A preparation method of protein body FNL

Abstract

The present invention provides a method for preparing protein-containing FNL (FNL), belonging to the field of biomedicine. The method comprises: dissolving soy lecithin and cholesterol in a solvent and rotary evaporating to obtain a thin film; dissolving sodium alginate, allantoin, and fibronectin in a buffer to obtain a protein solution; mixing the protein solution with the obtained thin film and rotating to obtain a rotating solution; and ultrasonicating the obtained rotating solution and extruding it through a microporous filter membrane to obtain protein-containing FNL. Sodium alginate and allantoin enhance the transdermal absorption rate of fibronectin, reaching over 70% after 24 hours, an increase of over 22% compared to the rate without sodium alginate and allantoin.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for preparing protein FNL. Background Art

[0002] Fibronectin, also known as fibronectin, adhesion protein, or fibronectin (abbreviated as FN), is primarily produced by the liver and vascular endothelial cells and is present in animal tissues and tissue fluids. It is a large glycoprotein with a dimer molecular weight of approximately 450 kDa and exhibits multiple biological activities. Fibronectin is highly conserved throughout evolution, and fibronectin in various animal body fluids has very similar structures, properties, and biological functions, making fibronectin from different sources interchangeable. Fibronectin can promote cell growth, increase cell attachment, enhance cell metabolism, shorten cell growth time, and improve cell fusion rates in hybridoma technology.

[0003] Fibronectin can remove dark spots, provide sun protection, moisturize, fill skin sags, and scavenge excess free radicals. Fibronectin has multiple benefits, including removing dark spots, providing sun protection, post-sun repair, moisturizing, anti-aging, wrinkle reduction, and repair. Therefore, fibronectin has potential applications in cosmetics. However, as a high-molecular-weight glycoprotein, fibronectin cannot be absorbed directly into the skin and thus fails to achieve its intended effects. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for preparing protein FNL. The preparation method provided by the present invention improves the skin absorption rate of protein FNL.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing protein FNL, comprising the following steps:

[0007] 1) dissolving soybean lecithin and cholesterol in a solvent and rotary evaporating the solvent to obtain a thin film;

[0008] 2) dissolving sodium alginate, allantoin, and fibronectin in a buffer solution to obtain a protein solution, and mixing the protein solution with the film obtained in step 1) and rotating to obtain a rotating solution;

[0009] 3) Ultrasonic treatment is performed on the rotating solution obtained in step 2), and the solution is squeezed through a microporous filter membrane to obtain protein bodies FNL.

[0010] Preferably, in step 1), the mass ratio of soybean lecithin to cholesterol is 8-12:3-5.

[0011] Preferably, in step 1), the volume ratio of the soybean lecithin to the solvent is 8-12 g: 250-300 ml.

[0012] Preferably, the solvent comprises ethanol and / or chloroform.

[0013] Preferably, in step 2), the mass ratio of sodium alginate to allantoin and fibronectin is 0.5-0.8:0.1-0.3:0.012-0.018.

[0014] Preferably, in step 2), the volume ratio of fibronectin to buffer is 12-18 mg:50-100 ml;

[0015] The mass ratio of the soybean lecithin to the fibronectin is 8-12:0.012-0.018.

[0016] Preferably, the conditions for the rotary evaporation in step 1) include: a temperature of 25 to 35° C. and a rotation speed of 150 to 250 rpm;

[0017] The conditions for the rotation in step 2) include: a temperature of 40-45° C., a time of 20-25 min, and a rotation speed of 100-200 rpm.

[0018] Preferably, the pore size of the microporous filter membrane in step 3) is 0.45 μm.

[0019] The present invention also provides a protein body FNL prepared by the preparation method described in the above technical solution.

[0020] The present invention also provides the use of the protein FNL described in the above technical solution in the preparation of drugs or cosmetics for improving skin transdermal absorption rate.

[0021] The beneficial effects of the present invention are:

[0022] Sodium alginate and allantoin increased the transdermal absorption rate of fibronectin, reaching more than 70% after 24 hours, which was an increase of more than 22% compared with not using sodium alginate and allantoin. DETAILED DESCRIPTION

[0023] The present invention provides a method for preparing protein FNL, comprising the following steps:

[0024] 1) dissolving soybean lecithin and cholesterol in a solvent and rotary evaporating the solvent to obtain a thin film;

[0025] 2) dissolving sodium alginate, allantoin, and fibronectin in a buffer solution to obtain a protein solution, and mixing the protein solution with the film obtained in step 1) and rotating to obtain a rotating solution;

[0026] 3) Ultrasonic treatment is performed on the rotating solution obtained in step 2), and the solution is squeezed through a microporous filter membrane to obtain protein bodies FNL.

[0027] The present invention dissolves soybean lecithin and cholesterol in a solvent and performs rotary evaporation to obtain a film. In the present invention, the mass ratio of soybean lecithin to cholesterol is preferably 8-12 g:3-5. In the present invention, the volume ratio of the mass of soybean lecithin to the solvent is preferably 8-12 g:250-300 ml. In the present invention, the solvent preferably includes ethanol and / or chloroform. In the present invention, the rotary evaporation conditions preferably include: a temperature of 25-35° C. and a rotation speed of 150-250 rpm.

[0028] The present invention dissolves sodium alginate, allantoin and fibronectin in a buffer solution to obtain a protein solution, mixes the protein solution with the obtained film, and rotates to obtain a rotating solution. In the present invention, the mass ratio of the sodium alginate, allantoin and fibronectin is preferably 0.5-0.8:0.1-0.3:0.012-0.018. In the present invention, the volume ratio of the mass of the fibronectin to the buffer solution is preferably 12-18 mg:50-100 ml. In the present invention, the mass ratio of the soy lecithin to the fibronectin is preferably 8-12:0.012-0.018. In the present invention, the buffer solution is preferably a phosphate buffer solution, and the pH value of the phosphate buffer solution is preferably 6.5-7.0.

[0029] The present invention ultrasonically processes the resulting rotating solution and extrudes it through a microporous filter membrane to obtain protein FNL. In the present invention, the ultrasonication conditions preferably include: a power of 250 to 350 W and a duration of 2 to 5 minutes. In the present invention, the pore size of the microporous filter membrane is preferably 0.45 μm.

[0030] The present invention also provides the protein body FNL prepared by the preparation method described in the above technical solution.

[0031] The present invention also provides the use of the protein FNL described in the above technical solution in the preparation of drugs or cosmetics with improved skin transdermal absorption rate. The present invention does not specifically limit the preparation method of the drug or cosmetic, and those skilled in the art can use conventional methods.

[0032] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] A method for preparing protein FNL, comprising the following steps:

[0035] 1) Dissolve 8 g of soybean lecithin and 5 g of cholesterol in 250 ml of chloroform and rotary evaporate at 25° C. and 150 rpm to obtain a thin film;

[0036] 2) dissolving 0.5 g of sodium alginate, 0.1 g of allantoin, and 12 mg of fibronectin in 100 ml of phosphate buffer (pH 6.5) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 40° C. and 100 rpm for 20 min to obtain a rotating solution;

[0037] 3) The rotating solution obtained in step 2) was sonicated at a power of 250 W for 5 min, and the whole particles were squeezed through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0038] Example 2

[0039] A method for preparing protein FNL, comprising the following steps:

[0040] 1) Dissolve 12 g of soybean lecithin and 4 g of cholesterol in 300 ml of chloroform and perform rotary evaporation at 30° C. and 200 rpm to obtain a thin film;

[0041] 2) dissolving 0.8 g of sodium alginate, 0.3 g of allantoin, and 15 mg of fibronectin in 80 ml of phosphate buffer (pH 6.7) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 40° C. and 150 rpm for 20 minutes to obtain a rotating solution;

[0042] 3) The rotating solution obtained in step 2) was sonicated at a power of 300 W for 4 min, and granulated by extrusion through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0043] Example 3

[0044] A method for preparing protein FNL, comprising the following steps:

[0045] 1) Dissolve 6 g of soybean lecithin and 4 g of cholesterol in 250 ml of chloroform and rotary evaporate at 25° C. and 150 rpm to obtain a thin film;

[0046] 2) dissolving 0.6 g of sodium alginate, 0.2 g of allantoin, and 15 mg of fibronectin in 100 ml of phosphate buffer (pH 7.0) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 45° C. and 150 rpm for 20 minutes to obtain a rotating solution;

[0047] 3) The rotating solution obtained in step 2) was sonicated at a power of 300 W for 3 min, and squeezed through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0048] Example 4

[0049] A method for preparing protein FNL, comprising the following steps:

[0050] 1) Dissolve 8 g of soybean lecithin and 3 g of cholesterol in 250 ml of chloroform and rotary evaporate at 25° C. and 150 rpm to obtain a thin film;

[0051] 2) dissolving 0.5 g of sodium alginate, 0.3 g of allantoin, and 18 mg of fibronectin in 90 ml of phosphate buffer (pH 6.5) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 40° C. and 100 rpm for 20 minutes to obtain a rotating solution;

[0052] 3) The rotating solution obtained in step 2) was sonicated at a power of 250 W for 5 min, and the whole particles were squeezed through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0053] Example 5

[0054] A method for preparing protein FNL, comprising the following steps:

[0055] 1) Dissolve 10 g of soybean lecithin and 3 g of cholesterol in 300 ml of chloroform and rotary evaporate at 25° C. and 150 rpm to obtain a thin film;

[0056] 2) dissolving 0.4 g of sodium alginate, 0.2 g of allantoin, and 15 mg of fibronectin in 100 ml of phosphate buffer (pH 6.5) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 40° C. and 100 rpm for 20 minutes to obtain a rotating solution;

[0057] 3) The rotating solution obtained in step 2) was sonicated at a power of 280 W for 3 min, and the whole particles were squeezed through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0058] Comparative Example 1

[0059] A method for preparing protein FNL, comprising the following steps:

[0060] 1) Dissolve 6 g of soybean lecithin and 4 g of cholesterol in 250 ml of chloroform and rotary evaporate at 25° C. and 150 rpm to obtain a thin film;

[0061] 2) dissolving 15 mg of fibronectin in 100 ml of phosphate buffer (pH 7.0) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 45° C. and 150 rpm for 20 minutes to obtain a rotating solution;

[0062] 3) The rotating solution obtained in step 2) was sonicated at a power of 300 W for 3 min, and the whole particles were squeezed through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0063] Comparative Example 2

[0064] A method for preparing protein FNL, comprising the following steps:

[0065] 1) Dissolve 8 g of soybean lecithin and 3 g of cholesterol in 250 ml of chloroform and rotary evaporate at 25° C. and 150 rpm to obtain a thin film;

[0066] 2) dissolving 0.5 g of sodium alginate and 18 mg of fibronectin in 90 ml of phosphate buffer (pH 6.5) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 40° C. and 100 rpm for 20 minutes to obtain a rotating solution;

[0067] 3) The rotating solution obtained in step 2) was sonicated at a power of 250 W for 5 min, and the whole particles were squeezed through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0068] Comparative Example 3

[0069] A method for preparing protein FNL, comprising the following steps:

[0070] 1) Dissolve 10 g of soybean lecithin and 3 g of cholesterol in 300 ml of chloroform and rotary evaporate at 25° C. and 150 rpm to obtain a thin film;

[0071] 2) dissolving 0.2 g of allantoin and 15 mg of fibronectin in 100 ml of phosphate buffer (pH 6.5) to obtain a protein solution, mixing the protein solution with the film obtained in step 1), and rotating at 40° C. and 100 rpm for 20 minutes to obtain a rotating solution;

[0072] 3) The rotating solution obtained in step 2) was sonicated at a power of 280 W for 3 min, and the whole particles were squeezed through a 0.45 μm microporous filter membrane to obtain protein body FNL.

[0073] Example 6

[0074] Transdermal absorption of protein FNL:

[0075] Ex vivo skin preparation: 30 female mice were sacrificed by cervical dislocation. Abdominal hair was trimmed, and the abdominal skin was excised. Subcutaneous tissue and fat were carefully removed using clean absorbent cotton dipped in saline. After washing, the skin was blotted dry with filter paper, wrapped in aluminum foil, and stored in a refrigerator (-20°C). Prior to the experiment, the mouse skin was thawed naturally at room temperature and immersed in saline for 0.5 hours. Skin integrity was inspected before each experiment, and intact skin was selected for testing.

[0076] Transdermal absorption measurement: Cut the skin of the same mouse into two pieces of appropriate size, inject 6.87 mL of physiological saline receiving solution into the receiving chamber, and place the mouse skin at the junction of the supply tank and the receiving tank of the transdermal absorption test device, with one side of the stratum corneum in contact with the supply tank, so that the liquid surface is completely in contact with the inner layer of the skin, and fix the junction with a spring clip. Add equal amounts of the protein body FNL prepared in Examples 1 to 5 and Comparative Examples 1 to 3 to the two diffusion cells, respectively, and equilibrate the samples for 30 minutes. Turn on the electromagnetic constant temperature stirrer, set the temperature to 37°C ± 2°C, and stir at a constant speed of 200 r / min. Take 0.5 mL of samples from the receiving cell at 2, 6, 12 and 24 hours, respectively, and add 0.5 mL of physiological saline at the same time and expel all bubbles. Use the Abbott diagnostic kit to determine the protein content in the transdermal solution in the two receiving cells at different times, and calculate the cumulative permeation amount of fibronectin per square centimeter of skin (Q n ).

[0077] Q n =(C n V n +ΣC k V k ) / S×100%

[0078] Where: C n represents the protein concentration (mg / mL) measured at the nth sampling point; C k V represents the protein concentration (mg / mL) measured at all sampling points before the nth sampling point; n Indicates the volume of the receiving solution (mL); V k It represents the sampling volume (mL) corresponding to all sampling points before the nth sampling point; S is the effective diffusion area.

[0079] The results are shown in Table 1.

[0080] Table 1 Transdermal absorption rate of protein FNL

[0081] 2h(％) 6h(％) 12h(％) 24h(％) Example 1 20.4 40.5 60.4 70.5 Example 2 21.3 42.3 63.2 72.4 Example 3 20.6 41.7 62.4 71.9 Example 4 21.5 40.9 61.9 72.4 Example 5 20.8 41.4 62.5 70.5 Comparative Example 1 12.2 32.4 42.2 48.2 Comparative Example 2 15.4 35.6 48.6 52.3 Comparative Example 3 14.9 34.8 47.2 50.7

[0082] It can be concluded from Table 1 that sodium alginate and allantoin increased the transdermal absorption rate of fibronectin, reaching more than 70% after 24 hours, which was an increase of more than 22% compared with the case without the use of sodium alginate and allantoin.

[0083] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing protein FNL, characterized in that: The following steps are involved: 1) dissolving soybean lecithin and cholesterol in a solvent and rotary evaporating the solvent to obtain a thin film; 2) dissolving sodium alginate, allantoin, and fibronectin in a buffer solution to obtain a protein solution, and mixing the protein solution with the film obtained in step 1) and rotating to obtain a rotating solution; 3) ultrasonically treating the rotating solution obtained in step 2), and extruding the solution through a microporous filter membrane to obtain protein FNL; In step 1), the mass ratio of soybean lecithin to cholesterol is 8-12:3-5; In step 1), the volume ratio of soybean lecithin to solvent is 8-12 g: 250-300 ml; In the step 2), the mass ratio of sodium alginate, allantoin and fibronectin is 0.5-0.8:0.1-0.3:0.012-0.018; In step 2), the volume ratio of fibronectin to buffer is 12-18 mg: 50-100 ml; The mass ratio of the soybean lecithin to the fibronectin is 8-12:0.012-0.

018.

2. The preparation method according to claim 1, characterized in that The solvent includes ethanol and / or chloroform.

3. The preparation method according to claim 1, characterized in that The conditions of the rotary evaporation in step 1) include: a temperature of 25 to 35° C. and a rotation speed of 150 to 250 rpm; The conditions for the rotation in step 2) include: a temperature of 40-45° C., a time of 20-25 min, and a rotation speed of 100-200 rpm.

4. The preparation method according to claim 1, characterized in that The pore size of the microporous filter membrane in step 3) is 0.45 μm.

5. A protein body FNL prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the protein body FNL according to claim 5 in the preparation of cosmetics.