Decellularized umbilical cord extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a decellularized umbilical cord extract as well as a preparation method and application thereof. The preparation method of the decellularized umbilical cord extract comprises the following steps: by taking human umbilical cord tissue as a raw material, disinfecting the human umbilical cord tissue, cleaning and removing impurities, and then sequentially performing pretreatment, enzyme digestion and ultrafiltration treatment to obtain the decellularized umbilical cord extract. The pretreatment process comprises the following steps: sequentially freezing, ultrasonically homogenizing and treating the human umbilical cord tissues by using a pretreatment solution. According to the method, the human umbilical cord tissue is extracted by adopting a multi-step method, so that the loss rate of protein and collagen in the umbilical cord can be reduced, and the stability and activity of the protein and collagen in the umbilical cord can be ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular, relates to a decellularized umbilical cord extract and a preparation method and application thereof. Background Art

[0002] Umbilical cord extract is a mixture of cytokines extracted from the umbilical cord of a newborn, which includes a variety of biologically active ingredients such as cytokines, active peptides, enzymes and amino acids. These active ingredients have multiple functions such as promoting cell division, proliferation, and repairing damaged tissues. They can help the skin remove aging keratin, promote metabolism, and enhance skin immunity. Based on the active ingredients in umbilical cord extract, umbilical cord extract currently has the following main application areas: 1. Skin care products: Umbilical cord extract has been widely used as a raw material for skin care products. It can promote skin repair and regeneration, anti-inflammatory, anti-aging, antioxidant and other effects. At present, many brands on the market have launched skin care products with umbilical cord extract as the main ingredient, and they are welcomed by more and more consumers. 2. Hair care field: Umbilical cord extract can promote hair growth, enhance hair quality, and inhibit the formation of dandruff. Therefore, it also has broad application prospects in hair care. 3. Medical field: In addition to the beauty field, umbilical cord extract is also widely used in the medical field. Due to its good biocompatibility and regeneration ability, it can be used in wound repair and tissue engineering.

[0003] Umbilical cord extract, as a natural and efficient medical and aesthetic raw material, is gradually becoming the focus of research in related fields. However, the extraction and processing of umbilical cord extract currently provided is relatively complicated and costly, and the extraction of effective active ingredients is difficult to a certain extent, thus limiting its wide application. Summary of the invention

[0004] The purpose of the present invention is to provide a decellularized umbilical cord extract and a preparation method and application thereof, so as to achieve effective extraction of bioactive substances in the umbilical cord.

[0005] To this end, the present invention provides the following technical solutions.

[0006] One aspect of the present invention provides a method for preparing a decellularized umbilical cord extract, the method comprising the following steps: Human umbilical cord tissue is used as a raw material, the human umbilical cord tissue is disinfected, cleaned and impurities removed, and then pretreated, enzymatically digested and ultrafiltered in sequence to obtain a decellularized umbilical cord extract; The pretreatment process comprises: freezing, ultrasonic homogenizing and treating the human umbilical cord tissue in sequence.

[0007] As a preferred embodiment of the present invention, the disinfection process is: using 75% alcohol by volume to disinfect the fresh human umbilical cord, and then ultraviolet sterilization for 20 to 40 minutes.

[0008] As a preferred embodiment of the present invention, the cleaning and impurity removal process is: soaking the disinfected human umbilical cord in a primary treatment solution for 1 to 3 hours, then taking it out and using purified water to disperse and clean it to remove residual impurities on the surface.

[0009] As a preferred embodiment of the present invention, the primary treatment liquid is composed of: 10-20 mM 2,6-pyridinedicarboxylic acid, 5-10 mM p-hydroxyphenylethylamine, 15-25 mM citric acid, and the balance is water.

[0010] As a preferred embodiment of the present invention, the freezing conditions are: temperature: -30~-15°C, time: 6~10h.

[0011] As a preferred embodiment of the present invention, the ultrasonic homogenization process is as follows: the frozen umbilical cord tissue is immersed in a secondary treatment liquid, first placed in a dark environment and irradiated with a 365nm wavelength ultraviolet lamp for 5 to 10 minutes, and then placed in an ultrasonic oscillator for ultrasonic homogenization to obtain a mixture.

[0012] As a preferred embodiment of the present invention, the secondary treatment liquid is composed of: 1-5 g / L of amino compound, 0.5-1.5 g / L of halide salt and 0.01-0.03 g / L of polyphenol, and the balance is water.

[0013] As a preferred embodiment of the present invention, the amino compound is selected from any one of polyethyleneimine, polyacrylamide, carboxymethyl chitosan, polylysine, and polyethylenediamine.

[0014] As a preferred embodiment of the present invention, the halide salt is selected from any one of ferrous chloride, ferric chloride, calcium chloride, magnesium chloride, sodium chloride and potassium chloride.

[0015] As a preferred embodiment of the present invention, the polyphenol is selected from any one of hydroquinone, catechol, and resorcinol. As a preferred embodiment of the present invention, the ultrasonic conditions are: power of 300-350 W, frequency of 20-30 KHz, oscillation time of 10-20 min, and oscillation temperature of 25-40° C.

[0016] As a preferred embodiment of the present invention, the enzymatic digestion process is: adding digestive enzymes to the obtained mixture for enzymatic hydrolysis, inactivating the enzymes after the enzymatic hydrolysis, cooling, and centrifuging to obtain a supernatant.

[0017] As a preferred embodiment of the present invention, the enzymatic hydrolysis conditions are: temperature 50-70°C, time 10-12h.

[0018] As a preferred embodiment of the present invention, the composition and mass percentage of the digestive enzymes are: 50-66% papain, 12-20% neutral protease and 8-34% trypsin.

[0019] As a preferred embodiment of the present invention, the added amount of the digestive enzyme is 0.2-0.3% of the mass of the mixture.

[0020] As a preferred embodiment of the present invention, the ultrafiltration process is: filtering with an ultrafiltration membrane, the molecular weight of the ultrafiltration membrane is 10~20KDa, and the operating pressure is 2.0~4.0Mpa.

[0021] As a preferred embodiment of the present invention, the method further comprises concentrating the obtained filtrate, freeze-drying it and then pulverizing it to obtain a cell-free umbilical cord extract.

[0022] Another aspect of the present invention also provides a decellularized umbilical cord extract prepared according to the preparation method as described above.

[0023] Another aspect of the present invention also provides the use of the decellularized umbilical cord extract in preparing a skin wound repair product.

[0024] By means of the above technical solution, the present invention has at least the following advantages: 1. The present invention uses human umbilical cord tissue as raw material, disinfects, cleans and removes impurities from the human umbilical cord tissue, and then sequentially performs pretreatment, enzyme digestion and ultrafiltration treatment to obtain a decellularized umbilical cord extract. The obtained decellularized umbilical cord extract is rich in protein, collagen, glycogen and lipid. The present invention can reduce the loss rate of protein and collagen in the umbilical cord by using a single treatment liquid and a pretreatment process for human umbilical cord tissue, and ensure the stability and activity of umbilical cord protein and collagen.

[0025] 2. The umbilical cord extract obtained by the present invention has multiple functions such as promoting skin repair and regeneration, anti-inflammatory, anti-aging, anti-oxidation, good biocompatibility, etc. It can be widely used in the fields of skin care, hair care, wound repair and tissue engineering.

[0026] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a physical picture of the decellularized umbilical cord extract prepared in Example 1 of the present invention; Figure 2 The present invention compares the wound repair effects of the composite hydrogel containing the decellularized umbilical cord extract. DETAILED DESCRIPTION

[0028] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0029] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0030] Unless otherwise specified, the papain (P6321, purchased from Tianjin Xidian Chemical Technology Co., Ltd.) used in the following examples has an activity of 2×10 5 U / g; neutral protease (04942078001, Sigma) activity is 2×10 5 U / g; trypsin (T6424, Sigma) activity is 1×10 5 U / g, other materials and reagents can be obtained from commercial sources.

[0031] Embodiment 1: Take fresh human umbilical cord, first disinfect it with 75% alcohol by volume, and then sterilize it with ultraviolet light for 30 minutes. Soak the disinfected human umbilical cord in a primary treatment solution (composition: 15mM 2,6-pyridinedicarboxylic acid, 7.5mM p-hydroxyphenylethylamine, 20mM citric acid, and the balance is water) for 2 hours, then take it out and wash it with purified water to remove residual impurities on the surface. The cleaned and impurity-free human umbilical cord is frozen at -20℃ for 8 hours, then broken, and the broken umbilical cord tissue is soaked in a secondary treatment solution (composition: 3g / L polyethyleneimine, 0.5g / L ferrous chloride, 0.5g / L ferric chloride and 0.02g / L hydroquinone, the balance is water), first place it in a dark environment and irradiate it with a 365nm wavelength ultraviolet lamp for 7.5 minutes, then place it in an ultrasonic oscillator with a power of 320W, a frequency of 25KHz, and a temperature of 30℃ for 15 minutes of ultrasonic homogenization to obtain a mixture. Add 0.25% digestive enzymes (composition and mass percentage ratio: 50% papain, 20% neutral protease and 30% trypsin) to the obtained mixture and perform enzymolysis at 60°C for 11 hours. After the enzymolysis is completed, inactivate the enzyme, cool, and centrifuge at 20,000 rpm for 10 minutes to obtain a supernatant. The obtained supernatant is filtered using an ultrafiltration membrane with a molecular weight of 20KDa at an operating pressure of 3.0Mpa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate is concentrated, freeze-dried, and crushed to obtain a decellularized umbilical cord extract. The actual picture is as follows Figure 1 shown.

[0032] Embodiment 2: Take fresh human umbilical cord, first disinfect it with 75% alcohol by volume, and then sterilize it with ultraviolet light for 30 minutes. Soak the disinfected human umbilical cord in a primary treatment solution (composition: 10mM 2,6-pyridinedicarboxylic acid, 5mM p-hydroxyphenylethylamine, 25mM citric acid, and the balance is water) for 1 hour, then take it out and wash it with purified water to remove residual impurities on the surface. The cleaned and impurity-free human umbilical cord is frozen at -30℃ for 6 hours, then broken, and the broken umbilical cord tissue is soaked in a secondary treatment solution (composition: 5g / L polyacrylamide, 0.5g / L ferrous chloride and 0.03g / L catechol, the balance is water), first placed in a dark environment and irradiated with a 365nm wavelength ultraviolet lamp for 5 minutes, and then placed in an ultrasonic oscillator with a power of 300W, a frequency of 30KHz, and a temperature of 25℃ for ultrasonic homogenization for 20 minutes to obtain a mixture. Add 0.2% digestive enzyme (composition and mass percentage ratio: papain 66%, neutral protease 12% and trypsin 22%) to the obtained mixture, and perform enzymolysis at 70°C for 10 hours. After the enzymolysis is completed, the enzyme is inactivated, cooled, and centrifuged at 20,000 rpm for 10 minutes to obtain a supernatant. The obtained supernatant is filtered using an ultrafiltration membrane with a molecular weight of 20 KDa at an operating pressure of 3.0 MPa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate is concentrated, freeze-dried, and crushed to obtain a decellularized umbilical cord extract.

[0033] Embodiment 3: Take fresh human umbilical cord, first disinfect it with 75% alcohol by volume, and then sterilize it with ultraviolet light for 30 minutes. Soak the disinfected human umbilical cord in a primary treatment solution (composition: 2,6-pyridinedicarboxylic acid 20mM, p-hydroxyphenylethylamine 10mM, citric acid 15mM, the balance is water) for 3 hours, then take it out and wash it with purified water to remove residual impurities on the surface. The cleaned and impurity-free human umbilical cord is frozen at -15℃ for 10 hours, then broken, and the broken umbilical cord tissue is soaked in a secondary treatment solution (composition: polyethylenediamine 1g / L, ferrous chloride 0.5g / L, ferric chloride 1.0g / L and resorcinol 0.01g / L, the balance is water), first placed in a dark environment and irradiated with a 365nm wavelength ultraviolet lamp for 10 minutes, and then placed in an ultrasonic oscillator with a power of 350W, a frequency of 20KHz, and a temperature of 40℃ for ultrasonic homogenization for 10 minutes to obtain a mixture. Add 0.3% digestive enzyme (composition and mass percentage ratio: papain 60%, neutral protease 20% and trypsin 20%) to the obtained mixture, and perform enzymolysis at 50°C for 12 hours. After the enzymolysis is completed, the enzyme is inactivated, cooled, and centrifuged at 20000rpm for 10 minutes to obtain a supernatant. The obtained supernatant is filtered using an ultrafiltration membrane with a molecular weight of 20KDa at an operating pressure of 3.0Mpa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate is concentrated, freeze-dried, and crushed to obtain a decellularized umbilical cord extract.

[0034] Comparative Example 1: Take fresh human umbilical cord, first disinfect it with 75% alcohol by volume, and then sterilize it with ultraviolet light for 30 minutes. Wash the disinfected human umbilical cord with purified water to remove residual impurities on the surface. Freeze the cleaned and impurity-removed human umbilical cord at -20℃ for 8 hours, then break it, soak the broken umbilical cord tissue in secondary treatment liquid (composed of: 3g / L polyethyleneimine, 0.5g / L ferrous chloride, 0.5g / L ferric chloride and 0.02g / L hydroquinone, the balance is water), first place it in a dark environment and irradiate it with a 365nm wavelength ultraviolet lamp for 7.5 minutes, then place it in an ultrasonic oscillator with a power of 320W, a frequency of 25KHz, and a temperature of 30℃ for 15 minutes of ultrasonic homogenization to obtain a mixture. Add 0.25% digestive enzyme (composition and mass percentage ratio: 50% papain, 20% neutral protease and 30% trypsin) to the obtained mixture and perform enzymolysis at 60°C for 11 hours. After the enzymolysis is completed, the enzyme is inactivated, cooled, and centrifuged at 20,000 rpm for 10 minutes to obtain a supernatant. The obtained supernatant is filtered using an ultrafiltration membrane with a molecular weight of 20 KDa at an operating pressure of 3.0 MPa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate is concentrated, freeze-dried, and crushed to obtain a decellularized umbilical cord extract.

[0035] Comparative Example 2: Take a fresh human umbilical cord, first disinfect it with 75% alcohol by volume, and then sterilize it with ultraviolet light for 30 minutes. Soak the disinfected human umbilical cord in a primary treatment solution (composed of: 15mM 2,6-pyridinedicarboxylic acid, 7.5mM p-hydroxyphenylethylamine, 20mM citric acid, and the balance is water) for 2 hours, then take it out and wash it with purified water to remove residual impurities on the surface. Freeze the cleaned and impurity-free human umbilical cord at -20℃ for 8 hours, then crush it, soak the crushed umbilical cord tissue in water, first place it in a dark environment and irradiate it with a 365nm wavelength ultraviolet lamp for 7.5 minutes, then place it in an ultrasonic oscillator at a power of 320W, a frequency of 25KHz, and a temperature of 30℃ for 15 minutes of ultrasonic homogenization to obtain a mixture. Add 0.25% digestive enzyme (composition and mass percentage ratio: 50% papain, 20% neutral protease and 30% trypsin) to the obtained mixture and perform enzymolysis at 60°C for 11 hours. After the enzymolysis is completed, the enzyme is inactivated, cooled, and centrifuged at 20,000 rpm for 10 minutes to obtain a supernatant. The obtained supernatant is filtered using an ultrafiltration membrane with a molecular weight of 20 KDa at an operating pressure of 3.0 MPa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate is concentrated, freeze-dried, and crushed to obtain a decellularized umbilical cord extract.

[0036] Comparative Example 3: Take fresh human umbilical cords, first disinfect them with 75% alcohol by volume, and then sterilize them with ultraviolet light for 30 minutes. Soak the disinfected human umbilical cords in a primary treatment solution (composition: 15mM 2,6-pyridinedicarboxylic acid, 7.5mM p-hydroxyphenylethylamine, 20mM citric acid, and the balance is water) for 2 hours, then take them out and wash them with purified water to remove residual impurities on the surface. Freeze the cleaned and impurity-free human umbilical cords at -20℃ for 8 hours, then crush them, and soak the crushed umbilical cord tissue in water to obtain a mixture. Add 0.25% of the mixture by mass of digestive enzymes (composition and mass percentage ratio: 50% papain, 20% neutral protease, and 30% trypsin) to the obtained mixture, and enzymolyze them at 60℃ for 11 hours. After the enzymolysis is completed, inactivate the enzymes, cool them, and centrifuge them at 20000rpm for 10 minutes to obtain the supernatant. The obtained supernatant was filtered using an ultrafiltration membrane with a molecular weight of 20 KDa under an operating pressure of 3.0 MPa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate was concentrated, freeze-dried, and pulverized to obtain a decellularized umbilical cord extract.

[0037] Comparative Example 4: Take fresh human umbilical cord, first disinfect it with 75% alcohol by volume, and then sterilize it with ultraviolet light for 30 minutes. Wash the disinfected human umbilical cord with purified water to remove residual impurities on the surface. Freeze the cleaned and impurity-removed human umbilical cord at -20℃ for 8 hours, then break it, soak the broken umbilical cord tissue in water, first place it in a dark environment and irradiate it with a 365nm wavelength ultraviolet lamp for 7.5 minutes, then place it in an ultrasonic oscillator and ultrasonically homogenize it for 15 minutes at a power of 320W, a frequency of 25KHz, and a temperature of 30℃ to obtain a mixture. Add 0.25% of the mass of the mixture to the obtained mixture (composition and mass percentage ratio: 50% papain, 20% neutral protease and 30% trypsin) and enzymolyze it at 60℃ for 11 hours. After the enzymolysis is completed, the enzyme is inactivated, cooled, and centrifuged at 20000rpm for 10 minutes to obtain a supernatant. The obtained supernatant was filtered using an ultrafiltration membrane with a molecular weight of 20 KDa under an operating pressure of 3.0 MPa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate was concentrated, freeze-dried, and pulverized to obtain a decellularized umbilical cord extract.

[0038] Comparative Example 5: Fresh human umbilical cords were first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes. The disinfected human umbilical cords were washed with purified water to remove residual impurities on the surface. The cleaned and impurity-free human umbilical cords were frozen at -20°C for 8 hours, then crushed, and the crushed umbilical cord tissue was soaked in water to obtain a mixture. Digestive enzymes (composition and mass percentage ratio: 50% papain, 20% neutral protease and 30% trypsin) were added to the obtained mixture, and enzymolysis was performed at 60°C for 11 hours. After the enzymolysis was completed, the enzyme was inactivated, cooled, and centrifuged at 20000rpm for 10 minutes to obtain a supernatant. The obtained supernatant was filtered using an ultrafiltration membrane with a molecular weight of 20KDa under an operating pressure of 3.0Mpa to obtain an ultrafiltrate. Finally, the obtained ultrafiltrate was concentrated, freeze-dried, and crushed to obtain a decellularized umbilical cord extract.

[0039] Experiment 1: Analysis of components of umbilical cord extract 1. Detection of total protein content: refer to the Kjeldahl nitrogen determination method of Part IV 0731 Protein content determination method of the Chinese Pharmacopoeia (2015 edition); 2. Detection of total sugar content: refer to Appendix E of YY / T1453-2016 "Characterization Method of Type I Collagen in Tissue Engineering Medical Devices" for determination of total sugar content; 3. Fat content detection: refer to the acid hydrolysis method of GB 5009.6-2016 "Food Safety Standard Determination of Fat in Food"; 4. Detection of residue on ignition content: refer to the ignition residue test method in 0841 of the Chinese Pharmacopoeia (Volume 4 of the 2015 edition).

[0040] All test items were repeated three times in parallel and the average value was taken. The results are shown in Table 1.

[0041] Table 1 Analysis of components of different umbilical cord extracts

[0042] It can be seen from the results in Table 1 that, compared with comparative examples 1-5, the total protein content and total sugar content in the umbilical cord extracts of Examples 1-3 of the present invention are significantly higher, wherein the ultrasound-assisted secondary treatment liquid treatment process can further increase the content of effective substances in the umbilical cord extracts.

[0043] Experiment 2: Repair of skin damage Preparation of composite hydrogel for skin damage repair 1: Add phenyl-2,4,6-trimethylbenzoyl phosphite lithium to PBS to obtain a phenyl-2,4,6-trimethylbenzoyl phosphite standard solution with a mass volume ratio concentration of 0.5%. Add methacrylated gelatin (purchased from Hubei Langbowan Biopharmaceutical Co., Ltd.) to a phenyl-2,4,6-trimethylbenzoyl phosphite standard solution with a mass volume ratio concentration of 0.5%, heat and dissolve at 60°C to obtain a methacrylated gelatin hydrogel solution with a mass volume ratio concentration of 0.5%. Add the decellularized umbilical cord extract obtained in Example 1 to the methacrylated gelatin hydrogel solution with a mass volume ratio concentration of 0.5% and stir evenly to obtain a mixed solution. The obtained mixed solution is placed in a mold and subjected to a photocuring reaction under a blue light with a wavelength of 405 nm for 2 min to obtain a composite hydrogel.

[0044] Skin damage repair composite hydrogels 2 to 8 were prepared according to the above method: the decellularized umbilical cord extract obtained in Example 1 was replaced with the decellularized umbilical cord extracts obtained in Examples 2-3 and Comparative Examples 1-5, respectively, and the rest was consistent with the above method to prepare skin damage repair composite hydrogels 2 to 8.

[0045] Blank hydrogel: A blank hydrogel was prepared according to the above method, wherein the decellularized umbilical cord extract was not added, and the other procedures were consistent with the above method to obtain a blank hydrogel.

[0046] Animal experiment: 35 7-week-old SD rats, weighing 200±20g, half male and half female were selected. First, a rat skin lesion model was constructed: a circular wound with a skin lesion area of ​​2 cm in diameter was constructed on the back of the rat. After the model rats were successfully constructed, the model rats were divided into 9 groups on average. The embodiment groups 1-8 were applied with the above-prepared skin damage repair composite hydrogels 1~8 on the wounds, and the blank group was applied with blank hydrogels. The diameters of the wounds of each group of rats were measured on days 0, 1, 5, 10, and 15, and the wound repair rate (%) was calculated: Wound repair rate (%) = (original wound area - repaired wound area) / original wound area * 100% The results are shown in Table 2 and Figure 2 shown.

[0047] Table 2 Repair rate of wounds in rats with different composite hydrogels

[0048] From Table 2 and Figure 2 It can be seen from the results that the composite hydrogels with the umbilical cord extracts of Examples 1-3 of the present invention as the main active substance have a significantly better wound repair rate in rats than the composite hydrogels with the umbilical cord extracts of Comparative Examples 1-5 as the main active substance and the blank group. This result shows that the active substances contained in the umbilical cord extracts of Examples 1-3 can promote wound repair in rats.

[0049] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a decellularized umbilical cord extract, characterized in that: The method comprises the following steps: Human umbilical cord tissue is used as a raw material, the human umbilical cord tissue is disinfected, cleaned and impurities removed, and then pretreated, enzymatically digested and ultrafiltered in sequence to obtain a decellularized umbilical cord extract; The pretreatment process comprises: freezing, ultrasonic homogenizing and treating the human umbilical cord tissue in sequence.

2. The method for preparing the decellularized umbilical cord extract according to claim 1, characterized in that: The disinfection process is as follows: using 75% alcohol by volume to disinfect the fresh human umbilical cord, and then sterilizing it with ultraviolet light for 20 to 40 minutes; The cleaning and impurity removal process is: soaking the disinfected human umbilical cord in a primary treatment solution for 1 to 3 hours, then taking it out and using purified water to disperse and clean it to remove residual impurities on the surface.

3. The method for preparing the decellularized umbilical cord extract according to claim 2, characterized in that: The primary treatment solution comprises: 10-20 mM 2,6-pyridinedicarboxylic acid, 5-10 mM p-hydroxyphenylethylamine, 15-25 mM citric acid, and the balance is water.

4. The method for preparing the decellularized umbilical cord extract according to claim 1, characterized in that: The freezing conditions are: temperature: -30 to -15°C, time: 6 to 10 hours.

5. The method for preparing the decellularized umbilical cord extract according to claim 1, characterized in that: The ultrasonic homogenization process is as follows: the frozen umbilical cord tissue is immersed in a secondary treatment liquid, firstly placed in a dark environment and irradiated with a 365nm wavelength ultraviolet lamp for 5 to 10 minutes, and then placed in an ultrasonic oscillator for ultrasonic homogenization to obtain a mixture.

6. The method for preparing the decellularized umbilical cord extract according to claim 5, characterized in that: The secondary treatment liquid comprises: 1-5 g / L of amino compound, 0.5-1.5 g / L of halide salt and 0.01-0.03 g / L of polyphenol, and the balance is water.

7. The method for preparing the decellularized umbilical cord extract according to claim 6, characterized in that: The amino compound is selected from any one of polyethyleneimine, polyacrylamide, carboxymethyl chitosan, polylysine, and polyethylenediamine; The halide salt is selected from any one of ferrous chloride, ferric chloride, calcium chloride, magnesium chloride, sodium chloride and potassium chloride; The polyphenol is selected from any one of hydroquinone, catechol and resorcinol.

8. The method for preparing the decellularized umbilical cord extract according to claim 5, characterized in that: The ultrasonic conditions are: power of 300-350W, frequency of 20-30KHz, oscillation time of 10-20min, and oscillation temperature of 25-40°C; And / or, the enzymatic digestion process is: adding digestive enzymes to the obtained mixture for enzymatic hydrolysis, inactivating the enzymes after the enzymatic hydrolysis, cooling, and centrifuging to obtain a supernatant; And / or, the enzymatic hydrolysis conditions are: temperature 50-70°C, time 10-12h; And / or, the composition and mass percentage of the digestive enzymes are: 50-66% papain, 12-20% neutral protease and 8-34% trypsin; And / or, the added amount of the digestive enzyme is 0.2-0.3% of the mass of the mixture. And / or, the ultrafiltration process is: filtering with an ultrafiltration membrane, the ultrafiltration membrane has a molecular weight of 10 to 20 KDa, and an operating pressure of 2.0 to 4.0 MPa.

9. A decellularized umbilical cord extract prepared according to the preparation method of claims 1-8.

10. Use of the decellularized umbilical cord extract according to claim 9 in preparing a skin wound repair product.

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