Recombinant collagen liposome and preparation method and application thereof

Abstract

This invention discloses a recombinant collagen liposome, its preparation method, and its application. The recombinant collagen liposome comprises 1-10 parts recombinant type XVII collagen, 2-15 parts soybean lecithin, 0.3-2 parts tocopherol, 0.5-7 parts antioxidant, 20-54 parts glycerol, and Dendrobium officinale enzymatic hydrolysate to a total weight of 100 parts. This invention improves the stability and transdermal absorption of recombinant type XVII collagen by encapsulating it in liposomes. The prepared recombinant collagen liposomes not only have a high encapsulation efficiency but also retain excellent barrier repair and anti-wrinkle effects after 8 weeks of storage, making them suitable for use in cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic raw material technology, and specifically relates to a recombinant collagen liposome, its preparation method and application. Background Technology

[0002] Collagen is a fibrous protein composed of three intertwined polypeptide chains, widely found in the connective tissues of animals, and is the most widely distributed functional protein in mammals. To date, researchers have discovered more than 20 types of collagen, which can be divided into fibroblastic collagen and non-fibroblastic collagen based on their structure and function. Type XVII collagen belongs to the transmembrane collagen category of non-fibroblastic collagen, comprising three major structural regions: intracellular, transmembrane, and extracellular. Its molecular weight is approximately 180 kDa. Type XVII collagen can exist in vivo in transmembrane or detached forms, thus possessing the dual functions of cell membrane surface receptors and extracellular matrix components. Simultaneously, Type XVII collagen is a key component of hemidesmosomes in epidermal stem cells, participating in basement membrane adhesion, skin homeostasis regulation, and anti-aging processes, especially playing a central role in promoting cell adhesion, migration, and maintaining stem cell function. Type XVII collagen is present in extremely low amounts in the human body, and its extraction from human tissues is extremely difficult, making industrial-scale production impossible. In existing processes, recombinant type XVII collagen is prepared using synthetic biotechnology, resulting in relatively low production costs and enabling its market application. Although recombinant type XVII collagen only extracts a portion of the active region from the original type XVII collagen, and its molecular weight is significantly smaller, it still reaches tens of thousands of Daltons, leading to low transdermal absorption. When applied directly, only a small amount penetrates the stratum corneum, making it difficult to target epidermal stem cells or the dermis, significantly reducing bioavailability. Summary of the Invention

[0003] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a recombinant collagen liposome.

[0004] Another object of the present invention is to provide a method for preparing the above-mentioned recombinant collagen liposomes.

[0005] Another object of the present invention is to provide the application of the above-mentioned recombinant collagen liposomes.

[0006] The objective of this invention is achieved through the following solution:

[0007] A recombinant collagen liposome, by weight, comprises: 1-10 parts recombinant type XVII collagen, 2-15 parts soybean lecithin, 0.3-2 parts tocopherol, 0.5-7 parts antioxidant, 20-54 parts glycerol, and Dendrobium officinale enzymatic hydrolysate to a total weight of 100.

[0008] Optionally, in some embodiments of the present invention, the recombinant collagen liposomes comprise 2-8 parts of recombinant type XVII collagen, 3-12 parts of soybean lecithin, 0.4-1.2 parts of tocopherol, 1-4 parts of antioxidant, 25-45 parts of glycerol, and Dendrobium officinale enzymatic hydrolysate to a total weight of 100 parts.

[0009] Preferably, in some embodiments of the present invention, the recombinant collagen liposomes comprise 4 parts recombinant type XVII collagen, 8 parts soybean lecithin, 0.8 parts tocopherol, 3 parts antioxidant, 38 parts glycerol, and Dendrobium officinale enzymatic hydrolysate to a total weight of 100 parts.

[0010] Optionally, in some embodiments of the present invention, the antioxidant includes vitamin C ethyl ether and / or inositol.

[0011] Preferably, in some embodiments of the present invention, the vitamin C ethyl ether and inositol are included, wherein the mass ratio of the vitamin C ethyl ether and inositol is (1-3):(1-3).

[0012] Optionally, in some embodiments of the present invention, the preparation method of the Dendrobium officinale enzymatic hydrolysate includes:

[0013] S1. After mixing the crushed Dendrobium officinale stem segments with water, the mixture is subjected to ultra-high pressure treatment to obtain pretreated material;

[0014] S2. Add a pH adjuster to the pretreated material in step S1 to obtain a mixture, and divide the mixture into mixture A and mixture B.

[0015] S3. Add a breakdown enzyme to the mixture A described in step S2 and perform a single enzymatic hydrolysis to obtain hydrolysate A.

[0016] S4. Add the breakdown enzyme to the mixture B described in step S2 and perform a second enzymatic hydrolysis to obtain hydrolysate B.

[0017] S5. Mix the enzymatic hydrolysate A from step S3 and the enzymatic hydrolysate B from step S4 evenly, and then heat and filter to obtain the Dendrobium officinale enzymatic hydrolysate.

[0018] Optionally, in some embodiments of the present invention, the mass ratio of Dendrobium officinale stem segments and water in step S1 is 1:(50-120).

[0019] Optionally, in some embodiments of the present invention, the pressure of the ultra-high pressure treatment in step S1 is 50-250 MPa, and the time of the ultra-high pressure treatment is 15-30 min.

[0020] In this invention, ultra-high pressure treatment uses water as the transfer medium. The ultra-high pressure within the container disrupts the three-dimensional structure of the Dendrobium officinale stem segments, affecting non-covalent bonds such as hydrogen bonds and ionic bonds that constitute biomolecules. Specifically, the physicochemical properties of the cell walls and cell membranes, as well as active substances (such as high-molecular-weight Dendrobium officinale polysaccharides), are altered, thus facilitating further enzymatic hydrolysis. Simultaneously, ultra-high pressure treatment also promotes the release of small-molecule active substances such as saponins, flavonoids, and alkaloids from the Dendrobium officinale stem segments into the water.

[0021] Optionally, in some embodiments of the present invention, the pH adjuster in step S2 includes at least one of hydrochloric acid solution, sulfuric acid solution, phosphoric acid solution, citric acid solution, and lactic acid solution, wherein the concentration of the acid in the pH adjuster is 1.0-6.0 mol / L.

[0022] Optionally, in some embodiments of the present invention, the pH of the mixture in step S2 is adjusted to 5.0-6.0.

[0023] In this invention, a pH adjuster is used to regulate the pH of the pretreated material to provide a suitable pH for subsequent enzymatic hydrolysis by the decomposing enzyme, thereby improving the hydrolysis efficiency. Furthermore, under weakly acidic conditions, high molecular weight Dendrobium officinale polysaccharides are more easily hydrolyzed, thus improving their bioavailability.

[0024] Optionally, in some embodiments of the present invention, the mass ratio of mixture A and mixture B in step S2 is (1-2):(1-2).

[0025] Optionally, in some embodiments of the present invention, the amount of the breakdown enzyme added in step S3 is 0.001-0.01% of the mass of mixture A in step S2.

[0026] Optionally, in some embodiments of the present invention, the temperature of the first enzymatic hydrolysis treatment in step S3 is 35-45°C, and the time of the first enzymatic hydrolysis is 3-6 hours.

[0027] Optionally, in some embodiments of the present invention, the amount of the breakdown enzyme added in step S4 is 0.001-0.01% of the mass of mixture B in step S2.

[0028] Optionally, in some embodiments of the present invention, the temperature of the secondary enzymatic hydrolysis in step S4 is 35-45°C, and the time of the secondary enzymatic hydrolysis is 8-12 hours.

[0029] Optionally, in some embodiments of the present invention, the heating temperature in step S5 is 80-90°C, and the heating time is 15-30 min.

[0030] Optionally, in some embodiments of the present invention, the filtration method described in step S5 includes diatomaceous earth filter filtration.

[0031] This invention divides the mixture into two parts, each subjected to enzymatic hydrolysis with a breakdown enzyme for different durations. The aim is to better control the molecular weight of the *Dendrobium officinale* hydrolysate. A shorter first hydrolysis time yields higher molecular weight *Dendrobium officinale* polysaccharides. A longer first hydrolysis time yields lower or even ultra-low molecular weight *Dendrobium officinale* polysaccharides. After the hydrolysis step, hydrolysate A and hydrolysate B are combined. The resulting *Dendrobium officinale* hydrolysate contains both high and low molecular weight *Dendrobium officinale* polysaccharides, which is beneficial for the preparation of recombinant collagen liposomes and the enhancement of their efficacy.

[0032] The preparation method of the above-mentioned recombinant collagen liposomes includes:

[0033] (1) Mix recombinant type XVII collagen, antioxidant and Dendrobium officinale enzymatic hydrolysate evenly to obtain phase A;

[0034] (2) Mix soybean lecithin, tocopherol and glycerol evenly to obtain phase B;

[0035] (3) Add phase A to phase B and mix to obtain crude liposomes;

[0036] (4) The crude liposomes are subjected to high-speed shear dispersion and high-pressure homogenization to obtain recombinant collagen liposomes.

[0037] Optionally, in some embodiments of the present invention, the mixing temperature in steps (1) and (2) is 20-40°C relatively independently.

[0038] Optionally, in some embodiments of the present invention, the rate at which phase A is added in step (3) is 4-8 mL / min.

[0039] Optionally, in some embodiments of the present invention, the temperature of the mixing reaction in step (3) is 55-65°C, and the time of the mixing reaction is 40-90 min.

[0040] Optionally, in some embodiments of the present invention, the high-speed shear dispersion rate in step (4) is 5000-8000 rpm, and the high-speed shear dispersion time is 8-15 min.

[0041] Optionally, in some embodiments of the present invention, the pressure of the high-pressure homogenization process in step (4) is 50-120 MPa, and the number of cycles is 2-6.

[0042] The above-mentioned recombinant collagen liposomes are used in the preparation of cosmetics.

[0043] The amount of the recombinant collagen liposomes used in the cosmetic is 0.1-50% by mass.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] (1) This invention utilizes liposome design to create a liposome that encapsulates recombinant collagen. The recombinant collagen liposomes prepared by this invention exhibit high encapsulation efficiency and good embedding effect for recombinant type XVII collagen. Compared with the direct use of recombinant collagen, the liposomes formed by this invention can improve the transdermal absorption of recombinant collagen, solving the problem that recombinant collagen is difficult to penetrate the stratum corneum when used directly, and improving the efficacy and bioavailability of recombinant collagen.

[0046] (2) In the liposome design of this invention, Dendrobium officinale enzymatic hydrolysate is used instead of deionized water in the traditional liposome preparation process. The Dendrobium officinale polysaccharides in the enzymatic hydrolysate can modify the liposomes, resulting in liposomes modified with Dendrobium officinale polysaccharides. Dendrobium officinale polysaccharides can act as ligands for the liposome wall material, modifying the membrane fluidity and allowing the liposomes to form liposomes with high encapsulation efficiency and small particle size without the addition of ligands such as cholesterol or phytosterols. Furthermore, the Dendrobium officinale polysaccharides formed in this invention are acidic. When modifying liposomes, they can form complexes with negatively charged macromolecules through intermolecular electrostatic interactions, overcoming the poor stability of liposomes and improving their stability.

[0047] (3) The Dendrobium officinale enzymatic hydrolysate of the present invention is obtained after pretreatment and enzymatic hydrolysis of Dendrobium officinale stem segments. In addition to Dendrobium officinale polysaccharides, the Dendrobium officinale enzymatic hydrolysate also contains many active substances. The Dendrobium officinale enzymatic hydrolysate can not only be used directly as a solvent for liposomes, but the active substances, such as the ultra-low molecular weight Dendrobium officinale polysaccharides, can be directly absorbed through the skin due to their low molecular weight, thus exerting skin care effects. In other words, the recombinant collagen liposomes of the present invention can achieve skin care effects in multiple dimensions.

[0048] (4) This invention provides an in-depth cellular analysis of the skincare efficacy of recombinant collagen liposomes, revealing that they can promote the expression of the Caspase-14 and TIMP-1 genes. Caspase is a group of cysteine-aspartic acid-specific proteases; Caspase-14 can participate in the hydrolysis of pro-filaggrin to generate filaggrin, which in turn forms a natural moisturizing factor, thereby enhancing the skin barrier. TIMP-1 is a human matrix metalloproteinase inhibitor; its upregulation can inhibit collagenase activity, slow down collagen destruction, and thus exhibit an anti-wrinkle effect. The recombinant collagen liposomes of this invention have excellent barrier repair and anti-wrinkle effects, making them suitable for use in cosmetics. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0050] Unless otherwise specified, all reagents used in the examples are commercially available.

[0051] Unless otherwise specified, all percentages mentioned in this invention are percentages by mass.

[0052] The recombinant type XVII collagen, recombinant type I collagen, and recombinant type III collagen described in this invention are all derived from Guangdong Gexin Biotechnology Co., Ltd.

[0053] The hydroxypropyl cyclodextrin described in this invention is derived from Guangzhou Xiaojia Chemical Technology Co., Ltd.

[0054] The glyceryl stearate / PEG-100 stearate described in this invention is sourced from Croda Chemicals (Shanghai) Co., Ltd., and is traded under the name Arlacel 165.

[0055] The soybean lecithin used in this invention is derived from Shanghai Taiwei Pharmaceutical Co., Ltd.

[0056] The remaining amount mentioned in the table of this invention refers to the amount of the component to make up to 100 parts by weight of the total recombinant collagen liposomes. Taking Example 1 as an example, the remaining amount of Dendrobium officinale enzymatic hydrolysate refers to 46.2 parts of Dendrobium officinale enzymatic hydrolysate. The remaining examples and comparative examples are deduced by analogy.

[0057] Examples 1-10: Recombinant collagen liposomes and their preparation methods

[0058] Examples 1-10 provide a recombinant collagen liposome and its preparation method, wherein the content of each component in the recombinant collagen liposome of Examples 1-10 is shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062] The preparation methods of the recombinant collagen liposomes in Examples 1-10 are as follows:

[0063] (1) The recombinant type XVII collagen, antioxidant (vitamin C ethyl ether and / or inositol) and Dendrobium officinale enzymatic hydrolysate were mixed evenly at 25°C to obtain phase A;

[0064] (2) Mix soybean lecithin, tocopherol and glycerol evenly at 25°C to obtain phase B;

[0065] (3) Add phase A to phase B at a rate of 6 mL / min and mix and react at 60 °C for 60 min to obtain crude liposomes;

[0066] (4) The crude liposomes were sheared and dispersed at a rate of 6000 rpm for 10 min, and then homogenized under high pressure at 80 MPa for 4 cycles to obtain recombinant collagen liposomes.

[0067] In Examples 1-8, the preparation method of Dendrobium officinale enzymatic hydrolysate includes:

[0068] S1. After crushing the Dendrobium officinale stem segments with a pulverizer, mix them with 80 times their weight of water, and then treat them under ultra-high pressure at 150 MPa for 20 minutes to obtain pretreated material;

[0069] S2. Add 3 mol / L hydrochloric acid solution to the pretreated material in step S1 to adjust the pH to 5.5 to obtain a mixture. Divide the mixture into mixture A and mixture B, with a mass ratio of 1:1.

[0070] S3. Add 0.005% of the mass of the decomposition enzyme to the mixture A in step S2, and perform a single enzymatic hydrolysis treatment at 40°C for 5 hours to obtain hydrolysate A.

[0071] S4. Add 0.005% of the mass of the breakdown enzyme to the mixture B in step S2, and perform a second enzymatic hydrolysis treatment at 40°C for 10 hours to obtain hydrolysate B.

[0072] S5. Mix the enzymatic hydrolysate A from step S3 and the enzymatic hydrolysate B from step S4, heat at 85°C for 20 minutes, and filter through a diatomaceous earth filter to obtain the filtrate of Dendrobium officinale.

[0073] The only difference between the preparation method of Dendrobium officinale enzymatic hydrolysate in Example 9 and that in Examples 1-8 is that the mass ratio of mixture A and mixture B in step S2 is changed from 1:1 to 2:1. All other steps and parameters are the same as those in Examples 1-8.

[0074] The preparation method of Dendrobium officinale enzymatic hydrolysate in Example 10 differs from that in Examples 1-8 only in that the mass ratio of mixture A and mixture B in step S2 is changed from 1:1 to 1:2. All other steps and parameters are the same as those in Examples 1-8.

[0075] Example 11 Recombinant collagen liposomes and their preparation method

[0076] Example 11 provides a recombinant collagen liposome, comprising, by weight: 1 part recombinant type XVII collagen, 2 parts soybean lecithin, 2 parts tocopherol, 0.125 parts vitamin C ethyl ether, 0.375 parts inositol, 20 parts glycerol, and Dendrobium officinale enzymatic hydrolysate to a weight of 100.

[0077] The preparation method of recombinant collagen liposomes in Example 11 includes:

[0078] (1) The recombinant type XVII collagen, antioxidants (vitamin C ethyl ether and inositol) and Dendrobium officinale enzymatic hydrolysate were mixed evenly at 40°C to obtain phase A;

[0079] (2) Mix soybean lecithin, tocopherol and glycerol evenly at 40°C to obtain phase B;

[0080] (3) Add phase A to phase B at a rate of 4 mL / min and mix and react at 55 °C for 90 min to obtain crude liposomes;

[0081] (4) The crude liposomes were sheared and dispersed at a rate of 8000 rpm for 8 min, and then homogenized under high pressure at 120 MPa for 2 cycles to obtain recombinant collagen liposomes.

[0082] The preparation method of Dendrobium officinale enzymatic hydrolysate in Example 11 includes:

[0083] S1. After crushing the Dendrobium officinale stem segments with a pulverizer, mix them with 50 times their weight of water, and then treat them under ultra-high pressure at 250 MPa for 15 minutes to obtain pretreated material;

[0084] S2. Add 6 mol / L citric acid solution to the pretreated material in step S1 to adjust the pH to 5, and obtain a mixture. Divide the mixture into mixture A and mixture B, with a mass ratio of 1:1 between mixture A and mixture B.

[0085] S3. Add 0.01% by mass of the decomposition enzyme to the mixture A in step S2, and perform a single enzymatic hydrolysis treatment at 45°C for 6 hours to obtain hydrolysate A.

[0086] S4. Add 0.01% of the decomposition enzyme to the mixture B in step S2, and perform a second enzymatic hydrolysis treatment at 45°C for 12 hours to obtain hydrolysate B.

[0087] S5. Mix the enzymatic hydrolysate A from step S3 and the enzymatic hydrolysate B from step S4, heat at 80°C for 30 minutes, and filter through a diatomaceous earth filter to obtain the filtrate of Dendrobium officinale.

[0088] Example 12 Recombinant collagen liposomes and their preparation method

[0089] Example 12 provides a recombinant collagen liposome, comprising, by weight: 10 parts recombinant type XVII collagen, 15 parts soybean lecithin, 0.3 parts tocopherol, 5.6 parts vitamin C ethyl ether, 1.4 parts inositol, 54 parts glycerol, and Dendrobium officinale enzymatic hydrolysate to a weight of 100.

[0090] The method for preparing recombinant collagen liposomes in Example 12 includes:

[0091] (1) The recombinant type XVII collagen, antioxidants (vitamin C ethyl ether and inositol) and Dendrobium officinale enzymatic hydrolysate were mixed evenly at 20°C to obtain phase A;

[0092] (2) Mix soybean lecithin, tocopherol and glycerol evenly at 20°C to obtain phase B;

[0093] (3) Add phase A to phase B at a rate of 8 mL / min and mix and react at 65 °C for 40 min to obtain crude liposomes;

[0094] (4) The crude liposomes were sheared and dispersed at a rate of 5000 rpm for 15 min, and then homogenized under high pressure at 50 MPa for 6 cycles to obtain recombinant collagen liposomes.

[0095] The preparation method of Dendrobium officinale enzymatic hydrolysate in Example 12 includes:

[0096] S1. After crushing the Dendrobium officinale stem segments with a pulverizer, mix them with 120 times their weight of water, and then treat them under ultra-high pressure at 50 MPa for 30 minutes to obtain pretreated material;

[0097] S2. Add 1 mol / L phosphoric acid solution to the pretreated material in step S1 to adjust the pH to 6 to obtain a mixture. Divide the mixture into mixture A and mixture B, with a mass ratio of 1:1 between mixture A and mixture B.

[0098] S3. Add 0.001% of the decomposition enzyme to the mixture A in step S2, and perform a single enzymatic hydrolysis treatment at 35°C for 3 hours to obtain hydrolysate A.

[0099] S4. Add 0.001% of the decomposition enzyme to the mixture B in step S2, and perform a second enzymatic hydrolysis treatment at 35°C for 8 hours to obtain hydrolysate B.

[0100] S5. Mix the enzymatic hydrolysate A from step S3 and the enzymatic hydrolysate B from step S4, heat at 90°C for 15 minutes, and filter through a diatomaceous earth filter to obtain the filtrate of Dendrobium officinale.

[0101] Comparative Example 1: Recombinant Collagen Liposomes and Their Preparation Method

[0102] Comparative Example 1 provides a recombinant collagen liposome. The only difference between Comparative Example 1 and Example 1 is that the recombinant type XVII collagen in Comparative Example 1 is replaced with an equal part by weight of recombinant type I collagen. All other steps and parameters are the same as in Example 1.

[0103] Comparative Example 2: Recombinant Collagen Liposomes and Their Preparation Method

[0104] Comparative Example 2 provides a recombinant collagen liposome. The only difference between Comparative Example 2 and Example 1 is that the recombinant type XVII collagen in Comparative Example 2 is replaced with an equal part by weight of recombinant type III collagen. All other steps and parameters are the same as in Example 1.

[0105] Comparative Example 3: Recombinant Collagen Liposomes and Their Preparation Method

[0106] Comparative Example 3 provides a recombinant collagen liposome. The only difference between Comparative Example 3 and Example 1 is that the soybean lecithin in Comparative Example 3 is replaced with an equal part by weight of hydroxypropyl cyclodextrin. All other steps and parameters are the same as in Example 1.

[0107] Comparative Example 4: Recombinant Collagen Liposomes and Their Preparation Method

[0108] Comparative Example 4 provides a recombinant collagen liposome. The only difference between Comparative Example 4 and Example 1 is that the soybean lecithin in Comparative Example 4 is replaced with an equal part by weight of glyceryl stearate / PEG-100. All other steps and parameters are the same as in Example 1.

[0109] Comparative Example 5: Recombinant Collagen Liposomes and Their Preparation Method

[0110] Comparative Example 5 provides a recombinant collagen liposome. The only difference between Comparative Example 5 and Example 1 is that 1.5 parts by weight of vitamin C ethyl ether and 1.5 parts by weight of inositol in Example 1 are replaced with 3 parts by weight of tocopherol acetate. All other steps and parameters are the same as in Example 1.

[0111] Comparative Example 6: Recombinant Collagen Liposomes and Their Preparation Method

[0112] Comparative Example 6 provides a recombinant collagen liposome. The only difference between Comparative Example 6 and Example 1 is that 1 part by weight of vitamin C ethyl ether and 1 part by weight of inositol in Comparative Example 6 are replaced with 2 parts by weight of deionized water. All other steps and parameters are the same as in Example 1.

[0113] Comparative Example 7: Recombinant Collagen Liposomes and Their Preparation Method

[0114] Comparative Example 7 provides a recombinant collagen liposome. The only difference between Comparative Example 7 and Example 1 is that glycerol in Comparative Example 7 is replaced with an equal part by weight of 1,2-butanediol. All other steps and parameters are the same as in Example 1.

[0115] Comparative Example 8: Recombinant Collagen Liposomes and Their Preparation Method

[0116] Comparative Example 8 provides a recombinant collagen liposome. The only difference between Comparative Example 8 and Example 1 is that the Dendrobium officinale enzymatic hydrolysate in Comparative Example 8 is replaced with an equal part by weight of deionized water. All other steps and parameters are the same as in Example 1.

[0117] Comparative Example 9: Recombinant Collagen Liposomes and Their Preparation Method

[0118] Comparative Example 9 provides a recombinant collagen liposome. The only difference between Comparative Example 9 and Example 1 is that the Dendrobium officinale enzymatic hydrolysate in Comparative Example 9 is replaced with an equal weight of Dendrobium officinale extract (the preparation method is different from that of the Dendrobium officinale enzymatic hydrolysate in Example 1). All other steps and parameters are the same as those in Example 1.

[0119] The preparation method of Dendrobium officinale extract includes:

[0120] S1. Mix the crushed Dendrobium officinale stem segments with 80 times their weight of water, add 3 mol / L hydrochloric acid solution to adjust the pH to 5.5, and obtain a mixture. Divide the mixture into mixture A and mixture B, with a mass ratio of 1:1.

[0121] S2. Add 0.005% of the mass of the decomposition enzyme to the mixture A in step S2, and perform a single enzymatic hydrolysis treatment at 40°C for 5 hours to obtain hydrolysate A.

[0122] S3. Add 0.005% of the mass of the breakdown enzyme to the mixture B in step S2, and perform a second enzymatic hydrolysis treatment at 40°C for 10 hours to obtain hydrolysate B.

[0123] S4. Mix the enzymatic hydrolysate A from step S2 and the enzymatic hydrolysate B from step S3, heat at 85°C for 20 minutes, and filter through a diatomaceous earth filter to obtain the filtrate, which is the Dendrobium officinale extract.

[0124] Comparative Example 10: Recombinant Collagen Liposomes and Their Preparation Method

[0125] Comparative Example 10 provides a recombinant collagen liposome. The only difference between Comparative Example 10 and Example 1 is that the Dendrobium officinale enzymatic hydrolysate in Comparative Example 10 is replaced with an equal weight of Dendrobium officinale extract (the preparation method is different from that of the Dendrobium officinale enzymatic hydrolysate in Example 1). All other steps and parameters are the same as those in Example 1.

[0126] The preparation method of Dendrobium officinale extract includes:

[0127] S1. After crushing the Dendrobium officinale stem segments with a pulverizer, mix them with 80 times their weight of water, and then treat them under ultra-high pressure at 150 MPa for 20 minutes to obtain pretreated material;

[0128] S2. Add 3 mol / L hydrochloric acid solution to the pretreated material in step S1 to adjust the pH to 5.5, and obtain a mixture;

[0129] S3. Add 0.005% of the mass of the decomposition enzyme to the mixture in step S2, and enzymatically hydrolyze it at 40°C for 5 hours to obtain hydrolysate A.

[0130] S4. Heat the enzymatic hydrolysate A from step S3 at 85°C for 20 minutes, and filter it through a diatomaceous earth filter to obtain the filtrate, which is the Dendrobium officinale extract.

[0131] Comparative Example 11: Recombinant Collagen Liposomes and Their Preparation Method

[0132] Comparative Example 11 provides a recombinant collagen liposome. The only difference between Comparative Example 11 and Example 1 is that the Dendrobium officinale enzymatic hydrolysate in Comparative Example 11 is replaced with an equal weight of Dendrobium officinale extract (the preparation method is different from that of the Dendrobium officinale enzymatic hydrolysate in Example 1). All other steps and parameters are the same as those in Example 1.

[0133] The preparation method of Dendrobium officinale extract includes:

[0134] S1. After crushing the Dendrobium officinale stem segments with a pulverizer, mix them with 80 times their weight of water, and then treat them under ultra-high pressure at 150 MPa for 20 minutes to obtain pretreated material;

[0135] S2. Add 3 mol / L hydrochloric acid solution to the pretreated material in step S1 to adjust the pH to 5.5, and obtain a mixture;

[0136] S3. Add 0.005% of the mass of the decomposition enzyme to the mixture in step S2, and enzymatically hydrolyze it at 40°C for 10 hours to obtain hydrolysate B.

[0137] S4. Heat the enzymatic hydrolysate B from step S3 at 85°C for 20 minutes, and filter it through a diatomaceous earth filter to obtain the filtrate, which is the Dendrobium officinale extract.

[0138] Comparative Example 12: Recombinant Collagen Liposomes and Their Preparation Method

[0139] Comparative Example 12 provides a recombinant collagen liposome. The only difference between Comparative Example 12 and Example 1 is that the Dendrobium officinale hydrolysate in Comparative Example 12 is replaced with an equal weight of Tremella fuciformis hydrolysate (the preparation method is the same as that of Dendrobium officinale hydrolysate in Example 1). All other steps and parameters are the same as those in Example 1.

[0140] The preparation method of Tremella fuciformis enzymatic hydrolysate includes:

[0141] S1. After crushing the fruiting bodies of Tremella fuciformis with a pulverizer, mix them with 80 times their weight of water, and then treat them under ultra-high pressure at 150 MPa for 20 minutes to obtain pretreated material.

[0142] S2. Add 3 mol / L hydrochloric acid solution to the pretreated material in step S1 to adjust the pH to 5.5 to obtain a mixture. Divide the mixture into mixture A and mixture B, with a mass ratio of 1:1.

[0143] S3. Add 0.005% of the mass of the decomposition enzyme to the mixture A in step S2, and perform a single enzymatic hydrolysis treatment at 40°C for 5 hours to obtain hydrolysate A.

[0144] S4. Add 0.005% of the mass of the breakdown enzyme to the mixture B in step S2, and perform a second enzymatic hydrolysis treatment at 40°C for 10 hours to obtain hydrolysate B.

[0145] S5. Mix the enzymatic hydrolysate A from step S3 and the enzymatic hydrolysate B from step S4, heat at 85°C for 20 minutes, and filter through a diatomaceous earth filter to obtain the filtrate, which is the tremella enzymatic hydrolysate.

[0146] Comparative Example 13: Liposomes and their preparation method

[0147] Comparative Example 13 provides a collagen liposome. The only difference between Comparative Example 13 and Example 1 is that the recombinant type XVII collagen in Example 1 is replaced with an equal weight of Dendrobium officinale enzymatic hydrolysate. All other steps and parameters are the same as in Example 1.

[0148] Comparative Example 14: Recombinant Collagen Liposomes and Their Preparation Method

[0149] Comparative Example 14 provides a recombinant collagen liposome. The only difference between Comparative Example 14 and Example 1 is that the recombinant type XVII collagen in Comparative Example 14 is increased from 4 parts by weight to 15 parts by weight, and the number of parts of Dendrobium officinale hydrolysate is reduced accordingly. All other steps and parameters are the same as in Example 1.

[0150] Comparative Example 15: Recombinant Collagen Liposomes and Their Preparation Method

[0151] Comparative Example 15 provides a recombinant collagen liposome. The only difference between Comparative Example 15 and Example 1 is that the soybean lecithin in Comparative Example 15 is increased from 8 parts by weight to 20 parts by weight, and the amount of Dendrobium officinale hydrolysate is reduced accordingly. All other steps and parameters are the same as in Example 1.

[0152] Example 1: Stability Test

[0153] Test samples: recombinant collagen liposomes or liposomes prepared in Examples 1-12 and Comparative Examples 1-15; control group: recombinant type XVII collagen solution with a mass concentration of 4% in water as solvent.

[0154] Test procedure: Take 10 mL of test sample and put it into a transparent PE bottle. Each test sample is put into 2 bottles. One bottle is placed at 25℃ and the other bottle is placed at 45℃ for 8 weeks. Observe the stratification of the test sample in the PE bottle at week 0, week 4 and week 8 and record it to determine the stability of the test sample.

[0155] The test results at 25℃ are shown in Table 2, and the test results at 45℃ are shown in Table 3.

[0156] Table 2

[0157]

[0158] Table 3

[0159]

[0160] Test results show that the recombinant collagen liposomes of this invention have excellent stability. After being placed at 25℃ and 45℃ for 8 weeks, the appearance did not change significantly, no stratification or flocculent matter appeared, indicating that the recombinant collagen liposomes prepared in this invention have excellent stability. In the control group, recombinant type XVII collagen was directly prepared into a 4% (w / w) solution using water as a solvent. Flocculent matter appeared after being placed at 45℃ for 4 weeks and at 25℃ for 8 weeks, indicating that directly preparing the recombinant type XVII collagen solution resulted in poor stability. Preparing recombinant type XVII collagen into liposomes can greatly improve its stability. Comparing Examples 1-12 and Comparative Examples 1-2, it can be seen that replacing the recombinant type XVII collagen with similar recombinant type I and recombinant type III collagen resulted in varying degrees of flocculent matter appearing in the final product after 8 weeks, indicating that the liposomes of this invention are more suitable for encapsulating recombinant type XVII collagen. Comparative Examples 1-12 and 3-7 show that the wall material, antioxidants, and solvents in the oil phase of the liposome material all affect the stability of the final product. Replacing the wall material, antioxidants, or solvents results in varying degrees of flocculent matter forming in the final product after being placed at different temperatures for a period of time. The preferred wall material in this invention is soybean lecithin, the preferred antioxidants are vitamin C ethyl ether and / or inositol, and the preferred solvent is glycerol. Comparative Examples 1-12 and 8-12 show that the type of plant in the enzymatic hydrolysate and the preparation method of the enzymatic hydrolysate all affect the stability of the recombinant collagen liposomes. Dendrobium officinale polysaccharides in the Dendrobium officinale enzymatic hydrolysate can modify the liposomes and improve their stability. The liposomes prepared in Comparative Example 8 do not contain Dendrobium officinale polysaccharides, and the Dendrobium officinale enzymatic hydrolysate in Comparative Example 9 has not undergone ultra-high pressure pretreatment, resulting in low extraction efficiency of Dendrobium officinale polysaccharides and thus poor stability of the final liposomes. Comparative Example 11 contained an enzymatic hydrolysate with a longer hydrolysis time, specifically the Dendrobium officinale hydrolysate, which contained less high-molecular-weight Dendrobium officinale polysaccharides. High-molecular-weight Dendrobium officinale polysaccharides can form a wall material together with soybean lecithin. When high-molecular-weight Dendrobium officinale polysaccharides are lacking, the wall material is insufficient, leading to poor stability of the final liposomes. Comparative Example 12 used Tremella fuciformis polysaccharide instead of Dendrobium officinale polysaccharide. However, Dendrobium officinale polysaccharide has poor modification properties for the liposome wall material, which is detrimental to improving the stability of the final product.Comparative Examples 1-12 and Comparative Examples 14 and 15 show that excessive amounts of recombinant type XVII collagen or soy lecithin are not conducive to maintaining the stability of liposomes. Excessive amounts of recombinant type XVII collagen result in poor encapsulation of liposomes and easy precipitation of recombinant type XVII collagen. Excessive phospholipids may cause the solubility of recombinant type XVII collagen in the phospholipid bilayer to reach saturation, leading to easy precipitation of recombinant type XVII collagen. In this invention, the preferred amount of recombinant type XVII collagen is 1-10 parts, and the preferred amount of soy lecithin is 2-15 parts.

[0161] Example 2: Encapsulation efficiency test

[0162] Test samples: Recombinant collagen liposomes prepared in Examples 1-12, Comparative Examples 1-12, and Comparative Examples 14 and 15.

[0163] Test procedure: Recombinant collagen liposomes were taken, and after ultracentrifugation (centrifugation conditions: 50,000 rpm, 4℃, 1 h) and ultrafiltration, the content of recombinant collagen in the filtrate was determined by the BCA method. The result is the free protein concentration C. 游离 A small amount of recombinant collagen liposomes was taken, and 1% Triton X-100 solution was added. The mixture was vortexed and incubated at 37°C for 30 min to lyse the liposomes and release the encapsulated recombinant collagen. After lysing, the liposomes were subjected to ultracentrifugation (50,000 rpm, 4°C, 1 h) and ultrafiltration. The recombinant collagen content in the filtrate was determined by the BCA method. The result is the total protein concentration (after lysis) C. 总 The encapsulation efficiency (EE%) of recombinant collagen liposomes was calculated using formula (1). Formula (1) is as follows:

[0164]

[0165] In formula (1), C 游离 The concentration of free recombinant collagen; C 总 V represents the concentration of total recombinant collagen after lysis. 游离 V represents the volume of free recombinant collagen. 总 This represents the volume of the original liposomes.

[0166] The BCA method for protein quantification is referenced in the 2020 edition of the Chinese Pharmacopoeia, section 0731, Protein Content Determination Method - 2,2'-Biquinoline-4,4'-Dicarboxylic Acid Method (BCA Method).

[0167] The encapsulation efficiency test results are shown in Table 4.

[0168] Table 4

[0169]

[0170]

[0171] Test results show that the liposomes of the present invention have a good encapsulation effect on recombinant type XVII collagen, with an encapsulation rate of over 86%. Comparing Example 1 and Comparative Examples 1 and 2, it is evident that under the same weight fractions of collagen, wall material, antioxidant, and solvent, the liposomes exhibit poor encapsulation effect on recombinant type I and recombinant type III collagen, indicating that the liposomes of the present invention are more suitable for encapsulating recombinant type XVII collagen. Comparing Examples 1-12 and Comparative Examples 3-7, it is evident that the type of wall material, the presence or absence of antioxidants, the type of antioxidant, and the oil phase solvent all affect the encapsulation effect of the liposomes. Comparative Examples 3 and 4 replaced the wall material with similar materials such as hydroxypropyl cyclodextrin and glyceryl stearate / PEG-100; Comparative Example 5 replaced the antioxidant with tocopheryl acetate; Comparative Example 6 did not add an antioxidant; and Comparative Example 7 replaced glycerol with 1,2-butanediol. The encapsulation effect of liposomes on recombinant type XVII collagen deteriorated. In this invention, the preferred oil phase solvent is glycerol, the preferred wall material is soybean lecithin, and the preferred antioxidant is at least one of vitamin C ethyl ether and inositol. Comparing the encapsulation rates of Examples 1-3, it is clear that the antioxidant is more preferably a combination of vitamin C ethyl ether and inositol. Comparing Examples 1-12 and Comparative Examples 8-12, it is clear that the type of aqueous solvent affects the encapsulation effect of liposomes. In this invention, Dendrobium officinale enzymatic hydrolysate is used instead of deionized water. The Dendrobium officinale polysaccharides in the Dendrobium officinale enzymatic hydrolysate can modify liposomes, thereby improving the encapsulation effect of liposomes on recombinant collagen. Furthermore, different preparation methods of Dendrobium officinale enzymatic hydrolysate result in variations in the size and content of Dendrobium officinale polysaccharides, leading to different modification effects of Dendrobium officinale polysaccharides on liposomes and thus affecting the encapsulation effect. In this invention, the Dendrobium officinale enzymatic hydrolysate preferably undergoes an ultra-high pressure pretreatment step, and more preferably, it is a composite enzymatic hydrolysate subjected to two enzymatic hydrolysis treatments at different times. Comparative Examples 1-12 and Comparative Examples 14 and 15 show that the amount of recombinant type XVII collagen and soybean lecithin added affects the encapsulation effect of liposomes. Excessive addition of recombinant type XVII collagen and excessive phospholipids may cause the solubility of recombinant type XVII collagen in the phospholipid bilayer to reach saturation, leading to easy precipitation of recombinant type XVII collagen. In addition, high concentrations of phospholipids will occupy the effective loading sites of liposomes, preventing recombinant type XVII collagen from reaching the loading sites of liposomes, resulting in a poor encapsulation effect.

[0172] Example 3: Transdermal absorption effect test

[0173] Test samples: Recombinant collagen liposomes prepared in Examples 1-12, Comparative Examples 1-12, and Comparative Examples 14 and 15 were prepared into a 20% (w / w) solution using PBS buffer (pH 7.4±0.2) as the solvent. Control group: 4% recombinant type XVII collagen solution (same as the control group in Effect Example 1) was prepared into a 20% (w / w) solution using PBS buffer (pH 7.4±0.2) as the solvent.

[0174] Test Procedure: A vertical diffusion cell was used for the in vitro transdermal experiment, with porcine skin as the model. The receiving solution was phosphate buffered saline (PBS solution, pH 7.4±0.2). The skin patch was fixed between the supply and receiving cells with the skin layer facing upwards and equilibrated for 20 minutes. The test sample was added to the supply cell, and the receiving solution was collected after 24 hours. The receiving solution was treated with 1% Triton X-100 solution, and the content of recombinant collagen was determined by the BCA method. The permeability of recombinant collagen was calculated based on the initial content of recombinant collagen before the experiment and the content of recombinant collagen measured in the receiving solution.

[0175] The test results of transdermal absorption are shown in Table 5.

[0176] Table 5

[0177] Group Transmission rate (%) Group Transmittance (%) control group 4.36 Comparative Example 1 26.49 Example 1 52.51 Comparative Example 2 18.26 Example 2 50.17 Comparative Example 3 14.02 Example 3 49.46 Comparative Example 4 24.38 Example 4 46.62 Comparative Example 5 28.20 Example 5 48.18 Comparative Example 6 37.57 Example 6 45.74 Comparative Example 7 32.64 Example 7 47.15 Comparative Example 8 28.42 Example 8 42.76 Comparative Example 9 30.35 Example 9 51.08 Comparative Example 10 35.62 Example 10 48.96 Comparative Example 11 32.09 Example 11 43.25 Comparative Example 12 34.26 Example 12 40.73 Comparative Example 13 / / / Comparative Example 14 25.81 / / Comparative Example 15 35.52

[0178] Comparing Example 1 and the control group, it can be seen that the transdermal absorption of recombinant type XVII collagen was increased by 12 times after encapsulation with liposomes, and the transdermal absorption of recombinant type XVII collagen was significantly improved after encapsulation with liposomes. Examples 1-12 showed better transdermal absorption than Comparative Examples 1 and 2, indicating that the liposomes prepared in this invention are more conducive to the absorption of recombinant type XVII collagen. Examples 1-12 showed better transdermal absorption than Comparative Examples 3-7, indicating that the type of wall material in the liposomes, the presence or absence of antioxidants, the type of antioxidants, and the oil phase solvent affect the encapsulation effect of liposomes on recombinant type XVII collagen, further affecting the transdermal absorption effect of recombinant type XVII collagen. The preferred oil phase solvent in this invention is glycerol, the preferred wall material is soybean lecithin, and the preferred antioxidant is at least one of vitamin C ethyl ether and inositol. Examples 1-12 showed better transdermal absorption than Comparative Examples 8-11, indicating that using Dendrobium officinale enzymatic hydrolysate instead of deionized water to prepare liposomes resulted in a final product that was more conducive to the transdermal absorption of recombinant type XVII collagen. Furthermore, the Dendrobium officinale enzymatic hydrolysate preferably underwent ultra-high pressure pretreatment, and more preferably was a composite hydrolysate obtained through two different enzymatic hydrolysis treatments. Examples 1-12 showed better transdermal absorption than Comparative Example 12, indicating that liposomes modified with Dendrobium officinale polysaccharides were more conducive to the transdermal absorption of recombinant type XVII collagen. Examples 1-12 showed better transdermal absorption than Comparative Examples 14 and 15, indicating that liposomes prepared with appropriate amounts of recombinant type XVII collagen and soybean lecithin were more conducive to transdermal absorption. The preferred amounts of recombinant type XVII collagen were 1-10 parts, and the preferred amounts of soybean lecithin were 2-15 parts.

[0179] Example 4: Barrier Repair Effect Test

[0180] Test samples: Recombinant collagen liposomes or liposomes (samples after being placed at 25°C for 8 weeks) prepared in Examples 1-12 and Comparative Examples 1-15 were prepared into a 2% (w / w) solution using H-DMEM medium containing 10% FBS as the solvent; Control Group 1: 4% recombinant type XVII collagen solution (prepared fresh for use) was prepared into a 2% (w / w) solution using H-DMEM medium containing 10% FBS as the solvent; Control Group 2: 4% recombinant type XVII collagen solution (samples after being placed at 25°C for 8 weeks) was prepared into a 2% (w / w) solution using H-DMEM medium containing 10% FBS as the solvent.

[0181] Test Principle: Caspases are a group of cysteine-aspartate specific proteases, all of which are cysteine ​​proteases and possess specific aspartate cleavage sites, also known as the Caspase protease family. Caspase-14 is a member of this family. Caspase-14 exhibits skin and tissue specificity, primarily distributed in the stratum corneum and stratum granulosum of the epidermis. It plays a crucial role in maintaining epidermal homeostasis. Caspase-14 plays a key role in the terminal differentiation of keratinocytes. It is activated during stratum corneum formation, participating in the hydrolysis of pro-filaggrin to generate filaggrin, which in turn forms natural moisturizing factor (NMF), essential for skin hydration and barrier function. Therefore, the expression level of the Caspase-14 gene can be used to assess the barrier repair effect of the test sample.

[0182] Test procedure: HaCaT cells in the logarithmic growth phase were seeded into 6-well culture plates (2 × 10⁻⁶ cells / well). 5 HaCaT cells (1 cell / well) were added to H-DMEM medium containing 10% FBS and cultured at 37°C with 5% CO2 for 24 h. The culture medium was then discarded. The blank control group was treated with 1 mL of H-DMEM medium containing 10% FBS, and the sample group was treated with 1 mL of test sample. Cultures were continued for another 24 h. After culture, total RNA was extracted from HaCaT cells in each well, cDNA was synthesized, and q-PCR was used to detect Caspase-14 mRNA expression. Results were analyzed using 2... －△△CT The expression level of Caspase-14 mRNA in the test samples was calculated using the method described above. The expression level of Caspase-14 mRNA in the blank control group was set as 1.00.

[0183] The test results are shown in Table 6.

[0184] Table 6

[0185]

[0186] Test results show that the recombinant collagen liposomes of Examples 1-12 can promote the expression of Caspase-14 mRNA in HaCaT cells, indicating that the recombinant collagen liposomes of Examples 1-12 can promote skin barrier repair. Control group 1, which was a freshly prepared aqueous solution of recombinant collagen, showed a barrier repair effect. However, after 8 weeks of storage (Control group 2), the barrier repair effect of the aqueous solution of recombinant collagen decreased sharply, indicating that the recombinant collagen has poor stability; its skin-care effect is greatly reduced after being directly prepared as an aqueous solution and stored for a period of time. After recombinant collagen was encapsulated in liposomes (Examples 1-12), the recombinant collagen liposomes still showed a good barrier repair effect after 8 weeks of storage. The barrier repair effect of Examples 1-12 is better than that of Control Examples 1 and 2, indicating that the recombinant type XVII collagen liposomes encapsulated in this invention have better barrier repair efficacy than recombinant type I collagen liposomes and recombinant type XVII collagen liposomes, and the liposomes of this invention encapsulate recombinant type XVII collagen more effectively. The barrier repair effects of Examples 1-12 are superior to those of Comparative Examples 3-7, indicating that the type of liposome wall material, the type of antioxidant, and the type of oil phase solvent affect the stability of liposomes, and thus the barrier repair effect of the final product. Combined with the liposome stability test results, it is clear that the wall material, antioxidant, and oil phase solvent in the liposome material all affect the stability of the final product. Replacing the wall material, antioxidant, or solvent reduces the stability of the liposomes, causing flocculent matter to appear, thereby affecting the barrier repair effect of the final product. The preferred wall material of this invention is soybean lecithin, the preferred antioxidant is vitamin C ethyl ether and / or inositol, and the preferred solvent is glycerol. The barrier repair effects of Examples 1-12 are superior to those of Comparative Example 8, indicating that the addition of Dendrobium officinale enzymatic hydrolysate can improve the barrier repair effect of liposomes. Furthermore, the barrier repair effects of Examples 1-12 are superior to those of Comparative Examples 9-11, indicating that the preparation process of the Dendrobium officinale enzymatic hydrolysate affects the content of the final active substances in the Dendrobium officinale enzymatic hydrolysate, thus affecting the barrier repair effect of the final product. In this invention, the Dendrobium officinale enzymatic hydrolysate preferably undergoes an ultra-high pressure pretreatment step, and more preferably is a composite enzymatic hydrolysate subjected to two enzymatic hydrolysis treatments at different times. The barrier repair effects of Examples 1-12 are superior to those of Comparative Example 12, indicating that the type of plant enzymatic hydrolysate affects the barrier repair effect of liposomes. In this invention, recombinant collagen liposomes prepared from Dendrobium officinale enzymatic hydrolysate are preferred. The barrier repair effects of Examples 1-12 are superior to those of Comparative Example 13, indicating that the combined action of recombinant collagen and Dendrobium officinale enzymatic hydrolysate in this invention can improve the barrier repair effect of liposomes; without recombinant collagen, the barrier repair effect of liposomes deteriorates.The barrier repair effects of Examples 1-12 are better than those of Comparative Examples 14 and 15, indicating that the liposome barrier repair effect obtained by using appropriate amounts of recombinant type XVII collagen and soybean lecithin is better. In this invention, the recombinant type XVII collagen is preferably 1-10 parts and the soybean lecithin is preferably 2-15 parts.

[0187] Example 5: Anti-wrinkle effect test

[0188] Test samples: Recombinant collagen liposomes or liposomes (samples after being placed at 25°C for 8 weeks) prepared in Examples 1-12 and Comparative Examples 1-15 were prepared into a 2% (w / w) solution using H-DMEM medium containing 10% FBS as the solvent; Control group 1: 4% recombinant type XVII collagen solution (prepared fresh for use) was prepared into a 2% (w / w) solution using H-DMEM medium containing 10% FBS as the solvent; Control group 2: 4% recombinant type XVII collagen solution (samples after being placed at 25°C for 8 weeks) was prepared into a 2% (w / w) solution using H-DMEM medium containing 10% FBS as the solvent.

[0189] Test Principle: Skin aging is characterized by loss of elasticity, sagging, wrinkles, and a rough appearance. Numerous studies have demonstrated that UV radiation induces an increase in matrix metalloproteinases (MMPs) produced by human skin fibroblasts. MMPs degrade collagen and extracellular matrix proteins, leading to wrinkles. Inhibitors of human matrix metalloproteinases (TIMPs) are natural, specific, endogenous inhibitors of MMPs. By inhibiting the activity of MMPs (such as collagenase MMP-1 and gelatinase MMP-2 / MMP-9), they protect extracellular matrix (ECM) components (such as collagen, elastin, and hyaluronic acid) from excessive degradation. Upregulation of TIMP expression (such as inhibitor of human matrix metalloproteinases-1, TIMP-1) can directly reduce the damage of MMPs to collagen. Therefore, the anti-wrinkle effect of a test sample can be determined by testing the gene expression level of TIMP-1 in fibroblasts.

[0190] Test procedure: ESF-1 skin fibroblasts in the logarithmic growth phase were seeded into 6-well culture plates (2 × 10⁻⁶ cells / well). 5 Cells were added to each well (10% FBS) and cultured in H-DMEM medium at 37°C with 5% CO2 for 24 h. The culture medium was then discarded. 1 mL of H-DMEM medium was added to the blank control group, and 1 mL of test sample was added to the sample group. Cultures were continued for another 24 h. After culture, total RNA was extracted from each well of ESF-1 cells, cDNA was synthesized, and TIMP-1 mRNA expression was detected using q-PCR. Results were analyzed using 2... －△△CTThe calculation was performed using the method described above. The expression level of TIMP-1 mRNA in the blank control group was set as 1.00, and the expression level of TIMP-1 mRNA in the test sample was calculated accordingly.

[0191] The test results are shown in Table 7.

[0192] Table 7

[0193]

[0194] Test results show that the recombinant collagen liposomes of Examples 1-12 can promote the expression of the TIMP-1 gene. Enhanced TIMP expression can reduce the damage of MMPs to human skin collagen. Therefore, the recombinant collagen liposomes of the present invention exhibit an anti-wrinkle effect. Control group 1, which was a freshly prepared recombinant collagen aqueous solution, showed an anti-wrinkle effect. However, after 8 weeks of storage (Control group 2), the expression of the TIMP-1 gene in the recombinant collagen aqueous solution was comparable to that of the blank control group, indicating that the anti-wrinkle effect of the recombinant collagen aqueous solution after 8 weeks was very poor, suggesting poor stability of the recombinant collagen. After encapsulating recombinant collagen in liposomes (Examples 1-12), the recombinant collagen liposomes still showed a good anti-wrinkle effect after 8 weeks of storage. The anti-wrinkle effect of Examples 1-12 is better than that of Control Examples 1 and 2, indicating that the recombinant type XVII collagen liposomes encapsulated in the present invention have better anti-wrinkle efficacy than recombinant type I collagen liposomes and recombinant type XVII collagen liposomes, and the effect of the liposomes of the present invention encapsulating recombinant type XVII collagen is superior. The anti-wrinkle effects of Examples 1-12 are superior to those of Comparative Examples 3-7, indicating that the type of liposome wall material, the type of antioxidant, and the type of oil phase solvent affect the stability of liposomes, and thus the anti-wrinkle effect of the final product. Combined with the liposome stability test results, it is clear that the wall material, antioxidant, and oil phase solvent in the liposome material all affect the stability of the final product. Replacing the wall material, antioxidant, or solvent reduces the stability of the liposomes, causing flocculent matter to appear, thereby affecting the anti-wrinkle effect of the final product. The preferred wall material of this invention is soybean lecithin, the preferred antioxidant is vitamin C ethyl ether and / or inositol, and the preferred solvent is glycerol. The anti-wrinkle effects of Examples 1-12 are superior to those of Comparative Example 8, indicating that the addition of Dendrobium officinale enzymatic hydrolysate can improve the anti-wrinkle effect of the liposomes. Furthermore, the anti-wrinkle effects of Examples 1-12 are superior to those of Comparative Examples 9-12, indicating that the type of plant enzymatic hydrolysate and the preparation process of Dendrobium officinale enzymatic hydrolysate affect the content of the final active substances in the Dendrobium officinale enzymatic hydrolysate, thus affecting the anti-wrinkle effect of the final product. In this invention, the Dendrobium officinale enzymatic hydrolysate preferably undergoes an ultra-high pressure pretreatment step, and is preferably a composite enzymatic hydrolysate that has undergone two enzymatic hydrolysis treatments at different times. The anti-wrinkle effects of Examples 1-12 are superior to those of Comparative Example 13, indicating that the combined effect of recombinant collagen and Dendrobium officinale enzymatic hydrolysate in this invention can improve the anti-wrinkle effect of liposomes; without recombinant collagen, the anti-wrinkle effect of liposomes deteriorates. The anti-wrinkle effects of Examples 1-12 are superior to those of Comparative Examples 14 and 15, indicating that liposomes obtained with appropriate amounts of recombinant type XVII collagen and soybean lecithin have a better anti-wrinkle effect. In this invention, the recombinant type XVII collagen is preferably 1-10 parts, and the soybean lecithin is preferably 2-15 parts.

[0195] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A recombinant collagen liposome, characterized in that, By weight, it includes: 1-10 parts recombinant type XVII collagen, 2-15 parts soy lecithin, 0.3-2 parts tocopherol, 0.5-7 parts antioxidant, 20-54 parts glycerol, and Dendrobium officinale enzymatic hydrolysate to a total weight of 100. The antioxidants include vitamin C ethyl ether and / or inositol; The preparation method of the Dendrobium officinale enzymatic hydrolysate includes: S1. After mixing the crushed Dendrobium officinale stem segments with water, the mixture is subjected to ultra-high pressure treatment to obtain pretreated material; S2. Add a pH adjuster to the pretreated material in step S1 to obtain a mixture, and divide the mixture into mixture A and mixture B. S3. Add a breakdown enzyme to the mixture A described in step S2 and perform a single enzymatic hydrolysis to obtain hydrolysate A. S4. Add the breakdown enzyme to the mixture B described in step S2 and perform a second enzymatic hydrolysis to obtain hydrolysate B. S5. Mix the enzymatic hydrolysate A from step S3 and the enzymatic hydrolysate B from step S4 thoroughly, then heat and filter to obtain the Dendrobium officinale enzymatic hydrolysate; wherein... In step S1, the mass ratio of *Dendrobium officinale* stem segments to water is 1:(50-120); the pressure of the ultra-high pressure treatment in step S1 is 50-250 MPa, and the treatment time is 15-30 min; the pH adjuster in step S2 includes at least one of hydrochloric acid solution, sulfuric acid solution, phosphoric acid solution, citric acid solution, and lactic acid solution, and the concentration of the acid in the pH adjuster is 1.0-6.0 mol / L; the pH of the mixture in step S2 is adjusted to 5.0-6.0; the mass ratio of mixture A to mixture B in step S2 is (1-2):(1-2); the breakdown in step S3... The amount of catalytic enzyme added in step S2 is 0.001-0.01% of the mass of mixture A; the temperature of the first enzymatic hydrolysis treatment in step S3 is 35-45℃, and the time of the first enzymatic hydrolysis is 3-6h; the amount of catalytic enzyme added in step S4 is 0.001-0.01% of the mass of mixture B in step S2; the temperature of the second enzymatic hydrolysis treatment in step S4 is 35-45℃, and the time of the second enzymatic hydrolysis is 8-12h; the heating temperature in step S5 is 80-90℃, and the heating treatment time is 15-30min; the filtration method in step S5 includes diatomaceous earth filter filtration.

2. The recombinant collagen liposomes according to claim 1, characterized in that, The recombinant collagen liposomes, by weight, comprise 2-8 parts of recombinant type XVII collagen, 3-12 parts of soybean lecithin, 0.4-1.2 parts of tocopherol, 1-4 parts of antioxidant, 25-45 parts of glycerol, and Dendrobium officinale enzymatic hydrolysate to a total weight of 100.

3. The recombinant collagen liposomes according to claim 1, characterized in that, The antioxidants include vitamin C ethyl ether and inositol, wherein the mass ratio of vitamin C ethyl ether to inositol is (1-3):(1-3).

4. The method for preparing recombinant collagen liposomes according to any one of claims 1-3, characterized in that, include: (1) Mix recombinant type XVII collagen, antioxidant and Dendrobium officinale enzymatic hydrolysate evenly to obtain phase A; (2) Mix soybean lecithin, tocopherol and glycerol evenly to obtain phase B; (3) Add phase A to phase B and mix to obtain crude liposomes; (4) The crude liposomes are subjected to high-speed shear dispersion and high-pressure homogenization to obtain recombinant collagen liposomes.

5. The method for preparing recombinant collagen liposomes according to claim 4, characterized in that, include: Include at least one of the following (XII)-(XVI): (XII) The mixing temperatures described in steps (1) and (2) are relatively independent of each other, ranging from 20 to 40°C; (XⅢ) The rate at which phase A is added in step (3) is 4-8 mL / min; (XIV) The temperature of the mixing reaction in step (3) is 55-65℃, and the time of the mixing reaction is 40-90 min; (XV) The high-speed shear dispersion rate in step (4) is 5000-8000 rpm, and the high-speed shear dispersion time is 8-15 min; (XVI) The pressure of the high-pressure homogenization process in step (4) is 50-120 MPa, and the number of cycles is 2-6.

6. The application of the recombinant collagen liposome according to claim 1 in the preparation of cosmetics.

7. The cosmetic product according to claim 6, characterized in that, The amount of the recombinant collagen liposomes added to the cosmetic product is 0.1-50% by weight.

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