Hypoallergenic whitening recombinant humanized collagen composition and preparation method thereof

Abstract

The invention discloses a low-sensitivity whitening recombinant humanized collagen composition and a preparation method thereof. The low-sensitivity whitening recombinant humanized collagen composition is prepared from modified recombinant humanized collagen, tetrahydrocurcumin, ethyl ascorbic acid ether and dipotassium glycyrrhizinate, the modified recombinant humanized collagen comprises a recombinant humanized collagen chain segment, a polyethylene glycol chain segment modified on the recombinant humanized collagen chain segment, and tetrahydromethyl pyrimidine formyl modified at the other end of the polyethylene glycol chain segment. The modified recombinant humanized collagen can provide proper steric hindrance to achieve the effects of shielding residual antigen epitopes and improving the efficiency of combining target spot cell surface receptors, and cooperates with other mild whitening components to act on various pathways (including oxidation resistance, transport inhibition, enzyme inhibition and inflammation resistance), so that synergistic interaction and irritant hedging are realized, and the skin whitening effect is improved. Therefore, the whitening effect is achieved under low skin irritation.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a hypoallergenic whitening recombinant humanized collagen composition and its preparation method. Background Technology

[0002] Recombinant humanized collagen has become a core ingredient in high-end skincare products and medical dressings due to its advantages such as the absence of animal-derived pathogens and low immunogenicity. However, current technologies still face the following bottlenecks: The contradiction between singular function and irritation: Most products on the market focus on the repair and moisturizing functions of collagen. To achieve whitening, additional active ingredients such as vitamin C, niacinamide, and arbutin are usually added. These ingredients may not only irritate the skin (especially sensitive skin), but their compatibility and stability with collagen are also challenged, and they are prone to inactivation over long-term storage.

[0003] Insufficient hypoallergenicity: Although recombinant technology reduces immunogenicity, residual endotoxins in some expression systems (such as E. coli) or permeation enhancers added to improve permeability may still cause skin discomfort in some people.

[0004] Low synergistic efficiency: Simple physical compounding (collagen + whitening agent) is difficult to achieve synergistic effects between ingredients. The transdermal absorption rate and targeting of whitening ingredients need to be improved, resulting in insignificant effects.

[0005] Therefore, the market urgently needs an innovative collagen composition that combines low-allergen, highly effective whitening and skin repair, with stable ingredients and good synergistic effects.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] This invention proposes a hypoallergenic whitening recombinant humanized collagen composition and its preparation method. This hypoallergenic whitening recombinant humanized collagen composition integrates hypoallergenicity, high-efficiency whitening and skin repair, and has stable components and good synergistic properties, and has broad application prospects in the field of whitening sensitive skin.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a hypoallergenic whitening recombinant humanized collagen composition, comprising: modified recombinant humanized collagen, tetrahydrocurcumin, ethyl ascorbic acid ether, carnosine, and dipotassium glycyrrhizate; wherein the modified recombinant humanized collagen comprises recombinant humanized collagen segments, polyethylene glycol segments modified on the recombinant humanized collagen segments, and tetrahydromethylpyrimidine formyl groups modified at the other end of the polyethylene glycol segments.

[0009] The tetrahydromethylpyrimidine formyl group has the following structure, which is obtained by dehydroxylating the carboxyl group of ecoxib and then linking it to a polyethylene glycol segment: .

[0010] The hypoallergenic whitening recombinant humanized collagen composition provided by this invention contains hypoallergenic modified recombinant humanized collagen, which, in conjunction with other mild whitening ingredients, has a good whitening effect on sensitive skin. The modified recombinant humanized collagen used in the composition of this invention contains polyethylene glycol segments as a first shield, and tetrahydromethylpyrimidine formyl groups bonded to the ends of the polyethylene glycol segments as a second shield. This effectively forms a hydration layer, maintaining the stability of the protein in the composition environment and the epidermal layer, while prolonging its action time on the skin surface. Furthermore, it provides suitable steric hindrance to both shield residual antigenic epitopes (which helps reduce skin irritation) and improve the efficiency of binding to target cell surface receptors.

[0011] Ection, chemically named 2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid (or tetrahydromethylpyrimidinecarboxylic acid), possesses a strong hydration capacity due to its molecular structure. When present alone in a composition, it stabilizes proteins within the same system through a "preferential hydration" mechanism, resulting in higher protein structural stability and less exposure of antigenic epitopes. This means that the local water molecule concentration around the protein in the same system is increased, and the protein surface is surrounded by a more stable and ordered layer of water molecules, which can reduce the protein's contact with potential denaturants in the environment and also mask residual antigenic epitopes of the protein.

[0012] In the modified recombinant humanized collagen provided by this invention, the hydration capacity of the tetrahydromethylpyrimidine formyl group derived from edodein (which depends on the combination of the internal hydrophobic rigid six-membered ring and the external hydrophilic endamidinium HN-C=N, forcing water molecules to form an ordered layered structure around edodein instead of flowing freely) can reduce the length of polyethylene glycol segments. This maintains an effective hydration layer while reducing steric hindrance, promoting the binding of the modified recombinant humanized collagen to receptors on the surface of target cells (such as keratinocytes) to achieve the effect of both shielding residual antigenic epitopes and improving the efficiency of action on target cells.

[0013] The powerful hydration effect of modified recombinant humanized collagen, through the "preferential hydration" mechanism, enables the formation of a protective hydration layer around cells, enzymes, and proteins in the same system. This helps maintain the moisture balance of these substances and improves their ability to resist external environmental stress, thereby exerting a soothing, moisturizing, and anti-inflammatory effect and reducing the irritation of whitening ingredients to the skin.

[0014] Tetrahydrocurcumin is a natural functional whitening ingredient derived from the hydrogenation of curcumin isolated from the rhizome of the ginger plant *Curcuma longa*. It possesses potent tyrosinase-inhibiting activity and exhibits whitening effects superior to arbutin. It effectively inhibits the generation of oxygen free radicals and scavenges existing free radicals, demonstrating significant antioxidant properties. Currently, there are no reports of cosmetics containing tetrahydrocurcumin exhibiting toxicity or side effects. Ethyl ascorbic acid ether is a highly stable vitamin C derivative. It not only efficiently reduces existing melanin but also reduces melanin synthesis at its source by inhibiting tyrosinase activity and promotes collagen production, synergistically combating aging with recombinant humanized collagen in the composition. Dipotassium glycyrrhizate is a naturally derived active ingredient with powerful anti-inflammatory and soothing effects. It effectively prevents post-inflammatory hyperpigmentation (inflammation caused by external stimuli such as ultraviolet radiation is a major cause of pigmentation) and further enhances the product's gentleness. Carnosine plays multiple roles, including anti-glycation, anti-oxidation, and repairing the skin barrier, which can delay skin aging, improve dull skin tone, and enhance skin elasticity and resistance.

[0015] This invention combines four active substances that act on multiple pathways (including: anti-oxidation, transport inhibition, enzyme inhibition, and anti-inflammation) to form a "multi-dimensional attack" network, achieving a synergistic effect of "1+1+1+1 > 4" and offsetting irritation, thereby achieving whitening effects with low skin irritation.

[0016] In some embodiments, one end of the polyethylene glycol segment is modified with an α-amino group at the N-terminus of the recombinant humanized collagen segment. This modification at the end group can increase the thickness of the hydration layer of the protein, reduce the protein's irritation, and maintain the hydrophilicity-hydrophobicity differences at different positions of the protein molecule, thereby ensuring the efficient binding of the protein to target cells.

[0017] In some embodiments, the molecular weight of the polyethylene glycol segment is 400~2000 g / mol, preferably 400~1939 g / mol; the modified recombinant humanized collagen of the present invention is also modified with tetrahydromethylpyrimidine formyl group, which greatly reduces the molecular weight of the polyethylene glycol segment, thereby reducing steric hindrance and helping to maintain the hydrophilicity-hydrophobicity difference at different positions of the protein molecule, thereby ensuring the efficient binding of the protein to the target cell.

[0018] In some embodiments, the modified recombinant humanized collagen is prepared as follows: (1) Ectoin was esterified with OH-PEG-CHO to obtain Ectoin-PEG-CHO; (2) The Ectoin-PEG-CHO reacts with recombinant humanized collagen with Schiff base, and the modified recombinant humanized collagen is obtained after purification and drying.

[0019] Preferably, in step (1), the ectoin is first protected with an amino group to obtain P-Ectoin, which is then esterified with OH-PEG-CHO, and deamination protection is performed after the Schiff base reaction in step (2).

[0020] Preferably, the molecular weight of the OH-PEG-CHO is 500~2000 g / mol.

[0021] Preferably, the esterification reaction includes: dissolving P-Ectoin or Ectoin in N,N-dimethylformamide (DMF) and activating it, adding OH-PEG-CHO and reacting at 20~30℃ for 24~36h; the activation includes adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine and reacting at 5℃ until no obvious precipitate is formed, followed by filtration.

[0022] Preferably, the pH of the Schiff base reaction system is 6-7, and NaBH3CN needs to be added, with a reaction time of 1-3 hours. Preferably, the basic repeating unit of the recombinant humanized collagen is GPRGAPGERGRPGLPGAAGARGNDGARGSD, and the number of repetitions of the basic repeating unit is 10-18. The preparation method of the recombinant humanized collagen is described in CN 117986355 A. This basic repeating unit is selected from human type III collagen and can be freely assembled to form high-molecular-weight recombinant humanized type III collagen. The resulting recombinant humanized type III collagen strictly adheres to the Gly-XY repeating sequence and can be coiled and twisted into a triple helix, thus promoting the adhesion and proliferation of human fibroblasts and repairing the skin barrier.

[0023] Preferably, in step (2), the mass ratio of recombinant humanized collagen, Ectoin-PEG-CHO or P-Ectoin-PEG-CHO, and NaBH3CN is 1g:0.3~0.9g:1~3g.

[0024] Preferably, the amino group protection refers to the protection of the amino group of ectoin with 4-methyltriphenylmethyl (Mtt), and the preparation method is as follows: ectoin reacts with 4-methyltriphenylmethyl chloride under alkaline conditions to obtain P-Ectoin; the alkaline conditions refer to dissolving ectoin and 4-methyltriphenylmethyl chloride in dichloromethane (DCM) and adding triethylamine.

[0025] Preferably, the deamination protection refers to adding trifluoroacetic acid to the reaction solution obtained from the Schiff base reaction and stirring the reaction for 0.5 to 1.5 hours.

[0026] Preferably, the amino acid sequence of the recombinant humanized collagen is shown in SEQ ID NO:1.

[0027] In some embodiments, the hypoallergenic whitening recombinant humanized collagen composition comprises, by weight percentage: 0.47%~3.7% modified recombinant humanized collagen, 0.05%~0.1% tetrahydrocurcumin, 1%~2% ethyl ascorbic acid ether, 0.8%~1.5% dipotassium glycyrrhizate and 0.1%~0.3% carnosine, with the balance being a solvent, said solvent being a pH buffer.

[0028] In some embodiments, the hypoallergenic whitening recombinant humanized collagen composition is in the form of a gel. The three-dimensional gel network can lock in a large amount of water, preventing the components from flowing freely, thereby maintaining the physical stability of the system and giving the composition a refreshing gel-like texture.

[0029] In some embodiments, the hypoallergenic whitening recombinant humanized collagen composition comprises, by weight percentage: 0.47%~3.7% modified recombinant humanized collagen, 0.05%~0.1% tetrahydrocurcumin, 1%~2% ethyl ascorbic acid ether, 0.8%~1.5% dipotassium glycyrrhizate, 0.32%~1.1% chondroitin sulfate, and 0.1%~0.3% carnosine, with the balance being a solvent, wherein the solvent is a pH buffer solution.

[0030] Chondroitin sulfate is a natural skin component and a commonly used gelling agent in biomedical materials. It has a strong moisturizing effect, can enhance the skin barrier function, promote skin repair, and exert anti-inflammatory and anti-aging effects.

[0031] In some embodiments, the hypoallergenic whitening recombinant humanized collagen composition comprises, by weight percentage: (A): a gel containing modified recombinant humanized collagen, ethyl ascorbic acid ether, dipotassium glycyrrhizate and chondroitin sulfate; and (B): liposomes encapsulating modified recombinant humanized collagen, carnosine and tetrahydrocurcumin.

[0032] This invention first encapsulates protein peptide chains that are difficult to absorb through the skin and whitening components that are easily oxidized and degraded using liposomes. These encapsulated peptides are then loaded onto a three-dimensional gel network self-assembled from modified recombinant humanized collagen and chondroitin sulfate, forming a "gel-liposome complex system." This system offers the following advantages: the gel matrix allows active ingredients to remain on the skin surface for an extended period, giving liposomes ample time to slowly release the active ingredients and improve their effectiveness; the complex system simultaneously stabilizes two highly different components—water-soluble modified recombinant humanized collagen and fat-soluble tetrahydrocurcumin—solving the problems of easy layering and inactivation in traditional formulations; the gel network prevents liposome aggregation, precipitation, or oil-water separation, ensuring long-term stability of the entire system; the gel provides excellent immediate moisturizing and a pleasant application feel, while the loaded liposomes carry the active ingredients and efficiently penetrate deep into the skin.

[0033] When modified recombinant humanized collagen and chondroitin sulfate are mixed in solution, they self-assemble into a three-dimensional cross-linked network structure through electrostatic attraction. This portion of the modified recombinant humanized collagen not only provides a gel framework but also remains on the skin surface to form a film and provide moisturizing effects. Modified recombinant humanized collagen encapsulated in liposomes is delivered transdermally to the granular layer and spinous layer. The hydration environment, pH value, and enzyme activity in these areas have a significant impact on the stability of the peptide. The modified recombinant humanized collagen provided by this invention exhibits good stability, preventing enzymatic degradation and inactivation. Furthermore, the modification facilitates its penetration into the hydration layer and into keratinocytes, reduces irritation, and promotes skin barrier repair and the expression of moisturizing-related genes (such as FLG and LOR).

[0034] In some embodiments, the hypoallergenic whitening recombinant humanized collagen composition comprises, by weight percentage: 0.47%~3.7% modified recombinant humanized collagen, 0.05%~0.1% tetrahydrocurcumin, 1%~2% ethyl ascorbic acid ether, 0.8%~1.5% dipotassium glycyrrhizate, 0.32%~1.1% chondroitin sulfate, 0.1%~0.3% carnosine, 0.5%~3% liposome substrate, with the balance being a solvent, wherein the solvent is a pH buffer solution.

[0035] The liposome substrate comprises cholesterol and phospholipids. Preferably, the phospholipids comprise one or more of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM). More preferably, the phospholipids are hydrogenated soybean phosphatidylcholine (HSPC) and distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0036] The pH buffer solution is used to maintain the pH of the hypoallergenic whitening recombinant humanized collagen composition at 4.5~6.5. The pH buffer solution includes two or more of the following ions: citrate, sodium, chloride, phosphate, hydrogen phosphate, and dihydrogen phosphate.

[0037] Secondly, the present invention provides a method for preparing any of the hypoallergenic whitening recombinant humanized collagen compositions described above, comprising the following steps: S1: Preparation of liposomes: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, and phospholipids are dissolved in an organic solvent, heated to 50-70°C, and stirred until completely dissolved and transparent to form an oil phase; S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in buffer solution and stirred evenly to form an aqueous phase; S1.3 Hydration and homogenization: The oil phase is added to the aqueous phase for preliminary emulsification; subsequently, the resulting primary emulsion is homogenized under high pressure and concentrated to obtain a liposome suspension; S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate are dissolved in the liposome suspension. After stirring and dissolving, chondroitin sulfate solution is added dropwise while stirring. After the addition is complete, homogenization is performed, and finally the pH is adjusted to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0038] The preparation method provided by this invention involves the self-assembly of residual modified recombinant humanized collagen (not encapsulated by liposomes) with chondroitin sulfate to obtain a three-dimensional gel network. This cleverly utilizes the remaining free modified recombinant humanized collagen and stabilizes it with chondroitin sulfate, reducing the probability of deformation and inactivation. Therefore, under the same component ratio, the amount of free modified recombinant humanized collagen and the amount encapsulated by liposomes are inversely related; a higher encapsulation rate of liposomes results in more transdermal modified recombinant humanized collagen, but a lower gel viscosity leads to poorer component stabilization, and vice versa. Maintaining a suitable encapsulation rate is beneficial for balancing both aspects; therefore, the conditions for preparing liposomes affect the encapsulation rate of modified recombinant humanized collagen.

[0039] In some embodiments, the organic solvent is methanol or ethanol.

[0040] In some embodiments, the total mass ratio of the tetrahydrocurcumin, cholesterol, and phospholipids to ethanol is 10 mg: 0.5~1.5 mL.

[0041] In some embodiments, the pH of the buffer solution is 4.5 to 5.8, preferably a citrate-sodium citrate buffer solution.

[0042] In some embodiments, the total mass of the modified recombinant humanized collagen and carnosine is in the ratio of 2-3 mg to 1 mL of buffer solution.

[0043] In some implementations, the initial emulsification refers to stirring at 10,000 rpm for 3 to 6 minutes.

[0044] In some implementations, the high-pressure homogenization refers to cycling 5-10 times at a pressure of 100-150 MPa.

[0045] In some implementations, the concentration refers to removing ethanol by vacuum distillation at 60°C.

[0046] In some embodiments, the concentration of the chondroitin sulfate solution is 20-25 g / L.

[0047] In some implementations, the homogenization is performed at 3000 rpm for 2 minutes.

[0048] In some implementations, adjusting the pH means adding 1M sodium hydroxide solution or 1M hydrochloric acid to adjust the pH to 5-6.5.

[0049] Thirdly, the present invention provides the application of any of the hypoallergenic whitening recombinant humanized collagen compositions described herein, wherein the composition is used in the preparation of gel dressings.

[0050] A gel dressing, by weight percentage, comprises the following components: 90% to 98% hypoallergenic whitening recombinant humanized collagen composition, 2% to 10% small molecule moisturizer, 0% to 0.12% preservative and 0% to 0.1% pH adjuster.

[0051] In some embodiments, the small molecule moisturizer is at least one selected from glycerin, butylene glycol, dipropylene glycol, propylene glycol, and pentanediol; In some embodiments, the preservative is at least one of p-hydroxyacetophenone, phenoxyethanol, and ethylhexylglycerin.

[0052] In some embodiments, the pH adjuster is at least one of triethanolamine, sodium hydroxide, citric acid, and lactic acid.

[0053] The present invention has the following beneficial effects: (3) The hypoallergenic whitening recombinant humanized collagen composition provided by the present invention contains hypoallergenic modified recombinant humanized collagen, which, in conjunction with other mild whitening ingredients, has a good whitening effect on sensitive skin. The unique molecular structure design of this modified recombinant humanized collagen can provide suitable steric hindrance to achieve the dual effect of shielding residual antigen epitopes (which helps reduce skin irritation) and improving the efficiency of binding to target cell surface receptors.

[0054] (4) The low-sensitivity whitening recombinant humanized collagen composition provided by the present invention contains active substances that act on multiple pathways (including: anti-oxidation, inhibition of transport, enzyme inhibition, and anti-inflammation) to form a "multi-dimensional attack" network, achieving a synergistic effect of "1+1+1+1 > 4" and irritation offsetting, thereby achieving whitening effect with low skin irritation. Attached Figure Description

[0055] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0056] Figure 1 The mass spectrum (ESI-MS) of the modified recombinant humanized collagen in Example 1 is shown. Figure 2 The mass spectrum (ESI-MS) of the modified recombinant humanized collagen in Example 5 is shown. Figure 3 The mass spectrum (ESI-MS) of the modified recombinant humanized collagen in Example 8; Figure 4 This is the mass spectrum (ESI-MS) of the modified recombinant humanized collagen from Example 9. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0058] In the description of the embodiments of this application, the technical terms "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly indicating the number, specific order, or primary and secondary relationship of the indicated technical features.

[0059] The term "embodiment" in this document 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 in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments.

[0060] In the embodiments of this application, the term "or / and" is only a description of the relationship between related objects, indicating that there can be three kinds of relationships. For example, A or / and B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0061] Additionally, the character " / " in this article generally indicates that the objects before and after it are in an "or" relationship.

[0062] In the embodiments of this application, "multiple" means two or more (including two), similarly, "multiple groups" means two or more (including two groups), and "multiple layers" means two or more (including two layers), unless otherwise explicitly specified and limited.

[0063] In the embodiments of this application, "at least one" means one or more.

[0064] Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.

[0065] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0066] [Recombinant Humanized Collagen] The basic repeating unit of the recombinant humanized collagen used in this invention for preparing modified recombinant humanized collagen is GPRGAPGERGRPGLPGAAGARGNDGARGSD.

[0067] The basic repeating unit of recombinant humanized collagen has a repeating number of 10 to 18.

[0068] In some embodiments, the basic repeating unit of the recombinant humanized collagen is repeated 15 times, and its amino acid sequence is shown in SEQ ID NO:1, while its corresponding optimized gene sequence is shown in SEQ ID NO:2.

[0069] In some embodiments, the basic repeating unit of the recombinant humanized collagen is repeated 12 times, and its amino acid sequence is shown in SEQ ID NO:3, while its corresponding optimized gene sequence is shown in SEQ ID NO:4.

[0070] In some embodiments, the basic repeating unit of the recombinant humanized collagen is repeated 18 times, and its amino acid sequence is shown in SEQ ID NO:5, while its corresponding optimized gene sequence is shown in SEQ ID NO:6.

[0071] The preparation method of recombinant humanized collagen is described in CN 117986355 A. Taking SEQ ID NO:1 as an example, the preparation method includes the following steps: (1) Based on the amino acid sequence of SEQ ID NO:1, the gene sequence was reverse-engineered using the online codon optimization tool (ExpOptimizer) (https: / / www.novopro.cn / tools / codon-optimization.html) to select the preferred codons required for expression in the host Escherichia coli. During the design process, the NdeI and XhoI restriction sites were removed, which is beneficial for subsequent gene manipulation. The optimized gene sequence is shown in SEQ ID NO:2.

[0072] (2) The SEQ ID NO:2 gene sequence, i.e., a nucleotide fragment of 1350 bases, was handed over to Shanghai Sangon Biotech for whole-gene synthesis.

[0073] (3) The DNA fragment of SEQ ID NO:2 was cloned and amplified by PCR, and the target gene was recovered by agarose gel electrophoresis. Using the NdeI and XhoI restriction sites added to the primers, the gene fragment was cut into sticky ends by adding the corresponding restriction endonuclease, and co-incubated with the pET-28a expression vector that had been digested with the corresponding restriction enzymes. T4 DNA ligase was added, and the ligation was carried out at 4°C for 12 hours. The resulting fragment was transformed into E. coli DH5α. White spot clones were selected by screening with ampicillin sodium-LB plates. The plasmid was extracted by alkaline lysis or boiling method of molecular cloning. The positive clones were identified by restriction enzyme digestion and PCR. Finally, sequencing confirmed that the recombinant plasmid contained the correct gene sequence reading frame, and the pET-28a-1620 recombinant expression vector was successfully constructed.

[0074] (4) Transform the correct pET-28a-1620 plasmid into Escherichia coli BL21(DE3), screen single-spot clones using ampicillin sodium-LB plates, culture overnight in 5 ml LB medium, transfer at 1:100, culture in shake flasks at 37°C until OD600 is 0.4-0.6, add IPTG stock solution at 1:5000, culture at 20°C for 8-10 hours, collect the cells by centrifugation, and store at -20°C or proceed to the next purification step immediately.

[0075] (5) Wash the mixed bacterial pellet with PBS buffer, resuspend the pellet in 20-40 ml of 500 ml of bacterial solution, use lysozyme in combination with Triton X-100 to help lyse the bacteria, sonicate for 45 min in an ice-water mixture, centrifuge at 12000 rpm / min for 20 min, and collect the supernatant. At this point, the solution contains a large amount of the target recombinant humanized collagen.

[0076] (6) Preliminary purification was performed using ammonium sulfate precipitation and fractional salting-out. After exploring the saturation of ammonium sulfate, the optimal saturation for ammonium sulfate salting-out was found to be 0-20%. The nickel-NTA His-Bind Resin column was washed with PBS buffer, and then the column was mixed with the above-mentioned desalted and purified solution and incubated. The mixture was gently shaken at room temperature or on ice for 30 min, and then loaded onto the column. Impurities were washed with PBS solution containing 15 mM imidazole. An appropriate amount of His-tagged Prescission Protease was added to the column, and the mixture was gently shaken at room temperature or on ice for 2 hours. After elution with PBS solution, recombinant humanized collagen with a purity of over 95% (SEQ ID NO:1) was obtained and lyophilized into a powder for later use.

[0077] SEQ ID NO:1: GPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLP GAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSD.

[0078] SEQ ID NO:2:

[0079] SEQ ID NO:3: GPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSDGPRGAPGERGRPGLPGAAGARGNDGARGSD。

[0080] SEQ ID NO:4:

[0081] SEQ ID NO:5: 。

[0082] SEQ ID NO:6:

[0083] Example 1 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by mass percentage, comprises the following components: 1.87% modified recombinant humanized collagen, 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate and 0.2% carnosine, with the balance being PBS buffer.

[0084] The preparation method of modified recombinant humanized collagen is as follows: (1) The dried ectoin was dissolved in anhydrous dichloromethane (DCM), and 4-methyltriphenylmethyl chloride (Mtt) was added. The mixture was stirred under nitrogen protection at 5°C, and a DCM solution of triethylamine was added dropwise. After the addition was complete, the mixture was transferred to nitrogen protection at 25°C and reacted for 12 hours. The reaction solution was washed successively with saturated sodium bicarbonate solution, saturated saline solution, and water. After drying the organic phase, crude Mtt-Ectoin product was obtained.

[0085] (2) The crude Mtt-Ectoin product was dissolved in N,N-dimethylformamide (DMF), filtered, and then N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were added for the first activation reaction (5℃ ice bath). After no obvious precipitate was formed, the product was filtered, and OH-PEG-CHO was added to the filtrate for esterification reaction (25℃, 24h). The resulting reaction solution was dried and then redissolved in water. The solution was purified by dialysis and dried to obtain Mtt-Ectoin-PEG-CHO. The molar ratio of icotin, Mtt, triethylamine, DCC, DMAP, and OH-PEG-CHO is 1:1.3:2.6:1.2:0.2:1.3; the dialysis uses a molecular weight cutoff of 1000 Da, and the dialysis is performed for 3 days with the solution changed every 12 hours; the molecular weight of OH-PEG-CHO is 1000 Da.

[0086] (2) Add Mtt-Ectoin-PEG-CHO to the protein solution at pH=6, stir to dissolve, heat to 37℃, add NaBH3CN, keep warm and stir for 1h, then add trifluoroacetic acid (TFA) at 0.01gTFA / 1mL reaction solution, continue stirring for 1h to remove the Mtt protecting group, dialyze and concentrate the reaction solution, then load it onto an SP Sepharose FF chromatography column, and perform gradient elution with a buffer solution at pH=4 containing 0~0.5MNaCl. Detect the absorption peak at 220nm UV. Concentrate the collected main peak eluent, dialyze, and freeze dry to obtain modified recombinant humanized collagen.

[0087] The mass spectrum (ESI-MS) of the modified recombinant humanized collagen is attached. Figure 1As shown.

[0088] The concentration of the protein solution was 5 g / L, and the amino acid sequence of the protein was as shown in SEQ ID NO:1. The protein was obtained by dissolving it in a citric acid / sodium citrate buffer solution at pH=6. The feeding ratio of protein, Mtt-Ectoin-PEG-CHO, and NaBH3CN was 1 g: 0.6 g: 2 g.

[0089] This embodiment also provides a method for preparing the above composition, including the following steps: Tetrahydrocurcumin, ethyl ascorbic acid ether, and dipotassium glycyrrhizate were dissolved in 1×PBS buffer and heated to 40°C for high-speed shearing (stirring at 5000 rpm for 5 min). After cooling to room temperature, 1M sodium hydroxide solution or 1M hydrochloric acid solution was added to adjust the pH to 6. Then, modified recombinant humanized collagen and carnosine were added and stirred evenly to obtain the above-mentioned hypoallergenic whitening recombinant humanized collagen composition.

[0090] Example 2 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by mass percentage, comprises the following components: 1.87% modified recombinant humanized collagen (same as in Example 1), 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.32% chondroitin sulfate, and 0.2% carnosine, with the balance being buffer solution.

[0091] The buffer solution refers to a salt solution that maintains the pH of the composition at 6, including citrate ions, sodium ions, and hydrogen ions.

[0092] This embodiment also provides a method for preparing the above composition, including the following steps: (1) The modified recombinant humanized collagen solution and the chondroitin sulfate solution were mixed and stirred at 4°C to form a gel; wherein the modified recombinant humanized collagen solution contained 3.5% modified recombinant humanized collagen, pH=4.8, and the solvent was citrate-sodium citrate buffer; the chondroitin sulfate solution contained 22% chondroitin sulfate, pH=4.8, and the solvent was citrate-sodium citrate buffer.

[0093] (2) Dissolve tetrahydrocurcumin, ethyl ascorbic acid ether, and dipotassium glycyrrhizate in deionized water, heat to 40°C and shear at medium-high speed (stir at 5000 rpm for 5 min), cool to room temperature and add carnosine and stir evenly to obtain an active substance solution. Add the active substance solution obtained in step (2) to the gel in step (1), homogenize at 3000 rpm for 2 min, and adjust the pH to 6 by adding 1M sodium hydroxide solution or 1M hydrochloric acid solution to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0094] Example 3 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by mass percentage, comprises the following components: 1.87% modified recombinant humanized collagen (same as in Example 1), 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.2% carnosine, 2.4% liposome substrate, and the balance being PBS buffer.

[0095] The liposome substrate comprises cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 in a mass ratio of 10:32:3.

[0096] This embodiment also provides a method for preparing the above-mentioned hypoallergenic whitening recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine and DSPE-PEG2000 were dissolved in ethanol, heated to 60°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 was in a ratio of 10 mg to 1 mL of ethanol.

[0097] S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in PBS buffer at pH 1 and stirred until homogeneous to form an aqueous phase; The total mass of modified recombinant humanized collagen and carnosine was in a buffer solution ratio of 3 mg: 1 mL. S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 5 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 8 times at a pressure of 120 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate were dissolved in liposome suspension, stirred and dissolved, homogenized at 3000 rpm for 2 min, and the pH was adjusted to 6 by adding 1M sodium hydroxide solution or 1M hydrochloric acid solution to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0098] Example 4 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by weight percentage, comprises the following components: 1.87% modified recombinant humanized collagen (same as in Example 1), 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.32% chondroitin sulfate, 0.2% carnosine, 2.4% liposome matrix, and the balance being buffer solution.

[0099] The liposome substrate comprises cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 in a mass ratio of 10:32:3; the buffer solution refers to a salt solution that maintains the pH of the composition at 6, comprising citrate ions, sodium ions, and hydrogen ions.

[0100] This embodiment also provides a method for preparing the above-mentioned hypoallergenic whitening recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine and DSPE-PEG2000 were dissolved in ethanol, heated to 60°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 was in a ratio of 10 mg to 1 mL of ethanol.

[0101] S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in a citrate-sodium citrate buffer solution with a pH of 4.8 and stirred until homogeneous to form an aqueous phase; The total mass of modified recombinant humanized collagen and carnosine was in a buffer solution ratio of 3 mg: 1 mL. S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 5 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 8 times at a pressure of 120 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate were dissolved in liposome suspension and stirred until dissolved. Then, a 22% chondroitin sulfate aqueous solution was added dropwise and mixed. The mixture was stirred at 4°C to form a gel. The mixture was homogenized at 3000 rpm for 2 min, and 1M sodium hydroxide solution was added to adjust the pH to 6 to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0102] Example 5 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by weight percentage, comprises the following components: 1.87% modified recombinant humanized collagen, 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.32% chondroitin sulfate, 0.2% carnosine, 2.4% liposome matrix, and the balance being buffer solution.

[0103] The liposome substrate comprises cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 in a mass ratio of 10:32:3; the buffer solution refers to a salt solution that maintains the pH of the composition at 6, comprising citrate ions, sodium ions, and hydrogen ions.

[0104] The preparation method of modified recombinant humanized collagen is as follows: (1)~(2) Same as Example 1; (3) The difference from Example 1 is that Mtt-Ectoin-PEG-CHO was added to the protein solution at pH=7, stirred and dissolved, heated to 37°C, NaBH3CN was added, and the reaction was stirred for 1 h. Then, trifluoroacetic acid (TFA) was added at 0.01 g TFA / 1 mL of reaction solution, and stirring was continued for 1 h to remove the Mtt protecting group. The reaction solution was dialyzed, concentrated and then loaded onto an SPSepharose FF chromatography column. Gradient elution was performed with a buffer solution at pH=4 containing 0~0.5 M NaCl, and the absorption peak was detected by UV at 220 nm. The collected main peak eluent was concentrated, dialyzed and freeze-dried to obtain modified recombinant humanized collagen.

[0105] The protein solution was obtained by dissolving the protein in PBS buffer at pH 7; the ratio of protein (SEQ ID NO:1), Mtt-Ectoin-PEG-CHO, and NaBH3CN was 1g:0.6g:2g.

[0106] The mass spectrum (ESI-MS) of the modified recombinant humanized collagen is attached. Figure 2 As shown in the figure, the modified recombinant humanized collagen contains a variety of modified products, indicating that under these reaction conditions, the modifier (Mtt-Ectoin-PEG-CHO) has low selectivity for the N-terminal amino group, and multiple amino acid residues are also modified.

[0107] The preparation method of the above-mentioned hypoallergenic whitening recombinant humanized collagen composition is the same as that in Example 1.

[0108] Example 6 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by weight percentage, comprises the following components: 1.87% modified recombinant humanized collagen (same as in Example 1), 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.32% chondroitin sulfate, 0.2% carnosine, 2.4% liposome matrix, and the balance being buffer solution.

[0109] The liposome substrate comprises cholesterol and soybean lecithin in a mass ratio of 1:5; the buffer solution refers to a salt solution that maintains the pH of the composition at 6, including citrate ions, sodium ions, and hydrogen ions.

[0110] This embodiment also provides a method for preparing the above-mentioned hypoallergenic whitening recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, and soybean lecithin were dissolved in ethanol, heated to 60°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, and soy lecithin was in a ratio of 10 mg to 1 mL of ethanol.

[0111] S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in a citrate-sodium citrate buffer solution with a pH of 4.8 and stirred until homogeneous to form an aqueous phase; The total mass of modified recombinant humanized collagen and carnosine was in a buffer solution ratio of 3 mg: 1 mL. S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 5 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 8 times at a pressure of 120 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate were dissolved in liposome suspension and stirred until dissolved. Then, a 22% chondroitin sulfate aqueous solution was added dropwise and mixed. The mixture was stirred at 4°C to form a gel. The mixture was homogenized at 3000 rpm for 2 min, and 1M sodium hydroxide solution was added to adjust the pH to 6 to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0112] Example 7 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by weight percentage, comprises the following components: 1.87% modified recombinant humanized collagen (same as in Example 1), 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.32% chondroitin sulfate, 0.2% carnosine, 2.4% liposome matrix, and the balance being buffer solution.

[0113] The liposome substrate comprises cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 in a mass ratio of 10:32:3; the buffer solution refers to a salt solution that maintains the pH of the composition at 6, including citrate ions, sodium ions, hydrogen ions, and chloride ions.

[0114] This embodiment also provides a method for preparing the above-mentioned hypoallergenic whitening recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine and DSPE-PEG2000 were dissolved in ethanol, heated to 60°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 was in a ratio of 10 mg to 1 mL of ethanol.

[0115] S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in a citrate-sodium citrate buffer solution with a pH of 6.5 and stirred until homogeneous to form an aqueous phase; The total mass of modified recombinant humanized collagen and carnosine was in a buffer solution ratio of 3 mg: 1 mL. S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 5 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 8 times at a pressure of 120 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate were dissolved in liposome suspension and stirred until dissolved. Then, 22% chondroitin sulfate PBS solution was added dropwise and mixed. The mixture was stirred at 4°C to form a gel. The mixture was homogenized at 3000 rpm for 2 min, and 1M sodium hydroxide solution or 1M hydrochloric acid was added to adjust the pH to 6 to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0116] Example 8 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by weight percentage, comprises the following components: 0.35% modified recombinant humanized collagen, 0.1% tetrahydrocurcumin, 1.2% ethyl ascorbic acid ether, 1.5% dipotassium glycyrrhizate, 1.05% chondroitin sulfate, 0.3% carnosine, and 0.5% liposome matrix, with the balance being buffer solution.

[0117] The liposome substrate comprises cholesterol and egg yolk lecithin in a mass ratio of 1:6; the buffer solution refers to a salt solution maintaining the pH of the composition at 6.5, comprising citrate ions, sodium ions, and hydrogen ions. The preparation method of the modified recombinant humanized collagen differs from that in Example 1 in that the amino acid sequence of the recombinant humanized collagen is SEQ ID NO:3, and the molecular weight of OH-PEG-CHO is 500 Da.

[0118] The mass spectrum (ESI-MS) of the modified recombinant humanized collagen is attached. Figure 3 As shown.

[0119] This embodiment also provides a method for preparing the above-mentioned hypoallergenic whitening recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, and egg yolk lecithin are dissolved in ethanol, heated to 70°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, and egg yolk lecithin was in the ratio of 10 mg to 0.5 mL of ethanol.

[0120] S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in a citrate-sodium citrate buffer solution with a pH of 5.5 and stirred until homogeneous to form an aqueous phase; The total mass ratio of modified recombinant humanized collagen and carnosine to buffer solution was 2 mg: 1 mL; S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 3 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 5 times at a pressure of 150 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate were dissolved in liposome suspension and stirred until dissolved. Then, a 20% chondroitin sulfate aqueous solution was added dropwise and mixed. The mixture was stirred at 4°C to form a gel. The mixture was homogenized at 3000 rpm for 2 min, and 1M sodium hydroxide solution was added to adjust the pH to 6.5 to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0121] Example 9 This embodiment provides a hypoallergenic whitening recombinant humanized collagen composition, which, by weight percentage, comprises the following components: 2.1% modified recombinant humanized collagen, 0.05% tetrahydrocurcumin, 2% ethyl ascorbic acid ether, 0.8% dipotassium glycyrrhizate, 0.83% chondroitin sulfate, 0.1% carnosine, and 2.8% liposome matrix, with the balance being buffer solution.

[0122] The liposome substrate comprises cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 in a mass ratio of 15:32:3; the buffer solution refers to a salt solution maintaining the pH of the composition at 5.5, comprising citrate ions, sodium ions, and hydrogen ions. The preparation method of the modified recombinant humanized collagen differs from that in Example 1 in that the amino acid sequence of the recombinant humanized collagen is SEQ ID NO:5, and the molecular weight of OH-PEG-CHO is 2000 Da.

[0123] This embodiment also provides a method for preparing the above-mentioned hypoallergenic whitening recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine and DSPE-PEG2000 were dissolved in methanol, heated to 50°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 was in a ratio of 10 mg to 1.5 mL of methanol.

[0124] S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in a citrate-sodium citrate buffer solution with a pH of 4.5 and stirred until homogeneous to form an aqueous phase; The total mass of modified recombinant humanized collagen and carnosine was in a buffer solution ratio of 3 mg: 1 mL. S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 6 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 10 times under a pressure of 100 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate were dissolved in liposome suspension and stirred until dissolved. Then, a 25% chondroitin sulfate aqueous solution was added dropwise and mixed. The mixture was stirred at 4°C to form a gel. The mixture was homogenized at 3000 rpm for 2 min, and 1M sodium hydroxide solution was added to adjust the pH to 5.5 to obtain a hypoallergenic whitening recombinant humanized collagen composition.

[0125] Comparative Example 1 This embodiment provides a recombinant humanized collagen composition, which, by mass percentage, comprises the following components: 1.87% PEG-modified recombinant humanized collagen, 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.32% chondroitin sulfate, 0.2% carnosine, 2.4% liposome matrix and 2% ecoxib, with the balance being buffer solution.

[0126] The liposome substrate comprises cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 in a mass ratio of 10:32:3; the buffer solution refers to a salt solution that maintains the pH of the composition at 6, comprising citrate ions, sodium ions, and hydrogen ions.

[0127] The preparation method of PEG-modified recombinant humanized collagen is as follows: OH-PEG-CHO was added to a protein solution with pH=6, and after stirring to dissolve, the temperature was raised to 37℃, NaBH3CN was added, and the reaction was stirred and maintained for 1 hour. The resulting reaction solution was dialyzed, concentrated, and then loaded onto an SP Sepharose FF column for gradient elution with a buffer solution containing 0~0.5M NaCl at pH=4. The absorption peak was detected by UV at 220nm. The collected main peak eluent was concentrated, dialyzed, and freeze-dried to obtain PEG-modified recombinant humanized collagen.

[0128] The protein solution had a concentration of 5 g / L, and the amino acid sequence of the protein was as shown in SEQ ID NO:1. The protein was obtained by dissolving it in a citrate-sodium citrate buffer solution at pH=6. The feeding ratio of protein, OH-PEG-CHO, and NaBH3CN was 1 g: 0.6 g: 2 g. The molecular weight of OH-PEG-CHO was 1000 Da.

[0129] The mass spectrum (ESI-MS) of the modified recombinant humanized collagen is attached. Figure 4 As shown.

[0130] This embodiment also provides a method for preparing the above-mentioned recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine and DSPE-PEG2000 were dissolved in ethanol, heated to 60°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 was in a ratio of 10 mg to 1 mL of ethanol.

[0131] S1.2 Aqueous phase preparation: Recombinant humanized collagen and carnosine were dissolved in a citrate-sodium citrate buffer solution at pH 4.8 and stirred until homogeneous to form an aqueous phase; The total mass of recombinant humanized collagen and carnosine was in a buffer solution ratio of 3 mg: 1 mL. S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 5 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 8 times at a pressure of 120 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether, edodecanin and dipotassium glycyrrhizate were dissolved in liposome suspension and stirred until dissolved. Then, a 22% chondroitin sulfate aqueous solution was added dropwise and mixed. The mixture was stirred at 4°C to form a gel. The mixture was homogenized at 3000 rpm for 2 min and then 1M sodium hydroxide solution was added to adjust the pH to 6 to obtain the recombinant humanized collagen composition.

[0132] Comparative Example 2 The difference from Comparative Example 1 is that the OH-PEG-CHO used in the preparation of the PEG-modified recombinant humanized collagen contained in the composition has a molecular weight of 3000 Da.

[0133] Comparative Example 3 This embodiment provides a recombinant humanized collagen composition, which, by mass percentage, comprises the following components: 1.87% recombinant humanized collagen (amino acid sequence SEQ ID NO:1), 0.08% tetrahydrocurcumin, 1% ethyl ascorbic acid ether, 1.2% dipotassium glycyrrhizate, 0.32% chondroitin sulfate, 0.2% carnosine, 2.4% liposome matrix and 2% edodeoxyne, with the balance being buffer solution.

[0134] The liposome substrate comprises cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 in a mass ratio of 10:32:3; the buffer solution refers to a salt solution that maintains the pH of the composition at 6, comprising citrate ions, sodium ions, and hydrogen ions.

[0135] This embodiment also provides a method for preparing the above-mentioned recombinant humanized collagen composition, including the following steps: S1: Preparation of liposome suspension: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine and DSPE-PEG2000 were dissolved in ethanol, heated to 60°C, and stirred until completely dissolved and transparent to form an oil phase; The total mass of tetrahydrocurcumin, cholesterol, hydrogenated soybean phosphatidylcholine, and DSPE-PEG2000 was in a ratio of 10 mg to 1 mL of ethanol.

[0136] S1.2 Aqueous phase preparation: Recombinant humanized collagen and carnosine were dissolved in a citrate-sodium citrate buffer solution at pH 4.8 and stirred until homogeneous to form an aqueous phase; The total mass of recombinant humanized collagen and carnosine was in a buffer solution ratio of 3 mg: 1 mL. S1.3 Hydration and homogenization: Under shearing and stirring at 10,000 rpm, the oil phase was slowly added to the aqueous phase for initial emulsification for 5 min; then, the initial emulsion was passed through a high-pressure homogenizer and circulated 8 times at a pressure of 120 MPa, and concentrated to obtain a liposome suspension. S2: Ethyl ascorbic acid ether, edodecanin and dipotassium glycyrrhizate were dissolved in liposome suspension and stirred until dissolved. Then, a 22% chondroitin sulfate aqueous solution was added dropwise and mixed. The mixture was stirred at 4°C to form a gel. The mixture was homogenized at 3000 rpm for 2 min and then 1M sodium hydroxide solution was added to adjust the pH to 6 to obtain the recombinant humanized collagen composition.

[0137] Test Example 1: Component Compatibility Stability Test To investigate the compatibility and stability of the components of the composition, the compositions prepared in each specific embodiment were subjected to the following tests: (1) sealed and stood at 25°C for 30 days; (2) sealed and stood at 8°C for 10 days; (3) sealed and stood at 40°C for 10 days; and the appearance of the samples after standing was recorded. The results are listed in Table 1.

[0138] Table 1

[0139] As shown in Table 1, the compositions provided in Examples 4, 8, and 9 exhibit good component compatibility and stability. Example 1 did not encapsulate or load the active substance using liposomes and gels, resulting in poor component compatibility and susceptibility to environmental temperature. Example 2 did not encapsulate the oil-soluble tetrahydrocurcumin with liposomes, leading to decreased component compatibility and turbidity. Example 3 did not load the active substance into a gel network, causing decreased liposome stability and easy aggregation and precipitation when the temperature increased. In Example 7, the liposomes were prepared at a pH of 6.5, which is close to the isoelectric point of the modified recombinant human collagen, making it unsuitable for positive charging and thus hindering self-assembly with negatively charged chondroitin sulfate via electrostatic interactions, resulting in decreased composition stability. The PEG-modified recombinant human collagen used in Comparative Example 1 was not modified with tetrahydromethylpyrimidine formyl groups, but only with low molecular weight PEG, resulting in decreased hydration capacity. When the environmental temperature increased, the increased activity of water molecules led to protein exposure and aggregation and precipitation. The compatibility and stability of the components in the composition have a significant impact on the activity and bioavailability of each component. The appearance of precipitation indicates liposome aggregation or protein inactivation, thus making it difficult to exert its efficacy.

[0140] Unless otherwise specified, all test samples were freshly prepared and used immediately, and no precipitates were observed.

[0141] Test Example 2: Liposome Encapsulation Efficiency Test The encapsulation efficiency of the liposome suspensions prepared in Examples 4, 6, 7, 8, and 9 was tested as follows: A volume V of liposome suspension was subjected to ultracentrifugation (50,000 rpm, 4°C, 1 h) and ultrafiltration. The content of modified recombinant humanized collagen in the filtrate was determined by the BCA method and recorded as the free protein content W. 游离 A volume V of liposome suspension was taken and 1% Triton X-100 solution was added. The suspension was incubated at 37°C for 30 min to lyse the liposomes. After lysis, the liposomes were subjected to ultracentrifugation (50,000 rpm, 4°C, 1 h) and ultrafiltration. The content of modified recombinant humanized collagen in the filtrate was determined by the BCA method and recorded as the total protein content W. 总 Encapsulation efficiency is calculated using the following formula: (1 - W) 游离 ÷W 总 () × 100%, the results are listed in Table 2.

[0142] Table 2

[0143] As shown in Table 2, changing the conditions of the liposome suspension preparation method affects the encapsulation efficiency of modified recombinant humanized collagen. In the compositions of Examples 4, 6, and 7, the percentage of active ingredients was the same, but Example 6 changed the type and ratio of phospholipids, leading to a decrease in encapsulation efficiency, while Example 7 increased the pH of the liquid phase, resulting in an increase in encapsulation efficiency. Encapsulation efficiency affects both the free and encapsulated amounts of modified recombinant humanized collagen. Higher encapsulation efficiency of liposomes results in higher transdermal penetration of modified recombinant humanized collagen, but lower gel viscosity leads to poorer component stability and film-forming moisturizing properties, and vice versa. Therefore, maintaining a suitable encapsulation efficiency is beneficial for achieving both effects.

[0144] Test Example 3: Transdermal Absorption Efficacy Test Transdermal permeability testing was conducted using a vertical diffusion cell, with fresh pig skin (with subcutaneous fat removed) as the model. The treated skin sample was fixed between the supply and receiving chambers, with the skin layer facing the supply chamber. The test solution was added to the supply chamber, and 1×PBS solution was added to the receiving chamber. After 24 hours, the receiving fluid was collected. 1% Triton X-100 solution was added to the receiving fluid, and the mixture was incubated at 37°C for 30 min to lyse the liposomes. The lysed liposomes were then subjected to ultracentrifugation (50,000 rpm, 4°C, 1 h) and ultrafiltration. The content of modified recombinant humanized collagen was determined using the BCA method. Based on the initial content before the experiment (the method for determining the initial content before the experiment was the same as that in the receiving fluid) and the content measured in the receiving fluid, the permeability of the recombinant collagen was calculated. The results are shown in Table 3.

[0145] Table 3

[0146] As shown in Table 3, encapsulation of modified recombinant humanized collagen with liposomes improved the transdermal permeability of the protein. In Example 5, the reaction pH of Mtt-Ectoin-PEG-CHO with recombinant humanized collagen was 7, which reduced the selectivity of Mtt-Ectoin-PEG-CHO for the N-terminal amino groups of the protein, resulting in more modified side chains. Consequently, the hydrated shell of the recombinant humanized collagen was thicker and more hydrophilic, leading to increased transdermal permeability. The encapsulation efficiency of the modified recombinant humanized collagen in Example 6 decreased, resulting in a decrease in transdermal permeability. The modified recombinant humanized collagen in Example 8 had a lower molecular weight, resulting in stronger transdermal permeability. The modified recombinant humanized collagen in Example 9 had a higher molecular weight and a lower encapsulation efficiency, resulting in poorer transdermal permeability. The PEG-modified recombinant humanized collagen in Comparative Example 1 lacked a tetrahydromethylpyrimidine formyl group, resulting in weaker hydration capacity, smaller molecular radius, and stronger lipophilicity, thus achieving a higher transdermal permeability.

[0147] Test Example 4: Stimulation Test (1) The test was conducted according to SNT2329-2009 "Chicken Embryo Villi Allantoic Membrane Test for Eye Irritation / Corrosion of Cosmetics". IS < 1 indicates no irritation; 1 ≤ IS < 5 indicates mild irritation; and 5 ≤ IS < 9 indicates moderate irritation.

[0148] (2) Patch test: The test was conducted according to the relevant methods of human skin patch test in the "Cosmetic Safety Technical Specifications" (2015 edition). Twenty volunteers aged 25-45 years who met the inclusion criteria were selected as subjects, including 10 male and 10 female subjects. The composition of each specific implementation method or 1.87wt% recombinant humanized collagen (SEQ ID NO:1) PBS solution (pH=7.4) was added to the patch device, and the amount added was precisely controlled at 0.020±0.005g; no substance was added to the patch device of the blank control group. Using non-irritating adhesive tape, the patch device containing the sample to be tested was attached to the flexor side of the forearm of the subject, and then the palm was gently pressed to ensure that the patch device was evenly attached to the skin. The test was performed once every 24 hours. After each patch was removed, the skin was left to stand for 30 minutes until the indentation on the skin surface completely disappeared, and then the skin reaction was observed and recorded. The evaluation criteria are as follows: Grade 0 is a negative reaction: no irritation, no erythema; Grade 1 is a suspicious reaction: mild erythema; Grade 2 is a weak positive reaction: erythema; Grade 3 is a strong positive reaction: erythema, papules, vesicles; Grade 4 is an extremely strong positive reaction: severe edema, bullae.

[0149] The test results are shown in Table 4.

[0150] Table 4

[0151] As shown in Table 4, the compositions provided in the embodiments of the present invention are non-irritating. Compared with the unmodified recombinant humanized collagen used in the embodiments and Comparative Examples 1-2, the modified recombinant humanized collagen or PEG-modified recombinant humanized collagen used in Comparative Example 3 showed a significant reduction in irritation, indicating that modification of recombinant humanized collagen can reduce the irritation of the composition. The compositions of each embodiment are suitable for whitening sensitive skin. In addition, Comparative Example 1 used PEG-modified recombinant humanized collagen instead of modified recombinant humanized collagen. Due to the lower chain length of PEG, Comparative Example 1 could not completely mask the antigen epitope, resulting in increased irritation.

[0152] Test Example 5: Cell Experiment Human keratinocytes (Guangdong Boxi Biotechnology Co., Ltd.) were incubated in DMEM medium (containing 10% FBS and 1% penicillin and streptomycin) at 37°C in a 5% CO2 incubator until the cells grew to 80%~90% of the culture flask. The cells were then digested, transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in culture medium to obtain a cell suspension.

[0153] Human keratinocytes at 8×10 3 Cells were seeded per well (180 μL) into 96-well plates for cytotoxicity assay. For the cytotoxicity assay, 20 μL of the specific embodiment composition was added to each well, with 5 replicates per sample. An equal volume of culture medium was added to the blank control group. After gentle pipetting, the plates were incubated in an incubator (37°C, 5% CO2) for 24 h, followed by medium replacement and sample addition, and incubation for another 24 h. The supernatant was discarded, and 50 µL of 1 mg / mL MTT solution was added to each well, with incubation for another 2 h. After incubation, the supernatant was discarded, and 100 µL of LDMSO was added to each well, shaken for 10 min to completely dissolve the blue-purple crystals. The OD value was read at 570 nm using a microplate reader, and cell viability was calculated. The results are listed in Table 5.

[0154] Human keratinocytes at 8×10 3 Cells were seeded per well into 6-well plates and incubated at 37°C in a 5% CO2 incubator until the cell layering rate reached 50%–60%. The medium was changed, and 2 mL of the test sample was added to each well (the specific compositions or culture media from each example served as blank controls). Three replicates were performed per group. The cells were then gently placed in the incubator and incubated for another 24 h. The culture medium was aspirated, and the cells were washed twice with PBS. 1 mL of RNAiso Plus was added, and the cells were lysed by pipetting before collecting the sample. RNA extraction, reverse transcription, and quantitative real-time PCR were performed according to the kit instructions to detect the expression levels of barrier and moisture-related genes (LOR, TGM1, IVL, OCLN1, DSG1, FLG, Caspase14, K1). A 23 -△△CT The expression levels (relative expression rates) of each specific embodiment relative to the blank control were calculated using the method, and the results are listed in Table 5. RNAiso Plus, the reverse transcription kit, and the fluorescent dye were all purchased from TaKaRa, Japan.

[0155] Table 5

[0156] The t-test was used. * indicates a statistically significant difference compared with the blank control group (p<0.05), and ** indicates a significant difference compared with the blank control group (p<0.01).

[0157] As shown in Table 5, the composition of Example 4 promotes the proliferation of human keratinocytes and significantly promotes the expression of skin barrier and moisturizing-related genes, indicating that the composition has repair and moisturizing effects. Specific sequences (or specific residues exposed after folding) on ​​recombinant humanized collagen bind to specific receptors on the cell surface, thereby activating key signaling pathways such as FAK, MAPK, and PI3K / Akt within the cell, thus promoting proliferation and the expression of key genes.

[0158] Comparing Examples 1-3, it is evident that the shorter the PEG chain segment, the worse the hydration, and the less likely it is to mask residual antigenic epitopes, thus exhibiting stronger cytotoxicity. Comparative Example 2 uses PEG-modified recombinant humanized collagen instead of modified recombinant humanized collagen. While the larger PEG chain segment provides sufficient hydration radius for protein stability and low irritation, the flexible PEG chain, in its extended state in an aqueous environment, covers a larger area of ​​the protein, resulting in lower differences in hydrophilicity and hydrophobicity at various points on the protein molecule. This makes it difficult for the protein to bind to cell-specific receptors (proteins bind to cell receptors primarily through hydrophobic effects), thus lacking cytotoxicity and having no effect on cell proliferation and pathway regulation. In contrast, the composition provided by this invention contains a shorter PEG chain segment in the modified recombinant humanized collagen. The tetrahydromethylpyrimidine formyl group provides a sufficient hydration layer for the protein while maintaining certain differences in hydrophilicity and hydrophobicity at different sites, thereby promoting the binding of recombinant humanized collagen to specific receptors on the cell surface, promoting proliferation and activating key pathways.

[0159] Test Example 6: Tyrosinase Inhibition Rate Test Add 80 μL of substrate solution (1 mg / mL L-DOPA solution) to each well of a 96-well plate, followed by an equal volume of the test sample. After incubation for 5 min, measure the absorbance at 475 nm (OA1). Then add 40 μL of tyrosinase solution (500 U / mL), incubate at 37°C for 15 min, and measure the absorbance at 475 nm (OA2) again. For the blank control group, replace the test sample with PBS solution. Equilibrate 8 wells per group. All solutions were prepared with 0.2 mol / L PBS buffer at pH 6.8. Tyrosinase inhibition rate = (1 - (A... T1 -A1) / (A T0 -A0))×100%; where A1 and A0 are the absorbance values ​​of the sample group and the blank group, respectively; A1 and A0 are the absorbance values ​​of the sample group and the blank group, respectively. T1 and A T0 The absorbance values ​​OA2 of the sample group and the blank group are shown in Table 6.

[0160] Table 6

[0161] As shown in Table 6, the composition provided by the present invention has a good tyrosinase inhibitory effect, indicating that it can reduce melanin production by inhibiting enzyme activity and achieve a whitening effect.

[0162] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hypoallergenic whitening recombinant humanized collagen protein composition, characterized in that, The hypoallergenic whitening recombinant humanized collagen composition comprises: modified recombinant humanized collagen, tetrahydrocurcumin, ethyl ascorbic acid ether, carnosine, and dipotassium glycyrrhizate; the modified recombinant humanized collagen comprises recombinant humanized collagen segments, polyethylene glycol segments modified on the recombinant humanized collagen segments, and tetrahydromethylpyrimidine formyl groups modified on the other end of the polyethylene glycol segments.

2. The hypoallergenic whitening recombinant humanized collagen protein composition of claim 1, wherein, One end of the polyethylene glycol segment is modified with an α-amino group at the N-terminus of the recombinant humanized collagen segment; And / or, the molecular weight of the polyethylene glycol segment is 400~2000 g / mol; And / or, the tetrahydromethylpyrimidineformyl group has the following structure: 。 3. The hypoallergenic whitening recombinant humanized collagen protein composition of claim 1, wherein, The method for preparing the modified recombinant humanized collagen is as follows: (1) Ectoin was esterified with OH-PEG-CHO to obtain Ectoin-PEG-CHO; (2) The Ectoin-PEG-CHO reacts with recombinant humanized collagen with Schiff base, and the modified recombinant humanized collagen is obtained after purification and drying.

4. The hypoallergenic whitening recombinant humanized collagen protein composition of claim 3, wherein, In step (1), the amino group of the ectoin is first protected to obtain P-Ectoin, which is then esterified with OH-PEG-CHO, and deamination protection is performed after the Schiff base reaction in step (2). And / or, the molecular weight of the OH-PEG-CHO is 500~2000 g / mol; And / or, the esterification reaction includes: dissolving Ectoin in N,N-dimethylformamide and activating it, adding OH-PEG-CHO and reacting at 20~30°C for 24~36 h; the activation includes adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine and reacting at 5°C until no obvious precipitate is formed, followed by filtration; And / or, the reaction system of the Schiff base reaction has a pH of 6-7, and NaBH3CN needs to be added, with a reaction time of 1-3 hours; And / or, the basic repeating unit of the recombinant humanized collagen is GPRGAPGERGRPGLPGAAGARGNDGARGSD, and the number of repetitions of the basic repeating unit is 10 to 18.

5. The hypoallergenic whitening recombinant humanized collagen protein composition of claim 4, wherein, In step (2), the mass ratio of recombinant humanized collagen, Ectoin-PEG-CHO, and NaBH3CN is 1g: 0.3~0.9g: 1~3g. And / or, the amino group protection refers to the protection of the amino group of ectoin with 4-methyltriphenylmethyl, and the preparation method is as follows: ectoin reacts with 4-methyltriphenylmethyl chloride under alkaline conditions to obtain P-Ectoin; the alkaline conditions refer to dissolving ectoin and 4-methyltriphenylmethyl chloride in dichloromethane and adding triethylamine; And / or, the deamination protection refers to adding trifluoroacetic acid to the reaction solution obtained from the Schiff base reaction and stirring the reaction for 0.5 to 1.5 hours; And / or, the amino acid sequence of the recombinant humanized collagen is shown in SEQ ID NO:

1.

6. The hypoallergenic whitening recombinant humanized collagen composition according to claim 1, characterized in that, The hypoallergenic whitening recombinant humanized collagen composition, calculated by weight percentage, comprises: 0.47%~3.7% modified recombinant humanized collagen, 0.05%~0.1% tetrahydrocurcumin, 1%~2% ethyl ascorbic acid ether, 0.8%~1.5% dipotassium glycyrrhizate, and 0.1%~0.3% carnosine, with the balance being solvent.

7. The hypoallergenic whitening recombinant humanized collagen composition according to claim 1, characterized in that, The hypoallergenic whitening recombinant humanized collagen composition, calculated by weight percentage, comprises: 0.47%~3.7% modified recombinant humanized collagen, 0.05%~0.1% tetrahydrocurcumin, 1%~2% ethyl ascorbic acid ether, 0.8%~1.5% dipotassium glycyrrhizate, 0.32%~1.1% chondroitin sulfate, and 0.1%~0.3% carnosine, with the balance being solvent.

8. The hypoallergenic whitening recombinant humanized collagen composition according to claim 1, characterized in that, The hypoallergenic whitening recombinant humanized collagen composition, calculated by weight percentage, comprises: (A): a gel containing modified recombinant humanized collagen, ethyl ascorbic acid ether, dipotassium glycyrrhizate and chondroitin sulfate; and (B): liposomes encapsulating modified recombinant humanized collagen, carnosine and tetrahydrocurcumin.

9. The hypoallergenic whitening recombinant humanized collagen composition according to claim 8, characterized in that, The hypoallergenic whitening recombinant humanized collagen composition, calculated by weight percentage, comprises: 0.47%~3.7% modified recombinant humanized collagen, 0.05%~0.1% tetrahydrocurcumin, 1%~2% ethyl ascorbic acid ether, 0.8%~1.5% dipotassium glycyrrhizate, 0.32%~1.1% chondroitin sulfate, 0.1%~0.3% carnosine, 0.5%~3% liposome matrix, and the balance being solvent.

10. The method for preparing the hypoallergenic whitening recombinant humanized collagen composition according to any one of claims 8 to 9, characterized in that, Includes the following steps: S1: Preparation of liposomes: S1.1 Oil phase preparation: Tetrahydrocurcumin, cholesterol, and phospholipids are dissolved in an organic solvent, heated to 50-70°C, and stirred until completely dissolved and transparent to form an oil phase; S1.2 Aqueous phase preparation: Modified recombinant humanized collagen and carnosine were dissolved in pH buffer solution and stirred evenly to form an aqueous phase; S1.3 Hydration and homogenization: The oil phase is added to the aqueous phase for preliminary emulsification; subsequently, the resulting primary emulsion is homogenized under high pressure and concentrated to obtain a liposome suspension; S2: Ethyl ascorbic acid ether and dipotassium glycyrrhizate are dissolved in the liposome suspension. After stirring and dissolving, chondroitin sulfate solution is added dropwise while stirring. After the addition is complete, homogenization is performed, and finally the pH is adjusted to obtain a hypoallergenic whitening recombinant humanized collagen composition.

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