KR102344703B1 - Method of Trapa Natans ferments, a composition for improving skin wrinkles and anti-aging containing the Trapa Natans fermentation product or peptide produced therefrom as an active ingredient and a composition for improving skin aging comprising a fermented product - Google Patents

Abstract

Disclosed is a cosmetic composition capable of inhibiting the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promoting the generation of intracellular collagen, and proliferating epidermal cells. The present invention provides a cosmetic composition comprising a dried dried fermented product or a purified dried dried fermented product as an active ingredient.

Description

A method for manufacturing a fermented dried dried product and a composition for improving skin aging comprising a fermented product and a composition effective for skin wrinkle improvement and anti-aging containing a dried dried product or peptide prepared therefrom as an active ingredient {Method of Trapa Natans ferments, a composition for improving skin wrinkles and anti-aging containing the Trapa Natans fermentation product or peptide produced therefrom as an active ingredient and a composition for improving skin aging comprising a fermented product}

The present invention relates to a cosmetic composition, and more particularly, to a cosmetic composition comprising a dried dried fermented product or a fermented dried dried product.

One of the fields of greatest interest in cosmetics is aging-related wrinkles, pigmentation, loss of elasticity, dry skin, hair loss, and lack of hair gloss. The skin undergoes various changes through aging. First, the thickness of the epidermis, dermis, and subcutaneous tissue, which are components of the skin, becomes thinner and the extracellular matrix (ECM) component that gives elasticity to the skin changes. With age, its production decreases rapidly and has a close relationship with wrinkle formation, and collagen, elastin, proteoglycans, and glucosaminoglycans that make up the skin connective tissue , laminin, fibronectin, etc. are oxidized and lose their functions, so that the skin loses elasticity and wrinkles are excessively formed, changing to senile skin.

The connective tissue fibers of the extracellular matrix include glue fibers (collagen fibers), reticular fibers, and elastic fibers (elastic fibers), of which collagen accounts for about 70% of the skin connective tissue. is mostly formed in the fibroblasts of the skin. Collagen content in the skin connective tissue decreases with age, which is due to a decrease in collagen synthesis and promotion of decomposition. Therefore, the decrease in collagen biosynthesis and the promotion of collagen decomposition are the biggest causes of skin wrinkles.

One of the fields of greatest interest in cosmetics is aging-related wrinkles, pigmentation, loss of elasticity, dry skin, hair loss, and lack of hair gloss. The skin undergoes various changes through aging. First, the thickness of the epidermis, dermis, and subcutaneous tissue, which are components of the skin, becomes thinner and the extracellular matrix (ECM) component that gives elasticity to the skin changes. With age, its production decreases rapidly and has a close relationship with wrinkle formation, and collagen, elastin, proteoglycans, and glucosaminoglycans that make up the skin connective tissue , laminin, fibronectin, etc. are oxidized and lose their functions, so that the skin loses elasticity and wrinkles are excessively formed, changing to senile skin.

Registered Patent No. 10-1784201 discloses a composition for improving skin including fermented natural products as an active ingredient, but does not mention the composition for cosmetics using dry skin.

Accordingly, the present invention has been devised to solve the above problem, and provides a cosmetic composition capable of inhibiting the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promoting the generation of intracellular collagen, and proliferating epidermal cells want to

In order to solve the above problems, the present invention provides a cosmetic composition comprising a dried dried fermented product or a dried dried fermented product as an active ingredient.

In addition, the dried fermented product provides a cosmetic composition characterized in that it is fermented with a Bacillus sp. strain, a Lactobacillus sp. strain, or a mixed strain thereof.

In addition, the Bacillus genus ( Bacillus sp. ) The strain is Bacillus methylotrophicus ( Bacillus methylotrophicus ) or Bacillus subtilis ( Bacillus subtilis ), and the Lactobacillus genus ( Lactobacillus sp. ) The strain is Lactobacillus paracasei ( Lactobacillus paracasei ) ) provides a cosmetic composition, characterized in that.

In addition, the dried fermentation purified product is a tripeptide, and the tripeptide is glycine (Glycine, Gly)-proline (Proline, Pro)-serine (Serine, Ser), glycine (Glycine, Gly)-proline (Proline) , Pro)-Arginine (Arginine, Arg), Glycine (Glycine, Gly)-Proline (Proline, Pro)-Proline (Proline, Pro) or Glycine (Glycine, Gly)-Proline (Proline, Pro)-Methionine, Met) provides a cosmetic composition, characterized in that.

In addition, the cosmetic composition provides a cosmetic composition comprising 1 to 90% by weight of the dried dried fermented product or the fermented dried dried product.

In addition, the cosmetic composition provides a cosmetic composition characterized in that it inhibits the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promotes intracellular collagen production, and promotes the proliferation of epidermal cells.

For another object of the present invention, the method comprising: inoculating and fermenting the extract of the quince fruit extract with microorganisms to prepare a fermented product; and removing the microorganisms from the fermented product; provides a method for producing dried dried products comprising.

The present invention is a cosmetic composition capable of inhibiting the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promoting the generation of intracellular collagen, and proliferating epidermal cells by including a fermented dried dried dried product or a purified dried dried dried product as an active ingredient can provide

1 is a schematic diagram of a method for manufacturing a dried dried fermented product according to the present invention,
2 is a graph showing the results of Experimental Example 2 of the present invention;
3 is a photograph taken of the TLC result of Experimental Example 3 of the present invention;
4 is a photograph taken of the TLC result of Experimental Example 4 of the present invention;
5 is a graph showing the results of Experimental Example 5 of the present invention;
6 is a graph showing the results of Experimental Example 6 of the present invention;
7 is a graph showing the high performance liquid chromatography analysis results according to the present invention (FIG. 7a is comp2: Gly-Pro-Ser, FIG. 7b is comp1: Gly-Pro-Arg, FIG. 7c is comp3: Gly-Pro-Pro, Figure 7d shows comp4: Gly-Pro-Met),
8 is a graph showing the results of NMR analysis of Experimental Example 7 according to the present invention (FIG. 8a is comp2: Gly-Pro-Ser, FIG. 8b is comp1: Gly-Pro-Arg, FIG. 8c is comp3: Gly-Pro-Pro , Figure 8d is comp4: Gly-Pro-Met),
9 is a graph showing the MMP-1 inhibitory ability measurement result of Experimental Example 8 of the present invention;
10 is a graph showing the results of the intracellular collagen production promoting effect of Experimental Example 9 of the present invention;
11 and 12. Graphs showing the results of measuring the cell proliferation capacity of Experimental Example 10 of the present invention in 3T3 cells and HACAT cells
13 and 14 are graphs showing optical stability and thermal stability measurement results of Experimental Example 11 of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings, and the inventor should properly understand the concept of the term in order to best describe his invention. Based on the principle that can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention. Accordingly, since the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention and does not represent all the technical spirit of the present invention, various equivalents and modifications that can be substituted for them at the time of the present application It should be understood that there may be

In order to effectively prevent or improve skin aging and wrinkles, the present inventors have repeatedly studied to develop raw materials with confirmed antioxidant efficacy and cosmetic compositions effective for skin aging, as a result of fermenting the extract of the quince fruit, or fermenting the quince It was found that, when a peptide derived from water is used, it is possible to inhibit the activity of collagen degrading enzyme (Matrix Metalloproteinase-1), promote intracellular collagen production, and improve the efficacy of promoting epidermal cell proliferation, leading to the present invention.

Accordingly, the present invention discloses a cosmetic composition comprising a dried dried fermented product or a purified dried dried fermented product as an active ingredient.

In the present invention, the hydrangea ( Trapa japonica ) is an annual grass growing in a pond and can be mainly found in ponds or swamps. The rhombuses are called rhombuses because the leaves are rhombic triangles, and the fruits of the rhombuses are also called water chestnuts, open downward in the water, and have a unique shape with 2 to 4 horns.

In the present invention, the fermented watermelon refers to a fermented product obtained after drying and extracting the fruit of the seaweed, and the extract of seaweed is used as a drug effective for diseases such as gastric ulcer, and is rich in manganese to maintain skin moisture. has an effect on

The fermented dried dried or fermented dried dried product of the present invention is a raw material that acts on the skin to help improve skin wrinkles and alleviate skin aging, and by refining a specific effective ingredient, the effect can be further enhanced compared to before purification In addition, dried dried fermented products and dried dried fermented products can be used together.

According to another aspect, the present invention provides a method for producing a fermented watermelon product, the method comprising: inoculating and fermenting an extract of a watermelon fruit to prepare a fermented product; and removing the microorganisms from the fermented product.

Hereinafter, the present invention will be described in detail through the method for producing the dried fermented product.

In the present invention, the step of preparing a fermented product by inoculating and fermenting the extract of the quince fruit extract is a step for increasing the content of functional substances in the quince fruit.

The extract of the quince fruit may be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the preparation of the mixed extract using the solvent extraction method is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol and butanol) or a mixture thereof, which is a hydrous lower alcohol, propylene glycol, 1, 3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, dichloromethane, hexane, and mixtures thereof may be selected from the group consisting of, among which water, alcohol, or mixtures thereof When alcohol is used as the extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol, and even more preferably ethanol. have.

In addition, when extracting the quince fruit extract with ethanol, for example, 70% (v/v) ethanol may be added in an amount 5 to 20 times the weight of the quince fruit and extracted at room temperature for 18 to 30 hours, The sugar content of the extract of the quince fruit may be 4 to 10 brix, preferably 6 to 8 birx.

In addition, in the case of extracting the quince fruit extract by hot water extraction, for example, purified water may be added in an amount 5 to 20 times the weight of the quince fruit, and extracted at 100 to 140 ° C. for 4 to 8 hours, and the quince fruit The sugar content of the extract may be 4 to 10 brix, preferably 6 to 8 birx.

In the present invention, the microorganism may be fermented into a Bacillus genus (Bacillus sp . ) strain, a Lactobacillus sp . strain or a mixed strain thereof, and the Bacillus sp . strain is Bacillus methylotropicus ( Bacillus methylotrophicus ) Or Bacillus subtilis ( Bacillus subtilis ) It may be, the Lactobacillus genus ( Lactobacillus sp . ) The strain is Lactobacillus paracasei ( Lactobacillus paracasei ) It may be, preferably Bacillus subtilis and Bacillus methyl It may be a mixed strain of Rotropicus.

The fermentation refers to a process in which an enzyme possessed by microorganisms changes raw materials to create beneficial ingredients, and during the fermentation process, active ingredients are extracted and the content of functional substances increases. In addition, in the present invention, in the fermentation, the culture solution of the microbial strain ^{is adjusted to 10 7} to 10 ¹⁰ cfu/ml, and 0.1 to 10% by weight, preferably 0.5 to 5% by weight, can be inoculated with respect to the extract of the quince fruit. and 1 to 5 days, preferably 2 to 4 days, 25 to 45 ℃, preferably 30 to 40 ℃ can be carried out.

In addition, the composition of the medium for culturing and fermenting the microorganisms may be glucose 0.2 to 1% by weight, enzyme extract 0.1 to 0.5% by weight, soytone 0.05 to 0.3% by weight, and distilled water (residual amount).

In the present invention, the step of removing the microorganisms from the fermented product to prepare the dried fermented product is a step of removing microorganisms to prevent further fermentation of the dried fermented product, and the removal of the microorganisms may be removed through centrifugation and filtration, , For example, after separating the microorganisms through a centrifuge in the fermented product, it is possible to remove the microorganisms by filtering with a 0.1 ~ 0.3㎛ filter.

1 is a schematic diagram of a manufacturing method of a dried dried fermented product according to the present invention.

Referring to Figure 1, the solvent fractionation of the dried dried fermented product is a step of extracting peptides by purifying the dried dried ferment, and the solvent fractionation may be sequentially performed through hexane, dichloromethane, ethyl acetate, butanol and water. . In addition, it may be purified through a column after the solvent fractionation.

In addition, in the present invention, the purified water fermented watermelon may be a tripeptide derived from watermelon fruit, and the tripeptide is glycine (Glycine, Gly)-proline (Proline, Pro)-serine (Serine, Ser), Glycine (Gly)-Proline (Proline, Pro)-Arginine (Arginine, Arg), Glycine (Glycine, Gly)-Proline (Proline, Pro)-Proline (Proline, Pro) or Glycine (Glycine, Gly)-Proline (Proline, Pro)-methionine (Methionine, Met) may be.

In the present invention, the cosmetic composition may contain 1 to 90% by weight of the dried dried fermented product or the purified dried dried product, preferably 1 to 20% by weight. In addition, the cosmetic composition may include the dried dried fermented product and the dried dried fermented product, and may each contain 1 to 20% by weight.

It was confirmed that the cosmetic composition according to the present invention has a very excellent effect of inhibiting the activity of collagen-degrading enzymes such as collagenase and MMP-1 (Matrix Metalloproteinase-1), and also has an effect of promoting intracellular collagen production and skin It was confirmed that the effect of improving the cell regeneration ability was also very excellent.

The cosmetic composition according to the present invention may be applied in a gel type, skin type, cream type, ointment type, etc., but is not limited thereto. The above composition can be suitably prepared by a known method by adding an appropriate conventional softening agent, emulsifying agent, thickening agent or other substances known in the art depending on its type.

The gel-type composition may be prepared by adding a softening agent such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, or isostatic alcohol, and purified alcohol.

The skin type composition includes fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, butylene glycol, glycerin, allantoin, methyl paraben, EDTA- It can be prepared by adding 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenated castol oil, polysorbate 60 and purified water.

The cream-type composition includes fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol and isocetyl alcohol, lecithin, phosphatidylcholine, phosphatidylethanolamine, sofatizylserine, sofphatidyl. Lipids such as inositol; It can be prepared by adding a solvent such as propylene glycol and purified water.

The ointment-type composition may be prepared by adding a softening agent, an emulsifying agent, and waxes such as microcrystalline lead, paraffin, ceresin, beeswax, beeswax, and vaseline.

The cosmetic composition of the present invention may further contain one or more skin wrinkle-improving ingredients exhibiting the same or similar function in addition to the active ingredient. The skin wrinkle improvement component may be any one or more selected from the group consisting of vitamin C, retinoic acid, TGF, animal placental-derived protein, betulinic acid, and chlorella extract, but is not limited thereto. In addition, the cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes, humectants, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. can

In the present invention, the cosmetic composition includes softening lotion, astringent lotion, nourishing lotion, eye cream, serum, nourishing cream, massage cream, cleansing cream, cleansing lotion, cleansing foam, cleansing water, powder, essence, pack, hair tonic, hair treatment It can be formulated as a conditioner, shampoo or rinse.

The cosmetic composition may be formulated by a conventional method. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, International cosmetic ingredient dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association) , Inc, Washington, 1995) may be referred to.

Specifically, the cosmetic composition can be prepared in general emulsified formulations and solubilized formulations. For example, lotion, such as a softening lotion or a nourishing lotion; emulsions such as facial lotions and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleaning agent such as a cleansing foam, soap, body wash, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the cosmetic composition, in addition to the essential components in each formulation, other components may be appropriately blended within the range that does not impair the purpose according to the present invention according to the type or purpose of use, etc. of the formulation.

The cosmetic composition may include a conventionally acceptable carrier, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately blended, but is limited thereto no.

The acceptable carrier may vary depending on the formulation. For example, when formulated into an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or their Mixtures may be used.

When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof may be used as a carrier component, and in the case of a spray, chlorofluoro It may further contain a propellant such as hydrocarbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan may be used.

When the cosmetic composition is formulated as a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

The cosmetic composition is a fatty substance, organic solvent, solubilizer, thickening agent, gelling agent, softening agent, antioxidant, suspending agent, stabilizing agent, foaming agent, and fragrance commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, commonly used in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetics or dermatology, such as any other ingredients. However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

The cosmetic composition comprising, as an active ingredient, the fermented dried fermented product or the purified fermented dried fermented product according to the present invention contains the fermented dried fermented product or the purified fermented dried fermented product, thereby inhibiting the activity of the collagen-degrading enzyme (Matrix Metalloproteinase-1), and It promotes the production of inner collagen and can proliferate epidermal cells.

Hereinafter, specific examples according to the present invention will be described.

Preparation Example 1

After adding 2 kg of purified water to 200 g of dried quince, hot water extraction was performed at 120 °C for 6 hours. Extracted after Lactobacillus para Kasei (Lactobacillus paracasei, purchased from Korea Cell Line Bank) by culturing the strain and has a density measured with a spectrophotometer in the diamond fruit extract inoculated with 1.0 × 10 ⁹ cfu / ml of culture solution 1% by weight, at 37 ℃ It was fermented for 3 days, and the medium used for culture was a medium composed of glucose 0.6% by weight, enzyme extract 0.3% by weight, soytone 0.1% by weight, and distilled water. After fermentation, the fermentation broth was centrifuged at 4000 rpm for 10 minutes to separate the lactic acid bacteria and the culture broth, and then the culture broth was filtered through a 0.2 μm filter to prepare dry fermented products.

Preparation 2

As a microorganism in Preparation Example 1, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep Microrganism S001 ( purchased from the Korea Cell Line Bank) was used in the same manner as the dried dried fermented product.

Preparation 3

A dried dried fermented product was prepared in the same manner as in Preparation Example 1, except that Bacillus subtilis (Bacillus subtilis, Apep microrganism K007, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 4

As a microorganism in Preparation Example 1, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep microrganism S001, purchased from Korea Cell Line Bank) and Bacillus subtilis ( Apep ) Microrganism K007 ( purchased from Korea Cell Line Bank) was prepared in the same manner as a dried fermented product, except that a mixed strain mixed with 1:1 parts by weight was used.

Preparation 5

2 kg of 70% (v/v) ethanol was added to 200 g of dried quince fruits and extracted at 25°C for 24 hours. Extracted after Lactobacillus para Kasei (Lactobacillus paracasei, purchased from Korea Cell Line Bank) by culturing the strain and has a density measured with a spectrophotometer in the diamond fruit extract inoculated with 1.0 × 10 ⁹ cfu / ml of culture solution 1% by weight, at 37 ℃ It was fermented for 3 days, and the medium used for culture was a medium composed of glucose 0.6% by weight, enzyme extract 0.3% by weight, soytone 0.1% by weight, and distilled water. After fermentation, the fermentation broth was centrifuged at 4000 rpm for 10 minutes to separate the lactic acid bacteria and the culture broth, and then the culture broth was filtered through a 0.2 μm filter to prepare dry fermented products.

Preparation 6

As a microorganism in Preparation Example 5, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep Microrganism S001 ( purchased from the Korea Cell Line Bank) was used in the same manner as the dried dried fermented product.

Preparation 7

A dried dried fermented product was prepared in the same manner as in Preparation Example 5, except that Bacillus subtilis ( Apep microrganism K007, purchased from Korea Cell Line Bank) was used as a microorganism.

Preparation 8

As a microorganism in Preparation Example 5, Bacillus methylotrophicus (Bacillus methylotrophicus, Apep microrganism S001, purchased from Korea Cell Line Bank) and Bacillus subtilis ( Apep ) Microrganism K007 ( purchased from Korea Cell Line Bank) was prepared in the same manner as a dried fermented product, except that a mixed strain mixed with 1:1 parts by weight was used.

Preparation 9

After adding 2 kg of purified water to 200 g of dried quince fruit, hot water extraction was performed at 120° C. for 6 hours to prepare a quince fruit extract having a sugar content of 6-8 birx.

Preparation 10

2 kg of 70% (v/v) ethanol was added to 200 g of dried quince, and extracted at 25° C. for 24 hours to prepare a quince fruit extract having a sugar content of 6-8 birx.

refinement example

1) n-hexane, dichloromethane, ethyl acetate and butanol were sequentially added to the dried dried fermented product prepared in Preparation Example 4, and separated into an n-hexane fraction, a dichloromethane fraction, an ethyl acetate fraction, a butanol fraction and a water fraction. was prepared.

2) After concentrating the separated water fraction under reduced pressure, it is dissolved in a small amount of 70% (v/v) methanol and an open column filled with Sephadex LH-20 resin (Sigma) (a glass of 28 cm in diameter and 45 cm in length containing 50 g of resin) column) was used to separate SP1 (fr1~2), SP2 (fr3~5), SP3 (fr6~7), SP4 (fr8~14) and SP5 (fr15~21).

3) The separated SP2 (fr3~5) was subjected to silica column chromatography. At this time, a reverse phase silica column was used for the column, and the mobile phase solvent was changed by 1L in the order from 100% distilled water to distilled water: 99.5% (v/v) methanol 90: 10, 80: 20 in the order of fr1 ( 1 to 2), fr2 (3 to 5), fr3 (6 to 7), fr4 (8 to 14) and fr5 (15 to 21).

4) The separated fr2 (3~5) was purified by prep-HPLC (Xterra C18 ODS column, 3ml/min). Solvent A: Water in 0.1% TFA, solvent B: acetonitrile (ACN) in 0.1% TFA, solvent C: 99.5% (v/v) methanol in 95:2.5:2.5 conditions, comp1, comp2, The dried fermented products separated into comp3, comp4, comp5 and comp6 were purified.

Experimental Example 1

In order to confirm the skin wrinkle improvement effect of the dried fermented product prepared in Preparation Examples 1 to 10, an in vitro collagenase activity inhibition experiment was performed according to the following method, and the results are shown in Table 1 below. indicated.

<Experiment method>

(1) Prepare by dissolving collagen in phosphate buffer (100 ppm). At this time, adenosine is prepared at the same concentration as a positive control.

(2) 100mM Tris-cl buffer, 100mM CaCl ₂ and 0.25 mg/ml collagenase, prepare a mixture, and add each test substance and positive control to the mixture at a concentration of 5% (v/v) to proceed with the reaction.

(2) The reaction is terminated by adding 400 μl/tube of 25 mM citric acid.

(3) Thereafter, 1.5 ml/tube of ethyl acetate is added, and after vortexing, the supernatant is taken and transferred to sodium sulfate 150 mg/ep-tube, and centrifuged (10,000 rpm, 3 minutes) after vortexing.

(4) Take 300 μl/ep-tube of the supernatant, transfer it to a quartz 96-well plate, and measure the absorbance at 320 nm.

Referring to Table 1, the collagenase inhibitory ability can be confirmed in all fermented products (Preparation Examples 1 to 8). can be confirmed, and it can be confirmed that the collagenase inhibitory ability is superior to that in the case of not fermenting (Preparation Examples 9 and 10).

In addition, when using the Lactobacillus sp. strain (Preparation Examples 1 and 5) than when using the Bacillus sp. strain (Preparation Examples 2 to 4 and Preparation Examples 6 to 8), the collagenase inhibitory ability is excellent, and a single strain It can be seen that the collagenase inhibitory ability in the case of using the mixed strain (Preparation Examples 4 and 8) is superior to the case of using (Preparation Examples 1, 2, 3, 5, 6 and 7).

In addition, it can be seen that the case of hot water extraction (Preparation Examples 1 to 4) is superior to the case of ethanol extraction (Preparation Examples 5 to 8) than the collagenase inhibitory ability.

Experimental Example 2

The protein content of the extracts of the dried chestnut fruit extract of Preparation Example 9, the fermented dried dried radish extract of Preparation Example 4 and the fractions isolated in the purification example was measured using the lowry method, and the measurement results are shown in Table 2 and FIG. 2 below.

Referring to Table 2 and FIG. 2 , it can be seen that the quantitative value of the protein content of the fermented lychee (Preparation Example 4) is increased by about 12.3% compared to the extract of the lychee fruit (Preparation Example 9), Example) can confirm that the water fraction has the highest protein content.

In addition, it can be confirmed that the protein content of the dried dried fermented fraction (purified example) is twice or more than that of the dried dried fermented product (Preparation Example 4).

Experimental example 3

For SP1 to SP5 separated in the above purification example, silica gel thin layer chromatography (TLC) was performed in a mixed solvent of dichloromethane: 99.5% (v/v) methanol (1:1), and the TLC pattern according to this was performed 3 is shown.

Referring to FIG. 3 , as a result of confirming the separated material, it can be confirmed that it is a protein, and accordingly, protein purification conditions can be confirmed.

Experimental example 4

For fr1 to fr5 separated in the above purification example, silica gel thin layer chromatography (TLC) was performed in a mixed solvent of dichloromethane: 99.5% (v/v) methanol (1:1), and the TLC pattern according to this was performed 4 is shown.

Referring to FIG. 4 , as a result of confirming the separated material, it can be confirmed that it is a peptide, and accordingly, the conditions for purifying the peptide can be confirmed.

Experimental example 5

The protein content contained in water (water layer), SP2, fr2 and comp2 separated in the purification example was measured using the lowry method, and the measurement results are shown in Tables 3 and 5 below.

Referring to Table 3 and FIG. 5 , it was confirmed that the protein content increased during separation and purification through High Pressure Liquid Chromatography (HPLC), but it was confirmed that the protein content did not increase above a certain level.

Experimental Example 6

The content of the target peptide (Gly-Pro-Ser) contained in the dried fermented product according to Preparation Example 4, the water layer of the purification example, SP2, and comp1 to comp4 was measured using the same prep-HPLC as in the purification example, and the measurement result is shown in Table 4 and Figure 6 below.

Referring to Table 3 and Figure 6, in the case of dried fermented product (Preparation Example 4) and comp4 (Purified Example), the target peptide did not appear, but different peptides were present, and in the case of comp2 (Purified Example), the target peptide was It can be seen that the content is improved up to 92%.

Experimental example 7

The HPLC analysis results of comp1 to comp4 separated in the purification example are shown in FIG. 7 , and the experiment was performed according to the following experimental method to confirm the structure of comp1 to comp4, and the results are shown in FIG. 8 .

<Experiment method>

After preparing each sample at 100 ppm, the mass for each sample was measured using a mass (LCMS-IT-TOF, SHIMADZU). Then, using 600 MHz NMR (Agilent Technologies (DD2 600 MHz FT NMR)), 1H, 13C, HMBC, HMQC, COSY and DEPT were measured to confirm the structure.

Referring to Figure 8a, in the case of Tripeptide-G1 (comp2) ^The exact structure was identified through 1 H NMR, ¹³ C NMR, and 2D NMR, and _{CH 2} *2 peaks split by the characteristic surrounding environment of pyrrolidine at δ 1.8 to 2.173 in ^{1 H NMR.} was confirmed, and characteristic peaks of CH2-CH were confirmed at δ3.4 to 3.829. In addition, the total number of CH, CH ₂ , CH ₃ was identified by checking C*3, CH*2, CH ₂ *5 through DEPT, and pyrrolidine confirmed by H-NMR through 2D-NMR It was confirmed that it was bound to a double bond of and C=O, and a C=O peak bound in _{CH-CH 2 was also confirmed.} Through this, when it was confirmed with the molecular weight confirmed by MS, it was confirmed that it was a structure in which Ser was attached to Gly-Pro (Gly-Pro-Ser). After that, peptide synthesis was carried out, and it was confirmed that a peak appeared at the same time by comparing the synthesized peptide and the peptide separated and purified from the fermented product through HPLC.

Referring to FIG. 8b, in the case of Tripeptide-G2 (comp1), _{CH 2} *2 peaks split by the characteristic surrounding environment of pyrrolidine at δ 1.8 to 2.173 were confirmed through ^{1 H NMR, δ 1.510 to 1.8} The peaks of CH ₂ -CH ₂ -CH ₂ were confirmed in the band, and the CH peak was confirmed at δ4.2. Through this, it was confirmed that the molecular weight confirmed in Ms and the structure in which Arg is attached to Gly-Pro (Gly-Pro-Arg) were confirmed. After that, peptide synthesis was carried out, and it was confirmed that a peak appeared at the same time by comparing the synthesized peptide and the peptide separated and purified from the fermented product through HPLC.

Referring to FIG. 8c , in the case of Tripeptide-G3 (comp3), _{CH 2} *2 peaks split by the characteristic surrounding environment of pyrrolidine at δ 1.8 to 2.173 were confirmed through ¹ H NMR, and proline through this peak (proline) was confirmed. However, it was confirmed that this peak had a structure of pro-pro because it was doubled, and these peaks were also confirmed twice, confirming that it was a structure with two pros (Gly-pro-pro). After that, peptide synthesis was carried out, and it was confirmed that a peak appeared at the same time by comparing the synthesized peptide and the peptide separated and purified from the fermented product through HPLC.

Referring to Figure 8d, in the case of Tripeptide-G4 (comp4) ^The structure was identified through 1 H NMR, ¹³ C NMR, and 2D NMR, and _{CH 2} *2 peaks split by the characteristic surrounding environment of pyrrolidine were identified at δ 1.8~2.173 through ^{1 H NMR, and δ1.} The peak of _{S-CH 3} was confirmed in the 9th generation. In addition, the total number of Cs was determined by checking C*3, CH*2, CH ₂ *6, and CH ₃ *1 through DEPT, and pyrrolidine and C=O confirmed by H-NMR through 2D-NMR It was confirmed that it was bound to the double bond of, and _{the peak of CH-CH 2} was also confirmed. Through this, when it was confirmed with the molecular weight confirmed in Ms, it was confirmed that the structure in which Met was attached to Gly-Pro (Gly-Pro-Met). After that, peptide synthesis was carried out, and it was confirmed that a peak appeared at the same time by comparing the synthesized peptide and the peptide separated and purified from the fermented product through HPLC.

Experimental example 8

Substances that inhibit the production of matrix methaloprotease-1 (MMP-1, collagenase) protect collagen and maintain the mechanical properties of the skin tissue, thereby preventing elasticity and skin sagging. By measuring the active ingredients, it can be evaluated that it can be usefully used in the development of cosmetics for improving wrinkles and for elastic skin. Accordingly, the MMP-1 production inhibitory ability test of the fermented hyaluronidum fermented in Preparation Example 4, the extract of the quince fruit extract of Preparation Example 9 and the fractions of the purified example was carried out in the following manner, and the results are shown in FIG. 9 . Retinol was used as a control.

<Experiment method>

Human dermal fibroblasts (HDF) cells were cultured in DMEM (Dulbecco's modified Eagle's media, Sigma) supplemented with 10% fetal bovine serum (Sigma) at 37°C and 5% CO ₂ conditions. . Cells were cultured in a 24-well plate at ^{a concentration of 2 X 10 5} cells/well and cell adhesion was confirmed, and then only a solvent was added to the control group without any treatment. It was processed so that it became a density|concentration. After adding the test substance to each dish, it was cultured for 2 days. After 2 days, the medium was collected and the inhibition of MMP-1 production was measured using Matrix Metalloproteinase-1 (MMP-1), Human, biotrak, and ELISA kit (GE healthcare RPN2610).

Referring to FIG. 9 , it was confirmed that the amount of MMP-1 produced was decreased in most of the experimental groups, and it was confirmed that there was a difference according to the composition and the combination of functional raw materials in the composition even within the experimental group. In particular, it is judged that most of the substances inhibiting MMP-1 expression are distributed in the water layer (purified example) during fractionation after dry fermentation. From these results, when UV that induces MMP-1 production is applied to the skin as a factor, it is more useful to use a mixture of dried dried fermented product and dried dried fermented product to inhibit MMP-1 production to reduce skin aging. It is believed that it can be suppressed.

Experimental example 9

The intracellular collagen production capacity of the dried fermented product of Preparation Example 4, the water layer of the purified example, SP2, fr2 and comp2 was performed in the following manner, and the results are shown in FIG. 10 . Transforming growth factor beta (TGFβ) was used as a control.

<Experiment method>

Human dermal fibroblst (HDF) cells to be used for the test are medium 106 (cascade biologics, M-106-500) with 1X LSGS (Low Serum Growth Supplement, cascade biologics S-003-10) added to 37 ℃, 5% CO ₂ Incubated in conditions. Cells were cultured in a 24-well plate at ^{a concentration of 1X 10 5} cells/well and cell adhesion was confirmed, and TGFβ was treated to a concentration of 1 mg/ml and 10 μg/ml, respectively, as an experimental group and a control group. After adding the test substance to each dish, it was cultured for 2 days. After 2 days, the medium was collected and the amount of collagen produced was measured using the Procollagen Type I C-peptide (hereinafter PIP) EIA kit (takara MK101).

Referring to FIG. 10, it was confirmed that the amount of collagen production increased in all the experimental groups and the control group, and the purified (purified example water layer, SP2, fr2 and comp2) had an excellent collagen production effect than the unrefined (dried fermented product). It can be confirmed that there is, and it can be seen that the most purified comp2 has the highest collagen production effect.

From these results, it is presumed that a large amount of ingredients that promote collagen production in the skin are contained in the dried dried fruit. It is presumed that there will be

Experimental example 10

In order to confirm the cell proliferation ability of comp1 to 4 of the purification example, the experiment was conducted in the following manner, and the results are shown in FIGS. 11 and 12 . Cell proliferative capacity was measured using the MTT assay.

<Experiment method>

3T3 cells and HACAT cells were seeded in a 24-well plate at ^{a cell number of 4 X 10 4} cells/24well per well, and then incubated at 37° C. and 5% CO ₂ conditions for 24 hours. It was washed twice with PBS (phosphate buffer saline), and each sample was treated with each concentration (1mg ~ 1ug/mL) and incubated for 24 hours. After incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 0.5% in DPBS (Dulbecco's Phosphate-Buffered Saline) was mixed with the culture medium at 1:9 (v/v). After addition, incubate in a _{CO 2 incubator for 2 hours.} Then, the generated formazan (frmazan) was dissolved in DMSO (dimethyl sulfoxide) and absorbance was measured at 570 nm using ELISA.

11 and 12, for HACAT cells and 3T3 cells, it was confirmed that all four types of peptides (comp1 to 4) treated from low to high concentrations had cell proliferation ability in a concentration-dependent manner, and in particular, the highest cell in comp2 peptide. It was confirmed to show proliferative capacity, and no significant change was observed in the microscopic observation of the cells.

Through this, it can be seen that the cosmetic composition of the present invention has no toxicity to skin-related cells, and it appears that there is a skin cell proliferation effect. Therefore, it can be predicted that even if the cosmetic composition of the present invention is treated on the skin, it will not have a significant effect.

Experimental example 11

In order to confirm the thermal and photostability of comp1 to 4 of the tablet example, the experiment was conducted in the following manner, and the results are shown in FIGS. 13 and 14 .

<Experiment method>

Each sample is dissolved in 50 mM Tris-HCl (pH 8.0) buffer so that the sample is 10 mg/mL, aliquoted into a glass vial, and stored at 50° C. for 4 weeks, and the loss of peptide in the composition due to heat is measured. and stability under sunlight conditions was measured in the same manner.

13 and 14, as a result of measuring peptide loss due to heat and measuring stability in sunlight conditions, all four peptides (comp1 to 4) showed less than 10% peptide loss up to 4 weeks. And it was confirmed that it has high stability in the storage period in sunlight.

Example

Each of the components listed in Table 5 was added according to the ratio described below based on 100 wt% of the cosmetic composition, and 1 to 20 wt% of the dried dried fermented product and the dried dried fermented product was added to the cream type composition.

Preferred embodiments of the present invention described above are disclosed to solve the technical problem, and various modifications, changes, additions, etc. will be possible within the spirit and scope of the present invention by those of ordinary skill in the art to which the present invention pertains. , such modifications and changes should be regarded as belonging to the following claims.

Accordingly, the scope of the present invention is indicated by the claims described below rather than the above detailed description, and all changes or modifications derived from the meaning, scope, and equivalent concept of the claims are included in the scope of the present invention. should be interpreted

Claims (7)

As a cosmetic composition comprising a dried dried fermented product as an active ingredient,
The dried fermented product is a tripeptide, and the tripeptide is glycine (Glycine, Gly)-proline (Proline, Pro)-serine (Serine, Ser), glycine (Glycine, Gly)-proline (Proline, Pro) )-Arginine (Arginine, Arg), Glycine (Glycine, Gly)-Proline (Proline, Pro)-Proline (Proline, Pro) or Glycine (Glycine, Gly)-Proline (Proline, Pro)-Methionine (Methionine, Met) A cosmetic composition, characterized in that delete delete delete The method of claim 1,
The cosmetic composition is a cosmetic composition comprising 1 to 90% by weight of the dried fermented product. The method of claim 1,
The cosmetic composition inhibits the activity of a collagen degrading enzyme (Matrix Metalloproteinase-1), promotes intracellular collagen production, and promotes the proliferation of epidermal cells. delete

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