative Examples 1 to 3 and DPPH (2,2-diphenyl-1-picrylhydrazyl) were mixed in a 1: 1 ratio, and after reacting for 30 minutes, the absorbance was measured at 517 nm, and the control ascorbic acid (ascorbic acid) acid) is set to 100%, and the sulfuration power obtained is shown in Table 3 below.

division Antioxidant power (%, DPPH radical scavenging ability) Control (L-ascorbic acid) 100.0 Example 1 112.6 Example 2 125.4 Comparative Example 1 85.2 Comparative Example 2 86.9 Comparative Example 3 86.4

As shown in Table 3, the DPPH scavenging ability of Examples 1 and 2 was significantly higher than that of Comparative Examples 1 to 3, and it can be seen that the DPPH scavenging ability is higher than that of the control group.

Through this, it was confirmed that the mixture of the sage barley extract, the fermented vitamin tree fruit extract, and the vitamin tree fruit supercritical extract or liposomes thereof prepared according to Examples 1 and 2 had excellent antioxidant activity.

Test Example 3. Evaluation of whitening effect

Evaluation of Tyrosinase Inhibition

Tyrosinase is an enzyme involved in the most important initial rate-determining step in the melanin biosynthetic pathway in the human body. The ability to inhibit tyrosinase enzyme activity, which is a representative method used for evaluating whitening efficacy, was measured according to the following method.

A sample (0.5 mg/mL) was dissolved in ethanol or an appropriate solvent and diluted to an appropriate concentration range to inhibit tyrosinase activity, and was used as a sample solution. 140 μl of 100 mM Potassium Phosphate Buffer (pH 6.5), 40 μl of sample solution, and 20 μl of mushroom tyrosinase (1000 U/mL) were sequentially added to a 96-well plate and reacted at 37° C. for 10 to 15 minutes. Then, 50 μl of L-Tyrosine was added and reacted at 37 ° C for 10 to 15 minutes. The absorbance was measured at 490 nm using an ELISA reader, and 0.1 M phosphate buffer (pH 6.5) was added instead of the sample solution as a blank sample. At this time, using arbutin as a positive control group and using a buffer solution as a control group, the tyrosinase inhibitory ability (%) was measured according to Equation 1 below and shown in Table 4 below.

[Equation 1]

Tyrosinase inhibition ability (%) = {1-(sample absorbance/control absorbance)} × 100

division Tyrosinase inhibitory activity (%) Sample treatment concentration (mg/mL) 0.005 0.01 0.5 1.0 Positive control group (Arbutin) 42.89 44.76 48.57 53.15 Example 1 45.87 51.23 53.45 58.13 Example 2 48.54 54.36 58.47 63.25 Comparative Example 1 11.25 15.43 19.15 21.21 Comparative Example 2 11.46 15.89 20.46 25.13 Comparative Example 3 15.87 19.54 23.43 27.46

As shown in Table 4, it was confirmed that Examples 1 and 2 had significantly better tyrosinase inhibitory effects than Comparative Examples 1 and 3. In addition, when comparing Examples 1 and 2, it was confirmed that the tyrosinase inhibitory effect of the liposomalized Example 2 was superior.

Through this, a two-type mixture containing two extracts of sprout barley extract, vitamin tree fruit fermented extract, and vitamin tree fruit supercritical extract, or a three-type mixture containing all three extracts than liposomes thereof (Comparative Examples 1 to 3) Alternatively, it was confirmed that the tyrosinase inhibitory efficacy of the liposomes (Examples 1 to 2) was remarkably high, so it can be seen that Examples 1 to 2 can be used very effectively for skin whitening.

Test Example 5. Skin wrinkle improvement efficacy evaluation

HS68 cell culture

HS68 cells, which are human-derived skin fibroblasts, were purchased from Korea Cell Line Bank and used. HS68 cells were cultured in DMEM medium (Welgene) in a 37°C humidified CO ₂ incubator using a cell culture medium (complete DMEM culture medium) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin ( 5% CO ₂ /95% air). When the cells were about 80% full of the culture dish, the cell monolayer was washed with phosphate buffer saline (PBS, pH 7.4), and trypsin-2.65 mM EDTA was added to detach and subculture the cells, and the medium was changed every 2 days. .

Measurement of cell viability of HS68 cells

HS68 cells were dispensed in a 24-well plate to be 1 × 10 ⁵ cells/well, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, the cells were cultured for 24 hours by replacing them with serum-free cell culture medium containing various concentrations of each test substance. The absorbance of the cultured HS68 cells was measured at 570 nm, and the cell viability was calculated using the MTT Assay as shown in Equation 2 below, and the results are shown in Table 5 below.

[Equation 2]

Cell viability (%) = [absorbance of sample treatment group/absorbance of control group] x 100

division Cell viability (%) Sample treatment concentration (mg/mL) 0.005 0.01 0.5 1.0 Negative control group (no additives) 100 Example 1 99.1 98.9 98.5 98.3 Example 2 99.3 98.5 98.3 97.8 Comparative Example 1 93.1 93.7 94.3 94.5 Comparative Example 2 93.5 94.1 94.8 95.6 Comparative Example 3 93.0 93.2 93.8 94.5

As shown in Table 5, the mixture of the sprout barley extract, the fermented vitamin tree fruit extract and the vitamin tree fruit supercritical extract (Example 1) and the liposome thereof (Example 2) of the present invention can be safely used on human skin confirmed.

Measurement of type 1 procollagen content (%) produced and secreted by HS68 cells

In order to confirm the effect of improving skin wrinkles, the amount of production of type I procollagen protein, which maintains skin elasticity, was measured as follows.

HS68 cells were dispensed in a 24-well plate to be 1 × 10 ⁵ cells/well, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, the cells were cultured for 24 hours by replacing them with serum-free cell culture medium containing various concentrations of each test substance. After culturing the HS68 cells for 24 hours, the cell culture medium was collected and the type I procollagen content was measured using the Procollagen Type I C-Peptide (PIP) EIA Kit (Takara) according to the method suggested by the manufacturer, respectively. Negative control group ( untreated group) as 100% and calculated as a comparative value, and is shown in Table 6 below.

division Type I procollagen content (%) Sample treatment concentration (μg/mL) 100 500 1000 Negative control group (untreated group) 100 Positive control group (L-ascorbic acid 200 μM) 140.1 Example 1 95.1 120.4 153.2 Example 2 98.6 135.1 162.4 Comparative Example 1 81.2 91.2 98.1 Comparative Example 2 78.4 92.4 95.8 Comparative Example 3 77.1 86.1 92.5

As shown in Table 6, it can be seen that the production of type 1 procollagen was significantly promoted in Examples 1 and 2 compared to Comparative Examples 1 to 3. Through this, a two-type mixture of a mixture of two of a sprout barley extract, a vitamin tree fruit fermented extract, and a vitamin tree fruit supercritical extract, or a three-type mixture of all three extracts mixed than liposomes thereof (Comparative Examples 1 to 3), or a mixture thereof In liposomes (Examples 1 and 2), it was confirmed that the skin wrinkle improvement effect was doubled.

Measurement of MMP-1 content

In order to confirm the skin wrinkle improvement effect, the degree of inhibition of MMP-1 expression involved in wrinkle formation was measured according to the following method, and the negative control group (untreated group) was set as 100% and calculated as a comparative value and shown in Table 7 below was

HS68 cells were dispensed in a 24-well plate to be 1 × 10 ⁵ cells/well, and the cells were cultured for 24 hours. After culturing the cells for 24 hours, the cells were cultured for 24 hours by replacing them with serum-free cell culture medium containing various concentrations of each test substance. After culturing the cells for 24 hours, the cell culture medium was collected and the MMP-1 content was measured using the Human MMP-1 ELISA Kit (Sigma-Aldrich) according to the method suggested by the manufacturer.

division MMP-1 content (%) Sample treatment concentration (μg/mL) 100 500 1000 Negative control group (untreated group) 100 Positive control group (TGF-β1 10 ng/mL) 36.2 Example 1 75.1 48.1 35.5 Example 2 70.5 42.5 32.5 Comparative Example 1 98.5 72.5 67.5 Comparative Example 2 95.4 69.4 62.5 Comparative Example 3 94.5 70.5 65.2

As shown in Table 7, it was confirmed that Examples 1 to 2 inhibited MMP-1 protein expression to a more excellent level than Comparative Examples 1 to 3. Through this, it can be confirmed that Examples 1 and 2 can be effectively used for skin wrinkle improvement purposes that effectively prevent wrinkles and improve generated wrinkles.

Through the results of Test Example 5, a two-type mixture of a mixture of two of a sprout barley extract, a fermented vitamin tree fruit extract, and a vitamin tree fruit supercritical extract, or a mixture of all three extracts compared to liposomes thereof (Comparative Examples 1 to 3) The effect of improving skin wrinkles was significantly improved in one of the three extracts or their liposomes (Examples 1 and 2), and the inclusion of all of the barley sprout extract, the fermented extract of the fruit of the vitamin tree, and the supercritical extract of the fruit of the vitamin tree improved skin wrinkles. It can be seen that it is important for efficacy.

Test Example 6. Evaluation of Liposome Performance

1. Confirmation of the stability of liposomes (fine particles that promote transdermal absorption)

(1) Experiment method

Total polyphenol content was measured according to the Folin-Denis method. After adding 10 μL of Folin-Ciocalteau phenol reagent and 100 μL of distilled water to 5 μL of the sample, they were mixed at room temperature for 3 minutes. Then, 100 μL of a 20% Na ₂ CO ₃ solution was added to the mixed solution, followed by reaction at room temperature for 1 hour, and then absorbance was measured at 725 nm using a spectrophotometer. As a standard material, the total polyphenol content of the sample extract was calculated from the standard calibration curve obtained by analyzing gallic acid in the concentration range of 25 to 800 μg/mL in the same way.

(2) measurement of entrapment rate of liposome formulation

1 mL of the liposome suspension of Example 2 was taken and polyphenols not captured were removed using a sephadex G-50 column. Thereafter, an excessive amount of ethanol was added to break the membrane of the liposome, and then the ethanol was evaporated using a vacuum concentrator. Then, 1 mL of ethanol was added and dissolved to measure the polyphenol content captured in the liposome of Example 2. Next, the polyphenol content of the mixture of the sprout barley extract, the vitamin tree fruit fermented extract, and the vitamin tree fruit supercritical extract used in the liposome preparation of Example 2 and the polyphenol content captured in the liposome of Example 2 were calculated by the following equation The entrapment efficiency of liposomes was calculated by substituting in 3.

[Equation 3]

Entrapment efficiency (%) = (content of polyphenol captured in liposome / content of polyphenol in initial extract) x 100

The polyphenol content of the mixture of the barley sprout extract, the fermented vitamin tree fruit extract, and the vitamin tree fruit supercritical extract used in the preparation of liposomes in Example 2 was 3.12 mg GAE/g, and when captured in liposomes (Example 2), The phenol content was 2.56 mg GAE/g, indicating a capture rate of about 82%.

(3) Evaluation of stability of liposome formulation

Immediately after preparation of the liposome of Example 2 (0 month) and after storage for 3 months under accelerated conditions of temperature of 40±2° C. and relative humidity of 75±5%, the polyphenol content was measured and shown in Table 8.

sample Results (mg GAE/g) Example 2 (month 0) 2.56 Example 2 (3 months) 2.50

As shown in Table 8, after 3 months, the polyphenol content was 2.50 mg GAE / g, which was 98% or more compared to the amount of the initial liposome formulation, stably maintaining the formulation without significant change. .

2. Evaluation of skin permeability

In order to evaluate the skin permeability of a mixture of sprout barley extract, vitamin tree fruit fermented extract, and vitamin tree fruit supercritical extract and its liposome formulation, skin pampa, an artificial skin membrane, was used for comparison, and the permeability was analyzed by the polyphenol content, which is an indicator substance. did

(1) Evaluation method

The skin permeability evaluation test method was performed by the skin pampa method. A blank was prepared with prisma buffer and analyzed (reading). 1,000 μL of Prisma Buffer was put into a deep well plate according to the pH-map. Open the lid of the stock plate, take 5 μL of the sample using an 8 channel pipette, transfer it to the deep well plate, adjust the pipette to 500 μL, and mix 4 to 6 times. Then, the concentration of the solution was measured or calculated and the result was used as <reference> of the assay.

A donor plate for the skin test assay was prepared by transferring 200 μL of the sample solution from the deep well plate to the donor plate and covering with a lid. Then, adjust the 8 channel pipette to 200 μL, hydrate the well, prepare the donor plate assay, and transfer the top plate from the hydration solution reservoir to the support plate. moved Then, 200 μL of pH 7.4 Prisma buffer was added to each well of the acceptor plate, and the donor plate was removed. Taking care not to generate bubbles, slowly lift the acceptor plate onto the donor plate from the bottom row (H) to make a sandwich, and cover the acceptor plate with a lid to prepare a pampa sandwich with the acceptor plate placed on the donor plate. Then, to prevent drying of the skin membrane, the experiment should be started after finishing within 4 to 5 minutes. To minimize evaporation, sandwiches were placed in a humidity chamber or gut-box with wet sponges to maintain high relative humidity, and the sandwich plate was never tilted. Then, it was incubated for 5 hours at room temperature without agitation. After incubation, take out the PAMPA sandwich from the humidity chamber, open the lid, transfer 150 μL from the acceptor plate to the UV plate by using an 8-channel pipettor, and then set the range to 250 ~ 498 nm in a spectrophotometer and analyze it.

(2) Evaluation results

Permeation of the extract before permeation by the skin pampa method described above, the extract of barley sprout used in the preparation of the liposome of Example 2, the three mixtures of the fermented extract of the vitamin tree fruit and the supercritical extract of the fruit of the vitamin tree, and the liposome of Example 2 obtained by liposomizing the same. After that, the transmittance (%) was obtained by comparing the total polyphenol content and is shown in Table 9.

Total polyphenols (mg GAE/g) Transmittance (%) extract before permeation 3.17 3 types of extract 0.0327 1.03% Example 2 0.1027 3.24%

Referring to Table 9, it can be seen that the liposome of Example 2 exhibits a higher permeability when liposomalized (Example 2), so it can be predicted that the liposome of Example 2 will help to penetrate the skin and exert efficacy.

3. Clinical evaluation of skin compatibility

A patch experiment was conducted to confirm that the liposome of Example 2 had no skin irritation or side effects. According to the selection and exclusion criteria, 33 healthy female volunteers aged 20 to 60 were selected as subjects and a 48-hour single patch test was conducted according to Frosch & Kligman, ICDRG, and PCPC guidelines. At this time, the average age of the subjects was 44.6 ± 8.0 years old, the highest age was 58 years old, and the lowest age was 25 years old. The patch experiment was conducted according to the following conditions.

First, after disinfecting the patch site with 70% ethanol, Van der Bend (Van der Bend, Netherlands) to which 20 μl of the sample (800 μg/mL) of Example 2 was applied was applied. After 48 hours, the patch was removed, and the test site was marked with a skin marker (Chemotechnique Diagnostics AB, Sweden), and the reactivity of each test site was evaluated after 30 minutes and 24 hours. The evaluation criteria for the primary skin irritation test (Skin Primary irritation Test) were in accordance with the criteria of the PCPC Guidelines presented in Table 10 below, and the degree of skin reaction (average reactivity) evaluated twice was determined according to Equation 4 below. It was scored and the results are shown in Table 11.

[Equation 4]

Average reactivity = [{(reactivity × number of responding subjects)/(total number of subjects × maximum score (5 points))} × 100] × 1/2

reactivity Clinical symptoms (reaction) 0 unresponsive One faint or mild erythema 2 Moderate erythema and barely detectable edema and/or papule formation at the border of patches 3 moderate erythema; and edema spread throughout the patch area 4 severe erythema; and severe edema; or blistering 6 Severe reaction spreads beyond the patch site

Number of subjects who responded (persons) reactivity 30 minutes 24 hours average negative control group 0 0.0 0.0 0.0 Example 2 0 0.0 0.0 0.0

As shown in Table 11, as a result of the patch experiment, no skin reaction was observed in all subjects at the time of observation after removing the patch from all subjects, indicating that it was a hypoallergenic material.

Hereinafter, formulation examples of cosmetic compositions suitable for application of the present invention are presented.

Formulation Example 1. Lotion

Formulation examples of lotion containing a mixture of the sprout barley extract of Example 1, the fermented vitamin tree fruit extract, and the vitamin tree fruit supercritical extract are shown in Table 12 below.

ingredient Content (% by weight) Example 1 5 glycerin 7 1,3-butylene glycol 3 sodium hyaluronate 5 ethanol 10 Preservatives, Colors and Flavors a very small amount Purified water balance

Formulation Example 2. Lotion

Formulation examples of the lotion containing liposomes of the sprout barley extract, the fermented vitamin tree fruit extract, and the vitamin tree fruit supercritical extract of Example 2 are shown in Table 13 below.

ingredient Content (% by weight) Example 2 5 glycerin 5 1,3-butylene glycol 4 liquid paraffin 10 stearic acid 1.3 triethanolamine 1.2 Tocopheryl Acetate 3 squalane 2 Sorbitan sesquioleate One carboxyvinyl polymer One Preservatives, Colors and Flavors a very small amount Purified water balance

Formulation Example 3. Nutritional Cream

Formulation examples of the lotion containing the liposomes of the sprout barley extract, the fermented vitamin tree fruit extract, and the vitamin tree fruit supercritical extract of Example 2 are shown in Table 14 below.

ingredient Content (% by weight) Example 2 5 Pro-type glycerin monostearate 1.5 cetearyl alcohol 1.5 stearic acid 1.0 polysorbate 1.5 Sorbitan Stearate 0.6 squalane 5.0 isostearyl isostearate 5.0 mineral oil 35.0 dimethicone 0.5 Hydroxyethyl Cellulose 0.13 glycerin 6.0 triethanolamine 0.7 Preservatives, Colors and Flavors a very small amount Purified water balance

Claims (8)

Sprout barley extract;
Fermented vitamin tree fruit extract obtained by fermenting vitamin tree fruit with Lactobacillus rhamnosus HK-9 (accession number: KCCM11254P); and
Vitamin tree fruit supercritical extract; antioxidant, skin whitening and skin wrinkle improvement cosmetic composition containing as an active ingredient. The method of claim 1, wherein the cosmetic composition is antioxidant, skin whitening, characterized in that liposomes of the oily component containing the supercritical extract of the fruit of the vitamin tree, and the aqueous component including the extract of barley sprout and the fermented extract of the vitamin tree. and a cosmetic composition for improving skin wrinkles. The cosmetic composition for antioxidant, skin whitening and skin wrinkle improvement according to claim 1, wherein the sprout barley extract is extracted using 70 to 85% by weight of ethanol as a solvent. According to claim 1, wherein the vitamin tree fruit fermented extract is antioxidant, skin whitening and skin wrinkle improvement cosmetic composition, characterized in that extracted by fermenting vitamin tree fruit at 25 to 45 ℃ for 36 to 60 hours. The method of claim 1, wherein the vitamin tree fruit supercritical extract is extracted at a pressure of 300 to 450 bar and a temperature of 40 to 60 ° C. using carbon dioxide as a supercritical fluid for antioxidant, skin whitening and skin wrinkle improvement. cosmetic composition. The method of claim 1, wherein the cosmetic composition is characterized in that the sprout barley extract, the vitamin tree fruit fermented extract and the vitamin tree fruit supercritical extract are mixed in a weight ratio of 1: 0.5 to 1.5: 0.5 to 1.5. A cosmetic composition for whitening and improving skin wrinkles. The cosmetic composition for antioxidant, skin whitening and skin wrinkle improvement according to claim 1, wherein the cosmetic composition inhibits the activity of tyrosinase. According to claim 1, wherein the cosmetic composition inhibits MMP-1 expression, anti-oxidation, skin whitening and skin wrinkle improvement cosmetic composition, characterized in that increases the production of type I procollagen protein.

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