A preparation method and application of amniotic membrane extract

Abstract

The present invention belongs to the field of bioengineering technology, and discloses a preparation method and application of an amniotic membrane extract. Fresh amniotic membrane is disinfected, cleaned, and dried in sequence, and then crushed in an ice bath. Then, the crushed liquid is centrifuged and filtered to obtain the amniotic membrane extract. The dry matter of the amniotic membrane extract meets the following requirements: total protein content ≥78.02%, total sugar content ≥10.87%, fat content ≤3.53%, and ignition residue content ≤1.64%. The present invention improves the extraction efficiency of total protein and total sugar in the amniotic membrane extract by changing the existing production process of frozen crushing or liquid crushing, crushing the amniotic membrane in an ice-water mixture, and combining the control of process parameters such as crushing, which is beneficial to the promotion and application of the amniotic membrane extract in the preparation of ocular preparations such as ocular surface lubricating liquid, and improves the clinical use effect. In addition, the amniotic membrane extract can also be used as a functional component of biological amniotic membrane preservation liquid and combined with amniotic membrane covering surgery for the treatment of ocular surface diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for preparing an amniotic membrane extract. Background Art

[0002] The amniotic membrane is the innermost layer of the placenta, and its main components are 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. Most of the existing sheet-like amniotic membrane products require surgical sutures, which on the one hand increases the difficulty of surgical operations for doctors, and on the other hand causes secondary trauma to patients during suture surgery. In order to promote the application of amniotic membrane products and avoid the trauma caused to patients by surgical suture-type amniotic membrane transplants, suture-free amniotic membrane materials (such as amniotic membrane suspensions or amniotic membrane eye drops) have gradually been publicly reported in the literature.

[0003] For example, the paper "Amniotic membrane extract eye drops: a new approach to severe ocular surface pathologies" (Cell and tissue banking, 2022, 23(3), 473–481) reported a clinical follow-up study of amniotic membrane eye drops for the treatment of ocular surface diseases such as chronic dry eye disease and limbal stem cell deficiency (36 eyes of 25 patients). The amniotic membrane extract was crushed by liquid nitrogen freezing and purified by centrifugation to obtain the supernatant. The results showed that topical application of amniotic membrane eye drops was safe, well tolerated by patients, and could significantly improve ocular symptoms such as foreign body sensation, itching, and stinging. The preparation method of amniotic membrane eye drops was as follows: after informed consent from the donor, the placenta was collected. After flushing the placenta to remove blood clots, the amniotic membrane was peeled off with a blunt tool. The amniotic membrane was then flushed and cleaned with an antibiotic / antifungal solution. The next day, the amniotic membrane was rinsed with saline to remove antibiotics and then frozen in liquid nitrogen. The frozen amniotic membrane was ground and centrifuged at room temperature. The supernatant was divided into 1 mL vials, freeze-dried, and stored at room temperature until use. Each vial containing amniotic membrane powder was individually packaged. The final package included all the materials required to rehydrate the powder, i.e., a syringe, a needle, and a 10 mL bottle of sterile water. Prior to use, each vial of amniotic membrane powder was reconstituted with 4 mL of sterile water.

[0004] For example, the clinical study of amniotic membrane suspension conducted by Bonci P et al. proved that the method of using amniotic membrane suspension is less invasive, safe, can be used repeatedly for a long time, and has a good therapeutic effect on patients with superficial corneal defects (reference: Bonci P, Bonci P, Lia A (2005) Suspension made with amniotic membrane:clinical trial. Eur J Ophthalmol 15:441–445.). In addition, Lingyi Liang et al. proved through a prospective case study that topical application of amniotic membrane suspension can promote epithelialization in the treatment of ocular surface chemical burns (reference: LiangL, Li W, Ling S et al (2009) Amniotic membrane extraction solution for ocularchemical burns. Clin Exp Ophthalmol 37:855–863.).

[0005] In the prior art, the invention patent with application number 200910178422.7 discloses an amniotic eye drop and its preparation method. The amniotic extract crushing process of the patent is: PBS liquid crushing, and the purification process is: centrifugation to obtain the supernatant. The amniotic homogenate supernatant is the main active ingredient, and trehalose, hyaluronic acid or its salt, heparin or its salt, sodium chloride, benzalkonium bromide, vitamins and other substances are added. The amniotic homogenate supernatant is prepared by the following method: wash the amniotic membrane in PBS solution for 3 times on a clean bench for about 10 to 15 minutes, take a suitable size and weigh the wet weight, add PBS solution at a ratio of 1:1 and put it into a homogenizer, 5000 revolutions per minute, 10 strokes to grind the homogenate, and each stroke is 30 seconds. Take out the homogenate and add PBS solution at a ratio of 1:5, centrifuge it in a centrifuge tube at 2000 r / min for 10 minutes, and take the supernatant for use.

[0006] The invention patent with application number 202310856867.6 also discloses a tissue regeneration biomembrane tissue complex and its preparation method and application. The amniotic membrane extract crushing process of the patent is: physiological saline liquid crushing, and the purification process is: centrifugation to obtain the supernatant. It includes a biomembrane tissue extract and an adsorption-promoting gel sustained-release system, wherein the biomembrane tissue extract includes an amniotic membrane extract, and the preparation method is: after removing the blood clot with physiological saline, fresh human amniotic membrane is obtained by peeling with a blunt tool. Soak the fresh amniotic membrane in physiological saline containing amphotericin B (10 mg / mL), penicillin (100 u / mL), and streptomycin (10 mg / mL) for 2 hours. Keep the ratio of the wet weight of the amniotic membrane to the volume of the physiological saline at 1g / L, and rinse the amniotic membrane with physiological saline three times continuously under sterile conditions and neutral pH. Collect the supernatant after treatment with a tissue grinder and centrifugation: It is carried out under sterile conditions, the tissue grinder treatment time is 10 minutes, the centrifugation rate is 800rpm, and the centrifugation time is 10 minutes.

[0007] It can be seen that amniotic extract / amniotic suspension has good clinical use effect for a variety of ocular surface diseases. However, since the amniotic extract / amniotic suspension reported so far is limited to clinical research, in the process of industrial promotion, it is necessary to further improve the extraction efficiency and product quality of amniotic extract / amniotic suspension, such as purity improvement and impurity content control, etc. However, the preparation method reported in the above-mentioned public literature still has the following problems to be solved: First, the pulverization process uses liquid nitrogen freezing pulverization or adding PBS / saline liquid pulverization, and the pulverization effect is not good, which leads to poor extraction of effective components (such as total protein and total sugar) in amniotic extract; second, the purification process uses the method of taking the supernatant after centrifugation, and the obtained amniotic extract may still contain large particulate matter (>50μm), resulting in foreign body discomfort after dripping into the patient's eyes; third, the amniotic extract contains a high content of non-functional impurities (such as fat and ignition residue). Therefore, it is urgent to provide a preparation process for amniotic extract that can be applied industrially. Summary of the invention

[0008] The present invention aims to provide a method for preparing an amniotic membrane extract, which changes the existing production process of frozen pulverization or liquid pulverization. By pulverizing the amniotic membrane in an ice-water mixture, and combining and controlling process parameters such as the mass ratio of ice cubes to amniotic membrane, pulverization time, and pulverization times, the extraction efficiency of the total protein and total sugar in the amniotic membrane extract can be improved, which is conducive to the subsequent industrial promotion and application. To this end, the present invention also provides the use of the amniotic membrane extract prepared by the preparation method in the preparation of ophthalmic preparations.

[0009] The present invention is achieved by the following technical scheme: a method for preparing an amniotic membrane extract, wherein the fresh amniotic membrane is sterilized, cleaned, and dried in sequence, and then crushed in an ice bath, and then the crushed liquid is centrifuged and filtered to obtain the amniotic membrane extract.

[0010] During the ice bath crushing, the air-dried amniotic membrane and ice cubes are added into a container, the mass ratio of ice cubes to amniotic membrane is controlled to be 1-5:1, and then the mixture is allowed to stand for 5 minutes, and then the mixture is crushed by a crusher after obtaining the ice water amniotic membrane mixture, and the liquid temperature during the crushing process is controlled to be less than 8° C. After crushing for 10-60 seconds, the mixture is crushed again at an interval of 1 minute, and the process is repeated 30-90 times to complete the ice bath crushing.

[0011] The dry matter of amniotic membrane extract meets the following requirements: total protein content ≥78.02%, total sugar content ≥10.87%, fat content ≤3.53%, and residue on ignition content ≤1.64%. The dry matter of amniotic membrane extract described in the present invention refers to the amniotic membrane extract in a liquid state placed in a 50° C. forced air drying oven until it reaches a constant weight, usually dried for more than 2 days until a constant weight is reached.

[0012] During the disinfection, 75% alcohol is sprayed on the packaging material of the fresh amniotic membrane for disinfection, and then ultraviolet sterilization is performed for 30 minutes.

[0013] During the cleaning and impurity removal, the sterilized amniotic membrane is unpacked and placed in purified water to be dispersed and cleaned, and residual impurities on the surface are removed.

[0014] During the drying process, the cleaned and impurity-free amniotic membrane was spread on a sieve and dried for 1 hour, and turned over every 15 minutes.

[0015] The ice cubes are purified water ice cubes with a side length of 1 to 3 cm.

[0016] During the centrifugation, the crushed liquid was divided into 50 ml centrifuge tubes, each tube was filled with 50 ml of crushed liquid and then put into a centrifuge for centrifugation for 10 minutes, and the speed of the centrifuge was controlled to be 1000-3000 rpm.

[0017] The filtration is performed by vacuum filtration using filter paper with a pore size of 5 to 50 μm.

[0018] The dry matter of the amniotic membrane extract meets the following requirements: total protein content of 80.02-82.79%, total sugar content of 10.87-13.11%, fat content of 1.83-3.53%, and residue on ignition content of 0.94-1.64%.

[0019] The use of the amniotic membrane extract prepared by the above preparation method in the preparation of ophthalmic preparations, wherein the ophthalmic preparations include ocular surface lubricating liquid.

[0020] The amniotic membrane extract prepared by the above preparation method is used in the preparation of products for treating ocular surface diseases. For example, the amniotic membrane extract can be used as a functional component of biological amniotic membrane preservation fluid, or can be used in combination with amniotic membrane covering surgery in the treatment of ocular surface diseases.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] (1) The present invention improves the pulverization process of amniotic membrane extract. The existing pulverization process uses liquid nitrogen freezing pulverization or PBS / physiological saline liquid pulverization, which has poor pulverization effect, thereby resulting in poor extraction effect of effective components (such as total protein and total sugar) in amniotic membrane extract. The present invention uses ice bath pulverization and optimizes process parameters such as "the size of ice cubes used, the weight ratio of ice cubes to samples, pulverization time, and pulverization times", which greatly improves the extraction efficiency of amniotic membrane extract as an effective component of ophthalmic medical devices or drugs (i.e., total protein and total sugar), and is convenient for industrial use.

[0023] (2) The present invention also improves the purification process of amniotic membrane extract. The existing purification process adopts the method of taking the supernatant after centrifugation. The obtained amniotic membrane extract may still contain larger particles (>50μm), which causes discomfort after dripping into the patient's eyes. The amniotic membrane extract also contains a high content of non-functional impurities (such as fat and ignition residue). The present invention adopts centrifugation and filter paper vacuum filtration process. By selecting filter paper with a specific pore size (pore size of 5 to 50μm), large particles (>50μm) can be effectively filtered out, avoiding discomfort after dripping into the patient's eyes. At the same time, the content of non-functional impurities (fat and ignition residue) is greatly reduced. In addition, the vacuum filtration process accelerates the filtration speed and improves production efficiency with the assistance of an external vacuum pump.

[0024] In summary, the present invention aims to provide a production process of amniotic membrane extract, which can improve the extraction efficiency of effective ingredients in ocular surface lubricating fluid, reduce the content of non-functional impurities, and achieve an index system of 78.02-82.79% total protein content, 10.87-13.11% total sugar content, 1.83-3.53% fat content, and 0.94-1.64% residue on ignition in dry matter of amniotic membrane extract, which is conducive to the promotion and application of amniotic membrane extract in the preparation of ocular surface lubricating fluid and other ophthalmic preparations, and improves the clinical use effect. In addition, amniotic membrane extract can be used as a functional component of biological amniotic membrane preservation fluid, and combined with amniotic membrane covering surgery for the treatment of ocular surface diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a process flow chart of the preparation process of the amniotic membrane extract of the present invention;

[0026] Figure 2 A schematic diagram of a centrifugal process according to the present invention;

[0027] Figure 3 This is a physical picture of the amniotic membrane extract in Example 1 of the present invention;

[0028] Figure 4 This is a comparison test chart of the crushing process. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0030] Embodiment 1:

[0031] This embodiment relates to a method for preparing an amniotic membrane extract.

[0032] See also Figure 1 The preparation flow chart shown specifically comprises the following steps:

[0033] Step 1: Disinfection and cleaning

[0034] Take out the pre-frozen (-20°C) fresh amniotic membrane from the refrigerator, spray its packaging with 75% alcohol for disinfection, and put it into the workshop transfer window feed port for ultraviolet sterilization for 30 minutes. Then take out the disinfected amniotic membrane and unpack it. Use tweezers to take out the amniotic membrane and put it into a 304 tray or beaker. Add purified water so that the purified water covers the amniotic membrane by at least 2 cm. After 5 minutes, initially thaw and disperse the amniotic membrane by hand to avoid shrinkage. Change the water every 30 minutes, turn the amniotic membrane over and stir it gently each time to remove residual impurities on its surface. Repeat this step at least 3 times until the solution remains clear after adding purified water.

[0035] Step 2: Dry

[0036] The cleaned amniotic membrane is taken out by hand and placed on a sieve to dry. A tray is placed at the bottom to catch the moisture that is released. The amniotic membrane needs to be laid flat. If it cannot be laid flat, it should be cut and divided. The amniotic membrane cannot be stacked. When drying, the amniotic membrane needs to be turned over every 15 minutes and dried for a total of 1 hour.

[0037] Step 3: Ice bath crushing

[0038] Put the prepared beaker into the electronic balance to zero, put the amniotic membrane in the beaker and weigh it, and record its wet weight. Weigh the prepared purified water ice cubes (1cm×1cm×1cm), with a mass ratio of 1:1 to the amniotic membrane, and then pour the ice cubes into the beaker. Put it into the experimental crusher and let it stand for 5 minutes before crushing (at this time, part of the ice cubes melt to form an ice-water amniotic membrane mixture). While crushing, wrap the beaker with an ice bag, control the liquid temperature <8°C during the crushing process, and rest the equipment for 1 minute after each crushing for 10S. Repeat this process 30 times to complete the crushing.

[0039] Step 4: Centrifugation

[0040] The crushed liquid obtained by crushing in an ice bath (see Figure 2 The emulsion shown in A) was placed in 50 ml centrifuge tubes, and each tube was filled with 50 ml and placed in a centrifuge (see Figure 2 As shown in B), set the centrifuge speed to 1000 rpm, centrifuge for 10 minutes, and take out the centrifuged sample (see Figure 2 (as shown in C), take the supernatant and pour it into a beaker, and the centrifugation is completed.

[0041] Step 5: Vacuum filtration with filter paper

[0042] Place a medium-speed qualitative filter paper with a pore size of 5 to 10 μm on the Buchner funnel filtration bottle, pour in the supernatant, turn on the vacuum pump, and collect the filtrate, which is the amniotic membrane extract (see Figure 3 as shown).

[0043] Embodiment 2:

[0044] This embodiment is the same as Embodiment 1, both of which are methods for preparing amniotic membrane extracts, the only difference being that some parameters of ice bath crushing, centrifugation and filter paper vacuum filtration are slightly adjusted, and the remaining steps and process parameters are the same as those in Embodiment 1.

[0045] Specifically, in this embodiment, when crushing in an ice bath, purified water ice cubes with a size of 3 cm×3 cm×3 cm were used, and the mass ratio of purified water ice cubes to amniotic membrane was set to 5:1. Each time the crushing was performed for 60 seconds, the device rested for 1 minute, and this process was repeated 90 times in total. When centrifuging, the speed of the centrifuge was set to 3000 rpm. When vacuum filtering with filter paper, medium-speed qualitative filter paper with a size of 20 to 50 μm was used.

[0046] Embodiment 3:

[0047] This embodiment is the same as Embodiment 1, both of which are methods for preparing amniotic membrane extracts, the only difference being that some parameters of ice bath crushing, centrifugation and filter paper vacuum filtration are slightly adjusted, and the remaining steps and process parameters are the same as those in Embodiment 1.

[0048] Specifically, in this embodiment, when crushing in an ice bath, purified water ice cubes with a size of 2 cm×3 cm×2 cm were used, and the mass ratio of purified water ice cubes to amniotic membrane was set to 2:1. Each time the crushing was performed for 30 seconds, the equipment rested for 1 minute, and this process was repeated 60 times in total. When centrifuging, the speed of the centrifuge was set to 2000 rpm. When vacuum filtering with filter paper, medium-speed qualitative filter paper with a size of 10 μm to 20 μm was used.

[0049] Embodiment 4:

[0050] The present embodiment relates to an ocular surface lubricating liquid, specifically, an ocular surface lubricating liquid prepared using the amniotic membrane extract prepared in Example 1, and the formula thereof is as follows: 1% of the amniotic membrane extract from Example 1, 0.3% of sodium hyaluronate, 0.6% of sodium chloride, 0.4% of boric acid, 0.015% of borax, and the remainder being water for injection.

[0051] Embodiment 5:

[0052] This embodiment relates to a preservation solution for a membrane-like amniotic membrane product, specifically, the amniotic membrane extract prepared in Example 1 is used to prepare a biological amniotic membrane preservation solution. The formula is as follows: 10% of the amniotic membrane extract from Example 1, 40% of sterile physiological saline, and 50% of glycerol.

[0053] Embodiment 6:

[0054] This embodiment relates to a technical solution for the treatment of ocular surface diseases by combining amniotic membrane extract with amniotic membrane patching. The specific solution is as follows: after the ocular surface disease patient undergoes surgical suture of the membrane-like amniotic membrane product, the ocular surface lubricant prepared in Example 4 is dripped with 0.5 ml each time, and the dripping is repeated every 24 hours for 1 to 7 times.

[0055] Comparative Example 1:

[0056] The preparation method of comparative example 1 includes disinfection, cleaning and impurity removal, air-drying, PBS liquid grinding and purification. Among them, the disinfection, cleaning and impurity removal and air-drying are the same as those in Example 1; the PBS liquid grinding process is to wash the amniotic membrane in PBS liquid for 3 times on a clean bench for about 10 to 15 minutes, take a suitable size and weigh the wet weight, add PBS liquid at a ratio of 1:1 and put it into a homogenizer, 5000 r / min, 10 strokes to grind the homogenate, and each stroke is 30 seconds; the purification process is to add PBS liquid at a ratio of 1:5 into a centrifuge tube, centrifuge at 2000r / min for 10 minutes, take the supernatant, and obtain the amniotic membrane extract.

[0057] Comparative Example 2:

[0058] The preparation method of comparative example 2 includes washing, physiological saline liquid crushing and purification. The specific process is as follows: after using physiological saline to remove blood clots, fresh human amniotic membrane is peeled off by blunt tools, and the fresh amniotic membrane is soaked in physiological saline containing amphotericin B (10 mg / mL), penicillin (100 u / mL), and streptomycin (10 mg / mL) for 2 hours, and the ratio of the wet weight of the amniotic membrane to the volume of the physiological saline is maintained at 1g / L, and then the amniotic membrane is rinsed three times in a row with physiological saline under sterile conditions and neutral pH. The rinsed amniotic membrane is crushed for 10 minutes under sterile conditions using a tissue crusher, and the crushed slurry after treatment is added to a centrifuge tube, centrifuged at 800rpm for 10 minutes, and the supernatant is taken to obtain the amniotic membrane extract.

[0059] Comparative Example 3:

[0060] The preparation method of comparative example 3 includes washing, liquid nitrogen freezing, crushing and purification. The specific process is as follows: after flushing the placenta to remove blood clots, the amniotic membrane is peeled off with a blunt tool, and then the amniotic membrane is flushed and purified with an antibiotic / antifungal solution (1000 µ / mL penicillin and 20 mg / mL streptomycin in PBS). The next day, the amniotic membrane is rinsed with saline to remove antibiotics and then immersed in liquid nitrogen for freezing. The frozen amniotic membrane is ground and centrifuged at room temperature (centrifuged at a speed of 14000 r / min for 5 minutes), and the supernatant after centrifugation is taken to obtain an amniotic membrane extract (the supernatant is divided into 1 ml vials, freeze-dried, and stored at room temperature until use).

[0061] 1. Comparative test of crushing process

[0062] According to the relevant processes of Example 1 and Comparative Example 3, Example 1 adopts ice bath crushing, and Comparative Example 3 adopts liquid nitrogen freezing crushing. The crushing processes in Example 1 and Comparative Document 3 are compared, as shown in FIG. Figure 4 As shown, Figure 4 A, B, and C are all actual pictures of liquid nitrogen pulverization. It can be seen that the liquid nitrogen pulverization effect is not good, and the samples are prone to wall hanging and agglomeration; Figure 4 D in the middle is a real picture of ice bath crushing. It can be seen that the ice crushing effect is good, the sample fragments are not easy to stick to the wall, and there is no agglomeration phenomenon.

[0063] 2. Comparative test of purification process

[0064] According to the relevant process of Example 1, the purification process of Example 1 is centrifugation and vacuum filtration with filter paper. The state of the amniotic membrane extract before and after vacuum filtration with filter paper in Example 1 is compared. Figure 1 Middle C and Figure 2 As shown, Figure 1 Middle C is the amniotic membrane extract (i.e., supernatant) after centrifugation and before vacuum filtration with filter paper, which is an emulsion and still contains certain large-sized particles (>50 μm); Figure 2 The amniotic membrane extract (i.e., filtrate) obtained after vacuum filtration with filter paper is relatively clear, with particles smaller than 50 μm.

[0065] 3. Detection of total protein content in dry matter of amniotic membrane extract

[0066] The amniotic membrane extracts of Examples 1 to 3 and Comparative Examples 1 to 3 were respectively tested for total protein content. The test method was based on the Kjeldahl nitrogen determination method of Part IV 0731 of the Chinese Pharmacopoeia (2015 edition) for protein content determination.

[0067] The specific steps are as follows:

[0068] 1) Digestion: Weigh 0.1g of the dried constant-weight sample (amniotic membrane extract), weigh accurately, and carefully add it to the digestion tube (be careful to prevent the sample from sticking to the wall). Add 10mL sulfuric acid, 4.5g potassium sulfate, and 0.5g copper sulfate. Put it in the digestion furnace and heat it to 350℃ until the nitration is complete (that is, the solution turns transparent and light blue). After the nitration is complete, remove it and cool it. Add 30ml of distilled water to the nitration tube to dilute the sample and release heat, and cool it for use. Each batch of samples is measured once, and a blank parallel sample is prepared at the same time.

[0069] 2) Distillation, cancel the de-cooling sample, place it on the left side of the Kjeldahl nitrogen analyzer, seal it well, put a conical flask on the right side, and extend the liquid outlet tube to the bottom of the conical flask. Click the Kjeldahl nitrogen analyzer to start nitrogen determination, and the conical flask collects the distillate. After the distillation is completed, take out the conical flask that has collected the distillate.

[0070] 3) Titration: Take the sample distillate, add 2-3 drops of methyl red and bromocresol green indicator solution, and titrate with sulfuric acid titrant (0.05 mol / L) until the solution changes from green to red. Record the volume of titrant consumed.

[0071] 4) Calculation of results:

[0072] Nitrogen content: ,

[0073] Total protein content: ,

[0074] Where: M = 1.401, each 1mL sulfuric acid titration solution (0.05mol / L) is equivalent to 1.401mg of N;

[0075] W = sample weight (unit: g)

[0076] V 0 = consumption of standard acid for blank sample titration (unit: mL);

[0077] V = sample titration standard acid consumption (unit: mL);

[0078] C = protein conversion factor 6.25.

[0079] The experimental results are shown in Table 1:

[0080] Table 1 Data on total protein content of amniotic membrane extract dry matter

[0081]

[0082] The experimental results show that the total protein content in the dry matter of the amniotic membrane extracts in Examples 1 to 3 is 78.02-82.79%, and the total protein content in the dry matter of the amniotic membrane extracts in Comparative Examples 1 to 3 is 23.72-34.57%, that is, the total protein content in the dry matter of the amniotic membrane extracts obtained by the preparation process of the present invention is greatly improved.

[0083] The pulverizing processes of Comparative Examples 1 to 3 respectively use PBS liquid pulverizing, physiological saline liquid pulverizing, and liquid nitrogen freezing pulverizing, while Examples 1 to 3 of the present invention use ice bath pulverizing. By improving process parameters such as "the size of ice cubes used, the weight ratio of ice cubes to samples, pulverizing time, and the number of pulverizing times", the extraction efficiency of amniotic membrane extract as an active ingredient (total protein in dry matter) for ophthalmic medical devices or drugs is greatly improved.

[0084] 4. Detection of total sugar content in dry matter of amniotic membrane extract

[0085] The total sugar content of the amniotic membrane extracts of Examples 1 to 3 and Comparative Examples 1 to 3 was tested respectively. The test method refers to the total sugar content determination in Appendix E of YY / T1453-2016 "Characterization Method of Type I Collagen in Tissue Engineering Medical Devices".

[0086] The specific steps are as follows:

[0087] 1) Preparation of reference solution, D-anhydrous glucose reference stock solution: accurately weigh 10 mg of D-anhydrous glucose reference dried to constant weight at 105°C, place in a 50 mL volumetric flask, add water to dissolve and dilute to scale, shake well, and obtain the reference solution (each 1 mL contains 0.2 mg of D-anhydrous glucose). D-anhydrous glucose reference series solution: accurately measure 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of D-anhydrous glucose reference stock solution, place in stoppered test tubes, add water to each tube to 10 mL, and shake well.

[0088] 2) Preparation of test solution: weigh 0.01 g of dried amniotic membrane extract sample, place in a hydrolysis tube, add 2 mL of 6 mol / L hydrochloric acid, place at 75°C until completely hydrolyzed, cool, adjust pH to neutral, transfer to a 25 mL volumetric flask, add water to make up to volume, and the solution is ready. Measure once for each batch, and prepare a blank sample.

[0089] 3) Determination: Take 2 mL of blank solution, D-anhydrous glucose reference solution and test solution respectively, accurately add 1 mL of 5% phenol solution, shake well, quickly add 5.0 mL of concentrated sulfuric acid, shake well, keep in a 40℃ water bath for 40 minutes, take out and quickly put into ice water to cool for 20 minutes. Use blank as control, measure absorbance at 490 nm, make linear regression of absorbance with D-anhydrous glucose reference solution series concentration, obtain linear regression equation, and calculate the mass (mg) of D-anhydrous glucose in the test solution.

[0090] 4) Calculation of results: The total sugar content in the sample (measured as D-anhydrous glucose) is calculated according to the following formula:

[0091] Total sugar content in the sample (%) = ,

[0092] In the formula, m sa is the mass of D-anhydrous glucose in the test solution, in milligrams (mg); k is the dilution multiple; m It is the mass of the sample after drying and constant weight, in grams (g).

[0093] The experimental results are shown in Table 2:

[0094] Table 2 Total sugar content of amniotic membrane extract dry matter

[0095]

[0096] The experimental results show that the total sugar content in the dry matter of the amniotic membrane extract in Examples 1 to 3 is 10.87-13.11%, and the total sugar content in the dry matter of the amniotic membrane extract in Comparative Examples 1 to 3 is 3.67-5.30%, that is, the total sugar content in the dry matter of the amniotic membrane extract obtained by the preparation process of the present invention is greatly improved.

[0097] The pulverizing processes of Comparative Examples 1 to 3 respectively use PBS liquid pulverizing, physiological saline liquid pulverizing, and liquid nitrogen freezing pulverizing, while Examples 1 to 3 of the present invention use ice bath pulverizing. By improving process parameters such as "the size of ice cubes used, the weight ratio of ice cubes to samples, pulverizing time, and the number of pulverizing times", the extraction efficiency of amniotic membrane extract as an active ingredient (total sugar in dry matter) for ophthalmic medical devices or drugs is greatly improved.

[0098] (V) Detection of fat content in dry matter of amniotic membrane extract

[0099] The fat content of the amniotic membrane extracts of Examples 1 to 3 and Comparative Examples 1 to 3 was tested respectively. The test method was based on the acid hydrolysis method of GB 5009.6-2016 "National Food Safety Standard - Determination of Fat in Food".

[0100] The specific steps are as follows:

[0101] 1) Acid hydrolysis: weigh 0.1g of the dried constant weight sample, accurately weigh it, place it in a 50mL test tube, add 8mL of water, mix well, and then add 10mL of hydrochloric acid. Place the test tube in a 70~80℃ water bath, stir it with a glass rod every 5min~10min until the sample is completely digested, which takes about 3h. Each batch is measured three times in parallel.

[0102] 2) Extraction, take out the sample test tube, add 10mL ethanol, and mix. After cooling, transfer the mixture into a 100ml stoppered measuring cylinder, wash the test tube with 25ml anhydrous ether several times, and pour it into the measuring cylinder. After all the anhydrous ether has been poured into the measuring cylinder, add a stopper and shake for 1min, carefully unscrew the stopper, release the gas, rescrew it, let it stand for 12min, carefully unscrew the stopper, and rinse the stopper and the fat attached to the mouth of the measuring cylinder with ether. Let it stand for 10~20min, wait for the upper liquid to be clear, aspirate the supernatant into a weighing bottle that has been constant weight, add 5ml anhydrous ether into the stoppered measuring cylinder, shake, let it stand, and still aspirate the upper ether and put it into the original weighing bottle.

[0103] 3) Dry to constant weight: Place the weighing bottle containing the extract on a water bath to evaporate to dryness, and then dry at 100℃±5℃ to constant weight.

[0104] 4) Calculation of results:

[0105] ,

[0106] In the formula, X is the fat content in the sample, in grams per 100 grams (g / 100g); m 1 is the total weight of the weighing bottle and fat after constant weight, in grams (g); m 0 is the mass of the weighing bottle in grams (g); m 2 is the mass of the sample in grams (g); 100 is the conversion factor.

[0107] The experimental results are shown in Table 3:

[0108] Table 3 Fat content of amniotic membrane extract dry matter

[0109]

[0110] The experimental results show that the fat content in the dry matter of the amniotic membrane extract in Examples 1 to 3 is 1.83-3.53%, and the fat content in the dry matter of the amniotic membrane extract in Comparative Examples 1 to 3 is 6.15-6.85%, that is, the fat content in the dry matter of the amniotic membrane extract obtained by the preparation process of the present invention is greatly reduced.

[0111] The purification processes of Comparative Examples 1 to 3 all involve centrifugation to obtain the supernatant. In Examples 1 to 3 of the present invention, a centrifugation and filter paper vacuum filtration process is adopted. By selecting filter paper with a specific pore size (pore size of 5 to 50 μm), large particles (>50 μm) of impurities can be effectively filtered out, thereby greatly reducing the content of non-functional impurities (fat in dry matter).

[0112] (VI) Detection of the residue on ignition content in the dry matter of amniotic membrane extract

[0113] The amniotic membrane extracts of Examples 1 to 3 and Comparative Examples 1 to 3 were respectively tested for the residue on ignition content. The test method was based on the residue on ignition test method 0841 of the Chinese Pharmacopoeia (Volume 4, 2015 edition).

[0114] The specific steps are as follows:

[0115] 1) Empty crucible constant weight (W0): Take an empty crucible, ignite it in a muffle furnace at 500-600℃ for 2 hours, take it out and place it in a desiccator to cool for 1 hour, weigh it, and weigh it accurately. Then put it in the ignition for 30 minutes, take it out and let it cool for 1 hour, and weigh it. If the difference between the two weighings does not exceed ±0.3mg, it is considered constant weight, otherwise continue to ignite it until constant weight.

[0116] 2) Sample treatment: weigh 0.1g of the dried constant-weight sample, place it in a quartz crucible (W0) that has been ignited to constant weight, weigh it accurately, slowly ignite it until it is completely carbonized, and let it cool; add 0.5~1mL of sulfuric acid to moisten it, heat it at low temperature until the sulfuric acid vapor is completely removed, ignite it at 500~600℃ to completely ash it, move it to a desiccator, let it cool for 1 hour, ignite it again for 30 minutes, take it out and let it cool for 1 hour, and weigh it. If the interval between the two weighings does not exceed ±0.3mg, it is considered constant weight, otherwise continue to ignite it until it is constant weight.

[0117] 3) Calculation of results:

[0118] Residue on ignition (%) = (W1-W0) / m×100 (%),

[0119] Wherein, W0 is the total weight of the crucible before ignition, in g; W1 is the total weight of the residue and the crucible after ignition, in g; m is the mass of the sample, in g.

[0120] The experimental results are shown in Table 4:

[0121] Table 4 Data on the residue on ignition content of dry matter of amniotic membrane extract

[0122]

[0123] The experimental results show that the residue on ignition content in the dry matter of the amniotic membrane extract in Examples 1 to 3 is 0.94-1.64%, and the residue on ignition content in the dry matter of the amniotic membrane extract in Comparative Examples 1 to 3 is 3.06-3.67%, that is, the residue on ignition content in the dry matter of the amniotic membrane extract obtained by the preparation process of the present invention is greatly reduced.

[0124] The purification processes in Comparative Examples 1 to 3 all involve centrifugation to obtain the supernatant. In Examples 1 to 3 of the present invention, centrifugation and filter paper vacuum filtration processes are used. By selecting filter paper with a specific pore size (pore size of 5 μm~50 μm), large particles (>50 μm) of impurities can be effectively filtered out, thereby greatly reducing the content of non-functional impurities (residue on ignition in dry matter).

[0125] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing an amniotic membrane extract, characterized in that: The fresh amniotic membrane is sterilized, cleaned, dried, and then crushed in an ice bath. The crushed liquid is then centrifuged and filtered to obtain the amniotic membrane extract. During the ice bath crushing, the air-dried amniotic membrane and ice cubes are added into a container, wherein the ice cubes are purified water ice cubes with a side length of 1 to 3 cm, and the mass ratio of ice cubes to amniotic membrane is controlled to be 1 to 5:1, and then the mixture is allowed to stand for 5 minutes, and then the mixture of ice water and amniotic membrane is obtained and then crushed by a crusher, and the liquid temperature during the crushing process is controlled to be less than 8° C. After crushing for 10 to 60 seconds, the mixture is crushed again at an interval of 1 minute, and the process is repeated 30 to 90 times, thus completing the ice bath crushing. During the centrifugation, the crushed liquid was divided into 50 ml centrifuge tubes, each tube was filled with 50 ml of crushed liquid and then put into a centrifuge for centrifugation for 10 minutes, and the speed of the centrifuge was controlled to be 1000-3000 rpm. The filtration is performed by vacuum filtration using filter paper with a pore size of 5 to 50 μm. The dry matter content of amniotic membrane extract meets the following requirements: total protein content of 78.02-82.79%, total sugar content of 10.87-13.11%, fat content of 1.83-3.53%, and residue on ignition content of 0.94-1.64%.

2. The preparation method according to claim 1, characterized in that: During the disinfection, 75% alcohol is sprayed on the packaging material of the fresh amniotic membrane for disinfection, and then ultraviolet sterilization is performed for 30 minutes.

3. The preparation method according to claim 1, characterized in that: During the cleaning and impurity removal, the sterilized amniotic membrane is unpacked and placed in purified water to be dispersed and cleaned, and residual impurities on the surface are removed.

4. The preparation method according to claim 1, characterized in that: During the drying process, the cleaned and impurity-free amniotic membrane was spread on a sieve and dried for 1 hour, and turned over every 15 minutes.

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