KR102028954B1 - Skin cell activator for skin-beautifying agent , Manufacturing method thereof and skin-beautifying agent - Google Patents

Abstract

The present invention relates to a skin cell activator and a skin external agent, and more particularly, to a skin cell activator using a specific fraction of a biopeptone fermentation, a method for preparing the same, a skin care material using the same, a skin disease treatment / prevention material, and the like. It relates to an invention that can provide an external preparation for skin.

Description

Skin cell activator for skin care agent, preparation method thereof and skin care agent comprising the same {Skin cell activator for skin-beautifying agent, Manufacturing method

The present invention relates to a skin cell activator, and more particularly, to a skin cell activator using a specific fraction of a bio peptone fermentation, a method for preparing the same, and an application product using the same.

Soybeans, an important source of protein, are becoming increasingly popular due to their nutritional value and scientifically discovered variety. In recent years, soybeans are known to have a healing effect on cardiovascular disease, diabetes, cancer, overweight, and obesity, as well as improving skin such as promoting collagen production, inhibiting melanin production, inhibiting skin aging, and anti-inflammatory effects. . Soybeans are cultivated all over the world, so they are not only easy to source, but they are also a major source of plant protein containing 20-40% of protein in buildings. In addition, since it is a plant material that has secured the safety of the body through a long history, it is actively used for various physiological activities and functional studies.

Biologically active peptides (BPs) refer to small molecular weight amino acid sequences having biological activity derived from proteins. The amino acid sequences contained in the precursor protein are mostly inactive, but small peptides produced through degradation have various biological activities. Proteolysis is an essential process for producing physiologically active peptides, and the physiological activity of peptides produced by the degradation of proteins varies depending on the type of the original protein source, the type of enzyme, and the production conditions. In addition, the bioactive peptide produced through the decomposition action is not only a low molecular good penetration of the skin, but also easy to apply to various cosmetic materials due to the increased solubility in water.

Recently, with increasing interest in the function and importance of skin barriers, studies that have been conducted based on the existing antioxidant mechanisms have gradually diversified into studies on the restoration and control of skin barrier damage. The skin is an organ consisting of the epidermis, dermis, and hypodermis, and performs a barrier function to prevent water retention and invasion of external infectious agents, including pathogens. The skin barrier is low permeability and not only a physical barrier to protect the human body from the external environment but also various antibacterial substances including a moisturizing barrier that maintains moisture in the body through natural moisturizing factor (NMF) and an antimicrobial peptide. As a chemical barrier function that is responsible for skin immune function. Among them, various moisturizing factors (hyaluronic acid, collagen, phospholipid, free fatty acid, ceramide, urea, filaggrin, glycerol, etc.) that function as a moisturizing barrier are synthesized from keratinocytes and fibroblasts. By accumulating in the extracellular matrix, it performs various bioactive functions of the skin. If the synthesis of the moisturizing factor is reduced due to the deterioration of keratinocytes and fibroblasts, or the production of the moisturizing factor is reduced, defects of the skin barrier occur, resulting in atrophy of the epidermis, decrease of skin moisture, decrease of elasticity, wrinkle formation, etc. Various skin aging will occur. Recently, the association between atopic dermatitis and the filaggrin gene has been demonstrated, and chronic skin diseases such as psoriasis have also been reported to be associated with decreased expression of antimicrobial peptides. It is becoming an important issue.

Various raw materials to help skin cell activity, for example, soybean, judocho, dermis and other plant-derived extracts have been developed, but there is a problem of low cell regeneration ability or problems causing skin problems, There is an increasing demand for new natural materials and / or products that can be solved.

Korea Patent Publication No. 10-2014-0018666 (2014.02.13) Korean Laid-Open Patent No. 10-2007-0022936 (2007.02.28)

Nemes, Z. and Steinert, P. M. 1999. Brick and mortar of the epidermal barrier. Exp. Mol. Med. 31, 5-19. Lee, J. C., Lee, E., Lee, Y. C., Oh, H., Yoon, H. S., Ha, T. K. and Hong, E. H. 2007. Effects of Fructus foeniculi extract on recovering liver function. Kor. J. Herbology. 22,213-218.

In the process of identifying available materials for improving skin barrier function, the present inventors found that a specific fraction of a biopeptone ferment derived from soybeans, such as soybean, is composed of keratinocyte cells (HaCaT cells) and dermal cells, which constitute epidermal cells. Confirmed that the positive effect on the growth and proliferation of the fibroblast cell (Fibroblast cell), through which it was found to have a positive effect on the regulation of skin barrier to complete the present invention. That is, the present invention is to provide a skin cell activator using a specific fraction of the biopeptone fermentation, a preparation method thereof and an external skin preparation using the same.

In order to solve the above problems, the present invention relates to a skin cell activator, and includes a fraction obtained by fractionating a biopeptone fermented product by preparative liquid chromatography.

In one preferred embodiment of the present invention, the fraction is measured by high-performance liquid chromatography, the first fraction having the strongest peak at 1.96 ~ 2.30 minutes; The second fraction having the strongest peak at 23.40 to 23.70 minutes; The third fraction having the strongest peak at 1.60 to 1.75 minutes; Fourth fraction having the strongest peak at 9.0-9.3 minutes; The fifth fraction having the strongest peak at 7.20 to 7.70 minutes; And a sixth fraction having the strongest peak at 23.0 to 23.40 minutes; It may include one or more fractions selected from.

In a preferred embodiment of the present invention, the fraction may include one or more selected from the first fraction and the second fraction.

In one preferred embodiment of the present invention, the first fraction may further have peaks at 7.4 to 7.5 minutes, 12.45 to 12.55 minutes, and 23.0 to 23.12 minutes.

As a preferred embodiment of the present invention, the second fraction may further have a peak at 6.30 to 6.50 minutes, 15.2 to 16.0 minutes, and 20.2 to 20.7 minutes.

As a preferred embodiment of the present invention, the third fraction may further have peaks at 1.90-2.30 minutes, 4.9-5.2 minutes and 9.60-9.85 minutes.

As a preferred embodiment of the present invention, the fourth fraction may further have peaks at 1.50 to 1.80 minutes, 1.90 to 2.20 minutes, 9.40 to 9.70 minutes, 13.60 to 14.05 minutes, 14.10 to 14.30 minutes, and 20 to 20.90 minutes.

As a preferred embodiment of the present invention, the fifth fraction may further have peaks at 1.70 to 1.90 minutes, 2.0 to 2.20 minutes, 11.0 to 11.5 minutes, 13.8 to 14.2 minutes, 22.30 to 22.70 minutes, and 22.80 to 23.0 minutes.

As a preferred embodiment of the present invention, the sixth fraction may further have peaks at 10.70 to 11.00 minutes, 12.50 to 13.0 minutes, 17.30 to 17.70 minutes, 17.70 to 17.95 minutes, and 22.20 to 22.80 minutes.

As a preferred embodiment of the present invention, the bio peptone fermentation product is a fermentation product obtained by fermenting peptonated proteolytic products with lactic acid bacteria, and containing 100% by weight (including other inevitable impurities) of low molecular weight peptides having a weight average molecular weight of 200 to 500. can do.

In a preferred embodiment of the present invention, the small molecule peptide may include one or more selected from dipeptide, tripeptide and tetrapeptide.

As a preferred embodiment of the present invention, the lactic acid bacteria used in the fermentation may include a strain of the genus Lactobacillus ( Lactobacillus ).

In a preferred embodiment of the present invention, the peptonated proteolysate is at least one selected from soybeans, frosted, seomoktae, black Tae, kidney beans, hedge beans, green soybeans, green beans, red beans, soybeans, bean sprouts, seonbi beans and green soybeans Soybean containing may be peptonated using pepsin.

In a preferred embodiment of the present invention, the Lactobacillus strain is Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus casei and Lactobacillus planttanum ( Lactobacillus plantarum ), Lactobacillus brevis ( Lactobacillus brevis ) , Lactobacillus acidophilus ( Lactobacillus acidophilus ) may include one or more selected from.

In one preferred embodiment of the present invention, the skin cell activator of the present invention may comprise the fraction of 100 ppm to 10,000 ppm.

In addition, another object of the present invention relates to a method for preparing a skin cell activator, step 1 of preparing a bio peptone fermentation; And fractionating the biopeptone fermented product by preparative liquid chromatography to obtain a fraction.

In one preferred embodiment of the present invention, the biopeptone fermentation of step 3 is prepared by the step 1-1 to prepare a medium containing peptonated proteolysate; Inoculating the lactic acid bacteria in the medium step 1-2; A first step of preparing a biopeptide culture by culturing the inoculated lactic acid bacteria of the second step to ferment the peptonated proteolyte; And removing the cells and insoluble components from the low molecular peptide culture to obtain a biopeptide fermented product.

In a preferred embodiment of the present invention, the peptonated protease of step 1-1 may be a peptonated soy protease.

In a preferred embodiment of the present invention, the preparation of the bio peptone fermentation, the medium of step 1-1 is selected from glucose, sucrose, maltose, fructose, lactose, xylose, galactose and arabinose 1 Carbon sources containing at least species; And at least one inorganic element compound selected from magnesium sulfate, manganese sulfate, zinc sulfate, cooper sulfate, sodium acetate, potassium phosphate and calcium chloride.

In a preferred embodiment of the present invention, in the preparation of the bio peptone fermentation, the inoculation of the step 1-2 is characterized in that the lactic acid bacteria inoculated into the medium at lactic acid bacteria 1 × 10 ⁴ ~ 1 × 10 ⁷ cfu / ㎖ can do.

As a preferred embodiment of the present invention, when preparing the bio peptone fermentation, the fermentation of steps 1-3 may be characterized in that the fermentation is performed for 12 hours to 72 hours at 25 ° C to 35 ° C.

In addition, another object of the present invention relates to an external skin preparation using the skin cell activator, the external skin preparation may be a skin cosmetic or a skin disease treatment.

In a preferred embodiment of the present invention, the skin external preparation may be a cosmetic, an ointment or an ampoule.

In a preferred embodiment of the present invention, the cosmetic may be an emulsion, a lotion type, a cream type, a lotion type, an emulsion type, an essence type, a pack type, a gel type, or a mist type cosmetic.

As a preferred embodiment of the present invention, when the external preparation for skin is a cosmetic, and the cosmetic is a cream or lotion type, various types of skin cell activators described above, glycerin, butylene glycol, dipropylene glycol, carboxyvinyl polymer , Polysorbate, hardened castor oil, glyceryl stearate, cetearyl alcohol, cetyl ethyl hexanoate and licorice extract may be characterized.

As a preferred embodiment of the present invention, when the skin external preparation is a cosmetic, and the cosmetic is an emulsion type, a lotion type, an emulsion type, an ampoule type or an essence type, various types of skin cell activators described above, dipropylene glycol, 1 , 3-butylene glycol, polysorbate, dipotassium glycylizate, allantoin, panthenol and glycerin.

Skin cell activator of the present invention has a high cell proliferation effect, excellent skin aging inhibitory, skin cell regeneration, cell reconstruction ablility and cell migration ability bar physiological activity of skin cells and homeostasis of skin tissue Helps to maintain a variety of skin cosmetics and skin diseases can be applied as an agent.

1A to 1B are HLPC measurement results performed in Experimental Example 1, and are results of measurement of the biopeptone fermentation product prepared in Example 1. FIG. 1A shows the results from 0 to 60 minutes, and FIG. 1B shows the results from 0 to 25 minutes.
1 to 5 in FIG. 2 are MS spectrum measurement results performed in Experimental Example 1, and are results of confirming the molecular weight of the part shown in FIG. 1B.
3A and 3B are graphs of fractionation and measurement of the biopepton fermentation of Preparation Example 1 using liquid chromatography for preparation of the biopeptone fermentation performed in Example 1. FIG.
Figure 4 is a photograph of the six fractions obtained in Example 1.
FIG. 5 is an LC-MS analysis graph performed in Experimental Example 2, and the vertical axis of the graph is relative abundance, the ion detection intensity, and the higher the peak height, the higher the concentration of the component.
Figure 6 is a WST-1 assay cytotoxicity measurement results for HaCaT cells carried out in Experimental Example 3.
7A and 7B are results of measuring WST-1 assay cytotoxicity on HaCaT cells of the first and second fractions, respectively.
8A and 8B are results of WST-1 assay cytotoxicity measurement on fibroblasts of the first and second fractions, respectively.
9 is a phase contrast microscopy photograph of a cell regeneration test performed in Experimental Example 4. FIG.
10 is a result of measuring collagen synthesis promoting effect performed in Experimental Example 5.
11 is a result of measuring the tyrosinase enzyme inhibition effect carried out in Experimental Example 6.
12A and 12B show melanin pigment production inhibitory effect measured in Experiment 7.

The term "bio peptone fermented product" used in the present invention means a fermented product obtained by fermenting the protein-binding product of soybeans peptonized by pepsin, [protease] is a protein-lysate of soybeans unless otherwise specified Means. In addition, the peptonated proteolysate before fermentation refers to a mixture of oligopeptides and polypeptides having various molecular weights, and the biopeptone fermentation products obtained by fermentation thereof are di-peptide, tripeptide, and tetra It refers to a mixture composed of peptides having a low molecular weight of 200 to 500 weight average molecular weight such as peptide (tetra-peptide).

Hereinafter, the present invention will be described in more detail.

Skin cell activator of the present invention comprises the steps of preparing a bio peptone fermentation; And fractionating the biopeptone fermented product by preparative liquid chromatography to obtain a fraction.

Referring to the biopeptone fermentation of the first step is as follows.

The biopeptone fermentation of step 1 is a step 1-1 to prepare a medium containing the peptonated proteolysate; Inoculating the lactic acid bacteria in the medium step 1-2; A first step of preparing a soy peptide culture by culturing the inoculated lactic acid bacteria of the second step to ferment the peptonated proteolyte; And removing the cells and insoluble components from the soy peptide culture to obtain a bio peptone fermentation step 1-4.

In addition, the step 1-5 of drying and powdering the bio peptone fermentation; after further performing the process to powder, it may be used by liquefying it.

In the preparation of the biopeptone fermentation product, the medium of step 1-1 may include a carbon source containing one or more selected from glucose, sucrose, maltose, fructose, lactose, xylose, galactose and arabinose; And at least one inorganic element compound selected from magnesium sulfate, manganese sulfate, zinc sulfate, cooper sulfate, sodium acetate, potassium phosphate and calcium chloride.

And, the peptonated proteolysate is at least one selected from soybeans, frosted, seomoktae, blackened beans, kidney beans, hedge beans, green soybeans, green beans, red beans, soybeans, bean sprouts beans, sesame beans and green beans, preferably soybeans, Soybeans containing at least one selected from Seoryae, Seomoktae, kidney beans and mung beans may be characterized in that the peptonization using pepsin.

In addition, the lactic acid bacteria used in the fermentation of the step 1-2 in the preparation of the bio peptone fermentation products are Lactobacillus rhamnosus , Lactobacillus casei and Lactobacillus planttanum Lactobacillus plantarum , Lactobacillus brevis , Lactobacillus acidophilus ( Lactobacillus acidophilus ) may be used at least one strain of Lactobacillus ( Lactobacillus ) genus, preferably selected from Lactobacillus rhamnosus and Lactobacillus casei One or more strains of the genus Lactobacillus can be used.

In addition, the inoculation of step 1-2 is to inoculate the lactic acid bacteria into the medium at 1 × 10 ⁴ to 1 × 10 ⁷ cfu / ml, preferably at 1 × 10 ⁵ to 5 × 10 ⁶ cfu / ml. It may be characterized by. At this time, if the amount of lactic acid bacteria inoculated is less than 1 × 10 ⁴ cfu / ㎖ may be too long fermentation time, even if the amount of lactic acid bacteria inoculated more than 1 × 10 ⁷ cfu / ㎖ low molecular weight peptide in the bio peptone fermentation It is uneconomical because it no longer increases.

In the preparation of the biopeptone fermentation, the fermentation in steps 1-3 may be performed for 12 hours to 72 hours and preferably 24 hours to 48 hours under 25 to 35 hours. At this time, if the fermentation time is less than 12 hours, the low-molecular weight peptide content may be too low due to lack of decomposition time of the peptonated soybean protein, and the fermentation may be performed during the above time since the fermentation time exceeds 72 hours. It is good to do.

The bio peptone fermentation thus prepared includes a low molecular weight bio peptone having a weight average molecular weight of 200 to 500, and the low molecular weight peptide may include one or more selected from dipeptides, tripeptides, and tetrapeptides.

Next, the second step is a step of obtaining the fraction by fractionating the biopeptone fermentation product prepared as described above by preparative liquid chromatography (hereinafter referred to as pre-LC). An example of the pre-LC measurement result is shown in FIG. 3. And, there is no cytotoxicity of the fractions, by screening excellent fractions of skin aging, skin cell regeneration, reconstruction ablility and cell migration ability (see Fig. 4), and finally six kinds of fractions (see One fraction to sixth fraction) can be obtained.

In addition, the skin cell activator of the present invention may include one or more fractions selected from the first to sixth fractions, and may preferably include one or more selected from the first fraction and the second fraction. .

The first fraction of the fraction may have the strongest peak at 1.96-2.30 minutes when measured for 30 minutes by high-performance liquid chromatography, and preferably has a peak at 1.96-2.30 minutes, 7.4-7.5 minutes and 12.45-12.55 minutes. More preferably, 1.96-2.15 minutes, 7.4-7. 5 minutes, 11.6-12.0 minutes, 12.45-12.55 minutes and 22.98-23.15 minutes.

In addition, the second fraction of the fraction may have the strongest peak at 23.40 to 23.70 minutes when measured for 30 minutes by high-performance liquid chromatography, preferably 6.30 to 6.50 minutes, 15.2 to 16.0 minutes, 20.2 to 20.7 minutes and 23.40 It can have a peak at ~ 23.70 minutes, more preferably 2.20 ~ 2.40 minutes, 4.10 ~ 4.35 minutes, 6.30 ~ 6.50 minutes, 11.5 ~ 11.9 minutes, 14.2 ~ 14.8 minutes, 15.2 ~ 16.0 minutes, 17.60 ~ 17.90 minutes, 18.50 Peaks at ˜18.90 minutes, 19.40 to 19.80 minutes, 20.2 to 20.7 minutes, and 23.40 to 23.70 minutes and 23.71 to 23.80 minutes.

In addition, the third fraction of the fraction may have the strongest peak at 1.60 to 1.75 minutes when measured for 30 minutes by high-performance liquid chromatography, preferably 1.60 to 1.75 minutes, 1.90 to 2.30 minutes, 4.9 to 5.2 minutes and 9.60 It may have a peak at ~ 9.85 minutes, more preferably from 1.60 to 1.75 minutes, 1.90 to 2.30 minutes, 4.35 to 4.65 minutes, 4.9 to 5.2 minutes, 9.60 to 9.85 minutes and 11.55 to 11.90 minutes.

In addition, the fourth fraction of the fraction may have the strongest peak at 9.0 to 9.3 minutes when measured for 30 minutes by high-performance liquid chromatography, preferably 1.50 to 1.80 minutes, 1.90 to 2.20 minutes, 9.0 to 9.3 minutes, 9.4 Peaks at-9.7 minutes, 13.60-14.05 minutes, 14.10-14.30 minutes and 20-20.90 minutes, more preferably 1.50-1.80 minutes, 1.90-2.20 minutes, 4.9-5.2 minutes, 5.7-6.0 minutes, 8.15-8.40 Peaks at minutes, 9.0 to 9.3 minutes, 9.4 to 9.7 minutes, 13.60 to 14.05 minutes, 14.10 to 14.30 minutes, 20 to 20.90 minutes, and 23.0 to 23.35 minutes.

In addition, the fifth fraction of the fraction may have the strongest peak at 7.20 to 7.70 minutes when measured for 30 minutes by high-performance liquid chromatography, preferably 1.70 to 1.90 minutes, 2.0 to 2.20 minutes, 7.20 to 7.70 minutes, 11.0 Peaks at ˜11.5 minutes, 13.8 to 14.2 minutes, 22.30 to 22.70 minutes, and 22.80 to 23.0 minutes, more preferably 1.70 to 1.90 minutes, 2.0 to 2.20 minutes, 7.20 to 7.70 minutes, 11.0 to 11.5 minutes, 13.8 Peaks at ˜14.2 minutes, 15.7 to 16.2 minutes, 22.30 to 22.70 minutes, 22.80 to 23.0 minutes, and 23.25 to 23.55 minutes.

In addition, the sixth fraction of the fraction may have the strongest peak at 23.0 to 23.40 minutes when measured for 30 minutes by high-performance liquid chromatography, preferably 10.70 to 11.00 minutes, 12.50 to 13.0 minutes, 17.30 to 17.70 minutes, 17.70 ~ 17.95 minutes, 22.20 to 22.80 minutes and 23.0 to 23.40 minutes, more preferably 10.70 to 11.00 minutes, 12.20 to 12.45 minutes, 12.50 to 13.0 minutes, 17.30 to 17.70 minutes, 17.70 to 17.95 minutes, 22.20 to 22.80 Peaks at minutes, 23.0 to 23.40 minutes, 23.65 to 23.90 minutes, and 24.05 to 24.40 minutes.

The skin cell activator of the present invention may comprise such fractions at 100 ppm to 10,000 ppm, preferably at 300 ppm to 5,000 ppm, more preferably at 500 ppm to 2,500 ppm.

Such, the skin cell activator of the present invention can be used as a skin care material or a skin disease treatment and / or prevention material, and can be applied to a variety of formulations, in particular as an external skin preparation such as cosmetics, ointments or ampoules It can be applied.

The cosmetic may be a cosmetic such as an emulsion type, a lotion type, a cream type, a lotion type, an emulsion type, an essence type, a pack type, a gel type, or a mist type.

The present invention is not limited thereto, but specific examples thereof may be used when the skin cell activator is prepared in a cream or lotion type cosmetic product. The skin cell activator, glycerin, butylene glycol, dipropylene glycol, and carbon Compound vinyl polymer, polysorbate, hardened castor oil, glyceryl stearate, cetearyl alcohol, cetylethylhexanoate and licorice extract can be prepared by mixing, purified water, fragrance, preservative, etc. can be further mixed. .

In more detail, to prepare a first mixture by mixing carboxyvinyl polymer, purified water, glycerin, butylene glycol, dipropylene glycol and lecithin; Preparing a second mixture by mixing polysorbate, cured castor oil, glyceryl stearate, cetearyl alcohol, and cetylethylhexanoate; After heating the first mixture and the second mixture to 70 ℃ ~ 90 ℃, preferably 75 ℃ ~ 85 ℃ each, and then mixing and stirring the heated first mixture and the second mixture to prepare a third mixture step; Performing a process comprising a step of lowering the temperature to 35 ° C. to 50 ° C., preferably 38 ° C. to 45 ° C. while stirring the third mixture, and mixing skin cell activator, licorice extract and additives. A skin external preparation of cream type or lotion type can be prepared.

In this case, the glycerin serves as a skin moisturizing role, 400 to 800 parts by weight of glycerin, preferably 500 to 700 parts by weight based on 100 parts by weight of the skin cell activator. In this case, when the amount of glycerin is less than 400 parts by weight, there may be a problem of decreasing the moisturizing feeling, and when used in excess of 800 parts by weight, there may be a problem causing stickiness.

In addition, the butylene glycol serves as a skin moisturizing role, with respect to 100 parts by weight of the skin cell activator, butylene glycol 700 ~ 1,200 parts by weight, preferably 850 ~ 1,100 parts by weight, but, butylene glycol If the amount of less than 700 parts by weight may be a problem to reduce the moisturizing feeling, when used in excess of 1,200 parts by weight may be a problem to destabilize the formulation is recommended to use within the above range.

In addition, the dipropylene glycol serves as a skin moisturizing role, with respect to 100 parts by weight of skin cell activator, 800 parts by weight to 1,500 parts by weight of dipropylene glycol, preferably 1,000 to 1,350 parts by weight, wherein, If the amount of propylene glycol is less than 800 parts by weight, there may be a problem of deteriorating the moisturizing feeling, and when used in excess of 1,500 parts by weight, there may be an uneconomic problem, it is preferable to use within the above range.

In addition, the carboxyvinyl polymer serves as a thickener, and 30 to 80 parts by weight of the carboxyvinyl polymer, preferably 40 to 70 parts by weight, may be used based on 100 parts by weight of the skin cell activator. If the amount of the polyvinyl polymer used is less than 30 parts by weight, there may be a problem of dropping the viscosity, and when used in excess of 80 parts by weight, there may be a problem of causing a swelling phenomenon (not absorbed by the skin and appearance) within the above range. It is good to use.

In addition, the polysorbate serves as a solubilizer, and 50 to 150 parts by weight of polysorbate, preferably 70 to 130 parts by weight, may be used based on 100 parts by weight of a skin cell activator. If the amount is less than 50 parts by weight may be a problem to separate the formulation, when used in excess of 150 parts by weight may be a problem that causes the stickiness and bubbles during application of the skin is recommended to use within the above range.

In addition, the hardened castor oil serves as a solubilizer, with respect to 100 parts by weight of skin cell activator, hardened castor oil may be used 600 to 1,000 parts by weight, preferably 700 to 900 parts by weight, wherein the hardened castor oil If the amount of less than 600 parts by weight may be a problem to separate the formulation, when used in excess of 1,000 parts by weight may be a problem that causes foaming during application is recommended to use within the above range.

In addition, the glyceryl stearate serves as an emulsifier, 80 to 150 parts by weight, preferably 85 to 130 parts by weight of glyceryl stearate, based on 100 parts by weight of the skin cell activator, wherein glyceryl If the amount of stearate used is less than 80 parts by weight, there may be a problem of dropping the emulsion stability, and when used in excess of 150 parts by weight, there may be a problem of dropping the emulsion stability is good to use within the above range.

In addition, the cetearyl alcohol serves as an emulsifier, and 150 to 300 parts by weight of cetearyl alcohol, preferably 180 to 250 parts by weight, may be used based on 100 parts by weight of the skin cell activator. If the amount of reel alcohol is less than 150 parts by weight, there may be a problem of dropping the emulsion stability, if used in excess of 300 parts by weight may be a problem of dropping the emulsion stability is good to use within the above range.

The cetylethylhexanoate is used as an oil component to improve the feeling of use, and 400 to 800 parts by weight of cetylethylhexanoate, preferably 500 to 700 parts by weight, is used with respect to 100 parts by weight of a skin cell activator. In this case, when the amount of cetylethylhexanoate is less than 400 parts by weight, there may be a problem of deteriorating the feeling, and when it is used in excess of 800 parts by weight, there may be a problem of deodorization, so it is preferable to use it within the above range. .

In addition, the licorice extract acts as a skin moisturizing and anti-inflammatory agent, based on 100 parts by weight of the skin cell activator, licorice extract may be used 5 to 40 parts by weight, preferably 10 to 30 parts by weight, wherein, licorice extract If the amount is less than 5 parts by weight, there may be a problem that prevents skin moisturizing and anti-inflammatory action, and when used in excess of 40 parts by weight, there may be a problem causing skin irritation. .

In addition, the lecithin serves to improve the feeling of use as a natural emulsifier and to increase the skin absorption rate through the multi-layered liposome, with respect to 100 parts by weight of the skin cell activator, 2 to 50 parts by weight of lecithin, preferably 5 to 40 It can be used in parts by weight, in this case, if the amount of licorice extract is less than 2 parts by weight may have a problem to reduce the skin absorption rate, when used in excess of 50 parts by weight may have a problem to lower the stability of the formulation within the above range It is good to use.

In addition, the purified water plays a role of adjusting the solvent and viscosity, it can be used to adjust the amount used according to the skin external preparation type, preferably 8,000 ~ 20,000 parts by weight based on 100 parts by weight of the skin cell activator.

As another specific example of the external preparation for skin of the present invention, when the cosmetic of the present invention is prepared in an emulsion type, a lotion type, an emulsion type, an ampoule type or an essence type, the skin cell activator, dipropylene glycol, 1, It may be prepared by using 3-butylene glycol, polysorbate, dipotassium glycylizate, allantoin, panthenol and glycerin, or may be prepared by further mixing purified water, perfume and preservatives.

At this time, the dipropylene glycol is to act as a skin moisturizing, with respect to 100 parts by weight of skin cell activator, 1,000 to 1,700 parts by weight, preferably 1,200 to 1,500 parts by weight of dipropylene glycol, wherein, dipropylene glycol If the amount of less than 1,000 parts by weight may be a problem to reduce the moisturizing feeling, and when used in excess of 1700 parts by weight may be uneconomical it is recommended to use within the above range.

In addition, the 1,3-butylene glycol serves as a skin moisturizing role, with respect to 100 parts by weight of skin cell activator, 800 to 1,200 parts by weight, preferably 900 to 1,100 parts by weight of 1,3-butylene glycol In this case, when the amount of 1,3-butylene glycol is less than 800 parts by weight, there may be a problem of reducing the moisturizing feeling, and when used in excess of 1,200 parts by weight may cause a problem of destabilizing the formulation within the above range Good to use at

In addition, the polysorbate serves as a solubilizing agent, with respect to 100 parts by weight of the skin cell activator, 450 to 700 parts by weight of polysorbate, preferably 550 to 650 parts by weight, wherein the polysorbate If the amount is less than 450 parts by weight, there may be a problem to separate the formulation, and when used in excess of 700 parts by weight may cause a problem of causing stickiness and foaming when applying the skin, it is recommended to use within the above range.

In addition, as the dipotassium glycylizate plays a role in stimulating relaxation, the dipotassium glycylizate may be used in an amount of 60 to 130 parts by weight, preferably 80 to 120 parts by weight, based on 100 parts by weight of a skin cell activator. In this case, if the amount of dipotassium glycylizate is less than 60 parts by weight, there may be a problem that prevents the role of stimulation relief, and when used in excess of 130 parts by weight, there may be a problem of breaking the stability of the formulation. Good to use at

In addition, the allantoin acts as a stimulating alleviator, based on 100 parts by weight of the skin cell activator, allantoin 60 to 150 parts by weight, preferably 80 to 120 parts by weight, wherein the amount of allantoin is 60 parts by weight If less than there may be a problem that prevents the role of stimulation relief, and when used in excess of 150 parts by weight may be a problem of breaking the stability of the formulation is recommended to use within the above range.

In addition, the panthenol serves to supply nutrients to the skin with a stimulant and vitamin B5, and 50 to 150 parts by weight of panthenol, preferably 60 to 130 parts by weight, based on 100 parts by weight of a skin cell activator, In this case, when the amount of allantoin is less than 60 parts by weight, there may be a problem that prevents the role of irritation and skin nutrient supply, and when used in excess of 150 parts by weight, there may be a problem of breaking the stability of the formulation. Good to use at

In addition, the glycerin serves as a skin moisturizing role, 400 to 800 parts by weight of glycerin, preferably 500 to 700 parts by weight based on 100 parts by weight of the skin cell activator, wherein the amount of allantoin is 400 parts by weight If less than there may be a problem to reduce the skin moisturizing, if used in excess of 800 parts by weight may cause a problem causing stickiness it is recommended to use within the above range.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are not intended to limit the scope of the present invention, which will be construed as to help the understanding of the present invention.

EXAMPLE

Preparation Example 1 Preparation of Bio-Peptone Fermentation

Peptonated proteases were purchased from Fluka (Sigma-Aldrich, St. Quentin Fallavier, France). 500 g of the peptonated protein soybean peptone was added to the medium of the ingredients and contents shown in Table 1 below, mixed, and then sterilized by heating at 121 ° C. for 15 minutes. Next, after cooling, the inoculated Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) as lactic acid bacteria so that the initial number of bacteria 1 × 10 ⁶ cfu / ㎖, incubated and fermented for 48 hours at 30 ℃ while stirring at 50 rpm to bio Peptone fermentation cultures were obtained.

Next, the biopeptone fermentation culture obtained through the above process was heated to a final temperature of 95 ° C., followed by centrifugation (3,000 rpm, 15 min) to remove cells and insoluble components, thereby preparing a bio peptone fermentation product. . The freeze-dried product was used, and about 45 g of the lyophilized soy peptide fermented product was obtained from 1L of biopeptone fermented product.

No. Ingredient Name Content (w / w) One Soybean Peptone 5.000% 2 Glucose 0.500% 3 Potassium phosphate 0.2500% 4 Sodium acetate 0.120% 5 Ammonium citrate 0.120% 6 Magnesium Sulfate (MgSO ₄ ) 0.012% 7 Manganese Sulfate (MnSO ₄ ) 0.003% 8 Zinc Sulfate (ZnSO ₄ ) 0.001% 9 Copper Sulfate (CuSO ₄ ) 0.001% 10 Purified water 93.993% Sum 100.000%

Experimental Example  1: high performance liquid chromatography ( HPLC ) And mass spectrometry (MS) measurements Through bio  Molecular weight confirmation test of peptone fermentation broth

In order to confirm the biopeptone fermentation broth molecular weight of Preparation Example 1, High performance liquid chromatography and MS spectra were measured.

High-performance liquid chromatography measurement was performed using Accela UHPLC (manufacturer: Thermo Scientific). Lyophilized biopeptone fermentation was dissolved in 0.1% formic acid, and the temperature was set to RT (Room Temperature). The column used Water BEH C18 (2.1 × 100 mL, 1.7 μm) and the flow rate was maintained at 300 μl per minute. Sample analysis time was performed up to 60 minutes, the measurement results from 0 minutes to 60 minutes is shown in Figure 1a, the measurement results from 0 to 25 minutes are shown in Figure 1b.

In addition, MS spectral measurements were performed using an LTQ Orbitrap XL mass spectrometer (manufactured by Thermo Scientific). Five peaks having a large area among the single components separated by liquid chromatography were arbitrarily selected, and the molecular weight was confirmed through an MS spectrum. The results are shown in FIG. 2.

2 to 5 are data obtained by measuring the molecular weights of the peaks of the 1 to 5 display portions of FIG. 1B. Preferably, as a peptide having a weight average molecular weight of 200 to 300, it was estimated and / or confirmed that the peptide is composed of 2 to 4 amino acids.

Example  1: bio peptone From fermented products Fraction  acheive

Under the conditions of Table 2 and Table 3, the biopeptone fermentation product of Preparation Example 1 was partitioned into 6 fractions by fractionation by preparative liquid chromatography (Prep-LC), and 6 fractions were obtained. 3a is a graph when fractionated with ultrapure water (Di water), and FIG. 3b is a graph when fractionated with acetonitrile. In addition, photographs of six fractions are shown in FIG. 4, and the amounts of each of the obtained fractions are shown in Table 4 below.

device Waters delta preplc 300 software Waters empower column Kromasil 100-10C18, 250 mm ⅹ30mm menstruum A: Di water
B: Acetonitrile Flow rate 20 ml / min Injection of Samples for Purification 5 mL once

Time (minutes) Ultrapure Water (%) Acetonitrile (%) 0.0 100 0 20 100 0 30 80 20 35 0 100 40 0 100 41 100 0 50 100 0

division Fraction number
(Fraction No.) Weight (g) Third fraction 2-2 287.76 4th fraction 2-8 to 2-13 73.28 5th fraction 2-21 to 2-24 57.44 First fraction 2-25 to 2-26 140.59 Second fraction 2-28 to 2-30 162.59 6th fraction 2-31 to 2-33 121.78

Experimental Example  2: LC-MS (LC-Mass spectrometry) measurement

Under the conditions of Table 5 and Table 6, the first to six fractions obtained in Example 1 were subjected to LC-MS analysis for component profile pattern analysis for each, and the results are shown in FIG. 5.

menstruum Di water, acetonitrile (B & J) Reagent Formic acid (Aldrich)

Chromatography Thermo Accela UPLC Mass spectrometry Thermo LTQ-Orbitrap XL Column BEH (Ethylene Bridged Hybrid) C18,
1.7μm (particle size), 150mm × 2.1mm Solvent A: ultrapure water (0.1% formic acid)
B: acetonitrile (0.1% formic acid) Flow rate 0.4 ml / min Injection 2 μL Detection ion mode MS: positive ion [M ⁺ H] ⁺ Scan range MS: m / z 115-1500 Spray voltage 3.5 kV Capillary voltage 45 V Capillary temperature 300 ℃ software Xcalibur

Looking at the measurement results of Figure 5, it was confirmed that the first fraction has the strongest peak at 1.96 ~ 2.30 minutes, the second fraction has the strongest peak at 23.40 ~ 23.70 minutes. In addition, it was confirmed that the third fraction had the strongest peak at 1.60 to 1.75 minutes, the fourth fraction at 9.0 to 9.3 minutes, the fifth fraction at 7.20 to 7.70 minutes, and the sixth fraction at 23.0 to 23.40 minutes. The main peak results of each fraction were as in Table 7 below.

division Peak occurrence time (minutes) First fraction 1.96 ~ 2.15 min / 7.4 ~ 7.5 min / 11.6 ~ 12.0 min / 12.45 ~ 12.55 min / 22.98 ~ 23.15 min Second fraction 2.20 to 2.40 minutes / 4.10 to 4.35 minutes / 6.30 to 6.50 minutes / 11.5 to 11.9 minutes / 14.2 to 14.8 minutes / 15.2 to 16.0 minutes / 17.60 to 17.90 minutes / 18.50 to 18.90 minutes / 19.40 to 19.80 minutes / 20.2 to 20.7 minutes /
23.40 ~ 23.70 minutes / 23.71 ~ 23.80 minutes Third fraction 1.60 ~ 1.75 min / 1.90 ~ 2.30 min / 4.35 ~ 4.65 min /
4.9 to 5.2 minutes / 9.60 to 9.85 minutes / 11.55 to 11.90 minutes 4th fraction 1.50 ~ 1.80 min / 1.90 ~ 2.20 min / 4.9 ~ 5.2 min / 5.7 ~ 6.0 min /
8.15 to 8.40 minutes / 9.0 to 9.3 minutes / 9.4 to 9.7 minutes / 13.60 to 14.05 minutes / 14.10 to 14.30 minutes / 20 to 20.90 minutes / 23.0 to 23.35 minutes 5th fraction 1.70 to 1.90 minutes / 2.0 to 2.20 minutes / 7.20 to 7.70 minutes / 11.0 to 11.5 minutes / 13.8 to 14.2 minutes / 15.7 to 16.2 minutes / 22.30 to 22.70 minutes / 22.80 to 23.0 minutes / 23.25 to 23.55 minutes 6th fraction 10.70 to 11.00 minutes / 12.20 to 12.45 minutes / 12.50 to 13.0 minutes / 17.30 to 17.70 minutes / 17.70 to 17.95 minutes / 22.20 to 22.80 minutes / 23.0 to 23.40 minutes / 23.65 to 23.90 minutes / 24.05 to 24.40 minutes

Experimental Example  3: WST -1 assay Cytotoxicity and cell proliferation confirmation test

(1) Human epidermal Keratinocytes (Human Epidermal Keratinocytes) and Human Dermal Fibroblasts (Human Dermal Fibroblasts) were obtained from Innoprot (Bizkaia, Spain) and 10% FBS (Fetal bovine serum, Welgene, Daegu) (Dulbecco's modified Eagle's medium, Welgene, Daegu, Korea) were used to incubate at 37 ℃, 5% CO ₂ cell culture. The culture solution was changed every 3 days, and when the cell number showed more than 80% growth, passage was performed and the experiment was performed in a stable cell state for 2 weeks.

(2) Fraction HaCaT  Cytotoxicity and Cell Proliferation Activity Against Cells

To determine whether the fractions are toxic to HaCaT cells, 200 μL of 1 × 10 ⁴ cells / well per well were placed in a 96 well plate, followed by 24 hours in a 37 ° C., 5% CO ₂ cell incubator. Incubated for After 24 hours, the WST-1 assay was performed 24 hours after treatment with FMEM-containing DMEM medium and fractions (or biopeptone fermented or untreated). Into each well, 20 μL of WST-1 solution (Cell Proliferation Reagent WST-1, Cat no. 05 015 944 001, Roche) was added to 10% of the total, followed by 2 hours at 37 ° C and 5% CO ₂ cell incubator. Reacted.

Absorbance was measured at 450 nm and 620 nm using a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA) for absorbance measurement. The survival rate of the cells was calculated based on the following equation 1 based on the control group, 6 is shown.

[Equation 1]

Cell viability (% control) = (OD _S450 -OD _S620 ) / (OD _C450 -OD _C620 ) × 100 (%)

In Equation 1, OD _S45 is the absorbance value of the sample at 450nm, OD _S620 is the absorbance value of the sample at 620nm, OD _C450 is the absorbance value of the control at 450nm, and OD _C450 is the absorbance value of the control at 620nm.

The analytical data was expressed as mean ± standard error (mean ± SE), and the experimental results were statistically processed at p <0.05 level using SPSS 12.0K (SPSS Inc., Chicago, IL, USA) and t -test And Duncan's multiple range test.

In FIG. 6, the control group was not added any of the biopeptone fermentation product of Preparation Example 1 and the fractions of Example 1, and Bp of FIG. 6 was treated with 800 ppm concentration of the biopeptone fermentation product, and the fractions were each 800 ppm. It is treated with concentration. And, the survival ratio is expressed by percentage based on the control (100%).

Looking at Figure 6, both fractions were not toxic to HaCaT cells higher than the control, but rather showed a result of cell proliferation activity.

In particular, the first fraction and the second fraction was confirmed to have a better cell proliferation activity than the bio peptone fermentation.

(3) first and Second fraction HaCaT  Cytotoxicity and Cell Proliferation Activity Against Cells and Fibroblasts

Cytotoxicity against HaCaT cells and fibroblasts by concentration (100 ppm, 300 ppm, 500 ppm, 800 ppm, 1,000 ppm, 2,000 ppm) on the first and second fractions showing better cell proliferation activity than biopeptone fermentation And cell proliferation activity were measured in the same manner as in (2), and the results are shown in FIGS. 7 and 8.

Figure 7a is a first fraction for HaCaT cells, Figure 7b is a second fraction measurement results for HaCaT cells, Figure 8a is a first fraction for fibroblasts, Figure 8b is a second fraction measurement results for fibroblasts. And, the control of Figures 7 and 8 is not treated with fractions and bio peptone fermentation.

7A and 8A, the first fraction showed not only cytotoxicity but also significantly improved cell proliferation activity compared to the control group even at a low concentration of 100 ppm, especially the highest cell at 500 to 800 ppm. It showed proliferative activity. However, the cell proliferation activity was deteriorated from the time point exceeding 1,000 ppm.

8B and 8B, the second fraction also showed no cytotoxicity. However, even at a low concentration of 100 ppm, the cell proliferation activity was lower than that of the control group. The cell proliferation activity was remarkably increased from 500 ppm, and the highest cell proliferation activity was shown at 800 ppm. The use of ppm did not significantly reduce cell proliferation activity, and showed high cell proliferation activity.

Experimental Example  4 : Fraction Skin cells Mobility (cellular migration) and cell healing assay

The mobility of skin cells is a general physiological activity for continuously proliferating in the dermis, moving to the epidermis and differentiating into epidermal cells, and is one of the very important cell activating functions for skin reconstruction due to wounds. Experiments were conducted to determine the effect of fractions on the mobility of these skin cells.

In the experiment, the first fraction and the second fraction were used. After dispensing 1 × 10 ⁵ cells / ml of dermal cells into 6well plates, the cells were cultured at 37 ° C. and 5% CO ₂ for stabilization. Incubated for 24 hours. After 24 hours, the medium was removed, washed twice with 1X PBS, and then cultured with 10% FBS. Next, when the number of cells is about 80 ~ 90% using a 200 μl pipette tip (pipet tip) to the cells were wound. Then, the separated cells were washed with 1 X PBS, loaded with fresh medium, and captured through a phase contrast microscope, as shown in FIG. 9A.

Next, 10% FBS was added after washing with 1X PBS. Control group 1 was treated only with 10% FBS, control group 2 was treated with 10% FBS + 20 ppm of EGF, and the remaining groups were treated with 10% FBS + first fraction or second fraction to 500 ppm each. And, after treatment with EGF or fractions and incubated for 24 hours under 37 ℃, 5% CO ₂ culture conditions. After 24 hours, the medium was discarded, washed with 1 X PBS, and the medium was added and captured at a 200-fold rate through a phase contrast microscope, and the results are shown in B of FIG. 9.

Comparing the A and B of Figure 9, in the case of the control group 1, there is no cell regeneration effect, the control 2. The first fraction and the second fraction can be confirmed that the cell regeneration effect, in particular, the first fraction and agent The cell regeneration effect of the two fractions was confirmed to be relatively superior to the control 2.

Through this, it could be confirmed that the fraction of the present invention, that is, the skin cell activator, can increase the proliferation of cells generated due to the wound and effectively prevent or quickly regenerate the skin wound.

Experimental Example  5: Measurement of collagen type synthesis promoting effect

Collagen, which accounts for about 70-80% of the dry weight of skin epithelial cells, is known as a major factor related to skin elasticity and wrinkles along with hyaluronic acid and elastin. In general, the balance of collagen-type synthesis and the activity of matrix metalloproteinase-1 (MMP-1), which is one of the enzymes in the skin, is balanced, but collagen-type synthesis is degraded and the enzyme of MMP-1 is degraded in aging skin tissue. It is known that the activity is increased. Therefore, collagen synthesis promoting effect is an important factor for reducing elasticity and wrinkle prevention.

The collagen synthesis promoting effect of the skin cell activator was measured as follows.

Experiments were performed using PIP (Procollagen Type C-peptide) EIA kit (Cat no. MK101, Takara Bio) to confirm the type 1 collagen biosynthesis promoting effect of the skin cell activator.

Human Dermal Fibroblast, the same human dermal-forming cell line used in Experimental Example 3, was inoculated at 5 × 10 ⁵ cells / well in 6 well-plates and then cultured in a 37 ° C., 5% CO ₂ cell incubator. After 24 hours, 800 ppm of the first fraction, the second fraction, or the biopeptone fermentation of Preparation Example 1 were treated with Dulbeco's Modified Eagle's Media (DMEM) medium containing no serum (FBS), followed by 37 ° C, 5% CO. _The cells were cultured for 24 hours under ₂ conditions and the cell cultures were recovered.

Next, 100 μL of the Antibody-POD Conjugate Solution per well was added to the Anti-PIP Monoclonal Antibody Plate, and 20 μL of the recovered cell culture solution was added and reacted at 37 ° C. in a 5% CO ₂ cell incubator for 3 hours. After washing the wells four times using PBS, 100 μL of TMBZ (3,3 ', 5,5'-tetramethylbenzene) was added as a substrate solution and reacted at 25 to 27 ° C. Next, 100 μL of Stop solution (1N H ₂ SO ₄ solution) was added to stop the reaction, and the absorbance was measured. The results are shown in FIG. 10.

Absorbance was measured by absorbance at 450nm using a VERSAmax microplate reader (Molecular Devices, Sunnyvale, Calif.) And the synthesis rate of synthesized collagen (PIP) was calculated based on a standard curve. The analytical data were expressed as mean ± standard error (mean ± SE), and the experimental results were statistically processed at p <0.05 level using SPSS 12.0K (SPSS Inc., Chicago, IL, USA) and t -test And Duncan's multiple range test.

In Figure 10, control 1 is not treated with EGF, bio peptone fermentation or fractions, control 2 is treated with 20 ppm of EGF, Bp is the bio peptone fermentation of Preparation Example 1, the remaining group is the first fraction Alternatively, the second fractions were treated to 800 ppm each.

Referring to FIG. 10, it can be seen that Bp, the first fraction, and the second fraction have a superior collagen synthesis-promoting effect compared to the control group 1 or the control group 2, and the first and second fractions have relatively better collagen than the Bp. It was confirmed that the synthesis promoting effect. Through this, it was confirmed that the skin cell activator of the present invention can effectively increase the elasticity of the skin or effectively prevent the formation of wrinkles due to skin aging in the long term.

Experimental Example  6: tyrosinase enzyme ( Tyrosinase ) Inhibition effect measurement

Tyrosinase is an enzyme involved in the initial synthesis of the melanin biosynthetic pathway in the human body. When melanocytes are exposed to stress caused by external and endogenous factors such as ultraviolet rays, drugs, and α-MSH, the amino acid tyrosine is catalyzed by tyrosinase and thus L-DOPA. The melanin pigment is formed through the path of dopaquinone. Therefore, in order to confirm the inhibitory effect of tyrosinase, which acts as an important catalyst in melanin biosynthesis, we proceeded the inhibitory effect of tyrosinase enzymes involved in the melanin biosynthesis step for skin cell activators.

(1) Melanocyte B16F10 (Human Epidermal Melanocytes), a melanocyte-forming cell line, was sold by Innoprot (Bizkaia, Spain) and contained DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum, Welgene, Daegu, Korea). , Welgene, Daegu, Korea) were used to culture in 37 ℃, 5% CO ₂ cell culture. The culture solution was changed every 3 days, and when the cell number showed more than 80% growth, passage was performed and the experiment was conducted in a stable cell state for 2 weeks.

Next, 550 μL of distilled water (DW), 100 μL of 100 U tyrosine oxidase (Mushroom tyrosinase), 50 μL of 0.1 M KPB (potassium phosphate buffer), 50 μL of the melanocyte cells and the sample (albutin, biopeptone fermentate, first fraction or second) Fraction)) was charged at 800 ppm, mixed, and pre-incubated at 37 ° C. for 5 minutes. Next, 200 μL of 10 mM L-DOPA was added as a substrate and reacted at 37 ° C. for 10-20 minutes for enzyme activity. Next, the reaction solution was transferred to a 96 well-plate, and then absorbance was measured at 475 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, Calif.) And inhibition of tyrosinase enzyme activity was measured in comparison with the control group. Here, the control group was treated only with 1X PBS (Dulbecco`s Phosphate Buffer Saline, Cat.no 21300-025) instead of Example 1, the control group 2 was treated with arbutin (arbutin, 100 ppm), Bp Treated with the bio peptone fermentation of Preparation Example 1, the measurement results thereof are shown in FIG. Here, α-MSH (100 nM) acted as a positive control to stimulate melanin production by stimulating melanocytes, and the control group was 1X PBS (Dulbecco`s Phosphate Buffer Saline of Gibco, Cat.no 21300-025 ).

And, tyrosinase enzyme inhibition measurement was calculated based on the following equation 2 compared to the control.

[Equation 2]

% Tyrosinase enzyme inhibition = (Od _sample -OD _blank ) / (OD _control -OD _blank ) × 100

In Equation 2, OD _sample is the absorbance value of the _sample at 475nm, OD _control is the absorbance value of the _control at 475nm.

Referring to Figure 11, when compared with the control group 1, it can be seen that the biopeptone fermentation, the first fraction and the second fraction of the control group 2, Preparation Example 1 using the arbutin has a tyrosinase enzyme inhibitory effect.

In addition, it was confirmed that the first fraction and the second fraction, which are fractions fractionated from the biopeptone fermented product (Bp) of Preparation Example 1, were superior in inhibiting tyrosinase activity than Bp. It was confirmed that the skin cell activating agent of the present invention can effectively reduce the melanin production from tyrosine by inhibiting the activity of tyrosinase, so that the skin whitening effect can be expected.

Experimental Example  7: In melanocytes  Melanin Pigmentation Inhibitory Effect

Subsequent cultured and stabilized melanocytes B16F10 in the same manner used in Experimental Example 6 was dispensed in 6 well-plate at 2 × 10 ⁵ cells / well and incubated under 37 ° C. and 5% CO ₂ conditions. After 24 hours, the cells were treated with 0.1 mM IBMX (3-isobutyl-1-methylxanthine) and incubated for 48 hours at 37 ° C. and 5% CO ₂ cell incubator to induce intracellular melanin production, followed by arbutin (arbutin, 20 ppm). ), The biopeptone fermented product of Preparation Example 1 (800 ppm), the first fraction (800ppm) or the second fraction (800ppm). Then, the cells were washed with PBS, the cells were collected using a cell scraper, and centrifuged. Then, 800 μL of 1N-NaOH was added to a pellet obtained by removing the supernatant, and the cells were lysed at 80 ° C. in a thermostat. . Next, the absorbance was measured at 405 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA), and the melanin pigment amount was calculated based on Equation 3 below by comparing with a control, and the results are shown in FIG. 12A. In addition, a photograph of observing melanin production at 100 magnification using a phase contrast microscope is shown in FIG. 12B. Here, control 1 is none of the IBMX, arbutin and skin cell activator, and control 2 is treated only with IBMX.

Referring to FIG. 12A, while the melanin pigment was increased by 140% compared to the control group 1 in the IBMX-added cells (control 2), the production rate of the melanin pigment was increased by adding 800 ppm of the biopeptone fermentation product or fraction of IBMX. It was confirmed that the decrease to 115% compared to the control.

In addition, melanocytes were treated with IBMX to induce melanin pigment formation and then treated with biopeptone fermentation products and fractions to observe melanin pigments under a microscope (FIG. 12B), biopeptone fermentation products, first fractions, and the like. The two fractions inhibit melanin production in melanocytes, and through this result, it was confirmed that the skin cell activator of the present invention can effectively reduce melanin production in melanocytes and expect a whitening effect on the skin.

Production Example  1: Functionality Ampoule  Produce

Ampoules having the components shown in Table 8 were prepared using the skin cell activator of the first fraction. In more detail, dipropylene glycol, 1,3-butylene glycol, dipotassium glycyriate, allantoin, panthenol, and glycerin were added to purified water and mixed well to prepare a first mixture. Polysorbate 80 was added thereto, solubilized by mixing, followed by mixing with a preservative to prepare a second mixture. Next, preservatives, fragrances, and bio peptone fermented products were added to the second mixture, and stirred well to prepare an ampoule-type functional skin external preparation containing about 5,000 ppm of skin cell activator. Ampoule preparation was made at room temperature because it can be prepared at room temperature conditions without heating conditions.

division ingredient Manufacturing compound ratio
(Parts by weight) One Skin Cell Activator of Example 1 100 2 Dipropylene glycol 1,400 3 1,3-butylene glycol 1,000 4 Polysorbate 80 600 5 Dipotassium glycylizate 100 6 Allantoin 100 7 Panthenol 100 8 glycerin 600 9 Purified water 15,800 10 antiseptic 100 11 Spices 100

Production Example  2: Preparation of functional external preparation (cream)

Using a bio peptone fermentation of Example 1 to prepare a cream-type external preparation having the components shown in Table 8. In more detail, the purified water was added to the carboxyvinyl polymer and mixed to prepare the carboxyvinyl polymer. Then, glycerin, butylene glycol, dipropylene glycol, and lecithin were added thereto to prepare a first mixture. In addition, polysorbate 80, cured castor oil, glyceryl stearate, cetearyl alcohol, cetylethylhexanoate was added to prepare a second mixture. Then, each of the first and second mixtures was heated to 80 ° C., and then mixed and stirred to prepare a third mixture. Next, stir the third mixture evenly, drop the temperature to 40 ° C, add preservatives, fragrances, bio peptone fermentation and licorice extract, and then stir and mix to contain about 5,000 μg / mL biopeptone fermentation. A functional external preparation of a cream type having a composition of Table 9 was prepared.

division ingredient Manufacturing compound ratio
(Parts by weight) One Biopeptone Fermentation of Example 1 100 2 glycerin 600 3 Butylene glycol 1,000 4 Dipropylene glycol 1,200 5 Carboxyl Vinyl Polymer 60 6 Polysorbate 80 100 7 Cured Castor Oil 800 8 Glyceryl Stearate 100 9 Cetearyl Alcohol 200 10 Cetylethylhexanoate 600 11 Licorice Extract 20 12 lecithin 20 13 Purified water 15,000 14 antiseptic 100 15 Spices 100

Through the above examples and experimental examples, the skin cell activator of the present invention not only has an inhibitory effect on melanin pigment production through promoting skin collagen synthesis and inhibiting tyrosinase, but also without cytotoxicity, skin cell mobility and cell regeneration. Excellent effect, it was confirmed that it can be applied as a skin external preparation in various types of cosmetics, ampoules or ointments by applying as a skin cosmetic material or a skin disease treatment / prevention material.

Claims (13)

As a fraction obtained by preparative liquid chromatography, a biopeptone fermentation product containing a low molecular weight peptide having a weight average molecular weight of 200 to 500 in 100% by weight (including other unavoidable impurities) is prepared.
The bio peptone fermentation product is a fermentation product obtained by fermenting a peptonated proteolyte obtained by peptizing soybean with Lactobacillus rhamnosus.
The fraction is measured by high performance liquid chromatography for 30 minutes,
A first fraction having the strongest peak at 1.96-2.30 minutes; The second fraction having the strongest peak at 23.40 to 23.70 minutes; The fifth fraction having the strongest peak at 7.20 to 7.70 minutes; And a sixth fraction having the strongest peak at 23.0 to 23.40 minutes; At least one fraction selected from among,
The high-performance liquid chromatography measurement was performed using a column BEH (Ethylene Bridged Hybrid) C18, Thermo Accela UPLC equipped with 1.7 μm (particle size), scan range m / z 115 to 1500, injection potential 3.5 Skin cell activator for skin cosmetics, characterized in that measured under kV, Capillary voltage (Capillary voltage) 45V and Capillary Temperature (300 ℃).
The skin cell activator for skin cosmetics according to claim 1, wherein the fraction comprises at least one selected from a first fraction and a second fraction.
The skin cell activator according to claim 1, wherein the first fraction further has peaks at 7.4 to 7.5 minutes, 12.45 to 12.55 minutes, and 23.0 to 23.12 minutes.
The skin cell activator according to claim 1, wherein the second fraction further has peaks at 6.30 to 6.50 minutes, 15.2 to 16.0 minutes, and 20.2 to 20.7 minutes.
The method of claim 1, wherein the fifth fraction further has peaks at 1.70 to 1.90 minutes, 2.0 to 2.20 minutes, 11.0 to 11.5 minutes, 13.8 to 14.2 minutes, 22.30 to 22.70 minutes, and 22.80 to 23.0 minutes,
The sixth fraction further has peaks at 10.70 to 11.00 minutes, 12.50 to 13.0 minutes, 17.30 to 17.70 minutes, 17.70 to 17.95 minutes, and 22.20 to 22.80 minutes.
delete delete delete The skin cell activator for skin cosmetics according to claim 1, wherein the small molecule peptide comprises one or more selected from dipeptide, tripeptide and tetrapeptide.
The skin cell activator for skin cosmetics according to claim 1, wherein the fraction comprises 100 ppm to 10,000 ppm.
1 step of preparing a bio peptone fermentation product containing a low molecular weight peptide having a weight average molecular weight of 200 to 500 in 100% by weight (including other unavoidable impurities); And
And fractionating the biopeptone fermented product by preparative liquid chromatography to obtain a fraction.
The bio peptone fermentation product is a fermentation product obtained by fermenting a peptonated proteolyte obtained by peptizing soybean with Lactobacillus rhamnosus.
The fraction is measured by high performance liquid chromatography for 30 minutes,
A first fraction having the strongest peak at 1.96-2.30 minutes; The second fraction having the strongest peak at 23.40 to 23.70 minutes; The fifth fraction having the strongest peak at 7.20 to 7.70 minutes; And a sixth fraction having the strongest peak at 23.0 to 23.40 minutes; At least one fraction selected from among
The high-performance liquid chromatography measurement was performed using a column BEH (Ethylene Bridged Hybrid) C18, Thermo Accela UPLC equipped with 1.7 μm (particle size), scan range m / z 115 to 1500, injection potential 3.5 kV, Capillary potential (Capillary voltage) 45V and Capillary Temperature (Capillary Temperature) The method for producing a skin cell activator for skin cosmetics, characterized in that measured under 300 ℃:
Claims 1 to 5 and 9 to 10 of any one of the skin cosmetic agent comprising a skin cell activator selected from. delete

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