A dry biological amniotic membrane material and its preparation method and application as an ophthalmic material

Abstract

The present invention discloses a dry biological amniotic membrane material, a preparation method thereof, and an application thereof as an ophthalmic material. The preparation method of the dry biological amniotic membrane material comprises the following steps: (1) taking a placenta, separating the amniotic membrane from the placenta, and then removing the excess tissue on the surface of the amniotic membrane with a cell scraper; (2) washing the amniotic membrane treated in step (1) to obtain the washed amniotic membrane; (3) placing the washed amniotic membrane in a solution of a substance that promotes tissue regeneration for treatment; (4) drying, shearing, and irradiating and sterilizing the amniotic membrane treated in step (3) to obtain the dry biological amniotic membrane material. Studies have shown that the dry biological amniotic membrane material prepared by the method of the present invention not only has the effect of promoting tissue regeneration, but also has an antibacterial effect, and has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of ophthalmic material preparation, and in particular to a dry biological amniotic membrane material and a preparation method thereof and application thereof as an ophthalmic material. Background Art

[0002] The amniotic membrane is a thin membrane composed of a single layer of epithelial cells connected to each other. It is the innermost layer of the fetal membrane and is a translucent membrane that is connected to the amniotic membrane layer covering the placenta and umbilical cord. In ophthalmology clinics, the amniotic membrane can be used as a biomaterial to treat ocular surface injuries and has important application value.

[0003] For example, Chinese invention patent CN202310187395.X discloses a preparation method and application of dry amniotic membrane, the preparation steps of which include: toughening treatment, thickening and hydrophilic treatment, tissue regeneration promotion treatment and drying treatment, etc.; the dry amniotic membrane prepared by this method has enhanced suture strength, which is convenient for surgical suture; increased thickness and hydrophilicity, which is convenient for fitting to the wound surface during surgery; stronger tissue regeneration promotion performance; maintaining the flexibility of natural wet amniotic membrane and the activity of growth factors and collagen in the amniotic membrane.

[0004] However, the inventors found in their research that the dry amniotic membrane does not have antibacterial properties. In ophthalmic treatment, improper operation can easily cause bacterial infection of the eye. This problem needs to be further solved. Summary of the invention

[0005] In order to solve at least one of the technical problems existing in the prior art, the present invention provides a dry biological amniotic membrane material and a preparation method thereof and an application thereof as an ophthalmic material.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention provides a method for preparing a dry biological amniotic membrane material, which comprises the following steps:

[0008] (1) taking the placenta, separating the amniotic membrane from the placenta, and then using a cell scraper to remove excess tissue on the surface of the amniotic membrane;

[0009] (2) washing the amniotic membrane treated in step (1) to obtain a washed amniotic membrane;

[0010] (3) placing the cleaned amniotic membrane into a solution of a substance that promotes tissue regeneration for treatment;

[0011] (4) Drying, shearing and irradiation sterilizing the amniotic membrane treated in step (3) to obtain the dry biological amniotic membrane material.

[0012] The present invention provides a novel method for preparing dry biological amniotic membrane material; the method can make the prepared dry biological amniotic membrane material have the effect of promoting tissue regeneration by placing the cleaned amniotic membrane into a solution of a substance promoting tissue regeneration prepared by a novel method, and can also make the prepared dry biological amniotic membrane material have an antibacterial effect.

[0013] Preferably, the placenta described in step (1) refers to an ex vivo human placenta.

[0014] Preferably, the placenta described in step (1) is screened for pathogens before separation of the amniotic membrane.

[0015] Preferably, the cleaning in step (2) specifically refers to cleaning with physiological saline.

[0016] Preferably, the drying in step (3) specifically refers to freeze-drying or air-drying.

[0017] Preferably, the tissue regeneration promoting substance solution specifically refers to an aqueous solution with a mass concentration of the tissue regeneration promoting substance of 0.5 to 5%;

[0018] Preferably, the substance promoting tissue regeneration is prepared by the following method:

[0019] Ginseng and coptis chinensis are ground and then extracted with an organic solvent. After the extraction, the extract is taken and concentrated and dried to obtain a ginseng-coptis chinensis organic solvent extract;

[0020] The substance promoting tissue regeneration can be obtained by taking the organic solvent extract of ginseng and coptis root.

[0021] Preferably, the organic solvent is acetone.

[0022] Preferably, the weight ratio of the Chinese medicinal herbs ginseng and coptis root is 1:2-4.

[0023] Most preferably, the weight ratio of the Chinese medicinal herbs ginseng and coptis root is 1:3.

[0024] Preferably, the ratio of the total weight of the Chinese medicinal materials ginseng and coptis chinensis to the amount of the organic solvent is 1 kg:5-10 L.

[0025] Most preferably, the ratio of the total weight of the Chinese medicinal herbs ginseng and coptis chinensis to the amount of the organic solvent is 1 kg:8 L.

[0026] Preferably, the method for preparing the substance for promoting tissue regeneration further comprises the following steps:

[0027] Take the ginseng-coptis organic solvent extract and apply it to a macroporous resin column; first use a 6-8% volume fraction ethanol aqueous solution to elute and remove impurities; then use a 22-24% volume fraction ethanol aqueous solution to elute, collect the eluate eluted with the 22-24% volume fraction ethanol aqueous solution, and concentrate and dry to obtain the tissue regeneration promoting substance.

[0028] Most preferably, the method for preparing the substance for promoting tissue regeneration further comprises the following steps:

[0029] Take the ginseng-coptis organic solvent extract and apply it to a macroporous resin column; first use a 7% by volume ethanol aqueous solution to elute and remove impurities; then use a 23% by volume ethanol aqueous solution to elute, collect the eluate eluted with the 23% by volume ethanol aqueous solution, and concentrate and dry to obtain the tissue regeneration promoting substance.

[0030] The inventors found in their research that the preparation method of the tissue regeneration promoting substance of the present invention is very critical; in the preparation method of the present invention, the tissue regeneration promoting substance is prepared by further subjecting the ginseng-coptis organic solvent extract to the above-mentioned macroporous resin elution conditions of the present invention. Compared with using the ginseng-coptis organic solvent extract as the tissue regeneration promoting substance, the tissue regeneration promoting effect of the prepared dry biological amniotic membrane material can be greatly improved.

[0031] In addition, the inventors also found in the research that the macroporous resin elution conditions of the present invention are also very critical; it is necessary to use the tissue regeneration promoting substance prepared by the ginseng-coptis organic solvent extract through the macroporous resin elution conditions of the present invention, which can further significantly improve the tissue regeneration promoting effect of the dry biological amniotic membrane material compared to using the ginseng-coptis organic solvent extract as the tissue regeneration promoting substance; however, the tissue regeneration promoting substance prepared by the ginseng-coptis organic solvent extract through other macroporous resin elution conditions, compared to using the ginseng-coptis organic solvent extract as the tissue regeneration promoting substance, cannot further significantly improve the tissue regeneration promoting effect of the dry biological amniotic membrane material.

[0032] The present invention also provides a dry biological amniotic membrane material prepared by the above preparation method.

[0033] The present invention also provides a use of the dry biological amniotic membrane material as an ophthalmic material.

[0034] Beneficial effects: The present invention provides a novel method for preparing dry biological amniotic membrane material; the method not only enables the prepared dry biological amniotic membrane material to have the effect of promoting tissue regeneration, but also enables the dry biological amniotic membrane material to have antibacterial effect, and has important application value, by placing the cleaned amniotic membrane into a solution of a substance promoting tissue regeneration prepared by a novel method. DETAILED DESCRIPTION

[0035] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the protection scope of the present invention.

[0036] Example 1 Preparation method of dry biological amniotic membrane material

[0037] (1) obtaining an isolated human placenta after pathogen screening, separating the amniotic membrane from the placenta, and then removing excess tissue on the surface of the amniotic membrane with a cell scraper;

[0038] (2) washing the amniotic membrane treated in step (1) with physiological saline to obtain a washed amniotic membrane;

[0039] (3) Soaking the cleaned amniotic membrane in a solution of substances that promote tissue regeneration for 8 hours;

[0040] (4) freeze-drying, shearing and irradiating the amniotic membrane treated in step (3) to obtain the dry biological amniotic membrane material;

[0041] The tissue regeneration promoting substance solution specifically refers to an aqueous solution with a mass concentration of 1% of the tissue regeneration promoting substance;

[0042] The tissue regeneration promoting substance is prepared by the following method:

[0043] Ginseng and coptis chinensis are ground and then refluxed with an organic solvent at 56°C for 1 hour. After the extraction, the extract is concentrated and dried to obtain a ginseng-coptis chinensis organic solvent extract; wherein the weight ratio of ginseng and coptis chinensis is 1:3; and the total weight ratio of ginseng and coptis chinensis to the organic solvent is 1kg:8L;

[0044] The substance promoting tissue regeneration can be obtained by taking the organic solvent extract of ginseng and coptis root.

[0045] Example 2 Preparation method of dry biological amniotic membrane material

[0046] (1) obtaining an isolated human placenta after pathogen screening, separating the amniotic membrane from the placenta, and then removing excess tissue on the surface of the amniotic membrane with a cell scraper;

[0047] (2) washing the amniotic membrane treated in step (1) with physiological saline to obtain a washed amniotic membrane;

[0048] (3) Soaking the cleaned amniotic membrane in a solution of substances that promote tissue regeneration for 8 hours;

[0049] (4) freeze-drying, shearing and irradiating the amniotic membrane treated in step (3) to obtain the dry biological amniotic membrane material;

[0050] The tissue regeneration promoting substance solution specifically refers to an aqueous solution with a mass concentration of 1% of the tissue regeneration promoting substance;

[0051] The tissue regeneration promoting substance is prepared by the following method:

[0052] (1) Ginseng and coptis chinensis are ground and then refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is collected and concentrated and dried to obtain a ginseng-coptis chinensis organic solvent extract; wherein the weight ratio of ginseng and coptis chinensis is 1:3; and the total weight ratio of ginseng and coptis chinensis to the organic solvent is 1 kg:8 L;

[0053] (2) Take the ginseng-coptis chinensis organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 7% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 23% ethanol aqueous solution to elute, collect the eluate eluted with 23% ethanol aqueous solution, and concentrate and dry to obtain the tissue regeneration promoting substance.

[0054] Comparative Example 1 Preparation method of dry biological amniotic membrane material

[0055] (1) obtaining an isolated human placenta after pathogen screening, separating the amniotic membrane from the placenta, and then removing excess tissue on the surface of the amniotic membrane with a cell scraper;

[0056] (2) washing the amniotic membrane treated in step (1) with physiological saline to obtain a washed amniotic membrane;

[0057] (3) Soaking the cleaned amniotic membrane in a solution of substances that promote tissue regeneration for 8 hours;

[0058] (4) freeze-drying, shearing and irradiating the amniotic membrane treated in step (3) to obtain the dry biological amniotic membrane material;

[0059] The tissue regeneration promoting substance solution specifically refers to an aqueous solution with a mass concentration of 1% of the tissue regeneration promoting substance;

[0060] The tissue regeneration promoting substance is prepared by the following method:

[0061] (1) Ginseng and coptis chinensis are ground and then refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is collected and concentrated and dried to obtain a ginseng-coptis chinensis organic solvent extract; wherein the weight ratio of ginseng and coptis chinensis is 1:3; and the total weight ratio of ginseng and coptis chinensis to the organic solvent is 1 kg:8 L;

[0062] (2) Take the ginseng-coptis chinensis organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 15% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 30% ethanol aqueous solution to elute, collect the eluate eluted with 30% ethanol aqueous solution, and concentrate and dry to obtain the tissue regeneration promoting substance.

[0063] Comparative Example 2 Preparation method of dry biological amniotic membrane material

[0064] (1) obtaining an isolated human placenta after pathogen screening, separating the amniotic membrane from the placenta, and then removing excess tissue on the surface of the amniotic membrane with a cell scraper;

[0065] (2) washing the amniotic membrane treated in step (1) with physiological saline to obtain a washed amniotic membrane;

[0066] (3) Soaking the cleaned amniotic membrane in a solution of substances that promote tissue regeneration for 8 hours;

[0067] (4) freeze-drying, shearing and irradiating the amniotic membrane treated in step (3) to obtain the dry biological amniotic membrane material;

[0068] The tissue regeneration promoting substance solution specifically refers to an aqueous solution with a mass concentration of 1% of the tissue regeneration promoting substance;

[0069] The tissue regeneration promoting substance is prepared by the following method:

[0070] (1) Ginseng and coptis chinensis are ground and then refluxed with an organic solvent at 56° C. for 1 hour. After the extraction, the extract is collected and concentrated and dried to obtain a ginseng-coptis chinensis organic solvent extract; wherein the weight ratio of ginseng and coptis chinensis is 1:3; and the total weight ratio of ginseng and coptis chinensis to the organic solvent is 1 kg:8 L;

[0071] (2) Take the ginseng-coptis chinensis organic solvent extract and apply it to an HP-20 macroporous resin column; first use 5 times the column volume of 30% ethanol aqueous solution to elute and remove impurities; then use 5 times the column volume of 60% ethanol aqueous solution to elute, collect the eluate eluted with 60% ethanol aqueous solution, and concentrate and dry to obtain the tissue regeneration promoting substance.

[0072] The antibacterial rates of the dry biological amnion materials prepared in Examples 1 to 2 and Comparative Examples 1 to 2 against Staphylococcus aureus and Escherichia coli are shown in Table 1; the proliferation rates of the dry biological amnion materials prepared in Examples 1 to 2 and Comparative Examples 1 to 2 in promoting epidermal cell proliferation are shown in Table 1.

[0073] Table 1. Antibacterial effect of dry bio-amniotic membrane materials

[0074]

[0075] It can be seen from the antibacterial experimental data in Table 1 that the dry biological amniotic membrane materials prepared in Examples 1 and 2 have good antibacterial effects; this shows that the present invention can make the prepared dry biological amniotic membrane material have antibacterial effects by placing the cleaned amniotic membrane into a solution of a tissue regeneration promoting substance prepared by a new method.

[0076] Table 2. The proliferation rate of epidermal cells promoted by dry biological amniotic membrane materials

[0077]

[0078] It can be seen from the experimental results in Table 2 that the dry biological amniotic membrane material prepared in Example 1 promotes the proliferation rate of epidermal cells by 180%; this shows that: the present invention can make the prepared dry biological amniotic membrane material have the effect of promoting tissue regeneration by placing the cleaned amniotic membrane into a solution of a tissue regeneration promoting substance prepared by a new method.

[0079] It can be seen from the experimental results in Table 2 that the dry biological amniotic membrane material prepared in Example 2 promotes the proliferation rate of epidermal cells by 310%, which is significantly higher than that prepared in Example 1; this shows that: the preparation method of the tissue regeneration promoting substance of the present invention is very critical; in the preparation method of the present invention, the tissue regeneration promoting substance is prepared by further subjecting the ginseng-coptis organic solvent extract to the above-mentioned macroporous resin elution conditions of the present invention. Compared with using the ginseng-coptis organic solvent extract as the tissue regeneration promoting substance, the tissue regeneration promoting effect of the prepared dry biological amniotic membrane material can be greatly improved.

[0080] It can be seen from the experimental results in Table 2 that the dry biological amniotic membrane material prepared in Comparative Example 1 has an improved proliferation rate of epidermal cells compared with that in Example 1, but the improvement is not large; the dry biological amniotic membrane material prepared in Comparative Example 2 has not only not improved the proliferation rate of epidermal cells compared with that in Example 1, but has decreased; this shows that: the macroporous resin elution conditions of the present invention are also very critical; it is necessary to use the tissue regeneration promoting substance prepared by the ginseng-coptis organic solvent extract through the macroporous resin elution conditions of the present invention, which can further significantly improve the tissue regeneration promoting effect of the dry biological amniotic membrane material compared with the use of the ginseng-coptis organic solvent extract as the tissue regeneration promoting substance; however, the tissue regeneration promoting substance prepared by the ginseng-coptis organic solvent extract through other macroporous resin elution conditions, compared with the use of the ginseng-coptis organic solvent extract as the tissue regeneration promoting substance, cannot further significantly improve the tissue regeneration promoting effect of the dry biological amniotic membrane material.

Claims

1. A method for preparing a dry biological amniotic membrane material, characterized in that: The following steps are included: (1) Take the placenta, separate the amniotic membrane from the placenta, and then use a cell scraper to remove excess tissue on the surface of the amniotic membrane; (2) washing the amniotic membrane treated in step (1) to obtain washed amniotic membrane; (3) placing the cleaned amniotic membrane into a solution containing a substance that promotes tissue regeneration for treatment; (4) drying, shearing and irradiating the amniotic membrane treated in step (3) to obtain the dry biological amniotic membrane material; The tissue regeneration promoting substance solution specifically refers to an aqueous solution with a mass concentration of 0.5-5% of the tissue regeneration promoting substance; The tissue regeneration promoting substance is prepared by the following method: Ginseng and coptis chinensis are ground and then extracted with an organic solvent. After the extraction, the extract is taken and concentrated and dried to obtain a ginseng-coptis chinensis organic solvent extract; The substance promoting tissue regeneration can be obtained by taking the organic solvent extract of ginseng and coptis root.

2. The method for preparing a dry biological amniotic membrane material according to claim 1, characterized in that: The placenta described in step (1) refers to an isolated human placenta.

3. The method for preparing a dry biological amniotic membrane material according to claim 1, characterized in that: The placenta described in step (1) is screened for pathogens before separation of the amniotic membrane.

4. The method for preparing a dry biological amniotic membrane material according to claim 1, characterized in that: step The cleaning in step (2) specifically refers to cleaning with physiological saline.

5. The method for preparing a dry biological amniotic membrane material according to claim 1, characterized in that: step The drying in step (3) specifically refers to freeze-drying or air-drying.

6. The method for preparing a dry biological amniotic membrane material according to claim 1, characterized in that: The organic solvent is acetone.

7. The dry biological amniotic membrane material prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the dry biological amniotic membrane material according to claim 7 as an ophthalmic material.

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