Composition for improving darkness and dryness of sensitive skin as well as preparation method and application of composition

Abstract

The invention relates to a composition for improving darkness and dryness of sensitive skin and a preparation method and application thereof, and belongs to the technical field of cosmetics. The high-purity silk fibroin is prepared by adopting an ultrasonic-assisted enzymolysis method, the activity and purity of the silk fibroin are improved through combination of enzymolysis and ultrasonic waves, and dual improvement of transdermal absorption and formula compatibility is achieved through a peptidation technology. The composition for improving the darkness and dryness of the sensitive skin, provided by the invention, comprises the silk fibroin, the Ectoine, the oxyresveratrol and the anthemis nobilis extract prepared by the invention, and the four components are matched with one another, so that the composition can be used for improving the darkness and dryness of the sensitive skin in a precise synergistic manner from multiple dimensions such as moisturizing, skin color brightening, anti-inflammation, soothing and the like, and the problem of darkness and dryness of the sensitive skin is solved; and an efficient and safe new scheme is provided for sensitive skin nursing.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a composition for improving dullness and dryness of sensitive skin, its preparation method, and its application. Background Technology

[0002] Currently, many products in the industry simply mix various common moisturizing and whitening ingredients, lacking targeted and synergistic combinations. This fails to create an effective synergistic mechanism, failing to fully leverage the advantages of each ingredient. Consequently, their effectiveness in improving sensitive skin is limited, often failing to precisely address dullness and dryness. Furthermore, products manufactured using conventional processes often suffer from poor stability and permeability of active ingredients, making them difficult to absorb effectively onto the skin's surface and significantly reducing product efficacy. Moreover, some traditional ingredients may irritate sensitive skin, triggering allergic reactions and further exacerbating skin problems, thus failing to meet the needs of sensitive skin individuals for safe and highly effective skincare products.

[0003] With the continuous growth of consumer demand for "safe, efficient, and environmentally friendly" skincare, the demand for natural active ingredients in sensitive skin care has exploded. Silk fibroin, as a natural protein, possesses excellent biocompatibility and multiple skincare benefits, aligning with the trend of consumption upgrading. However, traditional extraction methods struggle to obtain high-purity silk fibroin with well-preserved activity. Therefore, developing a method for efficiently and environmentally friendly preparation of high-purity silk fibroin is of great significance. This will provide a higher-quality raw material for compositions that improve dullness and dryness in sensitive skin, thereby enhancing the overall performance and skincare effects of the product. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition for improving dullness and dryness of sensitive skin, its preparation method and application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a composition for improving dullness and dryness of sensitive skin, comprising the following components in parts by weight: 0.01-1 parts silk fibroin, 0.1-5 parts oxidized resveratrol, 0.1-5 parts ectoine, and 0.01-0.2 parts white chrysanthemum extract; wherein the silk fibroin is prepared from silkworm cocoons by ultrasonic-assisted enzymatic hydrolysis.

[0006] The composition provided by this invention for improving dullness and dryness in sensitive skin utilizes silk fibroin, which, with its highly hygroscopic polar amino acids, forms a moisturizing hydration film on the skin to lock in moisture, while ectoine constructs a "hydration shell" around cells to stabilize the barrier. The two work synergistically to lock in moisture from the outside and stabilize cells from the inside, significantly increasing the moisture content of sensitive skin, enhancing its moisturizing ability, and relieving tightness and peeling caused by dryness. This keeps the skin hydrated and soft, and long-term use can gradually repair the damaged skin barrier and enhance the skin's self-protective ability.

[0007] Oxidized resveratrol is a powerful antioxidant that neutralizes free radicals, reduces damage to skin cells, inhibits lipid peroxidation, and prevents skin from becoming dull due to oxidation. The amino acids tyrosine and tryptophan in silk fibroin can absorb ultraviolet light (UVA / UVB), block tyrosinase activity, and reduce melanin production. White chamomile flower extract contains various flavonoids and terpenes, possessing anti-inflammatory, antioxidant, and skin-soothing properties. It can reduce skin inflammation, prevent post-inflammatory hyperpigmentation, and further help brighten skin tone. These three ingredients work synergistically, addressing antioxidant activity, inhibiting melanin production, and reducing inflammatory hyperpigmentation to achieve a brightening effect on skin tone.

[0008] In addition to its moisturizing and cell-stabilizing effects, ectoine can regulate the skin's immune response, reduce skin sensitivity to external stimuli, and alleviate allergy symptoms. White chamomile flower extract has excellent anti-inflammatory properties; it can inhibit the release of inflammatory mediators and relieve discomfort such as redness, swelling, and itching. The mild nature and low immunogenicity of silk fibroin ensure it will not cause additional irritation to sensitive skin, while its cell-repairing properties also help reduce inflammation-induced skin damage. These three ingredients work together to exert anti-inflammatory and soothing effects, effectively reducing sensitivity reactions in sensitive skin.

[0009] Furthermore, this invention employs an enzymatic hydrolysis method combined with ultrasound-assisted extraction to prepare high-purity silk fibroin. Enzymatic hydrolysis, under relatively mild conditions, utilizes specific enzymes to precisely sever the bonds between silk fibroin and other substances, preserving its active structure to the greatest extent. Ultrasound-assisted extraction utilizes the cavitation and mechanical effects of ultrasound to accelerate the dissolution and diffusion of silk fibroin by the solvent, improving extraction efficiency and simultaneously helping to remove impurities, resulting in high-purity silk fibroin.

[0010] In summary, the composition of this invention, through the synergistic effect of multiple components and a unique preparation process, precisely improves the dullness and dryness of sensitive skin from multiple dimensions, providing a highly efficient and safe new solution for sensitive skin care.

[0011] Furthermore, the composition for improving dull and dry sensitive skin comprises the following components in parts by weight: 0.1-0.6 parts silk fibroin, 0.5-3 parts oxidized resveratrol, 1-3 parts ectoine, and 0.05-0.15 parts white chamomile flower extract.

[0012] Preferably, the composition for improving dull and dry sensitive skin comprises the following components in parts by weight: 0.2-0.3 parts silk fibroin, 1-1.2 parts oxidized resveratrol, 1.5-1.6 parts ectoine, and 0.09-0.11 parts white chrysanthemum extract.

[0013] Furthermore, the method for preparing the silk fibroin includes the following steps: S1. Add the cleaned and shredded silkworm cocoons to a Na2CO3 solution for boiling water bath treatment, rinse with distilled water until the rinsing solution is neutral, and obtain clean silkworm cocoon particles. Add the clean silkworm cocoon particles to a phosphate buffer solution and soak until the silk fibers swell. Take out the soaked silkworm cocoon particles, grind them, and obtain the reaction substrate. S2. First stage of enzymatic hydrolysis: Add 1%-3% alkaline protease solution according to the mass of the reaction substrate, adjust the pH to 9.0-11.0, react at 45-55℃ for 1-2 hours, and after the reaction, inactivate the enzyme in the hydrolysate to obtain the first hydrolysate; S3. Second stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 1%-2% neutral protease solution and 1%-2% trypsin solution to the first enzymatic hydrolysate, adjust the pH to 7.0-8.0, and sonicate at 40-50℃ for 1-3 hours. After completion, inactivate the enzyme in the enzymatic hydrolysate to obtain the second enzymatic hydrolysate. S4. Third stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 2%-4% papain solution to the second enzymatic hydrolysate, adjust the pH to 6.0-7.0, sonicate at 50-60℃ for 1-2 hours, and then inactivate the enzyme in the enzymatic hydrolysate to obtain the third enzymatic hydrolysate. S5. Centrifuge the third enzyme hydrolysate to obtain the supernatant. Then, subject the supernatant to salting out, dialysis, and freeze-drying to obtain silk fibroin.

[0014] Preferably, step S1 involves adding the cleaned and shredded silkworm cocoons to a 0.4-0.6 wt% Na2CO3 solution at a material-to-liquid ratio of 1 g:(20-30) mL, treating with a boiling water bath for 25-35 min, discarding the alkali solution, and washing the silkworm cocoon particles with distilled water at 55-65℃ until the washing solution is neutral; then adding the washed silkworm cocoon particles to a 0.2-0.4 wt% Na2CO3 solution at a material-to-liquid ratio of 1 g:(20-30) mL, treating with a boiling water bath for 15-25 min, rinsing with distilled water until the rinsing solution is neutral, obtaining clean silkworm cocoon particles; adding the clean silkworm cocoon particles to a phosphate buffer solution, soaking at 0-4℃ for 10-15 h, removing the soaked silkworm cocoon particles, crushing them, and obtaining the reaction substrate.

[0015] Preferably, the boiling water bath temperature in step S1 is 90±2℃.

[0016] Preferably, the pH of the phosphate buffer solution in step S1 is 6.5-7.5, and the mass ratio of silkworm cocoon particles to phosphate buffer solution is 1:15-20.

[0017] Preferably, the alkaline protease solution is a borate-sodium hydroxide buffer solution containing 4-6 g / L alkaline protease, wherein the pH value of the borate-sodium hydroxide buffer solution is 9.0.

[0018] Preferably, the neutral protease solution is a phosphate buffer containing 2-4 g / L neutral protease, and the trypsin solution is a phosphate buffer containing 1.5-3 g / L trypsin, wherein the pH of the phosphate buffer is 7.5.

[0019] Preferably, the papain solution is a disodium hydrogen phosphate-citric acid buffer containing 2-4 g / L papain, wherein the pH of the disodium hydrogen phosphate-citric acid buffer is 6.0.

[0020] Preferably, the method for inactivating the enzyme in the enzymatic hydrolysate in steps S2-S4 is as follows: after completion, heat to 80-90℃ and maintain for 10-15 minutes.

[0021] Preferably, the conditions for ultrasound in step S3 are as follows: pulse ultrasound mode is used, the ultrasound power is set to 100-200W, the frequency is 40kHz, and the ultrasound is performed for 5-10 seconds followed by a pause of 5-10 seconds.

[0022] Secondly, the present invention provides the use of the composition for improving dullness and dryness of sensitive skin as described in the first aspect in the preparation of cosmetics for improving dullness and dryness of sensitive skin.

[0023] Furthermore, the cosmetics include one of the following: toner, lotion, cream, mask, or spray.

[0024] Preferably, the cosmetic is a lotion.

[0025] Thirdly, the present invention provides an emulsion comprising the following components in weight percentage: 0.05%-0.5% thickener, 1%-10% moisturizer, 0.01%-0.3% pH adjuster, 1%-6% emulsifier, 5%-15% oil, 0.5%-3% preservative, 1%-10% of the composition described in the first aspect for improving dullness and dryness of sensitive skin, 0.01%-0.5% chelating agent, and the balance being deionized water.

[0026] Further, the emulsion includes at least one of the following: (a)-(e) (a) The thickener comprises at least one of xanthan gum, carbomer, hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer, ammonium acryloyl dimethyl taurate / VP copolymer, hydroxyethyl cellulose, and cetyl alcohol; (b) The moisturizer includes at least one of allantoin, sodium polyacrylate, panthenol, beta-glucan, trehalose, caprylyl glycol, dipropylene glycol, sodium hyaluronate, 1,2-butanediol, glycerin, and tremella polysaccharide; (c) The pH adjuster includes at least one of arginine, citric acid, and NaOH; (d) The emulsifier includes at least one of PEG-100 glyceryl stearate, glyceryl stearate citrate, cetearyl glucoside, cetearyl alcohol, PEG-20 methyl glucose sesquistearate, methyl glucose sesquistearate, cetyl phosphate potassium, and sodium stearoyl glutamate; (e) The oils include at least one of caprylic / capric triglyceride, polydimethylsiloxane, jojoba oil, grape seed oil, meadowfoam seed oil, squalane, macadamia nut oil, and camellia oil.

[0027] (f) The preservative includes at least one of 1,2-hexanediol, 1,2-pentanediol, ethylhexylglycerin, p-hydroxyacetophenone, phenoxyethanol, octanoyl hydroxamic acid, and sodium benzoate.

[0028] (g) The chelating agent is EDTA-2Na.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention employs an ultrasound-assisted enzymatic hydrolysis method to prepare high-purity silk fibroin. Enzymatic hydrolysis, under relatively mild conditions, utilizes specific enzymes to precisely sever the bonds between silk fibroin and other substances, preserving its active structure to the greatest extent. Ultrasound-assisted extraction utilizes the cavitation and mechanical effects of ultrasound to accelerate the dissolution and diffusion of silk fibroin by the solvent, improving extraction efficiency and simultaneously helping to remove impurities, resulting in high-purity silk fibroin.

[0030] 2. In the composition for improving dullness and dryness of sensitive skin provided by the present invention, the four components prepared by the present invention—silk fibroin, ectoine, oxidized resveratrol, and white chamomile extract—work together synergistically to precisely improve the problem of dullness and dryness of sensitive skin from multiple dimensions such as moisturizing, brightening skin tone, and anti-inflammatory soothing, providing a highly efficient and safe new solution for sensitive skin care. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] In the following embodiments, the materials are sourced from: Oxidized resveratrol was purchased from Maclean's, catalog number O860708-100mg; Ectocin was purchased from Guangzhou Muran Biochemical Technology Co., Ltd., product number E305040-1g; The extract of white chrysanthemum was purchased from Shaanxi Sinote Biotechnology Co., Ltd., and its trade name is white chrysanthemum extract. The alkaline protease was purchased from Maclean's, catalog number P750258, with an enzyme activity of 7-15 U / mg; Papain was purchased from Maclean's, product number P6321-25g, with an enzyme activity ≥10 U / mg; The neutral protease was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., product number D64920-500g, with an enzyme activity of 100U / mg. Trypsin was purchased from Guangzhou Jetway Biotechnology Co., Ltd., catalog number KGA1519-5, with an enzyme activity ≥250.0 U / mg; Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0033] The preparation methods of the enzyme solutions described in the following examples and comparative examples are as follows: Alkaline protease solution: Weigh a certain amount of alkaline protease and prepare an enzyme solution with an enzyme concentration of 5 g / L using a borate-sodium hydroxide buffer solution at pH 9.

[0034] Papain solution: Weigh a certain amount of papain and prepare an enzyme solution with an enzyme concentration of 3 g / L using disodium hydrogen phosphate-citric acid buffer at pH 6.

[0035] Neutral protease solution: Weigh a certain amount of neutral protease and prepare an enzyme solution with a concentration of 4 g / L using phosphate buffer at pH 7.5.

[0036] Trypsin solution: Weigh a certain amount of trypsin and prepare an enzyme solution with an enzyme concentration of 2 g / L using phosphate buffer at pH 7.5.

[0037] Example 1 This embodiment provides a method for preparing silk fibroin 1, including the following steps: S1. Cut the silkworm cocoons into small pieces with scissors, place them in a beaker, and wash them three times with ultrapure water. After each wash, drain the water to remove surface impurities. Cut the washed silkworm cocoons into smaller pieces and add them to a 0.5wt% Na2CO3 solution at a material-to-liquid ratio of 1g:25mL. Treat in a boiling water bath at 90±2℃ for 30min, discard the alkaline solution, and wash the silkworm cocoon particles with distilled water at 60℃ until the washing solution is neutral. Then add them to a 0.3wt% Na2CO3 solution at a material-to-liquid ratio of 1g:25mL and treat in a boiling water bath at 90±2℃ for 20min. Rinse with distilled water until the rinsing solution is neutral to obtain clean silkworm cocoon particles. Add the clean silkworm cocoon particles to 18 times their weight of a phosphate buffer solution with a pH of 7.0 and soak at 4℃ for 12h to allow the silk fibers to fully swell. Remove the soaked silkworm cocoon particles, grind them, and obtain the reaction substrate. S2. First stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 2% alkaline protease solution to the reaction substrate, adjust the pH of the system to 10.0, react at 50℃ for 1.5 h, and after the reaction is completed, raise the temperature to 85℃ and hold for 12 min to inactivate the enzyme, and obtain the first enzymatic hydrolysate; S3. Second stage enzymatic hydrolysis: Based on the mass of the reaction substrate, add 1.5% neutral protease solution and 1.5% trypsin solution to the first enzymatic hydrolysate, adjust the pH of the system to 7.5, and sonicate at 45℃ for 2 hours. After completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, obtaining the second enzymatic hydrolysate. The sonication is performed using pulsed ultrasound, with the ultrasound power set to 120W and the frequency set to 40kHz, with a 10s sonication interval followed by a 5s pause. S4. Third stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 3% papain solution to the second enzymatic hydrolysate, adjust the pH of the system to 6.5, sonicate at 55℃ for 1 hour, and after completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, to obtain the third enzymatic hydrolysate; the sonication is carried out in pulsed sonication mode, with the sonication power set to 120W and the frequency set to 40kHz, with a 10s sonication interval followed by a 5s pause; S5. Centrifuge the third enzymatic hydrolysate at 8000 r / min for 20 min, take the supernatant, and then perform salting out, dialysis and freeze-drying on the supernatant to obtain silk fibroin. The salting-out method is as follows: take the supernatant, slowly add ammonium sulfate powder to 70% saturation, let it stand at 4℃ for 12 hours, then centrifuge at 8000 r / min and 4℃ for 20 minutes, retain the precipitate, and obtain the preliminarily purified silk fibroin. The dialysis method is as follows: the preliminarily purified silk fibroin obtained by salting out is dissolved in deionized water at a material-to-liquid ratio of 1g:8mL, and placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa. Dialysis is performed with deionized water at 4°C, and the dialysis solution is changed every 8 hours until no white precipitate is found in the dialysis solution when tested with barium chloride (indicating that sulfate ions have been completely removed).

[0038] The freeze-drying method is as follows: the solution obtained after dialysis is poured into the sample tray of a freeze dryer, the condenser is set to a cooling temperature of -65°C, and dried until no moisture is present, resulting in a spongy white silk fibroin 1 with a loose and porous surface.

[0039] Example 2 This embodiment provides a method for preparing silk fibroin 2, which differs from Example 1 in that steps S2-S4 are specifically as follows: S2. First stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 3% alkaline protease solution to the reaction substrate, adjust the pH to 11.0, react at 55℃ for 2 hours, and after the reaction is completed, raise the temperature to 85℃ and hold for 12 minutes to inactivate the enzyme, and obtain the first enzymatic hydrolysate; S3. Second stage enzymatic hydrolysis: Based on the mass of the reaction substrate, add 2% neutral protease solution and 2% trypsin solution to the first enzymatic hydrolysate, adjust the pH of the system to 8.0, and sonicate at 50℃ for 3 hours. After completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, obtaining the second enzymatic hydrolysate. The sonication is performed using pulsed ultrasound, with the ultrasound power set to 120W and the frequency set to 40kHz, with an 8-second sonication interval followed by an 8-second pause. S4. Third stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 4% papain solution to the second enzymatic hydrolysate, adjust the pH to 7.0, and sonicate at 60℃ for 1.5h. After the end of the process, raise the temperature to 88℃ and hold for 12min to inactivate the enzyme, thus obtaining the third enzymatic hydrolysate. The sonication is performed using pulsed sonication, with the sonication power set to 120W and the frequency set to 40kHz, with an 8s interval between sonications and an 8s pause.

[0040] The remaining steps and parameters are the same as in Example 1, and silk fibroin 2 is prepared.

[0041] Example 3 This embodiment provides a method for preparing silk fibroin 3, which differs from Example 1 in that steps S2-S4 are specifically as follows: S2. First stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 1% alkaline protease solution to the reaction substrate, adjust the pH to 9.0, react at 45℃ for 1 hour, and after the reaction is completed, raise the temperature to 85℃ and hold for 12 minutes to inactivate the enzyme, and obtain the first enzymatic hydrolysate; S3. Second stage enzymatic hydrolysis: Based on the mass of the reaction substrate, add 1% neutral protease solution and 1% trypsin solution to the first enzymatic hydrolysate, adjust the pH to 7.0, and sonicate at 40℃ for 1 hour. After completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, obtaining the second enzymatic hydrolysate. The sonication is performed using pulsed ultrasound, with the ultrasound power set to 120W and the frequency to 40kHz, with a 5-second sonication interval followed by a 10-second pause. S4. Third stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 2% papain solution to the second enzymatic hydrolysate, adjust the pH to 6.0, sonicate at 50℃ for 0.5h, and after completion, raise the temperature to 88℃ and hold for 12min to inactivate the enzyme, to obtain the third enzymatic hydrolysate; the sonication is performed using pulsed sonication mode, with the sonication power set to 120W and the frequency set to 40kHz, with a 5s sonication interval followed by a 10s pause.

[0042] The remaining steps and parameters are the same as in Example 1, and silk fibroin 3 is prepared.

[0043] Comparative Example 1 The difference between this comparative example and Example 1 is that the first stage of enzymatic hydrolysis in step S2 is not performed, while the remaining steps and parameters are the same as in Example 1, and silk fibroin 1' is prepared.

[0044] Comparative Example 2 The difference between this comparative example and Example 1 is that the second-stage enzymatic hydrolysis in step S3 is not performed, while the remaining steps and parameters are the same as in Example 1, and silk fibroin 2' is prepared.

[0045] Comparative Example 3 The difference between this comparative example and Example 1 is that the third stage of enzymatic hydrolysis in step S4 is not performed. All other steps and parameters are the same as in Example 1, and silk fibroin 3' is prepared.

[0046] Comparative Example 4 The difference between this comparative example and Example 1 lies in the change of the enzymatic hydrolysis order in steps S2-S4. Specifically, the reaction parameters for the enzymatic hydrolysis stages in steps S2-S4 are as follows: S2. First stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 3% papain solution to the reaction substrate, adjust the pH of the system to 6.5, sonicate at 55℃ for 1 hour, and after completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, to obtain the first enzymatic hydrolysate; the sonication is carried out in pulsed sonication mode, with the sonication power set to 120W and the frequency set to 40kHz, with a 10s sonication interval followed by a 5s pause; S3. Second stage enzymatic hydrolysis: Based on the mass of the reaction substrate, add 1.5% neutral protease solution and 1.5% trypsin solution to the first enzymatic hydrolysate, adjust the pH of the system to 7.5, and sonicate at 45℃ for 2 hours. After completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, obtaining the second enzymatic hydrolysate. The sonication is performed using pulsed ultrasound, with the ultrasound power set to 120W and the frequency set to 40kHz, with a 10s sonication interval followed by a 5s pause. S4. Third stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 2% alkaline protease solution to the second enzymatic hydrolysate, adjust the pH of the system to 10.0, react at 50℃ for 1.5 h, and after the reaction is completed, raise the temperature to 85℃ and hold for 12 min to inactivate the enzyme, and obtain the third enzymatic hydrolysate; The remaining steps and parameters are the same as in Example 1, and silk fibroin 4' is prepared.

[0047] Comparative Example 5 The difference between this comparative example and Example 1 is that trypsin solution is not added in step S3, and the missing amount is made up with neutral protease solution. The remaining steps and parameters are the same as in Example 1, and silk fibroin 5' is prepared.

[0048] Comparative Example 6 The difference between this comparative example and Example 1 is that neutral protease solution is not added in step S3, and the missing amount is made up with trypsin solution. The remaining steps and parameters are the same as in Example 1, and silk fibroin 6' is prepared.

[0049] Comparative Example 7 This comparative example demonstrates the preparation of silk fibroin using traditional enzymatic hydrolysis methods. Ultrasound was not used in any of the three enzymatic hydrolysis stages (S2-S4). Specifically: S2. First stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 2% alkaline protease solution to the reaction substrate, adjust the pH of the system to 10.0, react at 50℃ for 1.5 h, and after the reaction is completed, raise the temperature to 85℃ and hold for 12 min to inactivate the enzyme, and obtain the first enzymatic hydrolysate; S3. Second stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 1.5% neutral protease solution and 1.5% trypsin solution to the first enzymatic hydrolysate, adjust the pH of the system to 7.5, react at 45℃ for 2 hours, and after the reaction is completed, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, and obtain the second enzymatic hydrolysate; S4. Third stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 3% papain solution to the second enzymatic hydrolysate, adjust the pH of the system to 6.5, react at 55℃ for 1 hour, and after the reaction is completed, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, and obtain the third enzymatic hydrolysate; The remaining steps and parameters are the same as in Example 1, and silk fibroin 7' is prepared.

[0050] Comparative Example 8 The difference between this comparative example and Example 1 is that all three enzymatic hydrolysis stages employ ultrasound-assisted enzymatic hydrolysis, specifically: S2. First stage enzymatic hydrolysis: Based on the mass of the reaction substrate, add 2% alkaline protease solution to the reaction substrate, adjust the pH of the system to 10.0, and sonicate at 50℃ for 1.5 h. After the reaction, raise the temperature to 85℃ and hold for 12 min to inactivate the enzyme, obtaining the first enzymatic hydrolysate; the sonication is performed using pulsed sonication, with the sonication power set to 120W and the frequency to 40kHz, with a 10s sonication interval followed by a 5s pause; S3. Second stage enzymatic hydrolysis: Based on the mass of the reaction substrate, add 1.5% neutral protease solution and 1.5% trypsin solution to the first enzymatic hydrolysate, adjust the pH of the system to 7.5, and sonicate at 45℃ for 2 hours. After completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, obtaining the second enzymatic hydrolysate. The sonication is performed using pulsed ultrasound, with the ultrasound power set to 120W and the frequency set to 40kHz, with a 10s sonication interval followed by a 5s pause. S4. Third stage of enzymatic hydrolysis: Based on the mass of the reaction substrate, add 3% papain solution to the second enzymatic hydrolysate, adjust the pH of the system to 6.5, sonicate at 55℃ for 1 hour, and after completion, raise the temperature to 88℃ and hold for 12 minutes to inactivate the enzyme, to obtain the third enzymatic hydrolysate; the sonication is carried out in pulsed sonication mode, with the sonication power set to 120W and the frequency set to 40kHz, with a 10s sonication interval followed by a 5s pause; The remaining steps and parameters are the same as in Example 1, and silk fibroin 8' is prepared.

[0051] Test Example 1: In vitro antioxidant DPPH free radical scavenging assay of silk fibroin Experimental principle: Silk fibroin contains amino acid residue side chain groups with antioxidant activity. By comparing the DPPH free radical scavenging rate of silk fibroin obtained by different extraction methods, the higher the scavenging rate, the more completely the active ingredients of the prepared silk fibroin are retained.

[0052] The test method is as follows: Preparation of DPPH solution: Weigh 4 mg of DPPH powder, dissolve it in 60% v / v ethanol aqueous solution, and make up to 100 mL to prepare a DPPH solution with a concentration of 40 mg / L. Store in the dark. Sample solution preparation: Using silk fibroin from Examples 1-3 and Comparative Examples 1-8 as samples, weigh the corresponding samples, dissolve and dilute with 60% v / v ethanol aqueous solution to prepare a sample solution with a concentration of 5 mg / mL. Preparation of positive group solution: Using vitamin C as the positive sample, dissolve and dilute with 60% v / v ethanol and water to prepare a positive solution with a concentration of 1 mg / mL.

[0053] b. Experimental Grouping: Blank control group (A0): 2 mL sample solution + 2 mL 60% v / v ethanol aqueous solution; Sample group (A1): 2 mL sample solution + 2 mL DPPH solution; Sample matrix group (A2): 2 mL DPPH solution + 2 mL 60% v / v ethanol aqueous solution; Reaction: After mixing each group evenly, let it stand in the dark for 30 min, centrifuge at 5000 r / min for 10 min, take the supernatant, and measure the absorbance at a wavelength of 517 nm.

[0054] c. Parallel experiments and replication Three parallel tubes were set up for each sample, and the average value of the results was taken.

[0055] d. Data calculation and result analysis The formula for DPPH free radical scavenging rate is: Scavenging rate (%) = [1 - (A1 - A0) / A2] × 100%; the results are shown in Table 1.

[0056] As can be seen from the data in Examples 1-3 of Table 1, the silk fibroin prepared by the preparation method provided in Examples 1-3 of this invention has significant antioxidant activity, indicating that the active components of the prepared silk fibroin are well preserved and have high activity. Comparing the data of Comparative Examples 1-4 with that of Example 1, it can be seen that the three enzymatic hydrolysis stages—alkaline protease hydrolysis, mixed hydrolysis of neutral protease and trypsin, and papain hydrolysis—and the order of hydrolysis significantly affect the preservation of the activity of the prepared silk fibroin. Comparing the data of Comparative Examples 5-6 with that of Example 1, it can be seen that the mixed hydrolysis of neutral protease and trypsin in the second hydrolysis stage can synergistically improve the antioxidant activity of the prepared silk fibroin. Comparing the data of Comparative Examples 7-8 with that of Example 1, the absence of ultrasound-assisted hydrolysis and the combination of ultrasound-assisted hydrolysis in all three stages have adverse effects on the antioxidant activity of the prepared silk fibroin; combining ultrasound-assisted hydrolysis only in the second and third stages has the best effect on preserving the activity of the prepared silk fibroin.

[0057] Table 1. Results of DPPH free radical scavenging rate

[0058] Application examples According to the results of Test Example 1, silk fibroin 1 exhibited the highest antioxidant activity. Therefore, silk fibroin 1 was subsequently used to prepare a composition for improving dullness in sensitive skin. Specifically, compositions for improving dullness in sensitive skin (hereinafter referred to as "compositions") were prepared according to the formulations shown in Table 2, resulting in compositions 1-13. Further, the obtained compositions were prepared into emulsions for improving dullness in sensitive skin (hereinafter referred to as "emulsions") according to the formulations shown in Table 3, resulting in emulsions 1-15 and a blank emulsion without the addition of compositions. Emulsions 1-7 were prepared by sequentially adding compositions 1-7, such as emulsion 1, which was prepared by adding composition 1, and so on. Emulsions 8 and 9 were prepared by adding composition 4. Emulsions 10-15 were prepared by sequentially adding compositions 8-13, such as emulsion 10, which was prepared by adding composition 8, and so on.

[0059] The method for preparing the emulsion includes the following steps: (1) Mix thickener, humectant and deionized water, heat at 75°C water bath temperature for 10 min, homogenize at 4000 rpm for 5 min in a homogenizer, keep warm at 75°C for later use, and obtain pre-prepared component A. (2) After mixing the oil and emulsifier, heat to 75°C, stir to dissolve evenly, keep warm for later use, and obtain pre-prepared component B; (3) Mix the pre-prepared component A and pre-prepared component B, and homogenize them in a homogenizer at 4000 rpm for 5 min to obtain the emulsified base material; (4) Stir the emulsion base material and cool it down to 50°C. Add the corresponding components of the composition, preservatives and chelating agents. Continue stirring until the material is uniform. Adjust the pH with a pH adjuster, stop stirring, and discharge the material to obtain the emulsion for improving dullness of sensitive skin.

[0060] Table 2. Component composition and dosage of the composition

[0061] Note: The total weight of compositions 1-13 is the same.

[0062] Table 3. Composition and dosage of emulsion components (weight percentage, %)

[0063] Note: "-" indicates that no water is added and the missing amount is made up with deionized water.

[0064] Test Example 2: In vitro anti-inflammatory test of the composition (1) Cell culture: RAW264.7 mouse macrophages were seeded in T75 culture flasks and cultured in DMEM medium containing 10% FBS at 37°C and 5v / v% CO2. When the cells reached the logarithmic growth phase, they were digested with 0.25% trypsin-EDTA to adjust the cell density to 2×10⁻⁶ cells / years.5 Cells / mL were seeded into 96-well plates, with 100 μL of cell suspension added to each well.

[0065] (2) Preparation of test samples: Take the composition 1-13 and dilute it with DMEM medium containing 10% FBS to prepare test samples 1-13 with a concentration of 100 μg / mL.

[0066] (3) Drug treatment and inflammation induction Group settings: Blank control group: 100 μL of DMEM medium containing 10% FBS.

[0067] LPS group: 50 μL of DMEM medium containing 10% FBS + 50 μL of 1 μg / mL LPS to induce inflammatory response.

[0068] Sample group: 50 μL of 100 μg / mL test sample 1-13, pretreated for 1 h, and then 50 μL of 1 μg / mL LPS was added.

[0069] (4) Specific operation: In a 96-well plate, first seed 100 μL of cell suspension (2 × 10⁻⁶ cells per well) into each well. 5 After culturing for 24 hours and allowing the cells to adhere, the culture medium was discarded, and 100 μL of DMEM medium containing 10% FBS was added again. The cells were then added according to the group settings (i.e., the final volume of each group was 200 μL). The treated cells were then placed in a 37°C, 5v / v% CO2 incubator for 24 h.

[0070] (5) Detection of inflammatory factors: Collect cell supernatant, and perform ELISA kit operation according to the instructions to detect the level of TNF-α inflammatory factor. Then, calculate the TNF-α inhibition rate using the following formula: TNF-α inhibition rate (%) = (1 - TNF-α concentration in the sample group / TNF-α concentration in the LPS group) × 100%.

[0071] The results are shown in Table 4. The compositions 1-7 provided by this invention exhibited an inhibition rate of 70.5%-87.4% against the inflammatory factor TNF-α, indicating that compositions 1-7 have significant in vitro anti-inflammatory effects. Comparing the data of compositions 8-11 with those of composition 4, it can be seen that the anti-inflammatory effect of compositions 8-11 is significantly lower than that of composition 4. The difference between compositions 8-11 and composition 4 lies in the absence of any one of silk fibroin 1, oxidized resveratrol, ectoine, or white chrysanthemum extract in the compositions. This indicates that silk fibroin 1, oxidized resveratrol, ectoine, and white chrysanthemum extract synergistically exert anti-inflammatory effects, jointly enhancing the anti-inflammatory activity of the compositions. Comparing the data of compositions 12-13 with those of composition 4, it can be seen that the ratio of the four components in the compositions also has a significant impact on the anti-inflammatory effect of the compositions.

[0072] Table 4 Results of the inhibitory rate of the composition on TNF-α

[0073] Test Example 3: Tyrosinase Inhibition Test This test case verifies the whitening effect of the composition through a tyrosinase inhibition experiment. The specific steps are as follows: Prepare a PBS buffer solution with pH 6.8 by mixing sodium dihydrogen phosphate and disodium hydrogen phosphate; dilute L-tyrosine to 3 mg / mL with PBS buffer as a substrate; dissolve mushroom tyrosinase in PBS buffer to prepare a 100 U / mL enzyme solution; prepare test samples (compositions 1-13) with a concentration of 5 mg / mL using PBS buffer. After incubating the buffer solution, test sample, and L-tyrosine at 37°C for 10 min, add tyrosinase to each group and continue the reaction at 37°C for 5 min. Measure the absorbance at 475 nm. Specific groupings are shown in Table 5. The inhibition rate is calculated after zeroing the blank control group using the following formula: Inhibition rate (%) = [1 - (OD of sample reaction group - OD of sample background group) / (OD of solvent reaction group - OD of solvent background group)] × 100%. The experimental results are shown in Table 6.

[0074] Table 5 Grouping of Tyrosinase Inhibition Experiments Grouping / Reagents Tyrosine (μL) Tyrosinase solution (μL) Sample (μL) Buffer (PBS, μL) Solvent (PBS, μL) Solvent background group 0 20 0 70 40 Solvent reaction group 40 20 0 30 40 Sample background group 0 20 40 70 0 Sample reaction group 40 20 40 30 0 Blank control group 40 0 0 30 60 As shown in Table 6, the data for compositions 1-7 indicate that compositions 1-7 provided by this invention have a significant inhibitory effect on tyrosinase, suggesting that compositions 1-7 have a good whitening effect. Comparing the data for compositions 8-13 with those for composition 4, it can be seen that the tyrosinase inhibition rate of compositions 8-11 is much lower than that of composition 4, indicating that silk fibroin 1, oxidized resveratrol, ectoine, and white chrysanthemum extract in the compositions synergistically enhance the whitening effect of the compositions. Comparing the data for compositions 12-13 with those for composition 4, it can be seen that the ratio of the four components in the compositions also has a significant impact on the whitening effect of the compositions.

[0075] Table 6 Results of Tyrosinase Inhibition Rate

[0076] Test Example 4: Human Patch Test Thirty participants were selected for the test according to the inclusion criteria, and the sample size was no more than 50 mm². 2 1. A qualified patch test apparatus with a depth of approximately 1 mm. Place the sample into the small chamber of the patch test apparatus, using approximately 0.020 mL to 0.025 mL. Apply the patch test apparatus containing the sample (emulsion 1-15, blank emulsion, with distilled water as the blank control group) to the flexor side of the subject's forearm using hypoallergenic adhesive tape. One patch test apparatus is applied to each arm of each volunteer. Each patch test apparatus has 10 holes, so each volunteer (both arms) can test a maximum of 20 samples simultaneously. Gently press the patch test apparatus with the palm of your hand to ensure even application to the skin, and leave it on for 24 hours. Observe the skin reaction according to the standards shown in Table 7 at 30 min (after the indentation disappears), 24 h, and 48 h after removing the patch test apparatus, and record the observation results.

[0077] Result determination Interpretation of results from occlusive skin patch testing: If more than 5 out of 30 subjects have a suspected Grade 1 skin reaction, or more than 2 out of 30 subjects have a weakly positive Grade 2 skin reaction, or if any one subject has a Grade 3 or higher adverse skin reaction, the test substance is considered to have an adverse skin reaction on humans.

[0078] Table 7 Skin Reaction Grading Standards Rating levels Skin reaction 0 negative reaction 1 Suspicious reaction, only slight erythema 2 Weak positive reaction (erythema reaction): erythema, infiltration, edema, and papules may be present. 3 Strong positive reaction (herpes reaction): erythema, infiltration, edema, papules; the reaction may extend beyond the test area. 4 Extremely strong positive reaction (confluent herpes simplex reaction): obvious erythema, severe infiltration, edema, confluent herpes simplex; reaction extends beyond the test area. After testing, the lotions 1-15 provided by this invention for improving dull and dry sensitive skin showed negative reactions after human patch testing, indicating that they are safe and non-irritating to human skin.

[0079] Test Example 5: Human Body Anti-dullness, Barrier Repair, and Moisturizing Efficacy Test Eighty healthy women aged 30-60 years were selected to participate in the test according to the inclusion criteria. They were divided into 16 groups of 5 people each. After washing their faces in the morning and evening, they applied 1.0 mL of the test product sample (lotion 1-15 (sample group) or blank lotion (blank group) to their entire face. The melanin content, TEWL value, and moisture content of the facial skin were measured before product use (day 0), and on days 14 and 28 after product use, using a narrow-band reflectance spectrophotometer, a Tewameter®TMHex transepidermal water loss probe, and a Corneometer CM 825 skin moisture meter. This allowed for a comprehensive evaluation of the anti-dullness efficacy, barrier repair, and moisturizing effect of the test product. The specific experimental parameters and equipment are shown in Table 8, and the evaluation parameters are shown in Table 9.

[0080] Table 8 Test Parameters and Equipment project Test time Test time point instrument Test metrics Number of people collected Anti-dullness 28 days 0 / 14 / 28 days Narrow-band reflection spectrophotometer melanin content 80 Repair 28 days 0 / 14 / 28 days Tewameter®™ Hex TEWL value 80 Moisturizing 28 days 0 / 14 / 28 days Corneometer CM 825 skin moisture content 80 Table 9 Evaluation Parameters Evaluation parameters Parameter meaning melanin content Melanin content refers to the amount of melanin in the skin, reflecting the skin's pigmentation status. The lower the melanin content, the whiter and brighter the skin. TEWL value TEWL refers to the amount of water that evaporates from the skin surface into the environment per unit time (usually expressed in g / (m²·h)), reflecting the skin's stratum corneum's ability to retain moisture. Damage to the stratum corneum barrier (such as dryness, inflammation, over-cleansing, or UV damage) accelerates moisture loss, resulting in a higher TEWL value; conversely, an intact stratum corneum structure (such as tightly packed keratinocytes and an intact lipid layer) effectively prevents moisture loss, leading to a lower TEWL value. skin moisture content Skin moisture content refers to the amount of water in the stratum corneum of the skin, reflecting the skin's ability to retain moisture. The higher the skin moisture content, the better the skin's moisturizing ability. The calculation formula is as follows: Melanin content improvement rate = [(Melanin content)] 样品组T0 -Melanin content 样品组T14 / 28 Melanin content 样品组T0 [×100%]-[(Melanin content)] 空白组T0 -Melanin content 空白组T14 / 28 Melanin content 空白组T0 [×100%]; Among them, melanin content 样品组T0 melanin content 样品组T14 / 28 These represent the melanin content measured in the sample groups on days 0, 14, and 28, respectively. 空白组T0 melanin content 空白组T14 / 28 These represent the melanin content measured in the control group on days 0, 14, and 28, respectively.

[0081] TEWL value improvement rate = [(TEWL value)] 样品组T0 -TEWL value 样品组T14 / 28 ) / TEWL value 样品组T0 [×100%]-[(TEWL value) 空白组T0 -TEWL value 空白组T14 / 28 ) / TEWL value 空白组T0 [×100%]; Among them, TEWL value 样品组T0 TEWL value 样品组T14 / 28 These represent the TEWL values ​​measured on days 0, 14, and 28 of the sample group, respectively. 空白组T0 TEWL value 空白组T14 / 28 These represent the TEWL values ​​measured in the blank group on days 0, 14, and 28, respectively.

[0082] Skin moisture content improvement rate = [(skin moisture content sample group T14 / 28 - skin moisture content sample group T0) / skin moisture content sample group T0 × 100%] - [(skin moisture content blank group T14 / 28 - skin moisture content blank group T0) / T skin moisture content blank group T0 × 100%]; Among them, skin moisture content 样品组T0 Skin moisture content 样品组T14 / 28 These represent the skin moisture content measured in the sample groups on days 0, 14, and 28, respectively. 空白组T0 Skin moisture content 空白组T14 / 28 These represent the skin moisture content measured in the control group on days 0, 14, and 28, respectively.

[0083] The results, taken as averages, are shown in Table 10. After using lotions 1-10, the subjects showed significant improvements in skin melanin content, TEWL value, and skin moisture content. The improvement became more pronounced with increasing application time, indicating that lotions 1-10 provided by this invention have significant anti-dullness, barrier repair, and moisturizing effects. Furthermore, the test results of lotions 10-15 and lotion 4 further verified the synergistic effect among silk fibroin, oxidized resveratrol, ectoine, and white chamomile extract in the composition provided by this invention. Simultaneously, within a specific ratio, they can significantly enhance the anti-dullness, barrier repair, and moisturizing effects of the composition.

[0084] Table 10 Results of tests on human anti-dullness, barrier repair, and moisturizing effects

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composition for improving dull and dryness of sensitive skin, characterized in that, The composition comprises the following components in parts by weight: 0.01-1 parts of silk fibroin, 0.1-5 parts of oxidized white resveratrol, 0.1-5 parts of ectoine, and 0.01-0.2 parts of white spring chamomile flower extract; the silk fibroin is prepared from cocoon by ultrasonic-assisted enzymatic hydrolysis.

2. The composition of claim 1, wherein The composition comprises the following components in parts by weight: 0.1-0.6 parts of silk fibroin, 0.5-3 parts of oxidized white resveratrol, 1-3 parts of ectoine, and 0.05-0.15 parts of white spring chamomile flower extract.

3. The composition of claim 1, wherein The composition comprises the following components in parts by weight: 0.2-0.3 parts of silk fibroin, 1-1.2 parts of oxidized white resveratrol, 1.5-1.6 parts of ectoine, and 0.09-0.11 parts of white spring chamomile flower extract.

4. The composition of claim 1, wherein The preparation method of the silk fibroin comprises the following steps: S1. Cleaned and chopped cocoon is added into a Na2CO3 solution for boiling water bath treatment, and then rinsed with distilled water until the rinsing liquid is neutral, to obtain clean cocoon particles, which are soaked in a phosphate buffer solution until the silk fibers are swollen, and then taken out, chopped, and reacted to obtain a reaction substrate; S2. First-stage enzymolysis: 1%-3% of an alkaline protease solution is added according to the mass of the reaction substrate, the pH is adjusted to 8.5-9.5, and then the reaction is carried out at 45-55℃ for 1-2h, after which the enzyme in the enzymolysis liquid is inactivated to obtain a first enzymolysis liquid; S3. Second-stage enzymolysis: 1%-2% of a neutral protease solution and 1%-2% of a trypsin solution are added to the first enzymolysis liquid according to the mass of the reaction substrate, the pH is adjusted to 7.0-8.0, and then the reaction is carried out under ultrasonic conditions at 40-50℃ for 1-3h, after which the enzyme in the enzymolysis liquid is inactivated to obtain a second enzymolysis liquid; S4. Third-stage enzymolysis: 2%-4% of a papain solution is added to the second enzymolysis liquid according to the mass of the reaction substrate, the pH is adjusted to 6.0-7.0, and then the reaction is carried out under ultrasonic conditions at 50-60℃ for 1-2h, after which the enzyme in the enzymolysis liquid is inactivated to obtain a third enzymolysis liquid; S5. The third enzymolysis liquid is centrifuged to obtain a supernatant, which is sequentially subjected to salting-out, dialysis, and freeze-drying treatment to obtain the silk fibroin.

5. The composition of claim 4, wherein In steps S2-S4, the alkaline protease solution is a boric acid-sodium hydroxide buffer solution containing 4-6g / L of alkaline protease; the neutral protease solution is a phosphate buffer solution containing 2-4g / L of neutral protease, and the trypsin solution is a phosphate buffer solution containing 1.5-3g / L of trypsin; and the papain solution is a disodium hydrogen phosphate-citric acid buffer solution containing 2-4g / L of papain.

6. The composition of claim 4, wherein In steps S3-S4, the ultrasonic conditions are as follows: pulse ultrasonic mode is adopted, the ultrasonic power is set to 80-150W, the frequency is set to 40kHz, each ultrasonic treatment lasts for 5-10s, and each pause lasts for 5-10s.

7. Use of the composition of any one of claims 1-6 in the preparation of a cosmetic product for improving the dull and dry appearance of sensitive skin.

8. Use according to claim 7, wherein the compound is ###0002### The cosmetic product is one of a cosmetic water, an emulsion, a cream, a mask, and a spray.

9. Use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The cosmetic product is an emulsion.

10. An emulsion characterized in that, A composition comprising the following components by weight percentage: thickening agent 0.05-0.5%, humectant 1-10%, pH adjuster 0.01-0.3%, emulsifier 1-6%, oil 5-15%, preservative 0.5-3%, the composition for improving dull and dry skin of sensitive skin according to any one of claims 1-6 1-10%, chelating agent 0.01-0.5%, deionized water the balance.

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