Decellularized amnion extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a decellularized amnion extract as well as a preparation method and application thereof. The preparation method of the decellularized amniotic membrane extract comprises the following steps: sequentially disinfecting, cleaning and removing impurities from a fresh amniotic membrane, then crushing, carrying out enzymolysis on the crushed product by adopting compound protease, and carrying out centrifugation and ultrafiltration on the enzymolysis product to prepare the amniotic membrane extract. According to the method, the steps of crushing, enzymolysis, ultrafiltration and the like are sequentially performed on the human amniotic membrane, so that effective extraction of effective components, namely total protein and total sugar, in the amniotic membrane extract can be realized, and the industrial production and wide application of the amniotic membrane extract are further realized.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular, relates to a decellularized amniotic membrane extract and a preparation method and application thereof. Background Art

[0002] The amniotic membrane is the innermost layer of the placenta, mainly containing proteins and total sugars (especially glycosaminoglycans). The amniotic membrane or amniotic membrane extracts are used as medical devices or drugs. The proteins in its components can serve as a natural scaffold for cell growth and adhesion, and have the function of promoting tissue repair. The glycosaminoglycans in the total sugars have a hydrophilic lubricating effect. Studies have shown that human amniotic membrane (hAM) contains rich growth factors and has immunomodulatory and anti-inflammatory effects. As a natural biomaterial, human amniotic membrane extract (hAME) is widely used in the treatment of ophthalmic diseases and burns because of its functions of promoting tissue regeneration, wound healing, and reducing inflammatory reactions and oxidative stress damage.

[0003] Therefore, the extraction of effective ingredients from amniotic membrane has become one of the current research directions. Summary of the invention

[0004] The purpose of the present invention is to provide a decellularized amniotic membrane extract and a preparation method and application thereof, so as to achieve efficient extraction of effective components in human amniotic membrane.

[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 amniotic membrane extract, the preparation method comprising: sterilizing, cleaning and removing impurities from fresh amniotic membrane in sequence, and then crushing the fresh amniotic membrane, hydrolyzing the crushed amniotic membrane with a composite protease, and centrifuging and ultrafiltering the hydrolyzed amniotic membrane to obtain the amniotic membrane extract.

[0007] As a preferred embodiment of the present invention, the disinfection process is: using 75% alcohol by volume to disinfect the fresh human amniotic membrane, 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: the sterilized human amniotic membrane is dispersed and cleaned with purified water to remove residual impurities on the surface.

[0009] As a preferred embodiment of the present invention, the drying process is: the cleaned and impurity-free human amniotic membrane is spread flat on a sieve to dry for 1 to 3 hours, and turned over every 10 to 20 minutes.

[0010] As a preferred embodiment of the present invention, the crushing process is: placing the dried human amniotic membrane in an ultrasonic reactor and adding modified diamond, stirring evenly and then performing ultrasonic treatment.

[0011] As a preferred embodiment of the present invention, the added amount of the modified diamond is 10-20% of the mass of the human amniotic membrane.

[0012] As a preferred embodiment of the present invention, the ultrasonic treatment method is intermittent ultrasound, and the ultrasonic conditions are: power 600-800W, and the ultrasound is carried out every 5-8 minutes and stopped for 1-3 minutes, and the total ultrasound time is 60-90 minutes.

[0013] As a preferred embodiment of the present invention, the preparation method of the modified diamond is:

[0014] Mixing quartz sand, tourmaline and diamond in a mass ratio of 5-10:1-2:3-5 and grinding to obtain a mixture;

[0015] The obtained mixture is placed in a muffle furnace, calcined at 600-1000°C for 1-5h, then cooled to 400-600°C at a cooling rate of 80-120°C / h and kept at that temperature for 30min, cooled to room temperature in air after the end of the heat preservation, and the obtained calcined product is crushed and passed through an 80-100 mesh sieve to obtain a modified diamond precursor powder;

[0016] The obtained diamond precursor powder is placed in a hydrofluoric acid solution with a volume concentration of 4-8% and stirred at a speed of 200-400 rpm for 5-10 minutes, and then filtered. The obtained solid product is washed and dried to obtain modified diamond.

[0017] As a preferred embodiment of the present invention, the grinding process conditions are: grinding three times in total, and the grinding conditions are:

[0018] First grinding: grinding at a speed of 3000-4000 rpm for 2-4 hours;

[0019] Second grinding: grinding at a speed of 2000-3000 rpm for 1-3 hours;

[0020] The third grinding: grinding at a speed of 1000-2000 rpm for 0.5-2h.

[0021] As a preferred embodiment of the present invention, the enzymatic hydrolysis process is: adding 0.01-0.05% of the total mass of the product to the product after ultrasonic treatment. The product is enzymatically hydrolyzed, and after the enzymatic hydrolysis is completed, the enzyme is inactivated, cooled, and centrifuged to obtain a supernatant.

[0022] As a preferred embodiment of the present invention, the composite protease is composed of neutral protease, papain and pancreatin, and the mass ratio of neutral protease, papain and pancreatin is 1:(1-3):(1-2).

[0023] As a preferred embodiment of the present invention, the enzymatic hydrolysis conditions are: temperature 50-70° C., time 10-12 h.

[0024] As a preferred embodiment of the present invention, the centrifugal conditions are: rotation speed 10000-20000 rpm, time 10-20 min.

[0025] 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 to 20 KDa, and the operating pressure is 2.0 to 4.0 MPa.

[0026] Another aspect of the present invention provides a decellularized amniotic membrane extract prepared according to the preparation method as described above.

[0027] Another aspect of the present invention provides the use of the decellularized amniotic membrane extract in preparing a product for promoting tissue regeneration.

[0028] By means of the above technical solution, the present invention has at least the following advantages:

[0029] 1. The present invention can realize the effective extraction of total protein and total sugar in the amniotic membrane extract by successively performing the steps of crushing, enzymolysis, ultrafiltration and the like on the human amniotic membrane, thereby further realizing the industrial production and wide application of the amniotic membrane extract.

[0030] 2. The present invention uses ultrasound-assisted modified diamond to crush human amniotic membrane during the crushing process, which can improve the crushing effect of human amniotic membrane tissue, thereby further improving the subsequent enzymatic hydrolysis efficiency. The ultrasound-assisted crushing material used in the present invention is modified diamond, which is made of quartz sand, tourmaline and diamond as raw materials and is subjected to calcination and etching to obtain a composite material with an irregular surface structure. The modified diamond of the present invention has a high hardness, and can form an irregular convex structure on the surface through hydrofluoric acid etching, thereby further improving the crushing effect, obtaining a crushed product with a smaller particle size, and making the final extract have a fine particle size and high protein and total sugar content.

[0031] 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

[0032] Figure 1 This is a physical picture of the amniotic membrane extract obtained in Example 1. DETAILED DESCRIPTION

[0033] 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.

[0034] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0035] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0036] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] 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.

[0039] Embodiment 1:

[0040] Preparation of modified diamond: Quartz sand, tourmaline and diamond are mixed in a mass ratio of 7.5:1.5:4, and the solvent ethanol is added in an amount of 0.5wt% of the total mass of the three substances of quartz sand, tourmaline and diamond. The mixture is ground three times, that is, firstly ground at a speed of 3500rpm for 3h, then ground at a speed of 2500rpm for 2h, and finally ground at a speed of 1500rpm for 1.25h, and a mixture is obtained after grinding. The obtained mixture is placed in a muffle furnace, calcined at 800℃ for 3h, then cooled to 500℃ at a cooling rate of 100℃ / h and kept warm for 30min, and then cooled to room temperature in air after the end of the heat preservation, and the obtained calcined product is crushed and passed through an 80-mesh sieve to obtain a modified diamond precursor powder. The obtained diamond precursor powder is placed in a hydrofluoric acid solution with a volume concentration of 6% and stirred at a speed of 300rpm for 7.5min, then filtered, and the obtained solid product is washed and dried to obtain modified diamond.

[0041] Preparation of human amniotic membrane extract: fresh human amniotic membrane was first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes; the disinfected human amniotic membrane was then dispersed and cleaned with purified water to remove residual impurities on the surface; finally, the cleaned and impurity-free human amniotic membrane was spread flat on a sieve to dry for 2 hours, and turned over every 15 minutes. The dried human amniotic membrane was placed in an ultrasonic reactor and 15% modified diamond of the mass of the human amniotic membrane was added and stirred evenly, and then ultrasonically treated for 70 minutes at 700W and 20KHz in a manner of 6 minutes of ultrasonication and 2 minutes of rest. After the ultrasonic treatment, 0.03% of the total mass of the product was directly added to the obtained product (composed of neutral protease, papain and pancreatin in a mass ratio of 1:2:1.5) and enzymolysis was performed at 60°C for 11 hours. After the enzymolysis was completed, the enzyme was inactivated and cooled to obtain an enzymolysis product. The obtained enzymolysis product was centrifuged at 20,000 rpm for 10 minutes to obtain the first supernatant. Add an appropriate amount of water to the obtained centrifugal precipitate, and centrifuge again under the same conditions to obtain a second supernatant. Combine the first and second supernatants obtained after two centrifugations to obtain a supernatant mixture. The obtained supernatant mixture is filtered using an ultrafiltration membrane with a molecular weight of 20KDa and at 3.0Mpa, and finally the obtained filtrate is concentrated and dried to obtain an amniotic membrane extract.

[0042] Embodiment 2:

[0043] Preparation of modified diamond: Quartz sand, tourmaline and diamond are mixed in a mass ratio of 5:1:3, and the solvent ethanol is added in an amount of 0.5wt% of the total mass of the three substances of quartz sand, tourmaline and diamond. The mixture is ground three times, that is, firstly ground at a speed of 3000rpm for 4h, then ground at a speed of 2000rpm for 3h, and finally ground at a speed of 1000rpm for 2h, and a mixture is obtained after grinding. The obtained mixture is placed in a muffle furnace, calcined at 600℃ for 5h, then cooled to 400℃ at a cooling rate of 100℃ / h and kept warm for 30min, and then cooled to room temperature in air after the end of the heat preservation, and the obtained calcined product is crushed and passed through an 80-mesh sieve to obtain a modified diamond precursor powder. The obtained diamond precursor powder is placed in a hydrofluoric acid solution with a volume concentration of 4% and stirred at a speed of 200rpm for 10min, then filtered, and the obtained solid product is washed and dried to obtain modified diamond.

[0044] Preparation of human amniotic membrane extract: fresh human amniotic membrane was first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes; the disinfected human amniotic membrane was then dispersed and cleaned with purified water to remove residual impurities on the surface; finally, the cleaned and impurity-free human amniotic membrane was spread flat on a sieve to dry for 2 hours, and turned over every 15 minutes. The dried human amniotic membrane was placed in an ultrasonic reactor and 10% modified diamond of the mass of the human amniotic membrane was added and stirred evenly, and then ultrasonically treated for 90 minutes at 600W and 20KHz in a manner of 8 minutes of ultrasonication and 3 minutes of stopping. After the ultrasonic treatment, 0.01% of the total mass of the product (composed of neutral protease, papain and pancreatin in a mass ratio of 1:3:1) was directly added to the obtained product for enzymolysis at 50°C for 12 hours. After the enzymolysis was completed, the enzyme was inactivated and cooled to obtain an enzymolysis product. The obtained enzymolysis product was centrifuged at 20,000 rpm for 10 minutes to obtain the first supernatant. Add an appropriate amount of water to the obtained centrifugal precipitate, and centrifuge again under the same conditions to obtain a second supernatant. Combine the first and second supernatants obtained after two centrifugations to obtain a supernatant mixture. The obtained supernatant mixture is filtered using an ultrafiltration membrane with a molecular weight of 20KDa and at 3.0Mpa, and finally the obtained filtrate is concentrated and dried to obtain an amniotic membrane extract.

[0045] Embodiment 3:

[0046] Preparation of modified diamond: Quartz sand, tourmaline and diamond are mixed in a mass ratio of 10:2:5, and the solvent ethanol is added in an amount of 0.5wt% of the total mass of the three substances of quartz sand, tourmaline and diamond. The mixture is first ground at a speed of 4000rpm for 2h, then ground at a speed of 3000pm for 1h, and finally ground at a speed of 2000rpm for 0.5h to obtain a mixture after grinding. The obtained mixture is placed in a muffle furnace, calcined at 1000℃ for 1h, then cooled to 600℃ at a cooling rate of 100℃ / h and kept warm for 30min. After the insulation is completed, it is cooled to room temperature in air, and the obtained calcined product is crushed and passed through an 80-mesh sieve to obtain a modified diamond precursor powder. The obtained diamond precursor powder is placed in a hydrofluoric acid solution with a volume concentration of 8% and stirred at a speed of 400rpm for 5min, then filtered, and the obtained solid product is washed and dried to obtain modified diamond.

[0047] Preparation of human amniotic membrane extract: fresh human amniotic membrane was first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes; the disinfected human amniotic membrane was then dispersed and cleaned with purified water to remove residual impurities on the surface; finally, the cleaned and impurity-free human amniotic membrane was spread flat on a sieve to dry for 2 hours, and turned over every 15 minutes. The dried human amniotic membrane was placed in an ultrasonic reactor and 20% modified diamond of the mass of the human amniotic membrane was added and stirred evenly, and then ultrasonic treatment was performed for 60 minutes at 800W and 20KHz in a manner of 5 minutes of ultrasonication and 1 minute of rest. After the ultrasonic treatment, 0.05% of the total mass of the product of the composite protease (composed of neutral protease, papain and pancreatin in a mass ratio of 1:1:2) was directly added to the obtained product for enzymolysis at 70°C for 10 hours. After the enzymolysis was completed, the enzyme was inactivated and cooled to obtain an enzymolysis product. The obtained enzymolysis product was centrifuged at 20,000 rpm for 10 minutes to obtain the first supernatant. Add an appropriate amount of water to the obtained centrifugal precipitate, and centrifuge again under the same conditions to obtain a second supernatant. Combine the first and second supernatants obtained after two centrifugations to obtain a supernatant mixture. The obtained supernatant mixture is filtered using an ultrafiltration membrane with a molecular weight of 20KDa and at 3.0Mpa, and finally the obtained filtrate is concentrated and dried to obtain an amniotic membrane extract.

[0048] Comparative Example 1:

[0049] Preparation of modified diamond: Diamond was ground three times under the condition that the amount of solvent ethanol added was 0.5wt% of the total mass of diamond, that is, firstly ground at a rotation speed of 3500rpm for 3h, then ground at a rotation speed of 2500rpm for 2h, and finally ground at a rotation speed of 1500rpm for 1.25h, and a mixture was obtained after grinding. The obtained mixture was placed in a muffle furnace, calcined at 800℃ for 3h, then cooled to 500℃ at a cooling rate of 100℃ / h and kept warm for 30min, and then cooled to room temperature in air after the insulation, and the obtained calcined product was crushed and passed through an 80-mesh sieve to obtain a modified diamond precursor powder. The obtained diamond precursor powder was placed in a hydrofluoric acid solution with a volume concentration of 6% and stirred at a rotation speed of 300rpm for 7.5min, and then filtered, and the obtained solid product was washed and dried to obtain modified diamond.

[0050] Preparation of human amniotic membrane extract: fresh human amniotic membrane was first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes; the disinfected human amniotic membrane was then dispersed and cleaned with purified water to remove residual impurities on the surface; finally, the cleaned and impurity-free human amniotic membrane was spread flat on a sieve to dry for 2 hours, and turned over every 15 minutes. The dried human amniotic membrane was placed in an ultrasonic reactor and 15% modified diamond of the mass of the human amniotic membrane was added and stirred evenly, and then ultrasonically treated for 70 minutes at 700W and 20KHz in a manner of 6 minutes of ultrasonication and 2 minutes of rest. After the ultrasonic treatment, 0.03% of the total mass of the product was directly added to the obtained product (composed of neutral protease, papain and pancreatin in a mass ratio of 1:2:1.5) and enzymolysis was performed at 60°C for 11 hours. After the enzymolysis was completed, the enzyme was inactivated and cooled to obtain an enzymolysis product. The obtained enzymolysis product was centrifuged at 20,000 rpm for 10 minutes to obtain the first supernatant. Add an appropriate amount of water to the obtained centrifugal precipitate, and centrifuge again under the same conditions to obtain a second supernatant. Combine the first and second supernatants obtained after two centrifugations to obtain a supernatant mixture. The obtained supernatant mixture is filtered using an ultrafiltration membrane with a molecular weight of 20KDa and at 3.0Mpa, and finally the obtained filtrate is concentrated and dried to obtain an amniotic membrane extract.

[0051] Comparative Example 2:

[0052] Preparation of modified diamond: Quartz sand, tourmaline and diamond are mixed in a mass ratio of 7.5:1.5:4, and the solvent ethanol is added in an amount of 0.5wt% of the total mass of the three substances of quartz sand, tourmaline and diamond, and ground three times, that is, firstly grind at a speed of 3500rpm for 3h, then grind at a speed of 2500rpm for 2h, and finally grind at a speed of 1500rpm for 1.25h, and then grind to obtain a mixture. The obtained mixture is placed in a muffle furnace, calcined at 800℃ for 3h, then cooled to 500℃ at a cooling rate of 100℃ / h and kept warm for 30min, cooled to room temperature in air after the end of the heat preservation, and the obtained calcined product is crushed and passed through an 80-mesh sieve to obtain a modified diamond precursor powder.

[0053] Preparation of human amniotic membrane extract: fresh human amniotic membrane was first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes; the disinfected human amniotic membrane was then dispersed and cleaned with purified water to remove residual impurities on the surface; finally, the cleaned and impurity-free human amniotic membrane was spread flat on a sieve to dry for 2 hours, and turned over every 15 minutes. The dried human amniotic membrane was placed in an ultrasonic reactor and 15% modified diamond of the mass of the human amniotic membrane was added and stirred evenly, and then ultrasonically treated for 70 minutes at 700W and 20KHz in a manner of 6 minutes of ultrasonication and 2 minutes of rest. After the ultrasonic treatment, 0.03% of the total mass of the product was directly added to the obtained product (composed of neutral protease, papain and pancreatin in a mass ratio of 1:2:1.5) and enzymolysis was performed at 60°C for 11 hours. After the enzymolysis was completed, the enzyme was inactivated and cooled to obtain an enzymolysis product. The obtained enzymolysis product was centrifuged at 20,000 rpm for 10 minutes to obtain the first supernatant. Add an appropriate amount of water to the obtained centrifugal precipitate, and centrifuge again under the same conditions to obtain a second supernatant. Combine the first and second supernatants obtained after two centrifugations to obtain a supernatant mixture. The obtained supernatant mixture is filtered using an ultrafiltration membrane with a molecular weight of 20KDa and at 3.0Mpa, and finally the obtained filtrate is concentrated and dried to obtain an amniotic membrane extract.

[0054] Comparative Example 3:

[0055] Preparation of human amniotic membrane extract: fresh human amniotic membrane was first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes; the disinfected human amniotic membrane was then dispersed and cleaned with purified water to remove residual impurities on the surface; finally, the cleaned and impurity-free human amniotic membrane was spread flat on a sieve to dry for 2 hours, and turned over every 15 minutes. The dried human amniotic membrane was placed in an ultrasonic reactor and 15% diamond of the mass of the human amniotic membrane was added and stirred evenly, and then ultrasonically treated for 70 minutes at 700W and 20KHz in a manner of 6 minutes of ultrasonication and 2 minutes of stopping. After the ultrasonic treatment, 0.03% of the total mass of the product was directly added to the obtained product (composed of neutral protease, papain and pancreatin in a mass ratio of 1:2:1.5) and enzymolysis was performed at 60°C for 11 hours. After the enzymolysis was completed, the enzyme was inactivated and cooled to obtain an enzymolysis product. The obtained enzymolysis product was centrifuged at 20,000 rpm for 10 minutes to obtain the first supernatant. Add an appropriate amount of water to the obtained centrifugal precipitate, and centrifuge again under the same conditions to obtain a second supernatant. Combine the first and second supernatants obtained after two centrifugations to obtain a supernatant mixture. The obtained supernatant mixture is filtered using an ultrafiltration membrane with a molecular weight of 20KDa and at 3.0Mpa, and finally the obtained filtrate is concentrated and dried to obtain an amniotic membrane extract.

[0056] Comparative Example 4:

[0057] Preparation of human amniotic membrane extract: fresh human amniotic membrane was first disinfected with 75% alcohol by volume, and then sterilized with ultraviolet light for 30 minutes; the disinfected human amniotic membrane was then dispersed and washed with purified water to remove residual impurities on the surface; finally, the washed and impurity-free human amniotic membrane was spread flat on a sieve to dry for 2 hours, and turned over every 15 minutes. The dried human amniotic membrane was placed in an ultrasonic reactor and ultrasonically treated for 70 minutes at 700W and 20KHz in a manner of 6 minutes of ultrasonication and 2 minutes of rest. After the ultrasonic treatment, a composite protease (composed of neutral protease, papain and pancreatin in a mass ratio of 1:2:1.5) with a total mass of 0.03% of the product was directly added to the obtained product for enzymolysis at 60°C for 11 hours. After the enzymolysis was completed, the enzyme was inactivated and cooled to obtain an enzymolysis product. The obtained enzymolysis product was centrifuged at 20,000 rpm for 10 minutes to obtain a first supernatant. An appropriate amount of water was added to the obtained centrifugal precipitate, and the product was centrifuged again under the same conditions to obtain a second supernatant. The first and second supernatants obtained after two centrifugations were combined to obtain a supernatant mixture, which was filtered through an ultrafiltration membrane with a molecular weight of 20 KDa at 3.0 MPa, and the filtrate was concentrated and dried to obtain an amniotic membrane extract.

[0058] Experiment 1: Analysis of components of amniotic membrane extract

[0059] 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);

[0060] 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;

[0061] 3. Fat content detection: refer to the acid hydrolysis method of GB 5009.6-2016 "National Food Safety Standard Determination of Fat in Food";

[0062] 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).

[0063] All test items were repeated three times in parallel and the average value was taken. The results are shown in Table 1.

[0064] Table 1: Component analysis

[0065]

[0066]

[0067] It can be seen from the results in Table 1 that compared with comparative examples 1-4, the total protein content and total sugar content in the amniotic membrane extracts of Examples 1-3 of the present invention are significantly higher, wherein the use of modified diamond-assisted ultrasonic crushing during the crushing process can significantly improve the subsequent enzymatic hydrolysis efficiency, thereby further increasing the protein and sugar content in the amniotic membrane extract.

[0068] Experiment 2: Effects on epidermal cell proliferation

[0069] First, the amniotic membrane extracts of Examples 1-3 and Comparative Examples 1-4 were respectively dissolved in water to obtain amniotic membrane extract solutions with a concentration of 1 g / L.

[0070] Mouse corneal epithelial cells TKE2 were cultured according to the instructions. When the cell confluence reached 80%, the supernatant was discarded, and the obtained cell precipitate was washed twice with PBS, and the amniotic membrane extract solutions of Examples 1-3 and Comparative Examples 1-3 were respectively used at a rate of 1×10 6 The cells were resuspended at a density of 100 / mL and then transferred to an incubator for culture at 37°C and 5% CO2, which was recorded as P1. When the cells were subcultured to P8, the cell proliferation in each group of culture bottles was detected using a cell proliferation detection kit (MTS) (Cat. No. ST1009, purchased from Shanghai Shangbao Biotechnology Co., Ltd.), and the results are shown in Table 2.

[0071] Table 2: Effects of different amniotic membrane extracts on cell proliferation

[0072] Group OD value Example 1 0.816 Example 2 0.795 Example 3 0.802 Comparative Example 1 0.789 Comparative Example 2 0.783 Comparative Example 3 0.727 Comparative Example 4 0.618

[0073] It can be seen from the results in Table 2 that, compared with Comparative Examples 1-4, the amniotic membrane extract of the present invention can promote the proliferation of mouse corneal epithelial cells with a significant effect.

[0074] 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 amniotic membrane extract, characterized in that: The preparation method comprises: sterilizing, cleaning and removing impurities from fresh amniotic membrane in sequence, then breaking it, hydrolyzing the broken product with composite protease, and centrifuging and ultrafiltering the hydrolyzed product to prepare the amniotic membrane extract.

2. The method for preparing the decellularized amniotic membrane extract according to claim 1, characterized in that: The disinfection process is as follows: using 75% alcohol by volume to disinfect the fresh human amniotic membrane, and then sterilizing with ultraviolet light for 20 to 40 minutes; The cleaning and impurity removal process is as follows: the sterilized human amniotic membrane is dispersed and cleaned with purified water to remove residual impurities on the surface; The drying process is as follows: spreading the dried human amniotic membrane on a sieve to dry it for 1 to 3 hours, and turning it over every 10 to 20 minutes.

3. The method for preparing the decellularized amniotic membrane extract according to claim 1, characterized in that: The crushing process is as follows: placing the cleaned and impurity-free human amniotic membrane in an ultrasonic reactor, adding modified diamond, stirring evenly, and then performing ultrasonic treatment; The added amount of the modified diamond is 10-20% of the mass of the human amniotic membrane.

4. The method for preparing the decellularized amniotic membrane extract according to claim 3, characterized in that: The ultrasonic treatment method is intermittent ultrasonic treatment, and the ultrasonic conditions are: power 600-800W, and ultrasonic treatment is carried out in a manner of 5-8 minutes of ultrasonic treatment and 1-3 minutes of ultrasonic treatment, and the total ultrasonic treatment time is 60-90 minutes.

5. The method for preparing the decellularized amniotic membrane extract according to claim 3, characterized in that: The preparation method of the modified diamond is: Mixing quartz sand, tourmaline and diamond in a mass ratio of 5-10:1-2:3-5 and grinding to obtain a mixture; The obtained mixture is placed in a muffle furnace, calcined at 600-1000°C for 1-5h, then cooled to 400-600°C at a cooling rate of 80-120°C / h and kept at that temperature for 30min, cooled to room temperature in air after the end of the heat preservation, and the obtained calcined product is crushed and passed through an 80-100 mesh sieve to obtain a modified diamond precursor powder; The obtained diamond precursor powder is placed in a hydrofluoric acid solution with a volume concentration of 4-8% and stirred at a speed of 200-400 rpm for 5-10 minutes, and then filtered. The obtained solid product is washed and dried to obtain modified diamond.

6. The method for preparing the decellularized amniotic membrane extract according to claim 5, characterized in that: The grinding process conditions are: grinding three times in total, and the grinding conditions are: First grinding: grinding at a speed of 3000-4000 rpm for 2-4 hours; Second grinding: grinding at a speed of 2000-3000 rpm for 1-3 hours; The third grinding: grinding at a speed of 1000-2000 rpm for 0.5-2h.

7. The method for preparing the decellularized amniotic membrane extract according to claim 1, characterized in that: The enzymatic hydrolysis process is as follows: adding 0.01-0.05% of the total mass of the product to the product after ultrasonic treatment for enzymatic hydrolysis, killing the enzyme after the enzymatic hydrolysis, cooling, and centrifuging to obtain a supernatant; The enzymatic hydrolysis conditions are: temperature 50-70° C., time 10-12 h.

8. The method for preparing the decellularized amniotic membrane extract according to claim 7, characterized in that: The composite protease is composed of neutral protease, papain and pancreatin, and the mass ratio of neutral protease, papain and pancreatin is 1:(1-3):(1-2); And / or, the centrifugal conditions are: rotation speed 10000-20000 rpm, time 10-20 min; And / or 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.

9. A decellularized amniotic membrane extract prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the decellularized amniotic membrane extract according to claim 9 in preparing a product for promoting tissue regeneration.

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