Preparation process for efficiently obtaining pig umbilical cord extract with high purity and activity

Abstract

The invention discloses a preparation process for efficiently obtaining a high-purity and high-activity pig umbilical cord extract. Comprising the following steps: S1, cleaning a pig umbilical cord; s2, crushing the pig umbilical cord; s3, filtering the ground pig umbilical cord; s4, performing ultrasonic treatment on the buffer solution and the pig umbilical cord tissue; s5, carrying out centrifugation to extract a supernatant; the novel pig umbilical cord is adopted to extract the extract, so that the precipitation rate of the extract can be increased; the pig umbilical cord is treated through an enzymolysis method, so that connecting substances between cells in the pig umbilical cord can be damaged, cells in umbilical cord tissue can be conveniently ground and broken, the precipitation rate of the pig umbilical cord extract is increased, the cells of the pig umbilical cord cannot be damaged by using the enzymolysis method, and the activity of the cells is kept; and moreover, ultrasonic treatment is adopted, so that cells can be destroyed, the extract can be efficiently separated out, and the obtaining rate of the extract is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of umbilical cord extracts, and specifically relates to a preparation process for efficiently obtaining high-purity and active pig umbilical cord extracts. Background Art

[0002] The umbilical cord is the connecting structure between the fetus and the placenta. It is shaped like a rope, with a smooth and transparent surface. It contains connective tissue, an umbilical vein, and a pair of umbilical arteries. The umbilical vein runs along the inner surface of the fetal abdominal wall through the hepatic sinusoids and umbilical arteries to connect with the fetal aorta. The other ends of these two blood vessels form a network of many interconnected capillaries distributed in the placental villi. Through the osmotic action of the placental villus epithelium, substances are exchanged between the fetal disc fluid and the maternal blood in the intervillous space.

[0003] Umbilical cord extract is a jelly-like binding tissue component extracted from the umbilical cord of animals. It is a water-soluble raw material obtained by adding a solvent. When used in skin care products, umbilical cord extract can help the skin fight aging, enhance the vitality of stem cells, promote collagen regeneration, help improve the health of the skin, increase the moisture content of the skin, and make the skin smoother and more elastic; umbilical cord extract has also been proven to increase epidermal density, the number of keratinocytes, type III collagen, and elastin, helping to keep the skin healthy and young. At the same time, it has also been verified to have the function of shrinking pores and fading dark circles; however, various pig umbilical cord extracts on the market still have various problems.

[0004] For example, the pig umbilical cord extract and its extraction method and application disclosed in the authorization announcement number CN115125193B, although it realizes the gradual removal of other components through physical methods such as freeze-thaw, ultrasound, filtration, etc. in combination with the structural characteristics of the umbilical cord, and finally obtains a sterile product rich in exosomes, and through identification experiments and functional verification experiments, it is fully confirmed that the present invention has established a preparation process for stable umbilical cord exosomes, but it does not solve the problem that freezing in the existing process easily causes cell death and the umbilical cord extract cannot be extracted, and the cell connection in the pig umbilical cord is destroyed by enzymatic hydrolysis, so that the cells in the umbilical cord tissue can be easily ground and broken, thereby improving the precipitation rate of the pig umbilical cord extract. For this reason, we propose a preparation process for efficiently obtaining high-purity and active pig umbilical cord extract. Summary of the invention

[0005] The object of the present invention is to provide a preparation process for efficiently obtaining a high-purity and active pig umbilical cord extract, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a preparation process for efficiently obtaining a high-purity and active pig umbilical cord extract, comprising the following steps:

[0007] S1. Cleaning the pig umbilical cord: Cleaning the pig umbilical cord, cleaning the blood, adhesions and mucus on the surface of the pig umbilical cord, and then disinfecting the pig umbilical cord;

[0008] S2, crushing the pig umbilical cord: cutting the pig umbilical cord to keep the size of the pig umbilical cord at 2*3 cm, soaking the small pieces of the pig umbilical cord by enzymatic hydrolysis, and then grinding the small pieces of the pig umbilical cord;

[0009] S3, filtering the ground pig umbilical cord: there is a large amount of blood and tissue fluid in the pig umbilical cord just after grinding, the ground pig umbilical cord is filtered through a filtering mechanism, and a buffer solution is added to the filtered pig umbilical cord tissue;

[0010] S4, ultrasonically treating the buffer solution and the porcine umbilical cord tissue: shaking the porcine umbilical cord tissue in the buffer solution at low temperature, and then ultrasonically treating the porcine umbilical cord tissue;

[0011] S5. Then centrifuge and extract the supernatant: centrifuge the pig umbilical cord tissue after ultrasonic treatment, then extract the supernatant, and filter the supernatant and continue to ultrasonicate for multiple times to obtain a pig umbilical cord extract, and the extract includes vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor.

[0012] Preferably, the surface of the porcine umbilical cord tissue in S1 is clean, without a large amount of blood and other impurities adhering, without decay and deterioration, and without peculiar smell, and the test results for Brucella and foot-and-mouth disease are negative, and both rational indicators and microbiological indicators are qualified.

[0013] Preferably, the rational indicators include at least pH value, protein content, lead, mercury, arsenic and cadmium, and the microbial indicators include total colony count, total mold and yeast count, heat-resistant coliform group, Pseudomonas aeruginosa and Staphylococcus aureus;

[0014] The pH value is controlled at 6.0-8.0, the protein content is ≥0.5mg / ml, the lead content is ≤0.1mg / kg, the mercury content is ≤0.3mg / kg, the arsenic content is ≤0.5mg / kg and the cadmium content is ≤1.0mg / kg;

[0015] The total colony count is ≤100CFU / g, the total number of molds and yeasts is ≤10CFU / g, and thermotolerant coliform bacteria, Pseudomonas aeruginosa and Staphylococcus aureus must not be detected.

[0016] Preferably, the pig umbilical cord cleaning in S1 is firstly cleaned with clean water to clean the blood, mucus and residues on the surface, and then immersed in 75% medical alcohol for three minutes, and then rinsed with distilled water or medical saline to clean the medical alcohol remaining on the surface of the pig umbilical cord.

[0017] Preferably, the enzymatic hydrolysis method in S2 uses a mixture of trypsin, collagenase and digestive enzymes, and when the enzymatic hydrolysis method is used, it is heated in a low-temperature water bath at 35-40°C, and the duration of the enzymatic hydrolysis method is 4-6 minutes. After the enzymatic hydrolysis treatment, the pig umbilical cord is rinsed with distilled water or medical saline to clean off the residual mixed agent on the surface of the pig umbilical cord.

[0018] Preferably, the buffer in S3 is PBS buffer, the main components of which are Na2HPO4, KH2PO4, NaCl and KCl, and the mass volume ratio of PBS buffer to pig umbilical cord tissue is 10%, that is, 0.1g of tissue sample is added with 1ml of liquid.

[0019] Preferably, the temperature of the low-temperature shaking in S4 is controlled at 30-38° C., and when shaking, the vibration frequency is set to 1000 times / minute and the vibration time is 5 minutes, so that the porcine umbilical cord tissue can be fully mixed in the PBS buffer solution, and then the porcine umbilical cord tissue is treated with an ultrasonic generator so that the cells in the porcine umbilical cord tissue solution can be broken to facilitate the precipitation of the extract.

[0020] Preferably, the centrifuge in S5 first places the centrifuge tube storing the pig umbilical cord tissue solution into a high-speed centrifuge under a low temperature environment, centrifuges at 4000 rpm for 30 minutes, takes out the obtained supernatant with a pipette, and filters it, then transfers the supernatant to a new centrifuge tube, performs multiple ultrasonic treatments, and finally uses a biomembrane filter for filtration.

[0021] Preferably, the first centrifugation of the high-speed centrifuge is set at 4000 rpm for 30 minutes, the second centrifugation is set at 4000 rpm for 1 hour, and the third centrifugation is set at 10000 rpm for 30 minutes, and the second and third centrifugations are for centrifuging the supernatant.

[0022] Preferably, the filtering mechanism adopts a multi-stage filtering device with decreasing filter mesh apertures, and the filter mesh apertures are filtered according to 5 μm, 1 μm and 0.45 μm in sequence to improve the purity of the supernatant.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] When the present invention is used, fresh pig umbilical cord is used to extract the extract, and the fresh pig umbilical cord can improve the precipitation rate of the extraction during the extraction, and the fresh pig umbilical cord can maintain the activity of cells, reduce the cell death caused by freezing, and the pig umbilical cord cannot be extracted, and prevent the problem of low extract yield after freezing;

[0025] The pig umbilical cord is treated by enzymatic hydrolysis, so that the connecting substances between cells in the pig umbilical cord can be destroyed, so that the cells in the umbilical cord tissue can be easily ground and broken, thereby improving the precipitation rate of the pig umbilical cord extract. The use of the enzymatic hydrolysis method will not damage the cells of the pig umbilical cord, and the activity of the cells is maintained, which is convenient for improving the precipitation of the extract. Ultrasonic treatment can destroy the cells, so that the extract can be precipitated efficiently, thereby improving the acquisition rate of the extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the steps of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0028] Embodiment 1:

[0029] See also Figure 1 The present invention provides a technical solution: a preparation process for efficiently obtaining a high-purity and active pig umbilical cord extract, comprising the following steps:

[0030] S1. Cleaning the pig umbilical cord: Cleaning the pig umbilical cord, cleaning the blood, adhesions and mucus on the surface of the pig umbilical cord, and then disinfecting the pig umbilical cord;

[0031] S2, crushing the pig umbilical cord: cutting the pig umbilical cord to keep the size of the pig umbilical cord at 2*3 cm, soaking the small pieces of the pig umbilical cord by enzymatic hydrolysis, and then grinding the small pieces of the pig umbilical cord;

[0032] S3, filtering the ground pig umbilical cord: there is a large amount of blood and tissue fluid in the pig umbilical cord just after grinding, the ground pig umbilical cord is filtered through a filtering mechanism, and a buffer solution is added to the filtered pig umbilical cord tissue;

[0033] S4, ultrasonically treating the buffer solution and the porcine umbilical cord tissue: shaking the porcine umbilical cord tissue in the buffer solution at low temperature, and then ultrasonically treating the porcine umbilical cord tissue;

[0034] S5. Then centrifuge and extract the supernatant: centrifuge the pig umbilical cord tissue after ultrasonic treatment, then extract the supernatant, and filter the supernatant and continue to ultrasonicate for multiple times to obtain a pig umbilical cord extract, and the extract includes vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor.

[0035] In order to improve the purity of the pig umbilical cord extract and prevent the pig umbilical cord from carrying pathogens, in this embodiment, preferably, the surface of the pig umbilical cord tissue in S1 is clean, without a large amount of blood and other impurities adhering, without rot and deterioration, and without odor, and the test results for Brucella and foot-and-mouth disease are negative, and the rational indicators and microbial indicators are qualified.

[0036] In order to improve the medical performance of pig umbilical cord and improve the purity of pig umbilical cord extract, in this embodiment, preferably, the rational indicators include at least pH value, protein content, lead, mercury, arsenic and cadmium, and the microbial indicators include total colony count, total mold and yeast count, heat-resistant coliform group, Pseudomonas aeruginosa and Staphylococcus aureus;

[0037] The pH value is controlled at 6.0-8.0, the protein content is ≥0.5mg / ml, the lead content is ≤0.1mg / kg, the mercury content is ≤0.3mg / kg, the arsenic content is ≤0.5mg / kg and the cadmium content is ≤1.0mg / kg;

[0038] The total colony count is ≤100CFU / g, the total number of molds and yeasts is ≤10CFU / g, and thermotolerant coliform bacteria, Pseudomonas aeruginosa and Staphylococcus aureus must not be detected.

[0039] In order to clean the pig umbilical cord, remove the blood, mucus and residues on the surface, and disinfect the pig umbilical cord to prevent contamination of the extract, in this embodiment, preferably, the pig umbilical cord cleaning in S1 first uses clean water to clean the blood, mucus and residues on the surface, and then soaks it in 75% medical alcohol for three minutes, and then uses distilled water or medical saline to rinse the pig umbilical cord to clean off the medical alcohol remaining on the surface of the pig umbilical cord.

[0040] In order to achieve the dissolution treatment of the pig umbilical cord and facilitate the grinding treatment of the pig umbilical cord, in this embodiment, preferably, the enzymatic hydrolysis method in S2 adopts a mixed agent of trypsin, collagenase and digestive enzyme, and when the enzymatic hydrolysis method is used, it is heated in a low-temperature water bath of 35-40°C, and the duration of the enzymatic hydrolysis method is 4-6 minutes. After the enzymatic hydrolysis treatment, the pig umbilical cord is rinsed with distilled water or medical saline to clean off the mixed agent remaining on the surface of the pig umbilical cord.

[0041] In order to dissolve the pig umbilical cord and achieve mixing treatment to facilitate the precipitation of the extract, in this embodiment, preferably, the buffer solution in S3 is PBS buffer solution, the main components of which are Na2HPO4, KH2PO4, NaCl and KCl, and the mass volume ratio of PBS buffer solution to pig umbilical cord tissue is 10%, that is, 0.1g of tissue sample is added with 1ml of liquid.

[0042] In order to achieve sufficient mixing of the pig umbilical cord in the buffer solution and maintain the uniformity of the mixing, in this embodiment, preferably, the temperature of the low-temperature shaking in S4 is controlled at 30-38°C, and when shaking, the vibration frequency is set to 1000 times / minute and the vibration time is 5 minutes, so that the pig umbilical cord tissue can be fully mixed in the PBS buffer solution, and then the pig umbilical cord tissue is treated with an ultrasonic generator so that the cells in the pig umbilical cord tissue solution can be broken to facilitate the precipitation of the extract.

[0043] In order to realize the centrifugal treatment of the pig umbilical cord so that the precipitate can be stratified with the cell wall, etc., in this embodiment, preferably, the centrifugation in S5 first places the centrifuge tube storing the pig umbilical cord tissue solution into a high-speed centrifuge under a low temperature environment, and centrifuges at a speed of 4000 rpm for 30 minutes, takes out the obtained supernatant with a pipette, and filters it, then transfers the supernatant to a new centrifuge tube, performs multiple ultrasonic treatments, and finally uses a biomembrane filter for filtration.

[0044] In order to achieve multiple filtration and centrifugation to make the extract have a higher purity, in this embodiment, preferably, the first centrifugation of the high-speed centrifuge is set to 4000 rpm for 30 minutes, the second centrifugation is set to 4000 rpm for 1 hour, and the third centrifugation is set to 10000 rpm for 30 minutes, and the second and third centrifugations are for centrifugation of the supernatant.

[0045] In order to achieve multiple filtration of the supernatant and improve the purity of the extract, in this embodiment, preferably, the filtration mechanism adopts a multi-stage filtration device with decreasing filter mesh apertures, and the filter mesh apertures are filtered according to 5μm, 1μm and 0.45μm in sequence to improve the purity of the supernatant.

[0046] Embodiment 2:

[0047] Experimental purpose: To extract VEGF, FGF-2 and HGF from pig umbilical cord with high efficiency and purity;

[0048] The experimental materials used include: fresh porcine umbilical cord, phosphate buffered saline (PBS), proteinase K, collagenase, heparin affinity chromatography column, ion exchange chromatography column (such as DEAE anion exchange column), gel filtration chromatography column, ultrafiltration centrifuge tube and protein quantification kit (such as BCA method);

[0049] Specific experimental steps:

[0050] Pretreatment: After the fresh pig umbilical cord is collected, it is quickly placed in PBS containing antibiotics precooled at 4°C; the surface of the umbilical cord is disinfected with 75% alcohol, and then rinsed with sterile PBS several times. The fetal membrane, blood vessels and connective tissue on the surface of the umbilical cord are removed, and cut into 1-2 cm small segments;

[0051] Cell lysis: Place the processed umbilical cord segments into a mixed enzyme solution containing 0.2% proteinase K and 0.1% collagenase, and digest them in a constant temperature shaker at 37°C for 2 hours; after digestion, filter with a sterile filter to remove undigested tissue residues and collect the filtrate; centrifuge the filtrate at 1500rpm for 15 minutes, discard the supernatant, resuspend the precipitate with PBS, centrifuge again, and repeat 3 times to fully wash the cells;

[0052] Salting out and ultrafiltration: add an appropriate amount of saturated ammonium sulfate solution to the washed precipitate, stir at 4°C for 1 hour, then centrifuge at 10,000 rpm for 30 minutes, and collect the supernatant; ultrafiltration and concentration of the supernatant using an ultrafiltration centrifuge tube to remove small molecule impurities;

[0053] Heparin affinity chromatography: Slowly load the ultrafiltered sample onto the heparin affinity chromatography column, perform gradient elution with PBS containing 0.5-2.0M NaCl after equilibration, and collect the eluate;

[0054] Ion exchange chromatography: After adjusting the pH and ionic strength of the eluent of heparin affinity chromatography, load it onto a DEAE anion exchange chromatography column and elute with a linear gradient containing 0-0.5M NaCl to collect the target protein peak;

[0055] Gel filtration chromatography: The eluate from the ion exchange chromatography is further loaded onto a gel filtration chromatography column, such as Sephadex G-75, eluted with PBS, and the target protein peak is collected;

[0056] Protein quantification and identification: The protein concentration was determined by BCA method; the extracted protein was identified and quantified by Western blot and ELISA;

[0057] Storage: The purified protein was packaged and stored in a -80℃ ultra-low temperature refrigerator for later use.

[0058] During the experiment, the experimental conditions, such as temperature, pH value, ionic strength, etc., must be strictly controlled, and the extraction effect of each step must be monitored and evaluated so that the experimental plan can be adjusted in time to improve the extraction efficiency and purity.

[0059] Embodiment three:

[0060] Pretreatment of pig umbilical cord: Wash the fresh pig umbilical cord, remove blood and impurities, cut it into small pieces, and disinfect the surface with 75% alcohol;

[0061] Homogenization and disruption: Place the processed umbilical cord segments into a homogenizer, add an appropriate amount of phosphate buffered saline (PBS), and perform homogenization to disrupt the cells;

[0062] Ultrasonic disruption: The homogenized sample is subjected to ultrasonic treatment to further disrupt the cells and release the proteins inside the cells;

[0063] Salting out precipitation: slowly add ammonium sulfate to the sample until it reaches a certain saturation, so that most of the impurities are precipitated, and then centrifuge to remove the precipitate;

[0064] Affinity chromatography: The supernatant is passed through a pre-equilibrated heparin affinity chromatography column and gradient eluted with eluents containing different concentrations of sodium chloride, and the eluent containing the target protein is collected;

[0065] Ion exchange chromatography: The eluate collected from the affinity chromatography is further purified by ion exchange chromatography, such as using a DEAE anion exchange column;

[0066] Gel filtration chromatography: The sample after ion exchange chromatography is then passed through a gel filtration chromatography column, such as Sephadex G-75, to remove small molecule impurities and obtain the purified target protein.

[0067] Embodiment 4:

[0068] Umbilical cord processing: clean, disinfect and cut fresh pig umbilical cord into pieces;

[0069] Enzyme digestion: Use a mixed enzyme solution of collagenase and hyaluronidase to digest the umbilical cord tissue;

[0070] Centrifugation: The digested mixture was centrifuged and the supernatant was collected;

[0071] Hydrophobic chromatography: The supernatant is passed through a hydrophobic chromatography column, such as a phenyl agarose gel column, and eluted with an eluent having a reduced salt concentration to collect the target protein fraction;

[0072] Metal chelate chromatography: The sample after hydrophobic chromatography is loaded onto a metal chelate chromatography column (such as a nickel ion chelate column) and eluted according to the binding characteristics of the target protein and the metal ions;

[0073] Concentration and dialysis: The eluate is concentrated by ultrafiltration and the remaining impurities and salts are removed by dialysis.

[0074] Embodiment five:

[0075] Pretreatment of pig umbilical cord: Wash the fresh pig umbilical cord, remove blood and impurities, cut it into small pieces, and disinfect the surface with 75% alcohol;

[0076] Repeated freezing and thawing: The processed umbilical cord segments are frozen and thawed multiple times to destroy the cell structure;

[0077] Hypotonic lysis: Place the sample in a hypotonic solution to lyse the cells;

[0078] Affinity magnetic bead method: Magnetic beads coupled with antibodies against VEGF, FGF-2 and HGF are incubated with the sample, and the magnetic bead-protein complex is collected by magnetic separation;

[0079] Elution: Elute the target protein from the magnetic beads with an appropriate elution buffer;

[0080] Purification: Purify the eluate by ultrafiltration or gel filtration chromatography to improve protein purity.

[0081] Embodiment six:

[0082] Experimental materials: fresh porcine umbilical cord, proteinase K, ammonium sulfate, affinity chromatography medium (specific ligands for three growth factors), and high performance liquid chromatography;

[0083] Experimental steps:

[0084] Pretreatment of pig umbilical cord: Wash the fresh pig umbilical cord, remove impurities, and cut it into small pieces;

[0085] Enzymatic hydrolysis: Add buffer containing proteinase K to the umbilical cord segments and incubate at 37°C for 2 hours;

[0086] Salting out: slowly add ammonium sulfate to the enzymatic hydrolyzate until the saturation reaches 60%, let it stand at 4°C overnight, and collect the precipitate by centrifugation;

[0087] Affinity chromatography: After the precipitate is dissolved, it is loaded onto the affinity chromatography medium and the target protein is eluted;

[0088] High-performance liquid chromatography (HPLC) purification: The affinity chromatography eluate was subjected to HPLC purification and the target protein peak was collected;

[0089] Protein analysis: The purity of the purified protein was analyzed by SDS-PAGE, and the concentration was determined using a quantitative kit.

[0090] Embodiment seven:

[0091] Several fresh pig umbilical cords were collected and immediately placed in sterile saline precooled at 4°C for temporary storage.

[0092] Required reagents: protease (such as trypsin, collagenase, etc.), phosphate buffered saline (PBS), cell lysis solution, chromatography column filler (such as heparin affinity chromatography column, ion exchange chromatography column, etc.), ultrafiltration centrifuge tube, standard products, etc.

[0093] Required instruments: low-temperature high-speed centrifuge, ultra-low temperature refrigerator, shaker, nucleic acid protein detector, high-performance liquid chromatography, etc.

[0094] Experimental process:

[0095] Pretreatment: Take the pig umbilical cord out of the saline solution, disinfect the surface with 75% alcohol, and then rinse with PBS three times; remove the fetal membrane, blood vessels and connective tissue on the surface of the umbilical cord, and cut it into small segments of 1-2 cm;

[0096] Cell separation: Place the processed umbilical cord segments into a digestion solution containing an appropriate amount of protease (such as 0.25% trypsin and 0.1% collagenase) and digest in a 37°C constant temperature shaker for 1-2 hours; after digestion, filter the digestion solution with a filter to remove undigested tissue residues and collect the filtrate; centrifuge the filtrate at 1000-1500 rpm for 10-15 minutes, discard the supernatant, resuspend the precipitate with PBS, centrifuge again, and repeat 2-3 times to fully wash the cells;

[0097] Cell lysis: Treat the precipitated cells with an appropriate amount of cell lysis buffer and place them on ice for 30-60 minutes, gently mixing every 10-15 minutes;

[0098] Initial centrifugation: After lysis is complete, centrifuge the lysate at 12,000-15,000 rpm for 20-30 minutes at 4°C and collect the supernatant;

[0099] Affinity chromatography: the supernatant was passed through a heparin affinity chromatography column, gradient eluted with PBS solution containing different concentrations of NaCl, and the eluate was collected;

[0100] Ion exchange chromatography: The eluate collected by affinity chromatography is subjected to ion exchange chromatography. The appropriate ion exchange column and elution conditions are selected according to the isoelectric point of the target protein for further separation and purification;

[0101] Ultrafiltration concentration: The eluate after ion exchange chromatography is ultrafiltered and concentrated using an ultrafiltration centrifuge tube with a suitable molecular weight cutoff to remove small molecule impurities;

[0102] Detection and identification: Enzyme-linked immunosorbent assay (ELISA), Western Blot or high performance liquid chromatography are used to perform quantitative and qualitative detection of the extracted proteins to determine the content and purity of VEGF, FGF-2 and HGF;

[0103] Storage: The purified protein was packaged and stored in a -80℃ ultra-low temperature refrigerator for later use.

[0104] The working principle and use process of the present invention:

[0105] Step 1: Clean the pig umbilical cord: clean the pig umbilical cord, remove the blood, adhesions and mucus on the surface of the pig umbilical cord, and then disinfect the pig umbilical cord;

[0106] Step 2: crushing the pig umbilical cord: cutting the pig umbilical cord to keep the size of the pig umbilical cord at 2*3 cm, soaking the small pieces of pig umbilical cord by enzymatic hydrolysis, and then grinding the small pieces of pig umbilical cord;

[0107] Step 3, filtering the ground pig umbilical cord: There is a large amount of blood and tissue fluid in the pig umbilical cord just after grinding. The ground pig umbilical cord is filtered through a filtering mechanism, and a buffer solution is added to the filtered pig umbilical cord tissue;

[0108] Step 4, ultrasonically treating the buffer solution and the porcine umbilical cord tissue: shaking the porcine umbilical cord tissue in the buffer solution at low temperature, and then ultrasonically treating the porcine umbilical cord tissue;

[0109] Step 5, then centrifuge to extract the supernatant: centrifuge the pig umbilical cord tissue after ultrasonic treatment, then extract the supernatant, and filter the supernatant and continue to ultrasonicate for multiple times to obtain the pig umbilical cord extract.

[0110] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for efficiently obtaining a high-purity and active pig umbilical cord extract, characterized in that: The following steps are included: S1. Cleaning the pig umbilical cord: Cleaning the pig umbilical cord, cleaning the blood, adhesions and mucus on the surface of the pig umbilical cord, and then disinfecting the pig umbilical cord; S2, crushing the pig umbilical cord: cutting the pig umbilical cord to keep the size of the pig umbilical cord at 2*3 cm, soaking the small pieces of the pig umbilical cord by enzymatic hydrolysis, and then grinding the small pieces of the pig umbilical cord; S3, filtering the ground pig umbilical cord: there is a large amount of blood and tissue fluid in the pig umbilical cord just after grinding, the ground pig umbilical cord is filtered through a filtering mechanism, and a buffer solution is added to the filtered pig umbilical cord tissue; S4, ultrasonically treating the buffer solution and the porcine umbilical cord tissue: shaking the porcine umbilical cord tissue in the buffer solution at low temperature, and then ultrasonically treating the porcine umbilical cord tissue; S5. Then centrifuge and extract the supernatant: centrifuge the pig umbilical cord tissue after ultrasonic treatment, then extract the supernatant, and filter the supernatant and continue to ultrasonicate for multiple times to obtain a pig umbilical cord extract, and the extract includes vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor.

2. The process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 1, characterized in that: The surface of the pig umbilical cord tissue in S1 is clean, without a large amount of blood and other impurities adhering, without decay and deterioration, and without peculiar smell, and the test results for Brucella and foot-and-mouth disease are negative, and both rational indicators and microbiological indicators are qualified.

3. A process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 2, characterized in that: The rational indicators include at least pH value, protein content, lead, mercury, arsenic and cadmium, and the microbial indicators include total colony count, total mold and yeast count, heat-resistant coliform group, Pseudomonas aeruginosa and Staphylococcus aureus; The pH value is controlled at 6.0-8.0, the protein content is ≥0.5mg / ml, the lead content is ≤0.1mg / kg, the mercury content is ≤0.3mg / kg, the arsenic content is ≤0.5mg / kg and the cadmium content is ≤1.0mg / kg; The total colony count is ≤100CFU / g, the total number of molds and yeasts is ≤10CFU / g, and thermotolerant coliform bacteria, Pseudomonas aeruginosa and Staphylococcus aureus must not be detected.

4. The process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 1, characterized in that: The pig umbilical cord cleaning in S1 is firstly cleaned with clean water to clean the blood, mucus and residue on the surface, and then immersed in 75% medical alcohol for three minutes, and then rinsed with distilled water or medical saline to clean the medical alcohol remaining on the surface of the pig umbilical cord.

5. The process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 1, characterized in that: The enzymatic hydrolysis method in S2 uses a mixture of trypsin, collagenase and digestive enzymes, and when the enzymatic hydrolysis method is used, it is heated in a low-temperature water bath at 35-40°C, and the duration of the enzymatic hydrolysis method is 4-6 minutes. After the enzymatic hydrolysis treatment, the pig umbilical cord is rinsed with distilled water or medical saline to clean off the residual mixed agent on the surface of the pig umbilical cord.

6. The process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 1, characterized in that: The buffer in S3 is PBS buffer, the main components of which are Na2HPO4, KH2PO4, NaCl and KCl, and the mass volume ratio of PBS buffer to pig umbilical cord tissue is 10%, that is, 0.1g of tissue sample is added with 1ml of liquid.

7. A process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 6, characterized in that: The temperature of the low-temperature shaking in S4 is controlled at 30-38° C., and when shaking, the vibration frequency is set to 1000 times / minute and the vibration time is 5 minutes, so that the pig umbilical cord tissue can be fully mixed in the PBS buffer solution, and then the pig umbilical cord tissue is treated with an ultrasonic generator so that the cells in the pig umbilical cord tissue solution can be broken to facilitate the precipitation of the extract.

8. The process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 1, characterized in that: The centrifugation in S5 first places the centrifuge tube storing the pig umbilical cord tissue solution into a high-speed centrifuge under a low temperature environment, centrifuges at a speed of 4000 rpm for 30 minutes, takes out the obtained supernatant with a pipette, and filters it, then transfers the supernatant to a new centrifuge tube, performs multiple ultrasonic treatments, and finally uses a biomembrane filter for filtration.

9. A process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 8, characterized in that: The high-speed centrifuge is set to centrifuge at 4000 rpm for 30 minutes for the first centrifugation, at 4000 rpm for 1 hour for the second centrifugation, and at 10000 rpm for the third centrifugation for 30 minutes, and the second and third centrifugations are performed on the supernatant.

10. The process for efficiently obtaining a high-purity and active pig umbilical cord extract according to claim 1, characterized in that: The filtering mechanism adopts a multi-stage filtering device with decreasing filter mesh apertures, and the filter mesh apertures are filtered in order of 5 μm, 1 μm and 0.45 μm to improve the purity of the supernatant.

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