KR20200083243A - 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 production of intracellular collagen, and proliferating epidermal cells. The present invention provides the cosmetic composition containing a Trapa japonica fermentation product or a Trapa japonica fermentation purified product as an active component.

Description

Method for manufacturing skin fermentation product and composition for improving skin wrinkles containing skin fermentation product or peptide produced therefrom as an active ingredient, composition for improving skin aging, comprising a composition effective for anti-aging and fermentation product {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 dry fermentation product or a dry fermentation tablet.

One of the areas of greatest interest in cosmetics is aging-related wrinkles, pigmentation, elasticity reduction, skin drying, hair loss, and lack of shine of hair. Skin undergoes various changes through aging. First, the components of the skin, the epidermis, dermis and subcutaneous tissue become thinner and the extracellular matrix (ECM) component that gives elasticity to the skin changes. Among them, collagen, which accounts for 70 to 80% of the extracellular matrix, As it ages, its production rapidly decreases and becomes closely related to wrinkle formation, and collagen, elastin, proteoglycans, and glucosaminoglycan that form skin connective tissue , Laminin (laminin), fibronectin, etc. are oxidized and lose their function, so the skin loses its elasticity and wrinkles are formed excessively, so it changes into senile skin.

The connective tissue fibers of the extracellular matrix include glue fibers (collagen fibers), reticular fibers, and elastic fibers (collar fibers, elastic fibers), of which collagen accounts for about 70% of skin connective tissue. Is mostly formed in the skin's fibroblasts. With age, the collagen content in skin connective tissue decreases, which is due to a decrease in collagen synthesis and acceleration of degradation. Therefore, lowering collagen biosynthesis and promoting collagen decomposition can be said to be the biggest cause of skin wrinkle formation.

One of the areas of greatest interest in cosmetics is aging-related wrinkles, pigmentation, elasticity reduction, skin drying, hair loss, and lack of shine of hair. Skin undergoes various changes through aging. First, the components of the skin, the epidermis, dermis and subcutaneous tissue become thinner and the extracellular matrix (ECM) component that gives elasticity to the skin changes. Among them, collagen, which accounts for 70 to 80% of the extracellular matrix, As it ages, its production rapidly decreases and becomes closely related to wrinkle formation, and collagen, elastin, proteoglycans, and glucosaminoglycan that form skin connective tissue , Laminin (laminin), fibronectin, etc. are oxidized and lose their function, so the skin loses its elasticity and wrinkles are formed excessively, so it changes into senile skin.

Patent No. 10-1784201 discloses a composition for skin improvement by fermenting a natural product and including it as an active ingredient, but does not mention the composition in a cosmetic using dry skin.

Accordingly, the present invention was devised to solve the above problem, and provides a cosmetic composition capable of inhibiting the activity of collagen degrading enzyme (Matrix Metalloproteinase-1), promoting the production of collagen in cells, and proliferating epidermal cells. I want to.

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

In addition, the dry fermentation product provides a cosmetic composition characterized by fermentation with Bacillus sp. strain, Lactobacillus sp. strain, or a mixed strain thereof.

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

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

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

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

For another purpose of the present invention, a step of inoculating microorganisms in the dried fruit extract and fermenting to produce a fermented product; And removing the microorganisms from the fermentation product.

The present invention inhibits the activity of collagen degrading enzyme (Matrix Metalloproteinase-1) by including a dry fermentation product or a dry fermentation purification product as an active ingredient, promotes the production of collagen in cells, and is a cosmetic composition capable of proliferating epidermal cells Can provide

1 is a schematic diagram of a method for producing a dried fermentation tablet according to the present invention,
Figure 2 is a graph showing the results of Experimental Example 2 of the present invention,
Figure 3 is a photograph of the TLC results of Experimental Example 3 of the present invention,
Figure 4 is a photograph taken the TLC results 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 results of high performance liquid chromatography analysis 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 is comp4: Gly-Pro-Met),
Figure 8 is a graph showing the results of NMR analysis of Experimental Example 7 according to the present invention (Figure 8a is comp2: Gly-Pro-Ser, Figure 8b is comp1: Gly-Pro-Arg, Figure 8c is comp3: Gly-Pro-Pro , Figure 8d comp4: Gly-Pro-Met),
9 is a graph showing the measurement results of MMP-1 inhibitory activity of Experimental Example 8 of the present invention,
10 is a graph showing the results of the collagen production promoting effect in cells of Experimental Example 9 of the present invention,
11 and 12 A graph 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 light 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 ordinary or lexical meanings, and the inventor appropriately explains the concept of terms to explain his or her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meanings and concepts consistent with the technical spirit of the present invention. Therefore, the configuration of the embodiments described herein is only one of the most preferred embodiments of the present invention, and does not represent all of the technical spirit of the present invention, and various equivalents and modifications that can replace them at the time of this application It should be understood that there may be.

The present inventors repeatedly conducted research to develop a raw material with antioxidant efficacy and a cosmetic composition effective in preventing skin aging in order to effectively prevent or improve skin aging and wrinkles. When using a peptide derived from water, it was found that the activity of inhibiting the activity of collagen degrading enzyme (Matrix Metalloproteinase-1), promoting the production of collagen in cells and promoting the proliferation of epidermal cells, was reached to the present invention.

Accordingly, the present invention discloses a cosmetic composition comprising a dry fermentation product or a dry fermentation purification product as an active ingredient.

In the present invention, the dry ( Trapa japonica) is an annual plant growing in a pond and can be mainly seen in ponds or swamps. The rhombus is referred to as rhombus because the leaves are rhombic triangles, and the fruit of the rhombus is also referred to as the water chestnut and opens downward in water and has a unique shape with 2 to 4 horns.

In the present invention, the dried fermented product means fermented product after drying and extracting the fruit of the dried fruit, and the dried fruit extract is used as a medicine effective in diseases such as gastric ulcer, and is rich in manganese components to maintain moisture in the skin. It is effective.

The dried fermented product or dried fermented tablet of the present invention acts on the skin, and is a raw material that helps to improve skin wrinkles and alleviate skin aging, and its effectiveness can be further enhanced compared to before purification by purifying certain effective ingredients. In addition, a dry fermentation product and a dry fermentation purification product may be used together.

In another aspect, the present invention provides a method for manufacturing a fermented fermentation product, wherein the method for manufacturing the fermentation product comprises inoculating microorganisms with a fruit extract and fermenting the fermentation product; And removing the microorganism from the fermentation product.

Hereinafter, the present invention will be described in detail through the manufacturing method of the dried fermentation product.

In the present invention, the step of inoculating microorganisms in the dried fruit extract and fermenting it to produce a fermented product is a step for increasing the content of the functional material of the dried fruit.

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

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

In addition, when extracting the dried fruit extract by hot water extraction, for example, purified water may be added at 5 to 20 times the weight of the dried fruit, and extracted at 100 to 140°C for 4 to 8 hours, and the dried 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 Bacillus sp . strain, Lactobacillus sp . strain, or a mixed strain thereof, and the Bacillus sp . strain is Bacillus methylotrophicus . ( Bacillus methylotrophicus ) or Bacillus subtilis , and the genus Lactobacillus sp . May be Lactobacillus paracasei , preferably Bacillus subtilis and Bacillus methyl It can be a mixed strain of rotopycus.

The fermentation refers to a process in which enzymes possessed by microorganisms change raw materials to produce beneficial ingredients. During the fermentation process, active ingredients are extracted and the content of functional substances increases. In addition, in the present invention, the fermentation can be inoculated with 0.1 to 10% by weight, preferably 0.5 to 5% by weight, with respect to the dried fruit extract, by adjusting the culture medium of the microorganism strain to 10 ⁷ to 10 ¹⁰ cfu/ml. It can be carried out at 1 to 5 days, preferably 2 to 4 days, 25 to 45 °C, preferably 30 to 40 °C.

In addition, the medium composition during the culture and fermentation of the microorganism may be 0.2 to 1% by weight of glucose, 0.1 to 0.5% by weight of an enzyme extract, 0.05 to 0.3% by weight of soytone, and distilled water (remaining amount).

In the present invention, the step of preparing the fermentation product by removing the microorganism from the fermentation product is a step of removing the microorganism to prevent further fermentation of the fermentation product, and the removal of the microorganism can be removed through centrifugation and filtration. , For example, after the microorganism is separated through a centrifuge, the fermentation product can be removed by filtering through a 0.1-0.3 μm filter.

1 is a schematic diagram of a method for manufacturing a dried fermentation tablet according to the present invention.

Referring to FIG. 1, the step of solvent fractionation of the dried fermentation product is a step of purifying the dried fermentation product to extract the peptide, and the solvent fraction may be sequentially performed through hexane, dichloromethane, ethyl acetate, butanol, and water. . In addition, it can be purified through a column after the solvent fraction.

In addition, in the present invention, the dried fermentation purification product may be a tripeptide derived from dried fruit, and the tripeptide is glycine (Glycine)-proline (Proline, Pro)-serine (Serine, Ser), Glycine (Gly)-proline (Proline, Pro)-arginine (Argine), glycine (Glycine, Gly)-proline (Proline, Pro)-proline (Proline, Pro) or glycine (Glycine, Gly-proline (Proline, Pro)-methionine (Methionine, Met).

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

The cosmetic composition according to the present invention has been confirmed to have a very good effect of inhibiting the activity of collagen degrading enzymes such as collagenase and matrix metalloproteinase-1 (MMP-1), and also promotes collagen production in cells and skin The effect of improving the cell's regenerative capacity was also confirmed to be very good.

The cosmetic composition according to the present invention may be applied in gel type, skin type, cream type, ointment type, etc., but is not limited to these. The above composition may be appropriately prepared by a known method by adding a conventional emollient, emulsifier, thickener or other materials known in the art according to its type.

The gel-type composition may be prepared by adding a softener such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, isocetic alcohol, or a purified liquor.

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

The cream-type composition may include stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, and other fatty alcohols, lecithin, phosphatidylcholine, phosphatidylethanolamine, sorbitylserine, and sopatidyl Lipids such as inositol, derivatives thereof, emulsifiers such as glyceryl stearate, sorbitan palmitate, and soribitan stearate, natural fats or oils such as avocado oil, apricot oil, barbasu oil, glassy oil, camellia oil, It may be prepared by adding a solvent such as propylene glycol and purified water.

The ointment type composition may be prepared by adding a wax such as a softener, an emulsifier, and microcrystalline lead, paraffin, ceresin, beeswax, lead, and vaseline.

The cosmetic composition of the present invention may additionally contain one or more skin wrinkle improvement components 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 placenta-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, water repellents, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. Can.

In the present invention, the cosmetic composition is a flexible cosmetic, convergent cosmetic, nutrient cosmetic, eye cream, serum, nutrition cream, massage cream, cleansing cream, cleansing lotion, cleansing foam, cleansing water, powder, essence, pack, hair tonic, hair treatment It can be formulated as a treatment, shampoo or rinse.

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

Specifically, the cosmetic composition may be prepared as a general emulsifying formulation and a solubilizing formulation. For example, lotion such as flexible lotion or nutrition lotion; Emulsions such as facial lotions and body lotions; Creams such as nutrition cream, moisture cream, and eye cream; essence; Cosmetic ointment; spray; Gel; pack; Sunscreen; Makeup base; Liquid type, solid type or spray type foundation; powder; Makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Or it may be formulated with a cleaning agent such as 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.

The cosmetic composition may be appropriately blended with other ingredients within a range that does not impair the purpose according to the present invention, depending on the type or purpose of use of the formulation, in addition to the essential ingredients in each formulation.

The cosmetic composition may include an acceptable carrier, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, and the like can be appropriately blended, but is not limited thereto. no.

The acceptable carrier may vary depending on the formulation. For example, when formulated as ointments, pastes, creams or gels, animal oils, vegetable oils, waxes, paraffins, starch, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or their Mixtures can be used.

When the cosmetic composition is formulated as 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 the case of spray, chlorofluoro Propellants such as hydrocarbon, propane, butane or dimethyl ether.

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

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

The cosmetic composition according to the quality or function of the final product, fatty materials, organic solvents, solubilizers, thickening agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances commonly used in the industry , Surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion blockers, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, commonly used in cosmetics It may further contain adjuvants commonly used in the cosmetic or dermatological field, such as any other ingredient. However, the adjuvant and its mixing ratio can be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

The cosmetic composition containing the dried fermented product or the dried fermented tablet according to the present invention as an active ingredient inhibits the activity of collagen degrading enzyme (Matrix Metalloproteinase-1) by containing the dried fermented product or dried fermented purified product, and the cells It promotes the production of collagen inside and can proliferate epidermal cells.

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

Preparation Example 1

2 kg of purified water was added to 200 g of dried dried fruit, followed by hot water extraction at 120° C. for 6 hours. After extraction, dried fruit extract with a sugar content of 6 to 8 birx was used. After extraction, the Lactobacillus paracasei ( Lactobacillus paracasei , purchased from the Korean Cell Line Bank) was cultured and inoculated with 1% by weight of a culture medium having a concentration of 1.0×10 ⁹ cfu/ml measured with a spectrophotometer on the dried fruit extract, and at 37° C. Fermentation was performed for 3 days, and the medium used for the culture was 0.6% by weight of glucose, 0.3% by weight of enzyme extract, 0.1% by weight of soytone, and a medium consisting of distilled water. After fermentation, the fermentation broth was centrifuged at 4000 rpm for 10 minutes to separate the lactic acid bacteria and the culture, and the culture broth was filtered through a 0.2 μm filter to prepare a dried fermentation product.

Preparation Example 2

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

Preparation Example 3

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

Preparation Example 4

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

Preparation Example 5

2 kg of 70% (v/v) ethanol was added to 200 g of dried dried fruit and extracted at 25° C. for 24 hours. After extraction, dried fruit extract having a sugar content of 6 to 8 birx was used. After extraction, the Lactobacillus paracasei ( Lactobacillus paracasei , purchased from the Korean Cell Line Bank) was cultured and inoculated with 1% by weight of a culture medium having a concentration of 1.0×10 ⁹ cfu/ml measured with a spectrophotometer on the dried fruit extract, and at 37° C. Fermentation was performed for 3 days, and the medium used for the culture was 0.6% by weight of glucose, 0.3% by weight of enzyme extract, 0.1% by weight of soytone, and a medium consisting of distilled water. After fermentation, the fermentation broth was centrifuged at 4000 rpm for 10 minutes to separate the lactic acid bacteria and the culture, and the culture broth was filtered through a 0.2 μm filter to prepare a dried fermentation product.

Preparation Example 6

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

Preparation Example 7

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

Preparation Example 8

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

Preparation Example 9

2 kg of purified water was added to 200 g of dried dried fruit, followed by hot water extraction at 120° C. for 6 hours to prepare dried fruit extract having a sugar content of 6 to 8 birx.

Preparation Example 10

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

Purification example

1) n-hexane, dichloromethane, ethyl acetate and butanol are sequentially added to the dried fermentation product prepared in Preparation Example 4 to separate n-hexane fraction, dichloromethane fraction, ethyl acetate fraction, butanol fraction and 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 filled with a Sephadex LH-20 resin (Sigma) open column (28 cm in diameter containing 50 g of resin, 45 cm in length glass) Column) to separate them into 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, the column used a reverse phase silica column (Reverse phase Silica column), the mobile phase solvent from 100% distilled water to distilled water: 99.5% (v / v) methanol 90: 10, 80: 20 in 1L in order of change to proceed fr1 ( 1~2), fr2(3~5), fr3(6~7), fr4(8~14) and fr5(15~21).

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 proceeds under 95:2.5:2.5 conditions, comp1, comp2, The dried fermentation tablets separated into comp3, comp4, comp5 and comp6 were purified.

Experimental Example 1

In order to confirm the effect of improving the skin wrinkles of the dried fermentation products prepared in Preparation Examples 1 to 10, an in vitro collagenase activity inhibition experiment was conducted according to the following method, and the results are shown in Table 1 below. Shown.

<Experiment method>

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

(2) 100mM Tris-cl buffer, 100mM CaCl ₂ And a 0.25 mg/ml collagenase-containing mixture was prepared, and each test substance and positive control were added to the mixture at a concentration of 5% (v/v) to advance the reaction.

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

(3) After that, ethyl acetate was added to 1.5 ml/tube, and after vortexing, the supernatant was taken and transferred to 150 mg/ep-tube of sodium sulfate, 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 absorbance at 320 nm.

Referring to Table 1 above, it is possible to confirm the collagenase inhibitory ability in all fermented products (Preparation Examples 1 to 8). Referring to Table 1 above, collagenase inhibitory ability in all fermented products (Preparation Examples 1 to 8) It can be confirmed, it can be confirmed that the collagenase inhibitory ability is better than the case where it is not fermented (Production Examples 9 and 10).

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

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

Experimental Example 2

The protein content of the dried fruit extract of Preparation Example 9, the fermented product of Preparation Example 4 and the fractions separated from the purification example were measured using lowry methods, and the measurement results are shown in Tables 2 and 2 below.

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

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

Experimental Example 3

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

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

Experimental Example 4

For the fr1 to fr5 separated in the purification example, silica gel thin layer chromatography (TLC) was performed under dichloromethane: 99.5% (v/v) methanol (1:1) mixed solvent, and thus the TLC pattern was obtained. It is shown in FIG. 4.

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

Experimental Example 5

The protein content contained in the water (water layer), SP2, fr2, and comp2 separated from the purification example was measured using lowry methods, 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 was increased during separation and purification through HPLC (High Pressure Liquid Chromatography), but it was confirmed that the protein content was not increased above a certain level.

Experimental Example 6

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

Referring to Table 3 and FIG. 6, in the case of the dry fermentation product (Preparation Example 4) and comp4 (Preparation Example), the target peptide was not shown, but different peptides were present, and in the case of comp2 (Preparation Example), the target peptide It can be seen that the content improved to 92%.

Experimental Example 7

The results of HPLC analysis of comp1 to comp4 separated from the purification example are shown in FIG. 7, and experiments were 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 Mass (LCMS-IT-TOF, SHIMADZU). Then, the structure was confirmed by measuring 1H, 13C, HMBC, HMQC, COSY, and DEPT using 600 MHz NMR (Agilent Technologies (DD2 600 MHz FT NMR)).

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. The characteristic peaks of CH2-CH were confirmed at δ3.4~3.829. In addition, C*3, CH*2, and CH ₂ *5 were checked through DEPT to determine the total number of CH, CH ₂ , and CH ₃ , and pyrrolidine confirmed by H-NMR through 2D-NMR. And C=O was confirmed to be attached to the double bond, and CH-CH ₂ bound to the C=O peak was also confirmed. Through this, when confirmed with the molecular weight confirmed by MS, it was confirmed that the structure (Gly-Pro-Ser) with Ser attached to Gly-Pro. After that, peptide synthesis was performed, and the peptides separated and purified from the synthesized peptides and fermented products were compared through HPLC, and it was confirmed that a peak appeared at the same time.

Referring to FIG. 8B, in the case of Tripeptide-G2 (comp1), CH ₂ *2 peaks separated by characteristic surrounding environment of pyrrolidine at δ 1.8 to 2.173 were identified through ¹ H NMR, and δ1.510 to 1.8. Peaks of CH ₂ -CH ₂ -CH ₂ were identified in the band, and CH peaks were identified in δ4.2. Through this, when confirmed with the molecular weight confirmed in Ms, it was confirmed that the structure (Gly-Pro-Arg) with Arg attached to Gly-Pro. After that, peptide synthesis was performed, and the peptides separated and purified from the synthesized peptides and fermented products were compared through HPLC, and it was confirmed that a peak appeared at the same time.

Referring to FIG. 8c, in the case of Tripeptide-G3 (comp3), CH ₂ *2 peaks separated by characteristic surrounding environment of pyrrolidine at δ 1.8 to 2.173 were identified through ¹ H NMR, and proline was observed through this peak. (proline) was confirmed. However, it was confirmed that the peak had a double pro-pro structure, and these peaks were identically doubled, confirming that the pro has two structures (Gly-pro-pro). After that, peptide synthesis was performed, and the peptides separated and purified from the synthesized peptides and fermented products were compared through HPLC, and it was confirmed that a peak appeared at the same time.

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 separated by characteristic surrounding environment of pyrrolidine at δ 1.8 to 2.173 were identified through ¹ H NMR, and δ1. The peak of S-CH ₃ was confirmed in 9 generations. In addition, C*3, CH*2, CH ₂ *6, and CH ₃ *1 were checked through DEPT to determine the total number of C, and pyrrolidine and C=O confirmed by H-NMR through 2D-NMR. It was confirmed that it is bound to the double bond of, and the peak of CH-CH ₂ was also confirmed. Through this, when confirmed with the molecular weight confirmed by Ms, it was confirmed that the structure (Gly-Pro-Met) with Met attached to Gly-Pro. After that, peptide synthesis was performed, and the peptides separated and purified from the synthesized peptides and fermented products were compared through HPLC, and it was confirmed that a peak appeared at the same time.

Experimental Example 8

Substances that inhibit the production of matrix methaloprotease-1 (MMP-1, collagenase) protect collagen to maintain the mechanical properties of skin tissue to prevent elasticity and skin sagging, thereby inhibiting the production of MMP-1 for raw materials By measuring, it can be evaluated that the raw material having activity can be used to improve wrinkles and to develop cosmetics for elastic skin. Thus, the fermentation product of Preparation Example 4, the extract of dried fruit of Preparation Example 9 and the fraction of the purification example were tested for inhibition of MMP-1 production 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 at 37°C and 5% CO ₂ conditions with 10% fetal bovine serum (Sigma) added to DMEM (Dulbecco's modified Eagle's media, Sigma). . After culturing the cells at a concentration of 2 X 10 ⁵ cells/well in a 24-well plate and confirming the adhesion of the cells, nothing was treated in the control group and only the solvent was added. In the dish, retinol was used as an experimental group and a control group of 10 ug/ml. It was treated to a concentration. Test dish was added to each dish and cultured for 2 days. After 2 days, the medium was collected, and the inhibitory ability of MMP-1 was measured through Matrix Metalloproteinase-1 (MMP-1), Human, biotrak, and ELISA kit (GE healthcare RPN2610).

Referring to FIG. 9, it was confirmed that the amount of expression of MMP-1 generated decreases in most of the experimental groups, and it was confirmed that there are differences depending on the composition and composition of functional raw materials in the composition within the experimental group. In particular, it is judged that a lot of substances that inhibit MMP-1 expression most are distributed in the water layer (tablets) during fractionation after drying. From these results, when UV, which causes MMP-1 production on the skin, is applied as a factor, it is more useful to mix and use dry fermented products and dry fermented purified products, thereby inhibiting MMP-1 production and inhibiting skin aging. It seems to be able to suppress.

Experimental Example 9

The dry fermentation product of Preparation Example 4, the water layer of the purification example, SP2, fr2, and comp2 were produced in cells in the following way, and the results were shown in FIG. 10. As a control, transforming growth factor beta (TGFβ) was used.

<Experiment method>

Human dermal fibroblst (HDF) cells to be used for the test are medium 106 (cascade biologics, M-106-500) and 1X LSGS (Low Serum Growth Supplement, cascade biologics S-003-10). C., cultured in 5% CO ₂ conditions. After culturing the cells at a concentration of 1X 10 ⁵ cells/well in a 24-well plate and confirming the adhesion of the cells, TGFβ was treated as concentrations of 1 mg/ml and 10 μg/ml, respectively, in the experimental group and the control group. Test dish was added to each dish and cultured for 2 days. After 2 days, the medium was collected, and the amount of collagen produced was measured through a Procollagen Type I C-peptide (hereinafter referred to as PIP) EIA kit (takara MK101).

Referring to FIG. 10, it was confirmed that the collagen production amount increased in all experimental groups and the control group, and the collagen production effect was superior to that of the unpurified (dry fermented product) (purified water layer, SP2, fr2, and comp2). It can be confirmed that the most purified comp2 has the highest collagen production effect.

From these results, it is presumed that the ingredients that promote the production of collagen in the skin are contained in a large amount in the dried fruit, and mixing and using dried fermented products and dried fermented tablets can increase the production of collagen and suppress skin aging of the skin. It seems to be.

Experimental Example 10

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

<Experiment method>

3T3 cells and HACAT cells were seeded in a 24-well plate so that the number of cells of 4 X 10 ⁴ cells/24well per well was incubated for 24 hours at 37°C and 5% CO ₂ conditions. Washed twice with PBS (phosphate buffer saline), and each sample was incubated for 24 hours by treatment by concentration (1 mg to 1 ug/mL). 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 in 1:9 (v/v) And then incubated 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, it was confirmed that all of the four peptides (comp1 to 4) treated with HACAT cells and 3T3 cells from low to high concentrations were cell-dependent in cell concentration, and in particular, the highest cells in the comp2 peptide. It was confirmed to show the ability to proliferate, and microscopic observation of the cells showed no significant change.

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

Experimental Example 11

In order to confirm the heat and light safety of comp1 to 4 of the purification example, experiments were conducted in the following manner, and the results are shown in FIGS. 13 and 14.

<Experiment method>

Each sample was dissolved in 50 mM Tris-HCl (pH 8.0) buffer solution, dispensed into a glass vial, and stored at 50°C for 4 weeks to measure the loss of peptide in the composition due to heat. And the stability was measured in the same way in daylight conditions.

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

Example

Each component listed in Table 5 was added according to the ratio described below with respect to 100% by weight of the cosmetic composition, and 1-20% by weight of the dried fermented product and the dried fermented purified product were added to the cream type composition.

Preferred embodiments of the present invention described above are disclosed to solve the technical problems, and those skilled in the art to which the present invention pertains may make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , Such modifications and changes should be considered to fall within the scope of the following claims.

Therefore, the scope of the present invention is indicated by the claims, which will be described later, rather than by the detailed description, and all modified or modified forms derived from the meaning, scope, and equivalent concepts of the claims are included in the scope of the present invention. Should be interpreted.

Claims (7)

A cosmetic composition comprising a dried fermented product or a dried fermented purified product as an active ingredient. According to claim 1,
The dry fermentation product is a Bacillus sp ( Bacillus sp . ) Strain, Lactobacillus sp ( Lactobacillus sp . ) Strain or a cosmetic composition characterized in that fermented with a mixed strain thereof. According to claim 2,
The Bacillus sp . strain is Bacillus methylotrophicus or Bacillus subtilis , and the Lactobacillus sp . strain is Lactobacillus paracasei Cosmetic composition, characterized in that. According to claim 1,
The dried fermentation purified product is tripeptide, and the tripeptide is glycine (Glycine)-proline (Proline, Pro)-serine (Serine, Ser), glycine (Glycine, Gly)-proline (Proline, Pro )-Arginine (Arg), Glycine, Gly-Proline, Pro-Proline, Pro or Glycine, Gly-Proline, Pro-Methionine, Met Cosmetic composition, characterized in that. According to claim 1,
The cosmetic composition is a cosmetic composition, characterized in that it comprises 1 to 90% by weight of the dried fermentation product or the dried fermentation tablets. According to claim 1,
The cosmetic composition is a cosmetic composition characterized in that it inhibits the activity of collagen degrading enzyme (Matrix Metalloproteinase-1), promotes the production of collagen in cells and proliferation of epidermal cells. Inoculating microorganisms in the dried fruit extract and fermenting to produce a fermented product; And
Removing the microorganism from the fermentation product;
Method of manufacturing a fermented fermentation product comprising a.

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