A leukocyte extract with repairing and anti-wrinkle effects and its preparation method and application

Abstract

The present invention provides a leukocyte extract with repair and anti-wrinkle effects, and a preparation method and application thereof. The method pre-treats leukocytes by intermittent high-frequency sonication, and then inoculates the leukocytes in a specific environment for culture. The obtained leukocyte extract has high purity and excellent repair and anti-wrinkle effects. In addition, compared with the method of obtaining leukocyte extracts by other pre-treatment methods or culture in other environments, the leukocyte extract obtained by the method of the present invention has higher purity and more excellent repair and anti-wrinkle effects.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture technology, and more specifically, relates to a leukocyte extract with repairing and anti-wrinkle effects, and a preparation method and application thereof. Background Art

[0002] Leukocyte extract (LEUKOCYTE EXTRACT) is a cell secretion product extracted from leukocytes. It contains exosomes, cytokines, proteins, nucleic acids and other bioactive ingredients. It can effectively activate the proliferation of fibroblasts and play a role in skin repair. It can also effectively promote the expression of type III collagen and inhibit elastase, thus playing an anti-wrinkle role. Therefore, leukocyte extract is widely used as an active ingredient in cosmetics or pharmaceuticals, and its preparation method has naturally become a research and development hotspot in the field of cosmetics and pharmaceuticals.

[0003] However, the purity of white blood cell extracts obtained by different pre-treatment methods and culture environments is significantly different, and the repair and anti-wrinkle effects are also quite different. Summary of the invention

[0004] The present invention aims to provide a method for preparing a leukocyte extract in view of the deficiencies in the prior art. The leukocytes are first pre-treated with intermittent high-frequency sound waves and then inoculated in a specific environment for culture. Compared with the method of obtaining leukocyte extracts by using other pre-treatment methods or culturing in other environments, the obtained leukocyte extract has higher purity and more excellent repairing, anti-wrinkle and other effects.

[0005] The first object of the present invention is to provide a method for preparing a leukocyte extract.

[0006] The second object of the present invention is to provide a leukocyte extract prepared by the above method.

[0007] The third object of the present invention is to provide the use of the leukocyte extract in preparing a cosmetic or medical composition.

[0008] A fourth object of the present invention is to provide a cosmetic or medical composition.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a method for preparing a leukocyte extract, which comprises: subjecting the leukocytes to intermittent high-frequency sonication treatment for 2 to 4 times, and then inoculating them in a medium containing 2 to 4 mM NH4Cl, 240 to 260 U / mL IL-2, and 8% ( v / v )~12%( v / v) FBS-containing RPMI-1640 medium; wherein, each intermittent high-frequency sonication treatment is: high-frequency sonication treatment is performed at a power of 3-6 W and a frequency of 8-12 MHz for 8-12 min, and then paused for 25-35 min.

[0011] The present invention uses intermittent high-frequency sonication to pre-treat leukocytes, and then inoculates them in a specific environment for culture. The obtained leukocyte extract has a high purity and also has excellent repair and anti-wrinkle effects. In addition, compared with the method of obtaining leukocyte extracts by other pre-treatment methods or culture in other environments, the leukocyte extract obtained by the method of the present invention has higher purity and more excellent repair and anti-wrinkle effects.

[0012] In the intermittent high-frequency sonication process of the present invention, a culture medium commonly used in the art that can maintain the survival of leukocytes can be used, such as a medium containing 8% ( v / v )~12%( v / v ) RPMI-1640 medium containing FBS, and the leukocyte inoculum was 5×10 6 ~2×10 7 Pieces / mL.

[0013] Preferably, the leukocytes are isolated from umbilical cord blood.

[0014] Further preferably, the leukocytes are isolated from umbilical cord blood of cattle, sheep or pigs.

[0015] Preferably, the inoculation amount of the leukocytes in the RPMI-1640 medium is 2×10 8 ~2×10 10 Pieces / L.

[0016] Preferably, the culture temperature is 35-39°C.

[0017] Preferably, the culture time is 3 to 5 days.

[0018] Preferably, after the culturing, centrifugation and purification are further performed.

[0019] More preferably, the centrifugation is performed at 3000-5000 rpm for 15-25 min.

[0020] Further preferably, the supernatant obtained by centrifugation is concentrated before the purification.

[0021] More preferably, the concentration is to concentrate the supernatant obtained by centrifugation by tangential flow filtration. The conditions of tangential flow filtration are preferably: the molecular weight cut-off of the hollow fiber column is 100-750 kD, the transmembrane pressure is 3-5 PSI, and the shear rate is 4000-6000 s-1 , Flux 6~15 L / m 2 ·h.

[0022] More preferably, the concentration is to concentrate the volume of the supernatant obtained by centrifugation by 10 to 100 times.

[0023] More preferably, the purification is performed by a protein purification system.

[0024] More preferably, the conditions of the protein purification system are: the chromatography column filler is DEAE weak anion exchange chromatography filler, the equilibrium buffer is 19-21 mM Tris-HCl solution (pH7.8-8.2), and the loading rate is 0.8-1.2 mL / min; the eluent is a solution containing 19-21 mM Tris-HCl and 0.8-1.2 M NaCl (pH7.8-8.2), and the elution rate is 1.8-2.2 mL / min.

[0025] More preferably, sterilization is performed after the purification. When the leukocyte extract is used in the cosmetic field, sterilization is required; when the leukocyte extract is used in the medical field, sterilization is not required.

[0026] More preferably, the sterilization is achieved by filtration, such as filtration sterilization using a filter with a pore size of 0.2 to 0.3 μM.

[0027] The leukocyte extract obtained by the above method has high purity and excellent repair and anti-wrinkle effects, and is suitable for preparing cosmetics or medical compositions. Therefore, the leukocyte extract obtained by the above method, the use of the leukocyte extract in preparing cosmetics or medical compositions, and a cosmetic or medical composition containing the leukocyte extract should all be within the protection scope of the present invention.

[0028] Preferably, the cosmetic or medical composition further contains cosmetically or medically acceptable excipients.

[0029] The present invention has the following beneficial effects:

[0030] The present invention uses intermittent high-frequency sonication to pre-treat leukocytes, and then inoculates them in a specific environment for culture. The obtained leukocyte extract has a high purity and also has excellent repair and anti-wrinkle effects. In addition, compared with the method of obtaining leukocyte extracts by other pre-treatment methods or culture in other environments, the leukocyte extract obtained by the method of the present invention has higher purity and more excellent repair and anti-wrinkle effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1This is a transmission electron micrograph of the leukocyte extract of Example 1.

[0032] Figure 2 This is a transmission electron micrograph of the leukocyte extract of Example 2.

[0033] Figure 3 This is a transmission electron micrograph of the leukocyte extract of Example 3.

[0034] Figure 4 This is a transmission electron micrograph of the leukocyte extract of Example 4.

[0035] Figure 5 This is the transmission electron microscopy image of the leukocyte extract of Comparative Example 1.

[0036] Figure 6 This is the transmission electron microscopy image of the leukocyte extract of Comparative Example 2.

[0037] Figure 7 This is the transmission electron microscopy image of the leukocyte extract of Comparative Example 3.

[0038] Figure 8 This is the transmission electron microscopy image of the leukocyte extract of Comparative Example 4.

[0039] Fig. 9 This is the transmission electron microscopy image of the leukocyte extract of Comparative Example 5.

[0040] Fig.10 This is the transmission electron microscopy image of the leukocyte extract of Comparative Example 6. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] RPMI-1640 culture medium was purchased from Wuhan Prosai Life Science Technology Co., Ltd.

[0044] Fresh umbilical cord blood of cattle was collected (there was no ethical controversy about the acquisition and processing methods), placed in a sterilized container, and anticoagulated with 6 volumes of 3.5% (v / v) trisodium citrate solution. After thorough mixing, the blood was filtered with sterilized gauze to remove fibers, blood clots and other impurities. The upper yellow clear plasma and the bottom red blood cells were discarded by centrifugation, and the middle layer (containing white blood cells, platelets and part of the red blood cell precipitate) was collected. Then, 8 volumes of red blood cell lysis solution (NH4Cl8.29 g, KHCO31.00 g, Na2EDTA 37.2 mg were dissolved in 1000 mL of distilled water, and the pH was adjusted to 7.3 with 1 mol / L HCl solution) was added. After stirring with a glass rod for 30 min, the blood was centrifuged at 2500 rpm for 10 min, the supernatant was discarded, and the precipitate was collected, which was the white blood cells.

[0045] In the pretreatment process of leukocytes in the present embodiment and the comparative example, the leukocyte culture medium used is 10% ( v / v ) RPMI-1640 medium containing FBS, and the leukocyte inoculum was 5×10 6 ~2×10 7 Pieces / mL.

[0046] Example 1 Preparation of leukocyte extract

[0047] After intermittent high-frequency sonication for 3 times, the leukocytes were inoculated in 1 L of 3 mM NH4Cl, 250 U / mL IL-2, 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2 h) concentrating the volume by 60 times, and then purifying it through a protein purification system (the chromatography column filler is a DEAE weak anion exchange chromatography filler, the equilibrium buffer is a 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), and the elution rate is 2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.22 μM to obtain the leukocyte extract;

[0048] Each intermittent high-frequency sonication treatment was as follows: high-frequency sonication treatment was performed at a power of 5 W and a frequency of 10 MHz for 10 min, followed by a pause of 30 min.

[0049] Example 2 Preparation of leukocyte extract

[0050] After intermittent high-frequency sonication for 2 times, the leukocytes were inoculated in 1 L of medium containing 4 mM NH4Cl, 260 U / mL IL-2, and 8% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 35 °C for 5 days and then centrifuged at 3000 rpm for 25 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 750 kD, a transmembrane pressure of 3 PSI, and a shear rate of 4000 s -1 , flux 15 L / m 2 h) concentrating the volume 100 times, and then purifying it through a protein purification system (the chromatography column filler is DEAE weak anion exchange chromatography filler, the equilibrium buffer is 21 mM Tris-HCl solution (pH 7.8), the loading rate is 0.8 mL / min; the eluent is a solution containing 21 mM Tris-HCl and 0.8 M NaCl (pH 7.8), and the elution rate is 1.8 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.2 μM to obtain the leukocyte extract;

[0051] Each intermittent high-frequency sonication treatment was as follows: high-frequency sonication treatment was performed at a power of 3 W and a frequency of 12 MHz for 8 minutes, followed by a pause of 35 minutes.

[0052] Example 3 Preparation of leukocyte extract

[0053] After intermittent high-frequency sonication for 4 times, the leukocytes were inoculated in 1 L of 2 mM NH4Cl, 240 U / mL IL-2, 12% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 39 °C for 3 days and then centrifuged at 5000 rpm for 15 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 100 kD, a transmembrane pressure of 5 PSI, and a shear rate of 6000 s -1 , flux 6 L / m 2h) concentrating the volume 10 times, and then purifying it through a protein purification system (the chromatography column filler is DEAE weak anion exchange chromatography filler, the equilibrium buffer is 19 mM Tris-HCl solution (pH 8.2), the loading rate is 1.2 mL / min; the eluent is a solution containing 19 mM Tris-HCl and 1.2 M NaCl (pH 8.2), and the elution rate is 2.2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.3 μM to obtain the leukocyte extract;

[0054] Each intermittent high-frequency sonication treatment was as follows: high-frequency sonication treatment was performed at a power of 6 W and a frequency of 8 MHz for 12 minutes, followed by a pause of 25 minutes.

[0055] Example 4

[0056] The same as Example 1, except that no filtration sterilization is performed. That is, the specific details of this example are as follows:

[0057] After intermittent high-frequency sonication for 3 times, the leukocytes were inoculated in 1 L of 3 mM NH4Cl, 250 U / mL IL-2, 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2 h) concentrating the volume of the extract by 60 times, and then purifying the extract by a protein purification system (the chromatographic column filler is a DEAE weak anion exchange chromatography filler, the equilibrium buffer is a 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), and the elution rate is 2 mL / min), thereby obtaining the leukocyte extract;

[0058] Each intermittent high-frequency sonication treatment was as follows: high-frequency treatment was performed at a power of 5 W and a frequency of 10 MHz for 10 min, followed by a pause of 30 min.

[0059] Comparative Example 1

[0060] The same as Example 1, except that the intermittent high-frequency sonication treatment is replaced by intermittent ultrasonication treatment, that is, the specific example of this comparative example is:

[0061] After intermittent sonication for 3 times, the leukocytes were inoculated in 1 L of 3 mM NH4Cl, 250 U / mL IL-2, 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2 h) after concentrating its volume 60 times, purifying it through a protein purification system (the chromatography column filler is DEAE weak anion exchange chromatography filler, the equilibrium buffer is 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), the elution rate is 2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.22 μM to obtain the leukocyte extract;

[0062] Each intermittent ultrasonic treatment was performed at a power of 50 W and a frequency of 20 kHz for 10 min, followed by a pause of 30 min.

[0063] Comparative Example 2

[0064] The same as Example 1, except that the intermittent high-frequency ultrasonic treatment is replaced by continuous ultrasonic treatment, that is, this comparative example is specifically as follows:

[0065] After ultrasonic treatment of leukocytes at 50 W and 20 kHz for 30 min, they were inoculated in 1 L of 3 mM NH4Cl, 250 U / mL IL-2, 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2·h) after concentrating its volume 60 times, purifying it through a protein purification system (the chromatography column filler is DEAE weak anion exchange chromatography filler, the equilibrium buffer is 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), and the elution rate is 2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.22 μM to obtain the leukocyte extract.

[0066] Comparative Example 3

[0067] The same as Example 1, except that the intermittent high-frequency acoustic wave treatment is replaced by intermittent low-level electric treatment (ET), that is, this comparative example is specifically as follows:

[0068] After intermittent low-level electroporation for 3 times, the leukocytes were inoculated in 1 L of 3 mM NH4Cl, 250 U / mL IL-2, 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2 h) concentrating the volume by 60 times, and then purifying it through a protein purification system (the chromatography column filler is a DEAE weak anion exchange chromatography filler, the equilibrium buffer is a 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), and the elution rate is 2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.22 μM to obtain the leukocyte extract;

[0069] Each intermittent low-level electrochemical treatment is as follows: the electrode is Ag-AgCl (surface area 2.5 cm 2 ), at a constant current of 0.35 mA / cm 2 After low-level electroporation for 10 min, the cells were paused for 30 min.

[0070] Comparative Example 4

[0071] The same as Example 1, except that the intermittent high-frequency acoustic wave treatment is replaced by a continuous low-level electrical treatment, that is, this comparative example is specifically as follows:

[0072] The electrode was Ag-AgCl (surface area 2.5 cm 2 ), at a constant current of 0.35 mA / cm 2 After low-level electroporation for 30 min, the leukocytes were inoculated in 1 L of 3 mM NH4Cl, 250 U / mL IL-2, 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2 ·h) after concentrating its volume 60 times, purifying it through a protein purification system (the chromatography column filler is DEAE weak anion exchange chromatography filler, the equilibrium buffer is 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), and the elution rate is 2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.22 μM to obtain the leukocyte extract.

[0073] Comparative Example 5

[0074] The same as Example 1, except that 3 mM NH4Cl is replaced by 40 μM chloroquine, that is, this comparative example is specifically:

[0075] After intermittent high-frequency sonication for 3 times, the leukocytes were inoculated in 1 L of 40 μM chloroquine, 250 U / mL IL-2, and 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2h) concentrating the volume by 60 times, and then purifying it through a protein purification system (the chromatography column filler is a DEAE weak anion exchange chromatography filler, the equilibrium buffer is a 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), and the elution rate is 2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.22 μM to obtain the leukocyte extract;

[0076] Each intermittent high-frequency sonication treatment was as follows: high-frequency sonication treatment was performed at a power of 5 W and a frequency of 10 MHz for 10 min, followed by a pause of 30 min.

[0077] Comparative Example 6

[0078] The same as Example 1, except that 3 mM NH4Cl is replaced by 10 μM leupeptin, that is, this comparative example is specifically:

[0079] After intermittent high-frequency sonication for 3 times, the leukocytes were inoculated in 1 L of medium containing 10 μM leupeptin, 250 U / mL IL-2, and 10% ( v / v ) FBS-containing RPMI-1640 medium (the inoculum size of leukocytes in RPMI-1640 medium is 2×10 8 ~2×10 10 The cells were cultured at 37 °C for 4 days and then centrifuged at 4000 rpm for 20 min. The supernatant was filtered by tangential flow filtration (hollow fiber column with a molecular weight cutoff of 300 kD, a transmembrane pressure of 4 PSI, and a shear rate of 5000 s -1 , flux 10 L / m 2 h) concentrating the volume by 60 times, and then purifying it through a protein purification system (the chromatography column filler is a DEAE weak anion exchange chromatography filler, the equilibrium buffer is a 20 mM Tris-HCl solution (pH 8), the loading rate is 1 mL / min; the eluent is a solution containing 20 mM Tris-HCl and 1 M NaCl (pH 8), and the elution rate is 2 mL / min), and finally filtering and sterilizing with a filter with a pore size of 0.22 μM to obtain the leukocyte extract;

[0080] Each intermittent high-frequency sonication treatment was as follows: high-frequency sonication treatment was performed at a power of 5 W and a frequency of 10 MHz for 10 min, followed by a pause of 30 min.

[0081] Test Example 1 Purity of Leukocyte Extract

[0082] The leukocyte extracts obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were observed by transmission electron microscopy.

[0083] Transmission electron microscopy images Figures 1 to 10 As shown, Figure 1 is a transmission electron micrograph of the leukocyte extract of Example 1, Figure 2 is a transmission electron micrograph of the leukocyte extract of Example 2, Figure 3 is a transmission electron micrograph of the leukocyte extract of Example 3, Figure 4 is a transmission electron micrograph of the leukocyte extract of Example 4, Figure 5 is the transmission electron microscopy image of the leukocyte extract of comparative example 1, Figure 6 This is the transmission electron microscopy image of the leukocyte extract of Comparative Example 2, Figure 7 is the transmission electron microscopy image of the leukocyte extract of comparative example 3, Figure 8 is the transmission electron microscopy image of the leukocyte extract of Comparative Example 4, Fig. 9 is the transmission electron microscopy image of the leukocyte extract of comparative example 5, Fig.10 The transmission electron microscope image of the leukocyte extract of comparative example 6. It can be seen that compared with comparative examples 1 to 6, the saucer-like morphology of the leukocyte extracts obtained in Examples 1 to 4 is obviously more typical, and there are fewer impurities, indicating that the present invention uses intermittent high-frequency sonication to pre-treat the leukocytes, and then inoculates them in a specific environment for culture, so that the obtained leukocyte extract has a higher purity.

[0084] Test Example 2 Protein content in leukocyte extract

[0085] 20 μL of the leukocyte extracts obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were added to a 96-well plate, and the protein content in the leukocyte extracts was determined using a BCA protein concentration assay kit (purchased from Beyotime Biotechnology Co., Ltd.). The results are shown in Table 1.

[0086] Table 1

[0087]

[0088] It can be seen that the protein content in the leukocyte extracts obtained in Examples 1 to 4 is significantly higher than that in Comparative Examples 1 to 6, indicating that the present invention uses intermittent high-frequency ultrasonic treatment to pre-treat leukocytes and then inoculates them in a specific environment for cultivation, which can significantly increase the protein content in the leukocyte extracts and thus significantly improve the biological activity of the leukocyte extracts.

[0089] Test Example 3 Antioxidant Effect of Leukocyte Extract

[0090] 1. Expression of glutathione (GSH) and superoxide dismutase (SOD)

[0091] The leukocyte extracts obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were added to human skin fibroblasts (purchased from Shanghai Fuheng Biotechnology Co., Ltd., and the culture medium used contained 10% ( v / v The concentration of leukocyte extract in human skin fibroblasts was 100 μL / mL in DMEM medium containing FBS, and the cells were cultured in an incubator at 37°C and 5% CO2 for 48 h to obtain culture medium. The expression levels of glutathione (GSH) and superoxide dismutase (SOD) in the culture medium were detected using an ELISA reader.

[0092] 2. DPPH free radical scavenging rate

[0093] Sample group: The leukocyte extracts obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were dissolved in distilled water (so that the final concentration of the leukocyte extracts was 1% (v / v)), 100 μL was added to a 96-well plate, and then 100 μL of 0.1 mM DPPH solution was added. After shaking in the dark for 10 min, the absorbance value A of the solution at 520 nm was tested using an ELISA instrument. x Three replicate wells were set up and the results were averaged.

[0094] Control group: 100 μL of distilled water was added to a 96-well plate, and then 100 μL of 0.1 mM DPPH solution was added. After shaking for 10 min in the dark, the absorbance value A0 of the solution at 520 nm was tested using an ELISA reader. Three replicate wells were set up and the results were averaged.

[0095] According to "Clearance (%) = [(A0-A x ) / A0]×100%” to calculate the DPPH radical scavenging rates of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 6, respectively.

[0096] 3. Antioxidant Activity Results

[0097] The results are shown in Table 2.

[0098] Table 2

[0099]

[0100] It can be seen that the expression levels of GSH and SOD and the DPPH free radical scavenging rate of the leukocyte extracts obtained in Examples 1 to 4 are significantly higher than those in Comparative Examples 1 to 6, indicating that the leukocyte extracts obtained by the present invention, after pre-treating the leukocytes by intermittent high-frequency ultrasonic treatment and then inoculating them in a specific environment for cultivation, have a more excellent antioxidant effect.

[0101] Test Example 4 Repairing effect of leukocyte extract

[0102] The leukocyte extracts obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were added to human skin fibroblasts (purchased from Shanghai Fuheng Biotechnology Co., Ltd., and the culture medium used contained 10% ( v / v ) DMEM medium containing FBS, the concentration of leukocyte extract in human skin fibroblasts was 100 μL / mL, and cultured in an incubator at 37 ℃ and 5% CO2 for 48 hours to obtain a culture solution. The absorbance value of human skin fibroblasts in the culture solution at a wavelength of 450 nm was detected by CCK8 method (to characterize the viability of human skin fibroblasts) to evaluate the repair activity of leukocyte extracts. The results are shown in Table 3.

[0103] Table 3

[0104]

[0105] It can be seen that the absorbance values ​​of human skin fibroblasts of the leukocyte extracts obtained in Examples 1 to 4 are significantly higher than those in Comparative Examples 1 to 6, indicating that the present invention uses intermittent high-frequency ultrasonic treatment to pre-treat leukocytes and then inoculates them in a specific environment for culture, and the obtained leukocyte extracts have more excellent repair effects.

[0106] Test Example 5 Anti-wrinkle effect of leukocyte extract

[0107] 1. Expression of type III collagen

[0108] The leukocyte extracts obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were added to human skin fibroblasts (purchased from Shanghai Fuheng Biotechnology Co., Ltd., and the culture medium used contained 10% ( v / v ) FBS-containing DMEM medium) was added to make the concentration of leukocyte extract in human skin fibroblasts 100 μL / mL, and cultured in an incubator at 37 ℃ and 5% CO2 for 48 h to obtain culture fluid. The supernatant was centrifuged and the expression of type III collagen in the supernatant was detected using a human type III collagen α1 (COL3A1) enzyme-linked immunosorbent assay kit (purchased from Wuhan Huamei Bioengineering Co., Ltd.).

[0109] 2. Elastase inhibition rate

[0110] Sample group: The leukocyte extracts obtained in Examples 1 to 4 and Comparative Examples 1 to 6 were diluted 20 times with physiological saline, 10 μL was added to a 96-well plate, and then 20 μL of 0.1 U / mL elastase (porcine pancreas) solution was added. After incubation in a 25°C constant temperature incubator for 15 min, 50 μL of 1 mg / mL substrate (98% purity N-succinyl-L-alanyl-L-alanine) was added. Finally, the absorbance value A of the solution at 410 nm was tested with an ELISA reader. xThree replicate wells were set up and the results were averaged.

[0111] Control group: 10 μL of water was added to a 96-well plate, and then 20 μL of 0.1 U / mL elastase (porcine pancreas) solution was added. After incubation in a 25°C constant temperature incubator for 15 min, 50 μL of 1 mg / mL substrate (98% purity N-succinyl-L-alanyl-L-alanine) was added. Finally, the absorbance value A of the solution at 410 nm was tested using an ELISA reader. x Three replicate wells were set up and the results were averaged.

[0112] According to "Inhibition rate (%) = [(A0-A x ) / A0]×100%" to calculate the elastase inhibition rates of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 6, respectively.

[0113] 3. Anti-wrinkle activity results

[0114] The results are shown in Table 4.

[0115] Table 4

[0116]

[0117] It can be seen that the expression level of type III collagen and the elastase inhibition rate of the leukocyte extracts obtained in Examples 1 to 4 are significantly higher than those in Comparative Examples 1 to 6, indicating that the leukocyte extracts obtained by the present invention, after pre-treating the leukocytes by intermittent high-frequency ultrasonic treatment and then inoculating them in a specific environment for culture, have a more excellent anti-wrinkle effect.

[0118] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a leukocyte extract, characterized in that: After intermittent high-frequency sonication treatment of leukocytes for 2 to 4 times, the leukocytes are inoculated in a RPMI-1640 medium containing 2 to 4 mM NH4Cl, 240 to 260 U / mL IL-2, and 8% (v / v) to 12% (v / v) FBS for culture; wherein the leukocytes are separated from bovine umbilical cord blood; each intermittent high-frequency sonication treatment comprises: performing high-frequency sonication treatment for 8 to 12 minutes at a power of 3 to 6 W and a frequency of 8 to 12 MHz, and then pausing for 25 to 35 minutes.

2. The preparation method according to claim 1, characterized in that: The inoculation amount of the leukocytes in the RPMI-1640 medium was 2×10 8 ~2×10 10 Pieces / L.

3. The preparation method according to claim 1, characterized in that: The culture temperature is 35-39°C.

4. The preparation method according to claim 1, characterized in that: The culture time is 3 to 5 days.

5. The preparation method according to claim 1, characterized in that: After the culture, centrifugation and purification are performed.

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