High-activity collagen skin care product based on pharmaceutical-grade process and preparation method of high-activity collagen skin care product
By employing techniques such as segmented temperature-controlled enzymatic hydrolysis, ultrasonic nano-coating, and chemical quenching, the problems of low activity, low purity, and low penetration efficiency in collagen skincare products have been solved. This has enabled the preparation of collagen skincare products with high activity, high purity, high penetration, and high safety, achieving pharmaceutical-grade safety standards.
Patent Information
- Application Number
- CN202610029127.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-09
- Publication Date
- 2026-03-06
AI Technical Summary
Existing collagen skincare products suffer from problems such as significant loss of activity, low purity, low penetration efficiency, and safety risks during the preparation process, making it difficult to meet the high safety standards of pharmaceutical grade.
A pharmaceutical-grade process is employed, which involves segmented temperature-controlled enzymatic hydrolysis, ultrasound-assisted nano-coating, micro-crosslinking, and chemical quenching. The molecular weight of collagen peptides is precisely controlled through segmented temperature-controlled enzymatic hydrolysis, and nanocarriers are formed by ultrasonic treatment. After crosslinking with a low-concentration crosslinking agent, unreacted aldehyde groups are quenched with amino acids, thus constructing a dual physical-chemical protection system.
It achieves high activity, high purity, high permeability and high safety in collagen skin care products, significantly improving the overall performance of the products. The activity retention rate is as high as 95% or more, the permeability reaches more than 30%, and the cross-linking agent residue is less than 0.1ppm.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of skincare technology, specifically to a highly active collagen skincare product based on pharmaceutical-grade processes and its preparation method. Background Technology
[0002] Collagen is an essential protein in human skin, widely used in skincare product manufacturing due to its skin-elasticity-promoting, repairing, and anti-aging effects. Existing methods for preparing collagen often employ enzymatic hydrolysis or physical purification, which frequently suffer from significant activity loss, low purity, and imprecise molecular weight control. This leads to three major technical challenges for collagen skincare products: first, difficulty in maintaining activity, as common enzymatic hydrolysis products are prone to degradation or denaturation during their shelf life; second, poor transdermal absorption, as large collagen molecules struggle to penetrate the stratum corneum, while excessively hydrolyzed small peptides lose their unique triple-helix biological functions; and third, safety concerns, as some technologies use high concentrations of aldehydes for cross-linking to stabilize the structure, resulting in residual cytotoxicity and failing to meet the high safety standards of "pharmaceutical grade," severely limiting the efficacy and commercial value of high-end skincare products. Based on the above, this invention proposes a highly active collagen skincare product based on pharmaceutical-grade processes and its preparation method. Summary of the Invention
[0003] To address the problems of low activity, low purity, and low penetration efficiency in the preparation of collagen skincare products in existing technologies, this invention proposes a highly active collagen skincare product based on pharmaceutical-grade processes and its preparation method.
[0004] In a first aspect, the present invention provides a method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes, employing the following technical solution: A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Disperse collagen in deionized water to form a collagen solution. First, add the first protease at 40-45°C for 1-3 hours to hydrolyze it. Then, raise the temperature to 50-55°C and add the second protease for 1-2 hours to hydrolyze it. Inactivate the enzyme, decolorize and filter to obtain a collagen peptide solution. S2. Add a polyhydroxy polymer and an auxiliary gelling agent to the collagen peptide solution, stir evenly, and then perform ultrasonic treatment to obtain a composite nano sol. S3. Add a crosslinking agent to the composite nanosol to carry out a crosslinking reaction. After the reaction is completed, add an amino acid quencher for quenching treatment for 30-60 minutes to obtain the reaction product. S4. The reaction product is purified by ultrafiltration and the pH value is adjusted to obtain a highly active collagen skin care product based on pharmaceutical-grade technology.
[0005] Preferably, the collagen solution in step S1 has a mass concentration of 5-10%.
[0006] Preferably, the first protease in step S1 is a neutral protease or papain, and its addition amount is 0.1-0.3% of the collagen solution mass.
[0007] Preferably, the second protease in step S1 is a flavor protease or an alkaline protease, and its addition amount is 0.1-0.3% of the collagen solution mass.
[0008] Preferably, in step S2, the ultrasonic treatment adopts a pulse mode, and the ratio of working time to intermittent time is 1-2:1.
[0009] Preferably, in step S2, the ultrasonic treatment frequency is 20-40kHz, the power density is 200-400W / L, and the treatment time is 10-30min.
[0010] Preferably, the polyhydroxy polymer in step S2 is selected from at least one of carboxymethyl chitosan, sodium alginate, polyvinyl alcohol, oxidized dextran, and pullulan.
[0011] Preferably, the auxiliary gelling agent in step S2 is calcium chloride or sodium tripolyphosphate.
[0012] Preferably, in step S2, the mass ratio of collagen, polyhydroxy polymer and auxiliary gelling agent is 8-12:2-5:1 on a dry weight basis.
[0013] Preferably, the crosslinking agent in step S3 is glutaraldehyde and / or genipin, and the amount added is 0.01-0.03% of the mass of the composite nanosol.
[0014] Preferably, the temperature of the crosslinking reaction in step S3 is 4-10℃, and the reaction time is 1-3h.
[0015] Preferably, the amino acid quencher in step S3 is glycine, lysine, or arginine, and its molar amount is 2-5 times that of the crosslinking agent.
[0016] Preferably, the ultrafiltration purification in step S4 specifically involves: first, using an ultrafiltration membrane with a molecular weight cutoff of 10-100 kDa to remove uncoated free polymers and macromolecular impurities, and then using an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to remove small molecule salts and reaction byproducts.
[0017] Preferably, adjusting the pH value in step S4 specifically involves adjusting the pH value to 5.6-6.5 using citric acid solution and / or sodium citrate solution.
[0018] Preferably, the average particle size of the skin care product is 50-200 nm.
[0019] Secondly, this invention provides a highly active collagen skincare product based on pharmaceutical-grade processes, employing the following technical solution: A highly active collagen skincare product based on pharmaceutical-grade processes is prepared using the above-mentioned method.
[0020] Preferably, the collagen peptides in the skin care product have a purity of ≥96.0%, an activity retention rate of ≥95.0%, and a relative penetration rate of ≥30% after 2 hours of skin application.
[0021] Preferably, the residual concentration of the crosslinking agent in the skin care product is <0.1ppm.
[0022] In summary, the present invention has the following beneficial effects: 1. Synergistically Achieving "High Activity, High Purity, High Permeability, and High Safety": This invention organically combines segmented temperature-controlled enzymatic hydrolysis, ultrasound-assisted nano-coating, micro-crosslinking, and chemical quenching technologies to construct a complete pharmaceutical-grade process loop. This solves the technical challenge of existing technologies simultaneously achieving collagen activity, purity, permeability, and safety, significantly improving the overall performance of the product.
[0023] 2. Constructing a dual physical-chemical protection system to significantly improve activity and stability: This invention utilizes the ultrasonic cavitation effect to induce the self-assembly of polyhydroxy polymers to form physical protection, and supplements it with trace cross-linking for chemical reinforcement, so that the collagen activity retention rate is as high as 95% or more.
[0024] 3. Precisely control molecular weight and optimize delivery to improve transdermal absorption efficiency: Through segmented temperature-controlled enzymatic hydrolysis, the molecular weight of collagen peptides is precisely controlled within a penetration window of 3-5 kDa, and nanocarriers with a particle size of 50-200 nm are constructed, which significantly improves the skin permeability compared to simple physical mixtures, enabling effective delivery of active ingredients to the deep layers of the skin.
[0025] 4. Introducing chemical quenching to solve safety hazards of crosslinking agents: After low-concentration crosslinking, this invention uses excess amino acids to quench unreacted aldehyde groups, so that the residual crosslinking agent in the final product is less than 0.1 ppm, which is non-irritating to the skin and achieves pharmaceutical-grade safety standards for the product, which is a significant improvement. Detailed Implementation
[0026] The present invention will be further described in detail below with reference to the embodiments.
[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.
[0028] The key raw materials used in this invention are sourced from the following sources: Collagen: CAS No. 9064-67-9, Product No. lnb-1162, Brand: Lanabai, Purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.
[0029] Papain: CAS No. 9001-73-4, 100,000 enzyme activity, brand Yuhe, purchased from Zhengzhou Yuhe Food Additives Co., Ltd.
[0030] Alkaline protease: CAS No. 9014-01-1, 100,000 enzyme activity, brand Yuhe, purchased from Zhengzhou Yuhe Food Additives Co., Ltd.
[0031] Carboxymethyl chitosan: CAS No. 83512-85-0, purity 99%, brand Huaxiang, purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd.
[0032] Glycine: CAS No. 56-40-6, content 99%, brand Chengfeng, Hubei Chengfeng Chemical Co., Ltd.
[0033] Examples 1-3 provide a highly active collagen skincare product based on pharmaceutical-grade processes and its preparation method.
[0034] Example 1 A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen was dispersed in deionized water to form a collagen solution with a mass concentration of 8%. Papain (0.2% of the collagen solution mass) was added at 43°C and hydrolyzed for 2 hours. The temperature was then raised to 52°C, and alkaline protease (0.2% of the collagen solution mass) was added and hydrolyzed for 1.5 hours. The hydrolysate was then rapidly heated to 95°C and kept at that temperature for 15 minutes to completely inactivate the protease. After cooling to room temperature, activated carbon from fruit shells (0.5% of the hydrolysate volume) was added. After stirring and adsorption for 30 minutes, the solution was filtered through a 0.45μm microporous membrane to remove activated carbon and insoluble matter, thus obtaining a collagen peptide solution. S2. Based on dry weight, control the mass ratio of collagen, carboxymethyl chitosan and calcium chloride to be 10:3.5:1. Add carboxymethyl chitosan and calcium chloride to the collagen peptide solution, stir for 30 min until uniformly mixed, and then perform ultrasonic treatment in pulse mode. Control the ratio of working time to intermittent time to be 1.5:1, the frequency to be 30 kHz, the power density to be 300 W / L, and treat for 20 min to obtain composite nanosol. S3. Add 0.02% of glutaraldehyde by mass to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 7℃ and the reaction time at 2h. After the reaction is completed, add 3.5 times the molar amount of glycine of glutaraldehyde for quenching treatment for 45min to obtain the reaction product. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 50kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0035] Example 2 A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen was dispersed in deionized water to form a 5% (w / w) collagen solution. Papain (0.1% by weight of the collagen solution) was added at 40°C and hydrolyzed for 1 hour. The temperature was raised to 50°C and alkaline protease (0.3% by weight of the collagen solution) was added and hydrolyzed for 2 hours. The hydrolysate was rapidly heated to 95°C and kept at that temperature for 15 minutes to completely inactivate the protease. Then it was cooled to room temperature and activated carbon (0.5% by volume of the hydrolysate) was added. After stirring and adsorption for 30 minutes, the activated carbon and insoluble matter were removed by filtration through a 0.45μm microporous membrane to obtain a collagen peptide solution. S2. Based on dry weight, control the mass ratio of collagen, carboxymethyl chitosan and calcium chloride to be 8:2:1. Add carboxymethyl chitosan and calcium chloride to the collagen peptide solution, stir for 20 minutes until uniformly mixed, and then perform ultrasonic treatment in pulse mode. Control the ratio of working time to intermittent time to be 1:1, the frequency to be 20kHz, the power density to be 200W / L, and treat for 30 minutes to obtain composite nanosol. S3. Add 0.01% of glutaraldehyde by mass to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 4℃ and the reaction time at 3h. After the reaction is completed, add glycine with a molar amount of glutaraldehyde twice the amount of glycine for quenching treatment for 60min to obtain the reaction product. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 10kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0036] Example 3 A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen is dispersed in deionized water to form a collagen solution with a mass concentration of 10%. First, at a temperature of 45°C, 0.3% of the collagen solution mass of papain is added for enzymatic hydrolysis for 3 hours. The temperature is then raised to 55°C, and 0.1% of the collagen solution mass of alkaline protease is added for enzymatic hydrolysis for 1 hour. The hydrolysate is then rapidly heated to 95°C and kept at that temperature for 15 minutes to completely inactivate the protease. Subsequently, it is cooled to room temperature, and 0.5% of the hydrolysate volume of activated carbon from fruit shells is added. After stirring and adsorption for 30 minutes, the solution is filtered through a 0.45μm microporous membrane to remove activated carbon and insoluble matter, thus obtaining a collagen peptide solution. S2. Based on dry weight, control the mass ratio of collagen, carboxymethyl chitosan and calcium chloride to be 12:5:1. Add carboxymethyl chitosan and calcium chloride to the collagen peptide solution, stir for 40 min until uniformly mixed, and then perform ultrasonic treatment in pulse mode. Control the ratio of working time to intermittent time to be 2:1, the frequency to be 40 kHz, the power density to be 400 W / L, and treat for 10 min to obtain composite nanosol. S3. Add 0.03% of glutaraldehyde by mass to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 10℃ and the reaction time at 1h. After the reaction is completed, add 5 times the molar amount of glycine of glutaraldehyde for quenching treatment for 30min to obtain the reaction product. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 100kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0037] To verify the comprehensive performance of the highly active collagen skincare product based on pharmaceutical-grade technology provided by this invention, comparative examples 1-5 were set up, wherein: Comparative Example 1 Comparative Example 1 is the same as Example 1, except that it uses only papain for enzymatic hydrolysis. Details are as follows: A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen was dispersed in deionized water to form a collagen solution with a mass concentration of 8%. Papain (0.4% by mass of the collagen solution) was added at 43°C and hydrolyzed for 3.5 hours. The hydrolysate was then rapidly heated to 95°C and kept at that temperature for 15 minutes to completely inactivate the protease. After cooling to room temperature, activated carbon from fruit shells (0.5% by volume of the hydrolysate) was added. After stirring and adsorption for 30 minutes, the solution was filtered through a 0.45μm microporous membrane to remove activated carbon and insoluble matter, thus obtaining a collagen peptide solution. S2. Based on dry weight, control the mass ratio of collagen, carboxymethyl chitosan and calcium chloride to be 10:3.5:1. Add carboxymethyl chitosan and calcium chloride to the collagen peptide solution, stir for 30 min until uniformly mixed, and then perform ultrasonic treatment in pulse mode. Control the ratio of working time to intermittent time to be 1.5:1, the frequency to be 30 kHz, the power density to be 300 W / L, and treat for 20 min to obtain composite nanosol. S3. Add 0.02% of glutaraldehyde by mass to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 7℃ and the reaction time at 2h. After the reaction is completed, add 3.5 times the molar amount of glycine of glutaraldehyde for quenching treatment for 45min to obtain the reaction product. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 50kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0038] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that it uses only alkaline protease hydrolysis. Details are as follows: A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen was dispersed in deionized water to form a collagen solution with a mass concentration of 8%. At 52°C, 0.4% (by mass) of alkaline protease was added to the collagen solution for 3.5 h of enzymatic hydrolysis. The hydrolysate was then rapidly heated to 95°C and kept at that temperature for 15 min to completely inactivate the protease. After cooling to room temperature, 0.5% (by volume) of activated carbon from fruit shells was added. After stirring and adsorption for 30 min, the solution was filtered through a 0.45 μm microporous membrane to remove activated carbon and insoluble matter, thus obtaining a collagen peptide solution. S2. Based on dry weight, control the mass ratio of collagen, carboxymethyl chitosan and calcium chloride to be 10:3.5:1. Add carboxymethyl chitosan and calcium chloride to the collagen peptide solution, stir for 30 min until uniformly mixed, and then perform ultrasonic treatment in pulse mode. Control the ratio of working time to intermittent time to be 1.5:1, the frequency to be 30 kHz, the power density to be 300 W / L, and treat for 20 min to obtain composite nanosol. S3. Add 0.02% of glutaraldehyde by mass to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 7℃ and the reaction time at 2h. After the reaction is completed, add 3.5 times the molar amount of glycine of glutaraldehyde for quenching treatment for 45min to obtain the reaction product. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 50kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0039] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that it involves simple polymer mixing without ultrasonic treatment. Details are as follows: A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen was dispersed in deionized water to form a collagen solution with a mass concentration of 8%. Papain (0.2% of the collagen solution mass) was added at 43°C and hydrolyzed for 2 hours. The temperature was then raised to 52°C, and alkaline protease (0.2% of the collagen solution mass) was added and hydrolyzed for 1.5 hours. The hydrolysate was then rapidly heated to 95°C and kept at that temperature for 15 minutes to completely inactivate the protease. After cooling to room temperature, activated carbon from fruit shells (0.5% of the hydrolysate volume) was added. After stirring and adsorption for 30 minutes, the solution was filtered through a 0.45μm microporous membrane to remove activated carbon and insoluble matter, thus obtaining a collagen peptide solution. S2. On a dry weight basis, control the mass ratio of collagen, carboxymethyl chitosan and calcium chloride to be 10:3.5:1. Add carboxymethyl chitosan and calcium chloride to the collagen peptide solution and stir for 50 minutes until the mixture is uniform to obtain the composite nano sol. S3. Add 0.02% of glutaraldehyde by mass to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 7℃ and the reaction time at 2h. After the reaction is completed, add 3.5 times the molar amount of glycine of glutaraldehyde for quenching treatment for 45min to obtain the reaction product. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 50kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0040] Comparative Example 4 Comparative Example 4 is the same as Example 1, except that calcium chloride is not added. Details are as follows: A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen was dispersed in deionized water to form a collagen solution with a mass concentration of 8%. Papain (0.2% of the collagen solution mass) was added at 43°C and hydrolyzed for 2 hours. The temperature was then raised to 52°C, and alkaline protease (0.2% of the collagen solution mass) was added and hydrolyzed for 1.5 hours. The hydrolysate was then rapidly heated to 95°C and kept at that temperature for 15 minutes to completely inactivate the protease. After cooling to room temperature, activated carbon from fruit shells (0.5% of the hydrolysate volume) was added. After stirring and adsorption for 30 minutes, the solution was filtered through a 0.45μm microporous membrane to remove activated carbon and insoluble matter, thus obtaining a collagen peptide solution. S2. Based on dry weight, control the mass ratio of collagen to carboxymethyl chitosan to be 10:3.5. Add carboxymethyl chitosan to the collagen peptide solution, stir for 30 minutes until uniformly mixed, and then perform ultrasonic treatment in pulse mode. Control the ratio of working time to intermittent time to be 1.5:1, the frequency to be 30kHz, the power density to be 300W / L, and treat for 20 minutes to obtain composite nanosol. S3. Add 0.02% of glutaraldehyde by mass to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 7℃ and the reaction time at 2h. After the reaction is completed, add 3.5 times the molar amount of glycine of glutaraldehyde for quenching treatment for 45min to obtain the reaction product. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 50kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0041] Comparative Example 5 Comparative Example 5 is the same as Example 1, except that a high concentration of glutaraldehyde was added for crosslinking, and glycine quenching treatment was not added, as detailed below: A method for preparing a highly active collagen skincare product based on pharmaceutical-grade processes includes the following steps: S1. Collagen was dispersed in deionized water to form a collagen solution with a mass concentration of 8%. Papain (0.2% of the collagen solution mass) was added at 43°C and hydrolyzed for 2 hours. The temperature was then raised to 52°C, and alkaline protease (0.2% of the collagen solution mass) was added and hydrolyzed for 1.5 hours. The hydrolysate was then rapidly heated to 95°C and kept at that temperature for 15 minutes to completely inactivate the protease. After cooling to room temperature, activated carbon from fruit shells (0.5% of the hydrolysate volume) was added. After stirring and adsorption for 30 minutes, the solution was filtered through a 0.45μm microporous membrane to remove activated carbon and insoluble matter, thus obtaining a collagen peptide solution. S2. Based on dry weight, control the mass ratio of collagen, carboxymethyl chitosan and calcium chloride to be 10:3.5:1. Add carboxymethyl chitosan and calcium chloride to the collagen peptide solution, stir for 30 min until uniformly mixed, and then perform ultrasonic treatment in pulse mode. Control the ratio of working time to intermittent time to be 1.5:1, the frequency to be 30 kHz, the power density to be 300 W / L, and treat for 20 min to obtain composite nanosol. S3. Add 0.5% by mass of glutaraldehyde to the composite nanosol to carry out a cross-linking reaction. Control the reaction temperature at 7℃ and the reaction time at 2h. After the reaction is completed, the reaction product is obtained. S4. The reaction product is first removed by ultrafiltration membrane with a molecular weight cutoff of 50kDa to remove uncoated free polymers and macromolecular impurities, and then by ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove small molecule salts and reaction byproducts. The pH value is adjusted to 6.2 by citric acid solution and sodium citrate solution to obtain a highly active collagen skin care product based on pharmaceutical-grade process.
[0042] The comprehensive performance of the highly active collagen skincare products prepared in Examples 1-3 and Comparative Examples 1-5 of this invention based on pharmaceutical-grade processes was tested respectively.
[0043] 1. Activity retention rate determination Hydroxyproline is a characteristic amino acid of collagen, and its content is directly proportional to the collagen content. The retention rate of collagen activity is assessed by measuring the change in hydroxyproline content before and after sample treatment.
[0044] According to the national standard GB / T 9695.23-2008, the highly active collagen skin care products in Examples 1-3 and Comparative Examples 1-5, as well as the untreated raw collagen peptide solution (as a 100% activity control), were freeze-dried at -30°C to constant weight to obtain the freeze-dried powder to be tested.
[0045] Accurately weigh the lyophilized powder to be tested, add it to a 6 mol / L HCl solution and hydrolyze it at 110℃ for 24 h. After cooling, take the hydrolysate and neutralize it with sodium hydroxide to pH 6.2, and then make up to 10 mL to obtain the final volume solution for each sample.
[0046] Preparation of standard curves: Prepare hydroxyproline standard solutions with concentrations of 0, 5, 10, 20, 40, and 80 µg / mL. Take 1 mL of each solution and react it with the colorimetric reagent, then measure the absorbance at 560 nm. Plot the standard curves, obtaining the regression equation y = 0.0125x + 0.0048 (R² = 0.9992), where y is the absorbance and x is the concentration of hydroxyproline.
[0047] Sample determination: Take 1 mL of the sample solution and perform the same colorimetric reaction and measure the absorbance. Substitute the absorbance into the standard curve equation to calculate the concentration of hydroxyproline in the sample.
[0048] Activity retention rate (%) = (hydroxyproline content in skin care product sample / hydroxyproline content in original collagen peptide solution) × 100%.
[0049] 2. Determination of molecular weight distribution The molecular weight distribution was determined according to the method in Appendix A of the national standard GB / T 22729-2008, and the molecular weight distribution of the highly active collagen skin care products in Examples 1-3 and Comparative Examples 1-5 of this invention was detected respectively.
[0050] 3. Purity determination High-performance liquid chromatography (HPLC) was used. The column was an Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution program was: 0–5 min, 5% B; 5–35 min, 5% → 60% B; 35–40 min, 60% → 5% B. Flow rate: 1.0 mL / min; column temperature: 30 °C; detection wavelength: 220 nm.
[0051] Take the lyophilized powder of the highly active collagen skincare products from Examples 1-3 and Comparative Examples 1-5, dissolve and dilute it to 1.0 mg / mL with mobile phase A, filter it through a 0.22 μm filter membrane, and inject 20 μL. The purity is calculated as the percentage of the collagen main peak area to the sum of all peak areas. A purity ≥96% is the pharmaceutical grade standard defined in this invention.
[0052] 4. Skin penetration test Volunteers were recruited to designate a test area (1 x 1 cm) on the inner side of the arm where the skin had uniform characteristics. 2 .
[0053] Before the test, the volunteers cleaned the skin on the inside of their arms with 75% alcohol. After cleansing, the highly active collagen skincare products from Examples 1-3 and Comparative Examples 1-5 were applied, at a dosage of 3 mg / cm². 2 Before application and 2 hours after application, spectra were collected at the skin surface (0 μm depth) and the dermal-epidermal junction (DEJ, approximately 80-100 μm depth) using a LabRAM Odyssey high-resolution confocal microscopic Raman spectrometer (HORIBA).
[0054] Relative penetration rate (%) = Raman spectral intensity of active ingredient entering the skin / Raman spectral intensity of active ingredient applied to the skin × 100%.
[0055] 5. Skin irritation (patch) test The patch test was conducted according to the human skin patch test method in the "Cosmetic Safety Technical Specifications" (2015 edition). Thirty healthy volunteers were recruited. A patch tester containing 0.02 mL of sample was applied to the skin on the back of each volunteer and removed after 24 hours. Skin reactions were observed at 0.5 h, 24 h, and 48 h after removal, and scored based on the degree of erythema and edema. A score of 0 indicated no reaction, and scores of 1-4 indicated mild, significant, and severe reactions, respectively.
[0056] The Integrated Rating Index (IRI) is calculated as follows: IRI = [Σ (sum of scores at all observation time points)] / (total number of subjects × number of observations). According to the standard, an IRI < 0.5 is considered essentially non-irritating.
[0057] The results are shown in Table 1 below.
[0058] Table 1: Comprehensive Performance Test Results of High-Activity Collagen Skincare Products As shown in Table 1, the high-activity collagen skincare products prepared in Examples 1-3 of this invention have significantly better overall performance than those in Comparative Examples 1-5.
[0059] Compared with Comparative Example 1 (using only papain) and Comparative Example 2 (using only alkaline protease), Example 1 showed a significantly higher proportion of molecules with molecular weights in the 3-5 kDa range. This demonstrates that the segmented temperature-controlled compound enzymatic hydrolysis process of the present invention has a synergistic effect on precisely controlling molecular weight distribution, which is a prerequisite for achieving efficient penetration.
[0060] Compared to Comparative Example 3 (simple mechanical stirring), Example 1 showed a significantly higher 2-hour permeability under identical conditions. This demonstrates that ultrasonic treatment is not simply mixing, but a crucial and necessary step in constructing an effective nanocarrier and achieving efficient transdermal absorption.
[0061] Compared with Comparative Example 4 (without calcium chloride), Example 1 showed significant advantages in both activity retention and permeability. This indicates that calcium chloride, as an auxiliary gelling agent, can effectively promote the formation of a denser and more stable nanoshell structure of carboxymethyl chitosan, which is crucial for protecting collagen activity and promoting permeation.
[0062] Comparative Example 5 used a high concentration of glutaraldehyde without quenching, achieving an activity retention rate as high as 97.1%, but it caused skin irritation. Example 1, while maintaining an extremely high activity retention rate of 96.3%, completely eliminated skin irritation through low-concentration cross-linking and glycine quenching.
[0063] In summary, the technical solution of this invention is not a simple superposition of steps, but rather an organic integration and synergistic effect of a series of technical features such as segmented enzymatic hydrolysis, ultrasonic nano-coating, ionic / chemical synergistic stabilization, and chemical quenching. This successfully prepares a collagen skin care product with high activity, high purity, high permeability, and high safety, demonstrating significant creativity and practicality.
[0064] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.
Claims
1. A method for preparing a high activity collagen skin care product based on a pharmaceutical grade process, characterized by, The method comprises the following steps: S1, dispersing collagen in deionized water to form a collagen solution, adding a first protease for enzymolysis at a temperature of 40-45°C for 1-3h, increasing the temperature to 50-55°C to add a second protease for enzymolysis for 1-2h, deactivating the enzyme, decolorizing and filtering to obtain a collagen peptide solution; S2, adding a polyhydroxy polymer and an auxiliary gelling agent to the collagen peptide solution, stirring uniformly, and then performing ultrasonic treatment to obtain a composite nanosol; S3, adding a crosslinking agent to the composite nanosol to perform a crosslinking reaction, after the reaction is completed, adding an amino acid type quenching agent for quenching treatment for 30-60min to obtain a reaction product; S4, purifying the reaction product by ultrafiltration, adjusting the pH value, and obtaining a high-activity collagen skin care product based on a pharmaceutical grade process.
2. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, The first protease in the step S1 is neutral protease or papain, and the addition amount is 0.1-0.3% of the mass of the collagen solution; the second protease is flavor protease or alkaline protease, and the addition amount is 0.1-0.3% of the mass of the collagen solution.
3. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, The ultrasonic treatment in the step S2 adopts a pulse mode, the ratio of working time to intermittent time is 1-2:1, the frequency is 20-40kHz, the power density is 200-400W / L, and the treatment time is 10-30min.
4. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, The polyhydroxy polymer in the step S2 is at least one selected from carboxymethyl chitosan, sodium alginate, polyvinyl alcohol, oxidized dextran, and pullulan.
5. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, The auxiliary gelling agent in the step S2 is calcium chloride or sodium tripolyphosphate.
6. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, In the step S2, the mass ratio of collagen, polyhydroxy polymer and auxiliary gelling agent is 8-12:2-5:1 in terms of dry weight.
7. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, The crosslinking agent in the step S3 is glutaraldehyde and / or genipin, and the addition amount is 0.01-0.03% of the mass of the composite nanosol.
8. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, The temperature of the crosslinking reaction in the step S3 is 4-10°C, and the reaction time is 1-3h.
9. The method for preparing a highly active collagen skincare product based on pharmaceutical-grade technology according to claim 1, characterized in that, The amino acid type quenching agent in the step S3 is glycine, lysine or arginine, and the addition molar amount is 2-5 times of the molar amount of the added crosslinking agent.
10. A high activity collagen skin care product based on pharmaceutical grade process characterized in that, The high-activity collagen skin care product based on a pharmaceutical grade process is prepared by the method of any one of claims 1-9.