An oil-soluble collagen, its preparation method and application

Abstract

This invention relates to the field of bioengineering technology, and more particularly to an oil-soluble collagen, its preparation method, and its applications. The oil-soluble collagen composition provided by this invention, based on a collagen content of 0.10 parts by weight, further comprises 52-75 parts by weight of emulsifier, 20-40 parts by weight of oily solvent I, 3-7 parts by weight of aqueous solvent, and 0.4-1 parts by weight of antioxidant. The oil-soluble collagen composition provided by this invention is clear and transparent, has a uniform texture, and a small particle size. It maintains good stability at different temperatures and exhibits strong adaptability and compatibility in pure oil systems, stably integrating into oily formulation systems and functioning effectively. The preparation method of the oil-soluble collagen composition provided by this invention employs low-energy emulsification, a gentle process that preserves the activity of the collagen.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an oil-soluble collagen, its preparation method, and its application. Background Technology

[0002] Collagen is a type of protein widely distributed in human connective tissue and is the most abundant protein in the human body, accounting for 25% to 35% of total body protein. Collagen in human skin is mainly composed of type I and type III collagen. Type III collagen is mostly located in the superficial dermis and determines skin elasticity. Type III collagen also plays a wide range of roles in repairing wounds, reducing inflammation, inhibiting scarring, promoting tissue regeneration, and improving skin condition. However, with age, the synthesis rate of type III collagen in the body gradually declines, causing the skin to gradually lose elasticity and radiance, resulting in wrinkles and sagging. Therefore, it is necessary to replenish the skin with exogenous type III collagen through skincare products and cosmetic procedures to optimize skin condition.

[0003] Most collagen ingredients currently on the market are water-soluble, which works well in water-based skincare systems. However, in recent years, the skincare concept of "oil-based skincare" has become increasingly popular, and the demand for oil-based skincare products, such as facial oils and repair oils, has continued to rise. Under this market trend, the problem of traditional water-soluble collagen being difficult to adapt to oil-based formula systems has gradually become apparent. Therefore, developing collagen ingredients with good oil solubility has become a key direction to meet the efficacy requirements of oil-based skincare products.

[0004] Currently, existing technologies using peptides, solubilizers, and oil-based solvents produce oil-soluble peptide compositions that are easily added to all-oil cosmetic formulations. However, these peptides typically have ≤5 amino acids, limiting their applicability to larger molecular weight peptides and proteins. Furthermore, they cannot achieve high transparency and lack stability under extreme temperature conditions (e.g., 40°C and above, -10°C and below). Additionally, oil-soluble systems significantly reduce the biological activity of proteins, preventing collagen from fully realizing its excellent biological activity and thus hindering its maximum potential.

[0005] Existing technologies also utilize recombinant collagen powder, an oily matrix, dispersants, stabilizers, and moisturizing ingredients to prepare oil-soluble liquid compositions containing recombinant collagen. However, this approach requires a high-shear homogenizer, consuming significant energy. Furthermore, it cannot precisely control or quantify the recombinant collagen content in the composition, and may suffer from excessively large particle sizes and a lack of uniformity. This presents significant limitations in the application of the compositions, resulting in poor reproducibility and stability.

[0006] Based on the existing technologies, there is an urgent need for an oil-soluble collagen product that does not require external energy, can spontaneously form a uniform and stable oily system, is applicable to different collagens, can achieve precise control of the content of recombinant collagen in the system, and ensures that its preparation method is simple to operate and highly reproducible. Summary of the Invention

[0007] The problem the invention aims to solve

[0008] To address the limitations of existing technologies, such as the fact that most collagen raw materials are water-soluble, thus restricting their application in oily products, this invention has conducted extensive research and provides an oil-soluble collagen composition, along with its preparation method and applications. This invention utilizes reverse microemulsion technology, employing the self-assembly of collagen aqueous solution, oily solvent emulsifiers, etc., to form a transparent or semi-transparent dispersion system. The preparation process employs a low-energy emulsification process, which is gentle and maintains the good activity of collagen. The resulting product has high transparency and good flowability, is compatible with most commonly used oils in oily product formulations, and is easily added and used in pure oil systems.

[0009] Solution for solving the problem

[0010] [1]. An oil-soluble collagen composition comprising the following components: collagen, emulsifier, oil solvent I, aqueous solvent and antioxidant; and, in the oil-soluble collagen composition, based on the content of collagen being 0.10 parts by weight, the content of the emulsifier being 52-75 parts by weight, the content of oil solvent I being 20-40 parts by weight, the content of aqueous solvent being 3-7 parts by weight, and the content of antioxidant being 0.4-1 parts by weight.

[0011] [2]. The oil-soluble collagen composition according to [1], wherein the emulsifier comprises at least two of sorbitan polyether-30 tetraoleate, polyglycerol-3 diisostearate, sorbitan oleate and polysorbate-80;

[0012] Preferably, the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate;

[0013] More preferably, if present, the mass ratio of the sorbitol polyether-30 tetraoleate to the polyglycerol-3 diisostearate in the oil-soluble collagen composition is 1:(0.7-1.3).

[0014] [3]. The oil-soluble collagen composition according to [1] or [2], wherein the oil solvent I comprises at least one of caprylic / capric triglyceride, isononyl isononanoate, isotretinoin isononanoate, dioctyl carbonate, squalane and coconut oil alcohol-caprylate decanoate;

[0015] Preferably, the oily solvent I comprises caprylic / capric triglyceride.

[0016] [4]. The oil-soluble collagen composition according to any one of [1]-[3], wherein the aqueous solvent comprises water and a polyol;

[0017] Preferably, the polyol comprises at least one of glycerol, butanediol, and 1,3-propanediol;

[0018] More preferably, the polyol is any one of glycerol, butanediol and 1,3-propanediol;

[0019] More preferably, in the oil-soluble collagen composition, the mass ratio of water to polyol is 1:(1.2-3).

[0020] [5]. An oil-soluble collagen composition according to any one of [1]-[4], wherein the antioxidant comprises at least one of tocopherol and tocopherol derivatives;

[0021] Preferably, the antioxidant comprises tocopherol acetate.

[0022] [6]. An oil-soluble collagen composition according to any one of [1]-[5], wherein the collagen comprises at least one of type I collagen, type III collagen, type IV collagen, type V collagen, type VII collagen and type XVII collagen;

[0023] Preferably, the average molecular weight of the type I collagen is 20-45 kDa;

[0024] Preferably, the average molecular weight of the type III collagen is 15-35 kDa;

[0025] Preferably, the average molecular weight of the type IV collagen is 25-45 kDa;

[0026] Preferably, the average molecular weight of the V-type collagen is 35-45 kDa;

[0027] Preferably, the average molecular weight of the type VII collagen is 25-45 kDa;

[0028] Preferably, the average molecular weight of the type XVII collagen is 15-35 kDa; more preferably, the average molecular weight of the type XVII collagen is 15-25 kDa or 25-35 kDa.

[0029] [7]. A method for preparing an oil-soluble collagen composition according to any one of [1]-[6], wherein the preparation method comprises the following steps:

[0030] S1: Mix the prescribed amount of collagen and the prescribed amount of aqueous solvent, stir to dissolve, and obtain mixture A;

[0031] S2: Add the prescribed amount of emulsifier, the prescribed amount of oily solvent I, and the prescribed amount of antioxidant to mixture A, stir, and the mixture is ready;

[0032] Preferably, in S2, the stirring is performed by a magnetic stirrer or a top stirrer;

[0033] Preferably, in S2, the stirring conditions include: a stirring speed of 300-800 rpm and a stirring temperature of 20-40℃.

[0034] [8]. The use of any one of the oil-soluble collagen compositions described in [1]-[6] in the preparation of oily products, wherein the oily products include at least one of facial essence oil, scalp essence oil, body care oil and makeup remover oil;

[0035] Preferably, the oily product comprises any one of the oil-soluble collagen composition described in [1]-[6] and oily solvent II, wherein the oily solvent II comprises at least one of caprylic / capric triglyceride, dioctyl carbonate, isotretinoinate, squalane, and cocoyl caprylate / capric acid ester.

[0036] [9]. An efficacy product, wherein the efficacy product comprises any one of [1]-[6] of the oil-soluble collagen composition, and wherein the amount of the oil-soluble collagen composition added in the efficacy product is 0.1wt%-5.0wt%.

[0037] Preferably, the functional product has any one or more of the following functions:

[0038] Hyaluronidase inhibitory activity,

[0039] elastase inhibitory activity,

[0040] Cell proliferation promoting effect

[0041] Cell migration promoting effect

[0042] It provides soothing and / or moisturizing, anti-wrinkle and firming effects, protects damaged skin, and delays skin aging.

[0043]

[10] . The efficacy product according to [9], wherein the efficacy product further comprises an oily matrix and / or an antioxidant;

[0044] Preferably, the oily matrix comprises at least one of squalane, silk oil, jojoba seed oil, coconut oil, lavender essential oil, caprylic / capric triglyceride, ethylhexyl palmitate, dioctyl carbonate, and isononyl isononanoate.

[0045] Preferably, the antioxidant comprises at least one of tocopherol, tocopherol derivatives, pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamic acid) ester, and rosemary extract.

[0046] The effects of the invention

[0047] By implementing the above technical solution, the beneficial effects of the present invention include at least the following:

[0048] 1. The oil-soluble collagen composition provided by the present invention is clear and transparent, has a uniform texture, small particle size, can form a good reverse microemulsion system, and can maintain good stability at different temperatures.

[0049] 2. The oil-soluble collagen composition formulation provided by the present invention is applicable to different types of collagen. The prepared oil-soluble collagen composition has high transparency and good fluidity. It exhibits strong adaptability and compatibility in pure oil systems with various different components and can be stably integrated into the product formulation system and play its role.

[0050] 3. The preparation method of the oil-soluble collagen composition provided by the present invention uses low-energy emulsification, which is a mild process and can maintain the activity of collagen; and the prepared oil-soluble collagen composition shows good and significant inhibitory effects on hyaluronidase activity and elastase activity, and exhibits excellent cell proliferation and migration promotion effects.

[0051] 4. The preparation method of the oil-soluble collagen composition provided by the present invention is simple and easy to implement, requiring no complex equipment or cumbersome procedures. Ordinary operators can quickly get started after simple training, which greatly reduces the production threshold.

[0052] 5. The preparation method of the oil-soluble collagen composition provided by the present invention uses environmentally friendly raw materials throughout the process, with no harmful waste emissions during production and energy consumption significantly lower than that of traditional processes. This reduces the burden on the environment from the source and achieves a synergy between economic and ecological benefits. Attached Figure Description

[0053] Figure 1 The images show a comparison of the transparency of oil-soluble type III collagen prepared in Example 1 (left) and Comparative Example 1 (right).

[0054] Figure 2-1 The particle size detection results are for Example 1.

[0055] Figure 2-2The particle size detection results are for Example 4.

[0056] Figure 2-3 The particle size detection results are for Comparative Example 1.

[0057] Figure 2-4 The particle size detection results are for Comparative Example 5.

[0058] Figure 3 The image shows the appearance of an oil-soluble type III collagen sample (left) and a microscope image (right), magnified at 600x.

[0059] Figure 4 Microscopic images of cell scratches taken for HaCat cell migration experiments, magnified 4x.

[0060] Figure 5 Photos of oil-soluble collagen samples prepared from different types of collagen. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention are described below, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below. The described embodiments are some embodiments of the present invention, but not all embodiments. Various modifications can be made within the scope of the claims, and the embodiments and implementations obtained by appropriately combining different implementation schemes and the technical means disclosed in each embodiment are also included in the technical scope of the present invention.

[0062] In this invention, "comprising," "having," "including," or "containing" can mean included or open-ended, and does not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0063] In this invention, the meaning of "may" includes both performing a certain process and not performing a certain process.

[0064] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0065] In this invention, the numerical ranges represented by "value A ~ value B", "value A - value B", and "value A above / below" refer to the ranges including the endpoint values ​​A and B.

[0066] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by the term "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0067] In this invention, terms such as "some specific / preferred embodiments," "other specific / preferred embodiments," and "implementation" refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to a particular embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0068] In this invention, "oil-based products" refers to a class of products that use oil or oily components as the main matrix.

[0069] In this invention, "efficacy product" refers to a type of product that can bring a specific, verifiable efficacy to an organism.

[0070] In this invention, the term "collagen" encompasses both natural collagen and recombinant collagen.

[0071] In this invention, "recombinant human collagen" refers to the full-length amino acid sequence encoded by a specific type gene of human collagen prepared by DNA recombination technology, and has a triple helix structure.

[0072] In this invention, "recombinant humanized collagen" refers to a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of human collagen.

[0073] In this invention, "recombinant collagen" refers to a designed and modified amino acid sequence or fragment thereof, or a combination of such functional amino acid sequence fragments, prepared using DNA recombination technology. Its gene-coding sequence or amino acid sequence has low homology with the gene-coding sequence or amino acid sequence of human collagen.

[0074] In this invention, "normal temperature" or "room temperature" refers to an indoor ambient temperature of "10~30℃".

[0075] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0076] I. Oil-soluble collagen composition

[0077] This invention provides an oil-soluble collagen composition, which refers to a composition formed by dispersing water-soluble collagen in an oily solvent using reverse microemulsion technology.

[0078] In some embodiments, the oil-soluble collagen composition provided by the present invention is a W / O (water-in-oil) reverse microemulsion.

[0079] In some embodiments, the oil-soluble collagen composition comprises the following components: collagen, emulsifier, oil solvent I, aqueous solvent, and antioxidant.

[0080] In some embodiments, the oil-soluble collagen composition comprises the following components: collagen, emulsifier, oil solvent I, aqueous solvent, and antioxidant.

[0081] [Collagen]

[0082] The formulation system of the oil-soluble collagen composition provided by the present invention is suitable for various types of collagen, including but not limited to natural collagen, recombinant collagen (such as recombinant human collagen, recombinant humanized collagen, recombinant collagen-like protein, etc.).

[0083] This invention does not impose any particular limitation on the source of collagen. For example, it can be extracted from animal tissues by acid, alkali, or enzymatic methods, synthesized by DNA recombination technology or microbial fermentation, or purchased as a finished product through commercial channels.

[0084] In some embodiments, in the oil-soluble collagen composition of the present invention, the collagen comprises at least one of type I collagen, type III collagen, type IV collagen, type V collagen, type VII collagen, and type XVII collagen.

[0085] In some embodiments, the average molecular weight of the type I collagen is 20-45 kDa.

[0086] In some embodiments, the type I collagen is derived from ZL202110968550.2, the contents of which are incorporated herein by reference.

[0087] In some embodiments, the amino acid sequence of the type I collagen is shown in SEQ ID NO.1.

[0088] In some embodiments, the average molecular weight of the type III collagen is 15-35 kDa.

[0089] In some embodiments, the type III collagen is derived from ZL201210482543.2, the contents of which are incorporated herein by reference.

[0090] In some embodiments, the amino acid sequence of the type III collagen is shown in SEQ ID NO.2.

[0091] In some embodiments, the average molecular weight of the type IV collagen is 25-45 kDa.

[0092] In some embodiments, the type IV collagen is derived from ZL202311391711.1, the contents of which are incorporated herein by reference.

[0093] In some embodiments, the amino acid sequence of the type IV collagen is shown in SEQ ID NO.3.

[0094] In some embodiments, the V-type collagen has an average molecular weight of 35-45 kDa.

[0095] In some embodiments, the V-type collagen is derived from ZL202210849498.3, the contents of which are incorporated herein by reference.

[0096] In some embodiments, the amino acid sequence of the V-type collagen is shown in SEQ ID NO.4.

[0097] In some embodiments, the average molecular weight of the type VII collagen is 25-45 kDa.

[0098] In some embodiments, the type VII collagen is derived from ZL202211017546.9, the contents of which are incorporated herein by reference.

[0099] In some embodiments, the amino acid sequence of the type VII collagen is shown in SEQ ID NO.5.

[0100] In some embodiments, the average molecular weight of the type XVII collagen is 15-35 kDa, more preferably, the average molecular weight of the type XVII collagen is 15-25 kDa or 25-35 kDa.

[0101] In some embodiments, the type XVII collagen is derived from ZL201911051106.3 and CN202410026356.6, the contents of which are incorporated herein by reference.

[0102] In some embodiments, the amino acid sequence of the type XVII collagen is shown in SEQ ID NO.6 or SEQ ID NO.7.

[0103] In some embodiments, the collagen in the oil-soluble collagen composition of the present invention comprises any one of type I collagen, type III collagen, type IV collagen, type V collagen, type VII collagen, and type XVII collagen.

[0104] In some embodiments, in the oil-soluble collagen composition of the present invention, the collagen is any one of type I collagen, type III collagen, type IV collagen, type V collagen, type VII collagen, and type XVII collagen.

[0105] In some embodiments, in the oil-soluble collagen composition of the present invention, the collagen content is 0.08-0.12 parts by weight, for example, 0.08 parts, 0.09 parts, 0.10 parts, 0.11 parts or 0.12 parts; preferably 0.09-0.11 parts; the collagen content is preferably calculated based on 100 parts by weight of the total mass of the oil-soluble collagen composition.

[0106] [Emulsifier]

[0107] In some embodiments, in the oil-soluble collagen composition of the present invention, the content of the emulsifier is 52-75 parts, based on 0.10 parts by weight of collagen, for example, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 parts, etc.; preferably 66-74 parts; the content of the emulsifier is preferably based on 100 parts by weight of the total mass of the oil-soluble collagen composition.

[0108] In some embodiments, in the oil-soluble collagen composition of the present invention, the emulsifier comprises at least two of sorbitan polyether-30 tetraoleate, polyglycerol-3 diisostearate, sorbitan oleate, and polysorbate-80.

[0109] In some embodiments, in the oil-soluble collagen composition of the present invention, the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate.

[0110] In some embodiments, in the oil-soluble collagen composition of the present invention, the emulsifier is sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate.

[0111] In some embodiments, in the oil-soluble collagen composition of the present invention, if present, the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3), for example, it can be 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2 or 1:1.3.

[0112] [Oil-based solvent I]

[0113] In some embodiments, in the oil-soluble collagen composition of the present invention, based on a collagen content of 0.10 parts by weight, the content of the oily solvent I is 20-40 parts, for example, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, 30 parts, 31 parts, 32 parts, 33 parts, 34 parts, 35 parts, 36 parts, 37 parts, 38 parts, 39 parts, or 40 parts, etc.; preferably 22-30 parts; the content of the above-mentioned oily solvent I is preferably based on a total mass of 100 parts by weight of the oil-soluble collagen composition.

[0114] In some embodiments, in the oil-soluble collagen composition of the present invention, the oil solvent I comprises at least one of caprylic / capric triglyceride, isononyl isononanoate, isotretinoinate, dioctyl carbonate, squalane, and coconut oil alcohol-caprylate decanoate.

[0115] In some embodiments, in the oil-soluble collagen composition of the present invention, the oil solvent I comprises at least one of caprylic / capric triglyceride, isononyl isononanoate, and dioctyl carbonate.

[0116] In some embodiments, in the oil-soluble collagen composition of the present invention, the oily solvent I comprises caprylic / capric triglyceride.

[0117] In some embodiments, in the oil-soluble collagen composition of the present invention, the oily solvent I is caprylic / capric triglyceride.

[0118] [Water-based solvent]

[0119] In some embodiments, in the oil-soluble collagen composition of the present invention, the content of the aqueous solvent is 3-7 parts, for example, 3, 4, 5, 6 or 7 parts, based on the collagen content of 0.10 parts by weight; preferably 3.3-4.8 parts; the content of the aqueous solvent is preferably based on 100 parts by weight of the total mass of the oil-soluble collagen composition.

[0120] In some embodiments, in the oil-soluble collagen composition of the present invention, the aqueous solvent comprises and polyols.

[0121] In some embodiments, in the oil-soluble collagen composition of the present invention, the polyol comprises a polyol having 2-4 carbon atoms, preferably a polyol having 3 or 4 carbon atoms.

[0122] In some embodiments, in the oil-soluble collagen composition of the present invention, the polyol comprises at least one of glycerol, butylene glycol, and 1,3-propanediol.

[0123] In some embodiments, in the oil-soluble collagen composition of the present invention, the mass ratio of water to polyol is 1:(1.2-3), for example, it can be 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2.0, 1:2.2, 1:2.4, 1:2.6, 1:2.8 or 1:3.0, etc.

[0124] In some specific embodiments, in the oil-soluble collagen composition of the present invention, the aqueous solvent comprises water and glycerin.

[0125] In some specific embodiments, in the oil-soluble collagen composition of the present invention, the aqueous solvent comprises water and butanediol.

[0126] In some specific embodiments, in the oil-soluble collagen composition of the present invention, the aqueous solvent comprises water and 1,3-propanediol.

[0127] In some specific embodiments, in the oil-soluble collagen composition of the present invention, the aqueous solvent is water and glycerin.

[0128] In some specific embodiments, in the oil-soluble collagen composition of the present invention, the aqueous solvent is water and butanediol.

[0129] In some specific embodiments, in the oil-soluble collagen composition of the present invention, the aqueous solvent is water and 1,3-propanediol.

[0130] [Antioxidants]

[0131] In some embodiments, in the oil-soluble collagen composition of the present invention, the content of the antioxidant is 0.4-1 parts, for example, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, or 1.0 parts, etc., based on 0.10 parts by weight of the collagen content; preferably 0.4-0.6 parts; the content of the antioxidant is preferably based on 100 parts by weight of the total mass of the oil-soluble collagen composition.

[0132] In some embodiments, in the oil-soluble collagen composition of the present invention, the antioxidant comprises at least one of tocopherol and tocopherol derivatives.

[0133] In some embodiments, the antioxidant in the oil-soluble collagen composition of the present invention comprises tocopheryl acetate.

[0134] In some embodiments, the antioxidant in the oil-soluble collagen composition of the present invention is tocopheryl acetate.

[0135] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on a collagen content of 0.10 parts by weight, the emulsifier content is 52-75 parts by weight, the oil solvent I content is 20-40 parts by weight, the aqueous solvent content is 3-7 parts by weight, and the antioxidant content is 0.4-1 parts by weight; the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, and the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3).

[0136] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on the collagen content of 0.10 parts by weight, the emulsifier content is 52-75 parts by weight, the oil solvent I content is 20-40 parts by weight, the aqueous solvent content is 3-7 parts by weight, and the antioxidant content is 0.4-1 parts by weight; the emulsifier is sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, and the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3).

[0137] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on a collagen content of 0.10 parts by weight, the emulsifier content is 52-75 parts by weight, the oil solvent I content is 20-40 parts by weight, the aqueous solvent content is 3-7 parts by weight, and the antioxidant content is 0.4-1 parts by weight; the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3); the aqueous solvent comprises water and a polyol, the polyol being any one of glycerol, butanediol, and 1,3-propanediol, the mass ratio of water to the polyol being 1:(1.2-3).

[0138] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on the collagen content of 0.10 parts by weight, the emulsifier content is 52-75 parts by weight, the oil solvent I content is 20-40 parts by weight, the aqueous solvent content is 3-7 parts by weight, and the antioxidant content is 0.4-1 parts by weight; the emulsifier is sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, and the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3); the aqueous solvent is water and a polyol, and the polyol is any one of glycerol, butanediol, and 1,3-propanediol, and the mass ratio of water to the polyol is 1:(1.2-3).

[0139] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on a collagen content of 0.10 parts by weight, the emulsifier content is 52-75 parts by weight, the oil solvent I content is 20-40 parts by weight, the aqueous solvent content is 3-7 parts by weight, and the antioxidant content is 0.4-1 parts by weight; the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3); the aqueous solvent comprises water and a polyol, the polyol being any one of glycerol, butanediol, and 1,3-propanediol, the mass ratio of water to the polyol is 1:(1.2-3); the oil solvent I comprises caprylic / capric triglyceride.

[0140] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on the collagen content of 0.10 parts by weight, the emulsifier content is 52-75 parts by weight, the oil solvent I content is 20-40 parts by weight, the aqueous solvent content is 3-7 parts by weight, and the antioxidant content is 0.4-1 parts by weight; the emulsifier is sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, and the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3); the aqueous solvent is water and a polyol, the polyol being any one of glycerol, butanediol, and 1,3-propanediol, and the mass ratio of water to the polyol is 1:(1.2-3); the oil solvent I is caprylic / capric triglyceride.

[0141] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on a collagen content of 0.10 parts by weight, the emulsifier content is 66-74 parts by weight, the oil solvent I content is 22-30 parts by weight, the aqueous solvent content is 3.3-4.8 parts by weight, and the antioxidant content is 0.4-0.6 parts by weight; the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, and the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3).

[0142] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on a collagen content of 0.10 parts by weight, the emulsifier content is 66-74 parts by weight, the oil solvent I content is 22-30 parts by weight, the aqueous solvent content is 3.3-4.8 parts by weight, and the antioxidant content is 0.4-0.6 parts by weight; the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3); the aqueous solvent comprises water and a polyol, the polyol being any one of glycerol, butanediol, and 1,3-propanediol, the mass ratio of water to the polyol being 1:(1.2-3).

[0143] In some specific embodiments, in the oil-soluble collagen composition of the present invention, based on a collagen content of 0.10 parts by weight, the emulsifier content is 66-74 parts by weight, the oil solvent I content is 22-30 parts by weight, the aqueous solvent content is 3.3-4.8 parts by weight, and the antioxidant content is 0.4-0.6 parts by weight; the emulsifier comprises sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3); the aqueous solvent comprises water and a polyol, the polyol being any one of glycerol, butanediol, and 1,3-propanediol, the mass ratio of water to the polyol is 1:(1.2-3); the oil solvent I comprises caprylic / capric triglyceride.

[0144] The oil-soluble collagen composition provided by the present invention can be dispersed and dissolved with any one or more of the following oily substances: caprylic / capric triglyceride, dioctyl carbonate, isotretinoinate, squalane, and coconut oil alcohol-caprylate / capric acid ester.

[0145] II. Preparation method of oil-soluble collagen composition

[0146] This invention provides a method for preparing the oil-soluble collagen composition, comprising the following steps:

[0147] S1: Mix the prescribed amount of collagen and the prescribed amount of aqueous solvent, stir to dissolve, and obtain mixture A;

[0148] S2: Add the prescribed amount of emulsifier, the prescribed amount of oily solvent I, and the prescribed amount of antioxidant to mixture A, stir, and the mixture is ready.

[0149] In some embodiments, in S2, the stirring is performed by a magnetic stirrer or a top stirrer; preferably by a magnetic stirrer.

[0150] In some embodiments, in S2, the stirring conditions include: a stirring speed of 300-800 rpm and a stirring temperature of 15-40°C; preferably, in S2, the stirring conditions include: a stirring speed of 400-700 rpm and a stirring temperature of 20-35°C.

[0151] III. Uses of Oil-Soluble Collagen Compositions

[0152] This invention provides the application of the oil-soluble collagen composition in the preparation of oily products, the oily products including at least one of facial essence oil, scalp essence oil, body care oil and makeup remover oil.

[0153] In some embodiments, the oily product comprises the oil-soluble collagen composition and oily solvent II, which may be the same as or different from oily solvent I. Oily solvent II comprises at least one of caprylic / capric triglyceride, dioctyl carbonate, isotretinoinate, squalane, and coconut oil alcohol caprylate / capric acid ester; preferably, oily solvent II comprises any one of caprylic / capric triglyceride, dioctyl carbonate, isotretinoinate, squalane, and coconut oil alcohol caprylate / capric acid ester.

[0154] IV. Efficacy products containing oil-soluble collagen compositions

[0155] This invention provides an efficacy product comprising the oil-soluble collagen composition, wherein the amount of the oil-soluble collagen composition added in the efficacy product is 0.1wt%-5.0wt%; preferably, the efficacy product has any one or more of the following effects: hyaluronidase inhibitory activity, elastase inhibitory activity, cell proliferation promoting effect, cell migration promoting effect, soothing and / or moisturizing, anti-wrinkle and firming effect, and protection of damaged skin and delaying skin aging.

[0156] In some embodiments, the efficacy product also includes an oily matrix and / or antioxidants.

[0157] In some implementations, the efficacy product also includes an oily matrix and antioxidants.

[0158] In some embodiments, the amount of the oily matrix added in the efficacy product is 92.0 wt%-97.0 wt%.

[0159] In some embodiments, the antioxidant is added in the efficacy product at an amount of 0.1wt%-3.0wt%.

[0160] In some embodiments, the oily matrix comprises at least one of squalane, silk oil, jojoba seed oil, coconut oil, lavender essential oil, caprylic / capric triglyceride, ethylhexyl palmitate, dioctyl carbonate, and isononyl isononanoate.

[0161] In some embodiments, the oily matrix comprises at least five of the following: squalane, silk oil, jojoba seed oil, coconut oil, lavender essential oil, caprylic / capric triglyceride, ethylhexyl palmitate, dioctyl carbonate, and isononyl isononanoate.

[0162] In some embodiments, the antioxidant comprises at least one of tocopherol, tocopherol derivatives, pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamic acid) ester, and rosemary extract.

[0163] In some embodiments, the antioxidant comprises any one of tocopherol, tocopherol derivatives, pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamic acid) ester, and rosemary extract.

[0164] In some embodiments, the antioxidant comprises tocopherol acetate.

[0165] In some embodiments, the antioxidant is tocopherol acetate.

[0166] In some specific embodiments, the efficacy product comprises the oil-soluble collagen composition, squalane, dioctyl carbonate, isononyl isononanoate, caprylic / capric triglyceride, jojoba seed oil, and tocopheryl acetate.

[0167] In some specific embodiments, the efficacy product comprises the oil-soluble collagen composition, coconut oil, dioctyl carbonate, isononyl isononanoate, lavender essential oil, jojoba seed oil, and tocopheryl acetate.

[0168] To more clearly illustrate the technical solution of the present invention, specific embodiments are described below, but these should not be construed as limiting the invention; they are merely some embodiments of the invention. Unless otherwise stated, the instruments, reagents, materials, laboratory animals, etc., used in the present invention can all be obtained through conventional commercial means.

[0169] The English and Chinese translations of the ingredients used in the examples (based on the International Cosmetic Ingredient Names (INCI)) are shown below:

[0170] (1) Sorbitol polyether-30 tetraoleate: SORBETH-30 TETRAOLEATE, trade name: RHEODOL 430V, purchased from Kao (Shanghai) Chemical Co., Ltd.;

[0171] (2) Polyglycerol-3 diisostearate: POLYGLYCERYL-3 DIISOSTEARATE, trade name: PLUROLDIISOSTEARIQUE CG, purchased from GATTEFOSS SAS;

[0172] (3) Caprylic / Capric Triglyceride: RADIA 7104, purchased from OLEON NV;

[0173] (4) Glycerin: GLYCERIN, trade name: MOON K GLYCERIN, purchased from Procter & Gamble International Operations SA, Singapore Branch;

[0174] (5) Butylene Glycol: BUTYLENE GLYCOL, trade name: 1,3-Butylene Glycol, purchased from Shandong Haike Xinyuan Materials Technology Co., Ltd.;

[0175] (6) 1,3-Propanediol: PROPANEDIOL, trade name: BIOBLANCA-1,3-PDO, purchased from Chifeng Zhihe Biotechnology Co., Ltd.

[0176] (7) Water: WATER;

[0177] (8) Tocopheryl acetate: trade name: D-α tocopherol acetate, purchased from Shaanxi Haisifu Biotechnology Co., Ltd.;

[0178] (9) Collagen: COLLAGEN.

[0179] Experimental Example 1: Preparation Method of Oil-Soluble Type III Collagen

[0180] (1) Weigh each component according to the formula in Table 1, mix type III collagen, water and glycerin, stir thoroughly to dissolve, and obtain mixture A;

[0181] (2) Add sorbitol polyether-30 tetraoleate, polyglycerol-3 diisostearate, caprylic / capric triglyceride and tocopheryl acetate to mixture A, and stir continuously for 30 minutes at 500 rpm using a magnetic stirrer to obtain oil-soluble type III collagen.

[0182] (3) The obtained oil-soluble type III collagen is a clear and transparent light yellow oily liquid.

[0183] Referring to GB / T 5525-2008, the transparency of vegetable oils was tested using a stoppered glass tube with a diameter of 25 mm and a height of 228 mm including the cap. The height of the liquid level was recorded when the bottom line of the tube was clearly visible. The results are shown in Table 1.

[0184] The conductivity values ​​of the samples measured at room temperature using a DDS-307A conductivity meter (see instruction manual for usage) are shown in Table 1 below.

[0185] The prepared sample was added to the sample cell and placed in a Malvern Zetasizer Nano As series laser particle size analyzer to determine the particle size. The measurement temperature was 25℃, and the number of measurement cycles was automatically set by the system (n=3). After the detection was completed, the average particle size of the measured sample was recorded. The results are shown in Table 1.

[0186] The type III oil-soluble collagen samples prepared in the examples were placed in high and low temperature cycling environments of 4℃ (for 24 hours and 3 months), room temperature (for 3 months), 40℃ (for 3 months), 50℃ (for 3 months), and -15℃ and 40℃ (for 3 months) to investigate their stability, and their transparency was tested again.

[0187] Table 1. Formulation and Stability of Oil-Soluble Type III Collagen

[0188]

[0189] After analyzing the transparency, particle size, conductivity, and stability of the samples from the above examples, it was found that the prepared oil-soluble type III collagen samples had excellent appearance and high transparency. Furthermore, the average particle size distribution of the prepared oil-soluble type III collagen was approximately 18–32 nm, and the conductivity was significantly lower than that of purified water (4.79 μS·cm). -1 ) and aqueous solution of type III collagen (736 μS·cm) -1 Therefore, it can be determined that the continuous phase of oil-soluble type III collagen is the oil phase, belonging to a reverse microemulsion. Stability testing results show that the samples prepared in the examples maintained good stability under different temperature conditions, remaining transparent liquid throughout the observation period without turbidity or stratification, indicating good stability. This demonstrates that it is a thermodynamically stable, isotropic, and transparent reverse microemulsion system, achieving oil solubility of collagen and enabling its application in oily products.

[0190] The samples from Comparative Examples 1-5 exhibited turbidity upon visual inspection under conditions of 4°C, room temperature, or high and low temperature cycling, indicating poor stability. Furthermore, the oil-soluble type III collagen prepared using these samples had a large average particle size, reaching up to approximately 25 times that of the examples, resulting in excessively large micelle sizes that made them unsuitable for use as a microemulsion system.

[0191] Specifically, after analyzing the transparency and particle size of Example 1 and Comparative Examples 1 and 2, it was found that, under the same conditions, the samples prepared in Comparative Examples 1 and 2 were cloudy in appearance and had significantly larger particle sizes than those in Example 1. This indicates that when sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate are used as emulsifiers, their ratio affects the transparency and particle size of the system. The optimal ratio of sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate is 1:(0.7~1.3).

[0192] Furthermore, analysis of the transparency and particle size of Example 1 and Comparative Examples 3-5 revealed that the glycerol content in the aqueous solvent significantly affected the low-temperature stability of the samples. Specifically, when the glycerol content was low, the samples performed well at room temperature, but became cloudy after being placed at 4°C, directly impacting the product's performance in actual use. Conversely, when the glycerol content was too high, the samples exhibited cloudiness and excessively large particle sizes, preventing the formation of a microemulsion system.

[0193] Figure 1 The results of the transparency comparison between the samples of Example 1 and Comparative Example 1 show that the transparency of the sample of Example 1 is good. In a glass tube with a diameter of 25 mm and a height of 228 mm including the cap, the bottom line of the tube is clearly visible. However, the bottom line of the tube of Comparative Example 1 is not visible at all under the same conditions.

[0194] Figures 2-1 to 2-4 The particle size detection results are shown for samples from Examples 1, 4, Comparative Example 1, and Comparative Example 5, respectively. As can be seen from the figure, the particle size of the examples is mainly distributed in the range of 20-30 nm, while the particle size of the comparative example samples is mainly distributed in the range of 300-400 nm. This particle size range exceeds the definition standard of microemulsion, indicating that the comparative example samples do not meet the particle size requirements of microemulsion.

[0195] Experiment Example 2: An Investigation into Emulsifiers

[0196] The formulation remains consistent with that of Example 1 above, except for the amount and type of emulsifier. Specific differences are shown in Table 2 below. The transparency and particle size were tested respectively.

[0197] Table 2. Different formulations of oil-soluble type III collagen and their corresponding stability.

[0198]

[0199] Results Analysis: The results of Comparative Examples 6-1 to 6-4 clearly demonstrate that the type and amount of emulsifier are key factors determining the performance of the microemulsion system in the system constructed in this invention. The experimental results show that using both sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate as emulsifiers enables the construction of a microemulsion system. The resulting oil-soluble type III collagen exhibits good stability, and the system is transparent and homogeneous. Furthermore, the particle size in the examples is controlled within the range of 19-20 nm. Conversely, using only one of sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, or a combination of other emulsifiers (such as polyglycerol-2 isostearate and sorbitol polyether-30 tetraisostearate), results in an opaque collagen system with poor stability under different temperature conditions. Moreover, the particle size of the comparative samples is significantly higher than that of the examples, reaching nearly 20-60 times.

[0200] In addition, the scheme of using a combination of sorbitol polyether-30 tetraoleate and ethoxydiethylene glycol as an emulsifier was verified. See Table 3 below for details. The experimental results of Comparative Examples 7-1 and 7-2 show that when the proportion of different components exceeds the range specified in this application (e.g., too little emulsifier or too much oily solvent), it is impossible to prepare isotropic, transparent, oil-soluble type III collagen.

[0201] Table 3. Different formulations of oil-soluble type III collagen and their corresponding stability.

[0202]

[0203] Experiment Example 3: An Investigation into Aqueous Solvents

[0204] The following examples and comparative examples validated different combinations of aqueous solvents, maintaining the same formulation as Example 1 above, except for the change in the combination of aqueous solvents. See Table 4 below for details. The results demonstrate that replacing glycerol in the aqueous solvents of Example 1 with butylene glycol and 1,3-propanediol resulted in oil-soluble type III collagen exhibiting an isotropic and transparent appearance. The results of Example 6 and Comparative Example 8, and Example 7 and Comparative Example 9, demonstrate that when the content of butylene glycol or 1,3-propanediol is too high, it directly leads to problems such as cloudy appearance and larger particle size.

[0205] Table 4. Different formulations of oil-soluble type III collagen and their corresponding stability.

[0206]

[0207] Experiment Example 4

[0208] The oil-soluble type III collagen sample obtained in Example 1 was tested, and its properties were characterized by observation of its appearance and solubility in an oily system. Efficacy verification included hyaluronidase activity inhibition assays, elastase activity inhibition assays, HaCaT cell proliferation assays, and HaCaT cell migration assays.

[0209] The results are as follows:

[0210] 1. Observation of appearance and morphology

[0211] like Figure 3 As shown, oil-soluble type III collagen appears as a pale yellow, clear, and homogeneous liquid. When observed using a MOTIC M200 inverted microscope, the microemulsion particles of oil-soluble type III collagen are uniformly spherical.

[0212] 2. Solubility experiment in oil phase system

[0213] The oil-soluble type III collagen prepared in Example 1 was added to different oil systems (Table 5 below) at a controlled addition amount of 10 wt%, and its dissolution state was observed.

[0214] Table 5. Solubility states of oil-soluble type III collagen in different lipid systems

[0215]

[0216] Experimental results show that oil-soluble type III collagen has good compatibility with synthetic oils such as caprylic / capric triglycerides and squalane, but poor compatibility with pure vegetable oils such as coconut oil and macadamia oil, and is incompatible with silicone oils containing cyclopentamethoxysiloxane.

[0217] 3. Hyaluronidase activity inhibition experiment

[0218] Hyaluronic acid (HA) is the most abundant and predominant component of the extracellular matrix (ECM). It maintains ECM volume, regulates the secretion of cell growth factors and cytokines, and influences cell adhesion, growth, proliferation, and differentiation. Therefore, it plays a crucial role in maintaining skin hydration and elasticity, wound healing, and angiogenesis. Hyaluronidase is a specific enzyme that cleaves hyaluronic acid; inhibiting HAase activity ensures normal HA content and function. HAase is strongly correlated with inflammation and allergies. Various drugs that release histamine from mast cells can regulate hyaluronidase activity, and some anti-allergy drugs strongly inhibit hyaluronidase activity. Therefore, inhibiting hyaluronidase activity can be used as an indicator for studying anti-allergic effects. Inhibiting HAase can achieve soothing and moisturizing effects by maintaining HA function.

[0219] This experiment evaluates the soothing / moisturizing efficacy of substances by detecting their inhibition rate of hyaluronidase.

[0220] A 96-well ELISA plate was configured with sample wells (T), sample background wells (T0), enzyme reaction wells (C), enzyme reaction background wells (C0), positive control wells (P), and positive control background wells (P0). The samples were oil-soluble III collagen reverse microemulsion prepared in Example 1, at concentrations of 50 mg / mL and 30 mg / mL, respectively. Samples were added according to the order and volume specified in Table 6 below. After the reaction was complete, the ELISA plate was transferred to an ELISA reader (02-MSP-30001, Thermo Fisher Scientific), and the absorbance was measured at 400 nm.

[0221] The hyaluronidase inhibition rate is calculated as follows: Inhibition rate (%) = [1 - (T0 - T) / (C0 - C)] × 100%

[0222] Table 6. Information on experimental groups for hyaluronidase activity inhibition

[0223]

[0224] Quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed, and p-values ​​were used to determine statistical significance; p < 0.05 was considered statistically significant. The inhibition rate of the positive control was 59.93%, indicating the effectiveness of the experimental system. The experimental results show that, compared with the enzyme reaction group, when the concentrations of oil-soluble type III collagen prepared in Example 1 were 50 mg / mL and 30 mg / mL, it significantly inhibited hyaluronidase activity (P < 0.05), with inhibition rates of 64.34% and 79.19%, respectively. The oil-soluble type III collagen prepared in Example 1 has soothing / moisturizing effects.

[0225] 4. Inhibition experiment of elastase activity

[0226] Elastin is the main component of elastic fibers. Elastic fibers are mainly found in ligaments and blood vessel walls. Elastic fibers coexist with collagen fibers, giving tissues elasticity and tensile strength. Elastase can digest and break down elastin in connective tissue proteins, causing elastic fibers to gradually break, thin, and even fragment. The elastic scaffold of the dermis is destroyed, and the skin loses its original retraction ability, resulting in dynamic wrinkles and even static wrinkles. Therefore, this experiment uses the elastase inhibition rate as an indicator to evaluate the anti-wrinkle and firming effects of substances.

[0227] A 96-well microplate was configured with sample wells (T), sample background wells (T0), enzyme reaction wells (C), enzyme reaction background wells (C0), positive control wells (P), and positive control background wells (P0). The sample was an oil-soluble type III collagen reverse microemulsion prepared in Example 1, with a concentration of 30 mg / mL. The sample was added according to the order and volume specified in Table 7 below. After addition, the microplate was transferred to a microplate reader and incubated at 25°C for 15 minutes. The absorbance was measured at 410 nm using a microplate reader (02-MSP-30001, Thermo Fisher Scientific).

[0228] The calculation method for elastase inhibition rate is: Inhibition rate (%) = [1-(T0-T) / (C0-C)]×100%.

[0229] Table 7. Information on experimental groups for elastase activity inhibition

[0230]

[0231] Quantitative data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed, and p-values ​​were used to determine statistical significance; p < 0.05 was considered statistically significant. The positive control showed an inhibitory effect, indicating the effectiveness of the experimental system. Experimental results showed that, compared with the enzyme reaction group, when the concentration of oil-soluble type III collagen prepared in Example 1 was 30 mg / mL, it significantly inhibited elastase activity (P < 0.05), with an inhibition rate of 26.99%. Oil-soluble type III collagen has anti-wrinkle and firming effects.

[0232] 5. HaCaT cell proliferation experiment

[0233] Human keratinocytes (HaCaT) are immortalized epidermal keratinocyte cell lines widely used to study the mechanisms of skin biology, oxidative stress, cell proliferation, and apoptosis. HaCaT cells exhibit typical keratinocyte characteristics, including expression of keratin and differentiation markers (such as involucrin), and can maintain a certain level of proliferation and migration ability in vitro. HaCaT cells adhere to epithelial cells, exhibit epithelial-like morphology, and express keratin, keratinocyte cross-linking outer membrane proteins, and intermediate filament-associated proteins, making them an important tool for studying epidermal keratinization.

[0234] Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were digested and centrifuged, and a cell suspension of appropriate density was prepared with complete culture medium. The suspension was then seeded into 96-well plates according to the experimental design and incubated at 37°C with 5% CO2 for 24 ± 2 h. The culture medium in the plates was discarded, and sample loading was performed according to Table 8 below. The plates were then incubated at 37°C with 5% CO2 for 24 ± 2 h and 48 ± 2 h. After incubation, the culture medium was discarded, and the absorbance of each well was read using a microplate reader according to the cytotoxicity assay procedure. The 24-hour mark was used as the starting point for the experiment, with a proliferation rate of 0, for subsequent statistical analysis of cell proliferation at different time points (e.g., 48 h, 72 h, etc.).

[0235] Cell proliferation rate (%) = [(OD value of test group - mean OD value of 24h blank control group) / mean OD value of 24h blank control group] × 100%

[0236] Table 8. Information on HaCaT cell proliferation experimental groups

[0237]

[0238] Compared with the blank control group, the cell proliferation rate of the sample group at a specific concentration was significantly increased (P<0.05), which means that under the test conditions, the sample at that concentration has the effect of promoting cell proliferation.

[0239] In the control group, the cell proliferation rate at 24 h was recorded as 0, and the cell proliferation rate at 48 h was 185.79%. In the sample group, when the oil-soluble type III collagen prepared in Example 1 was at a concentration of 25 mg / mL, the cell proliferation rates at 24 h and 48 h were 18.71% and 248.40%, respectively. Compared with the cell proliferation rate of the control group, the cell proliferation rates were significantly increased, demonstrating that oil-soluble type III collagen has a significant promoting effect on the cell proliferation of HaCaT cells.

[0240] 6. HaCaT cell migration assay

[0241] The cell scratch assay was used to simulate wound healing; it is a simple method for measuring cell migration and repair capabilities. Cells in the logarithmic growth phase were digested and centrifuged, and a cell suspension of appropriate density was prepared with complete culture medium. The suspension was then seeded into 6-well plates according to the experimental design and cultured at 37°C in a 5% CO2 incubator until cell confluence reached 98%. Before seeding cells, five straight parallel lines were drawn on the back of the 6-well plate with a marker, spaced 0.5 cm apart. Once cell confluence reached over 98%, a straight line was drawn perpendicular to the marker line at the bottom of each well using a sterile pipette tip. After drawing the line, the culture medium in the wells was discarded, and sample loading was performed according to Table 9. The plates were then incubated at 37°C in a 5% CO2 incubator for 24±2 h and 48±2 h. After incubation, the culture medium was discarded, and the cells were washed with DPBS. The scratch patterns were photographed at the same field of view as at 0 h. ImageJ was then used to analyze the scratch area and calculate cell migration rate.

[0242] Migration rate (%) = [(0h scratch area - scratch area at each time point) / 0h scratch area] × 100%

[0243] Table 9. HaCaT cell migration experimental group information

[0244]

[0245] The cell migration results for each group are shown below. Figure 4 The experimental results showed that in the sample group, when the concentration of oil-soluble type III collagen prepared in Example 1 was 2.5 mg / mL, the cell migration rate was 12.99% at 24 h and 57.43% at 48 h. Compared with the blank control group (3.13% at 24 h and 27.35% at 48 h), the cell migration rate of the 2.5 mg / mL concentration sample group was significantly increased (P < 0.05). Under the test conditions, this concentration of sample promoted cell migration, which is beneficial for skin barrier repair, synthesis of collagen and elastin, control or regulation of skin aging process, protection of damaged skin, and delay of skin aging.

[0246] The above experiments demonstrate that the oil-soluble system provided by this invention can fully utilize the biological activity of collagen without affecting the function of its active ingredient, collagen. The oil-soluble system does not diminish the efficacy of collagen; it still exhibits good hyaluronidase and elastase inhibitory activity, and has a good promoting effect on the proliferation and migration of HaCaT cells. It can exert soothing / moisturizing, anti-wrinkle, and firming effects, and can protect damaged skin and delay skin aging.

[0247] Experimental Example 5: Formulation Tables and Experimental Characterization of Different Types of Collagen

[0248] Based on Example 1, different types of collagen were used for preparation. The experimental formulation remained consistent with Example 1, except that different types of collagen (including type I, III, IV, V, VII, and XVII collagen) were used to verify the formulation of the oil-soluble collagen. The amino acid sequences of the different types of collagen and the average particle size of the prepared oil-soluble collagen are shown in Table 10 below.

[0249] Table 10 Amino acid sequence information of different types of collagen

[0250]

[0251] Experimental results are as follows Figure 5 As shown, using different types of collagen and the formulation described in Example 1, all the prepared oil-soluble collagen samples exhibited a pale yellow, clear, and homogeneous liquid state. They showed good stability under storage conditions of 4–40°C, with particle sizes controlled within the range of 20–50 nm, all <60 nm, and good compatibility with squalane and synthetic oils. This phenomenon fully demonstrates that the reverse microemulsion system constructed in this invention has good compatibility and applicability to various types of collagen, highlighting its broad value in practical applications.

[0252] Application Example 1: Preparation of Type III Collagen Essence Oil Samples

[0253] The formula for the Type III collagen essence oil is shown in Table 11.

[0254] Weigh each component according to the formula, add squalane, dioctyl carbonate, isononyl isononanoate, caprylic / capric triglyceride and jojoba seed oil to a mixing pot, stir evenly, then add tocopheryl acetate and the oil-soluble type III collagen prepared in Example 1, stir evenly, filter with a clean and dry filter screen to obtain the type III collagen essence oil.

[0255] The prepared essential oils were placed in 4℃, room temperature, 40℃, 50℃ and high and low temperature cycling environments for 1 month, and the product properties did not change significantly.

[0256] Table 11 Type III Collagen Essence Oil Formula

[0257]

[0258] Application Example 2: Preparation of XVII Type Collagen Scalp Essence Oil Sample

[0259] The formula for the XVII type collagen scalp essence oil is shown in Table 12.

[0260] Weigh each component according to the formula, add coconut oil, dioctyl carbonate, isononyl isononanoate and jojoba seed oil to a mixing pot, stir well, then add tocopheryl acetate, lavender essential oil and type XVII oil-soluble collagen, stir well, and filter with a clean and dry filter to obtain the type XVII collagen scalp essence oil.

[0261] The prepared scalp essence oil was kept at 4℃, room temperature, 40℃, 50℃ and high and low temperature cycling conditions for 1 month, and the product properties did not change significantly.

[0262] Table 12 Formula for Type XVII Collagen Scalp Essence Oil

[0263]

Claims

1. An oil-soluble collagen composition comprising the following components: collagen, emulsifier, oil solvent I, aqueous solvent, and antioxidant; and wherein, based on a collagen content of 0.10 parts by weight, the emulsifier content is 52-75 parts by weight, the oil solvent I content is 20-40 parts by weight, the aqueous solvent content is 3-7 parts by weight, and the antioxidant content is 0.4-1 parts by weight; The emulsifiers are sorbitol polyether-30 tetraoleate and polyglycerol-3 diisostearate, and in the oil-soluble collagen composition, the mass ratio of sorbitol polyether-30 tetraoleate to polyglycerol-3 diisostearate is 1:(0.7-1.3); The oily solvent I is caprylic / capric triglyceride; The aqueous solvent is water and a polyol, wherein the polyol is any one of glycerol, butanediol and 1,3-propanediol, and in the oil-soluble collagen composition, the mass ratio of water to the polyol is 1:(1.2-3).

2. The oil-soluble collagen protein composition according to claim 1, characterized in that, The antioxidant contains at least one of tocopherol and tocopherol derivatives.

3. The oil-soluble collagen protein composition according to claim 1, characterized in that, The antioxidant contains tocopherol acetate.

4. The oil-soluble collagen protein composition according to any one of claims 1 to 3, characterized in that, The collagen includes at least one of type I collagen, type III collagen, type IV collagen, type V collagen, type VII collagen, and type XVII collagen.

5. The oil-soluble collagen composition according to claim 4, characterized in that, The average molecular weight of the type I collagen is 20-45 kDa.

6. The oil-soluble collagen composition according to claim 4, characterized in that, The average molecular weight of the type III collagen is 15-35 kDa.

7. The oil-soluble collagen composition according to claim 4, characterized in that, The average molecular weight of the type IV collagen is 25-45 kDa.

8. The oil-soluble collagen composition according to claim 4, characterized in that, The average molecular weight of the V-type collagen is 35-45 kDa.

9. The oil-soluble collagen composition according to claim 4, characterized in that, The average molecular weight of the type VII collagen is 25-45 kDa.

10. The oil-soluble collagen composition according to claim 4, characterized in that, The average molecular weight of the XVII type collagen is 15-35 kDa.

11. The oil-soluble collagen composition according to claim 4, characterized in that, The average molecular weight of the XVII type collagen is 15-25 kDa or 25-35 kDa.

12. A method for preparing an oil-soluble collagen composition according to any one of claims 1-11, characterized in that, The preparation method includes the following steps: S1: Mix the prescribed amount of collagen and the prescribed amount of aqueous solvent, stir to dissolve, and obtain mixture A; S2: Add the prescribed amount of emulsifier, the prescribed amount of oily solvent I, and the prescribed amount of antioxidant to mixture A, stir, and the mixture is ready.

13. The preparation method according to claim 12, characterized in that, In S2, the stirring is performed by a magnetic stirrer or a top stirrer.

14. The preparation method according to claim 12, characterized in that, In S2, the stirring conditions include: a stirring speed of 300-800 rpm and a stirring temperature of 20-40℃.

15. The use of the oil-soluble collagen composition according to any one of claims 1-11 in the preparation of an oily product, said oily product comprising at least one of facial oil, scalp oil, body oil, and makeup remover.

16. The application according to claim 15, characterized in that, The oily product comprises the oil-soluble collagen composition according to any one of claims 1-11 and oily solvent II, wherein oily solvent II comprises at least one of caprylic / capric triglyceride, dioctyl carbonate, isotretinoinate, squalane, and cocoyl caprylate / capric acid ester.

17. A functional product, characterized in that, The efficacy product comprises the oil-soluble collagen composition according to any one of claims 1-11, and the amount of the oil-soluble collagen composition added in the efficacy product is 0.1wt%-5.0wt%.

18. The efficacy product according to claim 17, characterized in that, The product has any one or more of the following effects: Hyaluronidase inhibitory activity, elastase inhibitory activity, Cell proliferation promoting effect Cell migration promoting effect It provides soothing and / or moisturizing, anti-wrinkle and firming effects, protects damaged skin, and delays skin aging.

19. The efficacy product according to claim 17, characterized in that, The efficacy products also contain an oily base and / or antioxidants.

20. The efficacy product according to claim 19, characterized in that, The oily matrix comprises at least one of squalane, silk oil, jojoba seed oil, coconut oil, lavender essential oil, caprylic / capric triglyceride, ethylhexyl palmitate, dioctyl carbonate, and isononyl isononanoate.

21. The efficacy product according to claim 19, characterized in that, The antioxidant comprises at least one of tocopherol, tocopherol derivatives, pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamic acid) ester, and rosemary extract.

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