CN117442646B - Eye surface lubricating liquid and preparation process, quality control method and application thereof - Google Patents
Eye surface lubricating liquid and preparation process, quality control method and application thereof Download PDFInfo
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- CN117442646B CN117442646B CN202311765175.7A CN202311765175A CN117442646B CN 117442646 B CN117442646 B CN 117442646B CN 202311765175 A CN202311765175 A CN 202311765175A CN 117442646 B CN117442646 B CN 117442646B
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- amniotic membrane
- surface lubricating
- ocular surface
- liquid
- membrane extract
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- 230000001050 lubricating effect Effects 0.000 title claims abstract description 104
- 238000002360 preparation method Methods 0.000 title claims abstract description 43
- 238000003908 quality control method Methods 0.000 title abstract description 17
- 210000001691 amnion Anatomy 0.000 claims abstract description 200
- 239000000284 extract Substances 0.000 claims abstract description 118
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 69
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 39
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 29
- 230000001954 sterilising effect Effects 0.000 claims abstract description 23
- 235000000346 sugar Nutrition 0.000 claims abstract description 19
- 229920002385 Sodium hyaluronate Polymers 0.000 claims abstract description 18
- 229940010747 sodium hyaluronate Drugs 0.000 claims abstract description 18
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims abstract description 18
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Abstract
The invention discloses an eye surface lubricating liquid and a preparation process, a quality control method and application thereof, wherein fresh amniotic membrane is sequentially disinfected, cleaned, decontaminated and dried, then crushed by ice bath, the crushed liquid is centrifuged and suction filtered to obtain an amniotic membrane extract, and the eye surface lubricating liquid is prepared by the steps of preparing the prefabricated liquid according to the formula of 0.1-10% of the amniotic membrane extract, 0.1-0.4% of sodium hyaluronate, 5-10% of glycerol, 0.01-0.8% of sodium chloride, 0.2-0.6% of boric acid, 0.005-0.025% of borax and the balance of water for injection, filtering, packaging and sterilizing by irradiation. In the preparation process of the amniotic membrane extract, the amniotic membrane is crushed by utilizing ice water mixed solution, and then the total protein and total sugar optimized amniotic membrane extract is obtained by combining the control of technological parameters such as crushing and the like, and then the amniotic membrane extract is further used for preparing the ocular surface lubricating liquid, so that the ocular surface lubricating liquid can be better used in ocular surface environments, the safety and the effective performance are improved, and in addition, the ocular surface lubricating liquid can be jointly applied to the treatment of ocular surface diseases with other ocular surface dressing products.
Description
Technical Field
The invention belongs to the technical field of medical equipment and medicines, and in particular relates to an eye surface lubricating liquid and a preparation process, a quality control method and application thereof.
Background
The eye surface lubricating liquid can be used as a contact lens lubricating liquid, and can relieve uncomfortable symptoms such as dry eyes, fatigue and the like. The main component of the contact lens lubricating liquid sold in the market at present is usually sodium hyaluronate, which can play a role in lubrication and moisture retention. The amniotic membrane is the innermost layer of placenta, the components mainly contain protein and total sugar (especially glycosaminoglycan), the amniotic membrane or the amniotic membrane extract is used as a medical instrument or a medicine, the protein in the components can be used as a natural scaffold for cell growth adhesion, the function of promoting tissue repair is achieved, and the glycosaminoglycan in the total sugar has a hydrophilic lubrication function. At present, the amniotic membrane eye drops prepared from the amniotic membrane extract or the amniotic membrane suspension are used for postoperative treatment of various ophthalmic diseases and have better clinical use effects. However, it has been recently reported as a main component of a contact lens lubricating liquid.
Reference is made to the preparation process of amniotic membrane eye drops in the prior literature, such as: paper "New method for treating severe Ocular surface lesions", journal of cell and tissue library, 2022, 23 (3), 473-481 reports clinical follow-up studies of the preparation of amniotic membrane as an eye drop for treating patients with ocular surface diseases such as chronic dry eye, limbal stem cell deficiency (36 eyes of 25 patients). The amnion extract crushing process comprises the following steps: the liquid nitrogen freezing and crushing and purifying process comprises the following steps: centrifuging and taking supernatant. The results show that: the topical application of amniotic membrane eye drops is safe and well tolerated by patients, and can significantly improve ocular symptoms such as foreign body sensation, itching and stinging. The preparation method of the amniotic membrane eye drops comprises the following steps: placenta was collected after informed consent from the donor. After washing the placenta to remove the blood clot, the amniotic membrane is peeled off by a blunt tool. The amniotic membrane is then rinsed and purified with an antibiotic/antifungal solution. The next day, the amniotic membrane was rinsed with saline to remove antibiotics, then soaked in liquid nitrogen and frozen. The frozen amniotic membrane was ground at room temperature and centrifuged. The supernatant was divided into 1ml vials, freeze-dried and stored at room temperature until use.
Another example is: the invention patent application number 200910178422.7 discloses an amniotic membrane eye drop and a preparation method thereof, and the amniotic membrane extract crushing process comprises the following steps: PBS liquid is crushed and purified by the following steps: centrifuging and taking supernatant. The amniotic membrane homogenate supernatant is taken as a main active ingredient, and substances such as trehalose, hyaluronic acid or salts thereof, heparin or salts thereof, sodium chloride, benzalkonium bromide, vitamins and the like are added. The amniotic membrane homogenate supernatant is prepared by the following method: the amniotic membrane is washed in PBS for 3 times on an ultra clean bench for about 10 to 15 minutes, wet weight is weighed according to proper size, PBS is added according to the ratio of 1:1, the amniotic membrane is put into a homogenizer, 5000 revolutions per minute are carried out, homogenization is carried out through 10 strokes, and 30 seconds per stroke are obtained. Taking out the homogenate, adding PBS (phosphate buffered saline) solution into a centrifuge tube according to the ratio of 1:5, centrifuging for 2000r/min, 10 minutes, and taking the supernatant for later use.
The invention patent with application number 202310856867.6 also discloses a tissue regeneration type biomembrane tissue compound, a preparation method and application thereof, and the amniotic membrane extract crushing process of the patent comprises the following steps: the physiological saline liquid is crushed and purified by the following steps: centrifuging and taking supernatant. The preparation method comprises the steps of extracting biological membrane tissues and promoting an adsorption gel slow-release system, wherein the biological membrane tissues comprise amniotic membrane extracts, and the preparation method comprises the following steps: after the blood clot is removed by using normal saline, the fresh human amniotic membrane is obtained by peeling through a blunt tool. Fresh amniotic membrane was soaked in physiological saline containing amphotericin B (10 mg/mL), penicillin (100 u/mL) and streptomycin (10 mg/mL) for 2 hours. The ratio of the wet weight of the amniotic membrane to the volume of the physiological saline is kept at 1g/L, and the amniotic membrane is continuously rinsed three times by the physiological saline under the aseptic condition and the neutral pH value. After treatment with a tissue shredder and centrifugation, the supernatant was collected: the treatment was performed under aseptic conditions, with a tissue disruptor treatment time of 10 minutes, a centrifugation rate of 800rpm and a centrifugation time of 10 minutes.
As can be seen, the following problems still need to be solved in the prior art when preparing amniotic membrane extract for amniotic membrane eye drops: firstly, the crushing process adopts liquid nitrogen freezing crushing or PBS/physiological saline liquid crushing, the crushing effect is poor, and the extraction effect on the effective components (such as total proteins and total sugars) in the amniotic membrane extract is poor; secondly, the purification process adopts a method of taking supernatant after centrifugation, and the obtained amniotic membrane extract still possibly contains larger granular substances (more than 50 mu m), so that foreign matter discomfort appears after the amniotic membrane extract is dripped into eyes of a patient; third, the amniotic membrane extract contains higher levels of non-functional impurities (e.g., fat and burning residues).
Therefore, the quality of the amniotic membrane extract in the existing amniotic membrane eye drops is difficult to ensure, and the safety and effectiveness of the amniotic membrane extract in the use of the ocular surface are difficult to ensure. For this reason, in the application of amniotic membrane extract to ophthalmic preparation products such as contact lens lubricating liquid, it is highly necessary to provide an optimized preparation method of an ophthalmic preparation product containing amniotic membrane extract and establish relevant quality control standards.
Disclosure of Invention
The invention aims to provide a preparation process of an ocular surface lubricating liquid, which is characterized in that in the preparation process of an amniotic membrane extract, the amniotic membrane is crushed by utilizing an ice water mixed liquid, and then the amniotic membrane extract with optimized total protein and total sugar serving as active ingredients can be obtained by combining the matching and control of technological parameters such as the mass ratio of ice cubes to the amniotic membrane, the crushing time, the crushing times and the like, and then the amniotic membrane extract is used for preparing the ocular surface lubricating liquid, so that the ocular surface lubricating liquid can be better used in an ocular surface environment, and the safety and the effective performance are improved. Therefore, the invention also provides the eye surface lubricating liquid prepared by the preparation process and a quality control method of the eye surface lubricating liquid.
The invention is realized by the following technical scheme: a method for preparing eye surface lubricating liquid comprises sequentially sterilizing fresh amniotic membrane, cleaning, removing impurities, air drying, pulverizing in ice bath, centrifuging, vacuum filtering to obtain amniotic membrane extract, preparing into prefabricated liquid, filtering, packaging, sterilizing by irradiation to obtain eye surface lubricating liquid,
when the ice bath is used for crushing, the dried amnion and ice blocks are added into a container, the mass ratio of the ice blocks to the amnion is controlled to be 1-5:1, then the container is kept stand for 5min, the obtained ice water amnion mixed liquid is crushed by a crusher, the temperature of feed liquid in the crushing process is controlled to be less than 8 ℃, the crushing is carried out again at intervals of 1min after the crushing is carried out for 10-60S, the process is repeated for 30-90 times, and the ice bath crushing is completed,
the dry matter of the amniotic membrane extract meets the following conditions: the dry matter of the amniotic membrane extract is obtained by placing the amniotic membrane extract in a liquid state in a blast drying oven at 50 ℃ to constant weight, usually drying for more than 2 days to constant weight, wherein the total protein content is more than or equal to 78.02%, the total sugar content is more than or equal to 10.87%, the fat content is less than or equal to 3.53%, and the burning residue content is less than or equal to 1.64%,
the prefabricated liquid contains the following components in percentage by mass: the amniotic membrane extract is 0.1-10%, sodium hyaluronate is 0.1-0.4%, glycerin is 5-10%, sodium chloride is 0.01-0.8%, boric acid is 0.2-0.6%, borax is 0.005-0.025%, and the rest is water for injection.
And in the disinfection process, 75% alcohol is sprayed on a fresh amniotic membrane packaging material for disinfection, and then ultraviolet disinfection is carried out for 30min.
When the amnion is cleaned and decontaminated, the disinfected amnion is unpacked and put into purified water for dispersion and cleaning, and residual impurities on the surface of the amnion are removed.
And in the air drying process, the cleaned and decontaminated amniotic membrane is paved on a screen for air drying for 1h, and the amniotic membrane is turned over once every 15 min.
The ice cubes are purified water ice cubes with the side length of 1-3 cm.
During centrifugation, the crushed liquid is divided into 50ml of centrifugal tubes, each tube is filled with 50ml of crushed liquid and then is put into a centrifugal machine for centrifugation for 10min, and the rotating speed of the centrifugal machine is controlled to be 1000-3000 rpm.
The suction filtration is carried out by adopting filter paper with the aperture of 5-50 mu m.
The dry matter of the amniotic membrane extract meets the following conditions: the total protein content is 78.02-82.79%, the total sugar content is 10.87-13.11%, the fat content is 1.83-3.53%, and the burning residue content is 0.94-1.64%.
An eye surface lubricating liquid is prepared by the preparation method.
A quality control method of eye surface lubricating liquid comprises quality control of amniotic membrane extract in eye surface lubricating liquid and quality control of index system of eye surface lubricating liquid,
the quality control of the amniotic membrane extract in the ocular surface lubricating liquid comprises the following steps:
(1) Setting the mass concentration of the target total protein in the ocular surface lubricating liquid as W0;
(2) According to a first nitrogen determination method of 0704 in the 2020 edition of Chinese pharmacopoeia, the total protein mass concentration in the amniotic membrane extract is determined to be W1;
(3) Comparing W1 with the target total protein mass concentration W0 in the ocular surface lubricating liquid, and adding other ocular surface lubricating liquid components and water for injection until the total protein mass concentration is diluted to W0 when W1 is more than W0; when w1=w0, other ocular surface lubricating liquid components are directly added into the amniotic membrane extract; when W1 is less than W0, freeze drying the amniotic membrane extract until W1 is more than or equal to W0; if the compound solution after freeze drying can not reach W1 not less than W0, the amniotic membrane extract in the batch is judged to be unqualified in quality, the waste treatment is carried out,
the quality control of the eye surface lubricating liquid index system is to detect the eye surface lubricating liquid according to the following method, and the following performance index system is achieved:
(1) The pH value is 6.5-7.8 according to the pH value measurement method of 0631 in the 2020 edition of Chinese pharmacopoeia;
(2) According to the method for measuring the osmolality of 0632 in the 2020 edition of Chinese pharmacopoeia, the osmolality is 240-340 mOsm/kgH 2 O；
(3) The viscosity value is 1.0 to 50 mPas measured according to a 0633 viscometry method in the 2020 edition of Chinese pharmacopoeia;
(4) According to the method for measuring the content of 0731 protein in the 2020 edition of Chinese pharmacopoeia and the method for measuring 0704 first method nitrogen in the 2020 edition of Chinese pharmacopoeia, the total protein mass percentage concentration is 0.001-0.1%;
(5) The heavy metal content of the heavy metal is less than 10 mug/mL according to the detection method of 0821 heavy metal in the 2020 edition of Chinese pharmacopoeia;
(6) The bacteria are detected to be sterile according to a 1101 bacteria-free inspection method in the 2020 edition of Chinese pharmacopoeia;
(7) The bacterial endotoxin content of the leaching solution is less than 0.5 EU/mL according to the method specified by the gel limit test of the gel method in the method of 1143 bacterial endotoxin test method in the 2020 edition of Chinese pharmacopoeia.
The use of an ocular surface lubricating fluid as described above or in combination with other ocular surface dressings in the manufacture of a product for the treatment of ocular surface disorders. When the amniotic membrane covering operation is carried out, the prepared ocular surface lubricating liquid can be dripped on the ocular surface after the operation, so that the foreign body sensation after the operation can be effectively reduced, and the treatment effect is improved.
Compared with the prior art, the invention has the following advantages:
(1) The invention improves the preparation method of the amniotic membrane extract in the ocular surface lubricating liquid.
On one hand, the invention improves the crushing process of the amniotic membrane extract, changes the problems of poor crushing effect and poor extraction effect on the effective components (such as total proteins) in the amniotic membrane extract caused by adopting liquid nitrogen freezing crushing or adding PBS/physiological saline liquid crushing in the crushing process in the prior art, adopts ice bath crushing, optimizes the technological parameters such as the size of ice cubes, the weight ratio of ice cubes to samples, the crushing time, the crushing times and the like, and greatly improves the extraction efficiency of the amniotic membrane extract as the effective components (namely total proteins) of the ophthalmic medical instrument or medicine.
On the other hand, the invention also improves the purification process of the amniotic membrane extract, the existing purification process adopts a method of taking supernatant after centrifugation, and the obtained amniotic membrane extract still possibly contains larger granular substances (more than 50 mu m), so that foreign matter discomfort appears after the amniotic membrane extract is dripped into eyes of patients. The invention adopts the centrifugal and filter paper vacuum filtration process, and can effectively filter out large-particle (> 50 μm) size impurities by selecting filter paper with specific gap size (aperture of 5-50 μm), thereby avoiding foreign matter uncomfortable feeling after the filter paper is dripped into eyes of patients, and greatly reducing the content of impurities (fat and burning residues) without functions. In addition, the vacuum filtration process accelerates the filtration speed and improves the production efficiency by the assistance of an external vacuum pump.
(2) The invention improves the formula of the eye surface lubricating liquid. Compared with the conventional commercially available similar products of contact lens lubricating liquid (national mechanical injection 20163160145 or national mechanical injection 20173160321), the functional component of the contact lens lubricating liquid is generally sodium hyaluronate with lubricating effect, and the functional component in the ocular surface lubricating liquid disclosed by the invention comprises functional components (namely amniotic membrane extract) for promoting tissue repair besides sodium hyaluronate. The amniotic membrane extract contains various proteins, which are helpful for covering the ocular surface to form a protein scaffold, so that ocular surface epithelial cells of a patient can migrate onto the amniotic membrane extract to promote healing of the ocular surface epithelial cells. In addition, functional components (namely glycerin) which have less influence on viscosity due to irradiation are also added into the formula of the eye surface lubricating liquid. Experimental test results show that the viscosity value of the high molecular weight viscosity regulating substance (such as sodium hyaluronate with molecular weight larger than 1 MDa) is reduced to be less than one tenth of the initial value after irradiation sterilization (sterilization dose is 25-35 kGy), and the small molecular weight viscosity regulating substance (such as glycerol and molecular weight 92.09 Da) is added in the formula of the eye surface lubricating liquid, so that the influence of irradiation sterilization on the viscosity is effectively reduced. The ophthalmic preparation with higher viscosity can improve the residence time of the ophthalmic preparation on the ocular surface, thereby prolonging the time for the ophthalmic preparation to function.
(3) The quality control method of the amniotic membrane extract in the ocular surface lubricating liquid is established for the first time, the quality control method of the amniotic membrane suspension or the amniotic membrane eye drop product is not established in the prior publication, and the quality control of the amniotic membrane extract cannot be performed.
(4) The quality control method of the ocular surface lubricating liquid can limit key performance indexes of the ocular surface lubricating liquid, including pH value, osmotic pressure, viscosity, total protein content, heavy metal, sterility and bacterial endotoxin, and can effectively improve the safety and effectiveness of the ocular surface lubricating liquid in ocular surface use.
Drawings
FIG. 1 is a flow chart of a process for preparing an ocular surface lubricating fluid according to the present invention;
FIG. 2 is a graph showing the amount of amniotic membrane extract used in an ocular surface lubricating fluid sample;
fig. 3 is a graph comparing stability of an ocular surface lubricating fluid sample before and after irradiation sterilization.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
Example 1:
the formulation and the effects of the components of the ophthalmic surface lubricating liquid are shown in Table 1.
TABLE 1 eye surface lubricating fluid formulations and the effects of the ingredients
Referring to the preparation flow chart shown in fig. 1, the preparation steps of the ocular surface lubricating liquid are as follows:
step one: sterilizing, cleaning and removing impurities
Taking fresh amniotic membrane frozen (-20deg.C) in advance out of refrigerator, spraying 75% alcohol on the packaging material for sterilizing, and placing into workshop transfer window feed inlet for ultraviolet sterilization for 30min. Then taking out the sterilized amniotic membrane, removing the bag, taking out the amniotic membrane by using forceps, placing the amniotic membrane into a 304 tray or beaker, and adding purified water to enable the purified water to permeate at least 2cm. After 5min, the amniotic membrane is primarily thawed, and dispersed by hands to avoid shrinkage. The amniotic membrane was turned over every 30min with water and gently stirred to remove residual impurities on the surface. This step was repeated at least 3 times until the solution remained clear after the addition of purified water.
Step two: air-drying
The cleaned amniotic membrane is taken out by hands, is placed on a screen for airing, and is placed on the bottom to catch the separated water. The amniotic membrane needs to be laid flat, if the amniotic membrane cannot be laid flat and cut and divided, the amniotic membrane cannot be placed in a superposition way. When the amnion is dried, the amnion needs to be turned over once within 15min, and the amnion is dried for 1h.
Step three: crushing in ice bath
The prepared beaker is put into an electronic balance for zeroing, the amniotic membrane is weighed after being contained by the beaker, and the wet weight of the amniotic membrane is recorded. The prepared purified water ice cubes (1 cm. Times.1 cm) were weighed to the amniotic membrane in a mass ratio of 1:1, and then poured into a beaker. Placing into an experimental pulverizer for standing for 5 minutes, pulverizing (at this time, partial ice cubes are melted to form ice water amnion mixed liquid), simultaneously wrapping a beaker with an ice bag, controlling the temperature of feed liquid in the pulverizing process to be less than 8 ℃, taking rest for 1min by 10S equipment each time, and repeating the process for 30 times to finish the pulverizing.
Step four: centrifuging and vacuum filtering
Loading the crushed liquid obtained by ice bath crushing into 50ml centrifuge tubes, loading each tube into 50ml centrifuge tubes, setting the rotation speed of the centrifuge tubes to 1000rpm, centrifuging for 10min, taking out the centrifuged sample, and pouring the supernatant into a beaker, thus finishing the centrifugation. And then placing medium-speed qualitative filter paper with the aperture of 5-10 mu m on a buchner funnel suction filtration bottle, pouring supernatant, turning on a vacuum pump, and collecting filtrate, namely the amniotic membrane extract.
Step five: preparation of prefabricated liquid
According to the formula of the eye surface lubricating liquid, namely: 1% of amniotic membrane extract, 0.2% of sodium hyaluronate, 5% of glycerin, 0.4% of sodium chloride, 0.4% of boric acid, 0.015% of borax and the balance of water for injection.
Step six: filtration
Pouring the prefabricated liquid into a suction filtration bottle of a Buchner funnel, and selecting quick qualitative filter paper (20-25 mu m) for vacuum filtration.
Step seven: packaging
And (3) packaging the intermediate product of the ocular surface lubricating liquid in a secondary throwing pipe, sealing by using a sealing machine, and attaching a production label, namely finishing packaging.
Step eight: irradiation sterilization
And (3) carrying out irradiation sterilization on the packaged ocular surface lubricating liquid, wherein the sterilization dose is 25kGy.
Step nine: preservation of ocular surface lubricating liquid
And (3) storing the sterilized eye surface lubricating liquid in a refrigerator at the temperature of 2-8 ℃ to finish the preparation of the eye surface lubricating liquid.
Example 2:
the embodiment relates to an eye surface lubricating fluid, which comprises the following components in percentage by weight: 1% of amniotic membrane extract, 0.2% of sodium hyaluronate, 5% of glycerin, 0.4% of sodium chloride, 0.4% of boric acid, 0.015% of borax and the balance of water for injection.
The same preparation process flow of example 1 is adopted in this example, except that some parameters of ice bath pulverization, centrifugation and vacuum filtration of filter paper are slightly adjusted, and the rest steps and process parameters are the same as those of example 1.
Specifically, in this example, purified water ice cubes having a size of 3cm×3cm were used at the time of ice bath pulverization, the mass ratio of the purified water ice cubes to the amniotic membrane was set to 5:1, and the procedure was repeated 90 times for 1min each time of pulverization in 60S equipment. During centrifugation, the rotational speed of the centrifuge was set to 3000rpm. When the filter paper is vacuum filtered, medium speed qualitative filter paper with the diameter of 20-50 μm is adopted.
Example 3:
the present example relates to a quality control method for the amount of amniotic membrane extract in an ocular surface lubricating liquid and a quality control method for the final product of an ocular surface lubricating liquid.
Firstly, determining the mass concentration of target total protein in the ocular surface lubricating liquid as W0, then determining the mass concentration of the total protein in the amniotic membrane extract as W1, and comparing the W1 with the mass concentration of the target total protein in the ocular surface lubricating liquid as W0:
1) When W1 is more than W0, adding other components of the ocular surface lubricating liquid and water for injection until the total protein mass concentration is diluted to W0;
2) When w1=w0, other ocular surface lubricating liquid components are directly added into the amniotic membrane extract;
3) When W1 is less than W0, freeze-drying the amniotic membrane extract until W1 is more than or equal to W0, and if the compound solution cannot reach W1 is more than or equal to W0 after freeze-drying, judging that the amniotic membrane extract in the batch is unqualified, and performing waste treatment.
By testing the ocular surface lubricating liquids of example 1 and example 2, the index system satisfied:
(1) The pH value is 6.5-7.8, and is measured according to the four parts 0631pH value measuring method of Chinese pharmacopoeia (2020 edition);
(2) Osmolality 240-340 mOsm/kgH 2 O is measured according to four 0632 osmotic molar concentration measuring methods of Chinese pharmacopoeia (2020 edition);
(3) The viscosity value is 1.0-50 mPas, and is measured according to the four-part 0633 viscometry method of Chinese pharmacopoeia (2020 edition);
(4) The total protein mass percentage concentration is 0.001-0.1%, and is measured according to the four 0731 protein content measuring methods of Chinese pharmacopoeia (2020 edition) and the four 0704 first nitrogen measuring methods of Chinese pharmacopoeia (2020 edition);
(5) The heavy metal content is less than 10 mug/mL, and is measured according to four 0821 heavy metal inspection methods of Chinese pharmacopoeia (2020 edition);
(6) Sterile, according to the four 1101 sterile inspection methods of Chinese pharmacopoeia (2020 edition);
(7) The bacterial endotoxin content was less than 0.5. 0.5 EU/mL, and the extract was measured according to the gel limit test of gel method of method 1 of four 1143 methods for bacterial endotoxin detection of Chinese pharmacopoeia (2020 edition).
Example 4:
this example uses the ocular surface lubricating fluid prepared in example 1 in combination with amniotic membrane covering for treatment of ocular surface diseases. The treatment scheme is as follows: after the diaphragm-shaped amniotic membrane product is sutured by an operation, the ocular surface lubricating liquid is dripped into the patient with ocular surface diseases, wherein the dripping is repeated for 1-7 times at intervals of 24 hours with 0.5 milliliter each time.
The ocular surface lubricating fluids of examples 1 to 2 were compared with the existing commercial products of the same type (contact lens lubricating fluids), as shown in the table 2 comparing the performance of ocular surface lubricating fluids with contact lens lubricating fluids:
table 2 table of performance comparisons of ocular surface lubricating fluids and contact lens lubricating fluids
Comparative example 1:
comparative example 1 an amniotic membrane extract was prepared using the following steps: sterilizing, cleaning, removing impurities, air drying, pulverizing PBS liquid, and purifying. Wherein, the method of disinfection, cleaning and impurity removal and air drying is the same as that of the embodiment 1; the PBS liquid crushing process is to clean the amniotic membrane in PBS liquid for 3 times on an ultra clean bench for about 10-15 minutes, weigh the amniotic membrane with proper size and wet weight, add PBS liquid according to the ratio of 1:1, put the amniotic membrane into a homogenizer, grind the amniotic membrane into homogenate with 5000 r/min for 10 strokes, and grind the amniotic membrane for 30 seconds per stroke; the purification process comprises adding the homogenate into PBS (phosphate buffer solution) according to the ratio of 1:5, centrifuging at 2000r/min for 10min, and collecting supernatant to obtain the amniotic membrane extract.
The amniotic membrane extract prepared in comparative example 1 was prepared into an ocular surface lubricating fluid according to the same formulation and process flow as in example 1.
Comparative example 2:
comparative example 2 an amniotic membrane extract was prepared using the following steps: washing, crushing with physiological saline solution and purifying. The specific flow is as follows: after blood clots are removed by using physiological saline, a fresh human amniotic membrane is obtained by stripping through a blunt tool, the fresh amniotic membrane is soaked in the physiological saline containing amphotericin B (10 mg/mL), penicillin (100 u/mL) and streptomycin (10 mg/mL) for 2 hours, the volume ratio of the wet weight of the amniotic membrane to the physiological saline is kept to be 1g/L, then the process is carried out under the aseptic condition and the neutral pH value, and the amniotic membrane is rinsed three times continuously by using the physiological saline. Crushing the rinsed amnion for 10min under aseptic condition by adopting a tissue crusher, adding the crushed slurry into a centrifuge tube, centrifuging at 800rpm for 10min, and taking supernatant to obtain the amnion extract.
The amniotic membrane extract prepared in comparative example 2 was prepared into an ocular surface lubricating fluid according to the same formulation and process flow as in example 1.
Comparative example 3:
comparative example 3 amniotic membrane extract was prepared using the following steps: cleaning, freezing and crushing by liquid nitrogen and purifying. The specific flow is as follows: after washing the placenta to remove blood clots, the amniotic membrane was peeled off by a blunt tool, and then washed and purified with an antibiotic/antifungal solution (1000 [ mu ] m/mL penicillin and 20 mg/mL streptomycin PBS). The next day, the amniotic membrane was rinsed with saline to remove antibiotics, then soaked in liquid nitrogen and frozen. Grinding frozen amnion at room temperature, centrifuging (14000 r/min for 5 min), and collecting supernatant to obtain amnion extract (supernatant is separated into 1ml vials, freeze-dried, and stored at room temperature until use).
The amniotic membrane extract prepared in comparative example 1 was prepared into an ocular surface lubricating fluid according to the same formulation and process flow as in example 1.
Comparative example 4:
comparative example 4 is an amniotic membrane extract-free ocular surface lubricating fluid, the formulation of which differs from example 1 only in that no amniotic membrane extract is added, the formulation of which is as follows: sodium hyaluronate 0.2%, glycerin 5%, sodium chloride 0.4%, boric acid 0.4%, borax 0.015%, and water for injection the rest.
Comparative example 5:
comparative example 4 is a glycerin-free ocular surface lubricating liquid whose formulation differs from example 1 only in that no glycerin is added, and its formulation is as follows: 1% of amniotic membrane extract, 0.2% of sodium hyaluronate, 0.4% of sodium chloride, 0.4% of boric acid, 0.015% of borax and the balance of water for injection.
Detection of Total protein content in amniotic Membrane extract Dry Material
The amniotic membrane extracts of examples 1 and 2 and comparative examples 1 to 3 were taken for total protein content detection. Test methods refer to the Kjeldahl nitrogen determination method of the four 0731 protein content determination methods of the Chinese pharmacopoeia (2015 edition).
The method comprises the following specific steps:
1) Digestion, 0.1g of the dried constant weight sample (amniotic membrane extract) was weighed, precisely weighed, and carefully added to a digestion tube (care taken to prevent the sample from sticking to the walls). 10mL of sulfuric acid, 4.5g of potassium sulfate and 0.5g of copper sulfate were added. Placing into a digestion furnace, and heating to 350 ℃ until the nitrolysis is complete (i.e. the solution becomes transparent light blue). After complete nitration, the cooling was removed, 30ml distilled water was added to the nitration tube to dilute the sample and release heat, and the cooling was ready for use. Each batch of samples was assayed 1 time while 1 blank parallel was prepared.
2) Distilling, removing the liberated cooled sample, placing the sample on the left side of the Kjeldahl nitrogen determination instrument, sealing, placing the sample on the right side of the Kjeldahl nitrogen determination instrument into a conical flask, and extending a liquid outlet pipe to the bottom of the conical flask. The Kjeldahl nitrogen determination instrument was clicked to begin nitrogen determination, and the distilled liquid was collected in a conical flask. After the distillation is completed, the conical flask from which the distillate was collected is taken out.
3) Titration, sampling distilled liquid, adding 2-3 drops of methyl red bromocresol green indicator liquid, and titrating with sulfuric acid titration liquid (0.05 mol/L) until the solution changes from green to red. The volume of spent titrant was recorded.
4) And (3) calculating results:
nitrogen content: n (%) =，
Total protein content: p (%) =n (%) ×c
Wherein: m= 1.401, corresponding to 1.401mg of N per 1mL of sulfuric acid titration (0.05 mol/L);
w=sample weight (unit: g)
V 0 Standard acid consumption (unit: mL) was titrated for a blank;
v = standard acid consumption (unit: mL) of sample titration;
c=protein conversion factor 6.25.
The experimental results are shown in table 3, total protein content data of amniotic membrane extract dry matter:
TABLE 3 Total protein content data for amniotic membrane extract dry matter
Experimental results show that the total protein content in the dry matter of the amniotic membrane extracts in the examples 1 and 2 is 78.02-82.79%, and the total protein content in the dry matter of the amniotic membrane extracts in the comparative examples 1-3 is 23.72-34.57%, namely, the total protein content in the dry matter of the amniotic membrane extracts obtained by the preparation process is greatly improved.
The pulverization processes of comparative examples 1 to 3 sequentially employ PBS liquid pulverization, physiological saline liquid pulverization, and liquid nitrogen freezing pulverization, whereas in examples 1 and 2 of the present invention, ice bath pulverization was employed, and the extraction efficiency of the amniotic membrane extract as an ophthalmic medical device or a pharmaceutical active ingredient (total protein in dry matter) was greatly improved by improving the technological parameters of "size using ice cubes, ice cube to sample weight ratio, pulverization time, pulverization number of times", etc.
(II) detection of total sugar content in dry matter of amniotic membrane extract
The amniotic membrane extracts of examples 1 and 2 and comparative examples 1 to 3 were taken for total protein content detection. The test method refers to the determination of the total sugar content in annex E of YY/T1453-2016 collagen characterization method of tissue engineering medical appliance product type I.
The method comprises the following specific steps:
1) Preparing a reference substance solution, namely preparing a D-anhydrous glucose reference substance stock solution: precisely weighing 10mg of D-anhydrous glucose reference substance dried to constant weight at 105 ℃, placing in a 50mL volumetric flask, adding water for dissolution, diluting to scale, and shaking uniformly to obtain reference substance solution (0.2 mg of D-anhydrous glucose is contained in each 1 mL). D-anhydrous glucose control series liquid: precisely measuring 1mL, 2mL, 3mL, 4mL and 5mL of D-anhydrous glucose control stock solution, respectively placing into plug test tubes, respectively adding water to 10mL, and shaking.
2) Preparing a sample solution, weighing 0.01g of a dried constant-weight amniotic membrane extract sample, placing the sample in a hydrolysis tube, adding 2mL of 6mol/L hydrochloric acid, placing the sample in 75 ℃ until acidolysis is completed, cooling the sample, adjusting the pH to be neutral, transferring the sample into a 25mL volumetric flask, and adding water to fix the volume to obtain the amniotic membrane extract. Once per batch and a blank was prepared.
3) Measuring, namely taking 2mL of a blank solution, 2mL of a D-anhydrous glucose control series solution and 2mL of a test solution, precisely adding 1mL of a 5% phenol solution, shaking uniformly, rapidly adding 5.0mL of concentrated sulfuric acid, shaking uniformly, keeping in a water bath at 40 ℃ for 40min, taking out, and rapidly putting into ice water for cooling for 20min. The absorbance value is measured at 490nm by using blank as a control, the absorbance is linearly regressed by using the concentration of the D-anhydrous glucose control solution series, a linear regression equation is obtained, and the mass (mg) equivalent to the D-anhydrous glucose in the sample solution is calculated.
4) Results calculation the total sugar content (in terms of D-anhydrous glucose) in the sample was calculated as follows:
total sugar content (%) in sample=，
In the method, in the process of the invention,m sa the unit is milligrams (mg) of the mass of D-anhydrous glucose in the sample solution;kis dilution multiple;mthe mass of the sample after drying at constant weight is given in grams (g).
The experimental results are shown in table 4, total sugar content data for amniotic membrane extract dry matter:
TABLE 4 Total sugar content data for dry matter of amniotic membrane extract
Experimental results show that the total sugar content in the dry materials of the amniotic membrane extracts in the examples 1 and 2 is 10.87-13.11%, and the total sugar content in the dry materials of the amniotic membrane extracts in the comparative examples 1-3 is 3.67-5.30%, namely, the total sugar content in the dry materials of the amniotic membrane extracts obtained by the preparation process is greatly improved.
The pulverization processes of comparative examples 1 to 3 sequentially employ PBS liquid pulverization, physiological saline liquid pulverization, and liquid nitrogen freezing pulverization, whereas in examples 1 and 2 of the present invention, ice bath pulverization was employed, and the extraction efficiency of the amniotic membrane extract as an ophthalmic medical device or a pharmaceutical active ingredient (total sugar in dry matter) was greatly improved by improving the technological parameters of "size using ice cubes, ice cube to sample weight ratio, pulverization time, pulverization number of times", etc.
(III) detection of fat content in amniotic membrane extract dry matter
The amniotic membrane extracts of examples 1 and 2 and comparative examples 1 to 3 were taken for fat content detection. The test method refers to an acid hydrolysis method of GB 5009.6-2016 "determination of fat in food safety national Standard food".
The method comprises the following specific steps:
1) Acidolysis, weighing 0.1g of dried constant weight sample, precisely weighing, placing in a 50mL test tube, adding 8mL of water, mixing uniformly, and adding 10mL of hydrochloric acid. And (3) placing the test tube into a water bath with the temperature of 70-80 ℃, stirring the test tube with a glass rod for 1 time every 5-10 min until the test sample is completely digested, and taking about 3 hours. Each batch was assayed in triplicate.
2) The sample tube was removed, 10mL of ethanol was added, and mixed. After cooling, the mixture was transferred to a 100ml stoppered cylinder, the test tube was washed several times with 25ml of dry diethyl ether and poured into the cylinder. After all the anhydrous diethyl ether is poured into the measuring cylinder, adding the plug, shaking for 1min, carefully opening the plug, discharging gas, well plugging, standing for 12min, carefully opening the plug, and flushing the fat attached to the plug and the measuring cylinder opening with diethyl ether. Standing for 10-20 min, sucking out supernatant liquid in a constant-weight weighing bottle, adding 5ml of anhydrous diethyl ether in a measuring cylinder with a stopper, shaking, standing, sucking out upper diethyl ether, and placing in an original weighing bottle.
3) Drying to constant weight, evaporating the weighing bottle containing the extract to dryness in water bath, and drying to constant weight at 100+ -5deg.C.
4) And (3) calculating results:
，
in the method, in the process of the invention,Xthe content of fat in the sample is expressed in grams per hundred grams (g/100 g);m 1 constant weight rear weighing bottleAnd total weight of fat in grams (g);m 0 the unit is gram (g) for weighing the mass of the bottle;m 2 the mass of the sample is given in grams (g); 100 is a conversion coefficient.
The experimental results are shown in table 5 as fat content data for dry matter of amniotic membrane extract:
TABLE 5 fat content data for dry matter of amniotic membrane extract
Experimental results show that the fat content of the dry materials of the amniotic membrane extracts in the examples 1 and 2 is 1.83-3.53%, and the fat content of the dry materials of the amniotic membrane extracts in the comparative examples 1-3 is 6.15-6.85%, namely, the fat content of the dry materials of the amniotic membrane extracts obtained by the preparation process is greatly reduced.
The purification processes of comparative examples 1 to 3 are all centrifugation to obtain supernatant, and the centrifugation and filter paper vacuum filtration processes are adopted in examples 1 and 2 of the invention, so that large-particle (> 50 μm) size impurities can be effectively filtered out by selecting filter paper with a specific gap size (pore diameter of 5-50 μm), and the content of nonfunctional impurities (fat in dry matters) is greatly reduced.
(IV) detection of the content of burning residues in the dry matter of the amniotic membrane extract
The amniotic membrane extracts of examples 1 and 2 and comparative examples 1 to 3 were taken and the content of the ignition residue was measured. Test methods refer to the residue inspection method of 0841 glowing in Chinese pharmacopoeia (fourth 2015 edition).
The method comprises the following specific steps:
1) And (3) taking the empty crucible, burning the empty crucible in a muffle furnace at 500-600 ℃ for 2 hours, taking out the empty crucible, placing the empty crucible in a dryer, cooling the empty crucible for 1 hour, weighing, and precisely weighing. And putting the mixture into the kettle for burning for 30 minutes, taking out the kettle, cooling the kettle for 1 hour, and weighing the product. And the weight of the mixture does not exceed +/-0.3 mg between the two times of weighing, otherwise, the mixture continues to burn to constant weight.
2) Sample treatment, namely weighing 0.1g of a dried constant-weight sample, placing the dried constant-weight sample into a quartz crucible (W0) which is burnt to constant weight, precisely weighing, slowly burning to complete carbonization, and cooling; adding 0.5-1 mL of sulfuric acid to moisten, heating at a low temperature until sulfuric acid vapor is removed, burning at 500-600 ℃ to completely ash, placing in a dryer, cooling for 1 hour, then putting in the dryer for burning for 30 minutes, taking out, cooling for 1 hour, and weighing. And weighing for not exceeding +/-0.3 mg to be constant weight, or continuously igniting to be constant weight.
3) And (3) calculating results:
glowing residue (%) = (W1-W0)/mx100 (%)
Wherein W0 is the total weight of the crucible before ignition, and the unit g; w1 is the total weight of residue and crucible after ignition, and the unit g; m is the mass of the sample in g.
The experimental results are shown in table 6, data on the ignition residue content of the dry matter of the amniotic membrane extract:
TABLE 6 data on the ignition residue content of amniotic membrane extract dry matter
Experimental results show that the content of the ignition residues in the dry materials of the amniotic membrane extracts in the examples 1 and 2 is 0.94-1.64%, and the content of the ignition residues in the dry materials of the amniotic membrane extracts in the comparative examples 1-3 is 3.06-3.67%, namely, the content of the ignition residues in the dry materials of the amniotic membrane extracts obtained by the preparation process is greatly reduced.
The purification processes in comparative examples 1 to 3 are all centrifugation to obtain supernatant, and the centrifugation and filter paper vacuum filtration processes are adopted in examples 1 and 2 of the invention, so that large-particle (> 50 μm) size impurities can be effectively filtered out by selecting filter paper with a specific gap size (pore diameter of 5 μm to 50 μm), and the content of nonfunctional impurities (burning residues in dry matters) is greatly reduced.
(V) test of function of promoting epithelialization of ocular surface
The ocular surface lubricating liquids of example 1 and comparative examples 1 to 4 were each taken and tested for their functions of promoting epithelialization.
The testing method comprises the following steps: the blank cell culture solution was a conventional cell culture medium, and the ocular surface lubricating solutions of example 1 and comparative examples 1 to 4 were sequentially added to the cell culture solutions of experimental groups 1 to 5, each of which had a composition of 50% of the conventional cell culture medium and 50% of the ocular surface lubricating solution. 50mL human eye surface epithelial cell suspensions (40,000/mL) were added to each well of a 96-well cell culture plate, and cultured in a cell culture incubator for 24 hours. After incubation, the samples were rinsed 3 times for 5 minutes with PBS. 200 mL complete medium containing 10% CCK-8 reagent was then added and incubated in the cell incubator for 1 hour. The absorbance of the medium was then measured at 450 nm. High absorbance indicates a high number of surviving cells.
The experimental results are shown in table 7 as absorbance data for ocular surface lubricating medium:
TABLE 7 absorbance data for ocular surface lubricating fluid media
The experimental results show that compared with the blank control group and the experimental group 5 (comparative example 4), the absorbance of the experimental group 1 (example 1) at 450 nm is significantly improved, namely the ocular surface lubricating liquid prepared in example 1 has the function of promoting the epithelization of the ocular surface. The absorbance at 450 nm was also significantly increased in experimental group 1 (example 1) compared with experimental groups 2 to 4 (comparative examples 1 to 3), indicating that the amniotic membrane extract in the ocular surface lubricating fluid prepared in example 1 has a better function of promoting ocular surface epithelization.
Therefore, the functional components in the ocular surface lubricating liquid comprise sodium hyaluronate and functional components (namely, amniotic membrane extract) for promoting tissue repair. The amniotic membrane extract contains various proteins, which are helpful for covering the ocular surface to form a protein scaffold, so that ocular surface epithelial cells of a patient can migrate onto the amniotic membrane extract to promote healing of the ocular surface epithelial cells.
Functional test of viscosity Effect of irradiation
The products before and after irradiation in example 1 and comparative example 5 were used as samples, and the viscosity values thereof were measured.
The viscosity values were determined according to the four 0633 viscometry methods of the Chinese pharmacopoeia (2020 edition). The instrument was a rotational viscometer (apparatus model: NDJ-5 SE).
The experimental results are shown in the viscosity values before and after irradiation of the samples in table 8:
TABLE 8 viscosity values before and after sample irradiation
The experimental results show that example 1 has a smaller decrease in viscosity after irradiation (from 44.1 mPas to 21.7 mPas) due to the addition of glycerol compared to comparative example 5.
The viscosity value of the large molecular weight viscosity regulating substance (such as sodium hyaluronate with molecular weight larger than 1 MDa) is reduced to less than one tenth of the initial value (from 34.4 mPas to 2.6 mPas) after irradiation sterilization (sterilization dose is 25 kGy), and the viscosity regulating substance (namely glycerin and molecular weight 92.09 Da) of the small molecular weight viscosity regulating substance is added in the formula of the eye surface lubricating liquid, so that the influence of irradiation sterilization on the viscosity is effectively reduced. The ophthalmic preparation with higher viscosity can improve the residence time of the ophthalmic preparation on the ocular surface, thereby prolonging the functional time of the ophthalmic preparation.
Test of the amount of amniotic membrane extract in the ocular surface lubricating liquid
The formula is as follows: 11% of amniotic membrane extract, 0.2% of sodium hyaluronate, 5% of glycerol, 0.4% of sodium chloride, 0.4% of boric acid, 0.015% of borax and the balance of water for injection, and is used as an eye surface lubricating fluid sample of an experimental group 1; 8% of amniotic membrane extract, 0.2% of sodium hyaluronate, 5% of glycerin, 0.4% of sodium chloride, 0.4% of boric acid, 0.015% of borax and the balance of water for injection, and is used as an eye surface lubricating fluid sample of an experimental group 2.
After the ocular surface lubricating fluid samples of the experimental groups 1 and 2 were left standing for 7 days, as shown in fig. 2, the ocular surface lubricating fluid sample shown in the left graph (experimental group 1) in fig. 2 was a clear fluid, and no floc was seen.
Based on the above test results, the amount of amniotic membrane extract in the ocular surface lubricating liquid formulation can be set to less than 10%.
Test of boric acid and borax consumption in eye surface lubricating liquid
The formula is as follows: 1% of amniotic membrane extract, 0.2% of sodium hyaluronate, 5% of glycerol, 0.4% of sodium chloride and the balance of water for injection, and the water is used as an ocular surface lubricating fluid sample of an experimental group 1; 1% of amniotic membrane extract, 0.2% of sodium hyaluronate, 5% of glycerin, 0.4% of sodium chloride, 0.2% of boric acid, 0.2% of borax and the balance of water for injection, and the water for injection is used as an eye surface lubricating fluid sample of an experimental group 2; 1% of amniotic membrane extract, 0.2% of sodium hyaluronate, 5% of glycerol, 0.4% of sodium chloride, 0.4% of boric acid and 0.2% of borax are taken as eye surface lubricating fluid samples of experimental group 2.
The pH values of the ocular surface lubricating liquid samples of experimental groups 1 to 3 were measured respectively, the pH value of experimental group 1 was 6.18, the pH value of experimental group 2 was 7.37, and the pH value of experimental group 3 was 7.00.
Based on the above test results, it was found that the pH of the eye surface lubricating liquid could be adjusted to the safe range for eye surface use (pH range was set to 6.5 to 7.8) by adding a certain amount of boric acid and borax.
Test of stability before and after irradiation sterilization
Adopting an ocular surface lubricating liquid with the same formula as a sample, and carrying out irradiation sterilization on the sample in the experimental group 1; experiment group 2 did not employ irradiation sterilization.
The samples of the experiment group 1 and the experiment group 2 are taken out after being placed in a refrigerator at the temperature of 2-8 ℃ for 1 week, and the samples shown in the left graph (the experiment group 1) in the figure 3 have no sediment, flocculent, particle and heterochromatic condition after being seen in the figure 3; the sample shown in the first (experimental group 2) from the right in fig. 3 appears to be off-colored and has a bad smell.
Based on the results, the stability of the ocular surface lubricating liquid can be obviously improved after irradiation sterilization.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (9)
1. A preparation process of an eye surface lubricating liquid is characterized in that: sequentially sterilizing fresh amniotic membrane, cleaning, removing impurities, airing, crushing in ice bath, centrifuging the crushed solution, performing suction filtration on filter paper with the aperture of 5-50 mu m to obtain an amniotic membrane extract, preparing the amniotic membrane extract into a prefabricated solution, filtering, packaging, sterilizing by irradiation to obtain an ocular surface lubricating solution,
when the ice bath is used for crushing, the dried amnion and ice cubes are added into a container, the mass ratio of the ice cubes to the amnion is controlled to be 1-5:1, then the container is kept for 5min, the obtained ice water amnion mixed solution is crushed by a crusher, the temperature of feed liquid in the crushing process is controlled to be less than 8 ℃, the crushing is carried out again at intervals of 1min after the crushing is carried out for 10-60S, the process is repeated for 30-90 times, the ice bath crushing is finished, and dry matter of the amnion extract is as follows: the total protein content is more than or equal to 78.02%, the total sugar content is more than or equal to 10.87%, the fat content is less than or equal to 3.53%, the burning residue content is less than or equal to 1.64%,
the prefabricated liquid contains the following components in percentage by mass: 8% of amniotic membrane extract, 0.1-0.4% of sodium hyaluronate, 5-10% of glycerol, 0.01-0.8% of sodium chloride, 0.2-0.6% of boric acid, 0.005-0.025% of borax and the balance of water for injection.
2. The preparation process according to claim 1, characterized in that: and in the disinfection process, 75% alcohol is sprayed on a fresh amniotic membrane packaging material for disinfection, and then ultraviolet disinfection is carried out for 30min.
3. The preparation process according to claim 1, characterized in that: when the amnion is cleaned and decontaminated, the disinfected amnion is unpacked and put into purified water for dispersion and cleaning, and residual impurities on the surface of the amnion are removed.
4. The preparation process according to claim 1, characterized in that: and in the air drying process, the cleaned and decontaminated amniotic membrane is paved on a screen for air drying for 1h, and the amniotic membrane is turned over once every 15 min.
5. The preparation process according to claim 1, characterized in that: the ice cubes are purified water ice cubes with the side length of 1-3 cm.
6. The preparation process according to claim 1, characterized in that: during centrifugation, the crushed liquid is divided into 50ml of centrifugal tubes, each tube is filled with 50ml of crushed liquid and then is put into a centrifugal machine for centrifugation for 10min, and the rotating speed of the centrifugal machine is controlled to be 1000-3000 rpm.
7. The preparation process according to claim 1, characterized in that: the suction filtration is vacuum suction filtration.
8. The preparation process according to claim 1, characterized in that: the dry matter of the amniotic membrane extract meets the following conditions: the total protein content is 78.02-82.79%, the total sugar content is 10.87-13.11%, the fat content is 1.83-3.53%, and the burning residue content is 0.94-1.64%.
9. An eye surface lubricating fluid which is characterized in that: the process according to any one of claims 1 to 8.
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|---|---|---|---|---|
| KR20010098716A (en) * | 2000-04-19 | 2001-11-08 | 김재찬 | Amnionic extract for ophthalmopathy |
| CN1712020A (en) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | Production of amniotic extractive liquid and use thereof |
| CN101658491A (en) * | 2009-09-24 | 2010-03-03 | 哈尔滨医科大学 | Amnion eye drops for curing cornea alkali burn |
| CN104825493A (en) * | 2015-05-06 | 2015-08-12 | 广州优适清生物科技有限公司 | Biological sheep membrane for ocular surface treatment and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AU2003272536A1 (en) * | 2002-09-18 | 2004-04-08 | Emiliano Ghinelli | Use of a human amniotic membrane composition for prophylaxis and treatment of diseases and conditions of the eye and skin |
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|---|---|---|---|---|
| KR20010098716A (en) * | 2000-04-19 | 2001-11-08 | 김재찬 | Amnionic extract for ophthalmopathy |
| CN1712020A (en) * | 2004-06-23 | 2005-12-28 | 中山大学中山眼科中心 | Production of amniotic extractive liquid and use thereof |
| CN101658491A (en) * | 2009-09-24 | 2010-03-03 | 哈尔滨医科大学 | Amnion eye drops for curing cornea alkali burn |
| CN104825493A (en) * | 2015-05-06 | 2015-08-12 | 广州优适清生物科技有限公司 | Biological sheep membrane for ocular surface treatment and preparation method thereof |
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