Silk fibroin-containing composition as well as preparation method and application thereof

Abstract

The invention relates to a silk fibroin-containing composition as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. The composition provided by the invention comprises silk fibroin-tea polyphenol powder, myrothamnus flabellifolia leaf extract, alfalfa extract and hydrolyzed yeast protein, and the silk fibroin-tea polyphenol powder is prepared by grafting two tea polyphenol components, namely EGCG and ECG, onto silk fibroin, so that the original effects of the silk fibroin are retained, and meanwhile, the content of the tea polyphenol components in the silk fibroin-tea polyphenol powder is increased; and the application stability of the silk fibroin in the product can also be improved. Besides, the prepared silk fibroin-tea polyphenol powder, myrothamnus flabellifolia leaf extract, alfalfa extract and hydrolyzed yeast protein are reasonably compounded to prepare the composition, all the components in the composition have a synergistic effect, and the composition has good anti-inflammatory, soothing and barrier repairing effects.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a composition containing silk fibroin, its preparation method, and its application. Background Technology

[0002] Silk fibroin is a natural high-molecular-weight protein with good biocompatibility, biodegradability, and cell growth-promoting properties. It is rich in amino acids such as glycine, alanine, and serine, which can provide nutrition and hydration to the skin. Generally, silk fibroin conformations are considered to be divided into two types: Silk I and Silk II structures. Silk I structures include random coils and α-helices and are soluble in water, while Silk II structures are mainly composed of antiparallel β-sheets and are insoluble in water. Comparatively, Silk II structured silk fibroin materials have poorer hygroscopicity and flexibility, which is not conducive to biodegradation and absorption.

[0003] When silk fibroin is in a dilute solution with a low concentration, it initially exhibits a random coil structure, primarily existing as Silk I. However, with changes in the external environment, such as temperature and pH, silk fibroin will transform into a more stable β-sheet. Therefore, when silk fibroin is used in skincare products, changes in the external environment, such as temperature variations during product preparation or storage, may cause the silk fibroin to transform into a Silk II structure, leading to its precipitation from the product. Therefore, inhibiting the transformation of silk fibroin from a random coil to a β-sheet conformation, thus maintaining the silk I structure, is of great significance.

[0004] On the other hand, silk fibroin, due to its excellent biocompatibility, biodegradability, and cell-growth-promoting properties, has been increasingly applied in the beauty and skincare field. However, most product development remains at the level of basic moisturizing and carrier functions, failing to fully leverage its synergistic potential in promoting barrier repair, anti-inflammation, and soothing. Therefore, developing a multifunctional composite composition containing silk fibroin with a stable silk I structure, achieving synergistic effects through ingredient combination, simplifying the skincare process, and fundamentally improving skin health is of great significance. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition containing silk fibroin, its preparation method and application.

[0006] 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 containing silk fibroin, comprising the following components in parts by weight: 0.05-0.5 parts silk fibroin-tea polyphenol powder, 0.1-1 parts Myrothamnus fruticosa leaf extract, 0.1-2 parts alfalfa extract, and 0.2-2 parts hydrolyzed yeast protein; wherein the silk fibroin-tea polyphenol powder is obtained by grafting silk fibroin with tea polyphenol, and the tea polyphenol is a mixture of EGCG and ECG in a molar ratio of (1-3):1.

[0007] This invention grafts two tea polyphenol components, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), onto silk fibroin, thereby preserving the original efficacy of silk fibroin while improving its stability in product applications. Furthermore, a composition is prepared by mixing the obtained silk fibroin-tea polyphenol powder with Myrothamnus fruticosa leaf extract, alfalfa extract, and hydrolyzed yeast protein. This composition exerts anti-inflammatory, soothing, and barrier-repairing effects in multiple ways. Silk fibroin is rich in hydroxy amino acids and components similar to natural moisturizing factors (NMF), which can form hydrogen bonds with the stratum corneum of the skin, enhancing its moisture retention capacity and exerting a moisturizing effect. Effective moisturizing can strengthen the skin barrier function and reduce moisture loss.

[0008] Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), used for grafting silk fibroin, belong to the flavanone class of tea polyphenols. Catechins are the main compounds among flavanones, primarily including epigallocatechin gallate (EGCG), epicatechin (EC), and epicatechin gallate (ECG). Catechins can reduce cell damage caused by environmental pollution and ultraviolet radiation, exhibiting significant anti-aging and anti-inflammatory effects.

[0009] Myrothamnus flabellifolia leaf extract can protect the skin from the stress of high-energy visible light (HEV light), reduce ROS production in keratinocytes induced by blue light, and alleviate cell damage caused by oxidative stress.

[0010] Galactomannan in alfalfa extract can rebuild skin protein synthesis and promote the production of barrier proteins. In addition, alfalfa extract can also upregulate PPARα receptors, increase ceramide synthesis, and exert a soothing and repairing effect.

[0011] Hydrolyzed yeast protein can increase the expression of cysteine ​​aspartate protease-14 (Caspase-14), thereby increasing the skin's ability to resist UV rays and reducing UV-induced skin damage.

[0012] In summary, the above-mentioned ingredients can repair the skin barrier from multiple aspects, including moisturizing, relieving oxidative stress, protecting against UV rays, and promoting the generation of barrier proteins. This invention combines silk fibroin-tea polyphenol powder, Myrothamnus flabellifolia leaf extract, alfalfa extract, and hydrolyzed yeast protein to obtain a composition. The components in this composition work synergistically to enhance its anti-inflammatory, soothing, and barrier-repairing effects.

[0013] Furthermore, the preparation method of the silk fibroin-tea polyphenol powder includes the following steps: S1. Dissolve water-soluble silk fibroin in a phosphate buffer solution with a pH of 7.0~8.0 to obtain a silk fibroin solution; S2. Disperse tea polyphenols uniformly in a phosphate buffer solution with a pH of 7.3-7.6, then add tyrosinase and mix well to obtain a tea polyphenol solution; wherein the tea polyphenols are EGCG and ECG in a molar ratio of (1-3):1. S3. Mix the silk fibroin solution obtained in step S1 and the tea polyphenol solution obtained in step S2 at a mass ratio of (2~3):1 and react them. After the reaction is completed, adjust the pH of the system to 5.5~6.0, dialyze, filter, collect the filtrate, freeze dry and pulverize to obtain silk fibroin-tea polyphenol powder.

[0014] Preferably, the pH value of the phosphate buffer solution in step S1 is 7.4.

[0015] Preferably, the mass ratio of silk fibroin to phosphate buffer solution in step S1 is 1:(100~120).

[0016] Preferably, the mass ratio of tea polyphenols to phosphate buffer solution in step S2 is 1:(80~100).

[0017] Preferably, the concentration of tyrosinase in the tea polyphenol solution in step S2 is 8~10 U / mL.

[0018] Preferably, the mixing in step S2 is performed by stirring at 26~35℃ and 200~250r / min for 30~40min.

[0019] Preferably, the reaction in step S3 is carried out by stirring at room temperature and 200-250 r / min for 2-3 hours.

[0020] Preferably, the dialysis in step S3 is dialysis using a 14kDa dialysis bag for 48-72 hours; the filtration is filtration using a filter membrane with a pore size of 0.22-0.45μm; and the freeze-drying is pre-freezing the filtrate at -18 to -20°C for 20-30 hours, followed by freeze-drying at a vacuum of 6-10Pa and a temperature of -55 to -65°C for 60-80 hours.

[0021] Secondly, the present invention provides the use of the composition described in the first aspect in the preparation of cosmetics with soothing and repairing effects.

[0022] Furthermore, the composition accounts for 5% to 10% of the total mass of the cosmetic.

[0023] Furthermore, the cosmetic is at least one of toner, lotion, cream, gel, and mask.

[0024] Preferably, the cosmetic is a lotion.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention grafts two tea polyphenol components, EGCG and ECG, onto silk fibroin, so that the resulting silk fibroin-tea polyphenol powder retains the original efficacy of silk fibroin while improving the stability of silk fibroin in product applications.

[0026] (2) The present invention mixes the prepared silk fibroin-tea polyphenol powder with Myrothamnus fruticosa leaf extract, alfalfa extract and hydrolyzed yeast protein to obtain a composition. Each component in the composition has multiple functions such as moisturizing, relieving oxidative stress, resisting UV and promoting barrier protein generation. Each component in the composition works synergistically within a specific ratio range to improve the anti-inflammatory, soothing and barrier repair effects of the composition. Detailed Implementation

[0027] 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.

[0028] In the following embodiments, the components used are sourced from: Water-soluble silk fibroin was purchased from Guangzhou Qiyuan Biotechnology Co., Ltd., and its trade name is: hydrolyzed silk. EGCG was purchased from Shaanxi Senyuan Biotechnology Co., Ltd., and its trade name is: Epigallocatechin gallate EGCG. ECG was purchased from Shaanxi Huikang Biotechnology Co., Ltd., and its trade name is epicatechin gallate. Myrothamnus flabellifolia leaf extract was purchased from Xi'an Youlanda Biotechnology Co., Ltd., and its trade name is: Myrothamnus flabellifolia leaf extract; Alfalfa extract was purchased from Silab under the brand name INCLUSIUM®. The hydrolyzed yeast protein was purchased from Ashland under the trade name: Caspaline YST™.

[0029] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0030] Example 1 This embodiment provides a method for synthesizing silk fibroin-tea polyphenols under mild alkaline conditions, the method comprising the following steps: S1. Dissolve water-soluble silk fibroin in 110 times its weight of PBS solution with a pH of 7.4 to obtain a silk fibroin solution; S2. EGCG and ECG were mixed at a molar ratio of 2:1 to prepare tea polyphenol powder. The tea polyphenol powder was uniformly dispersed in 90 times its mass of PBS solution with a pH of 7.4. Tyrosinase was then added and the mixture was stirred at 30℃ and 230r / min for 35min to obtain a tea polyphenol solution containing 9U / mL tyrosinase. S3. The silk fibroin solution obtained in step S1 and the tea polyphenol solution obtained in step S2 are mixed at a mass ratio of 2.5:1. The mixture is stirred at room temperature and 230 r / min for 2.5 h. The pH is then adjusted to 5.8. The mixture is dialyzed using a 14 kDa dialysis bag for 60 h. After filtration using a filter with a pore size of 0.45 μm, the filtrate is collected and pre-frozen at -20℃ for 24 h. Then, it is freeze-dried at a vacuum of 8 Pa and a temperature of -60℃ for 72 h. Finally, the mixture is pulverized to obtain silk fibroin-tea polyphenol powder.

[0031] Example 2 This embodiment provides a method for synthesizing silk fibroin-tea polyphenols under mild alkaline conditions, the method comprising the following steps: S1. Dissolve water-soluble silk fibroin in 100 times its weight of PBS solution with a pH of 8.0 to obtain a silk fibroin solution; S2. Mix EGCG and ECG at a molar ratio of 1:1 to prepare tea polyphenol powder. Disperse the tea polyphenol powder evenly in 80 times its mass of PBS solution with pH 7.3, then add tyrosinase and stir at 26℃ and 200r / min for 40min to obtain a tea polyphenol solution containing 8U / mL tyrosinase. S3. The silk fibroin solution obtained in step S1 and the tea polyphenol solution obtained in step S2 are mixed at a mass ratio of 2:1. The mixture is stirred at room temperature and 250 r / min for 2 h. The pH is then adjusted to 5.5. The mixture is dialyzed using a 14 kDa dialysis bag for 48 h. After filtration using a filter with a pore size of 0.22 μm, the filtrate is collected and pre-frozen at -20℃ for 20 h. Then, it is freeze-dried at a vacuum of 6 Pa and a temperature of -65℃ for 60 h. Finally, the mixture is pulverized to obtain silk fibroin-tea polyphenol powder.

[0032] Example 3 This embodiment provides a method for synthesizing silk fibroin-tea polyphenols under mild alkaline conditions, the method comprising the following steps: S1. Dissolve water-soluble silk fibroin in 120 times its weight of PBS solution with a pH of 7.0 to obtain a silk fibroin solution; S2. Mix EGCG and ECG at a molar ratio of 3:1 to prepare tea polyphenol powder. Disperse the tea polyphenol powder evenly in 100 times the mass of PBS solution with pH 7.6. Add tyrosinase and stir for 30 min at 35℃ and 250 r / min to obtain a tea polyphenol solution containing 10 U / mL tyrosinase. S3. The silk fibroin solution obtained in step S1 and the tea polyphenol solution obtained in step S2 are mixed at a mass ratio of 3:1. After stirring and reacting at room temperature and 250 r / min for 3 h, the pH is adjusted to 6.0. The mixture is dialyzed using a 14 kDa dialysis bag for 72 h, filtered using a filter with a pore size of 0.45 μm, and the filtrate is collected. After pre-freezing at -18℃ for 30 h, it is freeze-dried at a vacuum of 10 Pa and a temperature of -55℃ for 80 h. The filtrate is then pulverized to obtain silk fibroin-tea polyphenol powder.

[0033] Comparative Example 1 The difference between this comparative example and Example 1 is that in step S2, the molar ratio of EGCG to ECG in the tea polyphenol powder is 1:2, while the remaining steps and parameters are the same as in Example 1.

[0034] Comparative Example 2 The difference between this comparative example and Example 1 is that in step S2, EGCG is not added to the tea polyphenol powder, and the missing amount of EGCG is made up with an equal amount of ECG. The remaining steps and parameters are the same as in Example 1.

[0035] Comparative Example 3 The difference between this comparative example and Example 1 is that in step S2, ECG is not added to the tea polyphenol powder, and the missing ECG is made up with an equal amount of EGCG. The remaining steps and parameters are the same as in Example 1.

[0036] Comparative Example 4 The difference between this comparative example and Example 1 is that the pH value of the PBS solution used in steps S1 and S2 is 8.5, while the remaining steps and parameters are the same as in Example 1.

[0037] Comparative Example 5 The difference between this comparative example and Example 1 is that tyrosinase is not added in step S2, while the remaining steps and parameters are the same as in Example 1.

[0038] Comparative Example 6 The difference between this comparative example and Example 1 is that the mass ratio of silk fibroin solution to tea polyphenol solution in step S3 is 1:2.5.

[0039] Test Example 1: Stability Determination of Silk Fibroin-Tea Polyphenol Powder The stability of silk fibroin-tea polyphenol powders prepared in Examples 1-3 and Comparative Examples 1-6 was determined using these powders as test samples.

[0040] Experimental Principle: Silk fibroin can be degraded by enzymes (such as protease XIV), and the Silk I conformation degrades much faster than the Silk II conformation. Therefore, by performing conformational changes on silk fibroin before and after grafting, and then testing the enzymatic degradation effect, the conformational stability of tea polyphenols grafted onto silk fibroin can be determined. Zn... 2+ It can induce the conversion of silk fibroin from silk I to silk II.

[0041] Experimental Methods: A certain amount of ZnCl2 was dissolved in deionized water, and a certain amount of silk fibroin-tea polyphenol powder was also dissolved in deionized water. The two solutions were mixed and then dissolved in Tri-HCl (pH=7.4, 5mM) solution until the concentration of silk fibroin-tea polyphenol was 2 mg / mL and the concentration of ZnCl2 was 15 μM, thus preparing the sample group. Untreated water-soluble silk fibroin was used as a control instead of the silk fibroin-tea polyphenol powder, and the same treatment was performed to prepare the control group. Both the sample group and the control group were incubated at room temperature for 48 h, and then dialyzed in deionized water for 2 h using a dialysis bag with a molecular weight cutoff of 10 kDa to remove free Zn. 2+ Add buffer salts, then place in a petri dish and air dry at room temperature for 24 hours to form a film, which is the sample to be tested; Accurately weigh a certain amount of the sample to be tested, denoted as m0, and dissolve it in 100 times its mass of PBS solution with pH=7.0. The PBS solution contains 1.0 U / mL proteinase XIV. Seal and place in a 37℃ environment with shaking for 72 hours. Centrifuge, collect the precipitate, wash with deionized water, centrifuge again, dry the precipitate, weigh it, and denoted as m1. Calculate the enzyme degradation rate according to the following formula: Enzyme degradation rate = (m0-m1) / m0×100%.

[0042] The results are shown in Table 1. The ungrafted silk fibroin had a lower degradation rate, indicating that it was less effective against Zn. 2+ Under induction, it transformed into the silk II conformation, which is not easily degraded by protease XIV treatment. From the enzymatic degradation rate data of Examples 1-3, it can be seen that the silk fibroin grafted with EGCG and ECG is less susceptible to degradation by Zn. 2+ After induction, the enzyme degradation rate was significantly increased after treatment with protease XIV, indicating that EGCG and ECG grafting inhibited the degradation of Zn. 2+ The induced conformational change of silk fibroin allowed it to remain in the silk I conformation as much as possible, resulting in a higher enzymatic degradation rate. Comparing the enzymatic degradation rate data of Comparative Examples 1-6 and Example 1, the enzymatic degradation rate of Comparative Examples 1-6 decreased, indicating that the silk fibroin in Comparative Examples 1-6 showed a lower degradation rate in Zn.2+ Under induction, a significant portion of the silk fibroin underwent a transformation to the silk II conformation. The difference between Comparative Examples 2-3 and Example 1 lies in the use of only EGCG or only ECG for the grafting reaction. The difference between Comparative Examples 4-5 and Example 1 lies in the reaction being carried out under higher alkaline conditions or without the addition of tyrosinase in the grafting reaction. This indicates that the simultaneous use of EGCG and ECG to graft silk fibroin can synergistically stabilize the silk I conformation of silk fibroin. Furthermore, the interaction between the mild alkaline reaction environment and the reaction system containing tyrosinase during the reaction process allows the prepared silk fibroin-tea polyphenols to stably maintain the silk I conformation of silk fibroin. The difference between Comparative Examples 1 and 6 and Example 1 lies in the ratio of EGCG to ECG or the ratio of silk fibroin solution to tea polyphenol solution, which is outside the limits of this invention. This indicates that the ratio of raw materials used in the preparation of silk fibroin-tea polyphenol powder also has a significant impact on the stability of the silk I conformation of silk fibroin.

[0043] Table 1. Results of stability test of silk fibroin-tea polyphenol powder Group Enzyme degradation rate (%) Untreated silk fibroin 5.7 Example 1 43.8 Example 2 38.6 Example 3 41.5 Comparative Example 1 21.9 Comparative Example 2 15.7 Comparative Example 3 17.3 Comparative Example 4 16.1 Comparative Example 5 14.4 Comparative Example 6 20.2 Application examples (1) Preparation of the composition According to Test Example 1, the silk fibroin-tea polyphenol powder prepared in Example 1 has the highest stability. Therefore, the silk fibroin-tea polyphenol powder prepared in Example 1 was subsequently used to prepare compositions. The components and amounts of the compositions are shown in Table 2, and compositions 1 to 3 were prepared.

[0044] The method for preparing the composition solution is as follows: weigh each component in the composition according to the weight parts, add 10 times the total mass of deionized water, mix evenly, and the composition solution is obtained.

[0045] Table 2. Component composition and dosage of the composition

[0046] Note: The total mass of compositions 1 to 3 is the same.

[0047] (2) Use compositions 1-3 to prepare soothing and repairing products. Specifically, prepare emulsions according to the formula shown in Table 3 to obtain emulsions 1-3.

[0048] The method for preparing the emulsion includes the following steps: 1) Mix each component of phase A with water and stir. Heat to 80°C and homogenize at 1200 rpm for 5 minutes. After homogenization, keep warm for later use to obtain the aqueous phase. 2) Mix the components of phase B, heat to 80°C, and homogenize at 1200 rpm for 5 minutes. After homogenization, keep warm for later use to obtain the oil phase. 3) Mix the components of phase C and heat to 60°C to melt them, thus obtaining the preservative; 4) Heat the aqueous phase to 80°C, add the oil phase at 300 rpm, stir and mix well, cool down to 60°C, add the preservative at 300 rpm and stir and mix well, cool down to 45°C, add the corresponding composition solution prepared in (1) and continue stirring for 5 min, finally add arginine to adjust the pH, then stop stirring, discharge the material, and obtain the emulsion.

[0049] Meanwhile, this application example also provides preparation of emulsions 4-5, wherein the difference between emulsion 4 and emulsion 2 is that the amount of composition solution added is 5 wt%; the difference between emulsion 5 and emulsion 2 is that the amount of composition solution added is 10 wt%, and the remaining components and preparation methods are the same as those of emulsion 2.

[0050] Table 3. Component composition and content of the emulsion

[0051] Comparative Application Example 1 This comparative application example provides a composition that differs from composition 2 in that it does not contain silk fibroin-tea polyphenol powder, but instead uses Myrothamnus fruticosa leaf extract, alfalfa extract, and hydrolyzed yeast protein in a weight ratio of 0.2:0.8:0.5 to make up for the missing amount, thus preparing composition 1'.

[0052] This comparative application example also provides an emulsion, which differs from emulsion 2 in that an equal amount of composition 1' is used to replace composition 2 to prepare emulsion 1'.

[0053] Comparative Application Example 2 This comparative application example provides a composition that differs from composition 2 in that it does not contain Myrothamnus flabellifolia leaf extract, but instead uses silk fibroin-tea polyphenol powder, alfalfa extract, and hydrolyzed yeast protein in a weight ratio of 0.1:0.8:0.5 to make up for the missing amount, thus preparing composition 2'.

[0054] This comparative application example also provides an emulsion, which differs from emulsion 2 in that an equal amount of composition 2' is used to replace composition 2 to prepare emulsion 2'.

[0055] Comparative Application Example 3 This comparative application example provides a composition that differs from composition 2 in that it does not contain alfalfa extract, but instead uses silk fibroin-tea polyphenol powder, Myrothamnus fruticosa leaf extract, and hydrolyzed yeast protein in a weight ratio of 0.1:0.2:0.5 to make up for the missing amount, thus preparing composition 3'.

[0056] This comparative application example also provides an emulsion, which differs from emulsion 2 in that an equal amount of composition 3' is used to replace composition 2 to prepare emulsion 3'.

[0057] Comparative Application Example 4 This comparative application example provides a composition that differs from composition 2 in that it does not contain hydrolyzed yeast protein, but instead uses silk fibroin-tea polyphenol powder, Myrothamnus fruticosa leaf extract, and alfalfa extract in a weight ratio of 0.1:0.2:0.8 to make up for the missing amount, thus preparing composition 4'.

[0058] This comparative application example also provides an emulsion, which differs from emulsion 2 in that an equal amount of composition 4' is used to replace composition 2 to prepare emulsion 4'.

[0059] Comparative Application Example 5 This comparative application example provides a composition that differs from composition 2 in that an equal amount of untreated silk fibroin replaces the silk fibroin-tea polyphenol powder in the composition to prepare composition 5'.

[0060] This comparative application example also provides an emulsion, which differs from emulsion 2 in that an equal amount of composition 5' is used to replace composition 2 to prepare emulsion 5'.

[0061] Comparative Application Example 6 This comparative application example provides a composition with the same total mass as composition 2, comprising the following components in parts by weight: 0.8 parts silk fibroin-tea polyphenol powder, 1.5 parts Myrothamnus fruticosa leaf extract, 0.05 parts alfalfa extract, and 0.1 parts hydrolyzed yeast protein; the preparation method is the same as that of composition 2, to obtain composition 6'.

[0062] This comparative application example also provides an emulsion, which differs from emulsion 2 in that an equal amount of composition 6' is used to replace composition 2 to prepare emulsion 6'.

[0063] Comparative Application Example 7 This comparative application example provides a composition with the same total mass as composition 2, comprising the following components in parts by weight: 0.03 parts silk fibroin-tea polyphenol powder, 0.05 parts Myrothamnus fruticosa leaf extract, 2.5 parts alfalfa extract, and 2.5 parts hydrolyzed yeast protein; the preparation method is the same as that of composition 2, to obtain composition 7'.

[0064] This comparative application example also provides an emulsion, which differs from emulsion 2 in that an equal amount of composition 7' ​​is used to replace composition 2 to prepare emulsion 7'.

[0065] Test Example 2: Anti-inflammatory effect test of the composition Compositions 1-3 and compositions 1'-7' were used as test samples to determine their anti-inflammatory effects. The test procedures are as follows: S1. Seeding: Logarithmically growing RAW264.7 cells were seeded into 24-well plates at a cell density of 2 × 10⁻⁶ cells / well. 5 Cells / mL, 0.5mL per well, after plating the plate, mark the top cover of the cell plate and incubate in a CO2 incubator for 24 h; S2. Sample processing: After cell incubation for 24 hours, the supernatant was aspirated, and 0.5 mL of the following samples were added respectively: ① Model group: 1 μg / mL LPS (DMEM medium containing 1 μg / mL LPS); ② Positive control group: 0.001 wt% dexamethasone + 1 μg / mL LPS (DMEM medium containing 0.001 wt% dexamethasone and 1 μg / mL LPS); ③ Experimental groups 1-10: 0.5 wt% composition + 1 μg / mL LPS (DMEM medium containing 0.5 wt% of composition 1-3 or composition 1'-7', and 1 μg / mL LPS respectively). S3. Detection of inflammatory factors in cell supernatant of each group: After 24 h of sample treatment, the supernatant was pipetted into centrifuge tubes, centrifuged at 1000 r / min for 5 min, the precipitate was discarded and the supernatant was collected. The changes in the amount of inflammatory factors in the supernatant were detected using the Xinbosheng TNF-α inflammatory factor detection kit. The results were expressed as TNF-α inhibition rate. TNF-α inhibition rate % = (TNF-α amount in model group - TNF-α amount in experimental group / TNF-α amount in positive control group) / TNF-α amount in model group × 100%.

[0066] As shown in Table 4, the TNF-α inhibition rates of compositions 1-3 reached 41.7%-44.5%, close to the positive control group, indicating that compositions 1-3 have good anti-inflammatory effects. Comparing the TNF-α inhibition rate data of compositions 1'-5' with those of composition 2, it can be seen that the anti-inflammatory effect of compositions 1'-5' is significantly reduced. The difference between compositions 1'-5' and composition 2 is that one of the components is missing or untreated silk fibroin is used to replace the silk fibroin-tea polyphenol powder. This indicates that the silk fibroin-tea polyphenol powder prepared in this invention, together with the four components of Myrothamnus fruticosa leaf extract, alfalfa extract, and hydrolyzed yeast protein, synergistically improves the in vitro anti-inflammatory effect of the composition. Comparing the results of compositions 6'-7' with those of composition 2, it can be seen that the proportion of each component in the composition significantly affects the anti-inflammatory effect of the composition.

[0067] Table 4. Anti-inflammatory test results of the composition Group TNF-α inhibition rate (%) Composition 1 42.5 Composition 2 44.2 Composition 3 41.7 Composition 1' 27.9 Composition 2' 26.6 Composition 3' 28.3 Composition 4' 24.4 Composition 5' 29.2 Composition 6' 31.9 Composition 7' 32.1 Positive control group 46.8 Test Example 3: Skin Soothing and Repairing Effect Test The emulsions prepared using Application Examples 1-7 and Comparative Application Examples 1-7 were used as test samples to test their skin soothing and repair effects. The method is as follows: (1) Several volunteers aged 18-45 were selected and randomly divided into 12 groups of 5 people each. During the test, each group randomly used the lotion of the application example and the control example of this invention, and used it once in the morning and once in the evening. The method of use is to take 1 g of sample and apply it evenly to the whole face after cleansing and moisturizing in the morning and evening, and gently massage until fully absorbed. Follow-up visits were conducted on days 0 and 14 to test various indicators.

[0068] (2) On the days of follow-up visits on days 0 and 14, the subjects did not apply any skin care products. After cleansing, they sat quietly for 30 minutes in a constant temperature and humidity room with a temperature of 21±1℃ and a humidity of 50±10%. The initial TEWL value and moisture content of the subjects were tested using the Tewameter®™ Hex (Courage+Khazaka) transdermal moisture loss probe and the Corneometer® CM 825 (Courage + Khazaka) moisture probe. Facial images were taken using Visia-7, and the exported red area images were analyzed to obtain the a* value. (3) Analyze the improvement of each indicator, and take the average value of the data. The improvement is calculated as follows: TEWL value improvement rate (%) = (X 使用前 -X 使用后 ) / X 使用前 ×100% Moisture content improvement rate (%) = (X 使用后 -X 使用前 ) / X 使用前 ×100% a* value improvement rate (%) = (X 使用前 -X 使用后 ) / X 使用前 ×100% In the formula: X 使用前 Data on various indicators tested before subjects used the product; X 使用后 Data on various indicators for subjects before they use the product.

[0069] The test results are shown in Table 5: Emulsions 1-5 of the application examples of this invention can significantly improve the skin TEWL value, moisture content and redness a* value of the subjects, indicating that emulsions 1-5 of the application examples of this invention have significant barrier repair, moisturizing and anti-inflammatory soothing effects, and can effectively soothe and repair the skin. Comparing the data of emulsion 2 and emulsions 1'-7' in Table 6, it can be seen that the silk fibroin-tea polyphenol powder, myrothamnus flabellifolia leaf extract, alfalfa extract and hydrolyzed yeast protein in the composition provided by this invention can exert a synergistic effect within a specific ratio range, and jointly improve the soothing and repairing efficacy of the composition.

[0070] Table 5. Results of Skin Soothing and Repair Effect Test Group TEWL value improvement rate (%) Moisture content improvement rate (%) a* value improvement rate (%) Emulsion 1 12.4 24.4 7.6 Emulsion 2 13.9 26.2 8.5 Emulsion 3 11.6 23.1 7.1 Emulsion 4 12.5 25.8 8.2 Emulsion 5 14.1 27.3 9.0 Emulsion 1' 6.5 10.4 1.9 Emulsion 2' 5.4 9.7 3.1 Emulsion 3' 7.3 12.5 3.6 Emulsion 4' 6.7 11.9 2.5 Emulsion 5' 7.8 13.0 3.8 Emulsion 6' 8.6 15.1 4.9 Emulsion 7' 8.2 14.2 4.5 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 containing silk fibroin, characterized in that, The product comprises the following components in parts by weight: 0.05-0.5 parts silk fibroin-tea polyphenol powder, 0.1-1 parts Myrothamnus fruticosa leaf extract, 0.1-2 parts alfalfa extract, and 0.2-2 parts hydrolyzed yeast protein; wherein the silk fibroin-tea polyphenol powder is obtained by grafting silk fibroin with tea polyphenols, and the tea polyphenols are a mixture of EGCG and ECG in a molar ratio of (1-3):

1.

2. The composition according to claim 1, characterized in that, The preparation method of the silk fibroin-tea polyphenol powder includes the following steps: S1. Dissolve water-soluble silk fibroin in a phosphate buffer solution with a pH of 7.0~8.0 to obtain a silk fibroin solution; S2. Disperse tea polyphenols uniformly in a phosphate buffer solution with a pH of 7.3-7.6, then add tyrosinase and mix well to obtain a tea polyphenol solution; wherein the tea polyphenols are EGCG and ECG in a molar ratio of (1-3):

1. S3. Mix the silk fibroin solution obtained in step S1 and the tea polyphenol solution obtained in step S2 at a mass ratio of (2~3):1 and react them. After the reaction is completed, adjust the pH of the system to 5.5~6.0, dialyze, filter, collect the filtrate, freeze dry and pulverize to obtain silk fibroin-tea polyphenol powder.

3. The composition according to claim 2, characterized in that, The mass ratio of silk fibroin to phosphate buffer solution in step S1 is 1:(100~120).

4. The composition according to claim 2, characterized in that, The mass ratio of tea polyphenols to phosphate buffer solution in step S2 is 1:(80~100).

5. The composition according to claim 2, characterized in that, The concentration of tyrosinase in the tea polyphenol solution described in step S2 is 8~10 U / mL.

6. The composition according to claim 2, characterized in that, The reaction described in step S3 is carried out by stirring at room temperature and 200-250 r / min for 2-3 hours.

7. The composition according to claim 2, characterized in that, The dialysis in step S3 is dialysis using a 14kDa dialysis bag for 48-72 hours; the filtration is filtration using a filter membrane with a pore size of 0.22-0.45μm; the freeze-drying is pre-freezing the filtrate at -18 to -20℃ for 20-30 hours, followed by freeze-drying at a vacuum of 6-10Pa and a temperature of -55 to -65℃ for 60-80 hours.

8. The use of the composition according to any one of claims 1 to 7 in the preparation of cosmetics having soothing and repairing effects.

9. The application as described in claim 8, characterized in that, The composition accounts for 5% to 10% of the total mass of the cosmetic.

10. The application as described in claim 8, characterized in that, The cosmetic product is at least one of the following: toner, lotion, cream, gel, and mask.

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