Method for extracting and purifying leukocyte extract

Abstract

The invention relates to a leukocyte extract extraction and purification method, and belongs to the field of biology. Specifically, animal umbilical cord blood is selected as a raw material, a leukocyte extract is obtained through a series of extraction and purification steps, and polypeptide shown in SEQ ID NO.1-2 is obtained through further separation; the leukocyte extract and polypeptide prepared by the invention have remarkable antioxidant and anti-wrinkle effects and can be used for preparing medicines or cosmetics with special effects, and the application prospect of the leukocyte extract is expanded.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a method for extracting and purifying a leukocyte extract. Background Art

[0002] White blood cells are divided into neutrophils, eosinophils, basophils, monocytes, and lymphocytes. The first three are called granulocytes because they contain chromophilic granules in their cytoplasm. Under a microscope, they are relatively large and few in number among blood cells, and have cell nuclei. Their main function is to phagocytize bacteria and defend against diseases. White blood cells can phagocytize foreign bodies, and plasma cells produce antibodies, which play an important role in healing body injuries, resisting the invasion of pathogens, and immunity to diseases.

[0003] Leukocyte extract (LEUKOCYTE EXTRACT) is a complex secreted by cells extracted from leukocytes. Leukocyte extract contains a large number of cell-specific proteins (PDGF, VEGF, HGF, PDGF, bFGF, TGF-β, EGF and hundreds of other repair factors), lipids, nucleic acids (DNA, RNA), etc., which can directly act on the mucosal layer. It has a high transdermal absorption rate and has the effects of promoting proliferation and division, angiogenesis, mucosal repair and cell regeneration for fibroblasts, epithelial cells, endothelial cells, etc. These ingredients can effectively activate aging cells, promote cell regeneration, repair inflammatory damage, improve skin immunity, reduce pigmentation, etc., and help cells in the body restore normal function and vitality. Therefore, leukocyte extracts are often used as antioxidant drugs or added to skin care products.

[0004] Since leukocyte extracts are rich in a variety of growth factors, cytokines and immune factors, they have many biological effects and are widely used, such as: 1. Anti-inflammatory effect: leukocyte extracts contain a variety of cytokines and growth factors, which can regulate immune responses and reduce inflammatory responses. 2. Promote cell proliferation and repair: Growth factors in leukocyte extracts can promote cell proliferation and repair, and help treat injuries and diseases. 3. Antioxidant effect: Antioxidants in leukocyte extracts can reduce cell oxidative stress and help protect cells from free radical damage. 4. Anti-aging effect: The ingredients in leukocyte extracts can delay skin cell aging and help keep the skin healthy and young. The existing human leukocyte extracts (leukocyte factors) are obtained from human blood buffy coats through extraction, adsorption and pH gradient elution. The products do not contain proteins or polypeptides, and a single peak with 100% purity can be obtained by high-pressure liquid chromatography. Laboratory studies have shown that leukocyte extracts have an immune-enhancing effect and have significant clinical efficacy.

[0005] CN114191373A discloses a method for extracting leukocyte extract from umbilical cord blood quickly and simply, comprising the following steps: S1, collecting human umbilical cord blood; S2, centrifuging the umbilical cord blood obtained in step S1, obtaining the buffy coat supernatant, i.e., umbilical cord blood plasma containing the leukocyte layer, and freezing and preserving the plasma; S3, thawing the frozen plasma in step S2, and performing infectious disease antibody detection, virus RT-PCR fluorescence or PCR fluorescence quantitative detection; S4, detecting endotoxin in the plasma; S5, centrifuging the plasma that has passed the tests in steps S3 and S4, and filtering after centrifugation, and finally freezing and preserving. This method is to prepare human leukocyte extract from human blood that needs to be collected, and the yield is relatively low.

[0006] CN105153272A discloses a method for preparing an ostrich heterophil antimicrobial peptide, which comprises the following specific steps: (1) collecting ostrich heterophils; (3) extracting; (4) deacidifying; (5) purifying the antimicrobial peptide. The method of the invention realizes the extraction of polypeptide substances with strong antimicrobial activity from ostrich heterophils, and provides a new approach and method for producing antimicrobial peptides.

[0007] At present, there are also the following technical defects in the preparation of leukocyte extracts: first, the yield of leukocyte extracts is low, and there are a lot of impurities, which affect the activity effect of leukocyte extracts. In addition, leukocyte extracts are now mostly used in anti-inflammatory drugs or skin treatments, and the application of leukocyte extracts still needs to be expanded. Summary of the invention

[0008] In view of this, the purpose of the present invention is to overcome the shortcomings of the prior art. One of the purposes of the present invention is to provide a method for preparing a leukocyte extract. The leukocyte extract selected in the method for preparing the leukocyte extract is derived from animal umbilical cord blood, thereby solving the problems of yield and ethical restrictions and achieving scalable production. The leukocyte extract and polypeptide have good safety and significant antioxidant and anti-wrinkle effects.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] The invention provides a polypeptide with anti-oxidation and anti-wrinkle effects. The amino acid sequence of the polypeptide is FDDKRYGWHSWF (SEQ ID NO.1) and IVFNYSRGWDFNN (SEQ ID NO.2).

[0011] Furthermore, the polypeptide is extracted from animal umbilical cord blood cells.

[0012] Furthermore, the polypeptide is extracted from animal leukocytes.

[0013] Furthermore, the animal is selected from sheep, horses, pigs, cows, rats, mice, dogs, cats, rabbits, donkeys, deer, monkeys or humans; preferably, the animal is selected from goats.

[0014] The present invention also provides a method for preparing a leukocyte extract, comprising the following steps: collecting animal serum, first centrifugation, second centrifugation, collecting leukocytes, culturing leukocytes, third centrifugation, lysing leukocytes and collecting leukocyte extracts.

[0015] Furthermore, the method comprises the following steps:

[0016] (1) Collecting animal serum: Take 100 ml of umbilical cord blood from a healthy postpartum animal and inject 10 ml of anticoagulant (3.8% w / v sodium citrate).

[0017] (2) First centrifugation: Centrifuge the sheep umbilical cord blood containing the anticoagulant, discard the upper plasma layer, and retain the lower blood cell layer.

[0018] (3) Second centrifugation: Add physiological saline to the lower blood cell layer at a ratio of 1:1, mix well, then slowly add to 20 ml of cell separation solution and centrifuge. After centrifugation, the blood cell layer is separated into three layers: the supernatant layer, the middle white blood cell layer and the lower red blood cell layer.

[0019] (4) Collecting white blood cells: Collect the middle white blood cell layer, centrifuge the collected white blood cell layer again, and after centrifugation, discard the upper clear liquid. The cells at the bottom are the white blood cells.

[0020] (5) Leukocyte culture: The leukocytes were inoculated into serum-free culture medium, and 5% FBS and 300 U / ml IL-2 were added. The culture volume was fixed to about 50 ml with culture medium and cultured in a 37°, 5% CO2 incubator. Depending on the cell growth status, an appropriate amount of serum-free culture medium was added every two days.

[0021] (6) Third centrifugation: After 12 days of culture, all white blood cells were collected by centrifugation and supernatant 1 was retained.

[0022] (7) Leukocyte lysis: Add physiological saline to the leukocytes and adjust the concentration of leukocytes in the suspension to 5×10 6 / mL, transferred from room temperature to liquid nitrogen, lysed the leukocytes by repeated freezing and thawing 3 times, centrifuged, and collected the supernatant 2.

[0023] (8) Collecting leukocyte extract: Mix supernatant 1 and supernatant 2, filter the culture medium using a 0.22 μm sterile filter membrane to remove impurities. The resulting liquid is the supernatant containing the leukocyte extract, which is vacuum-dried and stored at -80°C for long-term use.

[0024] Furthermore, the animal is selected from sheep, horses, pigs, cows, rats, mice, dogs, cats, rabbits, donkeys, deer, monkeys or humans; preferably, the animal is selected from goats.

[0025] Furthermore, the preparation method further comprises the following steps:

[0026] (1) Taking the supernatant of the obtained leukocyte extract, performing ultrafiltration separation on it using a membrane filtration system with a molecular weight cutoff of 1 kDa, and separating the enzymatic hydrolysis products using ultrafiltration membranes with different molecular weight cutoff ranges to obtain a membrane-separated polypeptide sample solution.

[0027] (2) The polypeptide sample solution of step (1) was collected, separated by an SP-650M ion exchange column, and then separated and purified by a Sephadex LH-20 column (3.0×100 cm), with a mobile phase of 30% methanol, and the elution peak was measured at 215 nm.

[0028] (3) Further separation is performed using RP-HPLC, and the components are collected, dialyzed for desalination, and freeze-dried for later use, thereby obtaining a high-purity leukocyte extracted polypeptide.

[0029] (4) The peptides extracted from leukocytes were subjected to primary mass spectrometry and secondary mass spectrometry alternately using a mass spectrometer. With the aid of software analysis, it was found that most of the peptides were concentrated in a sequence length of 10-20 amino acids, among which the most abundant peptide was shown in SEQ ID NO. 1-2.

[0030] Furthermore, the chromatographic conditions of RP-HPLC were: C18 chromatographic column (4.6 mm×250 mm, 5 μm), mobile phase A was 0.05% trifluoroacetic acid in water (V / V), mobile phase B was acetonitrile, gradient elution conditions were: 0-10 min, 3% B, 10-20 min, 3%-15% B, 20-30 min, 15% B-20% B, 30-45 min, 20% B-30% B, flow rate was 1.0 mL / min, and detection wavelength was 215 nm.

[0031] The present invention also provides the use of the polypeptide and / or leukocyte extract in the preparation of drugs or cosmetics with anti-oxidation and / or anti-wrinkle effects.

[0032] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses animal umbilical cord blood as raw material, obtains leukocyte extract through a series of extraction and purification steps, and further separates and obtains the polypeptide described in SE Q ID NO.1-2. The leukocyte extract and polypeptide prepared by the present invention have significant antioxidant and anti-wrinkle effects, and can be used to prepare drugs or cosmetics with special effects, expanding the application prospects of leukocyte extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The antioxidant effects of the leukocyte extracts and polypeptides of the present invention;

[0035] Figure 2 The leukocyte extract and polypeptide of the present invention have the effect of promoting cell proliferation. DETAILED DESCRIPTION

[0036] The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] This embodiment provides a method for extracting a leukocyte extract, comprising the following steps:

[0039] (1) Serum collection: 100 ml of umbilical cord blood from a healthy postpartum goat was collected and injected with 10 ml of anticoagulant (3.8% w / v sodium citrate).

[0040] (2) First centrifugation: Centrifuge the sheep umbilical cord blood containing the anticoagulant at 4000 rpm for 15 minutes, discard the upper plasma layer, and retain the lower blood cell layer.

[0041] (3) Second centrifugation: Add physiological saline to the lower blood cell layer at a ratio of 1:1, mix well and slowly add to 20 ml of cell separation solution, centrifuge at 8000 rpm for 20 min; after centrifugation, it is divided into three layers: upper, middle and lower layers, namely the supernatant layer, the middle white blood cell layer and the lower red blood cell layer.

[0042] (4) Collecting white blood cells: Collect the middle white blood cell layer, and centrifuge the collected white blood cell layer again at 2000 rpm for 10 minutes. After the centrifugation is completed, discard the upper supernatant and the bottom cells obtained are the white blood cells.

[0043] (5) Leukocyte culture: The leukocytes were inoculated into serum-free culture medium, and 5% FBS and 300 U / ml IL-2 were added. The culture volume was fixed to about 50 ml with culture medium and cultured in a 37°, 5% CO2 incubator. Depending on the cell growth status, an appropriate amount of serum-free culture medium was added every two days.

[0044] (6) Third centrifugation: After 12 days of culture, all white blood cells were separated and collected by centrifugation at 8000 rpm for 20 min, and supernatant 1 was retained.

[0045] (7) Leukocyte lysis: Add physiological saline to the leukocytes and adjust the concentration of leukocytes in the suspension to 5×10 6 / mL, transferred from room temperature to liquid nitrogen, lysed the leukocytes by repeated freezing and thawing 3 times, centrifuged at 8000rpm for 20min, and collected the supernatant 2.

[0046] (8) Collecting leukocyte extract: Mix supernatant 1 and supernatant 2, filter the culture medium using a 0.22 μm sterile filter membrane to remove impurities. The resulting liquid is the supernatant containing the leukocyte extract, which is vacuum-dried and stored at -80°C for long-term use.

[0047] Example 2

[0048] This embodiment provides a method for purifying a leukocyte extract, comprising the following steps:

[0049] (1) The supernatant of the leukocyte extract obtained in Example 1 was taken and ultrafiltration separation was performed using a membrane filtration system with a molecular weight cutoff of 1 kDa. The enzymatic hydrolysis products were separated using ultrafiltration membranes with different molecular weight cutoff ranges to obtain a membrane separated polypeptide sample solution.

[0050] (2) The polypeptide sample solution of step (1) was collected, separated by an SP-650M ion exchange column, and then separated and purified by a Sephadex LH-20 column (3.0×100 cm), with a mobile phase of 30% methanol, and the elution peak was measured at 215 nm.

[0051] (3) RP-HPLC reverse phase high performance liquid chromatography was used for further separation. The chromatographic conditions were: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase A was 0.05% trifluoroacetic acid water (V / V), mobile phase B was acetonitrile, gradient elution conditions were: 0-10 min, 3% B, 10-20 min, 3%-15% B, 20-30 min, 15% B-20% B, 30-45 min, 20% B-30% B, flow rate was 1.0 mL / min, and detection wavelength was 215 nm. The components were collected, dialyzed for desalination, and freeze-dried for standby use to obtain high-purity leukocyte extracted polypeptides.

[0052] (4) The peptides extracted from leukocytes were subjected to alternating primary and secondary mass spectrometry using a mass spectrometer. With the help of software analysis, it was found that most of the peptides were concentrated in a sequence length of 10-20 amino acids, among which the most abundant peptides were FDDKRYGWHSWF (SEQ ID NO. 1) and IVFNYSRGWD FNN (SEQ ID NO. 2).

[0053] (5) The obtained two peptide amino acid sequences SEQ ID NO.1-2 were entrusted to Shanghai Bioengineering for synthesis and then used for future use.

[0054] Example 3 Antioxidant Activity Determination

[0055] Test samples: the leukocyte extract obtained in Example 1, the polypeptide of SEQ ID NO.1 and the polypeptide of SEQ ID NO.2. The three test samples were respectively prepared into solutions with a concentration of 0.5 mM; 0.5 mM vitamin C was used as a positive control.

[0056] Experimental groups: Experimental group 1: 0.5 mM leukocyte extract; Experimental group 2: 0.5 mM polypeptide of SEQ ID NO.1; Experimental group 3: 0.5 mM polypeptide of SEQ ID NO.2; Positive control group: 0.5 mM vitamin C.

[0057] (1) Determination of DPPH free radical scavenging ability

[0058] The concentration was 1×10 -5 mol / L DPPH ethanol solution, store away from light. Add 2mL, 0.1mM DPPH anhydrous ethanol solution to a clean test tube containing 2mL of the test solution and mix well. After leaving it at room temperature for 30 minutes, measure the absorbance at 517nm. The smaller the absorbance value, the stronger the free radical scavenging ability.

[0059] Clearance rate (%) = [1-(A i -A j ) / A0]×100%, where A0 is 2 mL, 0.1 mM DPPH anhydrous ethanol solution + 2 mL pure water, blank control; A i 2mL, 0.1mM DPPH anhydrous ethanol solution + 2mL test solution; A j 2mL of anhydrous ethanol + 2mL of test solution.

[0060] (2) Determination of ABTS free radical scavenging activity

[0061] Dissolve ABTS in deionized water to a concentration of 7 mmol / L, and add potassium persulfate to a concentration of 2.45 mmol / L. Then place the solution in a dark place at room temperature for 12 to 16 hours overnight. Dilute the generated ABTS free radical solution with phosphate buffer (PBS, 0.2 mol / L, pH 7.4) to an absorbance of 0.70 at 734 nm. Take 0.1 ml of the test solution and 2.9 ml of the ABTS free radical solution, mix them, shake for 30 seconds, react in the dark for 10 minutes, and then measure the absorbance of the reaction solution at 734 nm. Use distilled water instead of the hydrolyzate as a blank.

[0062] Clearance rate (%) = (A0-A j ) / A0×100%, where A0 is the absorbance of a mixture of 2.9 mL ABTS reagent and 0.1 mL distilled water; A j It is the absorbance value of the mixture of 2.9mL ABTS reagent and 0.1mL hydrolyzate.

[0063] The results are as follows Figure 1 As shown, the leukocyte extract extracted by the present invention and the polypeptides of SEQ ID NO.1-2 obtained have significant ability to scavenge DPPH and ABTS free radicals, among which the polypeptide has the best antioxidant effect, reaching the same level as the scavenging rate of vitamin C.

[0064] Example 4 Promoting Fibroblast Proliferation

[0065] Experimental groups: Experimental group 4: 0.5 mM leukocyte extract; Experimental group 5: 0.5 mM polypeptide of SEQ ID NO.1; Experimental group 6: 0.5 mM polypeptide of SEQ ID NO.2.

[0066] Experimental method: BALB / c 3T3 cells were inoculated into a 96-well cell culture plate (5000 cells / well) and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours; DMEM serum-free medium was used for continuous culture for 12 hours; 0.5 mM solution of the experimental group 4-6 and PBS (negative control group) were added respectively, and culture was continued for 48-72 hours; 10 μL CCK-8 reagent was added to each well, and the plate was incubated in a cell culture incubator at 37°C and 5% CO2 for 2 hours before being taken out; the absorbance of the 96-well plate at 450 nm and 630 nm was read with an ELISA reader, and the absorbance was measured at 450 nm with 630 nm as the reference wavelength, and the measurement results were recorded.

[0067] Promoting cell proliferation rate = (OD 450nm - Negative control group OD 450nm ) / negative control group OD 450nm × 100%. The result is Figure 2As shown, the leukocyte extract extracted by the present invention and the obtained polypeptide of SEQ ID NO.1-2 have obvious cell proliferation promoting activity and obvious anti-wrinkle effect.

[0068] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure may be implemented in different specific forms without changing its technical spirit and essential features. Therefore, it should be understood that the above-mentioned embodiments are not restrictive in all aspects, but illustrative. The scope of the present disclosure is limited by the appended claims, rather than by the preceding description thereof, and therefore all changes and modifications fall within the boundaries and scope of the claims, or the equivalents of such boundaries and scope are therefore intended to be covered by the claims.

Claims

1. A polypeptide having antioxidant and anti-wrinkle effects, characterized in that: The amino acid sequence of the polypeptide is selected from SEQ ID NO.1-2.

2. The polypeptide according to claim 1, characterized in that The polypeptide is extracted from the umbilical cord blood cells of animals.

3. The polypeptide according to claim 2, characterized in that The polypeptide is extracted from the leukocytes of animals.

4. The polypeptide according to any one of claims 1 to 3, characterized in that The animal is selected from sheep, horses, pigs, cows, rats, mice, dogs, cats, rabbits, donkeys, deer, monkeys or humans; preferably, the animal is selected from goats.

5. A method for preparing a leukocyte extract, characterized in that: The following steps are involved: Collect animal serum, perform the first centrifugation, perform the second centrifugation, collect leukocytes, culture leukocytes, perform the third centrifugation, lyse leukocytes, and collect leukocyte extracts.

6. The preparation method according to claim 5, characterized in that: (1) Collecting animal serum: Take 100 ml of umbilical cord blood from a healthy postpartum animal and inject it with 10 ml of anticoagulant containing 3.8% w / v sodium citrate; (2) First centrifugation: centrifuging the sheep umbilical cord blood containing the anticoagulant, discarding the upper plasma layer, and retaining the lower blood cell layer; (3) Second centrifugation: Add physiological saline to the lower blood cell layer at a ratio of 1:1, mix well, and then slowly add to 20 ml of cell separation solution and centrifuge. After centrifugation, the blood cell layer is separated into three layers: the supernatant layer, the middle white blood cell layer, and the lower red blood cell layer. (4) Collecting white blood cells: Collect the middle white blood cell layer, centrifuge the collected white blood cell layer again, and after the centrifugation is completed, discard the upper supernatant. The cells at the bottom are the white blood cells; (5) Leukocyte culture: The leukocytes were inoculated into serum-free medium, and 5% FBS and 300 U / ml IL-2 were added. The volume was fixed to about 50 ml with medium, and the cells were cultured in a 37°C, 5% CO2 incubator. Depending on the cell growth status, an appropriate amount of serum-free medium was added every two days. (6) Third centrifugation: After culturing for 12 days, all white blood cells were collected by centrifugation and supernatant 1 was retained; (7) Leukocyte lysis: Add physiological saline to the leukocytes and adjust the concentration of leukocytes in the suspension to 5×10 6 / mL, transferred from room temperature to liquid nitrogen, lysed the leukocytes by repeated freezing and thawing 3 times, centrifuged, and collected the supernatant 2; (8) Collecting leukocyte extract: Mix supernatant 1 and supernatant 2, filter the culture medium using a 0.22 μm sterile filter membrane to remove impurities. The resulting liquid is the supernatant containing the leukocyte extract, which is vacuum-dried and stored at -80°C for long-term use.

7. The preparation method according to any one of claims 5 to 6, characterized in that: The animal is selected from sheep, horses, pigs, cows, rats, mice, dogs, cats, rabbits, donkeys, deer, monkeys or humans; preferably, the animal is selected from goats.

8. The preparation method according to claim 5, characterized in that: The following steps are also included: (1) taking the supernatant of the obtained leukocyte extract, performing ultrafiltration separation on it using a membrane filtration system with a molecular weight cutoff of 1 kDa, and separating the enzymatic hydrolysis products using ultrafiltration membranes with different molecular weight cutoff ranges to obtain a membrane separated polypeptide sample solution; (2) collecting the polypeptide sample solution of step (1), separating it through an SP-650M ion exchange column, and then separating and purifying it through a Sephadex LH-20 column, with the mobile phase being 30% methanol, and measuring the elution peak at 215 nm; (3) further separation by RP-HPLC, collection of components, desalination by dialysis, and freeze-drying for later use, thereby obtaining high-purity leukocyte extracted polypeptides; (4) The peptides extracted from leukocytes were subjected to primary mass spectrometry and secondary mass spectrometry alternately using a mass spectrometer. With the aid of software analysis, it was found that most of the peptides were concentrated in a sequence length of 10-20 amino acids, among which the most abundant peptide was shown in SEQ ID NO.1-2.

9. The preparation method according to claim 8, characterized in that: Chromatographic conditions of RP-HPLC: C18 chromatographic column, mobile phase A is 0.05% trifluoroacetic acid water (V / V), mobile phase B is acetonitrile, gradient elution conditions are: 0-10 min, 3% B, 10-20 min, 3%-15% B, 20-30 min, 15% B-20% B, 30-45 min, 20% B-30% B, flow rate is 1.0 mL / min, and detection wavelength is 215 nm.

10. Use of the polypeptide according to any one of claims 1 to 4 and / or the leukocyte extract according to any one of claims 5 to 9 in the preparation of medicines or cosmetics having antioxidant and / or anti-wrinkle effects.

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