Oil-soluble recombinant collagen composition with cell-grade anti-aging effect as well as preparation method and application of oil-soluble recombinant collagen composition

Abstract

The invention provides an oil-soluble recombinant collagen composition with a cell-grade anti-aging effect as well as a preparation method and application thereof, and particularly relates to the technical field of cosmetic intermediates. The exterior of the cell-grade oil-soluble recombinant collagen composition with the anti-aging effect is coated with a phospholipid membrane, and a recombinant collagen-supramolecular complex is embedded into the phospholipid membrane; the recombinant collagen-supramolecular complex is obtained by coating a supramolecular solution of recombinant collagen with first phospholipid and then freeze-drying. Hydrophilic groups are shielded through the recombinant collagen-supramolecular complex, so that the dispersity and the stability in an oil phase are enhanced; after the complex is coated with a phospholipid membrane, composite particles with a liposome-like structure are constructed, complete dissolution and long-term stability in an oil phase are achieved, layering or precipitation is avoided, and by means of the synergistic permeation enhancing effect of phospholipid and supramolecular components, cuticle lipid arrangement is interfered, skin permeability is improved, and the skin infiltration capacity of recombinant collagen is enhanced; and high bioavailability is realized.

Description

Technical Field

[0001] This invention relates to the field of cosmetic intermediates technology, and in particular to an oil-soluble recombinant collagen composition with cell-level anti-aging effects, its preparation method, and its application. Background Technology

[0002] Recombinant humanized collagen has become a core active ingredient in high-end skincare products due to its excellent biocompatibility and anti-aging and skin repair effects. However, its large molecular weight and rich hydrophilic groups result in significant water solubility, making it difficult to dissolve in pure oil systems. It is prone to aggregation, precipitation, and inactivation, severely limiting its application in "oil-based skincare" products such as facial oils, ointments, and makeup.

[0003] In existing technologies, while liposome encapsulation can enhance the transdermal penetration of collagen, the resulting system remains water-dispersible and cannot be directly compatible with pure oil systems. While water-in-oil emulsion technology can introduce recombinant collagen into the oil phase, it requires precise matching of the emulsion system to the oil polarity, resulting in poor compatibility, complex processes, and a lack of verification regarding the actual penetration effect of recombinant collagen. Currently, there is no oil-soluble recombinant collagen solution that is both widely compatible with various cosmetic oils of different polarities and possesses highly efficient transdermal penetration.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide an oil-soluble recombinant collagen composition with cellular-level anti-aging effects, its preparation method, and its application, aiming to solve at least one of the above-mentioned technical problems in the prior art.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of the present invention provides an oil-soluble recombinant collagen composition with cellular-level anti-aging effects, which is externally coated with a phospholipid membrane and has a recombinant collagen-supramolecular complex embedded inside the phospholipid membrane; wherein the recombinant collagen-supramolecular complex is obtained by freeze-drying a first phospholipid coated with a supramolecular solution of recombinant collagen; the supramolecular solvent is composed of a hydrogen donor and a hydrogen acceptor; and the phospholipid membrane is formed by a second phospholipid.

[0007] Furthermore, the oil-soluble recombinant collagen composition with cell-level anti-aging effects comprises, by weight, 5-30 parts of second phospholipid and 15.01-86 parts of recombinant collagen-supramolecular complex.

[0008] In the recombinant collagen-supramolecular complex, the recombinant collagen is 0.01 to 1 part by weight, the supramolecular solvent is 10 to 60 parts by weight, and the first phospholipid is 5 to 25 parts by weight.

[0009] Preferably, in the recombinant collagen-supramolecular complex, the weight ratio of recombinant collagen to supramolecular solvent is 1:100~200.

[0010] Preferably, the weight ratio of the recombinant collagen to phospholipids is 1:80~148.

[0011] Furthermore, the recombinant collagen includes at least one of type I recombinant collagen, type III recombinant collagen, type XVII recombinant collagen, type I small molecule recombinant collagen, type III small molecule recombinant collagen, and type XVII small molecule recombinant collagen.

[0012] Preferably, the recombinant collagen is a mixture of type III small molecule recombinant collagen and type I recombinant collagen in a mass ratio of 1:8.5.

[0013] Preferably, the supramolecular solvent is composed of a hydrogen donor and a hydrogen acceptor in a molar ratio of (0.5~2):1.

[0014] Preferably, the hydrogen donor is a fatty acid.

[0015] Preferably, the fatty acid includes isostearic acid and / or decanoic acid.

[0016] Preferably, the hydrogen acceptor is an alkaloid.

[0017] Preferably, the alkaloid includes at least one of matrine, oxymatrine, and L-carnitine.

[0018] Further, the phospholipids include at least one of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, phosphatidylcholine, phosphatidylethanolamine, dipalmitoylphosphatidylcholine (DPPC), and distearate phosphatidylcholine (DSPC).

[0019] Preferably, the soybean lecithin contains ≥90% phosphatidylcholine.

[0020] Preferably, the soybean lecithin contains ≥95% phosphatidylcholine.

[0021] Furthermore, the oil-soluble recombinant collagen composition with cell-level anti-aging effects also includes a stabilizer in parts by weight of 20 to 70.

[0022] Preferably, the stabilizer comprises at least one of caprylic / capric triglyceride, isononyl isononanoate, triglyceride (ethylhexanoate), propylene glycol carbonate, and diethyl sebacate.

[0023] Preferably, the oil-soluble recombinant collagen composition with cell-level anti-aging effects further includes 2 to 10 parts by weight of an adjuvant.

[0024] Preferably, the additive includes at least one selected from butanediol, propylene glycol, hexanediol, and pentanediol.

[0025] The second aspect of this invention provides a method for preparing the oil-soluble recombinant collagen composition with cellular-level anti-aging effects, comprising: dissolving recombinant collagen in a supramolecular solvent to obtain a supramolecular solution of recombinant collagen; adding a first phospholipid to the supramolecular solution of recombinant collagen and mixing evenly; then freeze-drying to obtain the recombinant collagen-supramolecular complex; mixing a second phospholipid, optional adjuvants, and optional stabilizers to obtain a phospholipid solution; and finally adding the recombinant collagen-supramolecular complex to the phospholipid solution and mixing evenly to obtain the oil-soluble recombinant collagen composition with cellular-level anti-aging effects.

[0026] Furthermore, the supramolecular solvent is prepared by grinding the hydrogen donor and hydrogen acceptor and dispersing them in an alcohol-water solution, and then reacting them under nitrogen protection to obtain the supramolecular solvent.

[0027] Preferably, the concentration of alcohol in the aqueous alcohol solution is 50-70 wt%.

[0028] Preferably, the alcohol in the aqueous alcohol solution includes at least one of methanol, ethanol, propylene glycol, and butanediol.

[0029] Preferably, the reaction temperature is 20~45℃ and the time is 20~36h.

[0030] A third aspect of the present invention provides the application of the oil-soluble recombinant collagen composition with cellular-level anti-aging effects in the preparation of cosmetics.

[0031] Furthermore, the amount of oil-soluble recombinant collagen composition with cell-level anti-aging effects added to cosmetics is 0.01~10wt%.

[0032] Preferably, the oil-soluble recombinant collagen composition with cell-level anti-aging effects is added during the cold preparation stage.

[0033] Preferably, the temperature during the cold preparation stage is below 50°C.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides an oil-soluble recombinant collagen composition with cellular-level anti-aging effects. By interacting recombinant collagen with a specific hydrogen donor-acceptor supramolecular system and then freeze-drying it, a stable recombinant collagen-supramolecular complex is formed, effectively shielding its hydrophilic groups and significantly enhancing its dispersibility and stability in an oil-phase environment. After being coated with a phospholipid membrane, this complex further constructs liposome-like composite microparticles, achieving complete solubility and long-term stability in various commonly used cosmetic oil phases, such as polyols, polar oils, and non-polar oils, avoiding stratification or precipitation. Simultaneously, through the synergistic penetration-enhancing effect of phospholipids and supramolecular components, it interferes with the lipid arrangement of the stratum corneum, improving skin permeability, thereby significantly enhancing the skin penetration capacity of recombinant collagen and achieving high bioavailability. This structural design balances oil solubility, universal compatibility, and efficient delivery performance, solving the technical challenges of recombinant collagen's poor solubility, instability, and poor permeability in pure oil systems.

[0035] The preparation method provided by this invention first dissolves recombinant collagen in a supramolecular solvent to effectively shield the hydrophilic groups of the recombinant collagen and stabilize its molecular state. Then, the recombinant collagen-supramolecular complex interacts fully with phospholipids to form a solid composite structure with oil-phase affinity. Next, it is dispersed in a phospholipid solution, ultimately forming an oil-soluble microstructure in which the recombinant collagen-supramolecular complex is completely encapsulated by a phospholipid membrane. This preparation method features reasonable steps and mild conditions, avoiding the destruction of protein activity caused by high temperatures or strong shear. It also ensures high transparency, long-term stability, and excellent permeability of the composition in various oil systems, achieving efficient loading and functional delivery of recombinant collagen in pure oil formulations, and possesses promising prospects for industrial application.

[0036] The application provided by this invention can be stably and transparently applied to various cosmetic systems, especially suitable for pure oil or high oil phase formulations such as essential oils, facial oils, ointments and makeup. It can achieve good compatibility without emulsification adjustment of existing oil systems, significantly improving the convenience of recombinant collagen application in actual products. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 The results of the 24-hour stability test for Example 1 are shown. Figure 2 The results are the 30-day stability test results for Example 1; Figure 3 The results are the 90-day stability test results for Example 1; Figure 4 The results of fluorescence permeation quantitative analysis in Example 1; Figure 5 The results of fluorescence permeation quantitative analysis are for Comparative Example 1; Figure 6 This is a comparison of the in vitro permeation amounts of Example 1 and Comparative Example 1; Figure 7 A bar chart showing the COL1 MFI values ​​in fibroblasts; Figure 8 A summary diagram of COL1 MFI content in fibroblasts (magnification 200). Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0040] In the following, the terms “comprising,” “having,” and their cognates, which may be used in various embodiments of the invention, are intended only to indicate a particular feature, number, step, operation, element, component, or combination thereof, and should not be construed as excluding, firstly, the presence of one or more other features, numbers, steps, operations, elements, components, or combinations thereof, or adding the possibility of one or more features, numbers, steps, operations, elements, components, or combinations thereof.

[0041] The first aspect of the present invention provides an oil-soluble recombinant collagen composition with cellular-level anti-aging effects, which is externally coated with a phospholipid membrane and has a recombinant collagen-supramolecular complex embedded inside the phospholipid membrane; wherein the recombinant collagen-supramolecular complex is obtained by freeze-drying a first phospholipid coated with a supramolecular solution of recombinant collagen; the supramolecular solvent is composed of a hydrogen donor and a hydrogen acceptor; and the phospholipid membrane is formed by a second phospholipid.

[0042] This invention provides an oil-soluble recombinant collagen composition with cellular-level anti-aging effects. By interacting recombinant collagen with a specific hydrogen donor-acceptor supramolecular system and then freeze-drying it, a stable recombinant collagen-supramolecular complex is formed, effectively shielding its hydrophilic groups and significantly enhancing its dispersibility and stability in an oil-phase environment. After being coated with a phospholipid membrane, this complex further constructs liposome-like composite microparticles, achieving complete solubility and long-term stability in various commonly used cosmetic oil phases, such as polyols, polar oils, and non-polar oils, avoiding stratification or precipitation. Simultaneously, through the synergistic penetration-enhancing effect of phospholipids and supramolecular components, it interferes with the lipid arrangement of the stratum corneum, improving skin permeability, thereby significantly enhancing the skin penetration capacity of recombinant collagen and achieving high bioavailability. This structural design balances oil solubility, universal compatibility, and efficient delivery performance, solving the technical challenges of recombinant collagen's poor solubility, instability, and poor permeability in pure oil systems.

[0043] Cellular-level anti-aging efficacy refers to methods that differ from physical covering and moisturizing that only act on the skin surface. It targets skin cells (such as dermal fibroblasts and epidermal keratinocytes) as the core target and achieves the effect of delaying the aging process and reversing the decline of cell function at the cellular level by regulating cellular physiological metabolism, repairing aging-related structural damage to cells, or activating cellular anti-aging signaling pathways. This results in improving signs of aging such as wrinkles, sagging, and dullness.

[0044] Furthermore, the oil-soluble recombinant collagen composition with cell-level anti-aging effects contains, by weight, 5-30 parts of second phospholipid and 15.01-86 parts of recombinant collagen-supramolecular complex. If the phospholipid content is too low, the encapsulation system will be unstable, and collagen will leak out during storage, thus precipitating out of the formula and making the formula opaque; while if the phospholipid content is too high, there is a risk of discoloration.

[0045] In the recombinant collagen-supramolecular complex, the recombinant collagen comprises 0.01 to 1 part by weight, the supramolecular solvent comprises 10 to 60 parts by weight, and the first phospholipid comprises 5 to 25 parts by weight. If the supramolecular solvent content is too low, the collagen will not dissolve completely, thus preventing subsequent phospholipid encapsulation processes. Furthermore, a low supramolecular solvent content will result in insufficient collagen permeability. Conversely, a high supramolecular solvent content will cause instability phenomena such as system delamination and discoloration.

[0046] Preferably, in the recombinant collagen-supramolecular complex, the weight ratio of recombinant collagen to supramolecular solvent is 1:(100~200).

[0047] Typically, but not limitingly, in the oil-soluble recombinant collagen composition with cellular-level anti-aging effects, the weight percentage of the second phospholipid can be, for example, 5 parts, 8 parts, 10 parts, 20 parts, or 30 parts, or any value within the range of 5 to 30 parts; the weight percentage of the recombinant collagen-supramolecular complex can be, for example, 15.01 parts, 15 parts, 20 parts, 30 parts, 40 parts, 50 parts, 60 parts, or 86 parts, or any value within the range of 15.01 to 86 parts.

[0048] In the recombinant collagen-supramolecular complex, the weight fraction of recombinant collagen can be, for example, 0.01 parts, 0.1 parts, 0.2 parts, 0.4 parts, 0.6 parts, 0.8 parts, or 1 part, or any value within the range of 0.01 to 1 part; the weight fraction of supramolecular solvent can be, for example, 10 parts, 20 parts, 30 parts, 40 parts, 50 parts, or 60 parts, or any value within the range of 10 to 60 parts.

[0049] Furthermore, the recombinant collagen includes at least one of type I recombinant collagen, type III recombinant collagen, type XVII recombinant collagen, type I small molecule recombinant collagen, type III small molecule recombinant collagen, and type XVII small molecule recombinant collagen.

[0050] Preferably, the recombinant collagen is a mixture of type III small molecule recombinant collagen and type I recombinant collagen in a mass ratio of 1:8.5. This ratio is based on the natural proportions found in healthy adult skin, which is closer to the collagen ratios in healthy skin. It has higher compatibility and affinity with the skin, lower immunogenicity, and higher bioavailability.

[0051] Preferably, the supramolecular solvent is composed of hydrogen donors and hydrogen acceptors in a molar ratio of (0.5~2):1. This not only dissolves recombinant collagen but also, through freeze-drying, further increases the interaction between the numerous hydrogen bonds in the supramolecular structure and collagen, making it compatible with phospholipids and thus with a wider range of oils, enabling its application in pure oil systems. Furthermore, the supramolecular solvent can reduce the resistance caused by skin interactions during the penetration process of recombinant collagen, increasing its permeability. Simultaneously, as a penetration enhancer, when applied to the skin, the supramolecular solvent can affect skin permeability by interfering with the lipid distribution of the stratum corneum, thereby improving the permeability of recombinant collagen.

[0052] Typical, but not limiting, ratios of hydrogen donors to hydrogen acceptors can be 0.5:1, 0.8:1, 1:1, 1.2:1, 1.5:1, 1.8:1, or 2:1, or any value in the range of 0.5 to 2.

[0053] Preferably, the hydrogen donor is a fatty acid.

[0054] Preferably, the fatty acid includes isostearic acid and / or decanoic acid.

[0055] Preferably, the hydrogen acceptor is an alkaloid.

[0056] Preferably, the alkaloid includes at least one of matrine, oxymatrine, and L-carnitine.

[0057] Further, the phospholipids include at least one of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, phosphatidylcholine, phosphatidylethanolamine, dipalmitoylphosphatidylcholine (DPPC), and distearate phosphatidylcholine (DSPC).

[0058] Preferably, the soybean lecithin contains ≥90% phosphatidylcholine; More preferably, the soybean lecithin has a phosphatidylcholine content of ≥95%; high-purity phospholipids have higher interfacial stability and higher penetration-enhancing ability.

[0059] Preferably, the weight ratio of the recombinant collagen to phospholipids is 1:(80~148).

[0060] It should be noted that, unless otherwise specified, when “the mass of phospholipids” is mentioned alone in this article, it refers to the total amount of the first phospholipid and the second phospholipid, and when “phospholipids” is mentioned alone, it refers to the first phospholipid and / or the second phospholipid.

[0061] Furthermore, the oil-soluble recombinant collagen composition with cell-level anti-aging effects also includes a stabilizer in parts by weight of 20 to 70.

[0062] Typically, but not limitingly, the stabilizer may be present in parts by weight of, for example, 20, 30, 40, 50, 60 or 70 parts, or any value in the range of 20 to 70 parts.

[0063] Preferably, the stabilizer includes at least one of caprylic / capric triglyceride, isononyl isononanoate, triglyceride (ethylhexanoate), propylene glycol carbonate, and diethyl sebacate. The stabilizer has high stability with commonly used oils and phospholipids in cosmetics, which is beneficial to improving the stability and compatibility of oil-soluble collagen in different oil systems.

[0064] Preferably, the oil-soluble recombinant collagen composition with cell-level anti-aging effects further includes 2 to 10 parts by weight of an adjuvant.

[0065] Typically, but not limitingly, the number of parts by weight of the adjuvant can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts, or any value in the range of 2 to 10 parts.

[0066] Preferably, the additive includes at least one of butanediol, propylene glycol, hexanediol, and pentanediol, and the additive has good compatibility with phospholipids, which helps maintain the stability of the phospholipid film.

[0067] The second aspect of this invention provides a method for preparing the oil-soluble recombinant collagen composition with cellular-level anti-aging effects, comprising: dissolving recombinant collagen in a supramolecular solvent to obtain a supramolecular solution of recombinant collagen; adding a first phospholipid to the supramolecular solution of recombinant collagen and mixing evenly; then freeze-drying to obtain the recombinant collagen-supramolecular complex; mixing a second phospholipid, optional adjuvants, and optional stabilizers to obtain a phospholipid solution; and finally adding the recombinant collagen-supramolecular complex to the phospholipid solution and mixing evenly to obtain the oil-soluble recombinant collagen composition with cellular-level anti-aging effects.

[0068] The preparation method provided by this invention first dissolves recombinant collagen in a supramolecular solvent to effectively shield the hydrophilic groups of the recombinant collagen and stabilize its molecular state. Then, the recombinant collagen-supramolecular complex interacts fully with phospholipids to form a solid composite structure with oil-phase affinity. Next, it is dispersed in a phospholipid solution, ultimately forming an oil-soluble microstructure in which the recombinant collagen-supramolecular complex is completely encapsulated by a phospholipid membrane. This preparation method features reasonable steps and mild conditions, avoiding the destruction of protein activity caused by high temperatures or strong shear. It also ensures high transparency, long-term stability, and excellent permeability of the composition in various oil systems, achieving efficient loading and functional delivery of recombinant collagen in pure oil formulations, and possesses promising prospects for industrial application.

[0069] Furthermore, the supramolecular solvent is prepared by grinding the hydrogen donor and hydrogen acceptor and dispersing them in an alcohol-water solution, and then reacting them under nitrogen protection to obtain the supramolecular solvent.

[0070] Preferably, the concentration of alcohol in the aqueous alcohol solution is 50-70 wt%.

[0071] Preferably, the alcohol in the aqueous alcohol solution includes at least one of methanol, ethanol, propylene glycol, and butanediol.

[0072] Preferably, the reaction temperature is 20~45℃ and the time is 20~36h.

[0073] Typically, but not limitingly, the reaction temperature can be, for example, 20°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C, or any value within the range of 20°C to 45°C; the reaction time can be, for example, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, or 36h, or any value within the range of 20h to 36h.

[0074] A third aspect of the present invention provides the application of the oil-soluble recombinant collagen composition with cellular-level anti-aging effects in the preparation of cosmetics.

[0075] The application provided by this invention can be stably and transparently applied to various cosmetic systems, especially suitable for pure oil or high oil phase formulations such as essential oils, facial oils, ointments and makeup. It can achieve good compatibility without emulsification adjustment of existing oil systems, significantly improving the convenience of recombinant collagen application in actual products.

[0076] Furthermore, the amount of oil-soluble recombinant collagen composition with cell-level anti-aging effects added to cosmetics is 0.01~10wt%.

[0077] Typical, but not limiting, amounts of oil-soluble recombinant collagen compositions with cellular-level anti-aging effects added to cosmetics may be, for example, 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 4 wt%, 6 wt%, 8 wt%, or 10 wt%, or any value in the range of 0.01 to 10 wt%.

[0078] Preferably, the oil-soluble recombinant collagen composition with cell-level anti-aging effects is added during the cold preparation stage.

[0079] Preferably, the temperature during the cold preparation stage is below 50°C.

[0080] The present invention is further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for illustrative purposes and should not be construed as limiting the invention in any way. Unless otherwise specified, the raw materials used in the embodiments and comparative examples of the present invention were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0081] Example 1 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The specific preparation steps are as follows: 1. Weigh 13.66 parts of matrine and 14.65 parts of isostearic acid, then grind them for 30 minutes at room temperature using a vibratory mill. The ground sample is then dispersed in 50 parts of an aqueous ethanol solution (70 wt%) to obtain a uniform liquid. The mixture is then stirred continuously at 40°C under nitrogen purging for 24 hours to obtain a viscous liquid, thus obtaining a supramolecular solvent.

[0082] 2. Mix supramolecular solvent with 0.2375 parts of recombinant collagen (including 0.2125 parts of type I recombinant collagen and 0.0250 parts of type III recombinant small molecule protein), and stir continuously at room temperature for 4 hours to ensure that the recombinant collagen is completely dissolved. Then add 10 parts of soybean lecithin and stir thoroughly to dissolve it completely. Then freeze-dry it using a vacuum freeze dryer to obtain recombinant collagen-supramolecular complex.

[0083] 3. Mix and dissolve 15 parts soybean lecithin, 2 parts hexanediol and 44.4525 parts caprylic / capric triglyceride (GTCC) at 40°C to obtain a transparent liquid, which is a phospholipid solution.

[0084] 4. Under rapid stirring conditions, the recombinant collagen-supramolecular complex is slowly added to the phospholipid solution, and stirred continuously at room temperature for 1 hour to obtain an oil-soluble recombinant collagen composition with cell-level anti-aging effects.

[0085] Example 2 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that matrine is 18.5 parts and isostearic acid is 9.81 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0086] Example 3 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that matrine is 17.71 parts and isostearic acid is 10.6 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0087] Example 4 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that matrine is 8.62 parts and isostearic acid is 19.69 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0088] Example 5 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that matrine is 7.72 parts and isostearic acid is 20.59 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0089] Example 6 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the amount of matrine is 5.46 parts and the amount of isostearic acid is 5.86 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0090] Example 7 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the amount of matrine is 6.83 parts and the amount of isostearic acid is 7.32 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0091] Example 8 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the amount of matrine is 21.86 parts and the amount of isostearic acid is 23.44 parts. The remaining raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0092] Example 9 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the amount of matrine is 22.36 parts and the amount of isostearic acid is 25.60 parts. The remaining raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0093] Example 10 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the lecithin in step 3 is 8 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0094] Example 11 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the lecithin in step 3 is 9.5 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0095] Example 12 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the lecithin in step 3 is 24.9 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0096] Example 13 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the lecithin in step 3 is 25.6 parts. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0097] Example 14 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that matrine is replaced with L-carnitine. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0098] Example 15 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that isostearic acid is replaced with decanoic acid. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0099] Example 16 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that the soybean lecithin is replaced with egg yolk lecithin. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0100] Example 17 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that hexanediol is replaced with pentanediol. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0101] Example 18 This embodiment provides an oil-soluble recombinant collagen composition with cell-level anti-aging effects. The difference from Example 1 is that caprylic / capric triglyceride is replaced with isononyl isononanoate. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0102] Comparative Example 1 This comparative example provides a water-soluble recombinant collagen solution, the components of which are: 0.2125 parts of type I recombinant collagen, 0.0250 parts of type III small molecule recombinant collagen, 2 parts of hexanediol, and 97.7625 parts of deionized water. The above components are mixed evenly to obtain the water-soluble recombinant collagen solution.

[0103] Comparative Example 2 This comparative example provides a recombinant collagen composition. Compared with Example 1, matrine is replaced with arginine. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0104] Comparative Example 3 This comparative example provides a recombinant collagen composition. Compared with Example 1, isostearic acid is replaced with levulinic acid. The other raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0105] Comparative Example 4 This comparative example provides a recombinant collagen composition. Compared with Example 1, isostearic acid and matrine are removed in step 1. The remaining raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0106] Comparative Example 5 This comparative example provides a recombinant collagen composition. Compared with Example 1, the phospholipids in step 3 are removed. The remaining raw materials and preparation methods are the same as in Example 1, and will not be repeated here.

[0107] Comparative Example 6 This comparative example provides a water-in-oil recombinant collagen solution, the components of which are: 0.2125 parts of type I recombinant collagen, 0.0250 parts of type III small molecule recombinant collagen, 7 parts of deionized water, 4 parts of hexanediol, 20 parts of Span 80, 2 parts of soybean lecithin, and 66.7625 parts of caprylic / capric triglyceride. Type I recombinant collagen and type III small molecule recombinant collagen are dissolved and then slowly added to the oil phase system containing hexanediol, Span 80, and caprylic / capric triglyceride. The mixture is stirred continuously for 60 minutes to obtain a water-in-oil recombinant collagen solution.

[0108] Test case The products obtained from the examples and comparative examples were subjected to performance tests, as detailed below: 1. Appearance method: Take the product into a 25mL sample bottle and visually observe the appearance of the product at room temperature and in the absence of direct sunlight. See Table 1 for specific data.

[0109] Among them, "transparent" means that the solution is uniform from top to bottom, and the object behind it can be directly observed through the sample bottle; "layered" means that the solution is not uniform from top to bottom, and the boundary between the two different solutions can be clearly observed; "turbid" means that the solution is not uniform and may contain suspended matter, and the object behind it cannot be directly observed through the sample bottle; "precipitate" means that the upper layer of the solution is transparent and the bottom layer shows obvious solids.

[0110] 2. Stability Test Method: The product was placed in a sample bottle and placed under four conditions: room temperature, -20℃, 45℃, and 4℃, for 72 hours. The appearance changes of the product were visually observed at room temperature and under non-direct sunlight. Specific data are shown in Table 1.

[0111] Among them, stability means that the color, odor, state, transparency and apparent viscosity of the sample did not change visibly after being placed under various conditions and then restored to room temperature, while precipitation means that the appearance becomes cloudy or obvious precipitation occurs.

[0112] Table 1. Appearance and stability test results of each embodiment and comparative example.

[0113] 3. Oil Compatibility Test: The oil-soluble recombinant collagen composition with cell-level anti-aging effects prepared in Example 1 was dispersed in different types of commonly used cosmetic oils at an addition ratio of 5%. The commonly used cosmetic oils used in this experiment were GTCC, squalane, hexanediol, propylene glycol carbonate, dioctyl ether, camellia oil, diisopropyl sebate, jojoba seed oil, rice bran oil, perilla seed oil, and octyl dodecanol, a total of 11 types, covering polyols, polar oils, and non-polar oils. The clarity and transparency of the solution and the accelerated stability after 72 hours of high temperature (45℃) and low temperature (4℃) were observed. As a comparison, the control group used the same amount of the same recombinant collagen raw material and the water-in-oil recombinant collagen from Comparative Example 6 were directly added to these oils to compare compatibility and stability. Specific data are shown in Table 2.

[0114] Table 2 Comparison of compatibility between recombinant collagen raw materials and Examples 1 and 6

[0115] 4. Formulation stability test: The oil-soluble recombinant collagen composition with cell-level anti-aging effects prepared in Example 1 was added to camellia seed oil at a ratio of 1%, and then placed under four conditions of room temperature, -20℃, 45℃ and 4℃ for 90 days. It was also placed under the conditions of room temperature, -20℃, 45℃ and 4℃, light exposure and cold and heat cycling for 24 hours and 30 days. After the test, the appearance of the product was visually observed at room temperature and under non-direct sunlight.

[0116] Figure 1 The results of the 24-hour stability test for Example 1 are as follows: Figure 1 It can be seen that, under the above test conditions, the oil-soluble recombinant collagen composition with cell-level anti-aging effects all maintained a pale yellow transparent liquid state.

[0117] Figure 2 The results of the 30-day stability test for Example 1 are as follows: Figure 2It can be seen that, under the above test conditions, the oil-soluble recombinant collagen composition with cell-level anti-aging effects all maintained a pale yellow transparent liquid state.

[0118] Figure 3 The results of the 90-day stability test for Example 1 are as follows: Figure 3 It can be seen that, under the above test conditions, the oil-soluble recombinant collagen composition with cell-level anti-aging effects all maintained a pale yellow transparent liquid state.

[0119] Combining Table 1 and Table 2, Figure 1 , Figure 2 and Figure 3 It can be seen that the oil-soluble recombinant collagen composition with cell-level anti-aging effects prepared using the method of Example 1 has good stability, formulation suitability, and high bioavailability. Compared with Comparative Examples 2-5, it is evident that a stable system cannot be obtained without the addition of supramolecular solvents or phospholipids; compared with Comparative Example 6, the oil-soluble recombinant collagen composition with cell-level anti-aging effects obtained in Example 1 has better compatibility with various oils and can be widely used in various pure oil systems.

[0120] Examples 2-5 demonstrate the effect of different hydrogen donor and hydrogen acceptor ratios on product stability. Within the preferred range, the product remains transparent and stable under all conditions. Too low a hydrogen donor ratio may cause turbidity, while too high a hydrogen acceptor ratio may cause precipitation in solution. Exceeding the preferred range will reduce product stability. Examples 6-9 demonstrate the effect of different supramolecular solvents relative to the amount of recombinant collagen on product stability. Too low a supramolecular solvent content may cause recombinant collagen to expose hydrophilic groups and precipitate in solution, resulting in system instability. Too high a supramolecular solvent content may pose a risk of layering and discoloration. Examples 10-13 demonstrate the effect of different phospholipid ratios on product stability. Too low a phospholipid content may result in incomplete encapsulation, potentially leading to leakage of recombinant collagen and precipitation during testing. Too high a phospholipid content may increase costs and pose a potential risk of discoloration.

[0121] Figure 1 , Figure 2 and Figure 3 This demonstrates that the oil-soluble recombinant collagen composition with cell-level anti-aging effects prepared in Example 1 can be conveniently applied to transparent essential oils and passes conventional cosmetic stability tests.

[0122] 5. Permeability Test: Bama pig skin was fixed between the diffusion chamber and the receiving chamber of a Franz cell diffusion cell, with the stratum corneum facing the diffusion chamber and the dermis facing the receiving chamber. After fixing the Bama pig skin, 8 mL of receiving solution (7.4% PBS) was added to the receiving chamber to ensure close contact between the Bama pig skin and the receiving solution. After the diffusion instrument water bath temperature stabilized, 0.1 g of samples containing the samples from Example 1 and Comparative Example 1 (Type I recombinant collagen was labeled with FITC, and Type III recombinant collagen was labeled with CY5) was added to the surface of the Bama pig skin and spread evenly. Sample Collection from the Receiving Chamber: After the permeability experiment, residual samples on the skin surface were cleaned. Pig skin from each group was placed in a -80℃ freezer. After tissue fixation, dehydration, sectioning, slide preparation, DAPI staining, mounting, and microscopic examination, images were observed and acquired under a fluorescence scanner. The fluorescence signal intensity was analyzed using ImageJ software to quantitatively analyze the permeability of recombinant collagen.

[0123] Figure 4 The results of fluorescence permeation quantitative analysis in Example 1 are as follows. Figure 5 The results of fluorescence permeation quantitative analysis for Comparative Example 1 are shown in the figure. Blue represents DAPI, green represents FITC-labeled type I recombinant collagen, and red represents CY5-labeled type III small recombinant collagen. Figure 6 This is a comparison of the in vitro permeation amounts of Example 1 and Comparative Example 1.

[0124] Figure 4 , Figure 5 and Figure 6 This indicates that the permeability of Example 1 is about 8 times higher than that of Comparative Example 1, which can significantly improve the bioavailability of recombinant collagen.

[0125] 6. Efficacy Experiment: To verify the promoting effect of this invention on the synthesis and secretion of type I collagen (COL1) in human dermal fibroblasts, the localization of COL1 protein in cells was observed and the changes in expression level were quantitatively analyzed by specifically labeling COL1 protein. Example 1 and Comparative Example 6 of this invention were prepared with a collagen concentration of 6 μg / mL as experimental groups; simultaneously, blank control group, positive control group, and negative control group were set up as control groups. The specific settings of each control group are as follows: the blank control group was a control group with only sterile cell culture medium and no UVA treatment; the positive control group was a control group with 7 μg / mL VE solution; the negative control group was a blank control group with only sterile cell culture medium and UVA treatment.

[0126] Each experimental group and control group was incubated for 24 hours in human skin fibroblast culture dishes specifically labeled with COL1. The blank control group received no UVA treatment, while the experimental groups, positive control group, and negative control group received UVA at 30 J / cm². 2Cells were processed and cultured for another 24 hours. The cultured cells were then stained with fluorescence. The fluorescence signal was quantified using weighted brightness calculated by ImageJ software. Images were processed using ImageJ (version 1.54p, National Institutes of Health, USA). The cumulative optical density value IntDen (integrated optical density, IOD) was analyzed, and the mean fluorescence intensity (MFI) and UV damage repair enhancement rate were calculated.

[0127]

[0128]

[0129] The experimental results are shown in Table 3. Figure 7 and Figure 8 (Compared with the UVA group, the positive group, Example 1, and Comparative Example 6 showed significant differences.) This means that a p-value < 0.05 indicates that... A p-value < 0.01 indicates that... The significance of the difference between the blank group and the model group is expressed as &, with P < 0.05 as & and P < 0.01 as &&. The MFI values ​​of the positive control group, Example 1 group, and Comparative Example 6 group were all significantly higher than those of the UVA damage model group (P < 0.01), and all three could effectively reverse the UVA-induced inhibition of COL1 synthesis. The MFI value of Example 1 group was significantly higher than that of Comparative Example 6 group (P < 0.01), and close to the level of the positive control group, with an UV damage repair improvement rate of 3492.86%, which is 1.5 times that of Comparative Example 6. The oil-soluble recombinant collagen composition with cell-level anti-aging effects provided by this invention has a significantly better UV damage repair ability than the water-in-oil recombinant collagen solution, and can repair cell damage caused by UVA to a certain extent, effectively achieving anti-aging repair of skin cells.

[0130] Table 3 Summary of COL1 MFI value data analysis results in fibroblasts

[0131] In summary, this invention dissolves recombinant collagen in a specific ratio of supramolecular solvent, uses phospholipids as an interface stabilizer, and, with the help of adjuvants and stabilizers, successfully applies recombinant collagen to pure oil systems. It exhibits compatibility with various oils and, more importantly, simultaneously improves the permeability of recombinant collagen, greatly enhancing its bioavailability. This allows it to simultaneously meet the application and efficacy requirements in oil products, offering the following advantages: (1) Oil phase solubilization: After surface treatment by supramolecular technology, the exposure of hydrophilic groups on the surface of recombinant collagen is reduced, so that it can exist stably in phospholipids without precipitation. Phospholipids serve as an interface medium, and it can be further applied to various oils. Under the premise of ensuring the transparency of the oil, the solubilization and application of recombinant collagen in various oils such as polyols, polar oils, and non-polar oils are realized. Its stability in transparent essence has been verified through long-term investigation, so that recombinant collagen can be truly applied to pure oil transparent systems.

[0132] (2) Achieving penetration enhancement while solubilizing the oil phase: As a hydrophilic molecule, recombinant collagen is difficult to penetrate the dense lipid stratum corneum, resulting in low bioavailability. This invention stabilizes it in the oil through supramolecular and phospholipid mediators, improving its affinity for the lipid stratum corneum. Furthermore, supramolecular and phospholipids, as penetration-enhancing components, achieve penetration enhancement by disrupting the orderly arrangement of the lipid layer, thus significantly improving the permeability of the recombinant collagen in this invention and greatly enhancing its bioavailability. This invention does not introduce any toxic or harmful organic solvents, nor does it introduce chemical penetration enhancers such as azone, which are unwelcome in cosmetics, achieving the safe and effective application of recombinant collagen in a pure oil system.

[0133] Finally, it should be noted that the above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the technical scope disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An oil-soluble recombinant collagen composition having a cell-level anti-aging effect, characterized by, The outer part is coated with a phospholipid film, and a recombinant collagen-ultramolecular complex is embedded in the inner part of the phospholipid film; The recombinant collagen-ultramolecular complex is a first phospholipid coated with a supermolecular solution of recombinant collagen, and then freeze-dried; The supermolecular solvent is composed of a hydrogen donor and a hydrogen acceptor; The phospholipid film is formed by a second phospholipid.

2. The oil-soluble recombinant collagen composition having cell-level anti-aging efficacy according to claim 1, characterized by, The second phospholipid is 5-30 parts by weight, and the recombinant collagen-ultramolecular complex is 15.01-86 parts by weight; The recombinant collagen-ultramolecular complex contains 0.01-1 parts by weight of recombinant collagen, 10-60 parts by weight of supermolecular solvent, and 5-25 parts by weight of first phospholipid; The weight ratio of recombinant collagen to supermolecular solvent in the recombinant collagen-ultramolecular complex is 1:100-200; The weight ratio of recombinant collagen to phospholipid is 1:80-148.

3. The oil-soluble recombinant collagen composition having cell-level anti-aging efficacy according to claim 2, characterized by, The recombinant collagen includes at least one of type I recombinant collagen, type III recombinant collagen, type XVII recombinant collagen, type I small molecule recombinant collagen, type III small molecule recombinant collagen, and type XVII small molecule recombinant collagen; The recombinant collagen is a mixture of type III small molecule recombinant collagen and type I recombinant collagen with a mass ratio of 1:8.5; The supermolecular solvent is composed of a hydrogen donor and a hydrogen acceptor with a molar ratio of (0.5-2):1; The hydrogen donor is a fatty acid; The fatty acid includes isostearic acid and / or decanoic acid; The hydrogen acceptor is an alkaloid; The alkaloid includes at least one of sophoridine, oxysophoridine, and L-carnitine.

4. The oil-soluble recombinant collagen composition having cell-level anti-aging efficacy according to claim 2, characterized by, The phospholipid includes at least one of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, phosphatidylcholine, phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, and distearoyl phosphatidylcholine; The soybean lecithin contains ≥90% of phosphatidylcholine; The soybean lecithin contains ≥95% of phosphatidylcholine.

5. The oil-soluble recombinant collagen composition having a cell-level anti-aging effect according to any one of claims 1 to 4, wherein The stabilizer is 20-70 parts by weight; The stabilizer includes at least one of caprylic capric triglyceride, isononyl isononanoate, glyceryl tri(ethylhexanoate), propylene glycol carbonate, and diethyl sebacate; The oil-soluble recombinant collagen composition with cell-level anti-aging effect further includes 2-10 parts by weight of an auxiliary agent; The auxiliary agent includes at least one of butanediol, propylene glycol, hexanediol, and pentanediol.

6. A method for preparing the oil-soluble recombinant collagen composition having a cell-level anti-aging effect according to any one of claims 1 to 4, characterized by, The recombinant collagen is dissolved in a supermolecular solvent to obtain a supermolecular solution of recombinant collagen, the first phospholipid is added to the supermolecular solution of recombinant collagen and mixed uniformly, and then freeze-dried to obtain the recombinant collagen-ultramolecular complex; The second phospholipid, the optional auxiliary agent, and the optional stabilizer are mixed to obtain a phospholipid solution, and then the recombinant collagen-ultramolecular complex is added to the phospholipid solution and mixed uniformly to obtain the oil-soluble recombinant collagen composition with cell-level anti-aging effect.

7. The production method according to claim 6, characterized by, The preparation method of the supramolecular solvent is: grinding the hydrogen donor and the hydrogen acceptor, then dispersing them in an alcohol aqueous solution, and then reacting under the protection of nitrogen to obtain the supramolecular solvent.

8. The production method according to claim 7, characterized by, In the alcohol aqueous solution, the concentration of alcohol is 50-70wt%; And / or, the alcohol in the alcohol aqueous solution includes at least one of methanol, ethanol, propylene glycol, and butylene glycol; And / or, the reaction temperature is 20-45℃, and the reaction time is 20-36h.

9. Use of the oil-soluble recombinant collagen protein composition with cell-level anti-aging effect according to any one of claims 1-4 in the preparation of a cosmetic product.

10. Use according to claim 9, characterized in that, The addition amount of the oil-soluble recombinant collagen protein composition with cell-level anti-aging effect in the cosmetic product is 0.01-10wt%; And / or, the oil-soluble recombinant collagen protein composition with cell-level anti-aging effect is added in a cold preparation stage; And / or, the temperature in the cold preparation stage is below 50℃.

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