Collagen-containing composite material and application thereof in preparation of corneal injury repair material

Abstract

The invention relates to the technical field of ophthalmic material preparation, and particularly discloses a collagen-containing composite material and application thereof in preparation of a corneal injury repair material. The collagen-containing composite material is prepared from collagen, polysaccharide and a plant extract, wherein the weight ratio of the collagen to the polysaccharide to the plant extract is 100: (5-15): (1-5). Researches show that the collagen-containing composite material disclosed by the invention has the effect of resisting pseudomonas aeruginosa; therefore, the corneal injury repair material prepared from the collagen-containing composite material can be used for preventing the occurrence of pseudomonas aeruginosa keratitis after being used for corneal surgical repair; the important application value is realized.

Description

Technical Field

[0001] The invention relates to the technical field of ophthalmic material preparation, and in particular to a collagen-containing composite material and application thereof in preparing corneal damage repair material. Background Art

[0002] The cornea is a transparent fiber membrane without blood vessels located in the front 1 / 6 of the eyeball wall. Corneal injury refers to the damage to the cornea caused by external factors or body factors, including corneal interlayer tear, corneal penetration and rupture, corneal abrasion, corneal contusion, deep corneal tissue loss, etc. If the cornea is seriously injured, surgical repair is required. Corneal injury repair materials are indispensable materials in the process of corneal injury surgical repair.

[0003] Chinese invention patent CN202011482728.4 discloses a rapid epithelialization collagen-based corneal regeneration and repair material and its preparation method. The method comprises: adjusting the pH value of the collagen solution to acidic and mixing it with a sodium hyaluronate solution, stirring to prepare a blended solution, stirring to remove bubbles, pouring it into a special mold with curvature, air-drying it into a film, and obtaining a collagen hyaluronic acid composite film with curvature; demoulding, taking out the collagen film, placing it in a drying tower, cross-linking and modifying it with the glutaraldehyde gas volatilized from the glutaraldehyde solution, rinsing, and air-drying it to obtain this product. The collagen-based repair material of the present invention has good water-containing properties, biocompatibility, suture resistance, and can promote the adhesion and proliferation of corneal epithelial cells on the surface of the material, and can be used for the repair and replacement of damaged corneas in the field of biomedical materials. The inventor has found in existing research that after corneal surgery, due to the presence of wounds, it is easier to be infected by bacteria and cause keratitis. The pathogenic bacteria of keratitis include Staphylococcus, Streptococcus, and Pseudomonas aeruginosa. In particular, Pseudomonas aeruginosa keratitis is an extremely serious acute suppurative keratitis, which usually sweeps the entire cornea in a very short time and causes devastating damage, with extremely serious consequences. However, the antibacterial effect of the collagen-based corneal regeneration and repair material disclosed in the above patent needs to be further improved. Chinese invention patent CN202180021872.4 discloses a biocomposite material comprising probiotics, collagen and bacterial extracellular polysaccharides and its use. The biocomposite material contains polysaccharides with antibacterial effects. The inventors found in the study that although it has good antibacterial effects on common Staphylococci and Streptococci; However, its antibacterial effect on Pseudomonas aeruginosa is poor and needs to be further improved. Summary of the invention

[0004] In order to solve at least one of the technical problems existing in the prior art, the present invention provides a composite material containing collagen and its application in preparing corneal damage repair material.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention first provides a collagen-containing composite material, which comprises collagen, polysaccharide and plant extract; wherein the weight ratio of collagen, polysaccharide and plant extract is 100:5-15:1-5.

[0007] Preferably, the collagen-containing composite material comprises collagen, polysaccharide and plant extract; wherein the weight ratio of collagen, polysaccharide and plant extract is 100:12:3.

[0008] Preferably, the collagen is type I collagen.

[0009] Preferably, the polysaccharide is selected from dextran.

[0010] Most preferably, the glucan is α-glucan.

[0011] Preferably, the plant extract is prepared by the following method:

[0012] The Chinese medicinal herb Senecio radiata is extracted with a solvent, the extract is separated, and the extract is concentrated and dried to obtain the Senecio radiata solvent extract; the Senecio radiata solvent extract is taken to obtain the plant extract.

[0013] The inventors found in their research that adding a plant extract obtained by extracting Senecio paniculata as a raw material to a composite material containing collagen can greatly enhance the anti-Pseudomonas aeruginosa effect of the composite material containing collagen.

[0014] Preferably, the usage ratio of Senecio grandiflora to solvent is 1kg:8-15L.

[0015] Most preferably, the usage ratio of Senecio grandiflora to solvent is 1 kg:12 L.

[0016] Preferably, the obtained solvent is a solvent consisting of n-propanol and ethyl acetate.

[0017] Preferably, the volume ratio of n-propanol to ethyl acetate is 3 to 6:1.

[0018] Most preferably, the volume ratio of n-propanol to ethyl acetate is 4:1.

[0019] The inventors found in further research that in the process of preparing plant extracts using Senecio radiata as raw material, the choice of solvent is very critical; studies have shown that, in the collagen-containing composite material, adding plant extracts obtained by extraction with water or ethanol or a single n-propanol or a single ethyl acetate cannot significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite material; only by adding plant extracts obtained by extraction with a solvent composed of n-propanol and ethyl acetate can the anti-Pseudomonas aeruginosa effect of the collagen-containing composite material be significantly improved.

[0020] Preferably, the extraction is heating reflux extraction; the time of the heating reflux extraction is 1 to 3 hours.

[0021] Most preferably, the heating reflux extraction time is 1.5 h.

[0022] Preferably, the method for preparing the plant extract further comprises the following steps:

[0023] The solvent extract of Senecio radiata is placed on a macroporous resin column, and impurities are first eluted with 5 to 8 times the column volume of an ethanol aqueous solution with a volume fraction of 37 to 39%; then it is eluted with 5 to 8 times the column volume of an ethanol aqueous solution with a volume fraction of 52 to 54%, and the eluate eluted with the 52 to 54% ethanol aqueous solution is collected, concentrated and dried to obtain the plant extract.

[0024] Most preferably, the solvent extract of Senecio grandiflora is loaded onto a macroporous resin column and first eluted with 6 times the column volume of a 38% ethanol-water solution to remove impurities; then eluted with 6 times the column volume of a 53% ethanol-water solution, and the eluate eluted with the 53% ethanol-water solution is collected and concentrated and dried to obtain the plant extract.

[0025] Preferably, the macroporous resin column is a D101 macroporous resin column.

[0026] The inventors found in further research that adding a plant extract obtained by further elution through the above-mentioned macroporous resin column to the collagen-containing composite material can further significantly enhance the anti-Pseudomonas aeruginosa effect of the collagen-containing composite material; thus, the prepared collagen-containing composite material has a very excellent anti-Pseudomonas aeruginosa effect.

[0027] The inventors also found in their research that the conditions for eluting the macroporous resin of the present invention are very critical; in the collagen-containing composite material, a plant extract eluted under the above-mentioned macroporous resin column elution conditions of the present invention must be added to further significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite material; however, adding a plant extract eluted under other macroporous resin column elution conditions cannot further significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite material.

[0028] The present invention also provides an application of the above-mentioned composite material containing collagen in preparing corneal damage repair material.

[0029] Preferably, in a specific application, the above-mentioned composite material containing collagen is used as a raw material to prepare a film to obtain a composite film containing collagen, that is, the corneal damage repair material.

[0030] Beneficial effects: The present invention provides a new collagen-containing composite material; studies have shown that the collagen-containing composite material of the present invention has an anti-Pseudomonas aeruginosa effect; therefore, the collagen-containing composite material of the present invention is further used to prepare a corneal damage repair material, which can prevent the occurrence of Pseudomonas aeruginosa keratitis after corneal surgical repair; it has important application value. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1 Preparation of composite materials containing collagen

[0033] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0034] The plant extract is prepared by the following method:

[0035] The Chinese medicinal herb Senecio radiata is heated to boiling with a solvent and then extracted for 1.5 hours, followed by separation of the extract, and the extract is concentrated and dried to obtain a Senecio radiata solvent extract; the Senecio radiata solvent extract is taken to obtain the plant extract; wherein the dosage ratio of Senecio radiata to the solvent is 1kg:12L; the solvent is composed of n-propanol and ethyl acetate in a volume ratio of 4:1.

[0036] Example 2 Preparation of composite materials containing collagen

[0037] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0038] The plant extract is prepared by the following method:

[0039] (1) taking a Chinese medicinal herb, Senecio radiata, and heating it to boiling with a solvent to extract it for 1.5 hours, then separating the extract, and concentrating and drying the extract to obtain a Senecio radiata solvent extract; wherein the ratio of Senecio radiata to the solvent is 1 kg:12 L; and the solvent is composed of n-propanol and ethyl acetate in a volume ratio of 4:1;

[0040] (2) The solvent extract of Senecio radiata is placed on a D101 macroporous resin column, and impurities are first eluted with 6 times the column volume of a 38% ethanol aqueous solution; then, the impurities are eluted with 6 times the column volume of a 53% ethanol aqueous solution, and the eluate eluted with the 53% ethanol aqueous solution is collected, concentrated and dried to obtain the plant extract.

[0041] Comparative Example 1 Preparation of a composite material containing collagen

[0042] Type I collagen and α-glucan are mixed evenly at a weight ratio of 100:15 to obtain the composite material containing collagen.

[0043] Comparative Example 2 Preparation of composite materials containing collagen

[0044] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0045] The plant extract is prepared by the following method:

[0046] The Chinese medicinal herb Senecio radiata is heated to boiling with a solvent and then extracted for 1.5 hours, followed by separation of the extract, and the extract is concentrated and dried to obtain a Senecio radiata solvent extract; the Senecio radiata solvent extract is taken to obtain the plant extract; wherein the usage ratio of Senecio radiata to the solvent is 1kg:12L; and the solvent is water.

[0047] Comparative Example 3 Preparation of composite material containing collagen

[0048] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0049] The plant extract is prepared by the following method:

[0050] The Chinese medicinal herb Senecio radiata is heated to boiling with a solvent and then extracted for 1.5 hours, followed by separation of the extract, and the extract is concentrated and dried to obtain a Senecio radiata solvent extract; the Senecio radiata solvent extract is taken to obtain the plant extract; wherein the dosage ratio of Senecio radiata to the solvent is 1kg:12L; and the solvent is an ethanol aqueous solution with a volume fraction of 95%.

[0051] Comparative Example 4 Preparation of a composite material containing collagen

[0052] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0053] The plant extract is prepared by the following method:

[0054] The Chinese medicinal herb Senecio radiata is heated to boiling with a solvent and then extracted for 1.5 hours, followed by separation of the extract, and the extract is concentrated and dried to obtain a Senecio radiata solvent extract; the Senecio radiata solvent extract is taken to obtain the plant extract; wherein the dosage ratio of Senecio radiata to the solvent is 1kg:12L; and the solvent is n-propanol.

[0055] Comparative Example 5 Preparation of composite material containing collagen

[0056] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0057] The plant extract is prepared by the following method:

[0058] The Chinese medicinal herb Senecio radiata is heated to boiling with a solvent and then extracted for 1.5 hours, followed by separation of the extract, and the extract is concentrated and dried to obtain a Senecio radiata solvent extract; the Senecio radiata solvent extract is taken to obtain the plant extract; wherein the dosage ratio of Senecio radiata to the solvent is 1kg:12L; and the solvent is ethyl acetate.

[0059] Comparative Example 6 Preparation of a composite material containing collagen

[0060] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0061] The plant extract is prepared by the following method:

[0062] (1) taking a Chinese medicinal herb, Senecio radiata, and heating it to boiling with a solvent to extract it for 1.5 hours, then separating the extract, and concentrating and drying the extract to obtain a Senecio radiata solvent extract; wherein the ratio of Senecio radiata to the solvent is 1 kg:12 L; and the solvent is composed of n-propanol and ethyl acetate in a volume ratio of 4:1;

[0063] (2) The solvent extract of Senecio radiata is placed on a D101 macroporous resin column, and impurities are first eluted with 6 times the column volume of a 25% ethanol aqueous solution; then, the impurities are eluted with 6 times the column volume of a 40% ethanol aqueous solution, and the eluate eluted with the 40% ethanol aqueous solution is collected, concentrated and dried to obtain the plant extract.

[0064] Comparative Example 7 Preparation of a composite material containing collagen

[0065] Type I collagen, α-glucan and plant extract are mixed evenly in a weight ratio of 100:12:3 to obtain the collagen-containing composite material;

[0066] The plant extract is prepared by the following method:

[0067] (1) taking a Chinese medicinal herb, Senecio radiata, and heating it to boiling with a solvent to extract it for 1.5 hours, then separating the extract, and concentrating and drying the extract to obtain a Senecio radiata solvent extract; wherein the ratio of Senecio radiata to the solvent is 1 kg:12 L; and the solvent is composed of n-propanol and ethyl acetate in a volume ratio of 4:1;

[0068] (2) The solvent extract of Senecio radiata is placed on a D101 macroporous resin column, and impurities are first eluted with 6 times the column volume of a 50% ethanol aqueous solution; then, the impurities are eluted with 6 times the column volume of a 65% ethanol aqueous solution, and the eluate eluted with the 65% ethanol aqueous solution is collected, concentrated and dried to obtain the plant extract.

[0069] Experimental Example 1 Anti-Pseudomonas aeruginosa Experiment

[0070] Take the collagen-containing composite materials prepared in Examples 1 and 2 and Comparative Examples 1 to 7, add 8 times the weight of 0.01 mol / L hydrochloric acid aqueous solution and mix evenly; then take 50 mL of each and evenly apply it on a plastic plate with a diameter of 10 cm, and after natural drying, obtain a collagen-containing composite film with a diameter of 5 cm.

[0071] The collagen-containing composite film prepared according to the above method was spread in a culture dish, and then the culture medium containing the same number of live Pseudomonas aeruginosa colonies was coated on the collagen-containing composite film, and then it was placed in an incubator and cultured for 24 hours; after the culture was completed, the number of Pseudomonas aeruginosa colonies in each culture dish was calculated; the inhibition rate was calculated based on the number of colonies; wherein, the inhibition rate = ((original number of colonies - number of colonies after 24 hours) / original number of colonies) x 100%; the results are shown in Table 1.

[0072] Table 1. Anti-Pseudomonas aeruginosa test results

[0073] Material to be tested Pseudomonas aeruginosa inhibition rate Example 1: The collagen-containing composite material prepared 65.4％ Example 2: Composite material containing collagen 99.9％ Comparative Example 1: Composite material containing collagen 36.8％ Comparative Example 2: Collagen-containing composite material prepared 40.2％ Comparative Example 3: Collagen-containing composite material prepared 42.1％ Comparative Example 4: Composite material containing collagen 44.9％ Comparative Example 5: Collagen-containing composite material prepared 48.3％ Comparative Example 6: Composite material containing collagen 72.1％ Comparative Example 7: Collagen-containing composite material prepared 76.8％

[0074] From the experimental results in Table 1, it can be seen that the collagen-containing composite material prepared in Comparative Example 1 has an antibacterial rate of only 36.8% against Pseudomonas aeruginosa; this indicates that the collagen-containing composite material obtained by combining type I collagen and α-glucan has a poor antibacterial effect against Pseudomonas aeruginosa.

[0075] It can be seen from the experimental results in Table 1 that the antibacterial rate of the collagen-containing composite material prepared in Example 1 against Pseudomonas aeruginosa is significantly higher than that of the collagen-containing composite material prepared in Comparative Example 1; this indicates that adding a plant extract obtained by extracting Senecio paniculata as a raw material to the collagen-containing composite material can significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite material.

[0076] It can be seen from the experimental results in Table 1 that the antibacterial rate of the collagen-containing composite materials prepared in Comparative Examples 2 to 5 against Pseudomonas aeruginosa is improved compared with that of the collagen-containing composite materials prepared in Comparative Example 1, but the improvement is not large; this shows that: in the process of preparing plant extracts using Senecio radiata as raw material, the selection of solvent is very critical; studies have shown that, in the collagen-containing composite materials, adding plant extracts obtained by extraction with water or ethanol or single n-propanol or single ethyl acetate does not significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite materials; only by adding plant extracts obtained by extraction with a solvent composed of n-propanol and ethyl acetate can the anti-Pseudomonas aeruginosa effect of the collagen-containing composite materials be significantly improved.

[0077] It can be seen from the experimental results in Table 1 that the collagen-containing composite material prepared in Example 2 has an antibacterial rate of 99.9% against Pseudomonas aeruginosa, which is significantly higher than the collagen-containing composite material prepared in Example 1; it has a very excellent anti-Pseudomonas aeruginosa effect; this means that: in the collagen-containing composite material, adding the plant extract obtained by further elution through the above-mentioned macroporous resin column can further significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite material; so that the prepared collagen-containing composite material has a very excellent anti-Pseudomonas aeruginosa effect.

[0078] It can be seen from the experimental results in Table 1 that the antibacterial rate of the collagen-containing composite materials prepared in Comparative Examples 6 and 7 against Pseudomonas aeruginosa is not much improved compared with the collagen-containing composite materials prepared in Example 1; this shows that: the conditions for elution of the macroporous resin of the present invention are very critical; in the collagen-containing composite materials, the plant extract eluted under the above-mentioned macroporous resin column elution conditions of the present invention must be added to further significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite materials; however, the addition of plant extracts eluted under other macroporous resin column elution conditions cannot further significantly improve the anti-Pseudomonas aeruginosa effect of the collagen-containing composite materials.

Claims

1. A composite material containing collagen, characterized in that: It comprises collagen, polysaccharide and plant extract; wherein the weight ratio of collagen, polysaccharide and plant extract is 100:5-15:1-5.

2. The collagen-containing composite material according to claim 1, characterized in that It comprises collagen, polysaccharide and plant extract; wherein the weight ratio of collagen, polysaccharide and plant extract is 100:12:

3.

3. The collagen-containing composite material according to claim 1, characterized in that The collagen is type I collagen.

4. The collagen-containing composite material according to claim 1, characterized in that The polysaccharide is selected from dextran.

5. The collagen-containing composite material according to claim 1, characterized in that The plant extract is prepared by the following method: The Chinese medicinal herb Senecio radiata is extracted with a solvent, the extract is separated, and the extract is concentrated and dried to obtain the Senecio radiata solvent extract; the Senecio radiata solvent extract is taken to obtain the plant extract.

6. The collagen-containing composite material according to claim 1, characterized in that The usage ratio of Senecio radiata to solvent is 1kg:8~15L. Most preferably, the usage ratio of Senecio grandiflora to solvent is 1 kg:12 L.

7. The collagen-containing composite material according to claim 1, characterized in that: The obtained solvent used is a solvent consisting of n-propanol and ethyl acetate. Preferably, the volume ratio of n-propanol to ethyl acetate is 3 to 6:

1. Most preferably, the volume ratio of n-propanol to ethyl acetate is 4:

1.

8. The collagen-containing composite material according to claim 1, characterized in that The extraction is heating reflux extraction; the time of the heating reflux extraction is 1 to 3 hours. Most preferably, the heating reflux extraction time is 1.5 h.

9. Use of the collagen-containing composite material according to any one of claims 1 to 8 in the preparation of corneal damage repair materials.

10. The use according to claim 9, characterized in that: The corneal damage repair material is obtained by using the collagen-containing composite material as claimed in any one of claims 1 to 8 as a raw material to form a film to obtain a collagen-containing composite film.