A composite small molecule protein composition and its preparation method

Abstract

This invention relates to the field of cosmetic technology and provides a composite small molecule protein composition and its preparation method. The composition comprises a core-shell structured complex and a ROS-responsive active component. The core-shell structured complex consists of a core A and a shell B connected by dual dynamic covalent bonds of Schiff base bonds and disulfide bonds. The Schiff base bonds impart pH-responsive transdermal release, while the disulfide bonds impart ROS-responsive self-repair function. The ROS-responsive active component is a thiol-containing small molecule peptide, accounting for 0.5-5 wt%. This invention achieves pH-responsive transdermal release and ROS-responsive self-repair function through a stepwise addition of the thiol-containing small molecule peptide via a pH-driven dual curing and mild oxidation process. The raw materials of this invention are readily available, and the process is simple. It can be further formulated into various dosage forms such as hair growth essence, hair growth gel, shampoo, conditioner, and hair mask, suitable for dry, oily, sensitive, and combination skin, and has extremely high industrialization value and market prospects.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, and in particular relates to a complex small molecule protein composition and its preparation method, which is specifically applicable to multiple scenarios such as scalp and hair care, facial skin care, and body skin care, and can be used to prepare anti-aging, repair, and hair growth cosmetics. Background Technology

[0002] Small molecule active peptides and hydrolyzed collagen peptides are widely used in anti-aging, repair, and hair growth cosmetics due to their multiple effects, such as promoting collagen synthesis, repairing the skin barrier, and resisting photoaging. However, the following common technical bottlenecks exist in practical applications: 1) Low transdermal efficiency: Although small molecule active peptides have a small molecular weight (usually 200-2000 Da), they are highly hydrophilic and easily repelled by the surface charge of the skin, making it difficult for them to effectively penetrate the stratum corneum to reach the key anti-aging target at the dermal-epidermal junction (DEJ), resulting in low bioavailability.

[0003] 2) Poor stability: Small molecule active peptides are prone to degradation reactions such as hydrolysis, oxidation, and aggregation in aqueous solutions. Their stability drops sharply, especially under high temperature and light conditions, resulting in short product shelf life and rapid efficacy decay. This problem exists in various dosage forms such as aqueous solutions, emulsions, and gels.

[0004] 3) Uncontrollable release: Although traditional encapsulation technologies such as liposomes and polymer nanoparticles can improve stability to a certain extent, they often exhibit burst release phenomena and cannot achieve on-demand release according to the skin's physiological state (such as increased oxidative stress levels), making it difficult to meet precise skincare needs. Summary of the Invention

[0005] This invention provides a composite small molecule protein composition and its preparation method, aiming to solve the above-mentioned problems.

[0006] The present invention is achieved by providing a composite small molecule protein composition comprising a core-shell structured complex and a ROS-responsive active component. The core-shell structured complex is composed of a core A and an outer shell B connected by dual dynamic covalent bonds of Schiff base bonds and disulfide bonds. The Schiff base bonds endow the pH-responsive transdermal release function, while the disulfide bonds endow the ROS-responsive self-repair function. The core A is a small molecule active peptide with a molecular weight of 200-800 Da, selected from at least one of acetyl tetrapeptide-2, palmitoyl tripeptide-8, and palmitoyl tripeptide-5; The outer shell B is a low molecular weight hydrolyzed collagen peptide modified with plant polyphenols, with a molecular weight of 1000-2000 Da; The ROS-responsive active component is a thiol-containing small molecule peptide, accounting for 0.5-5 wt% of the total mass of the composition.

[0007] Furthermore, the thiol-containing small molecule peptide is reduced glutathione with a purity ≥98%.

[0008] Furthermore, the core A can be compounded from 300-500Da small molecule active peptides (such as acetyl tetrapeptide-2, palmitoyl tripeptide-8) and 200-300Da small molecule active peptides (such as palmitoyl tripeptide-5) in a mass ratio of 2:1-3:1, and the core A accounts for 5-15wt% of the total mass of the composition after compounding.

[0009] Furthermore, the plant polyphenols comprise chlorogenic acid, rosmarinic acid, and resveratrol in a mass ratio of 5:1:0.5-10:2:1, and the polyphenols account for 5-20 wt% of the total mass of shell B.

[0010] Furthermore, the mass ratio of the core A to the outer shell B is 1:2 to 1:5.

[0011] Plant polyphenols (such as chlorogenic acid and rosmarinic acid) contain catechol or pyrogallol groups in their molecular structure. Under weakly alkaline conditions (pH 8.0-8.5), they are easily oxidized to form orthoquinone structures. Orthoquinone structures have high reactivity and can undergo Michael addition reactions or Schiff base reactions with the free amino groups of lysine, arginine, and other residues on collagen peptide chains to form stable CN covalent bonds, thereby achieving "polyphenol covalent modification" of collagen. This covalent modification not only endows collagen with additional antioxidant functions but also provides an active site for subsequent Schiff base reactions with the core A.

[0012] The present invention also provides a method for preparing the above-mentioned composite small molecule protein composition, comprising the following steps: a) Preparation steps for shell B: Low molecular weight hydrolyzed collagen peptides were dissolved in deionized water (solid-to-liquid ratio 1:8, g / mL), and plant polyphenols and thiol-containing small molecule peptides were added. 10% Adjust the pH to 8.0-8.5 with NaOH solution, and stir the reaction at 35-45℃ and 200-300 r / min for 3-5 hours. The thiol-containing small molecule peptides are added in steps: first, 50-70% of the total mass of the thiol-containing small molecule peptides are added to participate in the preparation of shell B, and the remaining 30-50% is added in the mild oxidation stage of step c. The reaction solution is transferred to a dialysis bag (molecular weight cutoff 3500 Da), dialyzed in deionized water for 24 hours (water changed every 6 hours), and then freeze-dried at -50℃ and 0.01 MPa for 12 hours to obtain the polyphenol-collagen-thiol-containing peptide complex. The progress of polyphenol grafting can be tracked by monitoring the change of absorbance at 280 nm. When the increase in absorbance tends to be stable (usually 3-5 hours), it indicates that the grafting reaction is complete. The grafting rate is controlled within the range of 5-20 wt%, which is achieved by adjusting the initial amount of polyphenol.

[0013] b) Core-shell pre-assembly steps: The shell B obtained in step a and the core A are dissolved in a phosphate buffer solution with a mass ratio of 2:1-5:1 and reacted for 1-2 hours to form a pre-complex containing Schiff base bonds. c) Dual curing and gentle oxidation steps: The pH of the reaction solution was adjusted to 5.3-6.0 with citric acid. 0.005-0.02 wt% of a natural cross-linking agent was added, and 0.1-0.5 v / v% of sterile air was introduced. The reaction was carried out at 15-25°C for 2-3 hours. The reaction solution was then purified by ultrafiltration (molecular weight cutoff 10 kDa, pressure 0.15 MPa) to obtain a composite small molecule protein composition. The free sulfhydryl content could be monitored using the Ellman method (DTNB reagent). When the free sulfhydryl content decreased to 30-50% of the initial value, it indicated that disulfide bond formation had reached the expected level. The amount of sterile air introduced (0.1-0.5 v / v%) and the reaction time (2-3 hours) synergistically controlled the degree of oxidation. Furthermore, the natural crosslinking agent mentioned in step c is tannic acid or genipin.

[0014] The present invention also provides the application of the above-mentioned composite small molecule protein composition in the preparation of anti-aging, repair and hair growth cosmetics, wherein the cosmetics are selected from at least one of hair growth essence, hair growth gel, shampoo, conditioner and hair mask.

[0015] Furthermore, the hair growth essence comprises: 10-20wt% of a complex small molecule protein composition, 5-10wt% of squalane, 2-5wt% of glycerin, 0.3-0.8wt% of panthenol, 0.05-0.1wt% of zinc pyrrolidone carboxylate, with the balance being deionized water; during preparation, the complex small molecule protein composition is mixed with oil at a mass ratio of 5:1-10:1, and homogenized by microfluidic jet 2-3 times at 20-25℃ and 600-800 bar pressure (each homogenization time is 5-10 min) to obtain a surfactant-free Pickering nanoemulsion with a particle size of 50-80 nm and a PDI < 0.2; zinc pyrrolidone carboxylate is an oil-controlling conditioner, suitable for various care needs.

[0016] Furthermore, the hair growth gel comprises: 8-15 wt% of a complex small molecule protein composition, 5-8 wt% caprylic / capric triglycerides, 0.1-0.5 wt% caffeine, 0.2-0.5 wt% hyaluronic acid oligosaccharides, 0.4-0.8 wt% carbomer 940, 0.1-0.3 wt% triethanolamine, and the balance being deionized water; in preparation, the complex small molecule protein composition is mixed with the above ingredients, stirred at room temperature until homogeneous, and the pH is adjusted to 5.5-6.0 to obtain a transparent gel; carbomer 940 is a gel consistency regulator, and triethanolamine neutralizes to form a gel, suitable for topical application.

[0017] Furthermore, the hair mask comprises: 10-15 wt% of a complex small molecule protein composition, 3-5 wt% of squalane, 2-4 wt% of panthenol, 1-2 wt% of hydrolyzed keratin, 0.5-1 wt% of lanolin, with the balance being deionized water. During preparation, a conventional emulsification process is used, heating the oil phase (squalane, lanolin) and the aqueous phase (complex small molecule protein composition, panthenol, hydrolyzed keratin, deionized water) to 75-80°C respectively, mixing and emulsifying, and then cooling to below 35°C to produce a cream-like hair mask. The hydrolyzed keratin and lanolin serve as film-forming moisturizers, suitable for deep repair needs.

[0018] Furthermore, the shampoo comprises: 5-10 wt% of a complex small molecule protein composition, 3-5 wt% of potassium cocoyl glycinate, 2-4 wt% of glycerin, 0.2-0.5 wt% of panthenol, 0.1-0.3 wt% of polyquaternium-73, with the balance being deionized water; it is prepared using conventional shampoo manufacturing processes, dissolving the surfactant at 70-75°C, cooling to below 35°C, adding the complex small molecule protein composition and conditioning agent, and stirring until homogeneous; potassium cocoyl glycinate is a mild amino acid surfactant, and polyquaternium-73 is a barrier repair conditioning agent, avoiding the irritation of traditional products.

[0019] Furthermore, the conditioner comprises: 8-12 wt% of a complex small molecule protein composition, 2-4 wt% of squalane, 1-3 wt% of cetyl alcohol, 0.5-1 wt% of stearyltrimethylammonium chloride, 0.2-0.5 wt% of hyaluronic acid oligosaccharides, with the balance being deionized water; it employs a conventional emulsification process, emulsifying the oils and emulsifiers at 75-80°C, cooling to below 35°C, adding the complex small molecule protein composition, and stirring until homogeneous; cetyl alcohol is a thickening and softening agent, and stearyltrimethylammonium chloride is a cationic softening agent, suitable for smoothness requirements.

[0020] Compared with the prior art, the embodiments of this application have the following main advantages: The composite small molecule protein composition provided by this invention achieves intelligent response by constructing a dual dynamic covalent bond system of Schiff base bonds (pH response) and disulfide bonds (ROS response) to maintain stability in normal environments and release during oxidative stress. When the skin encounters oxidative stress (increased ROS), the disulfide bonds break, releasing thiol-containing small molecule peptides to clear ROS. After the environment recovers, the disulfide bonds can reform, and the core-shell structure achieves self-repair. The ternary polyphenol gradient synergistic system (chlorogenic acid + rosmarinic acid + resveratrol) simultaneously achieves cross-linking, anti-oxidation, and anti-aging functions, exhibiting a synergistic effect. By adding thiol-containing small molecule peptides stepwise, the ratio of bound to free states can be controlled, providing a technical means to balance structural stability and response sensitivity. At the same time, the composition of this invention can be adapted to various dosage forms such as hair growth essence, gel, shampoo, conditioner, and hair mask. The auxiliary formula is gentle and suitable for various skin types, including dry, oily, sensitive, and combination skin. Attached Figure Description

[0021] Figure 1 This is a flowchart of a method for preparing a composite small molecule protein composition provided by the present invention. Detailed Implementation

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0023] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0024] Example 1: Preparation of a complex small molecule protein composition (containing 1 wt% thiol small molecule peptides, pH 5.8) 1) Preparation of outer shell B: 100g of low molecular weight hydrolyzed collagen peptides (Mw≈1200Da) were dissolved in 800mL of deionized water (solid-to-liquid ratio 1:8, g / mL). 20g of chlorogenic acid, 4g of rosmarinic acid, 2g of resveratrol, and 5g of reduced glutathione were added (3g, accounting for 60% of the total mass of glutathione, was added first for the preparation of shell B, and the remaining 2g was reserved for addition in step 3). The pH was adjusted to 8.5 with 10% NaOH solution, and the reaction was stirred at 40℃ and 250r / min for 4 hours. The absorbance at 280nm was monitored during the reaction. After 4 hours, the absorbance tended to stabilize, indicating that the grafting reaction was complete. The reaction solution was transferred to a dialysis bag (molecular weight cutoff 3500Da), dialyzed in deionized water for 24 hours (water was changed every 6 hours), and then freeze-dried at -50℃ and 0.01MPa for 12 hours to obtain the polyphenol-collagen-glutathione complex (shell B).

[0025] The obtained complex and the unmodified collagen peptide were analyzed by Fourier transform infrared spectroscopy (FTIR). The results showed that the polyphenol-modified complex was spectral at 1650 cm⁻¹. -1 (Amide I band) and 1540cm -1 The characteristic peak at (amide II band) shifts, and at the same time, at 1600 cm⁻¹... -1 The appearance of a new absorption peak nearby (vibration of the aromatic ring C=C skeleton) confirms that polyphenols have formed a covalent bond with collagen.

[0026] 2) Core-shell pre-assembly: Take 30g of the outer shell B obtained in step 1 and 10g of the core A (acetyl tetrapeptide-2: palmitoyl tripeptide-5 = 2:1, Mw≈400Da+250Da), dissolve them in 500mL of pH 8.2 phosphate buffer, and pre-react for 1 hour to form a reversible pre-complex containing Schiff base bonds.

[0027] 3) Dual curing and gentle oxidation: The pH of the reaction solution was slowly adjusted to 5.8 using a 10% citric acid solution. 2g of reduced glutathione and 0.05g of tannic acid (0.01wt% of the total reaction solution mass) reserved in step 1 were added. Sterile air was introduced at 0.3v / v% (of the total reaction solution volume), and the reaction was stirred at 20°C for 2 hours. The free sulfhydryl content was monitored using the Ellman method. After 2 hours, the free sulfhydryl content decreased to 40% of the initial value, indicating that disulfide bond formation had reached the expected level. The reaction solution was purified by ultrafiltration (molecular weight cutoff 10kDa, pressure 0.15MPa) to obtain a composite small molecule protein composition (active ingredient content ≥15wt%).

[0028] Example 2: Preparation of a composite small molecule protein composition (containing 3 wt% thiol small molecule peptides, with high ROS response) 1) Preparation of outer shell B: 100g of hydrolyzed collagen peptides (Mw≈1200Da), 20g of chlorogenic acid, 4g of rosmarinic acid, 2g of resveratrol, and 15g of reduced glutathione (10g, accounting for 67% of the total mass of glutathione, was added first for the preparation of shell B, and the remaining 5g was reserved for addition in step 3) were dissolved in 800mL of deionized water. The pH was adjusted to 8.5 with 10% NaOH. The reaction was carried out at 40℃ and 250r / min for 4 hours. The absorbance at 280nm was monitored until it stabilized. The mixture was dialyzed (3500Da, 24h, water changed every 6h) and freeze-dried at -50℃ and 0.01MPa for 12h. FTIR characterization was the same as in Example 1.

[0029] 2) Core-shell pre-assembly: Shell B 30g, core A (acetyl tetrapeptide-2: palmitoyl tripeptide-5=2:1) ​​10g, pH 8.3 phosphate buffer 500mL, pre-reaction 1.5 hours.

[0030] 3) Dual curing and gentle oxidation: The pH of the reaction solution was slowly adjusted to 5.6 using a 10% citric acid solution. 5g of reduced glutathione and 0.08g of tannic acid (0.016wt% of the total reaction solution mass) reserved in step 1 were added. Sterile air was introduced at 0.4v / v% (of the total reaction solution volume), and the reaction was stirred at 20°C for 2.5 hours. The free sulfhydryl content was monitored using the Ellman method. After 2.5 hours, the free sulfhydryl content decreased to 35% of the initial value, indicating that disulfide bond formation had reached the expected level. The reaction solution was purified by ultrafiltration (molecular weight cutoff 10kDa, pressure 0.15MPa) to obtain a composite small molecule protein composition.

[0031] Example 3: Preparation of a composite small molecule protein composition (containing 0.5 wt% thiol small molecule peptides, pH 5.5) 1) Preparation of outer shell B: 100g of hydrolyzed collagen peptides (Mw≈1200Da), 26g of chlorogenic acid, 5.2g of rosmarinic acid, 2.6g of resveratrol, and 2.5g of reduced glutathione (1.5g, accounting for 60% of the total mass of glutathione, was added first for the preparation of shell B, and the remaining 1g was reserved for addition in step 3) were dissolved in 800mL of deionized water. The pH was adjusted to 8.2 with 10% NaOH. The reaction was carried out at 40℃ and 250r / min for 4 hours. The absorbance at 280nm was monitored until it stabilized. The mixture was dialyzed (3500Da, 24h, water changed every 6h) and freeze-dried at -50℃ and 0.01MPa for 12h. FTIR characterization was the same as in Example 1.

[0032] 2) Core-shell pre-assembly: Shell B 40g, core A (palmitoyl tripeptide-8, Mw≈450Da) 10g, pH 8.2 phosphate buffer 500mL, pre-reaction 1 hour.

[0033] 3) Dual curing and gentle oxidation: The pH of the reaction solution was slowly adjusted to 5.5 using a 10% citric acid solution. 1 g of reduced glutathione and 0.05 g of tannic acid (0.01 wt% of the total reaction solution mass) reserved from step 1 were added. Sterile air (0.2 v / v% of the total reaction solution volume) was introduced, and the reaction was stirred at 20°C for 2 hours. The free sulfhydryl content was monitored using the Ellman method. After 2 hours, the free sulfhydryl content decreased to 45% of the initial value, indicating that disulfide bond formation had reached the expected level. The reaction solution was purified by ultrafiltration (molecular weight cutoff 10 kDa, pressure 0.15 MPa) to obtain a composite small molecule protein composition.

[0034] Example 4 The only difference from Example 1 is that the pH is adjusted to 5.3 in step 3; otherwise, they are exactly the same.

[0035] Example 5 The only difference from Example 1 is that the pH is adjusted to 6.0 in step 3; otherwise, they are exactly the same.

[0036] Example 6 The only difference from Example 1 is that hydrolyzed collagen peptides with a molecular weight of 1800 Da are used, and the chlorogenic acid grafting amount is increased to 18 wt%, otherwise they are exactly the same.

[0037] Example 7 The difference from Example 1 is that hydrolyzed collagen peptides with a molecular weight of 1000 Da are used, but otherwise they are exactly the same.

[0038] Example 8 The difference from Example 1 is that hydrolyzed collagen peptides with a molecular weight of 1500 Da are used, but otherwise they are exactly the same.

[0039] Comparative Example 1: Physical mixture (no covalent, no curing) 10g of core A (acetyl tetrapeptide-2) and 30g of shell B (a physical mixture of collagen peptides and polyphenols prepared according to the method of Example 1 but without covalent grafting) were directly physically mixed and dissolved in 500mL of pH 8.2 phosphate buffer. The mixture was purified directly by ultrafiltration without pH adjustment, cross-linking agent addition, or air introduction.

[0040] Comparative Example 2: No thiol-containing small molecule peptides, no disulfide bonds (no ROS response) The difference from Example 1 is that no thiol-containing small molecule peptides are added in step 1, and sterile air is not introduced in step 3. Only Schiff base single bonds are formed, and no disulfide bonds are formed.

[0041] Comparative Example 3: Traditional liposome encapsulation (conventional technique) Liposomes were prepared using a lecithin:cholesterol ratio of 4:1 (mass ratio), encapsulating a core A (acetyl tetrapeptide-2) of the same mass as in Example 1, but without employing the core-shell structure of this invention, and purified by ultrafiltration.

[0042] Comparative Example 4: Single Polyphenol Modification (No Synergy) The difference from Example 1 is that only 20g of chlorogenic acid was added in step 1, and rosmarinic acid and resveratrol were not added, but the rest were exactly the same.

[0043] Comparative Example 5: Diphenol Modification (without Ternary Synergistic Effect) The difference from Example 1 is that 20g of chlorogenic acid and 4g of rosmarinic acid were added in step 1, but resveratrol was not added; otherwise, they were exactly the same.

[0044] Comparative Example 6: Thiol-containing peptides added in one step (no stepwise process) The difference from Example 1 is that 5g of all reduced glutathione was added in step 1, but not in step 3; otherwise, they are exactly the same.

[0045] Effect Verification Example 1. Testing Method (1) Transdermal penetration test Franz diffusion cells were used, with excised pig scalp (hair removed, but the stratum corneum intact) serving as the transdermal barrier. The receiving cell was a pH 5.5 phosphate buffer solution (with 0.01 wt% zinc pyrrolidone carboxylate added) that simulated the scalp microenvironment. Samples prepared in each example and comparative example were uniformly coated onto the surface of pig scalp. After 24 hours, the core A content in the receiving solution was measured, and the cumulative transdermal rate over 24 hours was calculated. Each group had three replicates, and the data are expressed as mean ± standard deviation.

[0046] (2) Stability test Each sample was placed in a 45℃ constant temperature incubator and samples were taken at 0, 30, 60 and 90 days. The retention rate of core A was determined by HPLC. Each group was set up in 3 replicates. Data are expressed as mean ± standard deviation.

[0047] (3) ROS response release test The samples were placed in a release medium simulating scalp follicle oxidative stress (pH 5.5 buffer containing 0.1 mM H2O2 + 0.05 wt% follicle extract), with a scalp microenvironment medium without H2O2 as a control. The core A release amount was measured within 2 hours, and the scalp follicle ROS response release fold (release amount in oxidative environment / release amount in normal environment) was calculated. Each group was set up in 3 replicates.

[0048] (4) Self-repair function test The samples were placed in the aforementioned hair follicle oxidative stress medium for 2 hours, and the release amount was measured. Then, they were transferred to a fresh scalp microenvironment medium and left to stand for 2 hours (without H2O2). H2O2 was added again to simulate oxidative stress, and the secondary release amount was measured. The percentage of the secondary release amount to the initial release amount was calculated to evaluate the self-repair ability. Each group had 3 replicates.

[0049] (5) Structural restoration verification The sample from Example 1 was subjected to a self-healing cycle test. After each cycle, the nanoemulsion particle size and PDI were measured, and the scalp stratum corneum adhesion was measured (OD value was determined by fluorescence labeling). The changes were recorded.

[0050] (6) Hair follicle penetration depth test Using isolated pig scalp (containing intact hair follicles), a fluorescently labeled composition (FITC-labeled) was uniformly coated onto the skin surface. Frozen sections of the skin were prepared 24 hours later, and the fluorescence penetration depth in the longitudinal section of the hair follicles was observed and measured using a confocal microscope; each group had 3 replicates.

[0051] (7) Hair papilla cell proliferation test Human dermal papilla cells were seeded into 96-well plates, and different concentrations (50, 100, 200 μg / mL) of the mixture were added. After culturing for 48 hours, the OD value was measured by CCK-8 assay, and the proliferation rate was calculated. Each group had 6 replicates.

[0052] (8) In vitro hair follicle culture test Human hair follicles (growth phase) were taken from the body and placed in Williams E medium. 100 μg / mL of the compound was added and cultured for 7 days. The hair shaft length was measured daily and the growth rate was calculated. 10 hair follicles were collected in each group.

[0053] (9) Scalp safety test According to the scalp patch test method in the "Cosmetic Safety Technical Specifications (2015 Edition)," 40 scalp subjects (10 dry skin, 10 oily skin, 10 sensitive skin, and 10 combination skin) were selected, and the scalp irritation and hair compatibility of each composition were tested at 24 hours and 48 hours.

[0054] (10) Scalp barrier repair rate test Forty scalp subjects were selected using a transdermal water loss (TEWL) meter, and test areas were marked along the hairline. The scalp barrier was first slightly disrupted using an adhesive tape peeling method (TEWL values ​​increased to 2-3 times the initial value), and then various compositions (2 mg / cm²) were applied. 2 The blank area served as the control; the TEWL value was measured after 24 hours, and the scalp barrier repair rate was calculated: Repair rate (%) = (TEWL decrease after treatment / TEWL increase after initial damage) × 100%; each group had 3 replicates.

[0055] (11) Antioxidant activity test of hair follicle cells Human hair follicle papilla cells were cultured in vitro and pretreated with a composition (100 μg / mL) for 2 hours. Then, H2O2 (0.1 mM) was added to induce oxidative stress. After 2 hours, the intracellular ROS content was measured, and the antioxidant rate (%) was calculated as (1 - ROS value of sample group / ROS value of model group) × 100%. Each group was divided into 3 replicates.

[0056] (12) Statistical analysis All data were analyzed using t-tests. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant.

[0057] The test results are shown in the table below: Table 1. Results of transdermal penetration rate and stability tests of the composition stock solution Note: The differences between the embodiments of the present invention and the comparative examples are all highly significant (P<0.01).

[0058] Table 2 Results of ROS Response Release and Self-Repair Test of the Composition Stock Solution in Scalp Hair Follicles Note: The differences between the embodiments of the present invention and the comparative examples are all highly significant (P<0.01).

[0059] Table 3. Verification results of the original composition's structure recovery (Example 1) Note: The higher the OD value, the better the adhesion of the composition to the scalp stratum corneum; the adhesion partially recovers after standing, confirming the self-repairing ability of the core-shell structure.

[0060] Table 4. Results of hair follicle penetration depth and dermal papilla cell proliferation of the stock solution Table 5 Results of in vitro hair follicle culture of the stock solution of the composition (7 days) Table 6. Results of scalp safety and hair follicle cell antioxidant tests of the stock solution. Note: The difference between the embodiments of the present invention and Comparative Example 3 is extremely significant (P<0.01).

[0061] Table 7 Effect of Polyphenol Type on Performance Note: The performance of the ternary polyphenol combination was significantly better than that of the single / binary combination (P<0.01).

[0062] Table 8. Effect of the method of adding thiol-containing peptides on performance. Note: The performance of the stepwise addition process is significantly better than that of the single addition process (P<0.01).

[0063] Table 9. Effect of outer shell B molecular weight on transdermal penetration rate Note: Within the range of 1000-2000 Da, increasing the molecular weight is beneficial to improving transdermal penetration.

[0064] Results Analysis As shown in Table 1, the transdermal penetration rate of the compositions in the embodiments of the present invention (32.5-36.1%) is significantly higher than that of the comparative compositions (10.7-21.4%), proving that the core-shell structure constructed in the present invention effectively improves the penetration efficiency of small molecule active peptides into the scalp stratum corneum. In terms of stability, the core A retention rate of the compositions in the embodiments of the present invention after 3 months of accelerated curing at 45°C is >92%, which is much higher than that of the comparative compositions (58.3-76.2%), indicating that the dual curing process significantly enhances the stability of the compositions.

[0065] As shown in Table 2, the compositions of the present invention exhibit significant scalp follicle ROS response release characteristics (2.6-3.5 times), while the comparative compositions show no response. Furthermore, the 24-hour scalp barrier repair rate of the compositions of the embodiments is >82%, proving that the compositions can not only achieve intelligent release under oxidative stress in hair follicles, but also effectively repair damaged scalp barriers. The self-repair test shows that after undergoing oxidative stress release, the secondary release amount of the compositions of the present invention still reaches 63.2-78.6% of the initial release, proving that the core-shell structure has self-repair function.

[0066] pH value has a bidirectional regulatory effect on the scalp care performance of the composition (Table 1, Table 2). The lower curing pH (5.3, composition D of Example 4) has the highest stability (94.7%) and the strongest self-repair ability (78.6%). The higher curing pH (6.0, composition E of Example 5) releases faster and can meet the needs of rapid repair.

[0067] The effect of the content of thiol-containing small molecule peptides on the ROS response fold is dose-dependent and synergistically regulated with pH; the process advantages of synergistic addition of thiol peptides by ternary polyphenols and stepwise addition are significantly reflected in the performance of the composition (Tables 7 and 8).

[0068] As shown in Tables 3-6, the compositions of the present invention have good hair follicle penetration ability (212-256μm), hair papilla cell proliferation promotion effect (128-145%), and in vitro hair follicle growth promotion effect (hair shaft lengthening 18-29%). They are also non-irritating to various types of scalp and skin, and the hair follicle cell antioxidant rate is over 84%, which is significantly better than the comparative example.

[0069] Table 9 shows that within the range of 1000-2000 Da, increasing the molecular weight is beneficial to improving transdermal penetration, and compositions with different molecular weights can be adapted to different scalp skin types.

[0070] In summary, the composite small molecule protein composition of the present invention has excellent scalp care properties, including high transdermal penetration, high stability, ROS-responsive release, self-repair, hair follicle targeting, and hair growth promotion.

[0071] To further verify the suitability of the compositions of the present invention in actual products, the compositions prepared in some examples were respectively made into the following scalp care products, and their performance was verified.

[0072] Application Example 1: Preparation of Hair Growth Essence Take 50g of the composition from Example 1, add 10g of squalane, 3g of glycerin, 1g of panthenol, and 0.08g of zinc pyrrolidone carboxylate, and add deionized water to make up to 100g. Homogenize the mixture three times (8min each time) at 25℃ and 800bar pressure to obtain the hair growth essence.

[0073] Application Example 2: Preparation of Hair Growth Gel Take 40g of the composition of Example 3, add 8g of caprylic / capric triglyceride, 0.3g of caffeine, 0.3g of hyaluronic acid oligosaccharide, 0.6g of carbomer 940, and 0.2g of triethanolamine, add deionized water to 100g, stir at room temperature until a transparent gel is formed, adjust the pH to 5.8, and obtain hair growth gel.

[0074] Application Example 3: Shampoo Preparation Take 80g of the composition of Example 4, add 40g of potassium cocoyl glycinate, 30g of glycerin, 4g of panthenol, and 732g of polyquaternium-7, add deionized water to make up to 1000g, dissolve the surfactant at 70°C, cool to 32°C and add the composition, stir evenly to obtain a mild repair shampoo.

[0075] Application Example 4: Preparation of Hair Conditioner Take 100g of the composition of Example 5, add 30g of squalane, 20g of cetyl alcohol, 8g of stearyltrimethylammonium chloride and 4g of hyaluronic acid oligosaccharide, add deionized water to 1000g, emulsify at 78°C, cool to 30°C and add the composition, stir evenly to obtain a smoothing and repairing hair conditioner.

[0076] Application Example 5: Preparation of Hair Mask Take 12g of the composition from Example 2, add 4g of squalane, 3g of panthenol, 1.5g of hydrolyzed keratin, and 0.8g of lanolin, and add deionized water to make 100g; heat squalane and lanolin as the oil phase to 78°C, and heat the composite small molecule protein composition, panthenol, hydrolyzed keratin, and deionized water as the aqueous phase to 78°C. Slowly add the aqueous phase to the oil phase while stirring, homogenize and emulsify for 5 minutes, cool to below 35°C and discharge to obtain a cream hair mask.

[0077] Application example: Product performance verification The scalp care products prepared in Application Examples 1-5 were compared with the corresponding composition stock solutions for transdermal penetration rate and core A retention rate at 45°C for 3 months. The results are shown in Table 10.

[0078] Table 10 Comparison of performance between the stock solution of the composition and the product containing the composition Note: There was no significant difference in performance between the stock solutions of each composition and the corresponding products (P>0.05), proving that the compositions of the present invention can still maintain their core performance in complex matrices and have good dosage form compatibility.

[0079] In summary, the composite small molecule protein composition and its preparation method provided by this invention have readily available raw materials, simple processes, and low equipment investment, enabling rapid technology transfer from laboratory to mass production. The prepared composition itself has multiple functions such as high transdermal penetration, high stability, ROS-responsive release, self-repair, hair follicle targeting, and hair growth promotion. It can be further developed into various scalp care products such as hair growth essence, hair growth gel, shampoo, conditioner, and hair mask to meet the needs of different scalp types such as dry, oily, sensitive, and combination skin, and has extremely high industrial practical value and market prospects.

[0080] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0081] 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 invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A complex small molecule protein composition, characterized in that, It contains a core-shell structured complex and a ROS-responsive active component; The core-shell structured complex is composed of a core A and an outer shell B connected by dual dynamic covalent bonds of Schiff base bonds and disulfide bonds. The Schiff base bonds endow the pH-responsive transdermal release function, while the disulfide bonds endow the ROS-responsive self-repair function. The core A is a small molecule active peptide with a molecular weight of 200-800 Da; The outer shell B is a low molecular weight hydrolyzed collagen peptide modified with plant polyphenols, with a molecular weight of 1000-2000 Da; The ROS-responsive active component is a thiol-containing small molecule peptide, accounting for 0.5-5 wt% of the total mass of the composition.

2. The composite small molecule protein composition according to claim 1, characterized in that, The thiol-containing small molecule peptide is reduced glutathione with a purity ≥98%.

3. The composite small molecule protein composition as described in claim 1, characterized in that, The small molecule active peptides are all selected from at least one of acetyl tetrapeptide-2, palmitoyl tripeptide-8, and palmitoyl tripeptide-5.

4. The composite small molecule protein composition according to claim 1, characterized in that, The plant polyphenols contain chlorogenic acid, rosmarinic acid, and resveratrol in a mass ratio of 5:1:0.5-10:2:1, and the polyphenols account for 5-20 wt% of the total mass of shell B.

5. A method for preparing the complex small molecule protein composition according to any one of claims 1-4, characterized in that, Includes the following steps: a) Preparation of outer shell B: Low molecular weight hydrolyzed collagen peptides were dissolved in deionized water at a material-to-liquid ratio of 1:8 g / mL. Plant polyphenols and thiol-containing small molecule peptides were added, and the pH was adjusted to 8.0-8.5 with 10% NaOH solution. The reaction was stirred at 35-45℃ and 200-300 r / min for 3-5 hours. The thiol-containing small molecule peptides were added in steps: first, 50-70% of the total mass of the thiol-containing small molecule peptides were added to prepare outer shell B, and the remaining 30-50% was added in the mild oxidation stage of step c. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed in deionized water for 24 hours, with the water changed every 6 hours. Then, the solution was freeze-dried at -50℃ and 0.01 MPa for 12 hours to obtain the polyphenol-collagen-thiol-containing peptide complex. b) Core-shell pre-assembly: The shell B obtained in step a is dissolved in phosphate buffer at pH 8.0-8.5 at a mass ratio of 2:1-5:1 and pre-reacted for 1-2 hours to form a pre-complex containing Schiff base bonds; c) Dual curing and mild oxidation: Adjust the pH of the reaction solution to 5.3-6.0 with citric acid, add 0.005-0.02 wt% of a natural crosslinking agent, introduce 0.1-0.5 v / v% of sterile air into the reaction solution, react at 15-25°C for 2-3 hours, and purify the reaction solution by ultrafiltration to obtain a composite small molecule protein composition.

6. The method for preparing the complex small molecule protein composition according to claim 5, characterized in that, The natural crosslinking agent mentioned in step c is tannic acid or genipin.

7. The use of the composite small molecule protein composition according to any one of claims 1-4 or the composite small molecule protein composition prepared by the method according to any one of claims 5-6 in the preparation of anti-aging, repair, and hair growth cosmetics.

8. The application according to claim 7, characterized in that, The cosmetic product is selected from at least one of the following: hair growth essence, hair growth gel, shampoo, conditioner, and hair mask.

9. The application according to claim 8, characterized in that: The hair growth essence comprises: 10-20wt% complex small molecule protein composition, 5-10wt% squalane, 2-5wt% glycerin, 0.3-0.8wt% panthenol, 0.05-0.1wt% zinc pyrrolidone carboxylate, and the balance being deionized water; The hair growth gel comprises: 8-15 wt% of a complex small molecule protein composition, 5-8 wt% of caprylic / capric triglycerides, 0.1-0.5 wt% of caffeine, 0.2-0.5 wt% of hyaluronic acid oligosaccharides, 0.4-0.8 wt% of carbomer 940, 0.1-0.3 wt% of triethanolamine, with the balance being deionized water; The hair mask comprises: 10-15 wt% of a complex small molecule protein composition, 3-5 wt% of squalane, 2-4 wt% of panthenol, 1-2 wt% of hydrolyzed keratin, 0.5-1 wt% of lanolin, and the balance being deionized water; The shampoo comprises: 5-10 wt% complex small molecule protein composition, 3-5 wt% potassium cocoyl glycinate, 2-4 wt% glycerin, 0.2-0.5 wt% panthenol, 0.1-0.3 wt% polyquaternium-73, and the balance being deionized water; The hair conditioner comprises: 8-12 wt% of a complex small molecule protein composition, 2-4 wt% of squalane, 1-3 wt% of cetyl alcohol, 0.5-1 wt% of stearyltrimethylammonium chloride, 0.2-0.5 wt% of hyaluronic acid oligosaccharides, and the balance being deionized water.

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