WO2014178501A1 - Functional cosmetic composition comprising enzymatic hydrolysate of tilapia mossambica scales - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising an enzymatic hydrolysate of Tilapia mossambica scales and, more particularly, to a functional cosmetic composition, having anti-oxidation, anti-wrinkle and whitening effects, comprising the ingredients as active ingredients. The enzymatic hydrolysate of Tilapia mossambica scales, which are active ingredients of the present invention, shows excellent antioxidant activity, anti-wrinkle and whitening effects, and is, as a natural material, harmless to the human body, and thus is suitable for a use of the functional cosmetic composition.

Description

Functional cosmetic composition comprising enzymatic hydrolyzate of tilapia scales

The present invention relates to a cosmetic composition comprising an enzymatic hydrolyzate of Tilapia ( Tilapia Mossambica ) scales, and more particularly to a functional cosmetic composition having antioxidant, anti-wrinkle and whitening effects comprising the ingredient as an active ingredient. .

Cosmetics refers to all things used for make-up, such as creams and lotions. Cosmetics prescribed by the Pharmacist Act are "articles used by painting, spraying or other similar methods to clean or beautify the human body. It is defined as, "and at the same time, products that are regulated in all aspects, including manufacturing, sales, and advertising.

Recently, as the interest on the skin increases, researches on functional cosmetics having various functions continue. Cosmetics with various functions such as cosmetics to moisturize the skin, cosmetics to reduce the size of pores, cosmetics to remove dead skin cells, cosmetics to relieve wrinkles and cosmetics with whitening function are being released. The demand for the same functional cosmetics is expected to continue to increase.

On the other hand, when processing most fish, by-products of fish processing occur about 75% of the fish body, of which fish bones and shells account for about 30%. Therefore, it is very important to develop a technology for separating new useful components such as gelatin and collagen using processed by-products generated during fish processing.

In a previous study, Kim et al. (1993) succeeded in industrializing extracting useful gelatin from tilapia, and extracting collagen from tilapia scales to produce and sell some of them for food and medicine. In addition, the production of peptides from food proteins using proteolytic enzymes is known to be applied to the production of fish skin gelatin hydrolysates and convert them into useful components having physiological activity.

However, there have been no studies on physiological activity using tilapia scales, and research on the preparation of hydrolyzate derived from tilapia scales is insufficient. In this regard, it is very useful to extract useful ingredients using tilapia scales, which are processed by-products, and to use them as materials for functional cosmetics.

The problem to be solved in the present invention is to provide a functional cosmetic composition comprising an enzymatic hydrolyzate extracted from tilapia scales.

In order to solve the above problems, the present invention provides a cosmetic composition comprising an enzymatic hydrolyzate of Tilapia mossambica scale extract.

The enzymatic hydrolyzate is dried (S1) tilapia scales in far infrared rays after washing with water; (S2) hydrothermally extracting the dried tilapia scales; (S3) enzymatic hydrolysis of the hydrothermally extracted tilapia scales; (S4) activated carbon filtration of the enzymatic hydrolyzate of the tilapia scales; (S5) filtering the activated carbon filtrate with a 0.4 to 0.5 μm filtration membrane; (S6) filtering the filtrate by the filter membrane of 0.4 ~ 0.5μm with an ion exchange resin membrane; (S7) fractionating the ion exchange resin membrane filtrate into an ultrafiltration membrane; And (S8) is preferably prepared comprising the step of lyophilization and pulverization of the fractions.

The enzymatic hydrolysis is preferably selected from the group consisting of α-chymotrypsin, alcalase, kojozyme, protamex and trypsin.

The cosmetic composition preferably exhibits antioxidant, anti-wrinkle and whitening effects.

The enzymatic hydrolyzate of tilapia scales, which is an active ingredient of the present invention, exhibits excellent effects on antioxidant activity, wrinkle improvement and whitening, and is suitable for use as a functional cosmetic composition as it is harmless to the human body as a natural material.

1 shows a tilapia scale extraction process chart.

2 is a result of measuring the degree of hydrolysis of the enzymatic hydrolyzate obtained by treating tilapia scales with various enzymes.

3 is a result of measuring the degree of hydrolysis of the enzymatic hydrolyzate obtained by treatment with various enzymes after hot water extraction of tilapia scales.

4 is a result of measuring the degree of hydrolysis of tilapia scale α-chymotrypsin hydrolyzate according to substrate / enzyme concentration.

5 is a result of measuring the degree of hydrolysis of the tilapia scale α-chymotrypsin hydrolyzate according to the substrate concentration.

6 is a result of measuring the DPPH radical scavenging activity in order to confirm the antioxidant activity of the enzymatic hydrolyzate of tilapia scales, T1 is α-chymotrypsin enzyme hydrolyzate, P1 is <5kDa Ultrafiltration fraction, P2 represents <10 kDa ultrafiltration fraction and P3 represents <30 kDa ultrafiltration fraction.

7 is a result of measuring the H ₂ O ₂ radical scavenging activity in order to confirm the antioxidant activity of the enzymatic hydrolyzate of tilapia scales, T1 is α-chymotrypsin enzyme hydrolyzate, P1 is Ultrafiltration fraction of <5 kDa, P2 represents <10 kDa ultrafiltration fraction and P3 represents <30 kDa ultrafiltration fraction.

Figure 8 is the result of measuring the Elastinase inhibition in order to confirm the anti-wrinkle effect of enzymatic hydrolysates of tilapia scales, T1 is the α-chymotrypsin enzyme hydrolyzate, P1 is <5kDa ultrafiltration The filtration fraction, P2, represents <10 kDa ultrafiltration fraction and P3 represents <30 kDa ultrafiltration fraction.

Figure 9 is the result of measuring the tyrosinase inhibition to confirm the whitening activity of the enzymatic hydrolyzate of tilapia scales, T1 is the α-chymotrypsin enzyme hydrolyzate, P1 is <5 kDa ultrafiltration Fraction, P2 represents <10 kDa ultrafiltration fraction and P3 represents <30 kDa ultrafiltration fraction.

Figure 10a to 10h shows the manufacturing process of the essence, nourishing cream, body lotion, rinse, shampoo, body cleanser, lotion, skin comprising the cosmetic composition of the present invention, respectively.

11A to 11E are photographs showing representative steps of the manufacturing process of the mask pack including the genuine composition of the present invention.

12 and 13 show the results of analysis of changes visually evaluated in the test group with the essence of the present invention.

14 to 18 show the wrinkle parameter analysis results of the test group with the essence of the present invention.

19 to 23 show the results of the overall efficacy analysis of the test group with the essence of the present invention.

24 is a result showing the satisfaction with the usability of the test group with the essence of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The inventors of the present invention confirmed that the enzymatic hydrolyzate obtained from the tilapia scales had antioxidant activity, anti-wrinkle effect and whitening activity, and developed enzymatic hydrolyzate of tilapia scales having such activity for the use of functional cosmetic composition. It was.

Accordingly, the present invention provides a cosmetic composition comprising an enzymatic hydrolyzate of Tilapia mossambica scale extract.

Tilapia, a freshwater fish native to Southeast Africa, is said to have been farmed in Egypt for about 2,000 years. The first scientific farming was carried out in Kenya in 1924 and soon spread throughout Africa, to the East in the 1940s, and to North America in the 50s. Currently, breeding is carried out in more than 100 countries around the world, and was introduced to the Republic of Korea in May 1955 through the Inland Water Research Institute in Thailand.

The species transplanted to Korea are Oreochromis niloticus and Oreochromis mossambicus , which are collectively called tilapia. Since it is used as an alternative to red dome, it is also known as reverse dome and freshwater dome depending on the region. In Korean Walk, it is said that it is transplanted in Thailand and it is written as 'Taerae'.

In 1983, Ethelwynn Trewavas was classified into three genera, Tilapia, Sarotherodon, and Oreochromis. A total of 100 species, including subspecies, are known worldwide. Due to its high environmental resistance, fast growth and low-quality feed, aquaculture is increasing year by year.

The limit of aquaculture water temperature includes all kinds in the range of 14-45 degreeC, and the optimum water temperature range is 24-32 degreeC. In general, when they are young, they eat mainly animal food, and as they grow, their diet becomes vegetable or omnivorous. When the water temperature is maintained above 21 ℃, it keeps spawning and the male makes spawning ground at the bottom and attracts the female to spawn. The fertilized egg is hatched by the female.

The enzymatic hydrolyzate of the tilapia scales of the present invention comprises (S1) drying the tilapia scales with far infrared rays after washing with water; (S2) hydrothermally extracting the dried tilapia scales; (S3) enzymatic hydrolysis of the hydrothermally extracted tilapia scales; (S4) activated carbon filtration of the enzymatic hydrolyzate of the tilapia scales; (S5) filtering the activated carbon filtrate with a 0.4 to 0.5 μm filtration membrane; (S6) filtering the filtrate by the filter membrane of 0.4 ~ 0.5μm with an ion exchange resin membrane; (S7) fractionating the ion exchange resin membrane filtrate into an ultrafiltration membrane; And (S8) is preferably prepared comprising the step of lyophilization and pulverization of the fractions.

Tilapia scales are easily available throughout Korea, and are preferably used to obtain enzymatic hydrolysates after washing and drying with fresh water. As for the drying, it is effective to use far infrared rays.

The enzymatic hydrolyzate may be obtained by treating the tilapia scales with a proteolytic enzyme as it is, or by first extracting the tilapia scales by hydrothermal extraction and treating the hydrothermal extract with a proteolytic enzyme. In order to increase the yield of the hydrolyzate, it is preferable to perform hydrothermal extraction first. The conditions of hot water extraction are not particularly limited, but preferably 1 to 5 hours at 100 ° C, more preferably 3 hours at 100 ° C.

Proteolytic enzymes that may be used in the present invention include, for example, α-chymotrypsin, alcalase, kojizyme, protamex, trypsin, and the like. But preferably α-chymotrypsin or alcalase. Hydrolysis conditions are not particularly limited, but may be subjected to hydrolysis for 3 to 24 hours at the optimum pH and temperature of each enzyme.

The hydrolyzate is filtered using activated charcoal to remove off-flavor and peculiar color after hydrolysis. The filtration method using activated carbon may be any known one.

After activated carbon filtration, foreign matters are removed through a filtration membrane of 0.4 to 0.5 μm in diameter, and filtration using an ion exchange resin membrane is performed to completely remove heavy metals or other foreign substances that may remain in the hydrolyzate.

The hydrolyzate, which has undergone such multi-step filtration, is fractionated according to the molecular weight using an ultrafiltration membrane, and it is preferable to obtain a fraction having a molecular weight of 5kDa, 10kDa and 30kDa through the ultrafiltration membrane. The molecular weight distribution of the enzymatic hydrolyzate of tilapia scales showing antioxidant activity, wrinkle improvement and whitening activity varies from 5 kDa or less to 30 kDa. The molecular weight of the enzymatic hydrolyzate suitable for the cosmetic composition of the present invention is most preferably 5 kDa or less.

Fractions obtained through the above process is dried through a concentration process, and then stored until milled.

The cosmetic composition of the present invention may be prepared in the form of, for example, an essence, nutrition cream, body lotion, rinse, shampoo, body cleanser, lotion, skin, mask pack, and the like, but is not limited thereto. Preparation in this form may be readily performed according to various manufacturing processes known to those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

EXAMPLE

1. Material

Tilapia scales were obtained from Cheongryong Fisheries Co., Ltd. located in Seogwipo-si, Jeju-do, and used after washing with fresh water.

2. Determination of common components of tilapia scales

Moisture, crude protein, crude fat and ash of the dried tilapia scales were measured. According to the AOAC method, the moisture content was measured by 105 ° C atmospheric pressure drying method with a significant difference of 0.002 g or less as a constant amount, and the crude protein content was measured by the Micro Kjeldahl method, and the ash content was measured by the dry method.

Table 1

ingredient	Measures(%)
Crude protein	97.06 ± 0.1
moisture	2.6 ± 0.01
Ash	0.3 ± 0.01
Other	0.04 ± 0.01

As shown in Table 1, the moisture content was 2.6 ± 0.01, the ash content was 0.3 ± 0.01, the crude protein content was 97.06 ± 0.1, and the remaining components were 0.04 ± 0.01. As a result of component analysis of carp scales of freshwater fish such as tilipea, considering that crude protein of carp is 78.88g among 100g of scales, the result shows that the protein content of tilapia scales of the same freshwater fish is very high. From this, it can be seen that the high content of protein contained in tilapia scales can be usefully used in the protein raw material industry.

3. Preparation of Enzymatic Hydrolysates from Tilapia Scales

As shown in Table 2, dried tilapia scales were prepared by enzymatic hydrolysates of tilapia scales under the appropriate conditions corresponding to the enzymes by using five proteolytic enzymes in the following two methods.

TABLE 2

enzyme	pH	Temperature (℃)	origin
α-chymotrypsin	7.8	25	Bovine pancreas
Alcalase	8.0	50	Bacillus licheniformis
Kojizyme	6.0	40	Bacillus sp.
Protamex	6.0	40	A. oryzae
Trypsin	7.6	25	Procine pancreas

In the first method, tilapia scales were hydrolyzed directly using enzymes without hot water extraction. That is, 10 g of tilapia scales were mixed with 100 ml of distilled water, and then, each enzyme was added and hydrolyzed over time (3, 6, 12, 18, 24 hours) at the optimum pH and temperature for each enzyme.

In the second method, 10 g of tilapia scales were mixed with 100 ml of distilled water, followed by hot water extraction at 100 ° C. for 3 hours, and then the addition of the respective enzymes, followed by time lapse (3, 6, 12, 18, 24 hours).

The hydrolyzate prepared according to each method described above was boiled at 100 ° C. for 10 minutes to stop the enzyme reaction, and after adjusting the pH to neutral, the supernatant was separated by centrifugation (3500 rpm, 10 minutes) and vacuum filtration. The separated supernatant was lyophilized and used as a sample.

4. Determination of the degree of hydrolysis of enzymatic hydrolysates derived from tilapia scales

The degree of hydrolysis of enzymatic hydrolysates derived from tilapia scales was measured by TCA (trichloroacetic acid) method. In other words, the reaction mixture after the reaction was centrifuged (12000 rpm, 15 minutes) to take 2 ml from the supernatant, 20% TCA was added thereto in the same amount, centrifuged (3500 rpm, 10 minutes), and then a predetermined amount of the supernatant was removed. 10% TCA soluble nitrogen was measured by Lowry method, and the degree of hydrolysis was calculated from the following equation.

Degree of Hydrolysis (%) = (10% TCA Soluble Nitrogen / Total Nitrogen) × 100

When only enzymatic hydrolysis was performed without hydrothermal extraction of tilapia scales, it was confirmed that the hydrolysis degree of all five enzymatic hydrolysates was increased in a time-dependent manner from 3 hours to 12 hours. It tended to decrease (FIG. 2).

On the other hand, after hydrothermal treatment of tilapia scales, hydrolyzates prepared by enzymatic hydrolysis showed a significantly higher degree of hydrolysis than those produced by enzymes alone. In addition, when enzymatic hydrolysis was attempted after hydrothermal treatment, the degree of hydrolysis rapidly increased up to 6 hours except for α-chymotrypsin, and the degree of hydrolysis remained constant thereafter. However, hydrolyzate prepared by α-chymotrypsin showed a continuous increase in degree of hydrolysis up to 12 hours, and showed a high degree of hydrolysis of about 55% at 12 hours of hydrolysis (FIG. 3). Thus, it can be seen that for the tilapia scales, treatment with α-chymotrypsin enzyme after hydrothermal treatment was most efficiently hydrolyzed.

5. Optimal Hydrolysis Conditions of Enzymatic Hydrolysates from Tilapia Scales

To determine the optimal hydrolysis conditions of the enzymatic hydrolyzate, hydrolysis was performed according to the substrate-to-enzyme ratio and substrate concentration of the α-chymotrypsin enzymatic hydrolyzate.

Substrate-to-enzyme ratios were 10, 20, 50, 100, 200, 500 (weight / weight) and enzymatic hydrolyzate concentrations of the substrate, tilapia scales, were 1%, 3%, 5%, 10%, and 20%.

The hydrolysis was carried out in the same manner as the method presented above, and the degree of hydrolysis of the hydrolyzate was measured for the examination of all hydrolysis conditions.

As a result of measuring the degree of hydrolysis with the α-chymotrypsin hydrolyzate at 12 hours as described above, as shown in FIG. 4, as the ratio of the substrate to the enzyme of tilapia scales increased, that is, the concentration of the enzyme decreased. As the degree of hydrolysis decreased. In addition, as the ratio of tilapia scales to enzymes increased from 10 to 200, the degree of hydrolysis rapidly decreased from about 65% to about 45%, and from 200 to 500, the degree of hydrolysis was almost similar. From these results, it can be seen that when the substrate-to-enzyme ratio of 100 shows economic efficiency on the degree of hydrolysis.

Next, as a result of measuring the α-chymotrypsin hydrolyzate of the tilapia scales at the substrate concentration at 100 (wt / wt), as shown in FIG. 5, the lower the concentration of the substrate, the higher the degree of hydrolysis was confirmed. .

6. Filtration of Enzymatic Hydrolysates from Tilapia Scales

The α-chymotrypsin hydrolyzate of the tilapia scales was first filtered through an activated carbon filtration membrane, and then secondarily filtered through a 0.45 μm diameter filtration membrane. Next, the filtrate was filtered through an ion exchange resin membrane in 3rd order to obtain a final filtrate.

7. Preparation of molecular weight fractions from enzymatic hydrolysates derived from tilapia scales

In order to prepare molecular weight fractions, the filtrate of tilapia enzymatic hydrolyzate treated with α-chymotrypsin was 5 kDa or less, 10 kDa or less, using an ultrafiltration membrane using 5 kDa, 10 kDa, 30 kDa membranes. Each fraction below 30 kDa was prepared.

8. Determination of Antioxidant Activity

DPPH is a stable model of free radicals. It can be seen that DPPH reduction during the reaction proceeds to scavenging free radicals and predicts the inhibition of the initial reaction of lipid peroxidation. Active oxygen, called harmful oxygen, is known to attack unsaturated fatty acids, which are components of cell membranes, to induce lipid peroxidation reactions and to accumulate lipid peroxides in the body, resulting in deterioration of biological function and aging and adult diseases. Antioxidant activity by plant components and extracts has been reported. In addition, H ₂ O ₂ is also a representative active oxygen can be measured the degree of antioxidant reaction to the extent of reduction of H ₂ O ₂ .

As shown in Figures 6 and 7, the tilapia scale enzymatic hydrolyzate and each ultrafiltration fraction of the present invention can be confirmed that the excellent DPPH radical scavenging function.

9. Wrinkle improvement effect

In the dermal tissue of the skin, collagen and elastin form the network structure and maintain the elasticity of the skin.The elasticity and the luster decrease due to internal and external stress such as age and ultraviolet rays, and the elastin mesh is caused by the overexpressed elastinase. If the structure is broken, the skin sags and wrinkles are generated, resulting in skin aging. Therefore, skin aging can be suppressed by inhibiting the activity of elastinase, which is an enzyme of elastin, one of the main causes of skin aging.

In order to test the antiwrinkle effect of tilapia scale gelatin enzyme hydrolyzate and four ultrafiltration fractions, the inhibitory activity of elastinase was measured using Cannell et al. Elastinase inhibitory activity was measured by elastinase substrate VIII as a substrate for 15 minutes at room temperature to determine the amount of p-nitoanilide produced. 160 μl of each test solution diluted with 0.2 M tris-HCl (pH 8.0) buffer, 20 μl of 5 mM elastinase substrate VIII (CALBIOCHEM) solution and 20 μl of 10 μl / ml elastinase (sigma) enzyme were added in this order and reacted at 25 ° C. for 15 minutes. After absorbance was measured at 410nm using an ELISA reader (TECAN, AT / sunrise R / C, Switzerland).

As shown in Figure 8, the tilapia scale enzymatic hydrolyzate and each ultrafiltration fraction of the present invention can be seen to exhibit excellent elastinase inhibitory activity.

10. Whitening effect

In order to test the whitening effect of tilapia scale alkalase hydrolysates and ultrafiltration fractions, the inhibitory activity of tyrosinase was measured using the method of Fuller. Add 220 μl of 0.1M phosphate buffer (pH 6.5), 20 μl of sample solution, and 20 μl of tyrosinase (sigma) solution (1500 U / ml ~ 2000 U / ml). 40μl of 1.5mM trysine (sigma) solution was added to the solution and reacted at 37 ° C. for 10-15 minutes, and the absorbance was measured at 490 nm using an ELISA reader (TECAN, AT / sunrise R / C, Switzerland). 0.1 M phosphate buffer (pH 6.5) was used instead of the sample solution as the blank sample solution.

Melanin synthesis is based on tyrosine, one of the amino acids, and the reaction after 3,4-dihydroxyphenylalanin (DOPA) and DOPA quinone by tyrosinase is largely pheomelanin, which determines reddish brown or yellow color, and eumelanin, which determines brown to brown color. It is divided into, and whitening effect can be expected by inhibiting melanin production by inhibiting the activity of tyrosinase, a major enzyme of melanin production.

In this experiment, the tyrosinase inhibitory activity of the gelatin hydrolyzate derived from tilapia scales and four ultrafiltration fractions obtained therefrom was measured. As shown in Figure 9, the tilapia scale enzymatic hydrolyzate and each ultrafiltration fraction of the present invention can be seen to exhibit excellent tyrosinase inhibitory activity.

11. Toxicity of Ultrafiltration Fractions (Protein Peptides) from Tilapia Scale Hydrolysates

Toxicity testing of tilapia scale protein peptides was commissioned by a Biotoxtec specialized institution. Detailed results are as follows.

11-1. Single Oral Dose Toxicity Test Using a Red Tilapia Scale Enzyme Hydrolyzate

OBJECTIVES: To investigate the toxic response of a single oral dose of Tilapia scale enzyme hydrolyzate as a test substance in male and female Sprague-Dawley rats.

Summary and Conclusion: The group consisted of 2,000 mg / kg of test substance and 2 groups of control group (injection water), and five male and female mice were orally administered once. For 14 days after administration, general symptoms and body weights were observed, and euthanized by euthanasia at the end of the observation period. No deaths were observed in the 2,000 mg / kg male and female groups. In addition, the effects of test substance administration on general symptoms, body weight and necropsy were not recognized. As a result of a single oral administration of tilapia scale enzyme hydrolyzate to rats under the conditions of this test, the approximate lethal dose is estimated to be greater than 2,000 mg / kg of male and female.

11-2. Skin irritation test using rabbit of Tilapia scale enzyme hydrolyzate

Purpose: To test the test substance tilapia scale enzyme hydrolyzate once in the skin of rabbits and to evaluate the presence and extent of skin irritation.

SUMMARY AND CONCLUSIONS: The skin irritation test of test substance tilapia scale enzyme hydrolyzate was examined using three 16-week-old male NZW rabbits. The administration sites of the left and right each 2 sites and the sum 4 sites were set to the back of the rabbit, and 2 of them were irradiated and the other 2 were used as abrasion sites. 0.5 g of the test substance raw material was applied to each non-abrasive and abrasion site for 24 hours to blockage patch. The skin response was assessed according to Draize's Skin Response Evaluation Table at 24, 48 and 72 hours after dosing, and the primary skin irritation index (PII) was determined for 24 and 72 hours after dosing to determine skin irritation. . Skin reactions such as erythema and edema were not observed in all animals at 24, 48 and 72 hours post-administration at non-treated and untreated control sites. The primary skin irritation index (P.I.I.) of the test substance was zero. During the observation period, no abnormalities were observed for general symptoms and body weight. From the above results, it is judged that the test substance tilapia scale enzyme hydrolyzate is not irritating to the skin of the rabbit under the present test conditions.

11-3. Ocular Membrane Stimulation Test Using Rabbits of Tilapia Scale Enzyme Hydrolysates

Purpose: To test the test substance tilapia scale enzyme hydrolyzate once in rabbit ocular mucosa and to evaluate the presence and degree of eye irritation.

SUMMARY AND CONCLUSIONS: Eye irritation testing of test substance tilapia scale enzyme hydrolyzate was reviewed using six 16-week-old male NZW rabbits. In the non-eye group, 0.1 g of test substance was injected into three rabbits' right eye conjunctival sac, and eye lesions such as cornea, iris and conjunctiva were observed at 1, 24, 48 and 72 hours after administration. The face wash group was additionally administered in the same manner as the non-wash face group using three animals, and after washing for 30 seconds after administration, the face wash effect was confirmed. The eye irritation of the test substance was evaluated according to the grade of the eye lesion of Draize, and the degree of eye irritation was classified by referring to Guillot's eye irritation evaluation table. In the non-eye group, conjunctival redness (score 1) and conjunctival edema (score 1) were observed 1 hour after administration of the test substance, but all disappeared at 48 hours after administration. I.A.O.I. The Index of Acute Ocular Irritation was 4.0 and the final assessment was classified as non-irritant. In the face-wash group, conjunctival redness (score 1) and conjunctival edema (score 1) were observed 1 hour after administration of the test substance, but all disappeared 24 hours after administration. The I.A.O.I. of the cleansing group was 4.0, and there was no difference in the stimulus degree compared to the non-cleansing group, but the cleansing effect was confirmed because there was a difference in the disappearance time of eye irritation. During the observation period, no abnormalities were observed for general symptoms and body weight of each group. From the above results, the test substance tilapia scale enzyme hydrolyzate under this test condition is judged to be non-irritant to the ocular mucosa of rabbits.

11-4. Skin sensitization test using guinea pigs of Tilapia scale enzyme hydrolyzate (Maximization method)

Objective: As part of the safety test of the test substance tilapia scale enzyme hydrolyzate, a skin sensitization test (Maximization method) using guinea pigs was conducted to examine the possibility of allergic to human skin.

SUMMARY AND CONCLUSIONS: The skin sensitization test of test substance tilapia scale enzyme hydrolyzate was reviewed by the Maximization method using Hartley guinea pig. The test group consisted of three groups: test substance group (5), negative control group (5) and positive control group (5). In addition, 10% sodium dodecyl sulfate (SDS) was applied to the sensitized area of each group except the positive control group 24 hours before the second sensitization because no skin reactions such as erythema and edema were observed at the percutaneous administration site of the preliminary test. The test substance group was firstly sensitized by intradermal administration of 50% test substance, and secondly sensitized by 100% test substance for 48 hours. As a result of 24-hour obstruction with 100% test substance and water for injection, skin reactions such as erythema and edema were not observed in all animals 24 and 48 hours after removal of the induced patch. Negative controls were primary and secondary sensitized with water for injection, and as a result of 100% test substance and water for injection, skin reactions such as erythema and edema were not observed in all animals 24 and 48 hours after the removal of the induced patch. Positive controls were first and second sensitization with 0.1 and 1.0% 1-Chloro-2,4-dinitrobenzene (CDNB), respectively, and caused by 0.1% CDNB and olive oil, resulting in 0.1% at 24 and 48 hours after removal of the Yagi patch. Grade 3 strong erythema and edema in the causal region of CDNB were observed in all animals. Skin reactions such as erythema and edema were not observed in all animals. During the observation period, no abnormalities were observed for the general symptoms and body weight of all animals in each group. From the above results, the test substance tilapia scale enzyme hydrolyzate under this test condition is judged to be a substance without skin sensitization.

11-5. Phototoxicity Test Using Guinea Pigs of Tilapia Scale Enzyme Hydrolysate

Objective: As part of the safety test of the test substance tilapia scale enzyme hydrolyzate, the possibility of phototoxicity was examined using skin of guinea pigs.

SUMMARY AND CONCLUSIONS: The presence and extent of phototoxicity of the test substance tilapia scale enzyme hydrolyzate to the skin were examined using Hartley guinea pigs. The test group consisted of three groups: test substance group (5), negative control group (5) and positive control group (5). 22 cm of administration sites were set on the right and left sides of the guinea-pig skin of the test substance group, the negative control group, and the positive control group. 90% test substance was applied to the test substance group, water for injection to the negative control group, and 0.1% 8-Methoxypsoralen (8-MOP) to the positive control group was applied to each administration site. About 30 minutes after application, UV-A was irradiated so that the final energy was about 10 J / cm 2 with the light irradiation site on the left side and the non-light irradiation site on the right side. Skin reactions were evaluated 24, 48 and 72 hours after light irradiation, and the presence or absence of phototoxicity was determined. Skin reactions such as erythema and edema were not observed in all animals at 24, 48 and 72 hours after irradiation at 90% of the test substance dose. At all observation times at the injection site of the negative control group, no skin reactions such as erythema and edema were observed in all animals. At the time of observation, 0.1% 8-MOP of the positive control group showed erythema (score 3) and edema (score 4) at all the irradiation sites, but skin reactions such as erythema and swelling at the non-irradiation site were observed. Was not observed in all animals. During the observation period, no abnormalities were identified for general symptoms and body weight of each group. From the above results, the test substance tilapia scale enzyme hydrolyzate is judged to be non-phototoxic under the present test conditions.

11-6. Photosensitization Test Using Guinea Pigs of Tilapia Scale Enzyme Hydrolysates (Adjuvant & Strip Method)

Purpose: To test the safety of the test substance tilapia scale enzyme hydrolyzate on the skin. Adjuvant & Strip method using guinea pigs was performed to investigate the possibility of photosensitization.

Summary and Conclusions: Photosensitization of Tilapia scale enzyme hydrolysates was examined by the Adjuvant & Strip method using Hartley-based male guinea pigs. The group consisted of three groups: test substance group (5), negative control group (5) and positive control group (5). On day 0, the water-FCA emulsion was injected into the four corners of the sensitized area, and then the sensitized area was stripped. 0.1% of 90% test substance, water for injection, and 1.0% chlorpromazine (CP) were applied to the sensitized area of the test substance group, the negative control group and the positive control group, respectively, and then UV-A was irradiated. Stripping, open coating of the administration material, and UV-A irradiation were performed once a day for 5 consecutive days. Yagi was caused by UV-A irradiation on day 21 after administration of 90% test substance and water for injection in test substance and negative control group and 0.1% CP and ethanol in positive control group. Skin reactions were evaluated 24 and 48 hours after firing light irradiation. In the test substance group and the negative control group, skin reactions such as erythema and edema were not observed in all animals at 24 and 48 hours after the irradiated light irrespective of the irradiated area of 90% test substance and water for injection. In the positive control group, erythema of grade 3 and edema of grade 3 were observed in all animals at the irradiation site of 0.1% CP. Skin reactions such as erythema and edema were not observed in all animals. In ethanol-causing areas, skin reactions such as erythema and edema were not observed in all animals with or without light irradiation. During the observation period, no abnormalities were identified for general symptoms and body weight of each group. From the above results, the test substance tilapia scale enzyme hydrolyzate is judged to have no photosensitization under these test conditions.

12. Statistical Processing

Statistical processing of the experimental and analytical results of the present embodiment was carried out using a one-way ANOVA-test using the SPSS program (SPSS Inc., Version 12.0), and the significance between the means by Duncan's multiple range test (Duncan, 1955, P <0.05 ). Was assayed.

13. Cosmetic Formulation

Cosmetics in the form of essences, nourishing creams, body lotions, rinses, shampoos, body cleansers, lotions, skins and mask packs were prepared comprising the tilapia scale hydrolyzate and ultrafiltration fractions of the present invention. Specific manufacturing processes are shown in FIGS. 10A to 10H and 11A to 11E.

14. Human Body Application Test

14-1. Test purpose

The purpose of this study is to evaluate the wrinkle-improving effect and skin safety of the product by using the test product for 12 weeks in women who are 30 years old or older or who have already developed wrinkles.

14-2. Trial product

14-2-1. Test group and control group

Test group (A): containing 1% of tilapia protein peptide

Control group (B): without tilapia protein peptide

14-2-2. Characteristics of the product

essence

14-2-3. Storage of the product

Room temperature storage

14-2-4. Active ingredient of the product

Tilapia Protein Peptide 1%

14-2-5. How to use the product

Twice a day (morning and evening) for 12 weeks, after washing the skin, the product A and B were pumped twice in the next step, divided into cheeks, forehead, and chin, and gently absorbed. Subjects were divided into two groups through block randomization, and group A used the test group (product A) on the left side, and the control group (product B) on the right side, and group B used the control group (product B) on the left side, and the test group ( Product A) was used.

14-3. Test Methods

14-3-1. Subject Selection

The study was conducted on 20 female subjects who met the criteria for screening subjects over 30 years and did not meet the exclusion criteria.

For skin wrinkles, wrinkles begin to be produced in accordance with SMA of the Dermatological Institute of Dermatology, or those who have indicated their intention to explain the purpose and method, expected efficacy and adverse reactions of the test should be filled out. Participated in the test.

14-3-2. Assessment Methods

Evaluation was performed by visual evaluation of crow's feet before and after 4, 8, and 12 weeks of product use and measurement of skin wrinkle parameters (R-value) using Skin Visiometer® SV600 (C + K, Germany), VISIA®. (Canfield, USA) was used to evaluate skin wrinkle improvement and skin safety by taking photographs, evaluating subjects, and investigator observation and questionnaires.

14-4. Statistical analysis

Visual and instrumental evaluation data were tested for significance using the SPSS® Package Program (IBM, USA). Repeated Measures ANOVA was applied to take into account the interdependence (interaction) present in repeated measurements from the same subject.

The comparison between groups at each time point was confirmed by correcting the difference between two groups before using the product using covariance analysis (ANCOVA), and the before and after comparison was confirmed using RM ANOVA.

In the analysis of survey evaluation, the Mann-Whitney U-test was used to compare the nonparametric mean values of the two groups, and the Chi-Square test was used for the comprehensive evaluation. Statistical significance level was set to p value less than 0.05.

14-5. Test result

14-5-1. Subject Skin Characteristics

In this study, a total of 24 female subjects (average 45.6 ± 4.2 years old) over 30 years of age according to the exclusion criteria and selection criteria were conducted in full. The skin characteristics of the subject were examined by a questionnaire, and the analysis results are as follows.

TABLE 3

Table 4

Table 5

14-5-2. Assessment Methods

14-5-2-1. Visual evaluation

The evaluation was performed in 10 stages (Grade 0 ~ 9). Two testers independently evaluated the wrinkled skin condition at the tail of the eye before and after 4, 8, and 12 weeks of use (Tables 6 and 7). RM ANOVA was used to verify whether there was an interaction between 'group' and 'weekly change' (Table 8, Figures 14, 15).

Table 6

TABLE 7

Table 8

Differences between groups at each time point were analyzed using ANCOVA. Changes before and after each time point were confirmed by RM ANOVA for statistical significance.

(1) Comparison between groups by each time point

As a result of the analysis, there was no significant difference between the test group and the control group at all evaluation points after product use (Table 9).

Table 9

(2) Comparison of changes before and after each time point

As a result of the analysis, skin wrinkles of the test group tended to decrease slightly after 12 weeks of use (Table 10).

Table 10

14-5-2-2. Wrinkle parameter (R-value) analysis

Skin wrinkles were evaluated by measuring the wrinkle parameters R-values (R1-R5) of the test and control groups using the Skin Visiometer® SV600 at 4, 8, and 12 weeks before and after the use of the product (Table 11).

Table 11

In order to verify that there is an interaction between 'group' and 'weekly change', it was analyzed using RM ANOVA (Table 12, FIGS. 16 to 20).

Table 12

In all five wrinkle parameters (R1 ~ R5), the interaction between Group * Week (group * weekly change) was not significant, but there was a significant difference by time (WEEK).

Differences between groups at each time point were analyzed using ANCOVA. Changes before and after each time point were confirmed by RM ANOVA for statistical significance.

(1) Comparison between groups by each time point

As a result, at 12 weeks after the product use, the R1 of the parameters was significantly decreased in the test group compared to the control group (p <0.05), and there was no significant difference between the groups in the remaining parameters (R2, R3, R4, R5) (Table 13, 16-20).

Table 13

(2) Comparison of changes before and after each time point

As a result, in the test group, R2 was significantly decreased at 4 weeks and 12 weeks after using the product, and R1, R3, R4 and R5 were significantly decreased at 12 weeks after using the product. Significant decrease was found after 12 weeks of product use (p <0.05, Table 14, FIGS. 16-20).

Table 14

14-5-2-3. Survey analysis on efficacy and usability

At each time point after the use of the product, the questionnaire about the efficacy of the subjects and the evaluation on the comprehensive product usability questionnaire were combined.

(1) Evaluation of questionnaire about efficacy

As a result of the evaluation, 79 to 88% of the subjects in the test group and the control group responded positively at the time of 'moist' and 'soft'. There was no significant difference between groups in all items (Tables 15-19, Figures 21-25).

Table 15

Table 16

Table 17

Table 18

Table 19

(2) Survey evaluation about usability

As a result of the evaluation, 58 to 88% of the subjects in the test group and the control group responded positively in the items of 'color', 'flavor', 'absorbency' and 'satisfaction' (Table 20, FIG. 26).

Table 20

14-5-3. Skin safety assessment

No skin adverse events were observed in all subjects during this study (Table 21).

Table 21

Claims (4)

1. Cosmetic composition comprising an enzymatic hydrolyzate of Tilapia mossambica scale extract.
2. The method of claim 1,

   The enzymatic hydrolyzate is a cosmetic composition comprising the following steps:

   (S1) drying the tilapia scales in far infrared rays after washing with water;

   (S2) hydrothermally extracting the dried tilapia scales;

   (S3) enzymatic hydrolysis of the hydrothermally extracted tilapia scales;

   (S4) activated carbon filtration of the enzymatic hydrolyzate of the tilapia scales;

   (S5) filtering the activated carbon filtrate with a 0.4 to 0.5 μm filtration membrane;

   (S6) filtering the filtrate by the filter membrane of 0.4 ~ 0.5μm with an ion exchange resin membrane;

   (S7) fractionating the ion exchange resin membrane filtrate into an ultrafiltration membrane; And

   (S8) lyophilization and milling of the fractions after concentration.
3. The method of claim 2,

   The enzymatic hydrolysis is characterized in that selected from the group consisting of α-chymotrypsin (α-chymotrypsin), alcalase (alcalase), Kojozyme, Protamex (Trytamin) and trypsin (Trypsin) Cosmetic composition.
4. The method according to any one of claims 1 to 3,

   The cosmetic composition is characterized in that the antioxidant, anti-wrinkle and whitening effect cosmetic composition.

Images