Recombinant collagen glyceride and methods of making the same

Abstract

The application discloses a kind of recombinant collagen glycerol body and preparation method thereof.The recombinant collagen glycerol body is 100 parts by total weight, and is composed of 2.7-3.7 parts of recombinant collagen, 5-15 parts of glycerol, 3.0-4.0 parts of L-alpha-phosphatidylcholine, 0.55-0.75 parts of emulsion regulator cetyl alcohol, 2 parts of preservative 1,2-pentanediol and 0.5 parts of 1,2-hexanediol, 0.05-0.10 parts of thickening agent xanthan gum, 0.1 parts of sodium chloride and 75.05~84.9 parts of purified water, and is prepared by water phase precipitation method.The application uses glycerol body as the carrier of recombinant collagen, which can improve the stability of recombinant collagen on one hand, and promote the transdermal absorption of recombinant collagen on the other hand.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and relates to a recombinant collagen glycerosome and a preparation method thereof. BACKGROUND

[0002] The glycerosome is a carrier prepared by adding a certain amount of glycerol into a liposome, and due to the strong interaction between glycerol and the polar head group of phospholipid molecules, the vesicle stability is enhanced. The glycerosome is more stable and has stronger fluidity than the liposome, and is thus mainly used for transdermal delivery. Meanwhile, glycerol can improve the deformation index of the liposome bilayer, thereby enhancing the skin permeability.

[0003] The recombinant collagen is a new recombinant human collagen type III sequence obtained by codon optimization and splicing recombination of a part of human skin collagen type III sequence with strong water solubility and high biological activity, and is expressed by using a biological fermentation technology (CN102443057B). The recombinant collagen has good water solubility and high biological activity, and its performance is superior to that of human natural collagen, and thus has a wide application prospect in the fields of biomedical materials, cosmetics, food health care and the like. However, the recombinant collagen is prone to hydrolysis in an aqueous solution, has poor stability, and has a molecular weight of 55KD, and thus has poor transdermal absorption, and after use, most of the recombinant collagen stays on the skin surface and cannot further play a role. SUMMARY

[0004] The present application provides a recombinant collagen glycerosome and a preparation method thereof.

[0005] The technical solution for achieving the present application is as follows:

[0006] The recombinant collagen glycerosome is composed of an active ingredient, a glycerosome framework ingredient, a guest ingredient and purified water, wherein the active ingredient is recombinant collagen, the glycerosome framework ingredient includes glycerol and phospholipid, and the guest ingredient includes an emulsion regulator, a thickening agent and a preservative.

[0007] Further, the recombinant collagen glycerosome according to the present application is composed of the following components in the total weight of 100 parts: 2.7-3.7 parts of recombinant collagen, 5-15 parts of glycerol, 3.0-4.0 parts of L-α-phosphatidylcholine, 0.55-0.75 parts of emulsion regulator cetyl alcohol, 2 parts of preservative 1,2-pentanediol and 0.5 parts of 1,2-hexanediol, 0.05-0.10 parts of thickening agent xanthan gum, 0.1 part of sodium chloride and 75.05-84.9 parts of purified water, and the recombinant collagen is produced by fermentation of Pichia pastoris with the preservation number of CGMCC No. 5021.

[0008] The recombinant collagen protein is fully disclosed in Chinese Patent CN102443057B.

[0009] Preferably, the recombinant collagen glycerosomes consist of 3.2 parts of recombinant collagen, 10 parts of glycerol, 3 parts of L-α-phosphatidylcholine, 0.65 parts of cetyl alcohol as emulsion regulator, 2 parts of 1,2-pentanediol and 0.5 parts of 1,2-hexanediol as preservatives, 0.08 parts of xanthan gum as thickening agent, 0.1 parts of sodium chloride and 80.47 parts of purified water, based on 100 parts by total weight.

[0010] Preferably, the average particle size of the recombinant collagen glycerosome vesicles is 150-300 nm.

[0011] The recombinant collagen glycerosomes are prepared by a water-phase precipitation method, comprising the following steps:

[0012] (1) The L-α-phosphatidylcholine is dissolved in 5 times the mass of ethanol at 65-85°C in a water bath, and then cetyl alcohol as emulsion regulator is added and stirred uniformly to obtain an organic solvent system;

[0013] (2) The glycerol, recombinant collagen, thickening agent xanthan gum, preservatives 1,2-pentanediol and 1,2-hexanediol, sodium chloride and purified water are dissolved completely and mixed uniformly at 50-60°C in a water bath to obtain a water-phase system;

[0014] (3) The organic solvent system is added dropwise to the water-phase system while being subjected to ultrasonic treatment to evaporate the ethanol, and then the glycerosomes are obtained and filtered through a 0.45 μm hydrophilic filter membrane to prepare uniform and stable recombinant collagen glycerosomes.

[0015] Compared with the prior art, the present application has the following advantages:

[0016] (1) The present application uses glycerosomes as the carrier of recombinant collagen, which significantly improves the encapsulation rate of recombinant collagen liposomes and significantly improves the stability thereof.

[0017] (2) The present application significantly promotes the transdermal absorption of recombinant collagen, and the solution contains various skin moisturizing agents, which, in combination with collagen, can provide additional moisturizing effect and enhance the skin care efficacy of collagen, thereby achieving the effects of moisturizing, tightening, repairing and anti-wrinkling. DETAILED DESCRIPTION

[0018] The present application will be further described in detail below in conjunction with specific examples.

[0019] The recombinant collagen used in the following examples is purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0020] In the following examples and comparative examples, the method for measuring subcutaneous permeability is as follows:

[0021] Ex vivo skin test: Using a micropipette, at a concentration of 100 μg / cm 2 (Based on recombinant collagen content), the product was applied to isolated pig skin, which was then exposed in a diffusion chamber for 7 hours at a temperature maintained at 35±1℃. After 7 hours of exposure, the skin was removed. The stratum corneum was removed using adhesive tape, and the epidermal basal layer was separated. All skin areas and the adhesive tape were immersed in 2.5 ml of 50% methanol aqueous solution (v / v). The samples were then sonicated for 30 minutes, and collagen was extracted from the skin, which was then quantitatively analyzed by HPLC.

[0022] Example 1

[0023] The recombinant collagen glycerol bodies in this embodiment are prepared through the following steps:

[0024] By weight, 3.2 parts recombinant collagen, 10 parts glycerol, 3 parts L-α-phosphatidylcholine, 0.65 parts cetyl alcohol, 2 parts 1,2-pentanediol, 0.5 parts 1,2-hexanediol, 0.08 parts xanthan gum, 0.1 parts sodium chloride, and 80.47 parts purified water were weighed. First, L-α-phosphatidylcholine was dissolved in 5 times its weight of ethanol in a 75°C water bath. Then, cetyl alcohol was added and stirred until homogeneous to obtain an organic solvent system. Glycerol, recombinant collagen, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride, and purified water were completely dissolved in a 55°C water bath and mixed thoroughly to obtain an aqueous phase system. The organic solvent system was added dropwise to the aqueous phase system while simultaneously sonicating to evaporate the ethanol, yielding glycerol bodies. These were then filtered through a 0.45 μm hydrophilic membrane to obtain homogeneous and stable recombinant collagen glycerol bodies.

[0025] Example 2

[0026] The recombinant collagen glycerol bodies in this embodiment are prepared through the following steps:

[0027] The following ingredients are weighed according to the weight fraction: 2.7 parts of recombinant collagen, 5 parts of glycerol, 4 parts of L-α-phosphatidylcholine, 0.75 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.05 parts of xanthan gum, 0.1 parts of sodium chloride and 84.9 parts of purified water. First, the L-α-phosphatidylcholine is dissolved in 5 times the mass of ethanol under the condition of a water bath at 85°C, then the cetyl alcohol is added and stirred uniformly to obtain an organic solvent system; the glycerol, recombinant collagen, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved and uniformly mixed under the condition of a water bath at 60°C to obtain an aqueous phase system; the organic solvent system is added dropwise to the aqueous phase system while ultrasonic treatment is performed to evaporate the ethanol, and a glycerosome is obtained, which is then filtered through a 0.45 μm hydrophilic filter membrane to obtain a uniform and stable recombinant collagen glycerosome.

[0028] Example 3

[0029] The recombinant collagen glycerosome of this example is prepared by the following steps:

[0030] The following ingredients are weighed according to the weight fraction: 3.7 parts of recombinant collagen, 15 parts of glycerol, 3.0 parts of L-α-phosphatidylcholine, 0.55 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.1 parts of xanthan gum, 0.1 parts of sodium chloride and 75.05 parts of purified water. First, the L-α-phosphatidylcholine is dissolved in 5 times the mass of ethanol under the condition of a water bath at 65°C, then the cetyl alcohol is added and stirred uniformly to obtain an organic solvent system; the glycerol, recombinant collagen, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved and uniformly mixed under the condition of a water bath at 50°C to obtain an aqueous phase system; the organic solvent system is added dropwise to the aqueous phase system while ultrasonic treatment is performed to evaporate the ethanol, and a glycerosome is obtained, which is then filtered through a 0.45 μm hydrophilic filter membrane to obtain a uniform and stable recombinant collagen glycerosome.

[0031] Comparative Example 1

[0032] By weight parts, respectively, 3.2 parts of recombinant collagen, 10 parts of glycerol, 3.0 parts of L-α-phosphatidylcholine, 0.65 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.08 parts of xanthan gum, 0.1 parts of sodium chloride and 80.47 parts of purified water. First, L-α-phosphatidylcholine is dissolved in 5 times the mass of ethanol under the condition of 95℃ water bath, then cetyl alcohol is added, stirred uniformly, to obtain an organic solvent system; glycerol, recombinant collagen, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved and uniformly mixed under the condition of 45℃ water bath, to obtain an aqueous phase system; the organic solvent system is added dropwise into the aqueous phase system, and ultrasonic treatment is carried out at the same time, so that ethanol is evaporated, to obtain a glycerosome, which is then filtered through a 0.45μm hydrophilic filter membrane, to finally obtain a recombinant collagen glycerosome.

[0033] Comparative Example 2

[0034] By weight parts, respectively, 2.7 parts of recombinant collagen, 5 parts of glycerol, 4.0 parts of L-α-phosphatidylcholine, 0.75 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.05 parts of xanthan gum, 0.1 parts of sodium chloride and 84.9 parts of purified water. First, L-α-phosphatidylcholine is dissolved in 5 times the mass of ethanol under the condition of 50℃ water bath, then cetyl alcohol is added, stirred uniformly, to obtain an organic solvent system; glycerol, recombinant collagen, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved and uniformly mixed under the condition of 80℃ water bath, to obtain an aqueous phase system; the organic solvent system is added dropwise into the aqueous phase system, and ultrasonic treatment is carried out at the same time, so that ethanol is evaporated, to obtain a glycerosome, which is then filtered through a 0.45μm hydrophilic filter membrane, to finally obtain a recombinant collagen glycerosome.

[0035] Comparative Example 3

[0036] By weight parts, respectively, 3.7 parts of recombinant collagen, 3.0 parts of L-α-phosphatidylcholine, 0.55 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.1 parts of xanthan gum, 0.1 parts of sodium chloride and 90.05 parts of purified water. First, L-α-phosphatidylcholine is dissolved in 5 times the mass of ethanol under the condition of 75℃ water bath, then cetyl alcohol is added, stirred uniformly, to obtain an organic solvent system; recombinant collagen, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved and uniformly mixed under the condition of 55℃ water bath, to obtain an aqueous phase system; the organic solvent system is added dropwise into the aqueous phase system, and ultrasonic treatment is carried out at the same time, so that ethanol is evaporated, to obtain an emulsion, which is then filtered through a 0.45μm hydrophilic filter membrane, to finally obtain a recombinant collagen emulsion.

[0037] Comparative Example 4

[0038] Take 3.2 parts of recombinant collagen, 10 parts of glycerol, 3 parts of phosphatidylglycerol, 0.65 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.08 parts of xanthan gum, 0.1 parts of sodium chloride and 80.47 parts of purified water by weight. First, dissolve the phosphatidylglycerol in 5 times the mass of ethanol at 75°C water bath, then add cetyl alcohol, stir evenly, get the organic solvent system; glycerol, recombinant collagen, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved at 55°C water bath, mixed evenly, get the water phase system; the organic solvent system is added dropwise into the water phase system, and ultrasonic treatment is carried out at the same time, so that ethanol is evaporated, and then glycerol body is obtained. Then filter through 0.45 μm hydrophilic filter membrane, finally get recombinant collagen glycerol body.

[0039] Comparative Example 5

[0040] Recombinant collagen glycerol body is prepared by thin film evaporation method, the specific steps are as follows:

[0041] Take 3.2 parts of recombinant collagen, 10 parts of glycerol, 3 parts of L-α-phosphatidylcholine, 0.65 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.08 parts of xanthan gum, 0.1 parts of sodium chloride and 80.47 parts of purified water by weight. First, dissolve L-α-phosphatidylcholine and glycerol in 5 times the mass of ethanol at 75°C water bath, rotary evaporation, get uniform glycerol body lipid film; the recombinant collagen, cetyl alcohol, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved at 55°C water bath, mixed evenly, get mixed liquid; add the mixed liquid to the glycerol body lipid film, ultrasonic crushing and mixing evenly, homogenization, then filter through 0.45 μm hydrophilic filter membrane, finally get glycerol suspension, that is, recombinant collagen glycerol body.

[0042] Comparative Example 6

[0043] Recombinant collagen glycerol body is prepared by lipid fusion method, the specific steps are as follows:

[0044] By weight parts, respectively, 3.2 parts of recombinant collagen, 10 parts of glycerol, 3 parts of L-α-phosphatidylcholine, 0.65 parts of cetyl alcohol, 2 parts of 1,2-pentanediol, 0.5 parts of 1,2-hexanediol, 0.08 parts of xanthan gum, 0.1 parts of sodium chloride and 80.47 parts of purified water. First, L-α-phosphatidylcholine and glycerol are dissolved in 5 times the mass of ethanol under the condition of 95℃ water bath, rotary evaporation, to obtain a uniform glycerol lipid film; the recombinant collagen, cetyl alcohol, 1,2-pentanediol, 1,2-hexanediol, xanthan gum, sodium chloride and purified water are completely dissolved and uniformly mixed under the condition of 45℃ water bath to obtain a mixed liquid; the mixed liquid is added to the glycerol lipid film, ultrasonically broken and uniformly mixed, homogenized, and then filtered through a 0.45μm hydrophilic filter membrane to obtain a glycerol suspension, i.e., a recombinant collagen glycerol body.

[0045] Table 1

[0046]

[0047]

[0048] Compared with Example 1, the water bath temperature of the organic solvent system of Comparative Example 1 is too high, and the water bath temperature of the aqueous system is too low; compared with Example 2, the water bath temperature of the organic solvent system of Comparative Example 2 is too low, and the water bath temperature of the aqueous system is too high. The products finally prepared in Comparative Examples 1 and 2 cannot form a uniform emulsion liquid in appearance, and the particle size is larger and the encapsulation rate is lower compared with the products of Examples 1 and 2. The degradation rate of collagen in the aqueous solution is faster, and the subcutaneous permeability is significantly weakened. The above results show that for the specific recombinant collagen glycerol body solution of the present application, a suitable water bath temperature is more conducive to the formation of regular particle size of liposomes. The water bath temperature has different degrees of influence on the appearance, encapsulation rate, stability of recombinant collagen in the aqueous solution and collagen transdermal permeability of the product. Therefore, suitable process conditions are required for the preparation of recombinant collagen glycerol body.

[0049] Compared with Example 3, Comparative Example 3 lacks glycerol, a component of the glycerol body framework; compared with Example 1, Comparative Example 4 replaces L-α-phosphatidylcholine in the glycerol body framework component with phosphatidylglycerol. The particle size of the products finally prepared in Comparative Examples 3 and 4 is very large, the encapsulation rate is low, the degradation rate of collagen in the product in the aqueous solution is too fast, and the transdermal permeability is poorer. The glycerol body framework component and the guest component can ensure the stability and transdermal permeability of the product. The above results show that the glycerol body framework component cannot be absent, and the selection of L-α-phosphatidylcholine in the framework component can significantly improve the stability and transdermal permeability of the glycerol body.

[0050] Compared with Example 1, the collagen glycerosomes synthesized by thin film evaporation method and lipid fusion method in Comparative Examples 5 and 6, respectively, have large particle size, low encapsulation efficiency, fast degradation of collagen in aqueous solution, and poor transdermal property. The above results show that the aqueous phase precipitation method is more suitable for preparing recombinant collagen glycerosomes.

[0051] In summary, the appropriate proportion of glycerosome framework components, guest components, suitable phospholipids and synthesis process can ensure the appearance, stability and transdermal property of the product. Meanwhile, the solution contains a variety of skin moisturizers, which can play the effects of moisturizing, firming, repairing and anti-wrinkling in the application end of the finished product together with collagen.

Claims

1. A recombinant collagen glyceride, characterized in that, By total weight parts for 100 parts, consisting of: recombinant collagen 2.7-3.7 parts, glycerol 5-15 parts, L-α-phosphatidylcholine 3.0-4.0 parts, emulsion regulator cetyl alcohol 0.55-0.75 parts, preservative 1,2-pentanediol 2 parts and 1,2-hexanediol 0.5 parts, thickening agent xanthan gum 0.05-0.10 parts, sodium chloride 0.1 parts and 75.05~84.9 parts of purified water, the said recombinant collagen is produced by Pichia pastoris fermentation of Pasteur with accession number CGMCC No. 5021, prepared by the following steps: (1) according to the proportion, L-α-phosphatidylcholine is dissolved in 5 times the mass of ethanol under the condition of 65-85℃ water bath, then emulsion regulator cetyl alcohol is added, stirred uniformly, and the organic solvent system is obtained; (2) glycerol, recombinant collagen, thickening agent xanthan gum, preservative 1,2-pentanediol, 1,2-hexanediol, sodium chloride and purified water are completely dissolved and uniformly mixed at 50-60℃ water bath, and the water phase system is obtained; (3) the organic solvent system is added dropwise into the water phase system, and ultrasonic treatment is carried out at the same time, so that ethanol is evaporated, and glycerol body is obtained, then filtered through 0.45μm hydrophilic filter membrane, and uniform and stable recombinant collagen glycerol body is prepared.

2. The recombinant collagen glyceride of claim 1, wherein, By total weight parts for 100 parts, consisting of: recombinant collagen 2.7-3.7 parts, glycerol 5-15 parts, L-α-phosphatidylcholine 3.0-4.0 parts, emulsion regulator cetyl alcohol 0.55-0.75 parts, preservative 1,2-pentanediol 2 parts and 1,2-hexanediol 0.5 parts, thickening agent xanthan gum 0.05-0.10 parts, sodium chloride 0.1 parts and 75.05~84.9 parts of purified water, the said recombinant collagen is produced by Pichia pastoris fermentation of Pasteur with accession number CGMCC No. 5021, prepared by the following steps:

3. The recombinant collagen glyceride of claim 1, wherein, The average particle size of recombinant collagen glycerol body vesicle is 150~300nm.

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