KR102090385B1 - Two Step-sequential Enzyme treated extract of Plant originated materials, its functional cosmetic composition comprising the same and method producing the same - Google Patents

Abstract

The present application relates to a two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles, a whitening, moisturizing, skin elasticity and wrinkle-improving functional cosmetic composition containing the extract, and a method for preparing the extract.

Description

Two-step sequential enzyme treated extract of plant originated materials, its functional cosmetic composition comprising the same and method producing the same}

The present application relates to a two-step sequential enzymatic treatment extract of vegetable material, in particular, a two-stage enzymatic treatment extract of carrot, oyster mushroom and pine needles, a manufacturing method thereof, and a functional cosmetic composition comprising the extract.

Recently, as the well-being culture is settled and the consumer's standard of living is increasing, cosmetics made from natural materials are in the spotlight compared to the past. In particular, the cosmetics industry, which avoids the use of cosmetics using animal materials such as animal collagen, and utilizes various whitening, anti-aging, and anti-wrinkle functions using plant protein or amino acid components, is drawing attention. In addition, over the past decades, the cosmetic industry has accommodated the use of plants and plant products in various cosmetic formulations and products. While this trend is expected to continue, there is a need for more refined and higher quality plant ingredients that consistently exhibit properties that are interesting to the cosmetic industry and consumers.

The sugar protein derived from carrot, which can be used as a component of vegetable cosmetics, contains a large amount of hydroxyproline used in collagen biosynthesis. Hydroxyproline is a protein that is difficult to find other than animal collagen. It is also known that the glycine content is similar to that contained in animal collagen peptides. Carrot sugar protein is an important constituent protein of the primary cell wall and serves as a defense against external stimuli.In terms of amino acid composition, the content of serine with hydroxyl groups in the molecular side chain is high, so it has excellent moisturizing ability and easy biosynthesis of collagen in the human body. It is said to have been done.

Oyster mushroom is a functional food with various functions, and is widely used not only for food but also for medicinal purposes. Oyster mushroom has a high protein and fat content, and contains amino acids such as alanine, methionine, arginine, aspartic acid, serine, isoleucine, and threonine. In addition, oyster mushroom contains a lot of ergosterol, the mother of vitamin D2, which lowers cholesterol levels and prevents high blood pressure and arteriosclerosis. In addition, oyster mushroom contains organic acids such as lactic acid, oxalic acid, fumaric acid, succinic acid and malic acid, and free sugars such as trehalose, glucose and fructose.

In addition, pine needles, which can be used as a raw material for cosmetics, are rich in essential amino acids, which are important protein sources that form the human body.

Donguibogam said that if you reproduce pine needles for a long time, you will not get old, you will feel vigorous, your head will be darkened, and you will not know cold and hungry. Introducing

Among these pine needle components, terpene, a volatile component that flies into the air, is currently mentioned, and there are more than 40 types of terpene in pine needles, the most of which are alpha-pinene, beta-pinene, beta-phellandrene, and beta. -Calyophyllene, myrcene, campaign, alpha-terpinolene, etc. Also, the fatty acid contained in the pine needle is about 20% of linolenic acid, which is different from vegetable fats such as animal fatty acids.

It contains a lot, followed by palmitic acid, which accounts for 10%. In addition, it contains a lot of polyunsaturated fatty acids, including 5-olefinic acids that are not easily oxidized.

The first-stage carbohydrate degrading enzyme used in the present invention is a woodyli-derived xylase (EC 3.2.18) and a mushroom strain-derived mannase (EC 3.2.25) protease derived from Papaya Latex (EC 3.1.22.2, Endopeptidase) As a Bromelain derived from Papain and Pineapple Stem (EC 3.4.22.4, Protease), we aimed to develop a plant-derived cosmetic material that replaces animal collagen using only 4 kinds of plant-derived degrading enzymes.

Under this background, the applicants completed the present application by confirming the functions of the enzyme-treated extract of the carrot, oyster mushroom and pine needles, and a cosmetic composition containing the same as a result of earnest research efforts to develop a functional cosmetic product using only vegetable raw materials.

Korean Patent Registration No. 10-0579170 is known as a prior art related to the present application, but it relates to a method for preparing a cosmetic composition comprising a carrot extract. There is also Republic of Korea Patent No. 10-0334608, which relates to a method for preparing a cosmetic composition comprising a ethanol extract of oyster mushroom. In addition, Korean Patent Registration No. 10-1773452 draws a cosmetic composition containing a fermented extract of pine needles using steam drying, and Patent Publication No. 10-2008-63711 passes an activated carbon layer after hot-water treatment of materials derived from animals and plants. Disclosed is a method for obtaining collagen obtained by enzymatic hydrolysis of a protein in an organic acid solution.

However, among the vegetable raw materials of the present application, three kinds of materials of carrot, oyster mushroom and pine needles are selected, and specific carbohydrate enzymes and specific proteolytic enzymes are selected to perform two-step sequential treatment to prepare an enzyme-treated extract of vegetable raw materials. The method and the cosmetic composition using the extract have not been disclosed or implied anywhere in the prior literature.

The present inventors have come to undertake and complete the present invention to solve the technical shortcomings of patent application No. 10-2017-0145543 filed on the basis of the government task report after the date of June 30, 2017. Particularly, the present inventors first treated the above-mentioned known plant material for cosmetics with a mixture of xylase and mannanase enzymes, which are carbohydrate enzymes, followed by sequential treatment of known proteolytic enzymes in two steps to completely degrade the pine needle-derived terpenes. It has succeeded in providing skin elasticity and wrinkle improvement effect by preventing color change during cosmetic storage and increasing the concentration of amino acids in sulfur and branch chains.

Therefore, the object of the present application is to select the three vegetable materials of carrot, oyster mushroom and pine needles among vegetable materials, and first pre-treat the most preferred combination ratio of carbohydrate degrading enzyme xylase and mannanase 1: 1 (w / w), then papain and It is to provide a method for preparing an extract that is secondarily processed in two steps sequentially with Bromelain protease.

Another object of the present application is to provide a functional cosmetic composition for improving whitening, moisturizing, skin elasticity and wrinkles containing a two-step sequential enzymatic treatment extract of three vegetable materials of carrot, oyster mushroom and pine needles prepared according to the above method and containing the same. .

Other objects and advantages of the present application will become more apparent by the following detailed description in conjunction with the appended claims and drawings. The contents not described in this specification can be sufficiently recognized and inferred by those skilled in the technical field or similar field of the present application, and the description thereof will be omitted.

Hereinafter, the contents of the present application will be described in detail as follows. On the other hand, the description and embodiments of one embodiment disclosed in the present application may be applied to the description and embodiments of other embodiments with respect to common matters. Also, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not considered to be limited by the specific descriptions described below.

In addition, in the present application, the extraction yield of collagen extract and whitening, moisturizing, skin elasticity, and wrinkle improvement functions are enhanced by pre-treatment of carbohydrate degrading enzymes of vegetable origin for decomposition of insoluble polysaccharides constituting the cell wall, i.e., cellulose and glycoproteins. .

The carbohydrate degrading enzyme used in the present application is xylanase (EC) of mushroom strain origin, such as sprouts, wood rot, and fungi of higher plants that promote the hydrolysis of insoluble polysaccharide cellulose constituting the plant cell wall. 3.2.18), mananase (EC 3.2.25) is most preferred.

The carbohydrate hydrolase selected in the present application may be used in a high purity of 1.5 unit / mg or more, and in the case of a low concentration, an enzyme concentration of 11% (w / w) or more may be used. Preferably, each carbohydrate-degrading enzyme may have a concentration of 5% (w / w) or more, and most preferably, a low-purity enzyme of 5% (w / w) or more.

As confirmed by the present inventors, the carbohydrate degrading enzyme is preferably used in purified water extract of each carrot, oyster mushroom, and pine needle, and the compounding ratio thereof may be 1: 1 (w / w).

In addition, proteolytic enzymes that can be selected to achieve the object of the present application are enzymes that make amino acid or peptide mixtures according to the results of the experiments of the present inventors, and are enzymes involved in the hydrolysis of peptide bonds. Among them, pepsin, peptidase, trypsin, etc. may be included, but most preferably, a protease of vegetable origin, most preferably Papain (EC 3.4.22.2) or Bromelain.

On the other hand, in order to increase the yield and moisturizing, whitening, skin elasticity and wrinkle improvement functions of the collagen peptide of the present invention, a pre-treatment to decompose the cellulosic cellulosic glycoprotein is performed prior to proteolytic enzyme treatment prior to protease treatment. It is characterized by being.

Protease used in the present application may be used at a concentration of 1.5 unit / mg or more for high purity, or at least 11% (w / w) of enzyme concentration for low concentration. Preferably, each protease may use a concentration of 5% (w / w) or more, and most preferably, a low-purity enzyme may be used in a concentration of 5% (w / w) or more.

In the present application, when the corresponding raw material is extracted using purified water for the production of a two-step sequential enzyme-treated extract, the blending ratio is most preferably 75% (w / w): 25% (w / w). The pH may be used in the range of 5.5 to 7.5, but most preferably it is the neutral pH 7.0. In addition, the extraction time can be extracted for 30 minutes to 2 hours, but most preferably for 1 hour.

The term “whitening functional ingredient” used in the present application refers to a component that exhibits a skin whitening effect such as melanin suppression, and there are mulberry extract, arbutin, ethyl ascorbyl ether, oil-soluble licorice extract, ascorbyl glucoside, and niacinamide. These ingredients can be recognized as functional cosmetic ingredients only when they have a certain concentration or more. In particular, niacinamide is a water-soluble vitamin B complex that helps to maintain skin health by clearing the skin and preventing oxidation, and is preferably recognized when it contains 2% or more.

The “moisturizing power evaluation” used in the present application measures moisture in the skin, and a Hydrometer, Tewanmeter, or Corneometer can be used. Preferably, the moisturizing efficacy can be measured through a Corneometer that constantly measures the moisture content within a depth of 30 to 40 micrometers below the stratum corneum.

As described above, this application has the effect of providing a two-step sequential enzymatic treatment extract of vegetable raw carrots, oyster mushrooms and pine needles and a method of manufacturing the same, as well as whitening, moisturizing, skin elasticity and wrinkle improvement functions containing the extract There is an excellent effect of providing a cosmetic composition.

1 is a schematic diagram showing a manufacturing process of a two-step sequential enzyme treatment extract of three kinds of vegetable materials such as carrot, oyster mushroom and pine needle of the present application.
Figure 2 is a two-step sequential enzyme treatment extract of three kinds of vegetable materials, such as carrot, oyster mushroom, pine needle of the present application, invalid treatment, each enzyme low purity (LP, low purity) and high purity (HP, High purity).
3 is a graph showing the total protein quantitative results according to the Lowry Assay method based on the Bovine Serum Albumin as a standard for 15 extracts obtained by two-step sequential enzymatic treatment of three kinds of vegetable materials such as carrot, oyster mushroom and pine needle of the present application. .
Figure 4 is a schematic diagram showing the manufacturing process of the cream applied to the two-step sequential enzyme treatment extract of three kinds of vegetable materials such as carrot, oyster mushroom, pine needle of the present application.
Figure 5 is a photograph showing the property change according to the temperature of the cream applied to the two-step sequential enzyme treatment extract of three kinds of vegetable materials such as carrot, oyster mushroom, pine needle of the present application.
6 is a chromatogram of quantitative determination of Niacinamide in a two-step sequential enzymatic treatment extract applied cream of three kinds of vegetable materials such as a standard product of Niacinamide and carrot, oyster mushroom, pine needle of the present application.
7 is a graph showing the change in water retention according to the measurement time of the control cream, an animal material (pig) collagen-added cream and the carrot-treated enzyme extract cream of the present application. (Product # 1: Control cream, # 2: Animal collagen added cream, # 3: Carrot SEL (Papain) cream, # 4: Carrot ANFLA (Bromelain) cream)

This application discloses each protease-treated extract of three kinds of vegetable materials such as carrot, oyster mushroom, pine needles, and a method for manufacturing the same.

More specifically. The present invention is a two-step enzymatic treatment to treat proteolytic enzymes after pre-treating plant-derived carbohydrate enzymes to remove carbohydrates such as terpenes contained in cells and plant cell wall decomposition before proteolytic treatment of each vegetable material. Changes in amino acid composition and? Amount (concentration mol%) of the extracts obtained by performing the sequence were confirmed.

Hereinafter, the present invention will be described in more detail through examples according to the present application. However, the following examples of the present application are only examples of the present application. These examples are intended to explain the present application in more detail, and it is obvious to those skilled in the art to which the present application pertains that the scope of the present application set forth in the appended claims is not limited by this embodiment. something to do.

Example 1. Preparation of two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

To prepare protein enzymatic extracts of carrots, oyster mushrooms and pine needles, the enzymes were Papain (EC 3.4.22.2) from Papaya Latex and Bromelain (EC 3.4.22.4) from Pineapple Stem. Hot water extract was used as a control. Two types of enzyme purity were used: high purity and low purity. Xylase, Mananase, Papain and Bromelain≥1.5 unit / mg Protein for high purity, Xylase, Mananase, Papain and Papain, and Bromelain≥ plant protease 11% for low purity. Was used. Enzyme treatment extraction conditions are shown in Table 1 below.

division Enzyme concentration (%) enzyme Low purity High purity Raw materials (carrot, oyster mushroom, pine needles) (%) 25 25 Purified water (%) 75 75 Enzyme treatment concentration (%) 5.0 5.0

Specifically, in order to prepare enzymatically processed extracts of carrots, oyster mushrooms and pine needles, the compounding ratio of each corresponding raw carrot, oyster mushroom and pine needles in purified water (extract solvent) is 75% (w / w): 25% (w / After dissolving to w), the mixture was extracted for 1 hour at pH 7.0 and 60 ° C under neutral conditions. The hot water extract was treated with 5% (w / w) of each low-purity and high-purity xylase and mananase weight ratio 1: 1 coenzyme, and stirred for 10 to 20 minutes to decompose carbohydrates. Subsequently, each low-purity and high-purity Papain or Bromelain was each sequentially treated with 5% (w / w) and stirred for 10 to 20 minutes, respectively. Treatment of the enzymes was omitted in the control sample. The enzyme-treated extract was stirred for 30 minutes at a temperature of 80 to 85 ° C for enzyme inactivation, and the extraction was terminated, followed by cooling at room temperature of 20 to 25 ° C. On the other hand, after removing and filtering the raw material from the cooled enzyme-treated extract, a two-step sequential enzyme-treated extract was obtained for extracts of carrot, oyster mushroom and pine needles (FIG. 1).

The obtained extract is a control extract to which each enzyme is not applied to three kinds of raw materials, high purity (High Purity) and low purity (Low Purity) of each Papain (SEL) enzyme-treated extract, high purity (High Purity) and low purity Five samples of each Bromelain (ANFLA) extract of (Low Purity), a total of 15 two-step sequential enzyme-treated extracts were produced, and as a result of the verification, all of the extracts treated with Papain or Bromelain protease in two-steps were added to the control sample. In comparison, the external color became darker and the degree of darkness of the color was different according to the type or purity of the protease treated (FIG. 2).

Example 2. Measurement of total protein content of two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

The total protein was quantified according to the Lowry Assay method in order to check the protein content difference between the extracts of 15 enzyme extracts prepared in Example 1. For this quantitation, BSA (Bovine Serum Albumin) was used as a standard product, and a standard curve for each protein concentration for concentration of BSA was used (FIG. 3). According to the calibration curve graph R ² value of the standard calibration curve for BSA was was quantify the total protein of the extract 15 kinds on the basis of the standard curve is identified as 0.9971 The results are shown in Table 2 to 4. (carrot, Pleurotus, pine needles) .

Carrot extract Carrot Extract Carrot SEL Extract LP Carrot SEL Extract HP Carrot ANFLA Extract LP Carrot ANFLA Extract HP Total Protein (ppm) 555/575 562/592 560/580 616/654 717/763

[Example] Protease treatment extract / Carbohydrate hydrolase treatment followed by two-step sequential enzyme treatment extract

Oyster mushroom extract Mushroom Extract Mushroom SEL Extract LP Mushroom SEL Extract HP Mushroom ANFLA Extract LP Mushroom ANFLA Extract HP Total Protein
(ppm) 536/578 539/585 582/632 559/598 547/572

 [Example] Same as [Table 2] above.

Pine needle extract Pine Extract Pine SEL Extract LP Pine SEL Extract HP Pine ANFLA Extract LP Pine ANFLA Extract HP Total Protein
(ppm) 586/612 554/594 558/598 548/593 671/702

 [Example] Same as [Table 2] above.

As a result of analyzing the protein content of 15 different protease-treated extracts targeting three different raw materials, the protein content of each extract was maintained at a relatively constant level. That is, the protein content of the carrot-based extract is 575 ~ 763 ppm (0.058 ~ 0.076%), the extract based on the oyster mushroom is 572 ~ 678 ppm (0.06 ~ 0.07%), and the extract based on the pine needle is 593 ~ It was confirmed as 702 ppm (0.06 to 0.07%). Among the extracts, the total protein content in the extracts using carrots and pine needles was highest when high purity Bromelain was applied, and in the case of extracts based on oyster mushroom, when high purity Papain was applied. As a result, since the purity of the enzyme applied in this experiment did not significantly affect the total protein content, an extract treated with a low-purity enzyme was applied when preparing the cream of the present invention. These results are a mixture of xylase (EC 3.2.18) and mannase (EC 3.2.25) 1: 1 (w / w) as a carbohydrate degrading enzyme before protease treatment for each of the above three types of raw materials. 5% (w / w) pre-treatment showed no significant difference, but it was confirmed that 15 kinds of total protein content (ppm) increased by 5-10% each.

Example 3. Measurement of amino acid content of two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

The amino acids in the two-step sequential enzyme extract prepared in Example 1 were quantified to determine how enzyme treatment affects amino acid composition and content changes.

First, according to the amino acid composition and quantitative results in the extract using carrot as a raw material, regardless of whether enzyme treatment is performed in three types of extracts (processing of Papain or Bromelain immediately after enzyme 1: 1 treatment with xylase and mannanase 1: 1) The most important amino acid was Alanine. Alanine in the enzyme-free sample (water extract) was about 48%, which was close to half of the total amino acids. When the protease was treated, the concentration decreased, so when processing Papain, for example, the concentration of Alanine having the highest content was about 34%, about 29% lower than the sample without enzyme, and about 29% when treated with Bromelain. It showed a decrease of about 40% compared to the enzyme-free sample. On the other hand, protease treatment increased the concentration of sulfuric amino acid MET by 5 to 7 times, and the concentrations of branched chain amino acids VAL, ILE and LEU also increased by 1.5 to 1.6, 1.7 to 2.7 and 2.7 to 6.6, respectively (Table 5). .

Amino Acid No enzyme treatment
(mol%) Protease
Single treatment (mol%) Carbohydrate degrading enzyme
Sequential treatment (mol%) Papain Bromelain Papain Bromelain CYS ★ 0.00 0.00 0.00 1.05 1.47 ASP 8.91 7.16 4.95 6.18 3.65 GLU 3.11 6.57 0.40 5.95 0.35 ASN 7.17 6.26 8.84 5.92 5.85 SER 9.31 6.55 6.86 6.11 5.88 GLN 0.87 2.36 2.17 2.31 2.15 GLY 1.10 4.83 2.49 4.15 2.53 HIS 0.39 0.51 0.56 0.67 0.65 ARG 0.53 1.94 3.27 2.03 3.32 THR 1.52 1.94 2.05 2.01 2.09 ALA 47.83 33.85 29.10 22.28 16.36 PRO 3.22 3.18 2.59 3.02 2.62 TYR 0.46 0.89 3.63 0.93 2.72 VAL * 2.91 4.49 4.68 8.98 9.37 MET ★ 0.35 1.54 2.45 4.12 5.08 ILE * 2.00 3.49 5.44 8.21 11.72 LEU * 0.85 2.32 5.57 5.78 12.84 PHE 5.44 2.60 5.61 2.68 3.64 TRP 0.32 1.38 1.75 1.44 1.83 LYS 3.72 8.14 7.58 6.18 5.88 TOTAL 100.00 100.00 100.00 100.00 100.00

[View] ★ sulfur amino acid, * branch chain amino acid.

In particular, when the protease papain or bromelain was sequentially processed in two steps after treatment of the tin hydrate degrading enzyme (xylase and mannanse 1: 1 (w / w) mixture), sulfuric amino acids CYS and MET were each 1 mol% And concentrations of 4 to 5 mol% or more, and the concentrations of the branch chain amino acids VAL, ILE, and LEU also increased more than 2 times, respectively.

In addition, according to the amino acid composition and quantitative results of the enzyme extract using oyster mushroom as a raw material, the Alanine content was the highest as in the water extract using carrot as the raw material, the concentration before enzyme treatment was about 37%, and when treated with Papain alone 33 %, It was confirmed that it gradually decreased to 25% when treated with Bromelain alone. The amino acid that occupies the highest specific gravity after Alanine was Tryptophan, which was 12% before enzyme treatment, lowered to 10% when treated with Papain alone, and 9% when treated with Bromelain alone, resulting in a decrease in concentration through protease alone, which is the same result as in Alanine. Showed. On the other hand, the second step sequential enzyme treatment extract of the present invention using oyster mushroom as a raw material also confirmed that the concentrations of cystine and methionine are 8 to 9 times higher, and valine, isoleucine, and leucine concentrations are higher than 2 to 3 times respectively. (Table 6).

Amino Acid enzyme
No treatment
(mol%) Protease
Single treatment (mol%) Carbohydrate degrading enzyme
Sequential treatment (mol%) Papain Bromelain Papain Bromelain Cys ★ 0.00 0.00 0.00 0.87 0.98 ASP 2.63 1.49 1.92 1.22 1.62 GLU 7.93 6.25 1.47 5.81 1.21 ASN 1.23 1.65 3.84 1.35 3.26 SER 2.93 3.37 3.78 2.64 2.91 GLN 2.79 5.54 4.19 4.82 3.84 GLY 1.91 4.26 2.74 3.75 1.99 HIS 1.90 1.98 1.60 1.62 1.23 ARG 4.85 5.00 4.87 3.89 3.65 THR 2.91 3.11 3.12 2.74 3.06 ALA 36.75 32.81 24.57 27.63 19.01 PRO 2.13 2.21 1.84 1.85 1.62 TYR 2.53 2.57 4.75 1.63 2.92 VAL * 3.85 4.17 5.73 8.54 11.72 MET ★ 0.21 0.65 2.07 2.22 8.24 ILE * 2.42 3.05 6.30 6.21 10.28 LEU * 3.49 4.52 6.76 9.14 13.18 PHE 4.27 3.91 5.37 2.79 1.94 TRP 12.02 10.20 8.73 8.32 5.13 LYS 3.23 3.25 6.36 2.96 2.21 TOTAL 100.00 100.00 100.00 100.00 100.00

[View] ★ Sulfur Amino Acid * Branch Chain Amino Acid

Lastly, according to the amino acid composition and quantitative results in the extract using pine needle as a raw material, there were many differences from the amino acid composition of the extract using carrot and oyster mushroom as a raw material. In this case, Arginine (about 31%) and Glycine (about 24%) accounted for the highest proportion of enzymes without treatment, and these two types of amino acids accounted for more than half of the total amino acids. When Papain was treated alone, the contents of Arginine (ARG) and Glycine (GLN) were significantly reduced, while the proportion of Alanine (ALA) and Proline (PRO) increased to about 13%, respectively. When Bromelain was treated alone, Arginine (ARG) and Glycine (GLN) contents decreased similarly as when treated with Papain, while Asparagine (ASP) and Arsene (ASN) contents increased by 2 and 3 times, respectively. (Table 7).

On the other hand, the two-step sequential enzymatic treatment extract of the present invention using pine needles also increased the concentration of sulfuric acid amino acids by 2-3 times for CYS and 2 ~ 2.5 times for MET, respectively, and the content of eggplant ammonia amino acid was about 40% each. , Increased by 20%, ILE about 30%, 60% and LEU about 50%, 50% or more.

Amino Acid enzyme
No treatment
(mol%) Protease
Single treatment (mol%) Carbohydrate degrading enzyme
Sequential treatment (mol%) Papain Bromelain Papain Bromelain Cys ★ 0.58 0.00 0.00 2.13 3.12 ASP 1.58 2.11 5.65 1.93 4.11 GLU 0.00 3.84 1.83 3.11 1.63 ASN 1.51 5.38 17.72 4.86 15.42 SER 6.66 3.37 4.21 3.15 3.68 GLN 8.41 1.34 1.32 1.03 1.13 GLY 23.54 14.10 3.36 12.8 3.25 HIS 6.10 0.89 0.74 0.64 0.51 ARG 30.66 5.48 5.57 4.92 4.26 THR 1.64 2.15 2.25 1.86 2.14 ALA 7.93 12.94 11.78 11.14 9.42 PRO 4.11 12.79 8.35 10.92 7.09 TYR 0.92 5.05 3.92 4.87 3.15 VAL * 0.49 5.23 6.53 4.35 7.98 MET ★ 0.64 3.89 1.95 8.13 5.21 ILE * 0.38 6.35 5.07 8.35 8.27 LEU * 0.36 4.63 6.36 7.31 9.24 PHE 1.36 4.83 4.66 3.56 3.17 TRP 1.37 3.33 3.32 3.05 3.03 LYS 1.77 2.30 5.41 1.89 4.19 TOTAL 100.00 100.00 100.00 100.00 100.00

[View] ★ Sulfur Amino Acid * Branch Chain Amino Acid

Preparation Example 1. Preparation of cream containing enzyme-treated extracts of carrot, oyster mushroom and pine needles

Using the extract obtained in Example 1, a cream containing a two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles was prepared. The total ingredients and the blending ratio contained in the cream were used as shown in Table 8 below, and three types of enzyme-free treatment, Papain sequential treatment, and Bromelain sequential treatment per three raw materials were used to prepare a total of 9 creams.

In order to prepare the cream of the two-step sequential enzyme treatment extract of the carrot, oyster mushroom and pine needles, the respective components corresponding to the preferential water phase and the oil phase are weighed to have different temperatures (60 to 70 ° C in the water phase, 70 in the oil phase) To 80 ° C) and uniformly mixed, and after adding the oil phase component to the aqueous phase, emulsification is performed at a stirring speed of 2000 to 3000 RPM for 7 to 13 minutes, and the finished sample is 35 to 40 ° C. After cooling in, a thickening process was performed. After the above-mentioned sample, the effective functional ingredient was added to prevent the loss of the original function of the raw material due to the addition during the high temperature emulsification process. Fig. 4).

INCI Name (Product Name) content (%) A Carrot extract / oyster mushroom extract / 2 stage sequential enzyme extract of pine needle ¹⁾ to 100 Glycerin 1 to 10 Butylene Glycol 1 to 10 1,2-Hexanediol 1 to 10 Niacinamide 1 to 10 Betaine Max 1.0 Adenosine Max 0.1 Disodium EDTA Max 0.1 B Stearic Acid 1 to 10 Cetostearyl Alcohol 1 to 10 Shea Butter 1 to 10  Polysorbate-60 Max 1.0 Sorbitan Stearate Max 1.0 Polyglyceryl-3 Methylglucose Distearate 1 to 10 Hydrogenated Polydecene 1 to 10 Caprylic / Capric Triglyceride 1 to 10 Macadamia nut oil 1 to 10 Olive Oil 1 to 10 JoJoba Oil Max 1.0 Dimethicone Max 1.0 Cyclopentasiloxane 1 to 10 C Sodium acrylate / Sodium Acryloyldimethyl taurate 1 to 10 D Sodium Hyaluronate 1 to 10 Sodium Polyglutamate 1 to 10 Centella Asiatica Max 0.1 Copper Tripeptide-1 Max 1.0 Centella Asiatica Extract Max 1.0 Ethylhexylglycerin Max 0.1 Fragrance Max 0.1 Total 100.00%

_{1) Applied extract-Carrot extract: Carrot Extract, Carrot SEL Extract, Carrot ANFLA Extract (Total 3 types)}

 _{-Oyster Mushroom Extract: Mushroom Extract, Mushroom SEL Extract, Mushroom ANFLA Extract (3 types in total)}

 _{-Pine Needle Extract: Pine Needle Extract, Pine Needle SEL Extract, Pine Needle ANFLA Extract (3 types in total)}

Preparation Example 2. Preparation of lotion containing two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

The lotion containing the two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles obtained in Example 1 was prepared as shown in Table 9 below.

Raw material Raw material Content (% by weight) One Two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needle 10.0 2 glycerin 3.0 3 Butylene glycol 2.0 4 Propylene glycol 2.0 5 Polyoxyethylene cured castor oil 1.0 6 ethanol 10.0 7 Triethanolamine 0.1 8 antiseptic a very small amount 9 Coloring a very small amount 10 Spices a very small amount 11 Purified water Balance

Preparation Example 3. Lotion containing two-step sequential enzymatic treatment extract of carrot, oyster mushroom and pine needle Lotion containing two-stage sequential enzymatic treatment extract of carrot, oyster mushroom and pine needle obtained in Example 1 above Table It was prepared as 10.

Raw material Raw material Content (% by weight) One Two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles 10.0 2 Wax 1.0 3 Polysorbate 60 1.5 4 Solvidan Sesquioleate 0.5 5 Floating paraffin 10.0 6 Sorbidan stearate 1.0 7 Lipophilic monostearine glycerin 0.5 8 Stearic acid 1.5 9 Glyceryl stearate / page-400 stearate 1.0 10 Propylene glycol 3.0 11 Carboxy polymer 0.1 12 Triethanolamine 0.2 13 antiseptic a very small amount 14 Coloring a very small amount 15 Spices a very small amount 16 Purified water Balance

Preparation Example 4. Essence preparation containing two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

This application was prepared as shown in Table 11 below, the essence containing the two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needle obtained in Example 1 above.

content Raw material Content (% by weight) One Two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles 10.0 2 Citosterol 1.7 3 Polyglyceryl 2-oleate 1.5 4 Ceramide 0.7 5 Steareth-4 1.2 6 cholesterol 1.5 7 Dicetyl phosphate 0.4 8 Concentrated glycerin 5.0 9 Macadamia oil 15.0 10 Carboxyvinyl polymer 0.2 11 Shotgun 0.2 12 antiseptic a very small amount 13 Spices a very small amount 14 젱 Jesus Balance

Experimental Example 1. Temperature stability and appearance measurement of a cream containing two-step sequential enzyme treatment extracts of carrot, oyster mushroom and pine needles

In order to measure the temperature stability and appearance of the cream containing the two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needle prepared in Example 4, the cream was at room temperature (25 ° C. constant temperature), constant temperature (-4-50). ℃), high temperature (50 ℃, constant temperature) was stored at different temperatures for 30 days to check the stability and appearance of these creams. The conditions of the constant temperature (變 溫) mean conditions that accompany temperature change from -4 ° C to 8 hours, 25 ° C to 8 hours, and 50 ° C to 8 hours each day (24 hours).

As a result of the experiment, the cream containing the two-step sequential enzyme-treated extract of carrot or oyster mushroom did not show a significant difference according to the temperature condition, and the cream containing the two-step sequential enzyme-treated extract of pine needles showed no change at room temperature and temperature. No, it was confirmed that the color changed to light yellow during high-temperature storage (FIG. 5).

It was judged that the terpenes in which unsaturated hydrocarbons, such as terpene contained in a large amount of pine needles, remained during the heating process during the preparation of the cream, which accompanied the color change during storage. However, in the nine types of creams developed in the present application, in which the present invention carbohydrate-degrading enzyme was pre-treated and the papain and bromelain were sequentially processed in two steps, no color change occurred during storage. It is judged that all the compounds of the terpenes are decomposed.

Experimental Example 2. Measurement of the viscosity of a cream containing two-step sequential enzyme treatment extracts of carrot, oyster mushroom and pine needles

Temperature (room temperature) for a total of 9 types of creams containing the two-step sequential enzyme-treated extracts of carrots, oyster mushrooms and pine needles prepared in Preparation Example 1 (Void treatment group, Papain, Bromelain treatment group) , Viscosity stability by temperature and high temperature conditions) was confirmed. In the present application, LVT (Manufacturer Brookfield) was used as a model for measuring viscosity (CPS), Measuring Temperature was 25 ° C, Spindle Number-67, and RPM was performed at 6. In the case of variable temperature and high temperature excluding storage at room temperature, after storing at different temperatures for 30 days, the viscosity was measured after storage by transferring to a 25 ° C incubator the day before measurement.

As a result of the experiment, the viscosity at ambient temperature was increased to less than 10% compared to room temperature storage in all samples, whereas the viscosity at high temperature was lowered in all samples. Among them, the cream with carrot and oyster mushrooms had a viscosity lowered to less than 10% compared to room temperature storage, but the cream with pine needles had a viscosity decreased to 10 to 20% compared to room temperature storage samples.

Under the changing temperature condition, the temperature changes from 4 to 50 ℃ and the hardness of the tissue decreases slightly at high temperature, but the hardness of the tissue increases at low temperature below 0 ℃, and it is judged that the viscosity of the final cream increased as this operation was repeated. On the other hand, it was found that the cream tissue softened as the temperature was continuously maintained at 50 ° C during high temperature storage, resulting in lower viscosity compared to the room temperature storage cream. In addition to the change in viscosity, it was confirmed that there was no significant abnormality in the stability of emulsification, which is the most problematic in general creams, because there is no change in physical properties that can be observed in creams such as phase separation.

 The optimal viscosity range of the cream containing the two-step sequential enzymatic treatment extract of carrot, oyster mushroom and pine needle of the present application is 45,000 ~ 65,000 cps, and the viscosity of all 9 types of creams is within this range, so the viscosity is a big problem even under severe temperature conditions. It was confirmed that was not (Table 12).

Raw Sample name Room temperature
(25 ℃, constant temperature) Variable temperature (-4 ~ 50 ℃) High temperature (50 ℃) Viscosity (cps) Viscosity change rate compared to room temperature Viscosity (cps) Viscosity change rate compared to room temperature ²⁾ carrot Carrot Cream ¹⁾ 57,300 59,300 3.49% 51,750 -9.69% Carrot SEL Cream 53,300 55,700 4.50% 48,500 -9.01% Carrot ANFLA Cream 52,200 54,800 4.98% 49,200 -5.75% Oyster mushroom Mushroom Cream 59,200 59,500 0.51% 54,000 -8.78% Mushroom SEL Cream 55,700 58,000 4.13% 51,500 -7.54% Mushroom ANFLA Cream 57,700 60,600 5.03% 55,300 -4.16% pine needles Pine Needle Cream 59,100 62,000 4.91% 48,600 -17.77% Pine Needle SEL Cream 55,000 55,500 0.91% 49,700 -9.64% Pine Needle ANFLA Cream 54,600 58,500 7.14% 47,500 -13.00%

_{[Note] 1) Cream-Control cream without added enzyme (Ex, Carrot Cream: Cream with hot water extract based on carrots)}

Experimental Example 3. pH measurement of cream containing two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

A total of 9 creams containing two-step sequential enzyme-treated extracts of carrot, oyster mushroom and pine needles prepared in Example 4 (Void-treated group, xylase and mananase 1: 1 (w / w) mixed for each raw material extract) The pH of each sample (sequential treatment group of enzyme, Papain, and Bromelain) was checked for each temperature (room temperature, temperature, and high temperature conditions). After storing at different temperatures for 30 days, the rest of the two product lines except for room temperature storage were transferred to a 25 ° C thermostat on the day before measurement to measure the pH.

As a result of the experiment, it was confirmed that all of the nine creams had lower pH when stored at room temperature and higher than when stored at room temperature, and the standard deviation at this time was about 0.1 to 0.4. The pH of each cream was maintained at a constant level of 6.0 to 7.2, and the standard deviation of the pH of the control cream was constant in the range of 0.12, whereas the cream containing the extract had a higher pH deviation than this. Among these, the pH of the cream based on pine needles was higher than that of carrots and oyster mushrooms, and the pH was lowered when stored at room temperature (-4 ~ 50 ℃) and high temperature conditions than room temperature. The optimal pH range for the cream containing the two-step sequential enzymatic treatment extract of 9 kinds of carrots, oyster mushrooms and pine needles in this application is 5.5 to 7.5, so all development creams are included in this range, so the pH by storage temperature is not a problem It was confirmed (Table 13).

Raw material Sample name Room temperature
(25 ℃, constant temperature) Temperature
(-4 to 50 ℃) High temperature
(50 ℃) Standard Deviation Control Cream (Std) ¹⁾ 6.8 6.59 6.6 0.12 carrot Carrot Cream ²⁾ 6.75 6.55 6.37 0.19 Carrot SEL Cream 6.98 6.77 6.3 0.35 Carrot ANFLA Cream 6.47 6.3 6.19 0.14 Oyster
mushroom Mushroom Cream 6.77 6.62 6.4 0.19 Mushroom SEL Cream 6.64 6.5 6.36 0.14 Mushroom ANFLA Cream 6.9 6.81 6.47 0.23 pine needles Pine Needle Cream 6.98 6.68 6.38 0.30 Pine Needle SEL Cream 6.87 6.61 6.39 0.24 Pine Needle ANFLA Cream 7.1 6.81 6.36 0.37

_{[Note] 1) Cream (Std): No extract}

     _{2) Cream-Control cream with no enzyme added (e.g., Carrot Cream is a carrot-based hot water extract cream)}

Experimental Example 4. Measurement of the whitening function component of the cream containing two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

 Temperature (room temperature, temperature and high temperature conditions) for a total of six samples (Papain, Bromelain treatment group per extract of each raw material) of the cream containing two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needle prepared in Example 4 above The amount of stars Niacinamide was confirmed. Niacinamide is one of the whitening functional ingredients contained in the development cream group, and refer to the "Niacinamide Cream" quantitative method in the "Functional Cosmetic Standards and Test Methods" published by the Ministry of Food and Drug Safety to determine whether changes in the concentration of functional ingredients are accompanied by temperature changes. Confirmed. For the measurement of Niacinamide, an ultraviolet absorbance photometer (measurement wavelength: 263 ㎚) was used for detection, and the column was filled with an octadecylylated silica gel for liquid chromatograph of 5 μm in a stainless tube having an inner diameter of about 4.6 μm and a length of about 25 cm. Was used. As a mobile phase, a 0.05M potassium dihydrogen phosphate solution (pH 7.0, 25% sodium hydroxide solution) and a methanol mixed solution (75:25) were used, and a flow rate of 1.0 ml / min was used. The change in the content of Niacinamide was quantified by HPLC, and the average value for three repeated measurements per sample was obtained, and the difference between the quantitative values was confirmed through standard deviation. The temperature used in this example was room temperature (25 ° C), variable temperature (-4 to 50 ° C), and high temperature (50 ° C) as in the previous experiment, and the storage period was 30 days. Samples stored at a temperature other than the room temperature storage sample were stored for 1 day in a 25 ° C. constant temperature incubator the day before measurement, and then functional quantification was performed.

As a result of the experiment, Niacinamide, a whitening function component included in the cream, was added to all creams at room temperature by about 2.0%, and the content of Niacinamide contained in the creams stored at different temperature conditions did not show a significant difference between samples. Among the carrots, the standard deviation range of the cream with Papain enzyme and two creams based on oyster mushroom was the smallest among the sample groups measured from 0.03 to 0.04. The standard deviation in other creams was less than 0.15, and the quantitative values of Niacinamide at room temperature, temperature change, and high temperature conditions did not show a significant difference. That is, the whitening functional ingredient added in the cream, Niacinamide, was kept constant at different temperature conditions, confirming that they are all chemically stable products (Table 14, FIG. 6).

Raw material Sample name Room temperature
(25 ℃, constant temperature) Cycling Temp.
(-4 to 50 ℃) High temperature
(50 ℃) Standard deviation 1 ⁾ No additives Cream 2.02 2.08 1.81 0.14 carrot Carrot SEL Cream 2.05 2.07 2.11 0.03 Carrot ANFLA Cream 2.22 1.94 2.13 0.14 Oyster mushroom Mushroom SEL Cream 2.07 2.09 2.15 0.04 Mushroom ANFLA Cream 2.04 2.03 2.11 0.04 pine needles Pine Needle SEL Cream 2.07 2.01 2.06 0.03 Pine Needle ANFLA Cream 2.08 1.91 1.97 0.09

_{[Note] 1) Standard deviation between quantitative values of Niacinamide in 3 types of creams stored at different temperatures}

Experimental Example 5. Evaluation of the moisturizing power of a cream containing two-step sequential enzyme treatment extract of carrot

 To evaluate the in vivo moisturizing efficacy of the cream containing the two-step sequential enzyme-treated extract of carrot prepared in Example 4, an evaluation agency, Elyd (325, Hwangsaeul-ro, Bundang-gu, Seongnam-si, Gyeonggi-do), uses a corneometer for 24 hours The lasting effect of skin moisturization was measured. Among the 20-45 year old women, 20 women were selected as the subjects to measure the lasting effect of moisturization, and 20 women were selected, and those who voluntarily participated in this experiment by understanding the purpose and contents of the test and skin diseases There was no sudden or chronic physical illness, including, and it was limited to those who did not use a moisturizer at the test site from 1 week before the test. Exclusion criteria include eczema skin disease (eczema, atopic dermatitis, etc.), cosmetics, medicines, and irritation or allergies to daily sun exposure. Was used.

In order to evaluate the moisturizing power of the cream containing the two-step sequential enzyme treatment extract of the carrot, a 24-hour skin moisturizing effect evaluation using a Corneometer was used.

The test site (forearm part) was used as the anterior extremity region 5 cm from the wrists of the left and right arms of the study subject, and a total of 5 horizontal and 3 cm squares were drawn at 1.5 cm intervals for each test site. One square was selected as the unapplied site, and each test site was randomly selected. The study subjects waited for 30 minutes in a waiting room with constant temperature and humidity (20 ~ 24 ℃, 40 ~ 60% RH) before the test, and the researchers measured each test site 3 times using Corneometer (CM825, Courage & Khazaka, Germany) Afterwards, the average of the measured values was obtained to check the moisturizing amount before product application (0 hour, baseline). After the baseline measurement, 2µl / cm 2 was applied to the product application site of the research subject, and the absorption amount was measured after 1 hour, 3 hours, and 6 hours of product application in the same manner as before the absorption time. After measuring for 6 hours, the study subjects were returned home, and then they were visited the next day and waited for 30 minutes in a waiting room with constant temperature and humidity (20 to 24 ° C, 40 to 60% RH), and then measured the change in moisturizing amount after 24 hours. The better the skin moisturizing condition, the higher the result value through the Corneometer, and the unit of measurement is A.U. (Arbitrary Unit) was used. Skin moisturizing results and standard deviations were confirmed for the unapplied samples to which no samples were applied and the four cream products used in the experimental examples shown in Table 15 below (Table 16).

No product name Product Information #One Cream Control cream #2 Collagen Cream Cream with animal collagen # 3 Carrot SEL Cream Cream with Papain-treated extract when extracting carrots #4 Carrot ANFLA Cream Cream added with Bromelain treated extract when extracting carrots

As a result of the experiment, the water retention of the unapplied sample without applying any sample was maintained at a constant level of 30 to 32 A.U. in all measurement time periods. Products # 1 and # 2 are control samples, of which # 1 is a normal cream without added protein (or amino acid) and extract, and # 2 is a cream using animal collagen (pork collagen). Both products showed the highest water retention after 1 hour of application. At this time, the water retention was 55.208 A.U. for product # 1 and 56.560 A.U for product # 2. It was confirmed to, and the water retention from 3 hours to 24 hours of application was sequentially lowered. There was no significant difference in water retention over time between the two products. On the other hand, after 1 hour of application of the second-stage sequential enzyme treatment products # 3 and # 4 of the present invention, the water retention was 64.724 AU and 67.221 AU, respectively, and the water retention was 18% or higher compared to products # 1 and # 2, After 3 ~ 24 hours of these products, moisture retention was also maintained at a constant high level by more than 10% compared to products # 1 and # 2.

As a result, the water retention of the cream applied with the extract using carrot as a raw material was steadily higher in all measurement periods than when using animal collagen. It was confirmed through this clinical study that there was a better moisturizing effect in the applied cream (Table 16, Figure 7).

Product type Before application 1 hour after application 3 hours after application 6 hours after application 24 hours after application Mean ± standard
Deviation
(AU) Unapplied 31.162 31.180 31.100 30.880 31.702 Product # 1 30.643 55.208 49.157 42.525 37.583 Product # 2 30.977 56.560 50.057 43.245 38.163 Product # 3 30.822 58.987 / 64.724 53.523 / 60.224 46.647 / 42.225 40.157 / 49.878 Product # 4 31.335 59.370 / 67.221 53.682 / 64.304 46.927 / 53.275 40.552 / 50.525

[View] Protein Enzyme Single Treatment / Carbohydrate Degrading Enzyme Sequential Treatment

Experimental Example 6. Skin irritation evaluation of a cream containing two-step sequential enzyme treatment extracts of carrot, oyster mushroom and pine needles

For the cream containing the two-step sequential enzyme treatment extract of carrots, oyster mushrooms and pine needles prepared in Example 4, a normal cream containing no active ingredient and an animal collagen applied cream were used to determine whether skin irritation was involved. As a control, a cream containing two-step sequential enzyme treatment extracts of carrot, oyster mushroom and pine needles was used as a sample. The evaluation of skin irritation was conducted at the evaluation agency Elyd (325 Hwangsaeul-ro, Bundang-gu, Seongnam-si, Gyeonggi-do). 31 subjects (average age 43.74 years) among adult men and women aged 20 ~ 55 years were selected for the study subjects used in this experimental example. Other selection criteria and criteria for excluding study subjects were the conditions of the study subjects participating in the evaluation of moisturizing ability of Experimental Example 5 above. It was the same. For skin irritation evaluation, safety evaluation by skin patch test (Patch Test) and human body application test were performed. Specifically, for each 31 subjects, Van der Bend chamber (Van der Bend, the Netherlands) was used to spine the back. A sample was applied between the shoulder blade and the waist line centering on. After washing the test site with distilled water and drying it, 8 types of creams (samples # 1 to # 8) to be tested were added dropwise to 35 µl each in a Van der Bend chamber and fixed to the test site. It was applied to the skin for a total of 24 hours. After removing the patch, the test site was marked with a marking pen, and after 30 minutes, 24 hours, and 48 hours, respectively, the test site was observed.

  For skin reaction, the degree of irritation was evaluated according to the evaluation criteria (Table 17) of the Draize Dermal Irritation Scoring System, and after calculating the Mean Irritation Index (MII) according to the formula for calculating the average skin irritation (Reaction Scheme 1), EPA (Environmental Protection Agency) ) Standard Evaluation Procedure The stimulation group was determined according to the result of the primary skin irritation test (Table 18) reflecting the criteria for stimulation classification of the dermal classification system.

Erythema and Eschar Formation
(Erythema and peel formation) Value Edema Formation
(Edema formation) Value No erythema 0 No edema 0 Very slight erythema
(barely perceptible) One Very slight edema
(barely perceptible) One Well-defined erythema 2 Slight edema (edges of area well-defined by definite raising) 2 Moderate to servere erythema 3 Moderate edema
(raised approximately 1 ㎜) 3 Severe erythema (best redness) to slight, eschar formation (injuries in depth) 4 Severe edema (raised more than 1㎜ and extending beyond the area of exposure) 4

[Note] [Scheme 1] Calculation formula of average skin irritation (M.I.I .: Mean Irritation Index)

Stimulation group M.I.I Negligible 0.00 ≤ M.I.I. ≤ 0.50 Stimulation (Slight) 0.50 ≤ M.I.I. ≤ 2.00 Moderate 2.00 ≤ M.I.I. ≤ 5.00 Strong Stimulation (Strong) 5.00 ≤ M.I.I. ≤ 8.00

As a result of skin irritation evaluation, the average skin irritation of 8 types of cream was less than 0.50. As a result of confirming in the primary skin irritation test result decision table (Table 18), it was determined as “Negligible” and all the creams used in this example were skin It was confirmed that it is a safe product that does not involve irritation. As a result, it was confirmed that the cream containing the two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles developed in this application is superior in moisturizing efficacy and safer to the human body than the cream applied with the existing animal collagen (Table 19).

number Sample name Number of respondents After 30 minutes After 24 hours 48 hours
After skin
Average
Irritation One 2 3 4 One 2 3 4 One 2 3 4 #One Cream Erythema 10 5 - - - 3 - - - 5 - - - 0.14 ¹⁾ edema 0 - - - - - - - - - - - - #2 Collagen Cream Erythema 9 2 - - - 5 - - - 5 - - - 0.13 edema 0 - - - - - - - - - - - - # 3 Carrot SEL Cream Erythema 6 2 - - - 4 - - - 5 - - - 0.12 edema 0 - - - - - - - - - - - - #4 Carrot ANFLA Cream Erythema 6 2 - - - 3 - - - 5 - - - 0.11 edema 0 - - - - - - - - - - - - # 5 Mushroom SEL Cream Erythema 10 3 - - - 5- - - - 4 - - - 0.13 edema 0 - - - - - - - - - - - - # 6 Mushroom ANFLA
Cream Erythema 7 2 - - - 4 - - - 4 - - - 0.11 edema 0 - - - - - - - - - - - - # 7 Pine Needles SEL
Cream Erythema 15 5 - - - 6 - - - 7 - - - 0.19 edema 0 - - - - - - - - - - - - #8 Pine Needles ANFLA
Cream Erythema 8 2 - - - 4 - - - 4 - - - 0.11 edema 0 - - - - - - - - - - - -

_{[Note] 1) Calculation formula = (5 + 3 + 5) / (31) = 0.14}

Experimental Example 7. Skin elasticity improvement effect of cream containing two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles

The effect of improving the skin of the human body was measured with respect to all six creams containing two-step sequential enzyme treatment extracts of carrot, oyster mushroom and pine needles prepared in Example 4. After applying for 8 weeks twice a day, centering around the eye area, for 40 healthy women over the age of 40 to 60 years, after using the skin elasticity measuring instrument (cutometer MPA 580, Courage khazaka electronic GmbH, Germany), after 8 weeks Skin elasticity was measured.

The experimental results were as shown in [Table 20], and the results showed a remarkable effect of about 2 times as the average value of improvement.

number Sample name Skin elasticity improvement (%) #One Cream 0.72 #2 Collagen Cream 16.32 # 3 Carrot SEL Cream 30.93 #4 Carrot ANFLA Cream 36.83 # 5 Mushroom SEL Cream 29.82 # 6 Mushroom ANFLA Cream 37.24 # 7 Pine Needles SEL Cream 30.55 #8 Pine Needles ANFLA Cream 38.27

Experimental Example 8. Skin wrinkle improvement effect of a cream containing two-step sequential enzyme treatment extract of carrot, oyster mushroom, pine needles

The wrinkle improvement effect of the human body was measured for all six types of creams containing two-step sequential enzyme treatment extracts of carrot, oyster mushroom and pine needle prepared in Example 4.

40 women aged 50 to 60 were randomly sampled, randomly divided into 4 groups of 10, and the bowel composition prepared twice a day was uniformly applied around the eyes and tested for a total of 12 weeks. Cosmetic composition The wrinkles around the eyes before and after applying the cream were measured for skin wrinkle improvement effect using a skin biometer (Skin Visicometer SV 600, CK Electonic GmbH, Germany).

In the experiment, the length of the wrinkles was measured by dividing the sample made of silicone rubber into 5 parts on the area where the wrinkles were to be measured, and showing the average value of the difference between the highest and lowest points of the wrinkle height at each of the five places.

The experimental results are shown in [Table 21], and showed a remarkable effect of wrinkle improvement of about 5 to 7 times.

number Sample name term Skin wrinkle depth
Decrease rate (%) #One Cream T0 0.0 T12 2.2 #2 Collagen Cream T0 0.0 T12 5.5 # 3 Carrot SEL Cream T0 0.0 T12 25.5 #4 Carrot ANFLA Cream T0 0.0 T12 33.5 # 5 Mushroom SEL Cream T0 0.0 T12 28.7 # 6 Mushroom ANFLA Cream T0 0.0 T12 29.5 # 7 Pine Needles SEL Cream T0 0.0 T12 35.9 #8 Pine Needles ANFLA Cream T0 0.0 T12 35.4

This application has the effect of providing a two-step sequential enzyme treatment extract of carrot, oyster mushroom and pine needles, a whitening, moisturizing, skin elasticity and wrinkle-improving functional cosmetic composition containing the extract, and a method for preparing the extract, in particular Since it has an excellent effect that can be used as a new material for cosmetics such as cream, it is a very useful invention in the natural vegetable cosmetics industry.

Claims (8)

Extracting hot water by adding purified water to at least one vegetable raw material of carrot, oyster mushroom and pine needles; Treating the hydrolase with the hot water extract obtained in the step; Heating the enzyme-treated extract obtained in the above step to inactivate the hydrolase; Filtering the inactivated enzyme-treated extract obtained in the above step; In the method for producing an enzyme-treated extract of carrot, oyster mushroom and pine needles consisting of the step of obtaining the filtrate obtained in the above step;
The treatment of the hydrolase is first treated with a carbolytic enzyme composed of a mixture of xylase and mannanase 1: 1 (w / w), and then sequentially sequenced with any protease selected from papain or Bromelain. Method for producing an enzyme-treated extract of vegetable raw materials characterized by processing.
delete Enzyme-treated extract prepared by the method of claim 1.
A functional cosmetic composition for improving whitening, moisturizing, skin elasticity and wrinkles containing the enzyme-treated extract of claim 3.
delete The composition of claim 4 is a cosmetic composition characterized in that the formulation of any one of lotion, lotion, cream and essence.
delete delete

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