Collagen-modified superoxide dismutase and preparation method and application thereof

Abstract

The application provides a collagen-modified superoxide dismutase as well as a preparation method and application thereof. The preparation method is as follows: collagen is dissolved in water, superoxide dismutase, EDC and NHC are sequentially added into the water, and stirring is carried out under water bath preservation, so that a flowable gel sample is obtained; the obtained gel sample is diluted with water and centrifuged, and a lower viscous liquid is collected; the obtained viscous liquid after centrifugation is added into a PBS buffer, and dialysis is carried out to obtain the collagen-modified superoxide dismutase. Trehalose and mannitol are added into the collagen-modified superoxide dismutase, stirring is uniformly carried out, the obtained mixture is poured into a mold for freeze-drying, extrusion and flattening are carried out, and a repair patch is obtained. The cross-linked modified superoxide dismutase obtained by the application has good stability, the obtained repair patch is convenient to use, promotes wound healing, and has wide application prospect and practical significance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a collagen-modified superoxide dismutase, its preparation method, and its application. Background Technology

[0002] Oral ulcers primarily occur locally on the oral mucosa and are characterized by self-limitation, periodicity, and recurrence. They are characterized by superficial ulcers, approximately the size of a grain of rice and roughly round or oval in shape, appearing on the mucosa of the gums, tongue, cheeks, palate, and lips. The number and size of the ulcers vary, with regular edges and uneven distribution, exhibiting the clinical characteristics of being yellow, red, concave, and painful. Although they have a certain degree of self-limitation, they are accompanied by significant pain and systemic symptoms such as low-grade fever, swollen lymph nodes, and headache. They severely affect daily activities such as eating, speaking, and sleeping, causing great physical and psychological suffering to patients. Currently, the etiology and pathogenesis of oral ulcers are not fully understood, and there is no ideal cure. However, active and effective interventions can help accelerate ulcer healing, reduce pain, and decrease recurrence. Treatment often combines local anti-inflammatory measures with systemic therapy, but the therapeutic effect is not significant. Currently, most clinical medications for treating oral ulcers are in the form of pills, capsules, external powders, or pastes. These dosage forms have low effective concentrations in mucosal tissues or are difficult to maintain, and are easily affected by saliva, making them unable to accumulate at the lesion site.

[0003] Superoxide dismutase (SOD), a protease widely found in organisms, is a major antioxidant enzyme in the human body, responsible for scavenging free radicals and maintaining oxidative balance. SOD converts harmful superoxide free radicals into hydrogen peroxide, which is then broken down into water by catalase (CAT) and peroxidase (POD). SOD is considered a powerful enzyme and a natural scavenger of free radicals. Furthermore, SOD effectively removes excess free radicals from ulcer surfaces, thus alleviating and healing ulcers. However, due to its high extraction cost, limited availability, large molecular weight, easy inactivation, and poor absorption, SOD production and application remain limited. With technological advancements, artificial synthesis of SOD using genetic engineering has become the primary production method. Compared to natural SOD, artificially synthesized SOD is less expensive, has a smaller molecular weight, is more stable, and exhibits higher activity; however, the activity of liquid SOD gradually decreases over time.

[0004] Therefore, it is of great significance to find long-acting ingredients and use appropriate dosage forms to obtain effective drugs to improve oral ulcer disease. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a collagen-modified superoxide dismutase, its preparation method and application.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] Technical Topic 1:

[0008] A collagen-modified superoxide dismutase is prepared as follows:

[0009] S1: Dissolve collagen in water to prepare a collagen solution, add superoxide dismutase, EDC and NHC in sequence, stir under water bath temperature to crosslink it, and obtain a flowable gel sample.

[0010] S2: Dilute the gel sample obtained in S1 with water and centrifuge, then collect the lower viscous liquid layer;

[0011] S3: Add the viscous liquid obtained after centrifugation in S2 to PBS buffer to make a mixture. Place the mixture in a dialysis bag for dialysis and collect the cross-linked liquid in the bag to obtain collagen-modified superoxide dismutase.

[0012] The collagen is selected from one or more of recombinant type I, II, IV, III and XVII humanized collagen.

[0013] As a further embodiment of the present invention, the concentration of the collagen solution in S1 is 50~100mg / mL.

[0014] As a further embodiment of the present invention, the final enzyme activity of the superoxide dismutase in S1 is 400~1000U / mL.

[0015] As a further embodiment of the present invention, the molar ratio of EDC to NHS in S1 is 1~3:1; the total content of EDC and NHS is 60~100mmol / L.

[0016] As a further embodiment of the present invention, the water bath temperature in S1 is 30~40℃; the stirring speed is 700~1500r / min; and the stirring time is 10~30min.

[0017] As a further embodiment of the present invention, the volume of water added in S2 is 8 to 12 times the volume of the gel sample; the centrifugation speed is 8000 to 12000 r / min.

[0018] As a further embodiment of the present invention, the volume of the PBS buffer in S3 is 8 to 12 times the volume of the viscous liquid.

[0019] Technical Topic Two:

[0020] A method for preparing collagen-modified superoxide dismutase as described in Technical Subject 1.

[0021] Technical Topic 3:

[0022] Application of a collagen-modified superoxide dismutase as described in Technical Subject 1 in the preparation of oral ulcer repair drugs.

[0023] As a further embodiment of the present invention, the application method is as follows: trehalose and mannitol are added to the obtained collagen-superoxide dismutase, stirred and mixed, then poured into a mold for freeze drying to obtain a sample. The freeze-dried sample is then pressed flat to obtain a repair patch. The mass of trehalose added is 5%~10% of the mass of the collagen-modified superoxide dismutase; the mass of mannitol added is 5%~10% of the mass of the collagen-modified superoxide dismutase.

[0024] The beneficial effects of adopting the above technical solution are as follows:

[0025] 1. The addition of collagen effectively improves the SOD enzyme activity time. After cross-linking, collagen binds to SOD, which can stabilize the SOD molecular conformation and prolong the SOD enzyme activity time.

[0026] 2. Collagen and SOD work together to not only better utilize the effects of exogenous SOD, but also promote the body to produce more SOD.

[0027] 3. The oral repair patch provided by this invention can prevent saliva from dissolving the drug and ensure one-way drug release. In addition, the patch can not only protect the ulcer surface, but also allow the drug to act directly on the ulcer surface, prolong the retention time, increase the effective concentration of local active substances, and promote wound healing. More importantly, it is convenient to use, requires fewer uses, and is more adaptable to patients. Attached Figure Description

[0028] Figure 1 This is the recombinant collagen-SOD composite sample obtained in Example 16 of the present invention.

[0029] Figure 2 This is the recombinant collagen-SOD composite sample obtained in Example 17 of the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0031] EDC was purchased from Beyotime, product number ST1299-5g, molecular weight 191.7;

[0032] NHS purchased it from Thermo Fisher Scientific, product number 24500, with a molecular weight of 115.09;

[0033] The superoxide dismutase (SOD) activity is 10000 U / mg.

[0034] Example 1

[0035] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0036] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0037] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0038] Example 2

[0039] S1: Dissolve 5g of recombinant type II humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0040] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0041] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0042] Example 3

[0043] S1: Dissolve 5g of recombinant type III humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink and obtain a flowable gel sample.

[0044] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0045] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0046] Example 4

[0047] S1: Dissolve 5g of recombinant type IV humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0048] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0049] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0050] Example 5

[0051] S1: Dissolve 5g of recombinant type XVII humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0052] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0053] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0054] Example 6

[0055] S1: Dissolve 10g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 100mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0056] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0057] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0058] Example 7

[0059] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 4mg of superoxide dismutase to make the final SOD activity 400U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0060] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0061] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0062] Example 8

[0063] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 10mg of superoxide dismutase to make the final SOD activity 1000U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0064] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0065] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0066] Example 9

[0067] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 0.865g of EDC and 0.173g of NHC to make the final concentration of EDC 45mmol / L and the final concentration of NHC 15mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0068] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0069] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0070] Example 10

[0071] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.435g of EDC and 0.287g of NHC to make the final concentration of EDC 75mmol / L and the final concentration of NHC 25mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0072] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0073] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0074] Example 11

[0075] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD enzyme activity 500U / mL. Then add 0.765g of EDC and 0.460g of NHC to make the final concentration of EDC 40mmol / L and the final concentration of NHC 40mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0076] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0077] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0078] Example 12

[0079] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD enzyme activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 30℃ water bath and stir at 1500r / min for 10 minutes to crosslink the sample and obtain a flowable gel sample.

[0080] S2: Dilute the gel sample obtained in S1 with 10 times its volume of water, centrifuge at 8000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0081] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0082] Example 13

[0083] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD enzyme activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 40℃ water bath and stir at 700r / min for 30 minutes to crosslink and obtain a flowable gel sample.

[0084] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 12000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0085] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0086] Example 14

[0087] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0088] S2: Dilute the gel sample obtained in S1 with 8 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0089] S3: Add 8 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0090] Example 15

[0091] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0092] S2: Dilute the gel sample obtained in S1 with 12 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0093] S3: Add 12 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0094] Comparative Example 1

[0095] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to the solution to make the final SOD enzyme activity 500U / mL. Keep the solution in a 37℃ water bath and stir at 1100r / min for 20 minutes to obtain a watery sample with high fluidity.

[0096] S2: Dilute the sample obtained in S1 with 10 times its volume of water, centrifuge at 10000 r / min, and repeat the dilution and centrifugation 3 times;

[0097] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, dialyze for 60 hours, change the dialysate every 12 hours, and collect the processed liquid in the bag.

[0098] Comparative Example 2

[0099] S1: Dissolve 3g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 30mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0100] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0101] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0102] Comparative Example 3

[0103] S1: Dissolve 15g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 150mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0104] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0105] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0106] Comparative Example 4

[0107] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 1mg of superoxide dismutase to make the final SOD enzyme activity 100U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0108] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0109] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0110] Comparative Example 5

[0111] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 16mg of superoxide dismutase to make the final SOD activity 1600U / mL. Then add 1.15g of EDC and 0.23g of NHC to make the final concentration of EDC 60mmol / L and the final concentration of NHC 20mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink and obtain a flowable gel sample.

[0112] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0113] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0114] Comparative Example 6

[0115] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD activity 500U / mL. Then add 0.575g of EDC and 0.115g of NHC to make the final concentration of EDC 30mmol / L and the final concentration of NHC 10mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink the sample and obtain a flowable gel sample.

[0116] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0117] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0118] Comparative Example 7

[0119] S1: Dissolve 5g of recombinant type I humanized collagen in 100mL of water to prepare a concentrated solution with a concentration of 50mg / mL. Add 5mg of superoxide dismutase to make the final SOD enzyme activity 500U / mL. Then add 1.725g of EDC and 0.345g of NHC to make the final concentration of EDC 90mmol / L and the final concentration of NHC 30mmol / L. Incubate in a 37℃ water bath and stir at 1100r / min for 20 minutes to crosslink, obtaining a block sample with poor flowability.

[0120] S2: Dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10000 r / min, repeat the dilution and centrifugation 3 times to remove cross-linking agent as much as possible;

[0121] S3: Add 10 times the amount of PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixture, put it into a dialysis bag, and dialyze for 60 hours. Change the dialysis solution every 12 hours. Collect the treated cross-linked solution in the bag to obtain SOD-recombinant collagen complex, which is collagen-modified superoxide dismutase.

[0122] Example 16

[0123] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 1, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0124] Example 17

[0125] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 2, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0126] Example 18

[0127] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 3, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0128] Example 19

[0129] Add 0.5g of trehalose and 0.5g of mannitol to 5g of the collagen cross-linked modified superoxide dismutase obtained in Example 4, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0130] Example 20

[0131] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 5, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0132] Example 21

[0133] Add 0.5g of trehalose and 0.5g of mannitol to 5g of the collagen cross-linked modified superoxide dismutase obtained in Example 6, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0134] Example 22

[0135] Add 0.5g of trehalose and 0.5g of mannitol to 5g of the collagen cross-linked modified superoxide dismutase obtained in Example 7, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0136] Example 23

[0137] Add 0.5g of trehalose and 0.5g of mannitol to 5g of the collagen cross-linked modified superoxide dismutase obtained in Example 8, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, place the freeze-dried solid under a pressure plate and press it flat to obtain a repair patch for oral ulcers.

[0138] Example 24

[0139] Add 0.5g of trehalose and 0.5g of mannitol to 5g of the collagen cross-linked modified superoxide dismutase obtained in Example 9, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0140] Example 25

[0141] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 10, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0142] Example 26

[0143] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 11, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, place the freeze-dried solid under a pressure plate and press it flat to obtain a repair patch for oral ulcers.

[0144] Example 27

[0145] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 12, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0146] Example 28

[0147] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 13, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0148] Example 29

[0149] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 14, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0150] Example 30

[0151] Add 0.5g of trehalose and 0.5g of mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Example 15, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0152] Example 31

[0153] Add 0.5g trehalose and 0.5g mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Comparative Example 1, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0154] Example 32

[0155] Add 0.5g trehalose and 0.5g mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Comparative Example 2, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0156] Example 33

[0157] Add 0.5g trehalose and 0.5g mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Comparative Example 3, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0158] Example 34

[0159] Add 0.5g trehalose and 0.5g mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Comparative Example 4, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0160] Example 35

[0161] Add 0.5g trehalose and 0.5g mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Comparative Example 5, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0162] Example 36

[0163] Add 0.5g trehalose and 0.5g mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Comparative Example 6, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0164] Example 37

[0165] Add 0.5g trehalose and 0.5g mannitol to 5g of collagen cross-linked modified superoxide dismutase obtained in Comparative Example 7, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, press the freeze-dried solid under a pressure plate to flatten it, and obtain a repair patch for oral ulcers.

[0166] Comparative Example 8

[0167] Add 0.5g of trehalose and 0.5g of mannitol to 5g of the collagen cross-linked modified superoxide dismutase obtained in Example 1, stir and mix well to obtain collagen modified superoxide dismutase gel.

[0168] Comparative Example 9

[0169] Add 0.5g trehalose and 0.5g mannitol to 5g superoxide dismutase, stir and mix well, pour into a mold, place in a freeze dryer, freeze dry at -30℃ for 7h, keep at -10℃ for 25h with a vacuum of 10Pa, keep at 30℃ for 30h with a vacuum of 10Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample. Place the freeze-dried solid under a pressure plate and press it flat to obtain a repair patch for oral ulcers.

[0170] Experimental Example 1: Cytotoxicity Test

[0171] L929 cells (P6) in the logarithmic growth phase were plated at a density of 1×10⁻⁶ cells / cells. 4 Cells were cultured at 100 μL per well, with 5 replicates per group, and cultured for 24 h. After co-culturing the sample solution with the cells for 24 h, the culture medium was discarded, and 50 μL of MTT (thiazolyl blue) solution (1 mg / mL) was added to each well. The plates were incubated at 37 ℃ for another 2 h. The MTT solution was discarded, and 100 μL of isopropanol solution was added to each well. The plates were mixed for 10 min. The absorbance was measured on a well titration plate spectrophotometer equipped with a 570 nm filter (reference wavelength 650 nm), and cell viability was calculated. The experimental results are shown in Table 1.

[0172] The blank control group sample solution was MEM complete culture medium without any added samples;

[0173] The experimental group sample solution was the SOD-recombinant collagen complex obtained in Example 1 (200 U / mL).

[0174] The positive control group sample solution was a 99.90% DMSO (dimethyl sulfoxide) solution.

[0175] Table 1 Results of Cytotoxicity Tests

[0176]

[0177] The results show that the collagen-modified superoxide dismutase obtained in this invention has no toxic side effects on human cells and can promote cell growth.

[0178] Experimental Example 2: Cell Migration Assay

[0179] After passaged HaCaT (human immortalized keratinocytes) cells for 2-3 generations, the cell suspension was centrifuged at 1000 rpm for 5 min; the cell suspension was then resuspended in complete culture medium and the density was adjusted to (5~15) × 10⁻⁶. 5Cells / well, 2 mL / well; after 24 h of cell culture, a 200 μL pipette tip was used to gently push downwards to create a longitudinal (horizontal) scratch. The cells were washed three times with PBS to remove the scratched cells. Sample solution was added to the scratch culture medium and the cells were incubated at 37 ℃ in a 5% CO2 incubator. After 0 h, 24 h, and 48 h, images were taken under a 40x microscope with the intersection of the horizontal and vertical scratches as the core. The area of ​​the "scratch" region in the images was quantified and statistically analyzed using software. The cell migration rate was calculated based on the cell area, and the effect of the sample on cell migration was evaluated. The experimental results are shown in Table 2.

[0180] The blank sample solution was serum-free basic DMEM medium without any added samples;

[0181] The experimental group sample solution was 200 U / mL of the SOD-recombinant collagen complex obtained in Example 1.

[0182] Table 2 Results of cell migration assay

[0183]

[0184] The results show that the collagen-modified superoxide dismutase obtained in this invention has a significant promoting effect on the migration of human immortalized keratinocytes, can promote the healing of human epidermal lesions, and has a positive effect on the healing of oral ulcers.

[0185] Experimental Example 3: Enzyme Activity Detection Assay

[0186] S1: Referring to GB / T41906, the method for detecting superoxide dismutase activity, at approximately 25℃, add 2.35 mL of 0.01 mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer solution, 2.00 mL of distilled water, and 0.15 mL of 4.5 mmol / L pyrogallol hydrochloride solution sequentially to a 10 mL colorimetric tube. Immediately after adding the 4.5 mmol / L pyrogallol hydrochloride solution, mix and pour into a cuvette. Measure the absorbance at 325 nm wavelength after 0 s and 1 min, respectively. The difference between the two values ​​is the pyrogallol auto-oxidation rate ΔA. 325 (min-1); This experiment determines △A 325 (min-1) is 0.060;

[0187] S2: Measurement of the sample's inhibition rate of pyrogallol auto-oxidation.

[0188] (1) Add 2.00 mL of sample solution to 2.35 mL of 0.01 mol / L tris(hydroxymethyl)aminomethane hydrochloric acid buffer solution, and then add 0.15 mL of 4.5 mmol / L pyrogallol hydrochloric acid solution to obtain test solution 1;

[0189] (2) After adding the pyrogallol hydrochloric acid solution, mix immediately and pour into a cuvette; measure the absorbance at 0 s and 1 min under a wavelength of 325 nm.

[0190] (3) Take 2.00 mL of test solution 1 and add 2.35 mL of 0.01 mol / L tris(hydroxymethyl)aminomethane hydrochloric acid buffer solution, then add 0.15 mL of 4.5 mmol / L pyrogallol hydrochloric acid solution to obtain test solution 2. After adding the pyrogallol hydrochloric acid solution, mix immediately and pour into a cuvette; measure the absorbance at 0 s and 1 min under a wavelength of 325 nm respectively.

[0191] (4) Repeat step (3) to obtain test solution n until the difference ΔA' between the absorbance values ​​of test solution n at 0s and 1min under a wavelength of 325nm is obtained. 325 (min-1) is 1 / 2△A 325 (min-1), i.e., △A' 325 The enzyme activity was calculated based on a (min-1) of 0.030, and the results are shown in Table 3.

[0192] The blank sample solution consisted of 2.00 mL of distilled water.

[0193] The control group sample solution consisted of 1.98 mL of distilled water and 20.0 μL of a mixture of SOD and recombinant collagen obtained in Comparative Example 1.

[0194] The control group sample solution consisted of 1.98 mL of distilled water and 20.0 μL of a mixture of SOD and recombinant collagen obtained from Comparative Example 1 after being stored at room temperature for 30 days.

[0195] The sample solution for experimental group 1 consisted of 1.98 mL of distilled water and 20.0 μL of the SOD-recombinant collagen complex obtained in Example 1.

[0196] The sample solution in experimental group 2 consisted of 1.98 mL of distilled water and 20.0 μL of the SOD-recombinant collagen complex obtained in Example 1 after being stored at room temperature for 30 days.

[0197] The sample solution in experimental group 3 consisted of 1.98 mL of distilled water and 20.0 μL of the SOD-recombinant collagen complex obtained in Example 1 after being stored at room temperature for 90 days.

[0198] (3) Calculation of results:

[0199]

[0200] In the formula:

[0201] U / mL — SOD enzyme activity units;

[0202] △A 325 —The rate of auto-oxidation of pyrogallol;

[0203] △A' 325 —The test solution inhibits the auto-oxidation rate of pyrogallol;

[0204] V—The volume of enzyme solution obtained from the comparative example or the actual example, in milliliters (mL);

[0205] D—The dilution factor of the enzyme solution obtained in the comparative example or the embodiment;

[0206] 4.5 — Total volume of the reaction solution, in milliliters (mL);

[0207] C—SOD enzyme concentration in the test solution.

[0208] Table 3 Results of enzyme activity detection test

[0209]

[0210] The results show that the enzyme activity of the uncrosslinked SOD and recombinant type I humanized collagen complex decreased rapidly after one month of storage at room temperature in a humid state, and was basically inactive after 30 days. However, after crosslinking, the enzyme activity stability time of the collagen-modified superoxide dismutase obtained in this invention was significantly prolonged. In a humid state, the enzyme activity decreased by about 5% after one month and by about 20% after three months, indicating that it has good stability and can maintain its activity in drugs for a long time to exert its therapeutic effect.

[0211] Example 1

[0212] (1) A traumatic oral ulcer animal model was used. The animal model was established by fixing a glass tube with an inner diameter of 0.6 cm and a length of 3 cm vertically on the oral mucosa of a rabbit. 0.2 mL of 30% glacial acetic acid was injected into the tube. After 30 seconds, the glacial acetic acid was removed with a cotton swab. Ulcers formed the next day, with a diameter of about 0.2~0.3 cm.

[0213] (2) Comparison of ulcer healing: After ulceration, the repair patches obtained in Examples 16-42, bFGF growth factor (control group), and no treatment were applied to the ulcer surface respectively. The ulcer healing area was observed, and the patch was changed and recorded every six hours. The standard for healing was that no mucosal ulceration and hyperemia were observed.

[0214] Table 4 shows the healing status of the repair patches, bFGF growth factor, and ulcers without treatment obtained in Examples 16-37 and Comparative Examples 8-9.

[0215]

[0216] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A collagen-modified superoxide dismutase, characterized in that, The preparation method is as follows: S1: Dissolve collagen in water to prepare a collagen solution, add superoxide dismutase, EDC and NHC in sequence, stir under water bath temperature to crosslink it, and obtain a flowable gel sample. S2: Dilute the gel sample obtained in S1 with water and centrifuge, then collect the lower viscous liquid layer; S3: Add the viscous liquid obtained after centrifugation in S2 to PBS buffer to make a mixture. Place the mixture in a dialysis bag for dialysis and collect the cross-linked liquid in the bag to obtain collagen-modified superoxide dismutase. The collagen is selected from one or more of recombinant type I, type II, type IV, type III and type XVII humanized collagen; The concentration of the collagen solution described in S1 is 50~100mg / mL; The final enzyme activity of the superoxide dismutase described in S1 is 400~1000 U / mL; The molar ratio of EDC to NHS in S1 is 1~3:1; the total content of EDC and NHS is 60~100 mmol / L.

2. The collagen-modified superoxide dismutase according to claim 1, characterized in that, The water bath temperature in S1 is 30~40℃; the stirring speed is 700~1500r / min; and the stirring time is 10~30min.

3. The collagen-modified superoxide dismutase according to claim 1, characterized in that, The volume of water added in S2 is 8 to 12 times the volume of the gel sample; the centrifugation speed is 8000 to 12000 r / min.

4. The collagen-modified superoxide dismutase according to claim 1, characterized in that, The volume of the PBS buffer described in S3 is 8 to 12 times the volume of the viscous liquid.

5. A method for preparing collagen-modified superoxide dismutase as described in any one of claims 1 to 4.

6. The use of a collagen-modified superoxide dismutase as described in any one of claims 1 to 4 in the preparation of an oral ulcer repair drug.

7. The application according to claim 6, characterized in that, The application method is as follows: trehalose and mannitol are added to the obtained collagen-modified superoxide dismutase, stirred and mixed, then poured into a mold for freeze-drying to obtain a sample. The freeze-dried sample is then pressed flat to obtain a repair patch. The mass of trehalose added is 5%~10% of the mass of collagen-modified superoxide dismutase; the mass of mannitol added is 5%~10% of the mass of collagen-modified superoxide dismutase.

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