Method for preparing oil-soluble modified polypeptide based on water-phase acylation method and application of oil-soluble modified polypeptide in cosmetics

Abstract

The invention discloses a method for preparing oil-soluble modified polypeptide based on a water phase acylation method and application of the oil-soluble modified polypeptide in cosmetics, and belongs to the technical field of protein modification and cosmetics. Hydrophilic protein is subjected to acylating chlorination in a water phase, the ratio of protein to acyl chloride and the adding rate of acyl chloride are improved, and a series of oil-soluble modified protein with improved oil solubility is prepared. The modified protein prepared by the invention not only can exert the original biological activity in cosmetics, but also can have other additional functions, meets the diversified requirements of consumers on cosmetics, and has extremely high practical value and economic value.

Description

Technical Field

[0001] This invention relates to a method for preparing oil-soluble modified peptides based on aqueous phase acylation and its application in cosmetics, belonging to the field of protein modification and cosmetics technology. Background Technology

[0002] Small molecule peptides, as a special form of protein, are gradually emerging in the cosmetics industry. This is mainly due to their small molecular weight, easy penetration, and the resulting multiple bioactivities. Compared with large molecule proteins, small molecule peptides exhibit more unique skincare potential in skincare products. They not only provide excellent moisturizing effects but also penetrate more easily below the stratum corneum, promoting skin repair and renewal, and providing deeper nourishment and care for the skin.

[0003] Despite the numerous advantages of small molecule peptides in cosmetics, their application also faces several challenges. Firstly, due to their hydrophilic nature, they are mostly limited to water-based products such as serums and masks. In oil-based systems like water-in-oil (W / O) emulsions, facial oils, or sunscreens, small molecule peptides are difficult to add stably, limiting the diversity of cosmetic formulations and preventing some oil-based products with specific functions from fully utilizing their advantages. For example, in sunscreens, if oil-soluble modification cannot be achieved, small molecule peptides will separate from the oil phase components, resulting in an uneven sunscreen texture, affecting the user experience and sun protection effectiveness.

[0004] Secondly, when small molecule peptides are physically mixed with oil-phase components, they are prone to aggregation and inactivation due to phase separation. Aggregated small molecule peptides not only fail to effectively exert their biological activity, but may also form particulate substances, affecting the appearance and texture of cosmetics and further reducing consumer trust in the product.

[0005] Furthermore, the skin's surface lipid membrane is lipophilic, making it difficult for hydrophilic small molecule peptides to penetrate this barrier, thus affecting their anti-wrinkle and repair effects. Studies have shown that only active ingredients that can penetrate the lipid membrane can better reach the deeper layers of the skin and exert their biological effects. Therefore, improving the oil solubility of small molecule peptides is crucial for enhancing their transdermal efficiency.

[0006] To address the hydrophilicity issue of small peptides, some existing technologies employ chemical phase modification methods, such as glutaraldehyde crosslinking and succinic anhydride acylation. However, these methods require the use of toxic organic solvents (such as DMF and THF), which not only pollute the environment but also pose potential health hazards, failing to meet cosmetic safety standards. Furthermore, the use of organic solvents increases production costs and operational complexity, limiting their large-scale application. Some existing technologies utilize liposomes or nanoemulsions to encapsulate small peptides, but these methods suffer from low encapsulation rates and susceptibility to demulsification under high temperatures, resulting in poor release and stability of the small peptides.

[0007] Therefore, developing a method to modify small molecule peptides under mild conditions to improve their oil solubility, so that the modified small molecule peptides can not only exert their original biological activity in cosmetics, but also have other additional functions, thus meeting the diverse needs of consumers for cosmetics, has extremely high practical and economic value. Summary of the Invention

[0008] To address the aforementioned problems, this invention involves acyl chlorination of hydrophilic peptides in an aqueous phase, improving the ratio of protein to acyl chloride and the rate of acyl chloride addition, thereby preparing a series of oil-soluble modified proteins with enhanced oil solubility.

[0009] The first objective of this invention is to provide a method for preparing oil-soluble modified peptides, the method comprising the steps of: (1) Disperse the polypeptide powder in water and stir to obtain a polypeptide solution; (2) Under pH 8~10 conditions, acyl chloride is added dropwise to the polypeptide solution and reacted; after the reaction, ultrafiltration is performed, and 30% hydrochloric acid aqueous solution is added to adjust the pH to 3~3.5 before filtration to obtain filter cake. The filter cake is dried to obtain oil-soluble modified polypeptide. The ratio of polypeptide powder to acyl chloride is 300~500 mg: 330~800 μL; The polypeptide is any one of mussel protein peptide, silk fibroin peptide, or sericin peptide.

[0010] In one embodiment, the molecular weight of mussel protein peptide is 4-10 kDa, the molecular weight of silk fibroin peptide is 1.5-5 kDa, and the molecular weight of sericin peptide is 3-10 kDa.

[0011] In one embodiment, the ratio of polypeptide to water in step (1) is 300~500 mg: 5~10 mL.

[0012] In one embodiment, the stirring in step (1) is carried out at 10~25 °C for 30~35 min.

[0013] In one embodiment, the dropping rate of acyl chloride in step (2) is 1.83~13.33 μL / min.

[0014] Optionally, the dropping rate of acyl chloride is 2~8 μL / min.

[0015] In one embodiment, the reaction in step (2) is carried out at 5~25 °C for 120~360 min.

[0016] In one embodiment, the acyl chloride is any one of palmitoyl chloride, lauroyl chloride, cocoyl chloride, myristoyl chloride, and octanoyl chloride.

[0017] In one embodiment, the acyl chloride is palmitoyl chloride.

[0018] A second object of the present invention is to provide an oil-soluble modified polypeptide prepared by any of the above methods.

[0019] A third object of the present invention is to provide the application of any of the methods described above or the oil-soluble modified peptides in the preparation of cosmetics.

[0020] The fourth objective of this invention is to provide a cosmetic product, characterized in that the cosmetic product contains the oil-soluble modified polypeptide as described in claim 7; the cosmetic product is an essential oil, sunscreen, foundation, or lipstick.

[0021] A fifth objective of this invention is to provide a method for improving the oil solubility of peptides, the method comprising the steps of: (1) Disperse the polypeptide powder in water and stir to obtain a polypeptide solution; (2) Under pH 8~10 conditions, acyl chloride is added dropwise to the polypeptide solution and reacted; after the reaction, ultrafiltration is performed, and 30% hydrochloric acid aqueous solution is added to adjust the pH to 3~3.5 before filtration to obtain filter cake. The filter cake is dried to obtain oil-soluble modified polypeptide. The ratio of polypeptide powder to acyl chloride is 300-500 mg: 5-10 mL. The polypeptide is any one of mussel protein peptide, silk fibroin peptide, and sericin peptide; the acyl chloride includes palmitoyl chloride, lauroyl chloride, cocoyl chloride, myristoyl chloride, and capryloyl chloride; In step (2), the dropping rate of acyl chloride is 1.83~13.33 μL / min; Optionally, the dropping rate of acyl chloride is 2~8 μL / min.

[0022] The ratio of polypeptide powder to acyl chloride is 300~500 mg: 330~800 μL; The polypeptide is any one of mussel protein peptide, silk fibroin peptide, or sericin peptide.

[0023] In one embodiment, the ratio of polypeptide to water in step (1) is 300~500 mg: 5~10 mL.

[0024] In one embodiment, the stirring in step (1) is carried out at 10~25 °C for 30~35 min.

[0025] In one embodiment, the reaction in step (2) is carried out at 5~25 °C for 240~480 min.

[0026] Beneficial effects of the present invention This invention involves acyl chlorination of hydrophilic peptides in an aqueous phase, and by improving the ratio of peptides to acyl chlorides and the addition rate of acyl chlorides, a series of oil-soluble modified peptides with improved oil solubility are prepared.

[0027] Specifically, (1) In this invention, hydrophilic peptides are acyl chlorided in an aqueous phase, and the ratio of peptides to acyl chloride and the addition rate of acyl chloride are improved to prepare a series of oil-soluble modified peptides with improved oil solubility.

[0028] (2) The oil-soluble modified polypeptide prepared by the present invention has excellent oil solubility and can be dissolved in common natural plant oils such as meadowfoam seed oil and camellia seed oil (solubility > 0.5% w / v). (3) The oil-soluble modified peptides prepared by this invention have improved stability. After being dissolved in the oil phase and subjected to an accelerated test at 60°C for 30 days, the peptide structure retention rate is >83%. (4) The oil-soluble modified peptide prepared in this invention has enhanced transdermal penetration. According to the Franz diffusion cell test, the skin penetration of the modified peptide increased by 9.4 times, and the cumulative penetration in 24 h increased from 0.672 μg / cm² to 6.33 μg / cm². (5) The oil-soluble modified peptides prepared by the present invention have excellent formulation compatibility and can be stably present in high oil content systems such as W / O emulsions and cold creams. Attached Figure Description

[0029] Figure 1 The images show the infrared spectra of the protein before and after modification.

[0030] Figure 2 The results show the penetration of oil-soluble modified peptides and unmodified peptides.

[0031] Figure 3 The results of human efficacy testing (wrinkle reduction) for the addition of oil-soluble modified peptide emulsion. Detailed Implementation

[0032] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0033] Raw materials used in the examples: Mussel protein peptides (molecular weight 6 kDa) were purchased from Shaanxi Mufan Biotechnology Co., Ltd. Silk fibroin peptides (molecular weight 1.5 kDa) were purchased from Condis (Chemical) Hubei Co., Ltd. Sericin peptides (molecular weight 5 kDa) were purchased from Jiangsu Enming Biotechnology Co., Ltd. Palmitoyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Octanoyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Lauroyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Myristoyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Cocoyl chloride was purchased from Hubei Xingdongcheng Chemical Co., Ltd. Stearoyl chloride was purchased from Hubei Chengfeng Chemical Co., Ltd.

[0034] Test method: 1. Detection method for promoting fibroblast proliferation The MTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide) method is a classic cell proliferation / toxicity assay that indirectly reflects cell number and viability by detecting the ability of mitochondrial dehydrogenases in living cells to reduce MTT to formazan.

[0035] Take fibroblasts in the logarithmic growth phase and adjust the density (5×10⁻⁶). 3 ~1×10 4 Cells / well, 96-well plate); 100 μL of complete culture medium was added to each well, and the cells were cultured for 24 h to allow them to adhere; the experimental groups are as follows: Blank control group (culture medium only, no cells), negative control group (untreated cells), experimental group (different concentrations of test samples), positive control group (e.g., 10% FBS or PDGF stimulation); add 10 μL of MTT solution (5 mg / mL, prepared in PBS) to each well (final concentration 0.5 mg / mL); incubate at 37 ℃ for 4 h, then discard the supernatant; add 100~150 μL of DMSO to each well, and shake slowly for 10 min to dissolve the crystals; measure the absorbance at 490 nm using a microplate reader.

[0036] 2. Transdermal efficacy testing methods The Franz Diffusion Cell is a standard in vitro model for studying the transdermal absorption of drugs / active ingredients and is suitable for evaluating the transdermal permeability of oil-soluble modified proteins.

[0037] Take fresh hairless pig skin, remove subcutaneous fat, wash with physiological saline, check for integrity (no damage), and soak in receptor fluid for 30 minutes to achieve equilibrium. The skin is fixed between the donor pool and the recipient pool (with the stratum corneum facing the donor side). The recipient pool is filled with preheated recipient fluid (37±0.5℃), air bubbles are removed, and the fluid is magnetically stirred (500~600 rpm) to maintain its homogeneity. Add 200-500 μL of oil-soluble modified protein preparation to the donor pool (ensure even skin coverage), while the control group contains an unmodified protein aqueous solution. At time points of 0, 5, 10, 15, 20, 22, and 24 h, 200–500 μL of sample was taken from the receptor pool (with isothermal and equal-volume receptor solution added simultaneously).

[0038] Cumulative permeability Q n =(C n ×V0+Σ(i=1ton-1)(C i ×V i )) / A Among them, Q n C represents the cumulative permeability at the nth sampling time point; n Vn represents the concentration of the drug in the receiving cell at the nth time point; V0 represents the total volume of the receiving cell; Cn represents the total volume of the receiving cell. i For the i-th sampling point, the volume of the sample taken from the receiving cell; ∑(i=1 to n-1) is the summation symbol; C i × V i This represents the summation of all terms from the 1st to the (n-1th)th time point; A is the effective permeation area of ​​the diffusion pool.

[0039] 3. Stability testing methods Group 1 was divided into two fully packaged samples. One sample was placed at room temperature, and the other was placed in the freezer at a temperature of (-18±1)℃. After 24 hours, the sample in the freezer was removed and brought back to room temperature. The sample was then compared with the sample at room temperature to observe the differences in characteristics. Group 2 was divided into two fully packaged samples. One sample was placed at room temperature, and the other was placed in a constant temperature chamber at (40±1)℃. After 24 hours, the sample in the constant temperature chamber was removed and brought back to room temperature. The sample was then compared with the sample at room temperature to observe the differences. Samples from group 3 were placed in centrifuge tubes, and the centrifuge speed was set to 4000 rpm. After 30 minutes of testing, the stratification and separation of the products were observed.

[0040] Example 1: Preparation of oil-soluble modified sericin peptides The steps for modifying oil-soluble sericin peptides using an aqueous phase acylation method are as follows: (1) Slowly disperse 300 mg of sericin peptide (5 kDa) powder in 5 mL of pre-cooled deionized water and stir continuously on a 10℃ constant temperature magnetic stirrer for 30 min until completely dissolved to form a clear and transparent sericin peptide solution. (2) Under pH 9.0 conditions, 360 μL of palmitoyl chloride (C18) was added dropwise to the sericin peptide solution at a flow rate of 3 μL / min using a micro-injection pump. After the addition was completed, the reaction was continuously stirred, and the temperature of the reaction system was maintained at 10℃±0.5℃ by an ice-water bath. (3) After the reaction was completed, the solution was purified by ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa (3500 rpm, 4 cycles). The retentate was collected and 30% hydrochloric acid aqueous solution was added to adjust the pH to 3~3.5. The filter cake was then filtered and the filter cake was treated by freeze dryer (pre-cooled at -80℃, vacuum degree ≤5 Pa) for 24 hours to obtain a white fluffy powder product (i.e., oil-soluble modified sericin peptide).

[0041] Comparative Example 1: Changing the ratio of sericin peptides to palmitoyl chloride (1) Based on Example 1, the ratio of sericin peptide to palmitoyl chloride was changed to 300 mg: 330 μL, and the remaining steps were the same as in Example 1.

[0042] (2) Based on Example 1, the ratio of sericin peptide to palmitoyl chloride was changed to 300 mg: 390 μL, and the remaining steps were the same as in Example 1.

[0043] (3) Based on Example 1, the ratio of sericin peptide to palmitoyl chloride was changed to 300 mg: 480 μL, and the remaining steps were the same as in Example 1.

[0044] Comparative Example 2: Using other acyl chlorides Based on Example 1, octanoyl chloride, lauroyl chloride, myristoyl chloride, cocoyl chloride, and stearoyl chloride were used respectively, and the remaining steps were the same as in Example 1.

[0045] Comparative Example 3: Using traditional acyl chloride modification methods Based on the literature "The effect of crosslinking of genipin, glutaraldehyde, or EDC / NHS on the construction of collagen / chitosan dermal scaffolds", the traditional water-organic solvent method was used to modify sericin peptides, and the steps are as follows: When modifying sericin peptides using a water-acetone system, the sericin peptides are first dissolved in 0.5 M acetic acid solution (pH 3.0) or PBS buffer (pH 7.4) to prepare a 3 mg / mL solution, which is then stirred overnight at 4°C to ensure complete dissolution. Pre-cooled acetone is then slowly added dropwise under ice bath conditions, gradually increasing the acetone ratio (water:acetone from 1:1 to 1:4) through a gradient dehydration process, while controlling the temperature to not exceed 10°C to avoid protein denaturation. Further crosslinking requires the addition of 0.01%–0.1% glutaraldehyde or EDC / NHS as a crosslinking agent. After the reaction is complete, the precipitate is collected by centrifugation and washed with 70% cold acetone to remove residual reagents. Finally, reconstitution or freeze-drying is performed according to application requirements.

[0046] Comparative Example 4: Changing the temperature Based on Example 1, the reaction temperature in step (2) was changed to 25°C, and the remaining steps were the same as in Example 1, to prepare oil-soluble modified sericin peptides.

[0047] Comparative Example 5: Physical Embedding Method Following the literature "Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery; DOI:10.1016 / j.peptides.2009.10.002.", the physical embedding method was used to embed sericin peptides, and the steps are as follows: Liposomes were prepared by mixing sericin peptides with lipid materials such as soybean lecithin and cholesterol using a high-pressure homogenization method. The particle size and encapsulation efficiency of the liposomes were controlled by optimizing parameters such as homogenization pressure, temperature, and time. The prepared liposomes were then added to the oil phase to form a water-in-oil emulsion.

[0048] First, the sericin peptide solution was mixed with lipid materials such as soybean lecithin and cholesterol in a certain proportion. High-pressure homogenization was performed at 60 MPa for 5-10 cycles, with the temperature controlled within 25 °C. By optimizing the homogenization parameters (pressure 60 MPa, temperature 15 °C, 8 cycles), liposomes with a particle size of 200 nm and an encapsulation efficiency of over 80% were obtained. Then, the prepared liposome dispersion was slowly added to an oil phase preheated to 35-45 °C, with the oil-to-water volume ratio controlled between 3:1 and 5:1. High-speed shear emulsification was performed at 14000 rpm for 3 min, ultimately forming a stable water-in-oil emulsion. The emulsion particle size was determined by dynamic light scattering, and the encapsulation efficiency was quantitatively analyzed using ultrafiltration centrifugation combined with the BCA method. The entire preparation process must be carried out under aseptic conditions, and the pH must be controlled within the range of 6.5-7.4 to maintain the stability of the sericin peptides.

[0049] Example 2: Performance testing of oil-soluble modified sericin peptides The properties of the oil-soluble modified sericin peptides prepared in Example 1 and Comparative Examples 1-5 were tested.

[0050] 1. Infrared Spectroscopy The oil-soluble modified sericin peptides prepared in Example 1 were analyzed by Fourier transform infrared spectroscopy (FTIR) (with unmodified sericin peptides as a control). The results are as follows: Figure 1 As shown.

[0051] 2. Oil solubility test results The solubility of oil-soluble modified sericin peptides and unmodified sericin peptides prepared in Examples 1 and Comparative Examples 1-5 in different oils was tested, and the results are shown in Table 1.

[0052] Table 1. Solubility test results of different oils

[0053] The results showed that the oil-soluble modified sericin peptides had a solubility of 0.65% (w / v, g / mL) in meadowfoam seed oil, while the unmodified sericin peptides could not be dissolved in the oil, indicating that modification has a significant effect on improving the solubility of peptides in oil.

[0054] Among them, comparing Example 1 and Comparative Example 1, it can be seen that the solubility of the modified protein in Example 1 in meadowfoam seed oil, camellia seed oil and sunflower seed oil is significantly better than the solubility of the products obtained in the three different proportions in Comparative Example 1. Comparing Example 1 and Comparative Example 2, it can be seen that octanoyl chloride (C8), lauroyl chloride (C12), myristoyl chloride (C14), cocoyl chloride (mixed C8-C18) and stearoyl chloride (C18) all have poor solubility in meadowfoam seed oil, camellia seed oil and sunflower seed oil; Comparing Example 1 and Comparative Example 4, it can be seen that a higher reaction temperature will significantly affect the solubility of the prepared oil-soluble modified sericin peptides. Comparing Example 1 with Comparative Examples 3 and 5, it can be seen that the sericin peptide modification method of Example 1 is significantly better than the traditional acyl chloride modification method and physical modification method in terms of solubility.

[0055] 3. Bioactivity The effects of oil-soluble modified sericin peptides and unmodified sericin peptides prepared in Example 1 and Comparative Examples 1-5 on promoting fibroblast proliferation were tested, and the results are shown in Table 2.

[0056] Table 2. Proliferation-promoting effect on fibroblasts

[0057] The results showed that, compared with Example 1, oil-soluble modified sericin peptides prepared by different ratios of sericin peptides and palmitoyl chloride had no effect on the proliferation of fibroblasts and were similar to unmodified sericin peptides. Comparing Example 1 with Comparative Examples 2-4, it can be seen that the oil-soluble modified sericin peptide prepared in Example 1 has a better proliferative effect on fibroblasts than the modified sericin peptide prepared using other acyl chlorides and conventional methods.

[0058] 4. Stability testing Sericin peptides (2.75 mg / mL) were dissolved in meadowfoam seed oil; control group: stored at 4℃ (untreated group); experimental group: placed in a constant temperature oven at 60℃ for 30 days; Temperature 60℃±1℃ (daily calibration temperature), time 30 days (it is recommended to record oven temperature fluctuations daily), sampling points 0 days (initial), 7 days, 15 days, 30 days (can be increased as needed).

[0059] Structural characterization of sericin peptide samples was performed using solid-state circular dichroism chromatography (SS-CD). A certain amount of sample powder was compressed into tablets, ensuring uniform compression without cracks. A J-1500 circular dichroism chromatograph was used to scan within the wavelength range of 190–250 nm (parameter settings: bandwidth 1 nm, scan speed 50 nm / min, isothermal at 25℃). Baseline correction with a blank quartz slide was performed before testing, and the sample was placed perpendicular to the optical path to minimize scattering interference.

[0060] The results showed that α-helix had two negative peaks at 208 nm and 222 nm, and a positive peak at 198 nm; β-sheet had a single negative peak at 218 nm and a strong positive peak at 195 nm; and random coil had a strong negative peak near 200 nm, with no characteristic secondary peaks.

[0061] Furthermore, the secondary structure of the sample was analyzed. Deconvolution calculations using far-ultraviolet circular dichroism spectroscopy with CDPro software revealed an initial random coil structure content of 83.5%. To assess its structural stability, an accelerated aging experiment was conducted at 60°C for 30 days.

[0062] The CD spectrum of the aged sample maintained 87.3% of its initial peak intensity at 222 nm. This result indicates that the sample retains most of its secondary structure under harsh conditions, and its stability (87.3%) exceeds the design requirement of 85%. All data were obtained through three repeated scans during testing, with a relative standard deviation (RSD) of less than 3%, ensuring data accuracy.

[0063] The results are shown in Table 3. The results indicate that the modified sericin peptide can still effectively maintain the stability of its secondary structure under solid conditions.

[0064] Table 3 Stability Testing

[0065] 5. Transdermal Results The transdermal effect of the oil-soluble modified sericin peptides prepared in Example 1 was tested, and the results are as follows: Figure 2 As shown, the results indicate that the cumulative penetration of the oil-soluble modified sericin peptide in pig skin increased by 9.4 times over 24 h.

[0066] The transdermal effects of the oil-soluble modified sericin peptides prepared in Comparative Examples 3 and 5 were tested. The results showed that the transdermal effect of the oil-soluble modified sericin peptides prepared by the traditional acyl chloride method (Comparative Example 3) was only 1.8 times that of the natural sericin peptides. At the same time, due to the use of organic solvents, trace amounts of DMF residue were present in the products, which did not meet the strict cosmetic safety standards. The transdermal efficiency of physically encapsulated modified sericin peptides (Comparative Example 5) is only 1 times that of natural sericin peptides. Furthermore, the physical encapsulation method is complex and has high production costs, making it unsuitable for large-scale production.

[0067] 6. Stability in water-in-oil emulsions The oil-soluble modified sericin peptides prepared in Example 1 were used to prepare water-in-oil emulsions, and the steps are as follows: 0.4% by mass of modified sericin peptide and 5% by mass of Span 80 were dissolved in meadowfoam seed oil as the oil phase (70% oil phase), and homogenized with 30% by mass of aqueous phase (0.9% by mass of NaCl solution) at 15000 rpm for 5 min to prepare an emulsion (70% oil phase and 30% aqueous phase).

[0068] The results showed that the emulsion exhibited excellent stability: no stratification or oil separation occurred after storage at 40℃ for 30 days, and the water separation rate by centrifugation was only 1.8%±0.4%; microscopic observation confirmed that the droplets were uniformly distributed and did not aggregate.

[0069] The modified sericin peptides prepared in Example 1 effectively anchor the oil phase through hydrophobic chains, giving the emulsion excellent anti-agglomeration properties and fully meeting the application requirements of cosmetics.

[0070] The liposomes prepared in Comparative Example 5 had an average particle size of approximately 160 nm and an encapsulation rate of 26%. They were initially stable in water-in-oil emulsions, but demulsification occurred after 14 days of storage, leading to the release of sericin peptides and the separation of the emulsion.

[0071] Example 3: Application of oil-soluble modified sericin peptides in the preparation of cosmetics The oil-soluble modified sericin peptides prepared in Example 1 were used to prepare a cold cream (W / O), and its efficacy was tested.

[0072] (1) Preparation of anti-aging and moisturizing cold cream The anti-aging and moisturizing cold cream components include: oil-soluble modified sericin peptide (0.3% by mass), jojoba oil (25%), meadowfoam seed oil (30%), stearyl alcohol polyether-21 (1%), stearyl alcohol polyether-2 (1%), glycerin (15%), and deionized water (27.7%). A cold cream without oil-soluble modified sericin peptide was used as a control.

[0073] (2) Detection effect Subject selection criteria, basic conditions: Healthy adults aged 18-45 (with signed informed consent), without systemic diseases or immunodeficiency, and who have not taken anti-inflammatory / immunosuppressive drugs within the past month. Skin condition requirements are in accordance with GB / T 29665-2013. Smoothness: Score ≥3 points using palpation method (0-5 level scale, 3 points = texture can be perceived with slight touch but no roughness). Glossiness: Measured under standard lighting using a digital image analyzer (such as CK-MPA), the specular reflectance of the forehead area is ≥35%.

[0074] Exclusion criteria: The test site showed visible erythema, desquamation, or acne (confirmed by Wood's lamp test). Transepidermal water loss (TEWL) > 15 g·h -1 ·m -2 (Measured using MPA580 probe); A history of cosmetic allergy or positive dermatographia.

[0075] A double-blind, controlled trial was conducted on 30 female participants aged 35-55 for 4 weeks. The participants used the anti-aging serum oil twice a day, morning and evening, with each application consisting of two pumps.

[0076] The test results of the essential oil in the experimental group with added oil-soluble sericin peptides are as follows: Figure 3 As shown, the results indicate that the anti-aging essence oil can significantly improve dynamic expression lines. The VC20 wrinkle depth analyzer showed that the average depth of crow's feet wrinkles decreased by 21.4% (p<0.01), and the subjective scores of the subjects showed that more than 90% of the participants felt an improvement in skin firmness.

[0077] The results of a control group serum oil test without added oil-soluble silk protein peptides showed that the serum oil had limited effect on improving dynamic expression lines. VC20 wrinkle depth analyzer results showed no significant change in the average depth of crow's feet wrinkles, decreasing by only 3.6% (p>0.05). Meanwhile, subjective ratings from participants indicated that only about 30% reported an improvement in skin firmness.

[0078] (3) Accelerated stability test Further validation of its commercial potential: After storage at 40℃ / 75% RH for 3 months, the formula remained homogeneous and transparent, with no oil precipitation or sedimentation, indicating that the long-chain acylation modification effectively enhanced the dispersion stability of sericin peptides in complex oil systems. This formula provides an innovative solution for developing gentle and effective naturally derived anti-wrinkle products, especially suitable for the anti-aging needs of sensitive and oily skin types.

[0079] Example 4: Preparation of oil-soluble modified mussel protein Based on Example 1, mussel protein was used to replace sericin peptide, and the remaining steps were the same as in Example 1 to prepare oil-soluble modified mussel protein.

[0080] Example 5: Preparation of oil-soluble modified silk fibroin Based on Example 1, silk fibroin was used to replace sericin peptides, and the remaining steps were the same as in Example 1, resulting in oil-soluble modified silk fibroin.

[0081] The properties of oil-soluble modified mussel protein, oil-soluble modified silk fibroin, and oil-soluble modified sericin prepared in Examples 4, 5, and 6 were tested.

[0082] (1) Oil-soluble The oil solubility of oil-modified mussel protein and oil-modified silk fibroin was tested according to the method in Example 2, and the test results are shown in Table 4.

[0083] The results showed that the solubility of oil-soluble modified mussel protein and oil-soluble modified silk fibroin was improved in meadowfoam seed oil and camellia seed oil.

[0084] Table 4. Solubility test results of different oils

[0085] (2) Stability The oil-soluble modified mussel protein and oil-soluble modified silk fibroin prepared in Examples 4 and 5 were tested using the following steps: The oil-soluble modified proteins prepared in Examples 4 and 5 were dissolved in meadowfoam seed oil and placed in a constant temperature incubator at 60℃ (±1℃) for 30 days. Irradiation: UVB (302 nm, 0.5 mW / cm²) irradiation for 48 hours (simulating photoaging); Control group: Unmodified protein, blank oil phase; Centrifuge and separate the layers (3,000 rpm, 15 min), and calculate the oil separation rate; Detect changes in appearance (color, sediment).

[0086] The results are shown in Table 5. The results indicate that oil-soluble modified mussel protein and oil-soluble modified silk fibroin also have good stability in meadowfoam seed oil.

[0087] Table 5 Stability Test Results

[0088] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method of preparing an oil-soluble modified polypeptide, characterized by, The method comprises the steps of: (1) dispersing polypeptide powder in water, stirring to obtain a polypeptide solution; (2) under the condition of pH 8-10, adding acyl chloride dropwise into the polypeptide solution, and reacting; after the reaction, ultrafiltration, adding 30% hydrochloric acid aqueous solution to adjust the pH to 3-3.5, filtering to obtain a filter cake, and drying the filter cake to obtain an oil-soluble modified polypeptide; The ratio of the amount of polypeptide powder to the amount of acyl chloride is 300-500 mg: 330-800 μL. The polypeptide is any one of mussel protein peptide, silk fibroin peptide, and silk sericin peptide.

2. The method of claim 1, wherein, The ratio of the amount of polypeptide to the amount of water in step (1) is 300-500 mg: 5-10 mL.

3. The method of claim 1, wherein, The stirring in step (1) is stirring at 10-25 ℃ for 30-35 min.

4. The method of claim 1, wherein, The dropping rate of acyl chloride in step (2) is 1.83-13.33 μL / min. Optionally, the dropping rate of acyl chloride is 2-8 μL / min.

5. The method of claim 1, wherein, The reaction in step (2) is reaction at 5-25 ℃ for 120-360 min.

6. The method of claim 1, wherein, The acyl chloride is any one of palmitoyl chloride, lauroyl chloride, cocoyl chloride, stearoyl chloride, myristoyl chloride, and octanoyl chloride. Optionally, the acyl chloride is palmitoyl chloride.

7. The oil-soluble modified polypeptide prepared by the method of any one of claims 1-6.

8. The use of the method of any one of claims 1-6 or the oil-soluble modified polypeptide of claim 7 in the preparation of a cosmetic.

9. A cosmetic product, characterized by, The cosmetic contains the oil-soluble modified polypeptide of claim 7; and the cosmetic is essence oil, sunscreen, foundation, or lip balm.

10. A method of improving the oil solubility of a polypeptide, comprising, The method comprises the steps of: (1) dispersing polypeptide powder in water, stirring to obtain a polypeptide solution; (2) under the condition of pH 8-10, adding acyl chloride dropwise into the polypeptide solution, and reacting; after the reaction, ultrafiltration, adding 30% hydrochloric acid aqueous solution to adjust the pH to 3-3.5, filtering to obtain a filter cake, and drying the filter cake to obtain an oil-soluble modified polypeptide; The ratio of the amount of polypeptide powder to the amount of acyl chloride is 300-500 mg: 330-800 μL. The polypeptide includes mussel protein peptide, silk fibroin peptide, and silk sericin peptide; and the acyl chloride includes palmitoyl chloride, lauroyl chloride, cocoyl chloride, myristoyl chloride, and octanoyl chloride. The dropping rate of acyl chloride in step (2) is 1.83-13.33 μL / min. Optionally, the dropping rate of acyl chloride is 2-8 μL / min.

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