KR20150060004A - Cosmetic composition for anti-aging comprising Ceratonia Siliqua Fruit Extract - Google Patents

Abstract

The present invention relates to an anti-aging cosmetic composition for preventing saccharification including an extract of Ceratonia siliqua which is transformed by enzyme reaction as an active ingredient where the extract can be made by comprising: treating the extract with enzyme for breaking saccharide binding; turning Cyclitol Derivatives into pinitol; and reacting with other active ingredients in the extract, thereby having an anti-saccharification, an anti-aging effect while inhibiting collagenase and having good anti-inflammatory activity, and can be used in technology for manufacturing anti-aging cosmetic composition.

Description

[0001] The present invention relates to an anti-aging cosmetic composition comprising an extract of Carrocoops having an excellent anti-glycation and anti-aging activity as an active ingredient (Cosmetic composition for anti-aging comprising Ceratonia Siliqua Fruit Extract)

The present invention relates to an anti-aging cosmetic composition containing an extract of Ceratonia Siliqua (carob) fruit which is bioconverted by an enzyme reaction and is excellent in anti-glycation and anti-aging activity as an active ingredient. Specifically, The present invention relates to a technique for producing an extract having excellent antihalation, antioxidation, inhibition of collagenase expression and anti-inflammatory activity by bioconversion by treating with a specific sugar chain-degrading enzyme, and using this extract as an anti-aging cosmetic.

Skin is the largest organ that covers all the organs and organs of the human body and occupies 15-20% of body weight. It protects the body from external invasion and plays an important role in regulating the homeostasis by preserving the body's moisture and temperature. . Skin aging is a naturally occurring change with age, and is caused by many external factors such as ultraviolet rays, tobacco, and environmental pollution. Skin aging can be divided into two major categories. One is the reduction of the synthesis of extracellular matrix proteins such as collagen and ellastin with age, the reduction of elasticity, the loss of moisture in skin cells, and the alteration of the structure of the stratum corneum. (ROS) by stimulating external stimuli such as ultraviolet rays of the sunlight, activating various signal transduction systems by stimulating the production of cytokines, activator protein-1 (AP -1) and nuclear factor κB (NF-κB), which are caused by inflammation of the skin, resulting in damage to lipids, proteins, nucleic acids and enzymes.

In particular, wrinkles, skin tone changes, pigmentation, and the like are changes associated with skin aging. Age-related skin changes are the result of genetically inherent changes in the skin (intrinsic factors) and environmental effects (extrinsic factors). Extrinsic factors are more likely to influence change in the way they affect skin structure and function. 80 to 99% of the skin aging we see is the result of exposure to sunlight associated with photoaging. The phenomena of photoaging are increased wrinkle formation, decreased tension and elasticity, degeneration of vascular supply and skin thickness, hyperpigmentation, different skin discolorations, capillary vasodilation (capillary vasodilation), and reduced skin moisture binding properties.

Scientists have recently seen exposure to ultraviolet light as a major factor in these structural changes. In recent years they have come to understand the actual biochemistry that drives this change.

The chemical reactions that occur within the skin include the formation of reactive oxygen species, also known as free radicals, the activation of metalloproteinases (MMPs) that degrade collagen biosynthetic processes, There is a glycation reaction that produces a glycation product (Advanced Glycation End-products or AGEs).

Reactive Oxygen Species (ROS) include oxygen ions, free radicals and peroxides. ROS is generally a very small molecule and has intense reactivity due to the presence of unpaired electrons. ROS consists of a generally metabolized natural by-product of oxygen. During periods of environmental stress, ROS levels rise dramatically as a major cause of cell structure damage. This is well known for oxidative stress, the leading cause of degenerative diseases associated with aging and disease. The figure shows that UV-induced damage of the skin is caused by reactive oxygen species. In addition, lipid peroxidation, which is a consequence of ROS damage to cellular membranes, leads to premature aging, skin cancer, and cell death.

Matrix metalloproteinases are enzymes. When it is activated, it regulates intradermal tissue-bound degradation. MMPs contain collagenase that specifically degrades certain collagen and other proteins in the dendritic cell-diploid matrix. Collagenase is an enzyme that can break down collagen and elastin into different forms. For example, collagenase-1, or MMP-1, acts on collagen I, II, III, VII and X. MMP-1 degrades the Helix strand of collagen into smaller fragments, which naturally become denatured as peptides composed of gelatin. These modified peptides are further degraded by other MMPs. These actions redefine connective tissue, an essential part of aging and wound healing, and MMPs play a decisive role.

Collagen and elastin proteins, on the other hand, are likely to be affected by chemical reactions inside the body, called glycation. This is a nonenzymatic reaction that leads to the binding of sugars, such as free amino groups and glucose, to proteins. Glucose, which provides the energy of the cell, can react with proteins such as collagen, resulting in the production of Advanized Glycation End-products and Reactive Oxygen Species. These affect the cross-linking of protein fibers, the loss of elasticity, and changes in the dermis associated with the aging process.

When AGEs in the skin are produced, they act on the receptor site and form a complex known as Receptor-AGE (R-AGE). R-AGE signals intracellular processes associated with inflammation and sequelae. These are important because inflammation is a major factor that catalyzes the aging process and many diseases. For example, diabetes is characterized by high blood sugar levels and is accompanied by many complications, such as cataracts and arteriosclerosis, which are manifested by the formation of AGEs in the body. For this reason, diabetes is seen as a disease that accelerates aging due to increased inflammation caused by AGEs formation. This is not limited to diabetes. Many diseases related to muscle weakness, heart disease and brain are associated with glycation. Scientists believe that reducing glycation has the potential to slow down the aging process and disease induction. The creation of Advanced Glycation End-products is one of the most notable areas of research that can determine the age of the skin and the mechanism of disease generation in our bodies.

Ceratonia (Ceratonia siliqua L.) is a bean in an open pod on a carp tree that has long been used as a human nutritional source. Carp is native to most of the Mediterranean, and also grows in some parts of the US, South Africa, and Austria. The main producers of carrots are Spain, Italy, Morocco, Portugal and Greece. It is industrially used to produce locust bean gum extracted from seeds in the oil of carrots, and is widely used as a thickening and stabilizing agent in food. The pulp, which accounts for more than 90% of the carob fruit, contains carbohydrates, minerals, proteins, insoluble dietary fiber and various polyphenolic compounds. The powder obtained from the pulp of the carrots is used for the production of confectionery, drinks, bread or pasta. Carab powder is often used as a substitute for cocoa, especially for low fat, low calorie, high fiber and high calcium products. It is also a source of decaffeinated, mucilage ovalin and cholesterol. Several bioactivities have been reported for carab pulp powder. This is especially used as a branch office for infants. In addition, hot water extract has high antioxidant ability and free radical scavenging ability and has antiproliferative effect in hepatocytes.

Korean Patent Registration No. 0753982 discloses a process comprising the steps of: (a) diluting carob syrup to an appropriate concentration and then removing the common sugar component by culturing the microorganism; (b) a pretreatment step in which the microbial cells are removed by centrifugation or filtration; (c) removing the produced ethanol component by distillation when the microorganism is yeast; (d) efficiently recovering the pinitol from the sample using an activated carbon column; And (e) a step of concentrating the sample and adding ethanol to form crystals of pinolide.

In the 20th century, the rapid development of the chemical industry has made a major contribution to the development of all industries, including the electronics, machinery and metal industries. However, the development of science to make human life richer and richer causes serious environmental problems. Therefore, research has begun to replace chemical processes requiring high-temperature, high-pressure, highly toxic reagents and solvents under severe reaction conditions with mild conditions using room temperature, normal pressure, and safe solvent systems. Chemical processes are being replaced by bioreactors. This technique, called biotransformation, bioconversion, biosynthesis, bio-catalysis, etc., can produce the desired product from precursors using enzymatic functions.

Biotransformation technologies are widely used in many fields, including bioremedation, conversion of harmful substances into nontoxic substances, manufacture of fine chemicals, food processing, and production of medicines. In particular, in the development of pharmaceuticals, the bioconversion process can be used in a variety of ways to improve the usefulness and effectiveness of pharmaceuticals. The bioconversion process not only has the advantage of being able to produce pharmaceuticals in optically pure form, but also produces derivatives of compounds with complex structures, such as existing beta-lactam antibiotics, cyclosporin and steroids, .

According to the technology trend of cosmetics, it is expected that the crossover phenomenon between items will be further strengthened at present, and it has been long since the start of the fever, but a more scientific and different method has been proposed, Materials containing anti-aging concepts have been released with advanced scientific methods such as anti-glycation, anti-aging, and gene activation. Therefore, there is a desperate need to develop an active ingredient which is safe to the living body, inhibits the liberation of free radicals, inhibits the expression of collagenase (MMP-1), inhibits glycation reaction and AGEs production.

Therefore, the inventors of the present invention have investigated a method of further increasing the aging effect of skin by using the extract of carrots with high blood sugar controlling effect among the naturally occurring plants. As a result, it has been found that the extract of carrots The present inventors have found that the specific active ingredient is enhanced when bioconverting, and that the antihalation, antioxidation, inhibition of collagenase expression and anti-inflammatory effect are significantly increased.

In order to solve various skin phenomena that may occur due to aging, the present invention provides a method of enhancing antioxidation, antioxidation, collagenase expression inhibition and anti-inflammatory activity by reacting a specific active ingredient of a carob It is an object of the present invention to provide a cosmetic composition which has excellent carrageenan extract and contains it as an active ingredient and has an antiherating and antiaging effect.

To achieve the above object, according to the present invention, there is provided a method for producing a fermented soybean curd extract, comprising: extracting a fermented soybean curd with at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ether, ethyl acetate and chloroform; An enzyme-digesting step of dissolving the above extract of Corynebacterium glutamicum in a solvent and adding a glycosidase; And an enzyme reaction step in which an enzyme reaction is performed while stirring the enzyme-added extract, is provided. The enzyme reaction is preferably carried out under the conditions of a reaction pH of 3.5 to 5.0, a reaction temperature of 25 to 50 ° C, and a reaction time of 12 to 60 hours. The glycolytic enzyme may be selected from the group consisting of glucosidase, xylosidase, xylanase, cellulase, galactosidase, pectinase, pectinase, and naringinase, preferably at least one selected from the group consisting of galactosidase, more preferably galactosidase and pectinase, in an amount of 0.5 to 2.0: 0.5 to 2.0 can be used.

Through the enzymatic reaction, the sugar bond of the cyclitol derivative (Cyclitol Derivatives) contained in the extract of carob bean is selectively removed to enhance the pinitol component, and the chiro-inositol, myo-inositol -inositol) and the like, and a carob kernel extract having anticarious activity, antioxidation, inhibition of collagenase expression and anti-inflammatory activity is produced by synergistic action with various active ingredients contained in the extract.

According to the present invention, there is provided a cosmetic composition comprising the extract of carob bean produced by the enzyme reaction in an amount of 0.005 to 50% by weight, preferably 0.01 to 20% by weight, based on the total weight of the composition. The above-mentioned cosmetic composition exhibits excellent anti-glycation, antioxidant, inhibition of collagenase expression and anti-inflammatory effect, and thus can be used as an anti-aging cosmetic composition.

The cosmetic composition containing the enzyme-treated carob bean extract as an active ingredient can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, And may be applied to any one form selected from the group consisting of a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a latex, a press powder, a loose powder and an eye shadow.

The enzyme treated carrot extract produced by the production method according to the present invention is enhanced in pinitol component by bioconversion and exhibits superior antialgalization, antioxidation, collagenase expression due to synergy with various active ingredients in the extract Inhibition and anti-inflammatory effect, it is possible to secure an excellent anti-aging activity even with a small use amount, so that it is useful as a cosmetic composition.

Hereinafter, the present invention will be described in detail.

The present invention relates to a technique for producing a cosmetic having an anti-aging activity through a specific enzyme treatment to a curd extract to enhance the content of a relatively large amount of pinitol compared to other plants and to synergize with other active ingredients in the extract .

Pinitol, which is represented by the following formula, is one kind of water-soluble carbohydrates, and is one of components contained in beans, pine leaves, and the like.

Since 1987, pinitol has been shown to be effective in regulating blood sugar levels in animal experiments in countries around the world. Pinitol is converted into chiro-inositol in the body upon ingestion. Chiro-inositol is known to be one of the mediators involved in the insulin signaling pathway. In addition, when pinitol is given to an experimental animal whose blood glucose regulating function has been impaired, the blood glucose control function is significantly improved . Pinitol is distributed in the leaves, leaves, roots, roots, seeds and so on of the soybean, and its content is about 1% of the dried soybeans and is a valuable raw material.

According to one embodiment of the present invention, the enzyme-treated curd extract of the present invention can be prepared by the following steps. Extracting a carob bean with at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ether, ethyl acetate and chloroform to prepare a carob bean extract; An enzyme-containing step of dissolving the above extract of Corynebacterium glutamicum in a solvent, preferably water, and adding a glycosidase; And an enzyme reaction step in which an enzyme reaction is carried out while stirring the enzyme-added extract. The enzyme reaction is preferably carried out under the conditions of a reaction pH of 3.5 to 5.0, a reaction temperature of 25 to 50 ° C, and a reaction time of 12 to 60 hours.

The method will be described in detail as follows.

The extract of carrots provided in the enzymatic reaction is prepared by mixing the ground carrots with one or more organic solvents selected from the group consisting of water or an organic solvent, preferably water, ethanol, methanol, butanol, ether, ethyl acetate and chloroform, , More preferably 30% ethanol, in a conventional extraction method.

The extract of Corynebacterium glutinosa is an enzyme which performs a sugar-binding degradation activity, preferably glucosidase, xylosidase, xylanase, cellulase, galactosidase ), Pectinase, and naringinase. ≪ / RTI > At this time, when the enzyme reaction is proceeded with the treatment with galactosidase, it exhibits an excellent effect in terms of anti-glycation and anti-aging activity. More preferably, the pectinase is mixed with the galactosidase at a ratio of 0.5 to 2.0: 0.5 to 2.0 to proceed the enzymatic reaction, which exhibits the most excellent effect in terms of anticarious activity and anti-aging activity.

The saccharide binding of the cyclitol derivatives (Cyclitol Derivatives) contained in the carob bean extract is selectively removed by the sugar chain degrading enzyme, thereby increasing the content of the pinitol component in the extract and converting the other active ingredients in the extract . The enzymes may be selected from the group consisting of aspergillus genus, basillus genus, penicillium genus, rhizopus genus, rhizomucor genus, talaromyces genera, Can be obtained from microorganisms of the genus Bifidobacterium, Mortierella.

As compared with extracts of simple carob bean extracts, the carob bean extracts biotransformed by the enzymatic reaction have increased activities thereof, and thus have excellent antihalation, antioxidation, collagenase expression inhibition and anti-inflammatory effect .

Thus, according to the present invention, an enzyme-treated carob bean extract prepared by the enzyme reaction and having excellent anticariicity, antioxidation, inhibition of collagenase expression and anti-inflammatory activity is provided. According to the present invention, there is also provided an anticalculus and anti-aging cosmetic composition comprising the enzyme-treated carob powder extract in an amount of 0.005 to 50% by weight, preferably 0.01 to 20% by weight, based on the total weight of the composition.

The cosmetic composition containing the enzyme-treated carob bean extract as an active ingredient can be used as a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, And may be applied to any one form selected from the group consisting of a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a latex, a press powder, a loose powder and an eye shadow.

[Example]

Hereinafter, the present invention will be described in detail by way of the following examples and test examples, but the present invention is not limited to these examples.

Comparative Example  One: Of carrots extract  Produce

100 g of carob bean was extracted from 600 g of 30% ethanol at 50 ° C for 3 hours to obtain a carob bean extract, which was then concentrated under reduced pressure and lyophilized to obtain 5.4 g of carob bean extract.

Example  1 ~ 7: Enzyme treatment Of carrots extract  Produce

400 ml of purified water and 50 ml of the enzyme solution shown in Table 1 were mixed with 50 g of the concentrate (2 g of solid content per 100 g) suspended in the crude extract prepared in Comparative Example 1, and enzyme reaction was carried out with stirring at pH 4.5 and 45 ° C for 48 hours , And concentrated under reduced pressure to prepare an extract of carob bean by enzyme reaction.

Enzyme Origin Example 1 Amylase Aspergillus oryzae Example 2 Beta-glucosidase Aspergillus niger Example 3 Cellulase Aspergillus niger Example 4 Beta-galactosidase Aspergillus oryzae Example 5 Beta-xylocidase Aspergillus niger Example 6 Pectinase Rhizopus sp Example 7 Narin Gina Penicillium decumbens

Test Example  One: Pinitol Generation rate  Measure

The production rate of the pinolol of the enzymatically treated barley extract prepared in Examples 1 to 7 was measured by using HPLC and the standard material was D-pinitol (Sigma-aldrich). The analytical condition was analyzed by using a differential refractometer to detect and remove 85% acetonitrile mobile phase with high performance carbohydrate column.

The production rate of pinitol was the increase rate of the content of the pinitol when the extract of Comparative Example 1 was used as a control, and was calculated as shown in the following formula.

Production rate (%) = D-pinitol Standard sample amount (mg) × peak area value of the example ÷ D-pinitol standard peak area value ÷ sample amount (mg)

The results are shown in Table 2 below.

Test Example  2: Total polyphenol content measurement

10 ml of distilled water was added to 1 ml of each of the extracts of Comparative Example 1 and Examples 1 to 7, followed by addition of 2 ml of Folin-Ciocalteu phenol reagent, followed by reaction at room temperature for 5 minutes. 2 ml of 20% sodium carbonate was added to the reaction mixture, and the mixture was reacted at room temperature for 1 hour and then absorbance was measured at 725 nm. At this time, tannic acid was used as the indicator material.

The results are shown in Table 2 below.

Test Example  3: Total flavonoid content measurement

0.1 ml of 10% aluminum nitrate, 0.1 ml of 1M potassium acetate and 4.3 ml of ethanol were added to 0.5 ml of each of the extracts of Comparative Example 1 and Examples 1 to 7, and the mixture was reacted at room temperature for 40 minutes, and the absorbance was measured at 415 nm . At this time, the indicator substance was quercetin.

The results are shown in Table 2 below.

Production rate of pinitol (%) Total polyphenol content ([mu] g / ml) Total flavonoid content ([mu] g / ml) Comparative Example 1 6.8 448 134 Example 1 12 1084 20 Example 2 25 727 228 Example 3 29 978 164 Example 4 34 1279 253 Example 5 8 584 15 Example 6 29 1207 226 Example 7 12 985 30

As shown in the above Table 2, when beta-galactosidase was used as a sugar chain-degrading enzyme, it was confirmed that the conversion of biotin was most excellent in the production rate of pinitol, total polyphenol content and total flavonoid content, - glucosidase, cellulase, and pectinase as catalysts.

Example  8 ~ 13: Enzyme treatment Of carrots extract  Produce

In order to find the most suitable enzymatic treatment conditions, beta-glucosidase, cellulase, beta-galactosidase, pecks of Examples 2, 3, 4 and 6, which are excellent in terms of the production rate of pinitol, total polyphenol content and total flavonoid content Carrageenan Extracts were prepared by enzymatic reaction using different enzyme catalysts. 50 g of the suspension concentrate (2 g of solid content per 100 g) of the extract of Calabria extract prepared in Comparative Example 1 was mixed with 400 ml of purified water and 0.5 g of beta-glucosidase (Multifect FE, Genencor), cellulase (Cellulase KN, DSM) The mixture was mixed with 50 ml of a mixed enzyme solution of lactocidase (Sumilact, Shin-Nippon Chemical) and pectinase (Pectinex 100 L, Novozyme) to prepare an enzyme solution at the mixing ratio shown in Table 3 below. And concentrated to produce enzyme-treated carob bean extract.

enzyme Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Beta-glucosidase 50% 50% 50% Cellular-A 50% 50% 50% Beta-galactosidase 50% 50% 50% Pectinase 50% 50% 50%

Test Example  4: Pinitol Generation rate  Measure

The production rates of pinolol of carob bean extract by enzymatic reactions prepared in Examples 8-13 were measured and the results are shown in Table 4 below.

Test Example  5: Total polyphenol content measurement

The total polyphenol contents of the carob bean extract by enzymatic reactions prepared in Examples 8-13 were measured and the results are shown in Table 4 below.

Test Example  6: Total flavonoid content measurement

The total flavonoids of the carob bean extract according to the enzymatic reactions prepared in Examples 8-13 were measured and the results are shown in Table 4 below.

Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Production rate of pinitol (%) 27 30 27 32 29 35 Total polyphenol content
(占 퐂 / ml) 852 1103 967 1128 1092 1273 Total flavonoid content
(占 퐂 / ml) 196 255 227 223 195 284

As shown in Table 4, when beta-glucosidase, cellulase, and pectinase were mixed with the co-enzyme with beta-galactosidase as the sugar chain degrading enzyme, the production rate of the pinolide, total polyphenol Content and total flavonoid content were found to be excellent. Among them, β - galactosidase and pectinase were mixed with each other and showed the best result by biotransformation.

Test Example  7. Antialcification  Check the effect

When the glycation reaction proceeds, a series of complex processes such as oxidation and condensation are performed to produce final glycation products (AGEs) in which various compounds are mixed. Some of the final glycation products are fluorescent, and are used as a measure of the degree of glycation reaction. Among them, fluorescence at 370nm / emission 440nm is generally known as the degree of glycosylation.

The experiment was conducted using the above principle. Preparation of the reaction was carried out by modifying the method of Mcpherson et al. (Role of fructose in glycation and cross-linking of proteins. Biochemistry 1998, 27, 1901-1907). (BSA, 5 mg / ml), 25 mM ribose, 1 mM diethylenetriaminepentaacetic acid (DTPA) and 0.02% sodium azide were mixed to a final concentration in a 0.1M phosphate buffer (pH 7.0) 180 占 퐇 was dispensed into each well plate (Nunc, Denmark), and then 20 占 퐇 of each sample (10 mg / ml) was added. At this time, a control group (A) in which glycosylation did not occur without mixing the ribose, a glycosylation control group (B) in which glycosylation occurred in the preparation, and a sample treatment group (C) were prepared. A sample treatment group (D) without treatment with ribose was also prepared to eliminate the interference of fluorescence by each sample. After completion of the reaction, the reaction was carried out for 7 days at 37 ° C in a constant-temperature and constant-humidity incubator to prevent drying during the reaction. After the reaction was completed, fluorescence was measured at ex = 370 nm / em = 440 nm using a spectrophotometer (VICTOR3, Perkin Elmer, Waltham, MA, USA). Each sample was repeated three times. The inhibitory activity of the final glycation end product was 100% for the control group (A) without saccharification reaction and 0% for the glycosylation control group (B) in which the glycation reaction occurred. (CD), which excluded the fluorescence of each sample in which no saccharification reaction occurred, was used. Aminoguanidine was used as a control, and samples were prepared by adding Comparative Example 1, Example 13, and pinitol to each concentration. The inhibition rate (%) of the final glycation end product formation was calculated by the following formula, and the results are shown in Table 5 below.

Production inhibition rate (%) = 100 - [(CD) / (AB) x 100]

Sample name (쨉 g / ml) Production inhibition rate (%) Aminoguanidine 375 52 Pinitol 200 10 Carrot extract 100 14 (Comparative Example 1) 200 27 Enzyme-treated carrots extract 100 25 (Example 13) 200 54

As shown in Table 5, it was confirmed that the enzyme-treated barley extract (Example 13) had a better effect of inhibiting the final glycation end product formation than the barley extract (Comparative Example 1) at the same concentration treatment.

In other words, it can be seen that the enzyme-treated carrots extract of the present invention has an effect of inhibiting the final glycation endproduct formation better than the extracts of pinitol and common carrots.

Test Example  8 : Xanthine oxidase  Inhibitory activity

Xanthine oxidase inhibitory activity was measured by SOD - like activity assay / NBT (Nitroblue tetrazolium) method. 100 μl, 200 μl, and 10 μl of each of the samples of the extract of Carrocoops and the extract of the enzymes treated with the enzymes treated with the extracts of Comparative Example 1 and Example 13, 140 μl of 50 mM sodium phosphate buffer (pH 7.5), 40 μl of xanthine (3 mM) mM NBT, and 10 μl of xanthine oxidase (0.13 U / ml) was added thereto. The mixture was incubated at 25 ° C for 40 minutes, and absorbance was measured at 560 nm.

The Xanthine oxidase inhibitory activity of the extract of the present invention is shown by the following equation, and the antioxidant activity value is expressed as% with absorbance of the addition group and no addition group of the sample. As a control group, ascorbic acid was used, and samples were measured by adding Comparative Example 1, Example 13, and pinitol to each concentration.

Xanthine oxidase inhibitory activity (%) = (1 - absorbance of sample added / absorbance of sample not added) x 100

The results are shown in Table 6 below.

Sample name (쨉 g / ml) Xanthine oxidase inhibitory activity (%) Ascorbic acid (control group) 500 58 Pinitol 200 13 Carrot extract
(Comparative Example 1) 100 48 200 63 Enzyme-treated carrots extract
(Example 13) 100 65 200 88

As can be seen in Table 6, the enzyme-treated carob bean extract according to the present invention is superior to ascorbic acid, which is a conventional antioxidant, and exhibits excellent antioxidative ability as compared with the extracts of pinole and common carob.

Test Example  9: Cytotoxicity test

Fibroblasts were inoculated into IMDM medium supplemented with 10% FBS at a cell concentration of 5 × 10 ⁵ cells and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. After the culture, the culture medium was removed and the extract of Carracoop of Comparative Example 1 and the enzyme-treated carrots extract prepared in Example 13 were diluted in dimethyl sulfoxide to a final concentration of 50, 100, 500 and 1000 μg / After incubation for 24 hours, 100 μl of MTT (3- [4,5-dimethylthiazol-2yl] -2,5-diphenyltetrazolium boromide, Sigma, USA) Ml) for 4 hours. After removing the supernatant, 150 μl of dimethylsulfoxide was added, and the resulting formazan was shaken by shaking for 30 minutes. Absorption was measured at 540 nm using a multimicroplate reader (Molecular device Spectra max 190). Cell viability was calculated according to the following equation and the results are shown in Table 7 below.

Cell survival rate (%) = absorbance of sample-added group / absorbance of control group 100

Sample concentration
(占 퐂 / ml) Cell survival rate (%) Carrageenan Extract (Comparative Example 1) Enzyme treated black soybean extract (Example 13) 0 100 100 50 100 100 100 100 100 500 100 100 1000 94.76 95.43

As shown in the results of Table 7, it was confirmed that all the samples of the extract of Carrageenan (Comparative Example 1) and the enzyme-treated Carrageenan Extract (Example 13) had no cytotoxicity at a concentration of 500 μg / ㎍ / ㎖, cell survival rate was decreased but there was no safety problem.

Test Example  10: Collagenase ( MMP -1) production inhibitory effect

Human normal skin cells, fibroblasts (Korean Cell Line Bank, Korea), were inoculated into 48-well microplates (Nunc, Denmark) to be 10 < ⁶ > cells per well and cultured in DMEM medium (Sigma, For 24 hours. Then, the carob, the enzyme-treated carob, and the pinolol produced in Comparative Example 1 and Example 13 were added to dimethyl sulfoxide so as to have final concentrations of 50, 100, 500 and 1000 μg / Diluted diluted solutions were added and cultured in serum-free DMEM for 48 hours. After the incubation, the supernatant of each well was collected and the amount (쨉 g / ml) of the newly synthesized MMP-1 was measured using a collagenase (MMP-1) assay kit (Amersham, USA) (MMP-1) production inhibition was calculated, and the results are shown in Table 8 below. At this time, TGF-beta (10 mg / ml, Roche, USA) was used as a positive control for inhibition of collagenase (MMP-1) production.

(%) = (1- amount of MMP-1 in the test group / amount of MMP-1 in the control group) 占 100

Sample concentration
(占 퐂 / ml) Inhibition rate of collagenase (MMP-1) production (%) Carrot extract
(Comparative Example 1) Enzyme-treated carrots extract
(Example 13) Pinitol TGF-beta 50 17.2 30.5 2.5 42.2 100 20.9 55.5 4.8 65.5 500 47.6 63.5 5.9 69.8 1000 57.1 76.5 6.2 88.8

As shown in the results of the above Table 8, the effect of inhibiting the production of collagenase (MMP-1) was higher in the sample to which the enzyme-treated carob bean extract (Example 13) was applied than the common carob bean extract (Comparative Example 1) The enzyme-treated black soybean extract showed a similar effect to that of the control TGF-β with better effect.

Test Example  11: Anti-inflammatory efficacy test

       In order to examine the anti-inflammatory activity of the extracts of carrots and fermented soybean curd extracts and pinitol prepared in Comparative Examples 1 and 13, the effect on the production of tumor necrosis factor (TNF-α), one of the early inflammatory molecules, .

The formation of pro-inflammatory cytokines such as TNF-a leads to the process of converting arachidonic acid into prostaglandin (prostaglandin) and NO formation due to the activity of phospholipase A2 (phospholipase A2) . The cells used were HaCaT (Human Skin Keratinocyte cell line), Dulbecco's modified Eagle's medium supplemented with 5% CO2, 10% fetal bovine serum (FBS, Lonza) and 50 μg / mL streptomycin (DMEM, Invitrogen). After incubation, the cells were stressed by UV irradiation for 1 hour. As a control, UV-irradiated cells were used without sample treatment. At this time, α-Bisabolol was used as a positive control for anti-inflammatory activity.

For RNA analysis, total RNA in cells was extracted from cell culture using Trizol reagent (Invitrogen, USA). The purity and integrity of the RNA was confirmed by A260 nm / A280 nm ratio measurement, and the RNA yield was measured by absorbance at 260 nm. For cDNA synthesis, 3 μg of total RNA was added at 25 ° C. for 10 minutes with addition of Oligo dT 15 (500 ng / μl) primer, dNTP (10 mM), RTase inhibitor (40 U / μl) and Powerscript II RTase (Clontech, USA) The cDNA was synthesized by primer annealing at 42 ° C for 60 min and RTase denaturation at 95 ° C for 5 min. To amplify TNF-α and GAPDH from cDNA, 3 μl of cDNA, 5 μl of 10 × taq polymerase buffer, 2 μl of 10 mM dNTP, 2 μl of each 10 pmol primer and 0.5 μl of taq polymerase were mixed and adjusted to 50 μl by adding distilled water Lt; / RTI > The primer sequences are shown in Table 9. The PCR product was electrophoresed on 1% agarose gel and analyzed with an image analyzer (UGEN, U: Genius). The density of each band was measured using a density measurement program (Gene Tools from Syngene) Respectively.

Gene  Primer sequence GAPDH Forward    5 '- AAC GAA TTT GGT CGA ACA GC-3' Reverse    5 '- TGA GGA GGG ATT CAG TG - 3' TNF-a Forward    5 '- CAG AGG GAA GAG TTC CCC AG - 3' Reverse    5 '- CCT TGG TCT GGT AGG AGA CG-3'

The results are shown in Table 10 below.

TNF-α expression (%) Unchecked UV irradiation 0.00% 0.00% 0.01% 0.05% 0.10% 0.50% Blank 6.18 100.00 Carrageenan Extract (Comparative Example 1) 82.99 65.50 44.06 36.76 Enzyme-treated carrots extract
(Example 13) 55.74 37.70 30.58 18.55 Pinitol 56.88 43.66 37.55 26.65 α-Bisabolol 28.75

As can be seen from the above Table 10, the enzyme charynx extract (Example 13) according to the present invention inhibits the expression of tumor necrosis factor (TNF-α) induced by UV in a concentration-dependent manner And suppressed the inflammatory reaction. Therefore, the enzyme-treated black soybean extract effectively inhibits skin inflammation reaction, and thus has an excellent effect of alleviating skin troubles. In addition, the enzyme-treated carob bean extract of the present invention (Example 13) was superior to the ordinary carob bean extract (Comparative Example 1).

The anticarcinogenic and anti-aging cosmetic composition containing the enzyme-treated carob seed extract of the present invention can be produced by the following various formulations.

Produce Example  One: emulsion  Base manufacturing

An emulsion base was prepared according to the composition shown in Table 11 below as an antihalcating agent and an anti-aging cosmetic agent containing the enzyme-treated carrageenan extract of the present invention.

ingredient Content (% by weight) glycerin 3.00 Disodium iodide 0.02 Polyglyceryl-3-methylglucoside distearate 1.50 Cetearyl alcohol 0.50 Caprylic / capric triglyceride 7.00 Polyacrylamide & C13-14 Isoparaffin & Laureth-7 0.60 Enzyme treated black soybean extract (Example 13) 0.2 Incense, preservative a very small amount Purified water Balance system 100

Produce Example  2: Flexible lotion

A flexible lotion containing an enzyme-treated carrot extract according to the present invention in the composition shown in the following Table 12 was prepared by a conventional method.

ingredient Content (% by weight) Enzyme treated black soybean extract (Example 13) 0.1 1,3-butylene glycol 5.2 Oleyl alcohol 1.5 ethanol 3.2 Polysorbate 20 3.2 Benzophenone-9 2.0 Carboxyl vinyl polymer 1.0 glycerin 3.5 incense a very small amount antiseptic a very small amount Purified water Balance system 100

Manufacturing Example 3: Nutritional lotion

A nutritional lotion containing an enzyme-treated carrots extract according to the present invention in the composition shown in the following Table 13 was prepared by a conventional method.

ingredient Content (% by weight) Enzyme treated black soybean extract (Example 13) 0.1 Glyceryl stearate SE 1.5 Stearyl alcohol 1.5 lanolin 1.5 Polysorbate 1.3 Sorbitan stearate 0.5 Hardened vegetable oil 1.0 Mineral oil 5.0 Squalane 3.0 Trioctanoin 2.0 Dimethicone 0.8 Tocopheryl acetate 0.5 Carboxyvinyl polymer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 Sodium hyaruronate 5.0 Triethanolamine 0.12 Preservative, fragrance, pigment a very small amount Distilled water Balance Sum 100

Produce Example  4: Nourishing cream

Nutritive creams containing the enzyme-treated carrots extract according to the present invention in the composition shown in the following Table 14 were prepared by a conventional method.

ingredient Content (% by weight) Enzyme treated black soybean extract (Example 13) 0.2 Pro-type glycerin monostearate 2.0 Stearic acid 1.6 Wax 1.0 Polysorbate 1.6 Sorbitan stearate 0.6 Hardened vegetable oil 1.0 Squalane 3.0 Mineral oil 5.0 Trioctanoin 5.0 Dimethicone 1.0 glycerin 5.0 Triethanolamine 1.0 Sodium hyaruronate 4.0 Preservative, fragrance, pigment a very small amount Distilled water Balance Sum 100

Production Example 5: Massage cream

Massage cream containing the enzyme-treated carob seed extract according to the present invention in the composition shown in the following Table 15 was prepared by a conventional method.

ingredient Content (% by weight) Enzyme treated black soybean extract (Example 13) 0.2 glycerin 4.0 vaseline 3.5 Triethanolamine 0.5 Liquid paraffin 24.0 Squalane 3.0 Wax 2.1 Tocopheryl acetate 0.1 Polysorbate 60 2.4 Carboxyl vinyl polymer 1.0 Sorbitan sesquioleate 2.3 incense a very small amount antiseptic a very small amount Purified water Balance system 100

Example 6:

A pack containing the enzyme-treated Codonopsis extract according to the present invention in the composition shown in Table 16 below was prepared in a conventional manner.

ingredient Content (% by weight) Enzyme treated black soybean extract (Example 13) 0.1 Ethyl alcohol 3.0 EDTA-2Na 0.02 Propylene glycol 5.1 glycerin 4.5 Carbopol 1.0 Polyoxide 0.1 antiseptic a very small amount incense a very small amount Purified water Balance system 100

Claims (10)

A cosmetic composition for anti-aging comprising a carob kernel extract which is treated with a glycolytic enzyme to enhance anti-glycation and anti-aging activity in an amount of 0.005 to 50% by weight based on the total weight of the composition. The anti-aging cosmetic composition according to claim 1, wherein the extract of carob bean has enhanced polyphenol and flavonoid content. The anti-aging cosmetic composition according to claim 1, wherein the extract of carob bean has an enhanced pinitol component. The anti-aging cosmetic composition according to claim 1, wherein the sugar chain-degrading enzyme is galactosidase. 2. The anti-aging cosmetic composition according to claim 1, wherein the saccharide-degrading enzyme is a mixture of galactosidase and pectinase in a ratio of 0.5 to 2.0: 0.5 to 2.0. [Claim 2] The method of claim 1, wherein the carrageenan extract having enhanced anti-glycation and anti-aging activity is extracted with at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ether, ethyl acetate and chloroform, A step of preparing a barley extract to produce a barley extract; An enzyme-containing step of dissolving the above extract of Corynebacterium glutamicum in a solvent and adding an enzyme obtained by mixing β-galactosidase and pectinase as a saccharide-binding cleavage enzyme at a volume ratio of 1: 1; And an enzyme reaction is carried out under agitation at a reaction pH of 3.5 to 5.0, a reaction temperature of 25 to 50 ° C and a reaction time of 12 to 60 hours while stirring the enzyme-added extract. The cosmetic composition for anti-aging according to claim 1, wherein the cosmetic composition has collagenase expression inhibiting activity. The anti-aging cosmetic composition according to claim 1, wherein the cosmetic composition has anti-inflammatory activity. The anti-aging cosmetic composition according to claim 1, wherein the cosmetic composition has an antioxidative activity. A cosmetic composition for anti-saccharification having an activity of inhibiting the production of a final glycation end product by containing 0.005 to 50% by weight of a carrots extract, which is treated with a glycolytic enzyme to enhance anti-glycation and anti-aging activity, relative to the total weight of the composition.