KR101339915B1 - Preparation method for ceratonia siliqua fruit extract and cosmetic composition for anti-aging comprising the same - Google Patents

Abstract

The present invention relates to a method for manufacturing extract of Ceratonia siliqua (Carob) fruit, which is bioconversed by enzymatic reaction, and a cosmetic composition for anti-glycation and anti-aging containing the same extract as an active ingredient and, more specifically, a technique for manufacturing anti-aging cosmetic composition which converts Cyclitol Derivatives into pinitol by treating the Ceratonia siliqua fruit extract with glycosyl hydrolases and manufactures the extract having excellent activities of anti-glycation, anti-oxidation, colagenase expression inhibition, and anti-inflamation by synergism with other actuve ingredients.

Description

Preparation method for Ceratonia Siliqua Fruit Extract and cosmetic composition for anti-aging comprising the same}

The present invention relates to a method for producing an extract of Carat bean (Ceratonia Siliqua (carob) Fruit) having excellent bioglycosylation and anti-aging activity by enzymatic reaction, and an anti-aging cosmetic composition containing the extract as an active ingredient. The present invention relates to a technique for producing an extract having excellent anti-glycosylation, antioxidant activity, collagenase expression inhibition and anti-inflammatory activity by treating a carbobean extract with a specific glycoside-degrading enzyme, and converting it into a bio-converter.

The skin is the organ that wraps all organs and organs of the human body. It is the largest organ, accounting for 15-20% of body weight. It protects the body from external invasion. Do it. Aging of the skin is a change that occurs naturally with age, and is caused by many external factors such as ultraviolet rays, tobacco, and environmental pollution. Skin aging can be divided into two types. One is endogenous aging that decreases the amount of synthesis of extracellular matrix proteins such as collagen and ellastin, decreases elasticity, loses moisture in skin cells, and changes the structure of the stratum corneum with age. The other is to generate reactive oxygen species (ROS) by repeating external stimuli such as ultraviolet rays of sunlight, and to promote the production of cytokines to activate various signaling systems to activate activator protein-1 (AP). -1) and the inflammatory response caused by the activation of nuclear factor κB (NF-κB) is induced, and the lipids, proteins, nucleic acids, and enzymes that make up the skin can be damaged.

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 embedded changes (intrinsic factors) and environmental influences (external factors) in the skin. Both of these influence the structure and function of the skin, and external factors contribute to the change. 80 to 99% of the skin aging we see is the result of exposure to sunlight associated with photoaging. Symptoms of photoaging include increased wrinkle formation, decreased tension and elasticity, vascular supply and skin thickness degeneration, hyperpigmentation, other skin discolorations, capillary dilatation (capillary dilatation), and decreased skin water-binding properties.

Recently, scientists see UV exposure as the main factor for these structural changes. In recent years they understand the real biochemical triggers that drive these changes.

Chemical reactions occurring in and involving the skin include the generation of reactive oxygen species, known as free radicals, activation of matrix metalloproteinases or MMPs that degrade during collagen biosynthesis, and There is a glycation reaction that produces glycation end-products or AGEs.

Reactive Oxygen Species (ROS) include oxygen ions, free radicals and peroxides. ROS are generally very small molecules and have intense reactivity due to the presence of unpaired electrons. ROS consists of naturally metabolized natural byproducts of oxygen. During times of environmental stress, ROS levels rise dramatically as a major cause of damage to cellular structure. This is well known as 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 as a result of ROS damage to cell membranes leads to premature aging, skin cancer, cell death and the like.

Matrix metalloproteinases are enzymes. When it is activated, it regulates tissue binding degradation in the dermis. MMPs contain collagenases that specifically degrade specific collagen and other proteins in the extracellular matrix in the dermis. Collagenase is an enzyme that can break down collagen and elastin into other forms. For example, collagenase-1, or MMP-1, acts on collagen I, II, III, VII and X. MMP-1 breaks down the helix strand of collagen into smaller pieces that are naturally denatured into peptides consisting of gelatin. And these denatured peptides are further degraded by other MMPs. These actions play a crucial role in reconstituting connective tissue, an essential part of aging and wound healing.

Collagen and elastin proteins, on the other hand, are more likely to be affected by a chemical reaction inside the body called glycation. This is a non-enzymatic reaction that results in the binding of sugars such as free amino groups and glucose to proteins. Glucose, which provides energy to cells, can react with proteins, such as collagen, which results in the production of Advaned Glycation End-products and Reactive Oxygen Species. These affect cross-linking of protein fibers, 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 glucose levels and is accompanied by many complications such as cataracts and atherosclerosis, which are manifested by the production of AGEs in the body. For this reason, diabetes is viewed as a disease that accelerates aging due to the increased inflammation caused by the formation of AGEs. This is not limited to diabetes. Muscle weakness, heart disease and many brain-related diseases are associated with glycation. Scientists believe that reducing glycation has the potential to slow down the aging process and disease outbreaks. The creation of Advanced Glycation End-products is one of the hottest research areas that can determine how skin ages and the mechanism of disease production in our bodies.

Carob beans (Ceratonia siliqua L.) are beans in pods that open on a carp tree and have long been used as a source of human nutrition. The carob tree grows mostly in the Mediterranean and grows in some parts of the United States, South Africa, and Austria. The main producers of carob beans are Spain, Italy, Morocco, Portugal and Greece. It is used industrially to produce Locust Bean Gum, which is extracted from seeds in carob fruit, and is widely used in food as a thickening and stabilizing agent. Pulps, which make up more than 90% of the carp fruit, contain carbohydrates, minerals, proteins, insoluble fiber and various polyphenolic compounds. Powder obtained from the pulp of the carbs is used in the manufacture of confectionery, beverages, bread or pasta. Carob powder is often used as a cocoa substitute to produce products of low fat, low calorie, high fiber and high calcium. It is also a source of caffeine-free, missobromine and cholesterol-free. Several bioactive efficacies have been reported for carob pulp powder. This is especially used as a branch office for infants. In addition, hot water extract has high antioxidant power and free radical scavenging ability and antiproliferative effect in hepatocytes.

In this regard, the Republic of Korea Patent No. 0755982 (a) diluting the carob syrup to an appropriate concentration and then removing the general sugar component through the culture of microorganisms; (b) a pretreatment step of removing the cells produced by the microbial culture through centrifugation or filtration; (c) removing distilled ethanol produced when the microorganism is yeast; (d) efficiently recovering finitol using the activated carbon column from the sample; And (e) concentrating the sample and then adding ethanol to form crystals of finitol.

The rapid development of the chemical industry in the 20th century has made a significant contribution to the development of all industries, including the electronics, machinery and metals industries. However, the development of science to enrich and enrich human life is causing serious environmental problems. As a result, early research into replacing chemical processes requiring extreme reaction conditions under high temperature, high pressure, highly toxic reagents and solvents with mild conditions using normal temperature, atmospheric pressure, and safe solvent systems began. Chemical processes are being replaced by bioreactors. Called biotransformation, bioconversion, biosynthesis, bio-catalysis, etc., this technique uses enzymatic functions to produce desired products from precursors.

Bioconversion technology is widely used in many fields, including bioremedation, the manufacture of fine chemicals, food processing, and the manufacture of pharmaceuticals, which convert harmful substances into non-toxic substances. In particular, in the development of pharmaceutical products, bioconversion processes can be used in various ways to contribute to improving the usefulness and effectiveness of pharmaceutical products. The bioconversion process not only has the advantage that the drug can be produced in optically pure form, but also improves physical properties and activity by making derivatives of complex structures such as beta-lactam antibiotics, cyclosporins and steroids. It has the advantage that it is.

With the recent development of science and technology, according to the technical trend of cosmetics, the crossover phenomenon between items is expected to be strengthened in Korea at present, and while the craze has been started for a long time, more scientific and different methods are proposed. Advanced scientific methods such as anti-glycosylation, anti-aging, and gene activation have been mobilized to release materials containing anti-aging concepts. Therefore, there is an urgent need for development of an effective ingredient that is safe for the living body and inhibits free radical scavenging activity and inhibits the expression of collagenase (MMP-1), glycosylation reaction and AGEs production.

Accordingly, the present inventors studied a method of further enhancing skin anti-aging effect by using a carob bean extract having excellent glycemic control effect among natural native plants, and the carob bean extract was subjected to enzymatic reaction by a specific method. In the case of bioconversion, the specific active ingredient is strengthened to confirm that anti-glycosylation, antioxidant, collagenase expression inhibition and anti-inflammatory effect are significantly increased, and the present invention was completed.

The present invention by strengthening the specific active ingredient of the carob bean extract by reacting the carob beans with a specific enzyme to solve various skin phenomena that may occur due to aging, anti-glycosylation, antioxidant, collagenase expression inhibition and anti-inflammatory activity It is an object of the present invention to provide a method for producing an excellent carob bean extract.

Another object of the present invention is to provide a cosmetic composition having an anti-glycosylation and anti-aging effect by containing the enzyme-treated carob bean extract as an active ingredient.

According to the present invention to achieve the above object, a carp bean extract manufacturing step of extracting carp beans with at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ether, ethyl acetate and chloroform; An enzyme addition step of dissolving the carob bean extract in a solvent and adding a sugar-binding enzyme; And there is provided a method for producing an enzyme-treated carob bean extract comprising an enzyme reaction step of performing an enzyme reaction while stirring the extract to which the enzyme is added.

The enzyme reaction is preferably carried out under the conditions of reaction pH 3.5 ~ 5.0, reaction temperature 25 ~ 50 ℃ and reaction time 12 ~ 60 hours. Glucosidase, glucosidase, xylosidase, xylanase, xylanase, cellulose, galactosidase, pectinase, pectinase, At least one selected from the group consisting of naringinase may be used, preferably galactosidase, more preferably galactosidase and pectinase from 0.5 to 2.0: 0.5 to 2.0 Mixing in the ratio of may be used.

The enzymatic reaction selectively removes the sugar bonds of the Cyclitol Derivatives contained in the carob bean extract, thereby enhancing the pinitol component, and further, chiro-inositol and myo-inositol (myo). -inositol), etc., by synergizing with the various active ingredients contained in the extract is prepared carob bean extract excellent in anti-glycosylation, antioxidant, collagenase expression inhibition and anti-inflammatory activity.

In order to achieve the other object of the present invention, according to the present invention is provided a cosmetic composition containing 0.005 ~ 50% by weight, preferably 0.01 ~ 20% by weight of the carob bean extract prepared by the enzyme reaction based on the total weight of the composition . The cosmetic composition can be used as an anti-aging cosmetic composition because it exhibits excellent anti-glycosylation, antioxidant, collagenase expression inhibition and anti-inflammatory effect.

The cosmetic composition containing the enzyme-treated carob bean extract as an active ingredient is a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, an essence , Pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, press powder, loose powder and eye shadow can be applied in any one formulation selected from the group consisting of.

Enzyme-treated carob bean extract prepared by the production method according to the present invention is enhanced by the biotransformation of pinitol (pinitol) component, excellent anti-glycosylation, antioxidant, collagenase expression by synergy with various active ingredients in the extract Because of the inhibitory and anti-inflammatory effects, it is useful as a cosmetic composition because it can secure excellent anti-aging activity even at a small amount.

Hereinafter, the present invention will be described in detail.

The present invention relates to a technique for preparing a cosmetic having excellent anti-aging activity by strengthening finitol, which is contained relatively more than other plants by treating specific extracts of carob beans, and synergizing with other active ingredients in the extract. .

Pinitol represented by the following chemical formula is a kind of water-soluble carbohydrates, and is one of components included in beans, pine needles, and the like.

Pinitol has been found to have a glycemic control effect in animal experiments around the world since 1987. Pinitol is converted to chiro-inositol in the body when ingested. Caro-inositol is known to be one of the mediators involved in the insulin signaling system. In addition, ingestion of pinitol in experimental animals with reduced glycemic control function significantly improved blood glucose control function. It became. Pinitol is distributed in soybean leaves, leaf stalks, stems, roots, roots, and seeds, and is a valuable ingredient, accounting for about 1% of dried beans.

According to the present invention, extracting carob beans with at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ether, ethyl acetate and chloroform to prepare a carp bean extract;

An enzyme addition step of dissolving the carob bean extract in a solvent, preferably water, and adding a sugar-binding enzyme; And

Provided is a method for preparing an enzyme-treated carob bean extract comprising an enzyme reaction step of performing an enzyme reaction while stirring the extract to which the enzyme is added.

The enzyme reaction is preferably carried out under the conditions of reaction pH 3.5 ~ 5.0, reaction temperature 25 ~ 50 ℃ and reaction time 12 ~ 60 hours.

The method is described in detail as follows.

The carob bean extract provided for the enzymatic reaction may be prepared by mixing the ground carob beans with water or one or more organic solvents selected from the group consisting of water, an organic solvent, preferably water, ethanol, methanol, butanol, ether, ethyl acetate, and chloroform. It can be prepared by extraction with a conventional extraction method using a mixture, more preferably 30% ethanol.

The carob bean extract is an enzyme that performs a glycolytic decomposition, preferably glucosidase, xylosidase, xylanase, xylanase, cellulose, galactosidase ), Pectinase and naringinase are bioconverted through a reaction by one or more enzymes selected from the group consisting of. At this time, preferably, when treated with galactosidase (galactosidase) proceeds the enzymatic reaction shows an excellent effect in terms of anti-glycosylation and anti-aging activity. In addition, 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 with the enzymatic reaction, which shows the best effect in terms of antiglycosylation and anti-aging activity.

The sugar-binding degrading enzyme selectively removes the sugar bonds of the Cyclitol Derivatives contained in the carob bean extract, thereby increasing the content of the pinitol component in the extract, and converting other active ingredients in the extract. . The enzymes include the genus Aspergillus, genus Bacillus, genus penicillium, genus rhizopus, genus rhizomucor, genus talomyces, genus Bifi Obtained from microorganisms of the genus bifidobactertium, mortierella.

Compared to the extracts of simple carob beans, the carob bean extract bioconverted by the enzymatic reaction is increased in activity, so that it has more excellent anti-glycosylation, antioxidant activity, collagenase expression inhibition and anti-inflammatory effect even at a small amount. Confirmed.

As described above, according to the present invention, an enzyme-treated carob bean extract is prepared by the enzymatic reaction and has excellent anti-glycosylation, antioxidant, collagenase expression inhibition, and anti-inflammatory effect. According to the present invention, there is provided a cosmetic composition for anti-glycosylation and anti-aging containing the enzyme-treated carob bean extract 0.005-50% by weight, preferably 0.01-20% by weight based on the total weight of the composition.

The cosmetic composition containing the enzyme-treated carob bean extract as an active ingredient is a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, an essence , Pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, press powder, loose powder and eye shadow can be applied in any one formulation selected from the group consisting of.

[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: Carob Bean Extract  Produce

100 g of carob beans were 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 freeze-dried to prepare 5.4 g of carob bean extract.

Example  1 ~ 7: enzyme treatment Carob Bean Extract  Produce

50 g of purified water and 50 ml of the enzyme solution of Table 1 were mixed with 50 g of the concentrated solution (solid content 2 g per 100 g) of the prepared carob bean extract prepared in Comparative Example 1, followed by enzymatic reaction under stirring at pH 4.5 and 45 ° C. for 48 hours. After concentration, the mixture was concentrated under reduced pressure to prepare carob bean extract 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-xylosidase Aspergillus niger Example 6 Pectinase Rhizopus sp Example 7 Naringinase Penicillium decumbens

Test Example  One: Finitol Generation rate  Measure

The analyzer used HPLC and the standard used D-pinitol (sigma-aldrich) to measure the rate of finitol production of the enzyme-treated carob bean extract prepared in Examples 1 to 7. Analysis conditions were analyzed by differential refractometer after separation of 85% acetonitrile mobile phase with high performance carbohydrate column.

The production rate of finitol indicates an increase rate of the content of finitol when the extract of Comparative Example 1 was used as a control, and was calculated as follows.

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

The results are shown in Table 2 below.

Test Example  2: Determination of Total Polyphenol Content

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

The results are shown in Table 2 below.

Test Example  3: Determination of total flavonoid content

0.5 ml of the extracts of Comparative Example 1 and Examples 1-7 were mixed with 0.1 ml of 10% aluminum nitrate, 0.1 ml of 1M potassium acetate, and 4.3 ml of ethanol, respectively, and then reacted at room temperature for 40 minutes, and then absorbance was measured at 415 nm. . At this time, the indicator material was used quercetin (quercetin).

The results are shown in Table 2 below.

Finitol production rate (%) Total Polyphenol Content (µg / mL) Total flavonoid content (㎍ / ㎖) 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 Table 2 above, when beta-galactosidase was used as the glycoside degrading enzyme, the results of bioconversion of the pinitol production rate, total polyphenol content, and total flavonoid content were most excellent. -Glucosidase, cellulase, and pectinase also showed excellent bioconversion results in the reaction.

Example  8 ~ 13: enzyme treatment Carob Bean Extract  Produce

Beta-glucosidase, cellulase, beta-galactosidase, peck of Examples 2, 3, 4, and 6, which are excellent in terms of finitol production rate, total polyphenol content and total flavonoid content in order to find the most suitable enzymatic conditions. Carap bean extract by enzyme reaction was prepared by varying the reaction catalyst with the enzyme mixed with tinase. 50 g of suspension concentrate (solid content 2 g per 100 g) of the carob bean extract prepared in Comparative Example 1 was purified with 400 ml of beta-glucosidase (Multifect FE, Genencor), cellulase (Cellulase KN, DSM), beta-gal After mixing with 50ml of the mixed enzyme solution of lactosidase (Sumilact, Nippon Chemical Co., Ltd.), pectinase (Pectinex 100L, Novozyme) to prepare the enzyme solution in the mixing ratio as shown in Table 3 below to proceed the enzyme reaction, Concentrated enzyme treated carob bean extract was prepared.

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

Test Example  4: Finitol Generation rate  Measure

Finitol production rate of the carob bean extract by the enzyme reaction prepared in Examples 8-13 was measured and the results are shown in Table 4 below.

Test Example  5: Determination of Total Polyphenol Content

The total polyphenol content of the carob bean extract by the enzyme reaction prepared in Examples 8 to 13 was measured and the results are shown in Table 4 below.

Test Example  6: Determination of total flavonoid content

The total flavonoids of the carob bean extract by the enzyme reaction 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 Finitol formation rate (%) 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 above, when the beta-galactosidase and beta-glucosidase, cellulase, and pectinase were mixed with each other as co-enzyme as the glycoside degrading enzyme, the production rate of finitol and total polyphenol content were used. It was confirmed that the total flavonoid content had excellent results, and among them, beta-galactosidase and pectinase were used for the enzymatic reaction, and showed the best result by bioconversion.

Test Example  7. Antiglycosylation  Check the effect

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

The experiment was conducted using the above principle. Preparation of the reaction was performed by partially modifying the method of Mcpherson et al. (Role of fructose in glycation and cross-linking of proteins. Biochemistry. 1998. 27. 1901-1907). Bovine serum albumin (BSA, 5 mg / ml), 25 mM ribose, 1 mM diethylene triamine pentaacetic acid (DTPA) and 0.02% sodium azide were mixed in 0.1 M phosphate buffer (pH 7.0) to a final concentration of 96 180 μl was dispensed into the well plate (Nunc, Denmark), and 20 μl of each sample (10 mg / ml) was tested. At this time, the control group (A), the glycosylation control group (B) in which the saccharification reaction occurred in the preparation, was not prepared because the ribose was not mixed, and each sample treatment group (C) was prepared. In order to exclude interference of fluorescence by each sample, a sample treatment group (D) not treated with ribose was also prepared. In order to prevent drying during the reaction, the reaction was carried out for 7 days in a 37 ° C. constant temperature and humidity incubator. After the reaction, the fluorescence was measured at ex = 370 nm / em = 440 nm using a spectrofluorometer (VICTOR3, Perkin Elmer, Waltham, MA, USA), and each sample was repeated three times. The final glycosylation product inhibitory activity was 100% in the control group (A) that did not cause glycation reaction and 0% of the glycation control group in which the glycosylation reaction occurred. In this case, a value (CD) was used to exclude fluorescence of each sample in which no glycosylation reaction occurred. Aminoguanidine was used as a control, and the sample was measured by adding Comparative Example 1, Example 13, and finitol. Final glycation product production inhibition rate (%) was calculated by the following formula, the results are shown in Table 5 below.

Production Inhibition Rate (%) = 100-[(CD) / (AB) × 100]

Sample Name (µg / mL) % Production inhibition Aminoguanidine 375 52 Pinitol 200 10 Carob Bean Extract 100 14 (Comparative Example 1) 200 27 Enzymatic Treated Carob Bean Extract 100 25 (Example 13) 200 54

As shown in Table 5, it was confirmed that the enzyme-treated carob beans extract (Example 13) has a better inhibitory effect on the final glycation product production than the carob beans extract (Comparative Example 1) at the same concentration treatment.

In other words, it can be seen that the enzyme-treated carob bean extract of the present invention has a superior inhibitory effect on the production of the final glycated product than the finitol or the general carob bean extract.

Test Example  8 : Xanthine oxidase  Inhibitory activity

Xanthine oxidase inhibitory activity was measured according to SOD-like activity measurement / NBT (Nitroblue tetrazolium) method. 140 μl of 50 mM sodium phosphate buffer (PH 7.5) and 40 μl of xanthine (3 mM) in 10 μl of the carob bean extract and the enzyme treated carob bean extract samples of Comparative Example 1 and Example 13 dissolved at 100 and 200 ppm concentrations, respectively. 10 μl of mM NBT was added and mixed, and finally 10 μl of xanthine oxidase (0.13 U / mL) was added thereto, followed by mixing at 25 ° C. for 40 minutes, and then absorbance at 560 nm was measured.

Xanthine oxidase inhibitory activity of the extract of the present invention showed the antioxidant activity value in% with absorbance of the addition and no addition groups of the sample as shown in the following equation. Ascorbic acid was used as a control, and the samples were measured by adding Comparative Example 1, Example 13 and finitol.

Xanthine oxidase inhibitory activity (%) = (absorbance of 1-sample added / absorbed sample-free) x 100

The results are shown in Table 6 below.

Sample Name (µg / mL) Xanthine oxidase Inhibitory Activity (%) Ascorbic acid (control) 500 58 Finitol 200 13 Carob Bean Extract
(Comparative Example 1) 100 48 200 63 Enzymatic Treated Carob Bean Extract
(Example 13) 100 65 200 88

As confirmed in Table 6, the enzyme-treated carob bean extract according to the present invention was superior to the existing antioxidant ascorbic acid, and showed superior antioxidant capacity as compared to finitol and general carob bean extract.

Test Example  9: cytotoxicity test

Fibroblasts were inoculated in IMDM medium with 10% FBS at a cell concentration of 5 × 10 ⁵ and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. After culturing, the medium was removed, and the dilution prepared by diluting the carob bean extract of Comparative Example 1 and the enzyme-treated carob bean extract prepared in Example 13 with dimethyl sulfoxide so as to have a final concentration of 50, 100, 500, and 1000 µg / ml. The solution was treated and incubated for 24 hours, and then 100 μl of MTT (3- [4,5-dimethylthiazol-2yl] -2,5-diphenyltetrazolium boromide, Sigma, USA) solution was added to each well (3 mg / Ml) and further incubated 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 formula and the results are shown in Table 7 below.

Cell viability (%) = absorbance of the sample addition group / absorbance of the control group × 100

Sample concentration
(占 퐂 / ml) Cell survival rate (%) Carob Bean Extract (Comparative Example 1) Enzymatically Treated Carob Bean 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, all samples of the carob bean extract (Comparative Example 1) and the enzyme-treated carob bean extract (Example 13) were found to have no cytotoxicity at a concentration of 500 µg / ml, and the concentration used was 1000. The cell viability was reduced at ㎍ / ㎖ but it was confirmed that there is no problem in safety.

Test Example  10: Collagenase ( MMP -1) suppression effect

Fibroblasts (Korean Cell Line Bank, Korea), which are human normal skin cells, were seeded in 48-well microplates (Nunc. Denmark) to 1st 10 ⁶ cells per well, and treated with DMEM medium (Sigma, USA) and 37 ° C. After incubating for 24 hours at the carob bean extract prepared in Comparative Example 1 and Example 13 and the enzyme-treated carob bean extract, and pinitol were added to dimethyl sulfoxide so that the final concentration was 50, 100, 500, and 1000 µg / ml, respectively. The diluted solution prepared by diluting was added and then incubated in serum-free DMEM medium for 48 hours. After incubation, the supernatants of each well were collected, and the amount of the newly synthesized MMP-1 (μg / ml) was measured using a collagenase (MMP-1) analysis kit (Amersham, USA), and cola was prepared according to the following formula. The inhibition of genease (MMP-1) production was calculated and the results are shown in Table 8 below. At this time, TGF-β (10 mg / ml, Roche, United States) was used as a positive control of the inhibition of collagenase (MMP-1) production.

MMP-1 Production Inhibition Rate (%) = (quantity of MMP-1 in 1-test group / amount of MMP-1 in control group) × 100

Sample concentration
(占 퐂 / ml) Collagenase (MMP-1) production inhibition rate (%) Carob Bean Extract
(Comparative Example 1) Enzymatic Treated Carob Bean Extract
(Example 13) Finitol 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 Table 8, the sample to which the enzyme-treated carob bean extract (Example 13) was applied to the collagenase (MMP-1) production inhibition effect was higher than the general carob bean extract (Comparative Example 1), Enzyme-treated carob bean extract showed an effect similar to the control TGF-β with a better effect.

Test Example  11: anti-inflammatory efficacy test

       In order to confirm the anti-inflammatory activity of the carob bean extract and enzyme-treated carob bean extract and pinitol prepared in Comparative Examples 1 and 13, the effect on the production of tumor necrosis factor (TNF-α), which is one of the initial inflammatory molecules, was examined. .

The formation of pro-inflammatory cytokine such as TNF-α leads to the conversion of arachidonic acid to prostaglandin and NO formation due to the activity of phospholipase A2. . The cells used in the experiment were a Human Skin Keratinocytes cell line (HaCaT), Dulbecco's modified Eagle's medium with 37 ° C, 5% CO2, 10% Fetal bovine serum (FBS, Lonza), and 50 μg / mL streptomycin (Sigma). (DMEM, Invitrogen) was incubated. The cells were stressed by UV irradiation for 1 hour after incubation. As a control, cells irradiated with UV without treating the sample were used. In this case, α-Bisabolol was used as a positive control of anti-inflammatory efficacy.

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 > 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-α 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 Carob Bean Extract (Comparative Example 1) 82.99 65.50 44.06 36.76 Enzymatic Treated Carob Bean Extract
(Example 13) 55.74 37.70 30.58 18.55 Finitol 56.88 43.66 37.55 26.65 α-Bisabolol 28.75

As confirmed in Table 10, the enzyme isolated carob bean extract according to the present invention (Example 13) by reducing the concentration-dependent expression of tumor necrosis factor (TNF-α) which is an inflammation-induced inflammatory response factor induced by UV It was found that the inflammatory response was suppressed. Therefore, the enzyme-treated carob bean extract effectively suppresses skin inflammatory response, thereby also excellent in reducing skin troubles. In addition, the enzyme-treated carob bean extract (Example 13) of the present invention was very excellent in efficacy compared to the general carob bean extract (Comparative Example 1).

The anti-glycosylation and anti-aging cosmetic composition containing the enzyme-treated carob bean extract of the present invention can be prepared in the following various formulations.

Produce Example  One: emulsion  Base manufacturing

An emulsion base was prepared according to the composition of Table 11 as an anti-glycosylation and anti-aging cosmetic composition containing the enzyme-treated carob bean 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 Enzymatically Treated Carob Bean 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 the enzyme-treated carob bean extract according to the present invention with the composition shown in Table 12 was prepared by a conventional method.

ingredient Content (% by weight) Enzymatically Treated Carob Bean 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

Preparation Example 3: Nutritional Lotion

The nutritional lotion containing the enzyme-treated carob bean extract according to the present invention with the composition shown in Table 13 was prepared by a conventional method.

ingredient Content (% by weight) Enzymatically Treated Carob Bean 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 Tocopherol Acetate 0.5 Carboxyvinyl polymer 0.12 glycerin 5.0 1,3-butylene glycol 3.0 Sodium hyaluronate 5.0 Triethanolamine 0.12 Preservative, fragrance, pigment a very small amount Distilled water Balance Sum 100

Produce Example  4: nutrition cream

A nutritious cream containing the enzyme-treated carob bean extract according to the present invention with the composition shown in Table 14 was prepared by a conventional method.

ingredient Content (% by weight) Enzymatically Treated Carob Bean 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 hyaluronate 4.0 Preservative, fragrance, pigment a very small amount Distilled water Balance Sum 100

Preparation Example 5 Massage Cream

A massage cream containing the enzyme-treated carob bean extract according to the present invention with the composition shown in Table 15 was prepared by a conventional method.

ingredient Content (% by weight) Enzymatically Treated Carob Bean 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 Pack

A pack containing the enzyme-treated carob bean extract according to the present invention with the composition shown in Table 16 was prepared by a conventional method.

ingredient Content (% by weight) Enzymatically Treated Carob Bean 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 (9)

Carp bean extract manufacturing step of extracting carp bean extract with at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ether, ethyl acetate and chloroform;
An enzyme addition step of dissolving the carob bean extract in a solvent and adding a sugar-binding degrading enzyme obtained by mixing galactosidase and pectinase in a volume ratio of 1: 1; And
Method for producing an enzyme-treated carob bean extract comprising an enzyme reaction step of performing the enzyme reaction under the conditions of reaction pH 3.5 ~ 5.0, reaction temperature 25 ~ 50 ℃ and reaction time 12 ~ 60 hours while stirring the extract to which the enzyme is added . delete delete delete delete delete delete delete delete