KR20130058107A - Isolation and preparation of small peptides from soybean and its cosmetic composition - Google Patents

Abstract

PURPOSE: A biji(soybean curd residue)-derived low molecular peptide extract is provided to suppress DPPH oxidation and collagenase expression, to promote procollagen generation, and to ensure excellent antioxidation, anti-wrinkling, and antiaging effects. CONSTITUTION: A cosmetic composition contains a biji(soybean curd residue)-derived low molecular peptide extract as an active ingredient. The molecular weight of the low molecular peptide is 500-3000 Da. The biji is prepared from one or more kinds selected from a group consisting of Glycine max (L.) Merr., Rhynchosia Nulubilis Loureiro, Phaseolus angularis (Willd.) W. Wight, Vigna radiata (L.) Wilczek, Vigna sinensis King, Pisum sativum L., Phaseolus vulgaris var. humilis, Viciafaba L., and Canavalia gladiata DC. A method for preparing the extract comprises: a step of adding an extraction solvent to the biji and extracting; a step of filtering the extract and performing a heat treatment; and a step of centrifuging. [Reference numerals] (AA) Intensity of light%; (BB) Mass/electric charge(m/z)

Description

Isolation and preparation of small peptides from soybean and its Cosmetic composition}

The present invention relates to a method for separating a low molecular peptide extract from soybean and a cosmetic composition containing the same, and more particularly to a cosmetic composition having an effect on antioxidant and anti-aging by containing an extract mainly containing a low molecular peptide from soy bean curd. It is about.

Free radicals are produced by various physical, chemical and environmental factors such as enzymes, reducing metabolism, chemicals, pollutants and photochemical reactions in the body, and are non-selective and irreversible for cell components such as lipids, proteins, sugars and DNA. Destructive action is known to cause various diseases including cell aging or cancer. In addition, various body peroxides, including lipids and oxides resulting from lipid peroxidation by these free radicals, can also cause various diseases by causing oxidative destruction of cells and various functional disorders. Therefore, antioxidants such as free radical scavengers or peroxide production inhibitors are expected to suppress or treat aging and various diseases caused by these oxides.

Human skin is changed by a number of internal and external factors as it ages. That is, internally, the secretion of various hormones that regulate metabolism decreases, and the function of immune cells and the activity of cells decreases, thereby reducing the biosynthesis of immune proteins and constituent proteins necessary for living organisms. Due to the increase in the amount of ultraviolet rays that reach the surface of the sun's rays and intensifying environmental pollution, free radicals and free radicals increase, thereby reducing the thickness of the skin, increasing wrinkles, reducing elasticity, The color of the skin becomes dull, skin problems frequently occur, and there are various changes such as blemishes, freckles, and black mushrooms.

As aging progresses, symptoms of alteration or decrease in the content and arrangement of collagen, elastin, hyaluronic acid and glycoproteins, which are constituents of the skin, appear and are subject to oxidative stress caused by free radicals and active harmful oxygen. In addition, cyclooxygenase-2 (Cox-2, cyclooxygenase), an enzyme that produces proinflammatory cytokine, which is known to cause inflammation in most cells constituting the skin due to aging or ultraviolet rays. Biosynthesis of NM increases, and biosynthesis of matrix metalloproteinase (MMP), an enzyme that degrades skin tissues by these inflammatory factors, increases NO (nitric oxide) production by inducible nitric oxide synthase (iNOS). It is known to increase. In other words, the biosynthesis of the substrate material is reduced by the reduction of cellular activity and micro-inflammation due to naturally occurring endogenous aging, and by external factors such as the increase of stress caused by various harmful environments and the increase of active coral species caused by sunlight. As a result, decomposition and degeneration are accelerated, and the skin matrix is destroyed and thinned, and various symptoms of skin aging appear. Therefore, many studies have been conducted on active ingredients that can prevent and improve these aging phenomena.

Peptides are polymerized by peptide bonds of various amino acids. Generally, 2-50 amino acids are bonded and molecular weight is 10,000 Da (dalton) or less. Oligopeptide refers to a peptide having 10 or less amino acids. Among them, the low molecular soybean peptide has high solubility, little precipitate by acid alkali, and various applications as a health food material due to low viscosity in aqueous solution. . In particular, oligopeptides with a molecular weight of 1,000 Da or more are released by binding to bile acids and cholesterol in the small intestine, thereby lowering serum cholesterol concentrations and physiological activities such as antitumor, antihypertensive and immune enhancement. No aging effect has been reported yet.

Therefore, the present inventors, while studying to prepare an antioxidant and anti-aging cosmetics containing low-molecular peptides that are safe for the skin, excellent in antioxidant and anti-aging effects, and excellent stability as a product, soybean extract of bean extract After the filtration and heat treatment, the extract prepared through the centrifugation method was found to have a high content of low molecular peptide, very excellent in antioxidant and anti-aging effects, and completed the present invention.

Therefore, an object of the present invention is to extract the bean busy; Performing heat treatment after filtration on the extract of the step; Centrifuging the extract heat-treated in the step; To provide a method for producing a low molecular soy peptide, the content of the low molecular peptide comprising a.

Another object of the present invention is to provide a cosmetic composition having excellent skin antioxidant and anti-aging efficacy, which contains a low molecular weight peptide extract from soybeans extracted by the preparation method.

In order to achieve the above object, the present invention comprises the steps of extracting soy bean curd; Performing heat treatment after filtration on the extract of the step; Centrifuging the extract heat-treated in the step; It provides a method for producing a low molecular soy peptide, the content of the low molecular peptide comprising an increased. The present invention also provides a cosmetic composition containing the extract as an active ingredient.

Soy bean curd-derived low molecular weight peptide extract according to the present invention has an excellent anti-DPPH antioxidant activity and antioxidant activity, and has been shown to promote procollagen production and inhibit collagenase expression. Effect and anti-aging effect.

1 is a graph showing the protein size contained in the soybean extract.
Figure 2 is a graph showing the protein size contained in soy bean curd extract.
Figure 3 is a graph showing the protein size contained in the low molecular peptide extract derived from soy bean curd.

The cosmetic composition provided by the present invention contains soy bean curd-derived low molecular weight peptide extract as an active ingredient.

Soy bean curd of the present invention white, frosted (black bean) ( Glycine max (L.) Merr.), Rhynchosia Nulubilis Loureiro, Red bean (P haseolus) angularis ( Willd .) W. Wight), Vigna radiata (L.) Wilczek), East ( Vigna sinensis King, Pea ( Pisum sativum L.), kidney beans ( Phaseol vulgaris var . humilis), Vicia ( Vicia) faba L. and green beans ( Canavalia) gladiata DC.) manufactured from one or more beans selected from the group consisting of can be used.

The low molecular peptide of the present invention may use a low molecular weight peptide of 100 Da or more and 3000 Da or less, preferably, 100 Da or more and 1000 Da or less.

The cosmetic composition of the present invention contains the soy bean curd-derived low molecular weight peptide extract in an amount of 0.0001 to 30% by weight based on the total weight of the composition. If the content is less than 0.0001% by weight, the antioxidant and anti-aging effects of the extract may not be obtained. If the content is more than 30% by weight, the increase in effect is not large.

The low molecular peptide extract derived from soy bean curd of the present invention contains the low molecular peptide in an amount of 10 to 30% by weight based on the total weight of the extract.

The soy bean curd of the present invention can be prepared by a conventional method used in the art, for example, after washing the soybeans with water and soaked in water overnight and soaked with millstone or grinder while adding the appropriate amount of water do. 3 to 4 times the volume of soybeans are added to the ground soybeans, and heated at 100 to 105 ° C. for 15 minutes.

Soy bean curd low molecular peptide extract used in the present invention is prepared by the following method.

1) stirring the extraction by adding the extraction solvent to soy bean curd;

2) filtering and heat-treating the bean curd extract of step 1); And

3) centrifuging the extract of step 2);

Method for producing a low molecular peptide extract derived from soy bean curd.

In the present invention, the extraction solvent of step 1) may be water or an organic solvent, and more specifically, in the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane. One selected can be used alone or in combination of two or more.

Extraction temperature of the step 1) is preferably 10 ~ 80 ℃, can be extracted for 6 to 24 hours. If the extraction temperature and extraction time is out of the extraction efficiency may decrease or change of components may occur.

Filtration in the step 2) may be used microfiltration, ultrafiltration, Nao filtration, reverse osmosis membrane, etc. in a conventional method known in the art, preferably microfiltration.

The heat treatment in the step 2) is preferably performed at 80 ~ 120 ℃, extraction efficiency may be lowered below 80 ℃, if it exceeds 120 ℃ may change the components or destroy the active ingredient.

The cosmetic composition containing soy bean curd-derived low molecular weight peptide extract prepared by the above method can be confirmed to have antioxidant effects through DPPH oxidation inhibition experiment, and it has an effect of promoting procollagen production and inhibiting collagenase expression. The complex synergy of eggplant activity can provide excellent skin wrinkle improvement.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited only to these examples.

[ Example  1] bean bean origin Low molecule Peptides  Preparation of extract

1 kg of bean curd made from white bean beans and 3 L of purified water were added to a container, followed by mixing. Thereafter, the residue and the filtrate are separated by filtration through a filter cloth, and the separated filtrate is concentrated under reduced pressure. The concentrated extract is subjected to heat treatment at 100 ° C. for 20 minutes, and the residue and filtrate are separated by centrifugation. By drying the resulting filtrate, 12 g of soy bean curd-derived low molecular peptide extracts were prepared.

[ Comparative example  1] Preparation of Soybean Extract

1 kg of white bean beans and 3 L of purified water were mixed in a container, followed by stirring. Thereafter, the residue and the filtrate were separated through filter cloth filtration, and the separated filtrate was dried to obtain 112 g of a soybean extract.

[ Comparative example  2] Preparation of Bean Soybean Extract

1 kg of bean curd made from white bean beans and 3 L of purified water were added to a container, followed by mixing. Thereafter, the residue and the filtrate were separated through filter cloth filtration, and the separated filtrate was dried to obtain 60 g of soy bean curd extract.

[ Test Example  One] Example  One, Comparative example  Protein Size Differences in 1-2 Extracts

Protein size differences of the extracts prepared in Example 1 and Comparative Examples 1 and 2 were analyzed using Maldi-Tof and the results are shown in FIGS. 1 to 3.

As can be seen from the results of FIGS. 1 to 3, in the case of the soybean extract of Comparative Example 1, the extracted protein mass is concentrated at 6900 to 8380 m / z, and the soy bean curd extract is concentrated at about 1459, 5100 and 7800 m / z. Compared to the low-molecular peptide extract derived from soybean, it can be confirmed that the small-molecular peptide having a small protein mass is extracted at a content of 10-30% at 500-100 m / z. As a result, it can be seen that an extract having a high content of the low molecular peptide can be obtained by the manufacturing method of the present invention.

[ Test Example  2] antioxidant effect test DPPH  Test)

The DPPH oxidation inhibitory effect of the soy bean curd-derived low molecular weight peptide extract of Example 1 and the natural product extracts of Comparative Examples 1 and 2 were measured in comparison with trolox.

Antioxidant activity through the change in absorbance produced by the reduction of the organic radical DPPH (1,1-diphenyl-2-picrylhydrazyl, 1,1-diphenyl-2-picryl hydrazyl) (an antioxidant is oxidized) The method of evaluating was used. Oxidation of DPPH for Example 1 and Comparative Examples 1 to 2, which were the extracts obtained above, was inhibited, and the degree of absorbance was decreased by comparing with the control group, and the concentration showing an absorbance of 50% or less compared to the control group was measured. It was evaluated by antioxidant concentration.

190 μl of 100 μM (in ethanol) DPPH solution and 10 μl of each of the Examples, Comparative Examples, and Control Samples obtained above were added thereto to make a reaction solution, and reacted at 37 ° C. for 30 minutes, and the absorbance was measured at 540 nm. As a control sample, Trolox, a widely used synthetic antioxidant, was used. The DPPH analysis results of each substance are shown in Table 1 below, and IC ₅₀ means the sample concentration when the absorbance was reduced by 50% by the added sample.

DPPH analysis result (% inhibition) sample IC ₅₀ (ppm) Trolox 45 Comparative Example 1 Bean extract 103 Comparative Example 2 Soy bean curd extract 99 Example 1 Soy bean curd-derived low molecular peptide extract 30

As can be seen in Table 1, it can be seen that Example 1, the soy bean curd-derived low molecular peptide extract according to the present invention is much superior to the antioxidant activity than Comparative Example 1 and soy bean curd extract Comparative Example 2.

[ Test Example  3] Collagenase  Expression inhibition effect measurement

Inhibition of collagenase expression of soy bean curd-derived low-molecular peptide extract of Example 1 and natural extracts of Comparative Examples 1 and 2 was measured by comparing with tocopherol and EGCG. The lower the expression level of collagenase, the higher the expression inhibiting ability of collagenase, less breakdown of collagen in the skin, and therefore, the smaller the amount of wrinkles produced. In addition, tocopherol and EGCG are antioxidants and are known to have the function of preventing skin aging by regenerating epidermal cells of the skin.

The test was performed by placing human fibroblasts at 5,000 cells / well in a 96-well microtiter plate containing Dulbecco's Modified Eagle's Media (DMEM) medium containing 2.5% fetal calf serum. Incubate until 90% growth. After culturing for 24 hours in serum-free DMEM medium, and then treated with Comparative Examples 1-2 and Example 1, tocopherol and EGCG dissolved in serum-free DMEM medium for 24 hours at 10 ^-4 molarity, cell culture solution Was taken.

The collected cell culture solution was measured for collagenase production using a commercially available collagenase measuring instrument (Amersham Pharma Co., Ltd., USA). First, the collected cell culture was placed in a 96-well plate uniformly coated with primary collagenase antibody, and the antigen-antibody reaction was performed in a thermostat for 3 hours.

After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-well plate and reacted for another 15 minutes. After 15 minutes, the coloring stimulant was added, causing color development at room temperature for 15 minutes, and 1M sulfuric acid was added again to stop the reaction (color development). The color of the reaction solution was yellow and the degree of yellow color was different according to the progress of the reaction.

The absorbance of the yellowish 96-well plate was measured at 405 nm using an absorbance meter, and the degree of synthesis of collagenase was calculated by Equation 1 below. At this time, the reaction absorbance of the cell culture liquid collected from the group not treated with the composition was used as a control. That is, the degree of expression of collagenase in the untreated group was set at 100, and the degree of expression of collagenase in the group treated with the composition was determined, and the results are shown in Table 2.

matter Collagenase
Expression degree (%) Untreated group 100 Tocopherol 75 EGCG 60 Comparative Example 1 Bean extract 96 Comparative Example 2 Soy bean curd extract 90 Example 1 Soy bean curd-derived low molecular peptide extract 56

In the results of Table 2, it can be confirmed that Example 1 of soy bean curd derived low molecular peptide extract according to the present invention inhibits the expression of collagenase in vitro . In addition, it can be confirmed that the expression inhibiting ability of collagenase is superior to tocopherol known as an antioxidant.

[ Test Example  4] Procollagen Promoting Efficacy Experiment

Procollagen production ability of the soybean extract of Comparative Example 1, the soy bean curd extract of Comparative Example 2 and the low-molecular peptide derived from soybean curd obtained in Example 1 were measured by comparing with vitamin C. Procollagen is a substance that induces collagen production and is necessary for preventing collagen production and aging. The higher the degree of procollagen production, the higher the degree of collagen production, and thus, the generation of skin wrinkles can be prevented. Vitamin C is also known as an essential ingredient in the synthesis of collagen.

The test was performed by placing human fibroblasts at 5,000 cells / well in a 96-well microtiter plate containing Dulbecco's Modified Eagle's Media (DMEM) medium containing 2.5% fetal calf serum. Incubate until 90% growth. After culturing for 24 hours in serum-free DMEM medium, and then treated with Comparative Examples 1-2 and Example 1, vitamin C in a serum-free DMEM medium for 24 hours at a molar concentration of 10 ^-4 for 24 hours, the cell culture solution Was collected. After 24 hours, the amount of free collagen in the medium was measured using the procollagen type-1 C-peptide EIA kit (MK101, Takara, Japan).

The result of the measurement was calculated by the following formula (2) the degree of production of procollagen, the procollagen production degree collected from the group not treated with the composition as a control. That is, the degree of procollagen production in the untreated group was set to 100, and the degree of procollagen production in the group treated with the composition was calculated, and the results are shown in Table 3.

matter Procollagen
% Of generation Untreated group 100 Vitamin C 120 Comparative Example 1 Bean extract 105 Comparative Example 2 Soy Bean Extract 86 Example 1 Soy bean curd-derived low molecular peptide extract 118

In the results of Table 3, it can be seen that Example 1, a soy bean curd-derived small molecule peptide extract, promotes the production of procollagen in vitro .

Claims (13)

Cosmetic composition containing a low-molecular peptide extract derived from soy bean curd as an active ingredient. The cosmetic composition according to claim 1, wherein the low molecular weight peptide has a molecular weight of 500-3000 Da. According to claim 1 or 2, wherein the bean curd is white, frosted ( Glycine max (L.) Merr.), Rhynchosia Nulubilis Loureiro, Red bean (P haseolus) angularis (Willd.) W. Wight, Vigna radiata (L.) Wilczek), East ( Vigna sinensis King), Pea ( Pisum sativum L.), Kidney Beans ( Phaseolus) vulgaris there is . humilis), broad bean (Vicia faba L. and green beans ( Canavalia) gladiata DC.) Cosmetic composition characterized in that it is prepared from one or more selected from the group consisting of. The cosmetic composition according to claim 1 or 2, wherein the low molecular peptide extract derived from soy bean curd is contained at 0.0001 to 30% by weight based on the total weight of the composition. The cosmetic composition according to claim 1 or 2, wherein the low molecular peptide extract derived from soy bean curd contains the low molecular peptide in an amount of 10 to 30% by weight based on the total weight of the extract. The cosmetic composition according to claim 1 or 2, wherein the composition is for antioxidant. The cosmetic composition according to claim 1 or 2, wherein the composition is for anti-aging. 1) stirring the extraction by adding the extraction solvent to soy bean curd;
2) filtering and heat-treating the bean curd extract of step 1); And
3) centrifuging the extract of step 2);
Method for producing a low molecular peptide extract derived from soy bean curds comprising a. The method of claim 8, wherein the low molecular peptide has a molecular weight of 100 ~ 3000 Da. 10. The method according to claim 8 or 9, wherein the soy bean curd is white, frosted ( Glycine max (L.) Merr.), Rhynchosia Nulubilis Loureiro, Red bean (P haseolus) angularis (Willd.) W. Wight, Vigna radiata (L.) Wilczek), East ( Vigna sinensis King, Pea ( Pisum sativum L.), kidney beans (Phaseolus vulgaris there is . humilis), Vicia ( Vicia faba L. and green beans ( Canavalia) gladiata DC.) A method for producing a low-molecular peptide extract derived from soy bean curd, characterized in that it is prepared from one or more selected from the group consisting of. 10. The method according to claim 8 or 9, wherein the extraction solvent of step 1) uses at least one selected from the group consisting of purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane. Method for producing a low molecular peptide extract derived from soy bean curd, characterized in that. 10. The method according to claim 8 or 9, wherein the extraction of step 1) is performed at a temperature of 10 to 80 ° C. 10. The method of claim 8 or 9, wherein the heat treatment of step 2) is carried out at a temperature of 80 ~ 120 ° C.

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