Type III collagen freeze-dried flocculus containing serum albumin, fibronectin and polypeptide as well as preparation method and application of type III collagen freeze-dried flocculus

Abstract

The invention relates to the technical field of cosmetics, and particularly discloses a three-type collagen freeze-drying flocculant containing serum albumin, fibronectin and polypeptide as well as a preparation method and application of the three-type collagen freeze-drying flocculant. The three-type collagen freeze-dried flocculus containing serum albumin, fibronectin and polypeptide is successfully prepared through the preparation method with scientific proportioning and careful design, the synergistic effect of all the components is achieved, and the three-type collagen freeze-dried flocculus has good biocompatibility and stability, can remarkably promote cell adhesion, proliferation and differentiation, and has good application prospects. The three-type collagen freeze-drying floccule can be used for accelerating the tissue repair process, has a remarkable repair effect, and also has a remarkable moisturizing effect and a remarkable anti-wrinkle effect. And the preparation method of the three-type collagen freeze-dried floccules is simple and easy to implement, is suitable for large-scale production, and has relatively high application value and market prospect.

Description

Technical Field

[0001] The present application relates to the field of cosmetic technology, and in particular to a type III collagen freeze-dried floc containing serum albumin, fibronectin and polypeptide, and a preparation method and application thereof. Background Art

[0002] As an important protein component in organisms, collagen plays a key role in maintaining the structure and function of skin, blood vessels, internal organs and other tissues. Among them, type III collagen has become a hot topic in biomedical material research due to its unique biological activity and advantages in promoting cell growth and repairing tissues.

[0003] At present, the preparation method of type III collagen is mainly based on gene recombination technology, which uses human genes for splicing and recombination to obtain the required protein in cell culture. However, the type III collagen materials prepared by traditional methods have problems such as low purity, easy loss of activity, and poor storage stability, which limit their further application. In the prior art, although single-component collagen materials have been widely used in the biomedical field, their effects in promoting comprehensive cell proliferation, differentiation and tissue repair are limited.

[0004] In order to improve the bioactivity and functionality of the material, researchers have begun to explore methods of incorporating multiple bioactive ingredients into the collagen matrix. However, how to effectively maintain the activity of these ingredients and avoid mutual interference during the preparation process remains a major challenge facing current technology. Summary of the invention

[0005] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a type III collagen freeze-dried floc containing serum albumin, fibronectin and polypeptide, and a preparation method and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by this application is:

[0007] The present application provides a type III collagen freeze-dried floc containing serum albumin, fibronectin and polypeptide, wherein the type III collagen freeze-dried floc comprises the following components in parts by weight:

[0008] Serum albumin 0.05-0.2 parts, soluble collagen 0.05-0.5 parts, DNA sodium 0.05-0.3 parts, fibronectin complex 0.5-12 parts, acetyl hexapeptide-8 0.02-0.5 parts, dipeptide diaminobutyryl benzylamide diacetate 0.01-0.6 parts, oligopeptide-1 complex 0.2-3 parts and oligopeptide-3 complex 0.2-3 parts.

[0009] The present application prepares a type III collagen freeze-dried floccules with good biocompatibility and stability through scientific proportioning, and contains serum albumin, fibronectin and polypeptides. The type III collagen freeze-dried floccules of the present application are rich in the nutrients of collagen, which can effectively stimulate the collagen synthesis of the dermis and increase the elasticity and firmness of the skin. In addition, it can also promote skin metabolism and increase the renewal rate of the skin. The various nutrients in the type III collagen freeze-dried floccules can penetrate into the skin layer, increase the moisture and nutrients of the skin, improve the skin texture and reduce wrinkles. The fibronectin and other ingredients in the type III collagen freeze-dried floccules can form a protective layer to help the skin lock in moisture and keep the skin moist and moisturized. The ingredients such as oligopeptide-1 complex and oligopeptide-3 complex in the type III collagen freeze-dried floccules can improve the elasticity and firmness of the skin, prevent and repair oxidative damage, accelerate the repair and healing of the skin, reduce irritation and redness, moisturize dry and rough skin, and keep the skin young and healthy.

[0010] Among them, the combination of serum albumin, soluble collagen, fibronectin complex and polypeptides (oligopeptide-1 complex and oligopeptide-3 complex) can effectively maintain the activity of these ingredients and avoid mutual interference during the preparation process, so that the type III collagen freeze-dried flocs have good biocompatibility and stability, can significantly promote cell adhesion, proliferation and differentiation, accelerate the tissue repair process, and can also improve the moisturizing and anti-wrinkle effects of type III collagen freeze-dried flocs.

[0011] Soluble collagen is added to the freeze-dried flocs of type III collagen to help the skin retain moisture, making it softer and more moisturized. It locks the moisture inside the skin, forming a protective layer to prevent moisture loss, thereby maintaining the hydration of the skin; it promotes the production of skin collagen, reducing skin sagging and wrinkles. It accelerates the skin repair process, alleviates the problems of skin aging and wrinkles; it improves the elasticity and firmness of the skin, making the skin more elastic and firm; it promotes the metabolism of skin tissue, improves the integrity of the skin's fiber structure, thereby improving the firmness of the skin; it improves skin tone and gloss, making the skin look healthier and more lustrous; it can activate subcutaneous cytokines, promote the growth and regeneration of skin cells, improve skin tone and gloss, and has the comprehensive effects of moisturizing, anti-wrinkle, firming the skin and improving skin tone.

[0012] The addition of serum albumin has excellent moisturizing effect, can penetrate deep into the skin, provide lasting moisture to the skin, improve dry skin, peeling and other problems, and keep the skin supple and shiny; the molecular structure of serum albumin allows it to be easily absorbed by the skin, thereby enhancing the skin's moisturizing ability; serum albumin has a repairing effect on the skin, and it contains a variety of repair growth factor ingredients, which can promote the regeneration and activation of skin cells, improve cell damage, and relieve problems such as red blood streaks caused by thin or damaged skin stratum corneum. Serum albumin is called a cell revitalizing factor, which can strengthen the natural barrier of the skin surface and protect the skin from external damage; serum albumin can replenish the collagen needed by the skin and improve wrinkles and other problems caused by collagen loss; the antioxidant components in serum albumin can resist the damage of free radicals, delay the aging process of the skin, and keep the skin young, so it has the comprehensive effects of moisturizing, skin repair and anti-aging.

[0013] The addition of sodium DNA has a strong moisturizing ability, which can absorb moisture from the air and lock in the moisture of the skin, thereby helping the skin stay moist and smooth; sodium DNA is rich in antioxidants, which can neutralize the damage of free radicals and reduce the damage to the skin caused by environmental pollution and ultraviolet radiation; it promotes cell repair and regeneration, accelerates the recovery of damaged skin tissue, reduces the appearance of fine lines and wrinkles, and makes the skin look younger and healthier; sodium DNA has anti-inflammatory and skin-soothing effects, which can reduce redness and discomfort caused by irritation and sensitivity, therefore, it has the comprehensive effects of moisturizing, nourishing, anti-oxidation, repair and regeneration, soothing and calming.

[0014] The addition of fibronectin complex promotes skin repair and helps the skin return to a healthy state; it improves the skin's resistance and makes the skin more tolerant to external environmental stimuli. This helps reduce skin sensitivity, redness and swelling, and keeps the skin healthy and stable; it improves the symptoms of thin stratum corneum. It helps thicken the stratum corneum and restore the skin's sensitivity to normal, while also preventing serious damage or infection to the skin. Therefore, it has the comprehensive effects of moisturizing, anti-aging, firming, repairing and soothing.

[0015] The addition of acetyl hexapeptide-8 has the effect of improving wrinkles and increasing skin elasticity; the addition of dipeptide diaminobutyryl benzylamide diacetate has the effect of antioxidant and anti-wrinkle; the addition of oligopeptide-1 has the effect of moisturizing, repairing and nourishing the skin; the addition of oligopeptide-3 has the effect of moisturizing, repairing and nourishing the skin.

[0016] As a preferred embodiment of the freeze-dried type III collagen flocs of the present application, the freeze-dried type III collagen flocs include the following components in parts by weight:

[0017] Serum albumin 0.08-0.12 parts, soluble collagen 0.12-0.18 parts, DNA sodium 0.1-0.2 parts, fibronectin complex 2-8 parts, acetyl hexapeptide-8 0.05-0.15 parts, dipeptide diaminobutyryl benzylamide diacetate 0.08-0.12 parts, oligopeptide-1 complex 0.8-1.2 parts and oligopeptide-3 complex 0.8-1.2 parts.

[0018] The type III collagen freeze-dried flocs of the present application use components in specific mass parts, which can more effectively stimulate collagen synthesis in the dermis, increase the elasticity and firmness of the skin, promote skin metabolism, and increase the skin's renewal rate; the above components can better penetrate into the skin layer, increase the moisture and nutrients of the skin, improve the skin texture and reduce wrinkles, and repair the damaged skin barrier. Among them, when serum albumin, soluble collagen, fibronectin complex and polypeptide (oligopeptide-1 complex and oligopeptide-3 complex) are matched in specific mass parts, the moisturizing, repairing, anti-wrinkle and other effects of type III collagen freeze-dried flocs can be improved at the same time.

[0019] As a preferred embodiment of the freeze-dried type III collagen flocs of the present application, the freeze-dried type III collagen flocs include the following components in parts by weight:

[0020] 0.1 part of serum albumin, 0.15 part of soluble collagen, 0.15 part of sodium DNA, 5 parts of fibronectin complex, 0.1 part of acetyl hexapeptide-8, 0.1 part of dipeptide diaminobutyryl benzylamide diacetate, 1 part of oligopeptide-1 complex and 1 part of oligopeptide-3 complex.

[0021] When the present application preferably uses the above-mentioned components in parts by mass, the prepared type III collagen freeze-dried flocs can better improve the elasticity and firmness of the skin, prevent and repair oxidative damage, accelerate the repair and healing of the skin, reduce irritation and redness, moisturize dry and rough skin, and keep the skin young and healthy.

[0022] As a preferred embodiment of the type III collagen freeze-dried flocs described in the present application, the fibronectin complex comprises fibronectin, 1,2-hexanediol, sodium chloride, sodium citrate and water;

[0023] Preferably, the fibronectin complex comprises the following components in weight percentage:

[0024] Fibronectin 0.03-0.08%, 1,2-hexanediol 0.9-1.1%, sodium chloride 0.89-0.91%, sodium citrate 0.29-0.31% and the balance water.

[0025] The oligopeptide-1 complex comprises oligopeptide-1, trehalose, disodium hydrogen phosphate, sodium dihydrogen phosphate and water;

[0026] Preferably, the oligopeptide-1 complex comprises the following components in weight percentage:

[0027] Oligopeptide-1 0.09-0.11%, trehalose 0.9-1.1%, disodium hydrogen phosphate 0.2-0.25%, sodium dihydrogen phosphate 0.05-0.07% and the balance water.

[0028] The oligopeptide-3 complex comprises oligopeptide-3, trehalose, disodium hydrogen phosphate, sodium dihydrogen phosphate and water.

[0029] Preferably, the oligopeptide-3 complex comprises the following components in weight percentage:

[0030] Oligopeptide-3 0.09-0.11%, trehalose 0.9-1.1%, disodium hydrogen phosphate 0.2-0.25%, sodium dihydrogen phosphate 0.05-0.07% and the balance water.

[0031] Among them, a representative fibronectin complex is MELMPRO-500G fibronectin solution, which includes the following components in weight percentage: 0.05 parts of fibronectin, 1 part of 1,2-hexanediol, 0.9 parts of sodium chloride, 0.3 parts of sodium citrate and 97.75 parts of water.

[0032] Among them, a representative oligopeptide-1 complex includes the following components in weight percentage: 0.1 parts of oligopeptide-1, 1 part of trehalose, 0.22 parts of disodium hydrogen phosphate, 0.06 parts of sodium dihydrogen phosphate and 98.62 parts of water.

[0033] Among them, a representative oligopeptide-3 complex includes the following components in weight percentage: 0.1 parts of oligopeptide-3, 1 part of trehalose, 0.22 parts of disodium hydrogen phosphate, 0.06 parts of sodium dihydrogen phosphate and 98.62 parts of water.

[0034] The present application adopts the above-mentioned fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex to more effectively improve the effects of type III collagen freeze-dried flocs in moisturizing, skin repairing and anti-wrinkle.

[0035] As a preferred embodiment of the freeze-dried type III collagen flocs described in the present application, the freeze-dried type III collagen flocs further include at least one of a moisturizer, a thickener and an antibacterial agent.

[0036] As a preferred embodiment of the type III collagen freeze-dried flocs described in the present application, the moisturizing agent includes at least one of mannitol, trehalose, Tremella fuciformis fruiting body extract and soluble proteoglycan.

[0037] The present application adopts the above types of moisturizers to improve the moisturizing effect of type III collagen freeze-dried flocs, and can also enhance skin elasticity, anti-oxidation, promote blood circulation, repair skin barriers, and resist skin aging. Among them, soluble proteoglycans have good water absorption and film-forming properties, and can form a uniform film on the skin surface to reduce water evaporation, thereby maintaining the moisture of the skin. This moisturizing effect helps maintain the moisture balance of the skin and keep the skin soft and moisturized.

[0038] Adding Tremella fruiting body extract, this substance combines with water molecules to form hydrogen bonds through its countless terminal hydroxyl groups, thereby combining a large amount of water and forming a molecular sponge-like structure in space. This structure can effectively lock in moisture and provide a lasting moisturizing effect. The film formed by Tremella heteropolysaccharide is not easy to shrink with changes in humidity, so there is no feeling of tightness when used in skin care products. Compared with sodium hyaluronate, the film of Tremella heteropolysaccharide is more elastic and less likely to form cracks, providing a more comfortable use experience. Tremella fruiting body extract can inhibit the activity of reactive oxygen, increase the activity of SOD enzyme in skin cells, play an antioxidant role, and help slow down the aging process of the skin. Activate keratinocytes, promote keratinocyte proliferation, normalize epidermal metabolism, thereby repairing damaged skin barriers and protecting the skin from external stimuli. Compared with sodium hyaluronate, Tremella heteropolysaccharide shows better performance in moisturizing ability and film-forming properties. Especially in terms of evaporation rate and water absorption, Tremella heteropolysaccharide shows a slower evaporation rate and higher water absorption capacity.

[0039] As a preferred embodiment of the freeze-dried type III collagen flocs described in the present application, the mass fraction of the moisturizing agent is 0.63-7.6 parts.

[0040] As a preferred embodiment of the freeze-dried type III collagen flocs described in the present application, the moisturizing agent includes the following components in parts by weight:

[0041] 0.5-5 parts of mannitol, 0.1-2 parts of trehalose, 0.02-3 parts of tremella fruiting body extract and 0.01-0.3 parts of soluble proteoglycan.

[0042] Among them, the representative soluble collagen is recombinant human collagen (BSS) v0.

[0043] Among them, the representative serum albumin is plant-derived serum albumin freeze-dried powder.

[0044] Acetyl hexapeptide-8 uses ZPC008- Wrinklend008P; dipeptide diaminobutyryl benzylamide diacetate is used Wrinklend017P snake venom peptide powder.

[0045] The present application also provides a method for preparing the above-mentioned type III collagen freeze-dried flocs, comprising the following steps:

[0046] S1, weighing the above components by mass;

[0047] S2, mixing and stirring the soluble collagen and the moisturizer, heating to 80-90° C. until the components are dissolved and transparent, sterilizing by high pressure, and then cooling to room temperature to obtain a mixture I;

[0048] S3. Mix serum albumin, sodium DNA, fibronectin, acetyl hexapeptide-8, dipeptide diaminobutyryl benzylamide diacetate, oligopeptide-1, oligopeptide-3 and the mixture I obtained in step S2 to obtain a mixture II, and then freeze-dry the mixture II to obtain freeze-dried type III collagen flocs.

[0049] As a preferred embodiment of the method for preparing type III collagen freeze-dried flocs described in the present application, the freeze-drying step comprises:

[0050] 1) Prefreezing: Prefreeze the mixture II to -40°C ± 5°C and keep warm for 180 minutes;

[0051] 2) First heating: The pre-frozen mixture II is heated for the first time at a temperature of -15°C for 60 minutes and kept at -15°C for 180 minutes;

[0052] 3) Second heating: The mixture II that has been heated for the first time is heated for the second time at a temperature of -5°C for 60 minutes and then kept at -5°C for 180 minutes;

[0053] 4) Third heating: The mixture II that has been heated for the second time is heated for the third time at 5°C for 60 minutes and kept at 5°C for 180 minutes;

[0054] 5) Fourth heating: The mixture II which has been heated for the third time is heated for the fourth time at 30°C for 60 min and kept at 30°C for 180 min.

[0055] The freeze-dried type III collagen flocs of the present application are simple and easy to prepare using the above method, and multiple heating steps can ensure uniform heating, are suitable for large-scale production, and have high application value and market prospects.

[0056] The present application also provides the use of the above-mentioned type III collagen freeze-dried flocs in cosmetics.

[0057] Compared with the prior art, this application has the following beneficial effects:

[0058] The present application successfully prepares type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptides through scientific proportions and a carefully designed preparation method, achieving the synergistic effect of each component. The type III collagen freeze-dried flocs of the present application have good biocompatibility and stability, can significantly promote cell adhesion, proliferation and differentiation, accelerate the tissue repair process, and have a significant repair effect. In addition, the type III collagen freeze-dried flocs also have significant moisturizing and anti-wrinkle effects. The preparation method of type III collagen freeze-dried flocs is simple and easy, suitable for large-scale production, and has high application value and market prospects. DETAILED DESCRIPTION

[0059] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments.

[0060] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0061] In the following examples and comparative examples:

[0062] Soluble collagen used recombinant human collagen (BSS) v0, which was purchased from Jiangsu Jiangshan Juyuan.

[0063] Tremella fuciformis fruiting body extract and soluble proteoglycan were purchased from Shanghai Huiwen Biological.

[0064] Serum albumin was purchased from Wuhan Heyuan Biotechnology Co., Ltd. using plant-derived serum albumin lyophilized powder (containing 95% serum albumin, 3% sodium chloride and 2% sodium bicarbonate, of which sodium chloride and sodium bicarbonate were negligible).

[0065] DNA sodium was purchased from Runhui Biotechnology (Weihai); fibronectin complex was MELMPRO-500G fibronectin solution, purchased from Meierjian (Shenzhen) Biotechnology; acetyl hexapeptide-8 was obtained from ZPC008- Wrinklend008P was purchased from Zhejiang Paipeptide Biotechnology; dipeptide diaminobutyryl benzylamide diacetate was used Wrinklend017P snake venom peptide powder was purchased from Zhejiang Paipeptide Biological.

[0066] The oligopeptide-1 complex includes the following components in parts by mass: 0.1 parts of oligopeptide-1, 1 part of trehalose, 0.22 parts of disodium hydrogen phosphate, 0.06 parts of sodium dihydrogen phosphate and 98.62 parts of water.

[0067] The oligopeptide-3 complex includes the following components in parts by mass: 0.1 parts of oligopeptide-3, 1 part of trehalose, 0.22 parts of disodium hydrogen phosphate, 0.06 parts of sodium dihydrogen phosphate and 98.62 parts of water.

[0068] Example 1-5 and Comparative Example 1-11, a type III collagen freeze-dried floc containing serum albumin, fibronectin and polypeptide and a preparation method thereof

[0069] The formulas (mass percentage %) of type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptide provided in the examples and comparative examples are shown in Tables 1 and 2.

[0070] A method for preparing type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptides, comprising the following steps:

[0071] S1. Weigh the components and clean the production equipment;

[0072] S2, soluble collagen, mannitol, trehalose, Tremella fuciformis fruiting body extract and soluble proteoglycan are sequentially added into a stirring pot, heated to 85°C with stirring until the components are completely dissolved and transparent, canned and sealed, sterilized by high pressure at 120°C for 40 minutes, and then naturally cooled to room temperature (below 33°C) to obtain a mixture I;

[0073] S3. Mix serum albumin, sodium DNA, fibronectin, acetyl hexapeptide-8, dipeptide diaminobutyryl benzylamide diacetate, oligopeptide-1, oligopeptide-3 and the mixture I obtained in step S2, and immediately fill the mixture to obtain a mixture II, and then freeze-dry the mixture II to obtain freeze-dried type III collagen flocs.

[0074] The freeze-drying steps include:

[0075] 1) Prefreezing: Prefreeze the mixture II to -40°C ± 5°C and keep warm for 180 minutes;

[0076] 2) First heating: The pre-frozen mixture II is heated for the first time at a temperature of -15°C for 60 minutes and kept at -15°C for 180 minutes;

[0077] 3) Second heating: The mixture II that has been heated for the first time is heated for the second time at a temperature of -5°C for 60 minutes and then kept at -5°C for 180 minutes;

[0078] 4) Third heating: The mixture II that has been heated for the second time is heated for the third time at a temperature of 5°C for 60 minutes and then kept at 5°C for 180 minutes;

[0079] 5) Fourth heating: The mixture II which has been heated for the third time is heated for the fourth time at 30°C for 60 min and kept at 30°C for 180 min.

[0080] Note: When the temperature rises, open the limited leakage, and the aeration pressure is 23Pa±3Pa. Close the limited leakage 60 minutes before the plugging.

[0081]

[0082]

[0083] Table 1

[0084] Table 2

[0085]

[0086] The appearance of the type III collagen freeze-dried flocs prepared in Examples 1-5 of the present application is a white flocculent solid, and the pH value (direct measurement method) is 5.0-7.5; it is stored under heat-resistant conditions (45±2°C) for 3 months and returned to room temperature, and there are no abnormalities; it is stored under cold-resistant conditions (-18±2°C) for 3 months and returned to room temperature, and there are no abnormalities; and it is stored under cyclic test conditions (45°C, room temperature, -18°C) for 3 months and returned to room temperature, and there are no abnormalities.

[0087] The appearance of the type III collagen freeze-dried flocs prepared in comparative examples 1-4 was a white flocculent solid, with a pH value (direct measurement method) of 5.0-7.5; they were stored under heat-resistant conditions (45±2°C) for 3 months and returned to room temperature, and their appearance shrank and deformed; comparative examples 8-11 were stored under cold-resistant conditions (-18±2°C) for 3 months and returned to room temperature, and their appearance shrank and deformed; comparative examples 5-8 were stored under cyclic test conditions (45°C, room temperature, -18°C) for 3 months and returned to room temperature, and their appearance obviously shrank and deformed.

[0088] Test Example 1: Determination of Elastase Inhibition Rate

[0089] As a highly selective and specific proteolytic enzyme, elastase catalyzes the hydrolysis of many amino acids such as glycine, leucine, alanine and other hydroxyl-containing polypeptide bonds, and can decompose elastin in connective tissue proteins. Inhibiting the activity of elastase can effectively slow down the degradation rate of elastin and play a role in tightening the skin. In this experiment, Congo red-elastin is used as the substrate. After porcine pancreatic elastase hydrolyzes elastin, Congo red dye falls off into the solution. At this time, the solution appears red. When the enzyme inhibition sample is added, the enzyme activity is inhibited, the red color becomes lighter, and the inhibition ability is negatively correlated with the depth of red. The inhibition rate can be indirectly obtained by measuring the absorbance at 405nm using a UV-visible spectrophotometer. This test method is an in vitro method and is suitable for the detection of the anti-wrinkle effect of cosmetics of various matrix types.

[0090] 90 μL buffer (purified water), 30 μL product solution (the type III collagen freeze-dried flocs of Examples 1-5 and Comparative Examples 1-11 were dissolved in the same amount of purified water. The positive control group used a sivelestat sodium solution) and 10 μL substrate were added to a 96-well plate, mixed and incubated at 25°C for 20 minutes. Then 20 μL of elastase solution was added, shaken and mixed immediately, and OD405nm was detected. OD405nm was detected again after incubation for 10 minutes to obtain the effect of the sample on the activity of elastase (P value <0.05). The results are shown in Table 3.

[0091] Table 3

[0092]

[0093]

[0094] It can be seen from Table 3 above that the elastase inhibition rate of the type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptide prepared in Examples 1-5 of the present application is greater than 30%, and the elastase inhibition rate of the type III collagen freeze-dried flocs is higher than the elastase inhibition rate of the blank control group and the statistical difference P value is <0.05, indicating that the type III collagen freeze-dried flocs of the present application have good anti-wrinkle effect.

[0095] Comparative Example 5, Comparative Examples 1-5 lack one of soluble collagen, serum albumin, fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex respectively, and the prepared type III collagen freeze-dried flocs or freeze-dried flocs have a low elastase inhibition rate, indicating that the synergistic combination of serum albumin, soluble collagen, fibronectin complex and polypeptides (oligopeptide-1 complex and oligopeptide-3 complex) can effectively maintain the activity of these components and avoid mutual interference during the preparation process, and effectively improve the anti-wrinkle effect of type III collagen freeze-dried flocs.

[0096] The type III collagen freeze-dried flocs in comparative examples 6-8 lack sodium DNA, acetyl hexapeptide-8, and dipeptide diaminobutyryl benzylamide diacetate, respectively, and the low elastase inhibition rate indicates that the addition of sodium DNA, acetyl hexapeptide-8, and dipeptide diaminobutyryl benzylamide diacetate can better improve the elastase inhibition rate of type III collagen freeze-dried flocs and enhance the anti-wrinkle effect of the product.

[0097] When the dosage of fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex in comparative examples 9-11 is excessive, the elastase inhibition rate of type III collagen freeze-dried flocs is lower than that in examples 1-5, indicating that by controlling the fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex within the scope of the present application, the elastase inhibition rate of the product can be further improved. If the dosage of fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex deviates from the scope of the present invention, the anti-wrinkle effect of the present application will be significantly reduced, indicating that at the dosage specified in the present application, the synergistic effect is more obvious.

[0098] Test Example 2: Moisturizing Test - Transepidermal Water Loss Test

[0099] Test method: The moisture content of the facial skin of the subjects before and after using the product (the freeze-dried flocs of type III collagen in Examples 1-5 and Comparative Examples 1-11 were dissolved in the same amount of pure water.) was measured. The moisture content of the facial skin was measured using a transdermal water loss measuring instrument Vapometer (model: SWL5201, Delfin, Finland). The test was performed by comparing the skin moisture loss before and after using the product through the TEWL parameter. Where △TEWL = (TEWL1-TEWL2) / TEWL1×100%, TEWL1 is the test value before using the product, and TEWL2 is the test value after using the product. The larger the △TEWL value obtained, the greater the reduction in transdermal water loss of the test skin after using the product, and the better the moisturizing effect of the product. The average value of each group was recorded and calculated, and the results are shown in Table 4 below.

[0100] Table 4

[0101]

[0102] As shown in Table 4 above, compared with Comparative Examples 1 to 11, the △TEWL data of the type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptide prepared in Examples 1 to 5 all reached more than 39%, which has a significant moisturizing effect. The transepidermal water retention rate of Example 5 is the highest, which shows that Example 5 is the best combination.

[0103] By comparing Example 5 and Comparative Examples 1-5, it can be seen that the soluble collagen, serum albumin, fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex described in the present application have a significant synergistic effect in moisturizing effect. If one of the above is missing, the △TEWL of type III collagen freeze-dried flocs or freeze-dried flocs is low, and the moisturizing effect will be significantly reduced.

[0104] When comparative examples 6-8 lack one of sodium DNA, acetyl hexapeptide-8, and dipeptide diaminobutyryl benzylamide diacetate, the moisturizing effect of the prepared type III collagen freeze-dried flocs is also inferior to that of examples 1-5, indicating that the type III collagen freeze-dried flocs containing serum albumin, fibronectin, and polypeptides of examples 1-5 have significant moisturizing effects.

[0105] When the dosage of fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex in comparative examples 9-11 is excessive, the elastase inhibition rate of type III collagen freeze-dried flocs is lower than that in examples 1-5, indicating that the moisturizing effect of the product can be further improved by controlling the fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex within the scope of the present application. If the dosage of fibronectin complex, oligopeptide-1 complex and oligopeptide-3 complex deviates from the scope of the present invention, the moisturizing effect of the present application will be significantly reduced, indicating that the synergistic effect is more obvious at the dosage specified in the present application.

[0106] Test Example 3: Safety Test - Patch Test

[0107] The type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptide prepared in the above Examples 1-5 were subjected to human safety patch tests, and the test method referred to the human skin patch test in the "2015 Cosmetic Safety Technical Specifications".

[0108] The method of skin occlusion patch test is as follows: 30 subjects aged 18-60 years old are selected, qualified patch test equipment with an area of ​​no more than 50 mm2 and a depth of about 1 mm is selected, 0.020 mL of the above embodiment is put into the patch test chamber, the patch tester is applied to the curved side of the forearm of the subject with a low-allergenic tape, and the patch is lightly pressed with the palm to evenly apply it to the skin for 24 hours. The skin reaction is observed according to the standards in the table below at 0.5 hours after removal (after the indentation disappears), 24 hours and 48 hours, and the observation results are recorded.

[0109] The grading standards for skin reactions in the skin occlusive patch test are shown in Table 5.

[0110] Table 5

[0111]

[0112] The results are shown in Table 6.

[0113] Table 6

[0114] Group 0.5h 24h 48h Overall results Example 1 0 0 0 All 30 people had no positive reaction Example 2 0 0 0 All 30 people had no positive reaction Example 3 0 0 0 All 30 people had no positive reaction Example 4 0 0 0 All 30 people had no positive reaction Example 5 0 0 0 All 30 people had no positive reaction

[0115] It can be seen from Table 6 above that the patch test results of the type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptides prepared in Examples 1 to 5 are all negative, indicating that the type III collagen freeze-dried flocs containing serum albumin, fibronectin and polypeptides prepared in the present application are mild and non-irritating, and will not cause adverse skin reactions to this batch of subjects.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A type III collagen freeze-dried floc containing serum albumin, fibronectin and polypeptide, characterized in that: The type III collagen freeze-dried flocs include the following components: Serum albumin 0.05-0.2 parts, soluble collagen 0.05-0.5 parts, DNA sodium 0.05-0.3 parts, fibronectin complex 0.5-12 parts, acetyl hexapeptide-8 0.02-0.5 parts, dipeptide diaminobutyryl benzylamide diacetate 0.01-0.6 parts, oligopeptide-1 complex 0.2-3 parts and oligopeptide-3 complex 0.2-3 parts.

2. The freeze-dried type III collagen flocs according to claim 1, characterized in that: The type III collagen freeze-dried flocs include the following components in parts by weight: Serum albumin 0.08-0.12 parts, soluble collagen 0.12-0.18 parts, DNA sodium 0.1-0.2 parts, fibronectin complex 2-8 parts, acetyl hexapeptide-8 0.05-0.15 parts, dipeptide diaminobutyryl benzylamide diacetate 0.08-0.12 parts, oligopeptide-1 complex 0.8-1.2 parts and oligopeptide-3 complex 0.8-1.2 parts.

3. The freeze-dried type III collagen flocs according to claim 2, characterized in that: The fibronectin complex comprises fibronectin, 1,2-hexanediol, sodium chloride, sodium citrate and water; The oligopeptide-1 complex comprises oligopeptide-1, trehalose, disodium hydrogen phosphate, sodium dihydrogen phosphate and water; The oligopeptide-3 complex comprises oligopeptide-3, trehalose, disodium hydrogen phosphate, sodium dihydrogen phosphate and water.

4. The freeze-dried type III collagen flocs according to claim 1, characterized in that: The type III collagen freeze-dried flocs also include at least one of a moisturizer, a thickener and an antibacterial agent.

5. The freeze-dried type III collagen flocs according to claim 4, characterized in that: The moisturizing agent comprises at least one of mannitol, trehalose, tremella fruiting body extract and soluble proteoglycan.

6. The freeze-dried type III collagen flocs according to claim 4, characterized in that: The mass fraction of the moisturizing agent is 0.63-7.6 parts.

7. The freeze-dried type III collagen flocs according to claim 6, characterized in that: The moisturizing agent comprises the following components in parts by weight: 0.5-5 parts of mannitol, 0.1-2 parts of trehalose, 0.02-3 parts of tremella fruiting body extract and 0.01-0.3 parts of soluble proteoglycan.

8. A method for preparing type III collagen freeze-dried flocs according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1, weighing the above components by mass; S2, mixing and stirring the soluble collagen and the moisturizer, heating to 80-90° C. until the components are dissolved and transparent, sterilizing by high pressure, and then cooling to room temperature to obtain a mixture I; S3. Mix serum albumin, sodium DNA, fibronectin, acetyl hexapeptide-8, dipeptide diaminobutyryl benzylamide diacetate, oligopeptide-1, oligopeptide-3 and the mixture I obtained in step S2 to obtain a mixture II, and then freeze-dry the mixture II to obtain freeze-dried type III collagen flocs.

9. The method for preparing type III collagen freeze-dried flocs according to claim 8, characterized in that: The freeze-drying step comprises: 1) Prefreezing: Prefreeze the mixture II to -40°C ± 5°C and keep warm for 180 minutes; 2) First heating: The pre-frozen mixture II is heated for the first time at a temperature of -15°C for 60 minutes and kept at -15°C for 180 minutes; 3) Second heating: The mixture II that has been heated for the first time is heated for the second time at a temperature of -5°C for 60 minutes and then kept at -5°C for 180 minutes; 4) Third heating: The mixture II that has been heated for the second time is heated for the third time at 5°C for 60 minutes and kept at 5°C for 180 minutes; 5) Fourth heating: The mixture II which has been heated for the third time is heated for the fourth time at 30°C for 60 min and kept at 30°C for 180 min.

10. Use of the freeze-dried type III collagen flocs according to any one of claims 1 to 7 in cosmetics.

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