Anti-wrinkle composition as well as preparation method and application thereof

Abstract

The invention provides an anti-wrinkle composition as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. The composition disclosed by the invention is prepared from 1 to 10 parts of ginsenoside, 1 to 10 parts of pilose antler peptide, 15 to 20 parts of resveratrol, 1 to 10 parts of cod collagen, 2 to 8 parts of sodium hyaluronate, 10 to 15 parts of superoxide dismutase and 10 to 20 parts of honey. The cod collagen protein comprises a peptide fragment YGCC. According to the present invention, the cod collagen and the pilose antler peptide are subjected to fine processing to obtain the peptide fragment YGCC with significant anti-wrinkle activity in the cod collagen and the pilose antler peptide with small molecular weight, such that the anti-wrinkle effect of the composition is significantly improved. The invention also provides application of the anti-wrinkle composition in preparation of anti-wrinkle products, and the anti-wrinkle composition has a good market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and in particular relates to an anti-wrinkle composition and a preparation method and application thereof. Background Art

[0002] With the improvement of people's living standards and the growing pursuit of beauty, the market demand for anti-wrinkle products continues to rise. The mechanism of wrinkles caused by skin aging is extremely complex, involving the dual effects of internal physiological function decline and external environmental factors. Internally, with age, the ability to synthesize collagen and elastic fibers in the skin decreases, and degradation accelerates, causing the skin's firmness and elasticity to gradually lose. This change is particularly obvious in exposed areas such as the face, and wrinkles gradually form. Among the external environmental factors, long-term exposure to ultraviolet rays is a key cause of skin photoaging, which not only increases the expression of matrix metalloproteinases and accelerates the degradation of collagen and elastic fibers, but also triggers inflammatory responses and destroys the skin's barrier function. In addition, environmental pollution and bad living habits such as staying up late and smoking will further aggravate the skin aging process, making the wrinkle problem more serious.

[0003] At present, there are many kinds of anti-wrinkle products on the market, with different ingredients and mechanisms of action. Common anti-wrinkle ingredients include retinol and its derivatives, which can promote collagen synthesis and improve skin texture, but have poor stability, and some people will experience adverse reactions such as skin irritation after use. Peptides stimulate collagen production and inhibit nerve conduction to reduce muscle contraction, thereby achieving anti-wrinkle effects. However, their effects are relatively limited, and the improvement of deeper wrinkles is not obvious. Plant extracts such as ferulic acid and green tea extract have antioxidant properties, can scavenge free radicals, and reduce ultraviolet damage to the skin, but when used alone, the anti-wrinkle effect is difficult to meet the high requirements of consumers. However, the existing anti-wrinkle compositions often have the problem of insufficient synergy of ingredients in formula design, and cannot give full play to the advantages of each ingredient, and it is difficult to achieve a full range of multi-level anti-wrinkle effects. Patent CN202110448619.9 discloses an anti-wrinkle composition made of ginsenosides, antler peptides, hydrolyzed collagen, resveratrol and other components. Although it shows certain anti-wrinkle effects, it is prepared by blending commercially available raw materials, and the raw materials also have some problems such as low content of active ingredients, and still cannot achieve satisfactory anti-wrinkle and anti-aging effects. Therefore, it is urgent to conduct more refined research on the raw material components in the anti-wrinkle composition and develop a method for preparing an anti-wrinkle composition with excellent efficacy to meet consumers' growing demand for anti-wrinkle products. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide an anti-wrinkle composition, which significantly improves the anti-wrinkle effect of the composition by fine processing of the raw materials of the composition, and is safe, non-irritating and does not produce other side effects.

[0005] The present invention also aims to provide an application of the anti-wrinkle composition in the preparation of anti-wrinkle and anti-aging products, so as to prepare anti-wrinkle and anti-aging products with significant effects.

[0006] The present invention also aims to provide an anti-wrinkle product with significant anti-wrinkle effect.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides an anti-wrinkle composition, which comprises, by weight, 1-10 parts of ginsenoside, 1-10 parts of pilose antler peptide, 15-20 parts of resveratrol, 1-10 parts of cod collagen, 2-8 parts of sodium hyaluronate, 10-15 parts of superoxide dismutase and 10-20 parts of honey; the cod collagen comprises a peptide segment YGCC.

[0009] Preferably, the preparation method of cod collagen comprises the following steps: washing cod skin with running water, adding sodium hydroxide solution to soak; washing the soaked cod skin, mixing with phosphate buffer, grinding and homogenizing to obtain cod skin homogenate; adding pepsin to the cod skin homogenate, hydrolyzing for 2 to 5 hours, then adding papain and flavor protease, hydrolyzing for 1 to 4 hours to obtain enzymatic solution; the enzymatic solution passes through a ceramic membrane and an organic membrane in sequence to obtain a filtrate; the filtrate passes through a cation exchange resin and an anion exchange resin in sequence, and the material after resin adsorption is concentrated, sterilized and dried to obtain cod collagen.

[0010] Preferably, the concentration of the sodium hydroxide solution is 0.2% to 1%; the soaking time is 1 to 3 hours; and the soaking temperature is 25 to 35°C.

[0011] Preferably, the mass volume ratio of the fish skin to the phosphate buffer is 1 g:5-15 mL.

[0012] Preferably, based on the mass of the cod skin homogenate, the amount of pepsin added is 1 to 2 million U / g, the amount of papain added is 800,000 to 1.2 million U / g, and the amount of flavor protease added is 1 to 1.5 million U / g.

[0013] Preferably, the cod collagen is separated and purified by preparative high performance liquid chromatography, with mobile phase A: water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile for gradient elution, and the elution fractions of 32 to 36 min are collected and dried to obtain purified cod collagen.

[0014] Preferably, the gradient elution program is: 0% to 5% B in 0 to 5 min, 5% B to 30% B in 5 to 15 min, and 30% to 75% B in 15 to 45 min.

[0015] Preferably, the preparation method of the velvet antler peptide comprises the following steps: crushing the velvet antler, adding 60% to 80% ethanol solution, and extracting under microwave conditions for 2 to 10 minutes; ultrafiltration of the extract using 10K ultrafiltration membrane and 1K ultrafiltration membrane in sequence, and freeze-drying the ultrafiltrate to obtain the velvet antler peptide.

[0016] The present invention also provides the use of the anti-wrinkle composition in the preparation of anti-wrinkle and anti-aging products.

[0017] The present invention also provides an anti-wrinkle and anti-aging product, which comprises, by weight, 15 to 25 parts of the anti-wrinkle composition, 5 to 12 parts of a penetration enhancer, 0.1 to 0.5 parts of a thickener, 2 to 6 parts of a moisturizer and 10 to 80 parts of deionized water.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0019] The anti-wrinkle composition provided by the present invention creatively adds cod collagen rich in peptide segment YGCC, and the peptide segment YGCC has a strong binding ability with MAPK1 and MMP9, important targets of skin photodamage, and can regulate MAPK signaling pathways, FoxO signaling pathways, etc., and give full play to the effects of anti-inflammatory and anti-oxidative stress. At the same time, the present invention also provides a preparation method of low molecular weight antler peptides, which can effectively improve the absorption performance of antler peptides. The present invention significantly improves the anti-wrinkle and anti-aging effects of the anti-wrinkle composition by improving the preparation process of cod collagen and antler peptides in the composition, and has a prominent effect in alleviating skin photoaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : 3D image of YGCC docking with protein target molecules;

[0021] Figure 2 : The results of the determination of MF's ability to scavenge DPPH free radicals;

[0022] Figure 3 :The results of the determination of superoxide anion free radical scavenging ability of MF;

[0023] Figure 4 :The results of the determination of MF's ability to scavenge hydroxyl free radicals;

[0024] Figure 5 : Determination results of MF total reducing capacity;

[0025] Figure 6 : The results of the determination of ABTS+ free radical scavenging ability of MF;

[0026] Figure 7 :The results of MF regulating the expression of HaCaT cell related genes;

[0027] Figure 8 :MF regulates the expression of HDF-α cell-related genes;

[0028] Fig. 9 :Effects of gel mask on the survival rate of HaCaT cells;

[0029] Fig.10 :Effect of facial cream on the survival rate of HaCaT cells;

[0030] Fig.11 :Effects of gel mask on the survival rate of HDF-α cells;

[0031] Fig.12 :Effects of facial cream on the survival rate of HDF-α cells;

[0032] Fig.13 :Effects of gel mask and cream on the secretion of SOD, GSH-Px, MDA and MMP9 levels in HaCaT cells;

[0033] Fig.14 :Effects of gel mask and cream on the secretion of SOD, GSH-Px, MDA and MMP9 levels in HDF-α cells;

[0034] Fig.15 :HE staining skin histopathology results 1 (200×);

[0035] Fig.16 : HE staining skin histopathological results 2 (200×). DETAILED DESCRIPTION

[0036] The present invention provides an anti-wrinkle composition, which comprises, by weight, 1-10 parts of ginsenoside, 1-10 parts of velvet antler peptide, 15-20 parts of resveratrol, 1-10 parts of cod collagen, 2-8 parts of sodium hyaluronate, 10-15 parts of superoxide dismutase and 10-20 parts of honey; preferably, the anti-wrinkle composition of the present invention comprises 3-6 parts of ginsenoside, 2-8 parts of velvet antler peptide, 16-18 parts of resveratrol, 5-7 parts of cod collagen, 3-5 parts of sodium hyaluronate, 12-14 parts of superoxide dismutase and 15-18 parts of honey.

[0037] The cod collagen described in the present invention includes the peptide segment YGCC (SEQ ID No.1). The present invention processes and extracts cod collagen through an innovative preparation method to obtain cod collagen rich in the peptide segment YGCC (tyrosine-glycine-cysteine-cysteine). The present invention finds that the peptide segment YGCC has a strong free radical scavenging ability. It has been verified by molecular docking that it has a strong binding ability with MAPK1 and MMP9, important targets of skin photodamage, and can regulate the MAPK signaling pathway, FoxO signaling pathway, etc., give full play to the effects of anti-inflammatory, anti-oxidative stress, etc., and has significant antioxidant and anti-aging effects. The present invention effectively improves the anti-wrinkle effect of the composition by improving the preparation process of cod collagen.

[0038] The preparation method of cod collagen protein of the present invention comprises the following steps: washing cod skin with running water, adding sodium hydroxide solution to soak; washing the soaked cod skin, mixing with phosphate buffer, grinding and homogenizing to obtain cod skin homogenate; adding pepsin to the cod skin homogenate, hydrolyzing for 2 to 5 hours, then adding papain and flavor protease, hydrolyzing for 1 to 4 hours to obtain enzymatic solution; the enzymatic solution passes through a ceramic membrane and an organic membrane in sequence to obtain a filtrate; the filtrate passes through a cation exchange resin and an anion exchange resin in sequence, and the material after resin adsorption is concentrated, sterilized and dried to obtain cod collagen protein (crude product).

[0039] The present invention rinses the cod skin with running water to remove surface impurities, and then soaks it in a sodium hydroxide solution to remove fat and foreign proteins contained in the fish skin, and has the effect of killing epidermal microorganisms. The concentration (mass fraction) of the sodium hydroxide solution of the present invention is 0.2% to 1%, preferably 0.5%; the soaking time is 1 to 3 hours, preferably 2 hours; the soaking temperature is 25 to 35° C., preferably 28 to 30° C. The soaking temperature of the present invention is preferably maintained by heating in a water bath.

[0040] The present invention preferably rinses the soaked cod skin with a phosphate buffer to wash away the residual sodium hydroxide solution on the surface, then mixes it with the phosphate buffer, grinds it, and homogenizes it to obtain a cod skin homogenate. The mass volume ratio of the cod skin to the phosphate buffer of the present invention is 1g:5-15mL, preferably 1g:10mL. The grinding is preferably performed by a colloid mill, the grinding time can be selected from 0.5 to 1.5h, preferably 1h, the colloid mill is preferably 3000 to 5000 rpm, and the filter screen is 50 to 60 mesh; the homogenization is preferably performed by a high-pressure homogenizer, and the pressure is 4 to 7Kpa, preferably 5Kpa.

[0041] The present invention performs two-step enzymolysis on the cod skin homogenate, wherein the first step of enzymolysis is to add pepsin for enzymolysis, and the amount of pepsin added is 1 to 2 million U / g based on the mass of the cod skin homogenate. The present invention can optionally adjust the pH of the cod skin homogenate, and add pepsin for enzymolysis after the pH is adjusted to 2 to 4. The first step of enzymolysis temperature of the present invention is preferably 35 to 40°C, more preferably 36 to 37°C; the enzymolysis time is preferably 2 to 5h, more preferably 3 to 4h. The present invention performs the second step of enzymolysis after the first step of enzymolysis, and the amount of papain added is 800,000 to 1.2 million U / g based on the mass of the cod skin homogenate, and the amount of flavor protease added is 1 to 1.5 million U / g. The second step of enzymolysis temperature of the present invention is preferably 50 to 65°C, more preferably 55 to 60°C; the enzymolysis time is preferably 1 to 4h, more preferably 2h.

[0042] The present invention filters the obtained enzymatic hydrolysate, and the enzymatic hydrolysate passes through a ceramic membrane and an organic membrane in sequence, wherein the ceramic membrane is preferably a 28000-30000Da ceramic membrane, and the organic membrane is preferably 1-1.5Kd. The enzymatic hydrolysate of the present invention passes through a ceramic membrane to remove protein macromolecules that have not been enzymatically hydrolyzed. The filtrate of the present invention is filtered through an organic membrane to remove some insoluble impurities, free amino acids, sodium chloride and other small molecules to ensure the purity of the target extract. The organic membrane of the present invention can be optionally a polyethersulfone membrane (PES membrane).

[0043] The present invention sequentially passes the fermentation filtrate through a cation exchange resin and an anion exchange resin to remove heavy metals and other substances in the filtrate. The filtrate preferably passes through the cation exchange resin and the anion exchange resin at a flow rate of 20 to 30 L / h; the cation exchange resin of the present invention is preferably 001×7 resin, and the anion exchange resin is preferably D301 resin. The cation exchange resin and anion exchange resin of the present invention are preferably pretreated before use, and resin regeneration treatment is performed after use.

[0044] The concentration of the present invention is preferably to increase the material concentration to 30-50%, and double-effect falling film concentration can be optionally used.

[0045] The sterilization of the present invention is preferably high-temperature sterilization, the sterilization temperature is 80-90°C, preferably 85°C, and the sterilization time is 10-35 minutes, preferably 20-30 minutes.

[0046] The drying of the present invention is preferably spray drying, and the drying air inlet temperature of the spray drying is 160-170°C, preferably 165°C; the feed rate is 10-11 mL / min, preferably 10.5 mL / min.

[0047] The cod collagen (crude product) obtained in the present invention is separated and purified by a preparative high performance liquid chromatograph, and gradient elution is performed with mobile phase A: water + 0.1% trifluoroacetic acid, and mobile phase B: acetonitrile, and the eluted fraction of 32 to 36 minutes is collected and dried to obtain purified cod collagen. The cod collagen (crude product) described in the present invention is preferably prepared with ultrapure water to obtain a 0.5 to 2 mg / mL solution, which is used as a loading solution, and the loading solution concentration is preferably 1 to 1.5 mg / mL. The gradient elution program of the present invention is: 0 to 5 min 0% to 5% B, 5 to 15 min 5% B to 30% B, 15 to 45 min 30% to 75% B; the drying of the present invention is preferably freeze drying, and the freeze drying conditions are preferably -40°C pre-freezing for 2 to 4 hours, and the cold trap temperature is -70°C freeze drying for 15 to 50 hours.

[0048] The preparation method of the velvet antler peptide of the present invention comprises the following steps: crushing the velvet antler, adding 60% to 80% ethanol solution, and extracting for 2 to 10 minutes under microwave conditions; ultrafiltration of the extract using 10K ultrafiltration membrane and 1K ultrafiltration membrane in sequence, and freeze-drying the ultrafiltrate to obtain velvet antler peptide (freeze-dried powder).

[0049] The present invention pulverizes the antler after drying to obtain antler coarse powder, preferably the antler is quick-frozen by liquid nitrogen, then pulverized at ultra-low temperature, and then sieved through a 100-200 mesh sieve after pulverization. The present invention mixes the obtained antler coarse powder with an ethanol solution and extracts it under microwave conditions. The concentration of the ethanol solution is preferably 70% to 75%, and the mass volume ratio of the antler coarse powder and the ethanol solution is 1g:10-30mL, preferably 1g:15-20mL. The microwave frequency of the present invention is 800-1000Hz, preferably 850-900Hz. The extraction time of the present invention is preferably 5-8min. The present invention extracts and filters to obtain an extract, and the extract is ultrafiltered by a 10K ultrafiltration membrane and a 1K ultrafiltration membrane in sequence to obtain an ultrafiltrate. The ultrafiltrate of the present invention is freeze-dried to obtain antler peptide freeze-dried powder, and the freeze-drying conditions are preferably -40°C pre-freezing for 2-4h, and a cold trap temperature of -70°C freeze-drying for 15-50h.

[0050] The present invention also provides the use of the anti-wrinkle composition in the preparation of anti-wrinkle products. The products of the present invention include skin care products, cosmetics, and cleaning products. The present invention uses the anti-wrinkle composition as the main active ingredient, and those skilled in the art can add other auxiliary materials (such as penetration enhancers, thickeners, moisturizers, fragrances, conditioners, etc.) according to the type of specific products.

[0051] The present invention also provides an anti-wrinkle product, which comprises, by weight, 15 to 25 parts of the anti-wrinkle composition, 5 to 12 parts of a penetration enhancer, 0.1 to 0.5 parts of a thickener, 2 to 6 parts of a moisturizer, and 10 to 80 parts of deionized water, preferably 20 parts of the anti-wrinkle composition, 8 parts of a penetration enhancer, 0.2 parts of a thickener, 4 parts of a moisturizer, and 15 to 75 parts of deionized water. Those skilled in the art can prepare different anti-wrinkle products such as creams, masks, lotions, essences, facial cleansers, etc. according to conventional preparation processes.

[0052] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The experimental data were processed using SPSS 21.0 software. P < 0.05 indicates a significant difference, P < 0.01 indicates a relatively significant difference, and P < 0.001 indicates an extremely significant difference.

[0054] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0055] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0056] Example 1

[0057] An anti-wrinkle composition comprises, by weight, 5 parts of ginsenoside, 8 parts of pilose antler peptide, 18 parts of resveratrol, 7 parts of cod collagen, 5 parts of sodium hyaluronate, 12 parts of superoxide dismutase and 18 parts of honey. The preparation method is as follows:

[0058] 1. Preparation of cod collagen:

[0059] (1) Select cod skin of good quality, without deterioration and odor, rinse it with running water to remove surface impurities and dirt, and then soak it in 0.5% sodium hydroxide solution, heat it in a water bath to maintain the soaking temperature at 30° C., and soak it for 1.5 hours.

[0060] (2) The soaked cod fillets were rinsed with phosphate buffer to remove the sodium hydroxide solution on the surface of the cod skin, and then phosphate buffer was added at a mass volume ratio of 1 g:10 mL. The mixture was ground in a colloid mill (4000 rpm, 50 mesh filter) for 1 h. After grinding, it was treated with a high-pressure homogenizer (pressure 5 Kpa) to obtain a cod skin homogenate.

[0061] (3) After the pH of the cod skin homogenate was adjusted to 3.5, 1.5 million U / g pepsin was added for enzymatic hydrolysis at 37°C for 3 h. After the enzymatic hydrolysis was completed, 1 million U / g papain and 1.2 million U / g flavor protease were added for further enzymatic hydrolysis at 55°C for 2 h.

[0062] (4) The enzymatic hydrolyzate passes through a 30,000Da ceramic membrane and a 1.2Kd polyethersulfone membrane to obtain a filtrate.

[0063] (5) The filtrate passes through cation exchange resin (001×7) and anion exchange resin (D301) in sequence:

[0064] Pretreatment of cation exchange resin: Weigh the cation exchange resin, compact it, transfer it to the resin column with deionized water, and keep 3-5 cm of water on the top of the resin. Pass 4 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 2 times the volume of resin per hour. After the resin is washed with pure water, stop washing when the outlet pH is neutral. Pass 4 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 2 times the volume of resin per hour. After the resin is washed with deionized water, stop washing when the outlet pH is neutral, and set aside.

[0065] Anion exchange resin pretreatment: Weigh the anion exchange resin, compact it, transfer it to the resin column with deionized water, and keep 3-5 cm of water on the top of the resin. Pass 3 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 2 times the volume of resin per hour. After that, wash the resin with pure water until the outlet pH is <9. Then pass 3 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 2 times the volume of resin per hour. After that, wash the resin with deionized water until the outlet pH is >5 and set aside.

[0066] Column passing: The feed liquid passes through the resin column at a flow rate of 25L / h, and the conductivity of the outlet effluent is detected. When the feed liquid concentration is 5%, the conductivity is higher than 500 microSiemens per centimeter (us / cm). If it does not meet the requirements, stop passing the feed liquid.

[0067] (6) The material was concentrated by double-effect falling film concentration to increase the material concentration to 40%, and then the temperature was raised to 85° C. and maintained for 25 min. After sterilization, it was dried at a feed rate of 10.5 mL / min and a drying air inlet temperature of 165° C. The cod skin collagen crude product was obtained by drying.

[0068] (7) The crude cod skin collagen product is separated and purified.

[0069] The crude product of cod skin collagen was prepared with ultrapure water to obtain a 1 mg / mL solution as the loading solution;

[0070] Detection wavelength: 214 nm; flow rate: 0.3 mL / min; time: 45 min; injection volume: 13.5 mL; column temperature: room temperature.

[0071] Chromatographic conditions: preparative liquid chromatography column (550 mm*40 μm, 20 μm), mobile phase A: water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile for gradient elution, elution program: 0-5 min 0%-5% B, 5-15 min 5%-30% B, 15-45 min 30%-75% B; collect the elution fractions of 32-36 min.

[0072] The obtained eluted fraction was pre-frozen at -40°C for 3 h, and freeze-dried at a cold trap temperature of -70°C for 20 h to obtain cod collagen.

[0073] 2. Preparation method of velvet antler peptide:

[0074] (1) Select velvet antlers of good quality, without deterioration and odor, and put them into a liquid nitrogen quick-freezing device to ensure that the velvet antlers are completely immersed in liquid nitrogen until the center temperature of the velvet antlers reaches below -150°C. Then take them out and place them in an ultra-low temperature pulverizer (the pulverizing chamber temperature is -150°C to -80°C, and the speed is 20,000 rpm) for pulverization for 25 minutes. After pulverization, pass through a 200-mesh sieve to obtain velvet antler powder.

[0075] (2) Deer antler powder was mixed with 75% ethanol solution at a mass volume ratio of 1 g:20 mL, extracted under microwave conditions (800 Hz) for 6 min, and filtered to obtain a filtrate.

[0076] (3) The filtrate is ultrafiltered using a 10K ultrafiltration membrane (0.2 MPa) and a 1K ultrafiltration membrane (0.1 MPa) to obtain an ultrafiltrate.

[0077] (4) The ultrafiltrate was pre-frozen at -40°C for 2 h, and freeze-dried at -70°C for 20 h to obtain velvet antler peptide.

[0078] 3. Mix ginsenoside, pilose antler peptide, resveratrol, cod collagen, sodium hyaluronate and superoxide dismutase in proportion, dissolve in deionized water, add honey after complete dissolution, and stir evenly to obtain the anti-wrinkle composition.

[0079] Example 2

[0080] An anti-wrinkle composition comprises, by weight, 1 part of ginsenoside, 10 parts of velvet antler peptide, 15 parts of resveratrol, 10 parts of cod collagen, 2 parts of sodium hyaluronate, 15 parts of superoxide dismutase and 20 parts of honey. The preparation method is as follows:

[0081] 1. Preparation of cod collagen:

[0082] (1) Select cod skin of good quality, without deterioration and odor, rinse it with running water to remove surface impurities and dirt, and then soak it in 0.5% sodium hydroxide solution, heat it in a water bath to maintain the soaking temperature at 35°C, and soak it for 1 hour.

[0083] (2) The soaked cod fillets were rinsed with phosphate buffer to remove the sodium hydroxide solution on the surface of the cod skin, and then phosphate buffer was added at a mass volume ratio of 1 g:15 mL. The mixture was ground in a colloid mill (4000 rpm, 50 mesh filter) for 1.5 h. After grinding, it was treated with a high-pressure homogenizer (pressure 5 Kpa) to obtain a cod skin homogenate.

[0084] (3) After the cod skin homogenate was adjusted to pH 3, 2 million U / g pepsin was added for enzymatic hydrolysis at a temperature of 36°C for 4 h. After the enzymatic hydrolysis was completed, 1 million U / g papain and 1.2 million U / g flavor protease were added for further enzymatic hydrolysis at a temperature of 55°C for 2 h.

[0085] (4) The enzymatic hydrolyzate passes through a 30,000Da ceramic membrane and a 1.2Kd polyethersulfone membrane to obtain a filtrate.

[0086] (5) The filtrate passes through cation exchange resin (001×7) and anion exchange resin (D301) in sequence:

[0087] Pretreatment of cation exchange resin: Weigh the cation exchange resin, compact it, transfer it to the resin column with deionized water, and keep 3 to 5 cm of water on the top of the resin. Pass 3 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 2 times the volume of resin per hour. After the resin is washed with pure water, stop washing when the outlet pH is neutral. Pass 3 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 2 times the volume of resin per hour. After the resin is washed with deionized water, stop washing when the outlet pH is neutral, and set aside.

[0088] Anion exchange resin pretreatment: Weigh the anion exchange resin, compact it, and transfer it to the resin column with deionized water. Keep 3-5 cm of water above the resin. Pass 4 times the volume of resin through the resin column with a 4% sodium hydroxide solution at a flow rate of 2 times the volume of resin per hour. After that, wash the resin with pure water until the outlet pH is <9. Then pass 4 times the volume of resin through the resin column with a 4% hydrochloric acid solution at a flow rate of 2 times the volume of resin per hour. After that, wash the resin with deionized water until the outlet pH is >5 and set aside.

[0089] Column passing: The feed liquid passes through the resin column at a flow rate of 30L / h, and the conductivity of the outlet effluent is detected. When the feed liquid concentration is 5%, the conductivity is higher than 500 microSiemens per centimeter (us / cm). If it does not meet the requirements, stop passing the feed liquid.

[0090] (6) The material was concentrated by double-effect falling film concentration to increase the material concentration to 35%, and then the temperature was raised to 85° C. and maintained for 25 min. After sterilization, it was dried at a feed rate of 10.5 mL / min and a drying air inlet temperature of 165° C. The cod skin collagen crude product was obtained by drying.

[0091] (7) The crude cod skin collagen product is separated and purified.

[0092] The crude product of cod skin collagen was prepared with ultrapure water to obtain a 1 mg / mL solution as the loading solution;

[0093] Detection wavelength: 214 nm; flow rate: 0.3 mL / min; time: 45 min; injection volume: 13.5 mL; column temperature: room temperature.

[0094] Chromatographic conditions: preparative liquid chromatography column (550 mm*40 μm, 20 μm), mobile phase A: water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile for gradient elution, elution program: 0-5 min 0%-5% B, 5-15 min 5%-30% B, 15-45 min 30%-75% B; collect the elution fractions of 32-36 min.

[0095] The obtained eluted fraction was pre-frozen at -40°C for 2 h, and freeze-dried at a cold trap temperature of -70°C for 20 h to obtain cod collagen.

[0096] 2. Preparation method of velvet antler peptide:

[0097] (1) Select velvet antlers of good quality, without deterioration and odor, and put them into a liquid nitrogen quick-freezing device to ensure that the velvet antlers are completely immersed in liquid nitrogen until the center temperature of the velvet antlers reaches below -150°C. Then take them out and place them in an ultra-low temperature pulverizer (the pulverizing chamber temperature is -150°C to -80°C, and the speed is 20,000 rpm) for pulverization for 25 minutes. After pulverization, pass through a 200-mesh sieve to obtain velvet antler powder.

[0098] (2) Deer antler powder was mixed with 70% ethanol solution at a mass volume ratio of 1 g:30 mL, extracted under microwave conditions (800 Hz) for 8 min, and filtered to obtain a filtrate.

[0099] (3) The filtrate is ultrafiltered using a 10K ultrafiltration membrane (0.2 MPa) and a 1K ultrafiltration membrane (0.1 MPa) to obtain an ultrafiltrate.

[0100] (4) The ultrafiltrate was pre-frozen at -40°C for 2 h, and freeze-dried at -70°C for 20 h to obtain velvet antler peptide.

[0101] 3. Mix ginsenoside, pilose antler peptide, resveratrol, cod collagen, sodium hyaluronate and superoxide dismutase in proportion, dissolve in deionized water, add honey after complete dissolution, and stir evenly to obtain the anti-wrinkle composition.

[0102] Example 3

[0103] An anti-wrinkle composition comprises, by weight, 10 parts of ginsenoside, 1 part of pilose antler peptide, 20 parts of resveratrol, 1 part of cod collagen, 2 parts of sodium hyaluronate, 10 parts of superoxide dismutase and 10 parts of honey. The preparation method is the same as that of Example 1.

[0104] Test Example 1

[0105] In this test example, a nanoliter liquid chromatography-Q EXACTIVE mass spectrometry system was used to perform purity and structural identification on the 32-36 min elution fraction obtained in Example 1.

[0106] Chromatographic conditions:

[0107] Mobile phase: Phase A: water + 0.1% formic acid; Phase B: acetonitrile;

[0108] Mobile phase flow rate: 300nl / min; injection volume: 1μL supernatant;

[0109] The elution procedure was: 0%-5% B for 0-5 min, 5%-35% B for 5-25 min, 35%-60% B for 25-35 min, 60%-90% B for 35-40 min, and 90%-100% B for 40-45 min.

[0110] A high-purity polypeptide was obtained from the cod collagen fraction by nanoliter liquid chromatography-Q EXACTIVE mass spectrometry, and its structure was identified as YGCC (tyrosine-glycine-cysteine-cysteine).

[0111] The mass percentage of the obtained YGCC single-chain peptide in the cod collagen mixture was determined by liquid chromatography-mass spectrometry technology to be 1.04%. The YGCC single-chain peptide was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and used as an active ingredient to perform molecular docking using Autodock software to verify the relationship between the target MAPK1, MMP9 and the YGCC molecular structure. The binding energy value of the molecular docking was recorded, and the results are shown in Table 1.

[0112] Table 1 Molecular docking values ​​of YGCC and targets

[0113] Serial number Receptor protein (target) Ligand (active ingredient) Binding energy (kcal / mol) 1 MAPK1 YGCC -6.37 2 MMP9 YGCC -4.89

[0114] The results showed that YGCC had a strong binding ability with the targets MAPK1 and MMP9, indicating that the ligand receptors could bind spontaneously and had a good binding affinity.

[0115] The above molecular docking results were visualized using Pymol software. Figure 1 The drug ligand small molecule (yellow) and the receptor protein are connected to the residues by hydrogen bonds (red), further illustrating that a relatively stable bond can be formed between the core target and its active ingredient, which has a better effect in resisting skin photoaging.

[0116] Test Example 2

[0117] 1. Study on the protective effect of photodamage on HDF-α cells (human dermal fibroblasts) and HaCaT cells (immortalized keratinocytes)

[0118] 1.1 Preparation of drug solution

[0119] Deer antler peptide (prepared in Example 1), cod collagen (prepared in Example 1), ginsenoside, resveratrol, sodium hyaluronate, honey and the anti-wrinkle composition of Example 1 (denoted as MF) were accurately weighed, added to DMEM high-glucose medium to prepare a 1 mg / mL solution, and then diluted to 200, 150, 100, 75, 50, 25 μg / mL solutions respectively; the positive control drug VE was added to DMEM high-glucose medium to prepare a 75 μg / mL solution. Each solution was stored at 4°C.

[0120] 1.2 Cell culture

[0121] After HaCaT cells and HDF-α cells were revived, they were washed with 10% fetal bovine serum and 1 × 10 5 The cells were cultured in DMEM high-glucose medium containing 100 U / L penicillin and 100 mg / L streptomycin at 37°C and 5% CO 2 When the cells adhere to the wall and grow, remove the culture medium, wash with PBS three times, and then add complete medium to continue culturing. When the cells fill 80% of the culture bottle, use 0.25% trypsin to digest and subculture. Subculture once every 2 to 3 days, and take cells in the logarithmic growth phase for experiments.

[0122] 1.3 MTT assay to determine cell viability

[0123] The cultured cells were divided into 2 × 10 4 The suspension was inoculated into a 96-well plate, with 200 μL in each well, and the plate was incubated at 37°C with 5% CO 2After the cells were cultured in an incubator and grown by adhering to the wall, they were divided into the following groups: the drug group (administered with velvet antler peptide, cod collagen, ginsenoside, resveratrol, sodium hyaluronate, honey, anti-wrinkle composition MF and positive control drug VE), the model group and the blank group. The cells in the drug group and the model group were placed under ultraviolet light (UVB) for 10 minutes to establish a light damage model, and the blank group was not irradiated. After the model was successfully established, the drug groups were given different concentrations of the drug solution, and 5 replicates were set up in each group. The culture was continued for 24 hours, and 20 μL of 5g / L MTT solution was added to each well. After 4 hours of culture, the supernatant was discarded, and 150 μL DMSO was added. The cells were shaken for 5 minutes, and the absorbance value (A) was measured at a wavelength of 490 nm. The experiment was repeated 3 times independently, and the cell survival rate of each group was calculated. The results are shown in Tables 2 and 3.

[0124] Cell survival rate (%) = A treatment group / A blank group × 100%.

[0125] Table 2 Effects of each single herb and MF on the survival rate of HDF-α cells

[0126]

[0127]

[0128] Table 3 Effects of each single herb and MF on the survival rate of HaCaT cells

[0129]

[0130] Note: Data are percentages, results are expressed as mean standard deviation, different lowercase letters indicate significant differences.

[0131] The results showed that there was a very significant difference between the blank group and the model group (P < 0.001), indicating that the model was successfully established. After intervention with different concentrations of drugs, the growth ability of HDF-α cells and HaCaT cells in the drug administration group was significantly increased compared with the model group, and it was concentration-dependent. When the MF administration concentration reached 75 μg / mL, the survival rate of HDF-α cells and HaCaT cells reached the highest, which was significantly different from the model group (P < 0.01). At this concentration, the survival rates of light-damaged HDF-α cells and HaCaT cells intervened by each single herb at different levels, but there were significant differences compared with the MF group (P < 0.05). It can be seen that at a low dose concentration of 75 μg / mL, the survival rate of HDF-α cells and HaCaT cells was increased to a certain extent compared with the single herb, and there was no significant difference compared with the positive control group (P > 0.05), showing a good anti-photoaging effect.

[0132] 1.4 ELISA determination of SOD, GSH-Px, MDA and MMP9 contents in HaCaT cells and HDF-α cells

[0133] On the basis of step 1.3, after the MF administration group and the model group established the cell light damage model, different concentrations of MF were added and cultured for 24 hours, and the cell supernatant of each group was collected. The ELISA method was used to detect the content of SOD, GSH-Px, MDA and MMP9 in the cell supernatant. The operation was carried out according to the instructions of the kit, and the absorbance value was measured at 450nm.

[0134] The results of MF determination of the secretion of SOD, GSH-Px, MDA and MMP9 in HaCaT cells are shown in Table 4.

[0135] Table 4 Effects of different doses of MF on the contents of SOD, GSH-Px, MDA and MMP9 in HaCaT cells

[0136]

[0137]

[0138] Compared with the blank group, ## indicates a significant difference (P<0.01); ### indicates an extremely significant difference (P<0.001); compared with the model group, * indicates a significant difference (P<0.05); ** indicates a significant difference (P<0.01); *** indicates an extremely significant difference (P<0.001). The results showed that the secretion levels of SOD and GSH-Px in the HaCaT cell model group were significantly lower than those in the blank group (P<0.001), and the secretion levels of MDA and MMP9 were significantly increased (P<0.01), indicating that UVB radiation can cause photodamage to HaCaT cells. Compared with the model group, when the MF dosage reached 75 μg / mL, the secretion levels of SOD and GSH-Px in the cell supernatant reached the highest level, with mass concentrations of 40.50 ng / mL and 43.63 ng / mL, respectively (P<0.01), and the secretion levels of MDA and MMP9 dropped to the lowest level, decreasing to 1.78 nmol / L and 6.28 ng / mL (P<0.01). This indicates that MF can repair the light damage caused by UVB radiation to HaCaT cells, confirming that MF has a protective effect on light-damaged HaCaT cells.

[0139] The results of MF determination of SOD, GSH-Px, MDA and MMP9 secretion in HDF-α cells are shown in Table 5.

[0140] Table 5 Effects of different doses of MF on the contents of SOD, GSH-Px, MDA and MMP9 in HDF-α cells

[0141]

[0142] Compared with the blank group, ## indicates a significant difference (P<0.01); ### indicates an extremely significant difference (P<0.001); compared with the model group, * indicates a significant difference (P<0.05); ** indicates a significant difference (P<0.01); *** indicates an extremely significant difference (P<0.001). The results showed that compared with the blank group, the secretion levels of SOD and GSH-Px in the HDF-α cell model group were significantly decreased, and the secretion levels of MDA and MMP9 were significantly increased, indicating that the model was successfully established. Compared with the model group, when the dosage reached 75μg / mL, the secretion of SOD and GSH-Px in the cell supernatant reached the best, rising to 38.45ng / mL and 44.06ng / mL; the secretion of MDA and MMP9 was significantly reduced, falling to 1.85nmol / L and 6.87ng / mL, indicating that MF has a significant improvement effect on the photodamage of HDF-α cells.

[0143] 2. Determination of antioxidant capacity in vitro

[0144] 2.1 Preparation of test solution

[0145] Accurately weigh 1.04 g of the anti-wrinkle composition of Example 1 (denoted as MF), and prepare it into a solution with a concentration of 1 mg / mL with a 2% ethanol solution. Then take 25, 50, 100, 200, 400, 600, 800, and 1000 μL of the solution and dilute it with a 2% ethanol solution to 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mg / mL solutions as test solutions, and prepare VE solutions with the same concentration gradient as positive controls.

[0146] 2.2 DPPH free radical scavenging ability determination

[0147] Determine the scavenging ability of MF on DPPH free radicals: weigh 2 mg of DPPH, place it in a 50 mL brown volumetric flask, add anhydrous ethanol to the mark, shake well, and obtain a DPPH solution with a mass concentration of 0.04 mg / mL. Mix 1 mL of the test solution of different mass concentrations and 1 mL of DPPH-anhydrous ethanol solution, then place it away from light for 30 minutes, and measure the absorbance value A at 517 nm. 1 The blank group was replaced with 2% ethanol solution instead of the test solution A. 0 The control group used anhydrous ethanol instead of DPPH anhydrous ethanol solution A 2 VE was used as the positive control group, and each sample was measured three times in parallel. Figure 2 shown.

[0148] DPPH clearance rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%.

[0149] The results showed that the DPPH free radical scavenging rates of MF and VE increased in a dose-dependent manner with the increase of the administration concentration. 50 The values ​​were 0.324 mg / mL and 0.278 mg / mL respectively. When the mass concentration of MF was between 0.4 mg / mL and 1 mg / mL, there was no significant difference in the DPPH free radical scavenging ability compared with VE. This shows that MF has good DPPH free radical scavenging ability within a certain range.

[0150] 2.3 Determination of superoxide anion free radical scavenging ability

[0151] The superoxide anion radical scavenging ability of MF was determined by the pyrogallol auto-oxidation method: 4 mL of 50 mmol / L pH 8.2 Tris-HCl buffer, 1.5 mL of the test solution with different mass concentrations, and 1 mL of 30 mmol / L preheated pyrogallol solution were added to the reaction system, mixed and reacted for 4 minutes; then 0.4 mL of 10 mol / L HCl solution was added to terminate the reaction, and the absorbance A was measured at a wavelength of 325 nm after full wavelength scanning. 1 ; The blank group was replaced with 2% ethanol solution instead of test solution A 0 The control group was treated with distilled water instead of pyrogallol solution A. 2 VE was used as the positive control group, and each sample was measured three times in parallel. Figure 3 shown.

[0152] ·O 2- Clearance rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%.

[0153] The results showed that the higher the concentration of MF, the stronger the scavenging ability, and the scavenging effect was in a dose-effect relationship. 50 The value is 0.143 mg / mL, while the IC 50 The value was 0.087 mg / mL. When the mass concentration of MF was 1 mg / mL, the maximum clearance rate was 72.97±1.64%, which was no significant difference compared with VE.

[0154] 2.4 Hydroxyl radical scavenging ability determination

[0155] Determine the scavenging ability of MF on hydroxyl free radicals: add 9mmol / LFeSO to the reaction system in sequence 4 7H 2 O0.5mL, 9mmol / L salicylic acid-ethanol solution 0.5mL, different concentrations of test solution 2.5mL and 8.8mmol / L H 2 O 20.5 mL of the solution was reacted in a 37°C constant temperature water bath for 30 min, and the absorbance value A was measured at 510 nm. 1 The blank group was replaced with 2% ethanol solution instead of the test solution A. 0 The control group was treated with distilled water instead of H 2 O 2 Solution A 2 VE was used as the positive control group, and each sample was measured three times in parallel. Figure 4 shown.

[0156] OH removal rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%.

[0157] The results showed that MF at different concentrations had the ability to scavenge hydroxyl free radicals. With the increase of the concentration of the test solution, the scavenging rate of hydroxyl free radicals gradually increased. When the MF concentration was 1 mg / mL, the highest scavenging rate was 79.69±1.42%. The IC 50 The value was 0.142 mg / mL, and that of VE was 0.083 mg / mL. Within a certain mass concentration range, the trend of MF's ability to scavenge hydroxyl free radicals was basically consistent with that of VE, showing a good hydroxyl free radical scavenging effect.

[0158] 2.5 Total reducing capacity determination

[0159] The total reducing capacity of MF was determined by potassium ferrocyanide method: 2.5 mL of PBS solution with pH 6.80.1 mol / L and 2.5 mL of 1% potassium ferrocyanide solution were added to the test solution of different concentrations, and mixed for 20 min in a 50°C water bath. 2.5 mL of 10% trichloroacetic acid solution was added to the reaction system, and the above solution was mixed, centrifuged at 3000 r / min for 10 min, and the supernatant was taken. 2.5 mL of supernatant, 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution were fully mixed and reacted for 10 min, and the absorbance value A of the reaction system was measured at 700 nm. 1 VE was used as the positive control group, and each sample was measured three times in parallel. The greater the absorbance, the stronger the reducing ability. Figure 5 shown.

[0160] The results showed that MF can make Fe 3+ Reduction to Fe 2+, the solution changes from yellow to blue. The stronger the reducing ability, the greater the absorbance value. As the concentration of the test solution increases, the absorbance values ​​of MF and VE gradually increase, and the overall trend remains consistent, with good reducing ability. Reducing ability can slow down or terminate free radical reactions. The experimental results show that MF has excellent antioxidant activity.

[0161] 2.6ABTS + Free radical scavenging ability assay

[0162] Determine the scavenging ability of ABTS+ free radicals of MF: Take 25mL 7mmol / LABTS + Solution and 25mL 2.5mmol / LK 2 S 2 O 8 The solutions were mixed in equal volumes and reacted at room temperature in the dark for 12 to 16 hours. After the reaction, the solution was diluted 20 times with anhydrous ethanol to make the absorbance at 734 nm 0.7 ± 0.02 to obtain ABTS. + Working solution: 3.5 mL ABTS + The working solution was mixed with 0.5 mL of sample solution of different concentrations, reacted at room temperature for 6 min, and the absorbance value A was immediately measured at a wavelength of 734 nm. 1 ; The blank group was replaced with 2% ethanol solution instead of test solution A 0 The control group was treated with anhydrous ethanol instead of ABTS + Working fluid A 2 VE was used as the positive control group, and each sample was measured three times in parallel. Figure 6 shown.

[0163] ABTS + Clearance rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%.

[0164] The results showed that as the concentration of the test solution increased, the ABTS + The free radical scavenging ability gradually increases; the IC 50 The value is 0.302mg / mL, and the IC 50 The value is 0.259mg / mL. When the concentration is 0.2mg / mL~1mg / mL, the ABTS of MF + The free radical scavenging ability is better than VE, showing good ABTS + Free radical scavenging effect.

[0165] 3. RT-PCR detection of the effects on the expression of CASP3, MAPK1, SIRT1, and MMP9 proteins in cells

[0166] 3.1 Cell culture

[0167] HaCaT cells and HDF-α cells were cultured at 5×10 4 The cell suspension of 100 μg / mL was inoculated in a 6-well plate and cultured for 24 hours. When the cells adhered to the wall and grew to about 80%, they were placed under ultraviolet light (UVB) to establish a skin cell light damage model, and the blank group was not irradiated. After the model was successfully established, the anti-wrinkle composition (denoted as MF) of Example 1 with different concentrations of 25, 50, and 75 μg / mL was added to the drug group, and VE with a concentration of 75 μg / mL was added to the positive control group. Each group was repeated 3 times, and the cells were collected for detection.

[0168] 3.2 Total RNA extraction

[0169] Extract total RNA from cells using Trizol method: After cell culture, discard the supernatant, place on ice, add 1ml Trizol cell lysis buffer to each well, aspirate after lysis, and place in EP tube. Add 1 / 5 chloroform and mix well, let stand at room temperature for 5min, centrifuge at 4℃, 12000r / min for 15min, collect supernatant, add an equal amount of isopropanol and mix well, let stand at room temperature for 10min, centrifuge at 4℃, 12000r / min for 10min, discard supernatant, add 1mL of 75% ethanol (prepared with DEPC water) to wash the precipitate, centrifuge for 5min, aspirate excess water, dry the precipitate at room temperature for 5min, add 20μLDEPC water to dissolve RNA, and store at -80℃.

[0170] 3.3 RNA Reverse Transcription Process

[0171] The total RNA content was detected by nucleic acid protein analyzer, and the OD of RNA samples was measured. 260 / OD 280 The value was used to detect RNA purity. The optimal ratio was between 1.8 and 2.0. The total RNA was reverse transcribed into cDNA according to the PrimeScriptTM RT-PCR kit and the subsequent PCR experiment was performed.

[0172] 3.4 RT-PCR detection of related gene expression

[0173] According to the results of network pharmacology and molecular docking, the relative expression of mRNA of CASP3, MAPK1, SIRT1 and MMP9 was detected by RT-PCR, using cDNA as template and GAPDH as reference gene according to SYBR Premix Ex Taq TM Ⅱ kit for PCR amplification reaction, the pre-denatured primer sequence is shown in Table 6. After the PCR amplification is completed, the melting curve is analyzed to determine the purity of the product, and the relative quantitative analysis is used—2 -ΔΔCtThe numerical values ​​given by the system were calculated by the method. The results of MF regulating the expression of HaCaT cell-related genes are shown in the following figure. Figure 7 As shown in Figure 2, MF regulates the expression of HDF-α cell-related genes. Figure 8 shown.

[0174] Table 6 PCR primer sequences

[0175]

[0176] The results showed that compared with the blank group, the expression of CASP3 in the model group was significantly decreased (P < 0.01), and the expression of MAPK1 was significantly increased (P < 0.01); after treatment with 75 μg / mL administration, compared with the model group, the expression of CASP3 was significantly increased (P < 0.05), and the expression of its downstream gene MAPK1 was significantly decreased (P < 0.05). The expression levels of CASP3 and MAPK1 were equivalent to those of the positive drug, indicating that MF had significant anti-inflammatory pharmacological activity. In the detection of genes related to the anti-oxidative stress pathway, compared with the blank group, the expression of SIRT1 in the model group was significantly decreased (P < 0.01), and the expression of MMP9 was significantly increased (P < 0.01). After treatment with 75 μg / mL administration, the expression of SIRT1 was significantly increased (P < 0.01), and the expression of MMP9 was significantly decreased (P < 0.01). The expression levels of SIRT1 and MMP9 of MF were closest to those of the positive drug and the positive control group, indicating that MF had significant antioxidant activity.

[0177] Compared with the blank group, the expression of CASP3 in the model group was significantly decreased (P < 0.01), and the expression of MAPK1 was significantly increased (P < 0.01), indicating that the model was successfully established and ultraviolet radiation had a certain photodamage effect on HDF-α cells. Compared with the model group, after treatment with positive drugs and different concentrations of drugs, the expression of CASP3 was significantly increased (P < 0.05), and the expression of its downstream gene MAPK1 was significantly decreased (P < 0.05). When the drug concentration reached 75 μg / mL, MF played an equivalent role to the positive drug, indicating that MF can play an anti-inflammatory role by regulating CASP3 and MAPK1. In the anti-oxidative stress pathway, compared with the blank group, the expression of SIRT1 in the model group was significantly decreased (P < 0.01), and the expression of MMP9 was significantly increased (P < 0.01). After treatment with positive drugs and different concentrations of drugs, the expression of SIRT1 was significantly increased (P < 0.01), and the expression of MMP9 was significantly decreased (P < 0.01). When the drug concentration reached 75 μg / mL, the expression of SIRT1 was higher than that of the positive control group, and the expression of MMP9 was equivalent to that of the positive control group, indicating that MF can play an antioxidant role by regulating SIRT1 and MMP9. It shows that MF can regulate CASP3 / MAPK1 and SIRT1 / MMP9 to play an anti-skin photoaging role.

[0178] Example 4

[0179] An anti-wrinkle product is a facial cream, comprising, by weight, 20 parts of the anti-wrinkle composition of Example 1, 10 parts of a penetration enhancer, 0.2 parts of a thickener, 4 parts of a moisturizer, and 24 parts of deionized water. The penetration enhancer is propylene glycol, the thickener is xanthan gum, and the moisturizer is glycerin.

[0180] Preparation: Divide deionized water into three equal parts.

[0181] Phase A: deionized water, glycerin, propylene glycol, xanthan gum, sodium hyaluronate;

[0182] Phase B: resveratrol, deionized water;

[0183] Phase C: ginsenoside, velvet antler peptide, cod collagen, superoxide dismutase, honey, deionized water.

[0184] Add glycerin, propylene glycol, xanthan gum, sodium hyaluronate and deionized water into an emulsifier, homogenize for 4 minutes, heat to 85°C, stir until completely dissolved, homogenize for 10 minutes, keep warm for 15 minutes, add phase B, stir and cool to below 45°C, add phase C, stir evenly, eliminate bubbles, and obtain an anti-wrinkle cream.

[0185] Example 5

[0186] An anti-wrinkle product is a gel mask, which comprises, by weight, 15 parts of the anti-wrinkle composition of Example 1, 5 parts of a penetration enhancer, 0.1 parts of a thickener, 6 parts of a moisturizer, and 30 parts of deionized water. The penetration enhancer is propylene glycol, the thickener is xanthan gum and carbomer (1:1), and the moisturizer is glycerin.

[0187] Preparation: Divide deionized water into three equal parts.

[0188] Phase A: deionized water, glycerin, propylene glycol, xanthan gum, sodium hyaluronate;

[0189] Phase B: Carbomer;

[0190] Phase C: resveratrol, deionized water;

[0191] Phase D: ginsenoside, velvet antler peptide, cod collagen, superoxide dismutase, honey, and deionized water.

[0192] Glycerin, propylene glycol, xanthan gum, sodium hyaluronate and deionized water were fully stirred to dissolve, and then phase B was slowly added to allow it to fully infiltrate. It was allowed to stand at room temperature for 15 hours to allow it to fully swell and disperse evenly. Resveratrol and deionized water were heated and stirred to dissolve separately, and then added to the system. Phase D was then squeezed in, homogenized for 10 minutes, and kept warm for 15 minutes to eliminate bubbles to obtain a gel mask.

[0193] Test Example 3

[0194] 1. Study on the effect of cosmetics on UV-induced photodamage of human skin cells

[0195] 1.1 Preparation of drug solution

[0196] MF gel mask and MF cream were dissolved in water to prepare freeze-dried powder. The freeze-dried powder was reconstituted with DMEM culture medium to form a 1 mg / mL solution, filtered through a 0.22 μm sterilized microporous filter membrane, and diluted to 200, 150, 125, 100, 75, 50, 25, and 12.5 μg / mL solutions for cell experiments.

[0197] 1.2 Cell culture

[0198] The method steps are the same as 1.2 of Experimental Example 1.

[0199] 1.3 MTT assay to determine cell viability

[0200] The method and steps are the same as those in 1.3 of Experimental Example 1. Effect of gel mask on the survival rate of HaCaT cells Fig. 9 The effect of facial cream on the survival rate of HaCaT cells is shown in Fig.10 The effect of gel mask on the survival rate of HDF-α cells is shown in Fig.11 The effect of facial cream on the survival rate of HDF-α cells is shown in Fig.12 shown.

[0201] The results showed that compared with the blank group, the survival rate of the cells in the model group was significantly reduced (P<0.001), indicating that the model was successfully established. With the increase of the drug concentration, the growth ability of HaCaT cells showed a trend of first increasing and then decreasing. When the dosage of the gel mask reached 75μg / mL, the survival rate of HaCaT cells reached the best, with a value of 74.80±0.77%, which was significantly different from the model group (P<0.01). When the dosage of the cream reached 50μg / mL, the proliferation effect of HaCaT cells was the most significant compared with the model group (P<0.01), and its survival rate was 77.21±1.13%, indicating that the gel mask and cream had a certain protective effect on the photodamage of HaCaT cells. In the range of 25-125μg / mL, the cells proliferated with the increase of the drug mass concentration, indicating that within this concentration range, the gel mask and cream were non-toxic to HaCaT cells, and the synergistic effect of the drug and the matrix exerted its efficacy.

[0202] Compared with the blank group, the survival rate of the cells in the model group was significantly reduced (P<0.001), indicating that the model was successfully established. With the increase of the dosage, the cells showed a trend of first rising and then slightly decreasing. When the concentration of the gel mask was 75μg / mL, the survival rate of HDF-α cells was 77.40±1.40% (P<0.01); when the dosage of the cream reached 75μg / mL, the survival rate of HDF-α cells was 79.08±1.45%. Compared with the model group, its proliferation effect was the most significant (P<0.01), indicating that the gel mask and cream can alleviate the light damage caused by HDF-α cells. When the dosage was between 25 and 100μg / mL, the cell survival rate increased significantly (P<0.05 or P<0.01), indicating that the excipients added in this concentration range were non-toxic, and the gel mask and cream had anti-photoaging effects.

[0203] 1.4 ELISA determination of SOD, GSH-Px, MDA and MMP9 contents in HaCaT cells and HDF-α cells

[0204] The method and steps are the same as 1.4 of Experimental Example 1. The effects of gel mask and cream on the secretion of SOD, GSH-Px, MDA and MMP9 levels of HaCaT cells are as follows Fig.13 The effects of gel mask and cream on the secretion of SOD, GSH-Px, MDA and MMP9 levels in HDF-α cells are shown in Fig.14 In the figure, different dosing concentrations are 12.5, 25, 50, 75, and 100 μg / mL from left to right, N represents gel mask, and S represents cream.

[0205] The results showed that the secretion levels of SOD and GSH-Px in the HaCaT cell model group were significantly decreased compared with the blank group (P<0.001), and the secretion levels of MDA and MMP9 were significantly increased (P<0.001), indicating that UVB radiation can cause photodamage to HaCaT cells. Compared with the model group, when the concentration of the gel mask and cream was 25-100 μg / mL, the secretion levels of SOD and GSH-Px were significantly increased (P<0.05 or P<0.01 or P<0.001); in the range of 25-75 μg / mL, the secretion levels of MDA and MMP9 were significantly reduced (P<0.05 or P<0.01), indicating that the gel mask and cream have certain antioxidant activity and can play a role in delaying skin photoaging.

[0206] Compared with the blank group, the secretion levels of SOD and GSH-Px in the HDF-α cell model group decreased significantly (P<0.001), and the secretion levels of MDA and MMP9 increased significantly (P<0.001), indicating that the model was successfully established. When the concentration of gel mask and cream was 25-100 μg / mL, the secretion levels of SOD and GSH-Px in the cell supernatant were significantly increased compared with the model group (P<0.05 or P<0.01), and the secretion levels of MDA and MMP9 in the cell supernatant were significantly decreased compared with the model group at 25-75 μg / mL (P<0.05 or P<0.01), indicating that the gel mask and cream have a significant improvement effect on the photodamage of HDF-α cells.

[0207] 2. Effect of multifunctional cosmetics on alleviating photoaging of mouse skin

[0208] 2.1 Mouse photoaging modeling experiment

[0209] The long hair on the back of the experimental mice was shaved with a shaver, and the short hair was removed with a depilatory agent. The mice were shaved once every two days and irradiated with a 365nm ultraviolet lamp at a distance of about 25cm from the back of the experimental group mice for 1 hour per day for 40 days to establish a UV damage model in mice.

[0210] 2.2 Experiment on mice using MF cosmetics to alleviate photoaging

[0211] 42 male SPF KM mice (6-8 weeks) were randomly divided into groups, and 6 mice were raised in each group. Under the same environment, regular drinking water and feeding, without adverse factors, the skin photoaging experiment was started after 3 weeks of feeding. During the experiment, the fur color, diet, drinking water and mental activity of the mice were observed daily. The back of the mice in the experimental group was irradiated to establish a UV damage model of mice. Starting from the 21st day of modeling, the mice were given drugs and divided into blank group, model group, positive control group, original drug group, blank matrix group, test gel mask group (high dose: low dose = 2:1) and test cream group (high dose: low dose = 2:1). The specific dosing regimen is shown in Table 7. On the 41st day of the experiment, the mice were fasted for 12 hours and killed by dislocation 1 hour after the last application of the drug. Skin tissue specimens of approximately 2 cm × 2 cm were collected from the hair-depilated area on the back of the mice, the remaining subcutaneous tissue was shaved off, and the samples were washed with physiological saline, fixed with 4% paraformaldehyde, and stored at room temperature.

[0212] Table 7 Dosage regimen for each group

[0213]

[0214]

[0215] 2.3 Pathological sections of mouse skin tissues to alleviate photoaging

[0216] The skin tissue specimens were dehydrated, paraffin-embedded, sliced, and stained with HE. The tissue sections were observed and pathological analysis was performed to evaluate whether the drug had an alleviating effect on the degree of photoaging of the mouse skin. The results of the HE staining skin tissue pathology (200×) are shown in Figure 15-16 As shown, the scale bar in the figure is 100μm.

[0217] The results showed that after HE staining, observation under a light microscope showed that the epidermis of the blank group mice was intact, the squamous epithelial cells were closely arranged, the morphology and structure were normal, the dermis was rich in collagen fibers, and no obvious inflammation was observed; compared with the blank group, the epidermis and shallow dermis of the skin tissue of the model group mice were necrotic in large areas after light exposure, with a large number of necrotic cell fragments (black arrows), and the epidermis at the edge of the necrotic area was significantly thickened; the collagen fibers were disordered in the deep dermis, and there was inflammatory cell infiltration (red arrows), indicating that the model was successful. The skin tissue of the blank matrix group mice had clear structures in each layer, the epidermis cells were closely arranged, and the morphology was regular; the dermis was rich in collagen fibers, and there was no obvious abnormality in the subcutaneous tissue, indicating that the blank matrix had no obvious toxic side effects on the mouse skin. Compared with the model group, the skin tissue of the mice in the combination (FM) group showed mild necrosis in the epidermis and the superficial layer of the dermis, with a small amount of necrotic cell fragments (black arrows), and thickening of the epidermis at the edge of the necrotic area (yellow arrows); a small amount of inflammatory cell infiltration was seen in the dermis (red arrows); a medium area of ​​epidermal thickening was seen in the skin tissue of the mice in the positive control group (black arrows); a small amount of fibroblast proliferation was seen in the superficial layer of the dermis (red arrows), and no obvious inflammatory cell infiltration was seen; a medium area of ​​moderate thickening of the epidermis was seen in the skin tissue of the mice in the low-dose administration group (black arrows), and no other obvious abnormalities were found; a small area of ​​mild thickening of the epidermis was seen in the skin tissue of the mice in the high-dose administration group (black arrows), and no other obvious abnormalities were found. The results showed that compared with the model group, the gel mask group and the cream group could significantly treat the photodamage of the mouse back skin irradiated by UV rays; compared with the composition (FM), the gel mask group and the cream group could increase the residence time of the active substances on the skin surface and improve the efficacy by utilizing the advantages of the dosage form; compared with the positive control group, the effects of the gel mask group and the cream group were comparable to those of VE; compared with the blank group, except for the thickening of the epidermis, there were almost no changes in other skin tissues on the back in the gel mask group and the cream group, indicating that the gel mask and the cream had a certain effect on alleviating photoaging of the mouse skin.

[0218] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-wrinkle composition, characterized in that: Calculated by weight, the invention comprises 1-10 parts of ginsenoside, 1-10 parts of velvet antler peptide, 15-20 parts of resveratrol, 1-10 parts of cod collagen, 2-8 parts of sodium hyaluronate, 10-15 parts of superoxide dismutase and 10-20 parts of honey; the cod collagen comprises the peptide segment YGCC.

2. The anti-wrinkle composition according to claim 1, characterized in that The preparation method of cod collagen comprises the following steps: washing cod skin with running water, adding sodium hydroxide solution to soak; washing the soaked cod skin, mixing with phosphate buffer, grinding and homogenizing to obtain cod skin homogenate; adding pepsin to the cod skin homogenate, hydrolyzing for 2 to 5 hours, then adding papain and flavor protease, hydrolyzing for 1 to 4 hours to obtain enzymatic solution; the enzymatic solution passes through a ceramic membrane and an organic membrane in sequence to obtain a filtrate; the filtrate passes through a cation exchange resin and an anion exchange resin in sequence, and the material after resin adsorption is concentrated, sterilized and dried to obtain cod collagen.

3. The anti-wrinkle composition according to claim 2, characterized in that The concentration of the sodium hydroxide solution is 0.2% to 1%; the soaking time is 1 to 3 hours; and the soaking temperature is 25 to 35°C.

4. The anti-wrinkle composition according to claim 2, characterized in that The mass volume ratio of the fish skin to the phosphate buffer is 1 g:5-15 mL.

5. The anti-wrinkle composition according to claim 2, characterized in that: Based on the mass of the cod skin homogenate, the added amount of pepsin is 1-2 million U / g, the added amount of papain is 800,000-1.2 million U / g, and the added amount of flavor protease is 1-1.5 million U / g.

6. The anti-wrinkle composition according to any one of claims 2 to 5, characterized in that: The cod collagen is separated and purified by using a preparative high performance liquid chromatograph, with mobile phase A: water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile for gradient elution, and the elution fraction of 32 to 36 minutes is collected and dried to obtain the purified cod collagen.

7. The anti-wrinkle composition according to claim 6, characterized in that The gradient elution program is: 0% to 5% B from 0 to 5 min, 5% B to 30% B from 5 to 15 min, and 30% to 75% B from 15 to 45 min.

8. The anti-wrinkle composition according to claim 1, characterized in that The preparation method of the velvet antler peptide comprises the following steps: crushing the velvet antler, adding 60% to 80% ethanol solution, and extracting for 2 to 10 minutes under microwave conditions; ultrafiltration of the extract using a 10K ultrafiltration membrane and a 1K ultrafiltration membrane in sequence, and freeze-drying the ultrafiltrate to obtain the velvet antler peptide.

9. Use of the anti-wrinkle composition according to any one of claims 1 to 8 in the preparation of anti-wrinkle and anti-aging products.

10. An anti-wrinkle and anti-aging product, characterized in that: Calculated by weight, the composition comprises 15 to 25 parts of the anti-wrinkle composition according to any one of claims 1 to 8, 5 to 12 parts of a penetration enhancer, 0.1 to 0.5 parts of a thickener, 2 to 6 parts of a moisturizer and 10 to 80 parts of deionized water.

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