WO2014178500A1

WO2014178500A1 - Functional cosmetic composition comprising enzymatic hydrolysate of branchiostegus japonicus scales

Info

Publication number: WO2014178500A1
Application number: PCT/KR2013/009997
Authority: WO
Inventors: 문영섭; 안용석; 문창훈; 고창익
Original assignee: (주)청룡수산
Priority date: 2013-05-01
Filing date: 2013-11-06
Publication date: 2014-11-06
Also published as: KR20140130596A; KR101551412B1

Abstract

The present invention relates to a cosmetic composition comprising an enzymatic hydrolysate of Branchiostegus japonicus 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 Branchiostegus japonicus 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 jade fish scales

The present invention relates to a cosmetic composition comprising an enzymatic hydrolyzate of jade ( braniostegus japonicus ) scales, and more particularly to a functional cosmetic composition having an antioxidant, anti-wrinkle and whitening effect containing 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.

However, there have been no studies on physiological activity using jade dome scales, and research on the preparation of hydrolysates derived from jade dome scales is insufficient. In this regard, it is very useful to extract useful components using the processed by-product jade dome scales and to use them as materials of functional cosmetics.

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

In order to solve the above problems, the present invention provides a cosmetic composition comprising the enzymatic hydrolyzate of jade ( braniostegus japonicus) scale extract.

The enzymatic hydrolyzate is dried (S1) jade dome scales after washing with far infrared rays; (S2) hydrothermal extraction of the dried jade fish scales; (S3) enzymatic hydrolysis of the hydrothermally extracted jade fish scales; (S4) activated carbon filtration of the enzymatic hydrolyzate of the jade fish 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, neutrase and trypsin.

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

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

Figure 1 shows a flow chart of jade fish scales extraction.

Figure 2 is a result of measuring the degree of hydrolysis of the enzymatic gelatin hydrolyzate obtained by using various enzymes after hydrothermal treatment of jade fish scales.

Figure 3 is the result of measuring the degree of hydrolysis of jade dome scale alkalase hydrolyzate by substrate / enzyme treatment concentration.

Figure 4 is the result of measuring the degree of hydrolysis of jade dome scale alkalase hydrolyzate by substrate concentration.

Figure 5 shows the results of the LC-MS analysis of the result of ultrafiltration of the jade dome scale alkalase hydrolyzate.

6A to 6D show the molecular weights of the four peaks on the LC-MS analysis of the result of ultrafiltration of the jade dome scale alcalase hydrolyzate.

7 is a result of measuring the DPPH radical scavenging activity to confirm the antioxidant activity of the enzymatic hydrolyzate of the jade dome scales, E1 is an alkalase enzyme hydrolyzate of the jade dome scales, A is an ultrafiltration fraction having a molecular weight of 260, B is Ultrafiltration fraction with a molecular weight of 189, C represents an ultrafiltration fraction with a molecular weight of 223 and D represents an ultrafiltration fraction with a molecular weight of 288.

8 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 the jade dome scales, E1 is an alkalase enzyme hydrolyzate of the jade dome scales, A is an ultrafiltration fraction having a molecular weight of 260 , B denotes an ultrafiltration fraction having a molecular weight of 189, C denotes an ultrafiltration fraction having a molecular weight of 223, and D denotes an ultrafiltration fraction having a molecular weight of 288.

Figure 9 is the result of measuring the Elastinase inhibition in order to confirm the wrinkle improvement effect of the enzymatic hydrolyzate of jade dome scales, E1 is an alkalase enzyme hydrolyzate of jade dome scales, A is an ultrafiltration fraction of molecular weight 260, B is the molecular weight 189 ultrafiltration fraction, C represents an ultrafiltration fraction with a molecular weight of 223 and D represents an ultrafiltration fraction with a molecular weight of 288.

Figure 10 is the result of measuring the tyrosinase inhibition in order to confirm the whitening activity of the enzymatic hydrolyzate of jade dome scales, E1 is an alkalase enzyme hydrolyzate of jade dome scales, A is an ultrafiltration fraction having a molecular weight of 260, B is 189 molecular weight Phosphorus ultrafiltration fraction, C denotes ultrafiltration fraction with molecular weight 223 and D denotes ultrafiltration fraction with molecular weight 288.

11 is a result of measuring the inhibitory activity of MMP-1 (Collagenase-1) of the enzymatic hydrolyzate of jade fish scales.

12a to 12h respectively show the manufacturing process of the essence, nourishing cream, body lotion, rinse, shampoo, body cleanser, lotion, skin comprising the cosmetic composition of the present invention.

13A to 13E are photographs showing representative steps of the manufacturing process of the mask pack including the cosmetic composition of the present invention.

14 and 15 show the results of analysis of the changes visually evaluated by the test group with the essence of the present invention.

16 to 20 show the wrinkle parameter analysis results of the test group with the essence of the present invention.

21 to 25 show the results of the overall efficacy analysis of the test group with the essence of the present invention.

26 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 jade dome scales has antioxidant activity, anti-wrinkle effect and whitening activity, and developed the enzymatic hydrolysates of the jade dome 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 jadedox ( Braniostegus japonicus ) scale extract.

It is called 'red horsehead' in Europe because it resembles a horse's head. It is also called 'japones tilefish' because it is always beautifully tiled. In Japan, it is called `` Akaamadai '' which means sweet fish.

Its maximum length is 45cm and its weight is 1.25kg, and the front of the head is very inclined so that it is almost vertical and resembles a horse's head. The body height is the highest at the very back of the head, tapered toward the back, the contour of the back is almost straight, and the lateral line passes above the center of the side of the body and is almost parallel to the contour of the back. The body is covered with relatively large rectangular comb scales, and the body is generally reddish, with two or three horizontal stripes appearing just above the tip of the pectoral fin. Triangular silver-white spots are clearly visible on the back of the eye, and caudal fin has 5-6 rows of clear yellow vertical stripes.

The bottom of 30 ~ 200m depth lives on the edge of the continental shelf of sand and mud, and there is a habit of digging holes in sand or sand bottom and living in it. It does not move much but moves north in autumn and south in spring and eats crustaceans such as shrimp, crabs and crayfish, as well as worms, fish and shellfish. The spawning season is from June to October, and eggs are laid in the seabed at the depth of 70-100m before and after the water temperature of 18 ℃, and the life span is 8-9 years.

Since it lives on the bottom of the sea, it is possible to attach several fishing lines to the fishing line so that it can sink to the bottom of the sea, hang a cover with a flag and float it in order to catch the fish and then remove the fish. It is also possible to catch the net by dragging the net with a fishing boat. As a fish species in November, it is treated as a high-quality fish. As a fish product of Jeju Island, dried and frozen products are common. The flesh is very white and tastes good. It is cooked with seasoning grilled, grilled with salt, seaweed soup and fish porridge.

Enzymatic hydrolyzate of the jade dome scales of the present invention comprises the steps of (S1) drying the jade dome scales in far infrared rays after washing with water; (S2) hydrothermal extraction of the dried jade fish scales; (S3) enzymatic hydrolysis of the hydrothermally extracted jade fish scales; (S4) activated carbon filtration of the enzymatic hydrolyzate of the jade fish 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.

Jade dome scales can be easily obtained throughout the Republic of Korea, it is preferable to use to obtain enzymatic hydrolyzate 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 domido scales with proteolytic enzymes as it is, or by first extracting the domido scales from hydrothermal and then treating the hydrothermal extract with proteolytic enzymes. 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 can be used in the present invention include, for example, α-chymotrypsin, alcalase, neutrase, 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 the jade dome 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.

[실시예]EXAMPLE

1. 재료1. Material

In order to obtain a gelatin hydrolyzate derived from jade dome scales, the jade dome scales used in the present invention were obtained from Cheongryong Fisheries Co., Ltd. located in Seogwipo-si, Jeju-do, and dried under far-infrared light after washing with fresh water.

2. 옥돔 비늘의 일반 성분 측정2. Measurement of common components of jade fish scales

Moisture, crude protein, crude fat and ash of dried jade fish scales were measured. According to the AOAC method, the moisture content was measured by 105 ℃ atmospheric pressure drying method with a significant difference of 0.002g or less, the crude protein content was measured by Micro Kjeldahl method, the ash content was measured by dry method, and the crude fat content was determined by Soxhlet extraction method. Measured.

Table 1

ingredient	Measures(%)
Crude protein	46.1 ± 0.02
moisture	8.5 ± 0.01
Ash	45.0 ± 1
Other	0.4 ± 0.001

As shown in Table 1, the water content was 8.5 ± 0.01, the ash content was 45.0 ± 1, the crude protein content was 46.1 ± 0.02, and the remaining components were 0.4 ± 0.001. Hamanda and Kumagai (1988) analyzed the scales of sardines, a marine fish species, and reported that their contents were similar, with 53.16g of 100g heavy ash and 41g of collagen protein. These results do not show a big difference from the scale of the dome of the present invention, it was confirmed that the scale of the dome is higher in the protein content than other components can be a useful raw material for gelatin extraction.

3. 젤라틴의 제조3. Preparation of Gelatin

In order to remove foreign substances in the jade dome scales, the non-collagen protein was removed by alkali treatment at 10 ° C. for 3 days using 1 N NaOH alkaline solution in 8 times jade dome scales. After alkaline treatment, water was washed with running water and neutralized with 6N HCl. Six times (v / w) of distilled water was added to the neutralized jade dome scales, and hot water was extracted three times at 60 ° C. for three hours. The hot water extracted solution was filtered under reduced pressure using a filter paper (5A 110mm, advantec, Japan), and the filtered gelatin extract solution was concentrated at 60 ° C using a vacuum concentrator, and then lyophilized to be pulverized into powder and used as a sample. .

4. 옥돔 비늘 유래 젤라틴의 효소적 가수분해물의 제조4. Preparation of Enzymatic Hydrolysates of Gelatin Derived from Jade Dome Scales

The dried and powdered oxime scale-derived gelatin was hydrolyzed by time using four proteolytic enzymes shown in Table 2 below. The hydrolyzate was further centrifuged (12000 rpm, 15 minutes) to separate the supernatant and the degree of hydrolysis was measured.

TABLE 2

enzyme	pH	Temperature (℃)	origin
α-chymotrypsin	7.8	25	Bovine pancreas
Alcalase	8.0	50	Bacillus licheniformis
Neutrase	6.0	50	Bacillus amyloliquefaciens
Trypsin	7.6	25	Procine pancreas

5. 옥돔 비늘 유래 젤라틴의 효소적 가수분해물의 가수분해도 측정5. Determination of the degree of hydrolysis of enzymatic hydrolysates of gelatin derived from jade fish scales

The degree of hydrolysis of the enzymatic hydrolyzate of jade-derived gelatin 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

As shown in FIG. 2, the hydrolysis degree of the hydrolyzate of each time zone extracted using four proteolytic enzymes showed a sharp increase in hydrolysis degree up to 6 hours of hydrolysis reaction in all four enzymes. Was almost constant. In addition, among the four hydrolysates, alkalase hydrolyzed the oxime scale gelatin most effectively, and showed a degree of hydrolysis of about 65% or more after 6 hours of the hydrolysis reaction.

6. 옥돔 비늘 유래 효소적 젤라틴 가수분해물의 최적 가수분해 조건6. Optimal Hydrolysis Conditions of Enzymatic Gelatin Hydrolysates Derived from Jade Dome Scales

In order to determine the optimal hydrolysis conditions of the alkalase hydrolyzate, hydrolysis was performed according to the substrate-to-enzyme ratio and substrate concentration of the enzymatic hydrolyzate.

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

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 shown in FIG. 3, the degree of hydrolysis was increased as the ratio of oxime scale-derived gelatin to enzyme decreased. In addition, as the ratio of gelatin to enzyme increases from 10 to 100, the degree of hydrolysis is over 60%, and from 200 it can be seen that the degree of hydrolysis decreases rapidly. From this result, it can be seen that 100 of the various substrate-to-enzyme ratios show the best efficiency for the degree of hydrolysis.

In the case of the substrate-to-enzyme ratio 100, the results of measuring the degree of hydrolysis according to the substrate concentration are shown in FIG. 4. The lower the concentration of the substrate was, the higher the degree of hydrolysis was. However, when the substrate concentration was 5% or less, the degree of hydrolysis was almost similar. In terms of mass production, a substrate concentration of 5% is considered to be the most appropriate.

7. 옥돔 비늘 유래 효소적 가수분해물의 여과7. Filtration of Enzymatic Hydrolysates from Odomes Scales

The alkalase hydrolyzate of the jade dome scale 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.

8. 옥돔 비늘 유래 효소적 가수분해물로부터 분자량별 분획물의 제조8. Preparation of Molecular Weight Fractions from Enzymatic Hydrolysates Derived from Odomes

In order to prepare molecular weight fractions, the filtrate of the hydrochloric acid-treated dome enzymatic hydrolyzate was 5kDa, 10kDa or less, 30kDa using an ultrafiltration membrane using 5kDa, 10kDa and 30kDa membranes. The following fractions were each prepared.

As a result of analyzing by LC-MS for fractions of 5 kDa or less, four peaks were obtained as shown in FIG. 5, and the molecular weights of the respective peaks were measured. As shown in FIGS. 6A to 6D, respectively, 260, 189, 223 and 288 were obtained. A fraction having a molecular weight of was identified.

9. 항산화 활성 측정9. 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 accumulate lipid peroxides, resulting in deterioration of biological function and at the same time causing aging and adult diseases. Antioxidant activity by plant components and extracts has been reported. In addition, H ₂ O ₂ can also measure the degree of antioxidant reaction by the degree of reduction of H ₂ O ₂ as a representative active oxygen.

As shown in Figure 7 and 8, it can be seen that the jade dome scale enzymatic hydrolyzate and each ultrafiltration fraction of the present invention has excellent DPPH radical scavenging function.

10. 주름 개선 효과 10. 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.

The inhibitory activity of elastinase was measured using Cannell et al. To examine the anti-wrinkle effect of oxime scale gelatin hydrolyzate and four ultrafiltration fractions. 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 9, it can be seen that the jade dome scale enzymatic hydrolyzate and each ultrafiltration fraction of the present invention shows excellent elastinase inhibitory activity.

11. 미백 효과11. Whitening effect

In order to examine the whitening effect of jade dome alkalase hydrolysates and ultrafiltration fractions, the inhibitory activity of tyrosinase was measured using Fuller's method. 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, tyrosinase inhibitory activity of gelatin hydrolysates derived from jade fish scales prepared using alcalase and four ultrafiltration fractions obtained therefrom was measured. As shown in Figure 10, it can be seen that the jade dome scale enzymatic hydrolyzate and each ultrafiltration fraction of the present invention shows excellent tyrosinase inhibitory activity.

12. MMP-1(Collagenase-1) 저해 활성12. MMP-1 (Collagenase-1) Inhibitory Activity

The inhibitory activity of MMP-1, which acts to degrade skin collagen, was measured to evaluate the stability of jade fish scale hydrolyzate.

After making agarose gel plate at the ratio of agarose: gelatine: buffer = 10: 2: 1, put 89μl buffer + 1μl + collagenase 10μl into 96well plate (by adjusting the amount of buffer and sample, the final concentration can be changed. ), And the reaction (prereaction) at 37 ℃ for 1 hour, and then put the paper disk on a pre-made gel plate. After 1 hour of reaction, drop 10 μl of each reaction onto the paper disk. After 12 to 24 hours of reaction, the paper disk was removed and stained with collagen staining solution for a certain time. After destaining with de-staining solution, the effect of the sample was confirmed to the extent of the ring size and discoloration, and the results are shown in FIG. 11.

As shown in FIG. 11, when all of the domido scale hydrolyzate was compared with the control, it was confirmed that there was almost no difference in the size of the ring that was decolorized (collagen was decomposed). Little inhibitory activity was confirmed.

13. 옥돔 비늘 가수분해물의 한외 여과 분획물(단백질펩타이드)의 독성검사13. Toxicity of Ultrafiltration Fractions (Protein Peptides) from Jade Dome Scale Hydrolysates

Toxic scale protein peptide toxicity test was commissioned by Biotoxtech specialized institution, detailed results are as follows.

13-1. 옥돔 비늘 효소가수분해물의 렛드를 이용한 단회 경구투여 독성시험13-1. Single Oral Dose Toxicity Test Using Reddish of Dome Scale Enzyme Hydrolysates

OBJECTIVES: To investigate the toxic response of oral administration of oxydome hydrolyzate, a test substance, in male and female Sprague-Dawley rats, and to obtain an approximate lethal dose.

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. Under the conditions of this test, a single oral dose of oxime scale hydrolyzate to rats is estimated to exceed approximately 2,000 mg / kg of male and female.

13-2. 옥돔 비늘 효소가수분해물의 토끼를 이용한 피부자극시험13-2. Skin irritation test using rabbit of jadefish scale enzyme hydrolyzate

Purpose: To evaluate the presence and extent of skin irritation after treatment of the test substance jade fish scale hydrolyzate with rabbit skin once.

SUMMARY AND CONCLUSIONS: The skin irritation test of test substance jade fish 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 after administration of the test substance 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 jade fish scale hydrolyzate under the present conditions is not irritating to the skin of rabbits.

13-3. 옥돔 비늘 효소가수분해물의 토끼를 이용한 안점막자극시험13-3. Ocular Membrane Stimulation Test Using Rabbits of Jade Dome Scale Enzyme Hydrolysates

Purpose: To evaluate the presence and degree of ocular irritation after the test substance jade fish scale hydrolyzate was treated once in the rabbit's eye mucosa.

SUMMARY AND CONCLUSIONS: Eye irritation testing of test substance jade fish 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 jade fish scale hydrolyzate under this test condition is judged to be non-irritant to the ocular mucosa of rabbit.

13-4. Skin sensitization test using guinea pigs from jade fish scale enzyme hydrolysis (Maximization method)

Objective: As part of the safety testing of the hydrolyzate of domido scale enzymes, test material (Maximization method) using guinea pigs was conducted to examine the possibility of allergic to human skin.

SUMMARY AND CONCLUSIONS: The skin sensitization test of the test substance jade fish 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, it is judged that the test substance jade fish scale hydrolyzate is a substance without skin sensitization under the present test conditions.

13-5. 옥돔비늘 효소 가수분해물의 기니픽을 이용한 광독성시험13-5. Phototoxicity test using guinea pigs from jadefish scale enzyme hydrolyzate

Purpose: To evaluate the possibility of phototoxicity using the skin of guinea pigs as part of the safety test of the hydrolyzate of domido scale enzyme.

SUMMARY AND CONCLUSIONS: The presence and extent of phototoxicity of the test substance jade fish 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, it is judged that the test substance jade fish scale hydrolyzate is non-phototoxic under the conditions of this test.

13-6. Photo-sensitization test using guinea pigs from jadefish scale enzyme hydrolysis (Adjuvant & Strip method)

Purpose: To test the safety of the oxidized scale hydrolyzate of the test substance, endodontic scale, and to investigate the possibility of photosensitization by the Adjuvant & Strip method using guinea pigs.

Summary and Conclusion: Photosensitization of the hydrolyzate of oxymedium scale enzyme 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, it is judged that the test substance jade fish scale hydrolyzate is a substance without photosensitization under this test condition.

14. 통계 처리14. Statistical Processing

Statistical processing of the experimental and analytical results of the present embodiment was performed by the 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.

15. 화장품 제형15. 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 jade fish scale hydrolyzate and ultrafiltration fractions of the present invention. Specific manufacturing processes are shown in FIGS. 12A-12H and 13A-13E.

16. 인체 적용 시험16. Human application test

16-1. 시험 목적16-1. 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.

16-2. 시험 제품16-2. Trial product

16-2-1. 시험군 및 대조군16-2-1. Test group and control group

Test Group (A): Contains 1% of Odome Protein Peptides

Control group (B): no domome protein peptide

16-2-2. 제품의 성상16-2-2. Characteristics of the product

essence

16-2-3. 제품의 보관16-2-3. Storage of the product

Room temperature storage

16-2-4. 제품의 유효 성분16-2-4. Active ingredient of the product

Odome Protein Peptide 1%

16-2-5. 제품의 사용 방법 16-2-5. 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.

16-3. 시험 방법16-3. Test Methods

16-3-1. 피험자 선정16-3-1. 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.

16-3-2. 평가 방법16-3-2. 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.

16-4. 통계 분석16-4. 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.

16-5. 시험 결과16-5. Test result

16-5-1. 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

16-5-2. 평가 방법16-5-2. Assessment Methods

16-5-2-1. 육안 평가16-5-2-1. 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, the skin wrinkles of the test group tended to decrease slightly after 12 weeks of product use (Table 10).

Table 10

16-5-2-2. 주름 파라미터 (R-value) 분석16-5-2-2. 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).

(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

16-5-2-3. 효능 및 사용성에 관한 설문 분석 16-5-2-3. 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

16-5-3. 피부 안전성 평가 16-5-3. Skin safety assessment

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

Table 21

[Revision under Rule 26 06.02.2014] Deleted

Claims

Cosmetic composition comprising an enzymatic hydrolyzate of jade ( braniostegus japonicus ) scale extract.
The method of claim 1,

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

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

(S2) hydrothermal extraction of the dried jade fish scales;

(S3) enzymatic hydrolysis of the hydrothermally extracted jade fish scales;

(S4) activated carbon filtration of the enzymatic hydrolyzate of the jade fish 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.
The method of claim 2,

The enzymatic hydrolysis is a cosmetic composition, characterized in that selected from the group consisting of α-chymotrypsin (α-chymotrypsin), alcalase (alcalase), neutrases (Trytrain) and trypsin (Trypsin).
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.